WO2020092924A1 - Combination therapy for the treatment of egfr tyrosine kinase inhibitor resistant cancer - Google Patents

Combination therapy for the treatment of egfr tyrosine kinase inhibitor resistant cancer Download PDF

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WO2020092924A1
WO2020092924A1 PCT/US2019/059424 US2019059424W WO2020092924A1 WO 2020092924 A1 WO2020092924 A1 WO 2020092924A1 US 2019059424 W US2019059424 W US 2019059424W WO 2020092924 A1 WO2020092924 A1 WO 2020092924A1
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pkc5
egfr
inhibitor
tki
cells
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PCT/US2019/059424
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French (fr)
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Mien-Chie Hung
Pei-Chih LEE
Yueh-Fu FANG
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Board Of Regents, The University Of Texas System
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates generally to the field of medicine. More particularly, it concerns the combination therapy for the treatment of epidermal growth factor (EGFR) tyrosine kinase inhibitor resistant cancer.
  • EGFR epidermal growth factor
  • EGFR-activating mutant non-small cell lung cancer often initially responds well to EGFR tyrosine kinase inhibitors (TKIs) (Haber et al, 2011); however, the disease almost always recurs about 10-33 months of therapy.
  • TKIs EGFR tyrosine kinase inhibitors
  • third-generation TKIs e.g., AZD9291 (osimertinib)
  • AZD9291 osimertinib
  • EGFR C797S mutation e.g., EGFR C797S mutation
  • activation of Akt and MAPK e.g., Akt and MAPK
  • amplification of HER-2, MET, or EGFR e.g., HER-2, MET, or EGFR
  • the present disclosure provides a method for treating cancer in a subject comprising administering an effective amount of a protein kinase C delta (PKC5) inhibitor and/or a phospholipase C gamma (PFCy) inhibitor in combination with an epidermal growth factor (EGFR) tyrosine kinase inhibitor (TKI) to the subject.
  • PLC5 protein kinase C delta
  • PFCy phospholipase C gamma
  • EGFR epidermal growth factor
  • TKI epidermal growth factor
  • the subject is human.
  • the subject is administered the PKC5 inhibitor and EGFR TKI. In certain aspects, the subject is administered the PFCy inhibitor and EGFR TKI. In some aspects, the subject is administered the PKC5 inhibitor, PFCy inhibitor and EGFR TKI.
  • the cancer is an EGFR-mutant cancer.
  • the cancer is an EGFR TKI -resistant cancer.
  • the EGFR-TKI resistant cancer comprises amplification or upregulation of Axl, Her-2, c-Met, Akt, Erk, and/or NF-KB signaling.
  • the EGFR-TKI resistant cancer may comprise an EGFR second-site mutation, such as T790M and/or C797S.
  • the PKC5 inhibitor is a pan-PKC inhibitor.
  • the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893.
  • the sotrastaurin may be administered at a dose of 300-500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day.
  • the PKC5 inhibitor is not enzastaurin.
  • the PFCy inhibitor is U73122.
  • the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib.
  • the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day.
  • the EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
  • the PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI are administered orally, intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.
  • the PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI may be administered intravenously.
  • the PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI may be administered more than once, such as multiple times a day, once daily, once every 2 days, once every 3 days, or once weekly.
  • the PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI are administered concurrently.
  • the PKC5 inhibitor may be administered before or after the EGFR TKI.
  • the method further comprises the step of administering at least one additional therapeutic agent to the subject.
  • the at least one additional therapeutic agent is selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, and immunotherapy.
  • the at least one additional therapeutic agent is an immunomodulator, growth factor, or cytokine.
  • a pharmaceutical composition comprising a PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI.
  • the composition comprises the PKC5 inhibitor and EGFR TKI.
  • the composition comprises the PFCy inhibitor and EGFR TKI.
  • the composition comprises the PKC5 inhibitor, PFCy inhibitor and EGFR TKI.
  • the PKC5 inhibitor is a pan- PKC inhibitor.
  • the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893.
  • the sotrastaurin may be administered at a dose of 300-500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day.
  • the PKC5 inhibitor is not enzastaurin.
  • the PFCy inhibitor is U73122.
  • the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib.
  • the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day.
  • the EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
  • a pharmaceutical composition of the embodiments for use in the treatment of EGFR- resistant cancer is provided herein.
  • Another embodiment provides the use of a therapeutically effective amount of a PKC5 inhibitor, PFOy inhibitor, and/or EGFR TKI for the treatment of EGFR-resistant cancer.
  • the use comprises the PKC5 inhibitor, PFCy inhibitor and EGFR TKI.
  • the PKC5 inhibitor is a pan-PKC inhibitor.
  • the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893.
  • the sotrastaurin may be administered at a dose of 300- 500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day.
  • the PKC5 inhibitor is not enzastaurin.
  • the PFOy inhibitor is U73122.
  • the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib.
  • the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day.
  • the EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
  • compositions comprising a therapeutically effective amount of a PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI for the treatment of EGFR- resistant cancer in a subject.
  • the composition comprises the PKC5 inhibitor and EGFR TKI.
  • the composition comprises the PFCy inhibitor and EGFR TKI.
  • the composition comprises the PKC5 inhibitor, PFCy inhibitor and EGFR TKI.
  • the PKC5 inhibitor is a pan-PKC inhibitor.
  • the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893.
  • the sotrastaurin may be administered at a dose of 300-500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day.
  • the PKC5 inhibitor is not enzastaurin.
  • the PFOy inhibitor is U73122.
  • the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib.
  • the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day.
  • the EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
  • a method of treating cancer a subject comprising administering an effective amount of a PKC5 inhibitor to the subject, wherein the subject has been identified to have PKC5 activation.
  • the EGFR-resistant cancer is NSCFC.
  • PKC5 activation is detected by increased nuclear PKC5 expression as compared to a control.
  • nuclear PKC5 is detected by immunohistochemistry, immunofluorescence, or western blot.
  • the method comprises administering the PKC5 inhibitor and EGFR TKI.
  • the composition comprises the PFCy inhibitor and EGFR TKI.
  • the method comprises administering the PKC5 inhibitor, PFCy inhibitor and EGFR TKI.
  • the PKC5 inhibitor is a pan-PKC inhibitor.
  • the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893.
  • the sotrastaurin may be administered at a dose of 300-500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day.
  • the PKC5 inhibitor is not enzastaurin.
  • the PFOy inhibitor is U73122.
  • the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib.
  • the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day.
  • the EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
  • a further embodiment provides a method of predicting response to an EGFR TKI comprising detecting the level of nuclear PKC5 in a sample, wherein an increased nuclear PKC5 as compared to a control indicates a subject is resistant to the EGFR TKI.
  • nuclear PKC5 is detected by immunohistochemistry, immunofluorescence, or western blot.
  • the method further comprises administering a PKC5 inhibitor and EGFR TKI to the subject identified to be resistant to the EGFR TKI.
  • the method comprises administering the PKC5 inhibitor and EGFR TKI.
  • the composition comprises the PLCy inhibitor and EGFR TKI.
  • the method comprises administering the PKC5 inhibitor, PLCy inhibitor and EGFR TKI.
  • the PKC5 inhibitor is a pan-PKC inhibitor.
  • the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893. The sotrastaurin may be administered at a dose of 300-500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day.
  • the PKC5 inhibitor is not enzastaurin.
  • the PFCy inhibitor is U73122.
  • the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib.
  • the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day.
  • the EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
  • an in vitro method of identifying an EGFR TKI resistant sample comprising: (a) obtaining a cancer sample; and (b) detecting a level of nuclear PKC5 in the sample, wherein an elevated level of nuclear PKC5 indicates the sample is EGFR TKI resistant.
  • nuclear PKC5 is detected by immunohistochemistry, immunofluorescence, or western blot.
  • the method further comprises detecting the level of PLCy.
  • an elevated level of PLCy further indicates the sample is EGFR TKI resistant.
  • FIGS. 1A-1E A TKI-insensitive role of activating-mutant EGFR maintains survival of NSCLC resistant to EGFR TKIs.
  • A Comparison of responses to EGFR depletion and to EGFR kinase inhibition in H1650 cells. Cells were counted after treatment with 1 mM gefitinib (Gef), 0.1 mM erlotinib (Erl), or an EGFR shRNA (El or E2) for the indicated time. Error bars are based on assays that were repeated at least in triplicate and are present for each time point, but nominal in some cases.
  • C Re-expression of either endogenous EGFR (del 19) or kinase-dead (dell9-kd) EGFR reversed EGFR depletion-induced H1650 cell death. H1650 cells infected with EGFR shRNA and re-expressed shRNA-resistant EGFR (rEGFR) variants were counted on day 7.
  • FIGS. 2A-2H is involved in TKI-insensitive pathways of mutant EGFR and confers resistance to EGFR TKIs.
  • A Top, flow diagram of strategies used for establishing (I) scrambled shRNA control (shCtrl), (II) EGFR-depleted, and (III) EGFR- depletion resistant (EDR) stable cells.
  • Bottom flow cytometric analysis of EGFR expression in shCtrl and EDR cells at the end of treatment.
  • B Schematic of antibody array analysis identifying potential mediators.
  • Stable shCtrl cells were treated with or without 1 mM gefitinib for 24 h (1+) and subjected to antibody array analysis for comparison with EGFR- depleted cells (II) and EDR cells (III). Spots of interest were identified by using the following three criteria: (1) the spot was expressed similar levels (between 0.8- 1.2 fold) in control (I) and TKI-treated groups (I+); (2) the difference in expression level of the spots between the EGFR-depleted group (II) and the control group (I) was ⁇ 0. l5-fold; (3) the observed difference in (2) changed in the opposite direction in the EDR group (III) (> 4-fold from the EGFR-depleted group). A total of 27 candidates were thus identified.
  • Phosphorylated and total EGFR status in the indicated groups was determined by Western blot.
  • C Gefitinib dose response in H 1650 cells expressing scrambled shRNA (control), two PKC5 shRNAs, and/or re-expression of shRNA-resistant PKC5 (rPKCd). Each stable cell was treated with gefitinib 36 for 10 days. PKC5 levels in each cell were determined by Western blot.
  • D Sensitivity to sotra in H 1650 cells harboring active (del 19) or inactive (dell9-kd) EGFR. The cells generated for the experiment shown in FIG. 1B were treated with sotra for 10 days and cell viability was assayed. Data are represented as mean ⁇ SD.
  • E Synergistic effects of gefitinib with PKC inhibitor (PKCi) in H1650 cells.
  • Cells were treated with PKCi, sotra (Sotra) or Go-6983 (Go) in combination with gefitinib at the indicated concentrations for 10 days.
  • F Quantification of tumor growth (as represented by luciferase intensity) in intrinsically TKI- resistant xenografts treated as indicated.
  • G The H-score of phosphorylation of Akt, RelA, and, Erk and levels of proliferation marker (Ki67), nuclear and cytosolic PKC5, and phosphorylation of EGFR in Hl650-derived xenograft tumors from mice treated as in (F).
  • H Dose response of drug treatment. Data are represented as mean ⁇ SEM.
  • FIGS. 3A-3H PKC8 is required and sufficient for EGFR TKI-resistance.
  • A The IC50 of gefitinib in GR cells expressing scrambled control shRNA (shCtrl), PKC5 shRNA (shPKCd) or re-expressed shRNA-resistant PKC5 (shPKC5-rPKC5) were measured after treatment with gefitinib for 10 days. PKC5 expression in indicated cells was determined by Western blot.
  • B The IC50 of gefitinib in GR cells was measured after 10 days of treatment with vehicle (control) or sotra. Data are represented as mean ⁇ SD.
  • C Quantification of tumor growth (represented by luciferase intensity) in lung orthotopic xenografts treated as indicated.
  • D, E Gefitinib dose response in TKI-sensitive H3255 (D) and HCC827 (E) cells ectopically expressing PKC5 in vitro.
  • FIGS. 4A-4F Nuclear localization of PKC8 is required for TKI-resistance.
  • PKC5 was identified by immunofluorescence staining as in (A). Botom, the percentage of H1650 cells with high, medium, and low levels of nuclear PKC5 (nPKCd). Relative intensity of nPKCd was determined by fluorescence microscopy. Bar, 10 pm.
  • D PKC5 expression in nuclei and cytosol of H1650 cells after sotra treatment. The levels of PKC5 in nuclear extract (NE) and cytosol extract (CE) were determined by Western blot analysis.
  • E Top, the ICso of gefitinib in cells expressing vector control, wide-type (WT) PKC5, or NLS-mutant PKC5 (NLSml and NLSm3).
  • FIGS. 5A-5D Nuclear localization of PKC8 is induced by EGFR heterodimers in TKI-resistant cells.
  • A Gefitinib induced EGFR interactions with Axl and Her-2. Untreated or gefitinib-treated GR4 and GR10 cell lysates were subject to immunoprecipitation (IP) with EGFR antibody. The IP (left) or cell lysates (right) were then bloted with the indicated antibodies.
  • IP immunoprecipitation
  • the IP left
  • cell lysates (right) were then bloted with the indicated antibodies.
  • B Western blot showing PKC5 expression in nuclear extracts of GR4 and GR10 cells treated with 1 pM gefitinib in combination with 2.5 pM R428 and 5 pM lapatinib (Lapa).
  • Lapa is known to target both Her-2 and EGFR.
  • C Western blot showing phospho-PLCy2 in cells treated as indicated. SE, short exposure; LE, long exposure.
  • D Western blot showing PKC5 expression in NE and phospho40 PLCy2, ERK in whole cell extracts (WCE) of GR4 and GR10 cells treated with U73122 (5 pM).
  • FIGS. 6A-6G nPKCd reduces progression-free survival in patients with naive EGFR-mutant NSCLC treated with a first-line EGFR TKI, and confers resistance of EGFR T790M+ NSCLCs to 3rd generation EGFR TKIs.
  • C Effects of nPKCd on PFS in patients with EGFR-mutantNSCLC treated with a first-line single agent TKI.
  • D, E H1975 (D) and TM0204 PDX (E) tumor growth. Mice bearing tumor were treated with AZD9291, sotra, or the combination. Data are represented as mean ⁇ SEM.
  • FIGS. 7A-7E related to FIG. 1.
  • A Western blot showing Y1068 and Y1086 phosphorylation of EGFR in H 1650 cells treated with gefitinib (gef) or erlotinib (erl) for 1, 5, and 7 days.
  • B KY of gefitinib in HCC827 sensitive and H1650, H1975, and H820 resistant cells.
  • C Re-expression of endogenous EGFR reversed EGFR depletion-induced cell death.
  • H1975 and HCC827 cells were infected with EGFR shRNA and re-expressed shRNA-resistant EGFR (rEGFR) variants, L858R+T790M (H1975) and del 19 (HCC827) in presence or absence of gefitinib and AZD9291, respectively. The cells were counted after treatments for 7 days.
  • D Cells were treated with gefitinib and afatinib for 3 days. Cell number were counted and expressed as percent of control cells and mean ⁇ SD of three independent experiments. ICY of gefitinib and afatinib in HCC827 parental and 15 GR cells were calculated and showed in right.
  • FIGS. 8A-8F (A) The numbers of canonical pathways involving the 27 candidates identified in FIG. 2B. Ingenuity pathway analysis identified a total 32 canonical pathways involving the 27 candidates (Table 3). The canonical pathways involving each individual candidate were counted. (B) The effects of sotrastaurin treatment on T505 phosphorylation of PKC5 in H1650 cells. Western blot showing T505 phosphorylation of PKC5 in H1650 cells treated with sotrastaurin. (C) Images of mice with H1650 tumors at day 14.
  • FIG. 2G Representative IHC images of pAkt, pRelA, pErk, Ki67, PKC5, and pEGFR in H1650 xenografts from mice treated as indicated. Arrows denote representative nuclear PKC5-positive cells. Bar, 50 pm.
  • F Representative IHC images of nPKCd positive PDX tumors. Arrow denotes representative nuclear PKC5-positive cells. Bar, 10 pm.
  • FIGS. 9A-9D (A) Western blot showing pAkt, pErk, pRelA in GR cells treated with the indicated inhibitor(s).
  • B Images of mice with GR6 tumors at day 28.
  • C, D Body weight changes (C) as well as indicators for liver and kidney functions (D) in each treatment group before and after drug treatment for 3 weeks.
  • the normal range of aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), blood urea nitrogen (BUN), and creatinine are 63-253 U/L, 35-90 U/L, 17-38 mg/dl, and 0.3-0.5 mg/dl, respectively.
  • FIGS. 10A-10J (A) Western blots showed total PKC5 expression in HCC827 parental (P) and GR cells. (B) Confocal microscopy analysis of PKC5 localization in H1650 cells in the presence or absence of sotrastaurin treatment with the LS-C 199448 antibody that recognizes the N-terminal region of PKC5 * Bar, 10 pm. (C) Sotrastaurin reduced nuclear PKC5 expression in GR4 and GR6 cells. (D) Sotrastaurin reduced total PKC5 expression in whole cell extracts (WCE) of H 1650, GR4, and GR6 cells.
  • WCE whole cell extracts
  • E Real-time PCR analysis of PKC5 messenger RNA levels in H1650 cells in the presence of sotrastaurin followed by the treatment with or without proteasome inhibitor MG132.
  • F G
  • H Top, sequence alignment of PKC5 nuclear localization signal (NLS) domain across species.
  • WT human wide-type
  • PKC5 catalytic fragment PKC5 catalytic fragment, 5CF.
  • This 5CF containing an NLS sequence, accumulates in the nucleus and plays a pro- apoptotic role (Reyland, 2007).
  • two antibodies, AM82126 (FIG. 4A, 4C, and 4D) and FS-C199448 were used which recognize the C-terminal (aa 500 to C-terminus) and N-terminal (aa 18-67) domain of PKC5, respectively, for immunofluorescence staining.
  • FIGS. 11A-11C (A) The median inhibitory concentrations (ICso) of gefitinib in Axl -positive GR4 and Her-2 -positive GR10 cells measured after 10 days of treatment of R428 (Axli) and Fapatinib (Fapa, Her2i) as well as sotrastaurin and Go6983 (PKCi) in GR4 and GR10 cells with gefitinib.
  • FIGS. 12A-12G (A and B) Western blot (A) and IHC staining (B) in H1650 cells showing the specificity of PKC5 antibody (abeam ab 182126) used for human IHC staining.
  • the samples were H1650 shRNA control (shControl), PKC5-depleted (shPKCd), and re-expressing shRNA-resistant PKC5 cells from left to right in that order. PKC5-depleted samples were used as negative control. Bar, 30 pm.
  • C Representative images of PKC5 by IHC staining in paired pretreatment and resistance specimens of cases 4, 5, 7, and 8 in Table 1. Bar, 50 pm.
  • H1975 cells were treated with AZD9291 for 24 h.
  • H1975 cells were treated with sotrastaurin for 24 h.
  • the cell extracts were subjected to Western bloting.
  • H Left, the H-score of Erk, RelA, and Akt phosphorylation, proliferation marker Ki67, nuclear and cytosolic PKC5, apoptosis (TUNEL), and gH2AC in El 1975 -derived xenograft tumors from mice treated as indicated.
  • Right representative IHC images of pErk, pRelA, pAkt, Ki67, PKC5, TUNEL, and gH2AC in H1975 xenografts from mice treated as indicated. Yellow arrows denote representative nuclear PKC5-positive cells. Bar, 50 pm.
  • FIGS. 13A-13E (A) Western blot showing Y1068 and Y1086 phosphorylation of EGFR in H1650 cells treated with gefrtinib (gef) or erlotinib (erl) for 1, 5, and 7 days. (B) IC50 of gefrtinib in HCC827 sensitive and H1650, H1975, and H820 resistant cells. (C) Re expression of endogenous EGFR reversed EGFR depletion-induced cell death.
  • H1975 and HCC827 cells were infected with EGFR shRNA and re-expressed shRNA-resistant EGFR (rEGFR) variants, L858R+T790M (H1975) and dell9 (HCC827) in presence or absence of gefrtinib and AZD9291, respectively. The cells were counted after treatments for 7 days.
  • D Cells were treated with gefitinib and afatinib for 3 days. Cell number were counted and expressed as percent of control cells and mean ⁇ SD of three independent experiments. IC50 of gefitinib and afatinib in HCC827 parental and 15 GR cells were calculated and showed in right.
  • E Parental and GR cell lysates were subjected to Western blots analysis with the indicated antibodies. Antibodies used correspond to previously reported features of known TKI resistance.
  • FIGS. 14A-14F (A) The numbers of canonical pathways involving the 27 candidates identified in FIG. 2B. Ingenuity pathway analysis identified a total 32 canonical pathways involving the 27 candidates (Table 3). The canonical pathways involving each individual candidate were counted. (B) The effects of sotrastaurin treatment on T505 phosphorylation of PKC5 in H1650 cells. Western blot showing T505 phosphorylation of PKC5 in H1650 cells treated with sotrastaurin. (C) Images of mice with H1650 tumors at day 14. (D) Mice survival in combination group compared to control, gefitinib (Gef) and sotrastaurin (Sotra) alone groups. (E) Related to FIG.
  • FIGS. 15A-15D (A) Western blot showing pAkt, pErk, pRelA in GR cells treated with the indicated inhibitor(s).
  • B Images of mice with GR6 tumors at day 28.
  • C, D Body weight changes (C) as well as indicators for liver and kidney functions (D) in each treatment group before and after drug treatment for 3 weeks.
  • the normal range of aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), blood urea nitrogen (BUN), and creatinine are 63-253 U/L, 35-90 U/L, 17-38 mg/dl, and 0.3-0.5 mg/dl, respectively.
  • FIGS. 16A-16J (A) Western blots showed total PKC5 expression in HCC827 parental (P) and GR cells. (B) Confocal microscopy analysis of PKC5 localization in H1650 cells in the presence or absence of sotrastaurin treatment with the LS-C 199448 antibody that recognizes the N-terminal region of PKC5 * Bar, 10 pm. (C) Sotrastaurin reduced nuclear PKC5 expression in GR4 and GR6 cells. (D) Sotrastaurin reduced total PKC5 expression in whole cell extracts (WCE) of H 1650, GR4, and GR6 cells.
  • WCE whole cell extracts
  • E Real-time PCR analysis of PKC5 messenger RNA levels in H1650 cells in the presence of sotrastaurin followed by the treatment with or without proteasome inhibitor MG132.
  • F G
  • H Top, sequence alignment of PKC5 nuclear localization signal (NLS) domain across species.
  • WT human wide-type
  • PKC5 catalytic fragment a constitutively active catalytic C-terminal fragment
  • 5CF constitutively active catalytic C-terminal fragment
  • This 5CF containing an NLS sequence, accumulates in the nucleus and plays a pro-apoptotic role (Reyland, 2007).
  • AM82126 FIG. 4A, 4C, and 4D
  • LS-C199448 which recognize the C-terminal (aa 500 to C-terminus) and N-terminal (aa 18-67) domain of PKC5, respectively, for immunofluorescence staining.
  • FIGS. 17A-17C (A) The median inhibitory concentrations (IC50) of gefitinib in Axl-positive GR4 and Her-2-positive GR10 cells measured after 10 days of treatment of R428 (Axli) and Lapatinib (Lapa, Her2i) as well as sotrastaurin and Go6983 (PKCi) in GR4 and GR10 cells with gefitinib. (B) Western blot showing phosphorylation of EGFR Y1173, Y845, Y1068, and Y1086 in cells treated as indicated. SE, short exposure; LE, long exposure. (C) Western blot showing phosphorylation of PLCyl in cells treated as indicated.
  • IC50 The median inhibitory concentrations (IC50) of gefitinib in Axl-positive GR4 and Her-2-positive GR10 cells measured after 10 days of treatment of R428 (Axli) and Lapatinib (Lapa, Her2i) as well as sotrasta
  • FIGS. 18A-18H (A and B) Western blot (A) and IHC staining (B) in H1650 cells showing the specificity of PKC5 antibody (abeam ab 182126) used for human IHC staining.
  • the samples were H1650 shRNA control (shControl), PKC5-depleted (shPKCo). and re-expressing shRNA-resistant PKC5 cells from left to right in that order. PKC5-depleted samples were used as negative control. Bar, 30 mih.
  • C Representative images of PKC5 by IHC staining in paired pretreatment and resistance specimens of cases 4, 5, 7, and 8 in Table 1. Bar, 50 mih.
  • D H1975 cells were treated with AZD9291 for 24 h.
  • H1975 cells were treated with sotrastaurin for 24 h.
  • the cell extracts were subjected to Western blotting.
  • H Left, the H-score of Erk, RelA, and Akt phosphorylation, proliferation marker Ki67, nuclear and cytosolic PKC5, apoptosis (TUNEL), and gH2AC in Hl975-derived xenograft tumors from mice treated as indicated.
  • Right representative IHC images of pErk, pRelA, pAkt, Ki67, PKC5, TUNEL, and gH2AC in H1975 xenografts from mice treated as indicated. Yellow arrows denote representative nuclear PKC5-positive cells. Bar, 50 mih.
  • Lung cancer is the leading cancer killer in both men and women in the United States. Nearly 40% of lung cancers are adenocarcinoma, in which EGFR is one of the addicted oncogenes. Lung adenocarcinoma with activating mutations in EGFR often responds to treatment with EGFR tyrosine kinase inhibitors (TKls), but the degrees of tumor regressions are variable and the therapeutic outcomes are invariably limited by the emergence of drug resistance. Although distinct resistant mechanisms were reported in a portion of patients, there is no effective therapy for individuals who develop such resistance.
  • TKls tyrosine kinase inhibitors
  • TKIs EGFR tyrosine kinase inhibitors
  • NSCLC non-small cell lung cancer
  • a clinically-used PKC5 inhibitor such as sotrastaurin
  • TKI in combination with TKI in xenograft mice induced tumor regression in tumors that were TKI resistant.
  • PKC5 activation which was determined by nuclear translocation of PKC5, as well as PLCy overexpression were observed in most TKI-acquired resistant cells, but not in parental cells.
  • nuclear PKC5 in human tumors with acquired resistance to TKI in comparison with their baseline tumors.
  • the nuclear PKC5 in naive tumors was negatively correlated with the tumor response and progression-free survival in patients treated with first-line TKls.
  • PKC5 activation and PLCy overexpression in human lung adenocarcinoma with activating EGFR mutation may serve as markers for resistance to TKIs and be able to stratify patients who will benefit most from combination therapy of the PKC inhibitor with TKI.
  • TKI-inactivated EGFR induces its dimerization with other membrane receptors implicated in TKI resistance to promote PKC5 nuclear translocation.
  • the level of nuclear PKC5 is associated with TKI response in patients.
  • the combined inhibition of PKC5 and EGFR was shown to induce marked regression of resistant tumors with EGFR mutation.
  • the present disclosure provides methods for treating cancer by administering a PKC5 inhibitor and/or PLCy inhibitor to the subject in combination with an EGFR TKI, such as a third generation EGFR TKI.
  • an EGFR TKI such as a third generation EGFR TKI.
  • methods for identifying a subject that is sensitive to EGFR TKI therapy by detecting the level of nuclear PKC5 and/or PFCy expression, wherein the subject is identified to be sensitive if the level of nuclear PKC5 and/or PFCy expression is elevated.
  • “a” or“an” may mean one or more.
  • the words“a” or “an” when used in conjunction with the word“comprising,” the words“a” or “an” may mean one or more than one.
  • the term“patient” or“subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof.
  • the patient or subject is a primate.
  • Non- limiting examples of human patients are adults, juveniles, infants and fetuses.
  • “Treating” or treatment of a disease or condition refers to executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease .
  • Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition,“treating” or“treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.
  • ‘Prevention” or“preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity.
  • Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as l,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalene sulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene- l-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and dicarboxybc acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cin
  • Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases.
  • Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide.
  • Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, L'-mcthylgl ucam i nc and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).
  • A“pharmaceutically acceptable carrier,”“drug carrier,” or simply“carrier” is a pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and/or transporting a chemical agent.
  • Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and/or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery to the specific target sites.
  • carriers include: liposomes, microspheres (e.g., made of poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.
  • determining an expression level means the application of a gene specific reagent such as a probe, primer or antibody and/or a method to a sample, for example a sample of the subject and/or a control sample, for ascertaining or measuring quantitatively, semi-quantitatively or qualitatively the amount of a gene or genes, for example the amount of mRNA.
  • a level of a gene can be determined by a number of methods including for example immunoassays including for example immunohistochemistry, ELISA, Western blot, immunoprecipitation and the like, where a biomarker detection agent such as an antibody for example, a labeled antibody, specifically binds the biomarker and permits for example relative or absolute ascertaining of the amount of polypeptide biomarker, hybridization and PCR protocols where a probe or primer or primer set are used to ascertain the amount of nucleic acid biomarker, including for example probe based and amplification based methods including for example microarray analysis, RT-PCR such as quantitative RT-PCR, serial analysis of gene expression (SAGE), Northern Blot, digital molecular barcoding technology, for example Nanostring mCounterTM Analysis, and TaqMan quantitative PCR assays.
  • immunoassays including for example immunohistochemistry, ELISA, Western blot, immunoprecipitation and the like
  • a biomarker detection agent such as an antibody for
  • mRNA in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples or cells can be applied, such as mRNA in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples or cells.
  • FFPE paraffin-embedded
  • QuantiGene®ViewRNA Affymetrix
  • This system for example can detect and measure transcript levels in heterogeneous samples; for example, if a sample has normal and tumor cells present in the same tissue section.
  • TaqMan probe-based gene expression analysis can also be used for measuring gene expression levels in tissue samples, and for example for measuring mRNA levels in FFPE samples.
  • TaqMan probe-based assays utilize a probe that hybridizes specifically to the mRNA target. This probe contains a quencher dye and a reporter dye (fluorescent molecule) attached to each end, and fluorescence is emitted only when specific hybridization to the mRNA target occurs.
  • the exonuclease activity of the polymerase enzyme causes the quencher and the reporter dyes to be detached from the probe, and fluorescence emission can occur. This fluorescence emission is recorded and signals are measured by a detection system; these signal intensities are used to calculate the abundance of a given transcript (gene expression) in a sample.
  • sample includes any biological specimen obtained from a patient.
  • Samples include, without limitation, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), ductal lavage fluid, nipple aspirate, lymph (e.g., disseminated tumor cells of the lymph node), bone marrow aspirate, saliva, urine, stool (i.e., feces), sputum, bronchial lavage fluid, tears, fine needle aspirate (e.g., harvested by fine needle aspiration that is directed to a target, such as a tumor, or is random sampling of normal cells, such as periareolar), any other bodily fluid, a tissue (e.g., tumor tissue) such as a biopsy of a tumor (e.g., needle biopsy) or a lymph node (e.g., sentinel lymph node biopsy), and cellular extracts thereof.
  • the sample is whole blood, plasma, serum, red blood cells, white blood cells (
  • a“fixed” sample refers to a sample which has undergone preservation.
  • the fixation can terminate any biochemical reactions and increase the tissue’ s stability.
  • Chemical fixation methods can include subjecting the sample to aldehydes, such as formaldehyde or glutaraldehyde, or alcohols, such as methanol or ethanol.
  • the terms “increased”, “elevated”, “overexpress”, “overexpression”, “overexpressed”,“up-regulate”, or“up-regulated” interchangeably refer to a biomarker that is present at a detectably greater level in a biological sample, e.g. plasma, from a patient with cancer, in comparison to a biological sample from a patient without cancer.
  • the term includes overexpression in a sample from a patient with cancer due to transcription, post-transcriptional processing, translation, post-translational processing, cellular localization (e.g, organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a sample from a patient without cancer.
  • Overexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins (i.e., ELISA, immunohistochemical techniques, mass spectroscopy, Luminex® xMAP technology). Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a sample from a patient without cancer. In certain instances, overexpression is l-fold, 2-fold, 3-fold, 4-fold 5, 6, 7, 8, 9, 10, or l5-fold or higher levels of transcription or translation in comparison to a sample from a patient without cancer.
  • the term“detecting” refers to observing a signal from a label moiety to indicate the presence of a biomarker in the sample. Any method known in the art for detecting a particular detectable moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. II. EGFR Tyrosine Kinase Inhibitor Resistance
  • the present disclosure concerns methods for detecting PKC5 and/or PLCy expression to determine if a subject is resistant to EGFR TKIs.
  • the methods concern the detection of PKC5 activation by measuring the level of nuclear PKC5.
  • Nuclear PKC5 may be detected by methods known in the art, such as immunohistochemistry, immunofluorescence, or western blot.
  • An elevated level of nuclear PKC5 and/or PLCy expression can indicate EGFR TKI-resistance, which may be overcome by the administration of a PKC5 and/or PLCy inhibitor.
  • any antibody-based method of detection is contemplated for use with the present methods.
  • the present methods could be used for the detection of immune checkpoint molecules such as by immunoblotting, quantitative EFISA, immunofluorescence (IF) imaging, and IHC staining.
  • IF immunofluorescence
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g. , Nakamura et al. (1987), incorporated herein by reference.
  • sample may refer to a whole organism or a subset of its tissues, cells or component parts.
  • a sample may also refer to a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof.
  • samples include urine, blood, cerebrospinal fluid (CSF), pleural fluid, sputum, and peritoneal fluid, bladder washings, secretions, oral washings, tissue samples, touch preps, or fine-needle aspirates.
  • a sample may be a cell line, cell culture or cell suspension.
  • a sample corresponds to the amount and type of expression products present in a parent cell from which the sample was derived.
  • a sample can be from a human or non-human subject.
  • the sample used for performing antibody-based detection is a formalin fixed paraffin embedded (FFPE) specimen.
  • the sample may comprise body fluids and tissue samples that include but are not limited to blood, tissue biopsies, spinal fluid, meningeal fluid, urine, alveolar fluid.
  • tissue samples that include but are not limited to blood, tissue biopsies, spinal fluid, meningeal fluid, urine, alveolar fluid.
  • the cells can be dissociated by standard techniques known to those skilled in the art. These techniques include but are not limited to trypsin, collagenase or dispase treatment of the tissue.
  • the present methods may be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al, 1990; Abbondanzo et al, 1990; Allred et al, 1990).
  • frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections.
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.
  • the slides may be dried at 40-45°C in an oven overnight and then incubated at 58-65 C for 1-3 hours.
  • the slides can then be deparaffmized with xylene and ethanol and hydrated in distilled H 2 0.
  • Antigen retrieval can be performed in 10 mM citric acid (pH 6.0) in a microwave for 10 min (2 min 1000W, 8 min 200W), cooled down at room temperature for 60 min, and washed with PBS twice.
  • the slides can then be blocked in 3% H 2 0 2 /methanol for 10 min at room temperature and washed with PBS three times.
  • Normal horse serum or goat serum (10% normal serum in PBS) is applied for 30 min in a humid chamber at room temperature and normal serum is wiped off.
  • the primary antibody in applied in a humid chamber at 4°C overnight and then washed with PBS three times.
  • the secondary antibody is applied in a humid chamber for 1 hour at room temperature and then washed with PBS three times.
  • Peroxidase conjugated avidin biotin complex is applied in a humid chamber for 1 hour at room temperature and then washed with PBS three times.
  • AEC chromogen substrate is applied for 5-10 min and washed with distilled H 2 0 three times. The sample is then counterstained with Mayer’ s hematoxylin for 30 seconds and washed with distilled H 2 0 three times.
  • the present disclosure provides methods for the treatment of cancer by the combination of an EGRF TKI and a PKC5 inhibitor, such as sotrastaurin.
  • the combination treatment may comprise the combination of an EGFR TKI and a PLCy inhibitor.
  • the sotrastaurin may be administered at a dose of 300-500 mg per day, with a dose of 800 mg/day as the maximum tolerable dose.
  • the EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib, 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
  • the EGFR TKI for use in the present methods may be selected from the group consisting of: erlotinib, afatinib, Cetuximab, panitumumab, Erlotinib HC1, Gefitinib, Lapatinib, Neratinib, Lifirafenib, HER2-nhibitor-l, Nazartinib, Naquotinib, Canertinib, Lapatinib, AG-490, CP- 724714, Dacomitinib, WZ4002, Sapitinib, CUDC-101, AG-1478, PD153035 HCL, pelitinib, AC480, AEE788, AP26113-analog, OSI-420, WZ3146, WZ8040, AST-1306, Rociletinib, Genisten, Varlitinib, Icotinib, TAK-285, WHI-P154, Daphnetin, PD16839
  • Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor.
  • Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.
  • Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastemas, myelomas, and the like.
  • cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung
  • cancer of the peritoneum gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer)
  • pancreatic cancer cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
  • the combination therapy can be administered once, for a limited time period, or is administered as maintenance therapy (for a longer period of time until the condition is improved, cure, or continuous lifetime subject). Finite periods of time may be 1 week, 2 weeks, 3 weeks, 4 weeks and up to one year, contained in any time period between these values, inclusive of the endpoints included. In some embodiments, the combination therapy may be administered about 1 day, about 3 days, about 1 week, about 10 days, about 2 weeks, about 18 days, about 3 weeks, or any range between any one of these values, inclusive of the endpoints included.
  • the composition is administered once a day to a subject in need thereof. In another embodiment, every other day, one week or once every three days composition. In another embodiment, the composition is administered twice a day. In yet another embodiment, four times a day or three times a day administration of the composition. In another embodiment, the composition is administered least once a day, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In yet another embodiment, the composition is administered least once a day for a long time, such as at least 4, 6, 8, 10, 12 or 24 months. In some embodiments, administration including but not limited to, at least daily frequency is 2, 3 or 4 times the dose of the composition administered l0-50mg. In some embodiments, once a week, once a month, once every other week, or is administered the composition.
  • the PKC5 or PLCy inhibitor and EGFR TKI may be administered orally, intravenously, intraperitoneally, directly by injection to a tumor, topically, or a combination thereof.
  • the PKC5 or PLCy inhibitor and EGFR TKI are administered as a combination formulation.
  • the PKC5 or PLCy inhibitor and EGFR TKI are administered as individual formulations.
  • the PKC5 or PLCy inhibitor and EGFR TKI r are administered sequentially.
  • the PKC5 or PLCy inhibitor and EGFR TKI are administered simultaneously.
  • the methods provided herein further comprise a step of administering at least one additional therapeutic agent to the subject.
  • All additional therapeutic agents disclosed herein will be administered to a subject according to good clinical practice for each specific composition or therapy, taking into account any potential toxicity, likely side effects, and any other relevant factors.
  • the additional therapy may be immunotherapy, radiation therapy, surgery (e.g., surgical resection of a tumor), chemotherapy, bone marrow transplantation, or a combination of the foregoing.
  • the additional therapy may be targeted therapy.
  • the additional therapy is administered before the primary treatment (i.e.. as adjuvant therapy).
  • the additional therapy is administered after the primary treatment (i.e., as neoadjuvant therapy.
  • the additional therapy comprises an immunotherapy.
  • the immunotherapy comprises an immune checkpoint inhibitor.
  • the PKC5 or PLCy inhibitor and EGFR TKI may be administered before, during, after, or in various combinations relative to an additional cancer therapy.
  • the administrations may be in intervals ranging from concurrently to minutes to days to weeks.
  • the PKC5 or PLCy inhibitor and EGFR TKI is provided to a patient separately from an additional therapeutic agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient.
  • chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogue
  • DNA damaging factors include what are commonly known as g-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation, and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • immunotherapies may be used in combination or in conjunction with methods of the embodiments.
  • immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • Rituximab (RITUXAN ® ) is such an example.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells
  • ADCs Antibody-drug conjugates
  • MAbs monoclonal antibodies
  • cell-killing drugs may be used in combination therapies.
  • This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in“armed” MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen.
  • Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index.
  • Exemplary ADC drugs inlcude ADCETRIS ® (brentuximab vedotin) and KADCYLA ® (trastuzumab emtansine or T-DM1).
  • the tumor cell must bear some marker that is amenable to targeting, i.e.. is not present on the majority of other cells.
  • Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, erb b2 and pl55.
  • An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
  • Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-l, MCP-l, IL-8, and growth factors, such as FLT3 ligand.
  • cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN
  • chemokines such as MIP-l, MCP-l, IL-8
  • growth factors such as FLT3 ligand.
  • immunotherapies include immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds); cytokine therapy, e.g., interferons a, b, and g, IL-l, GM-CSF, and TNF; gene therapy, e.g., TNF, IL-l, IL-2, and p53; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti- p 185. It is contemplated that one or more anti -cancer therapies may be employed with the antibody therapies described herein.
  • immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds
  • cytokine therapy e.g., interferons a, b, and g, IL-l, GM-CSF, and T
  • the immunotherapy may be an immune checkpoint inhibitor.
  • Immune checkpoints either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal.
  • Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3 -dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-l), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA).
  • the immune checkpoint inhibitors target the PD-l axis and/or CTLA- 4.
  • the immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies.
  • Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used.
  • alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure.
  • Such alternative and/or equivalent names are interchangeable in the context of the present disclosure.
  • lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.
  • the PD-l binding antagonist is a molecule that inhibits the binding of PD-l to its ligand binding partners.
  • the PD-l ligand binding partners are PDL1 and/or PDL2.
  • a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners.
  • PDL1 binding partners are PD-l and/or B7-1.
  • the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners.
  • a PDL2 binding partner is PD- 1.
  • the antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • the PD-l binding antagonist is an anti -PD-l antibody (e. g. , a human antibody, a humanized antibody, or a chimeric antibody) .
  • the anti-PD-l antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011.
  • the PD-l binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-l binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
  • the PD-l binding antagonist is AMP-224.
  • Nivolumab also known as MDX- 1106-04, MDX-l 106, ONO-4538, BMS-936558, and OPDIVO ® , is an anti-PD-l antibody that may be used.
  • Pembrolizumab also known as MK-3475, Merck 3475, lambrolizumab,
  • KEYTRUDA is an exemplary anti-PD-l antibody.
  • CT-011 also known as hBAT or hBAT-l, is also an anti-PD-l antibody.
  • AMP-224 also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • CD152 cytotoxic T-lymphocyte-associated protein 4
  • the complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006.
  • CTLA-4 is found on the surface of T cells and acts as an“off” switch when bound to CD80 or CD86 on the surface of antigen-presenting cells.
  • CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells.
  • CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells.
  • CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal.
  • Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA- 4, an inhibitory receptor for B7 molecules.
  • the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • an anti-CTLA-4 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody
  • an antigen binding fragment thereof e.g., an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
  • art recognized anti-CTLA-4 antibodies can be used.
  • An exemplary anti-CTLA- 4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen binding fragments and variants thereof.
  • the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab.
  • the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab.
  • the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the above-mentioned antibodies.
  • the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti -cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
  • agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment.
  • additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin.
  • FAKs focal adhesion kinase
  • compositions and formulations comprising a PKC5 or PLCy inhibitor and EGFR TKI and a pharmaceutically acceptable carrier.
  • compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22 nd edition, 2012), in the form of aqueous solutions, such as normal saline (e.g., 0.9%)and human serum albumin (e.g 10%).
  • active ingredients such as an antibody or a polypeptide
  • optional pharmaceutically acceptable carriers Remington's Pharmaceutical Sciences 22 nd edition, 2012
  • aqueous solutions such as normal saline (e.g., 0.9%)and human serum albumin (e.g 10%).
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
  • An article of manufacture or a kit comprising a method of detecting nuclear PKC5 and/or PLCy expression is also provided herein.
  • the kit may further comprise PKC5 and/or PLCy inhibitors.
  • the article of manufacture or kit can further comprise a package insert comprising instructions for using the inhibitors to treat or delay progression of cancer in an individual. Any of the inhibitors described herein may be included in the article of manufacture or kits.
  • Suitable containers include, for example, bottles, vials, bags and syringes.
  • the container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy).
  • the container holds the formulation and the label on, or associated with, the container may indicate directions for use.
  • the article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti -neoplastic agent).
  • Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
  • a TKI-insensitive role of EGFR maintains cell survival of EGFR- mutant NSCLC with TKI resistance: To corroborate the TKI-insensitive role of EGFR in TKI resistance, EGFR was depleted to compare treatment with TKIs in H1650 cells, which harbor EGFR-activating mutation and are resistant to TKIs via mechanisms unrelated to T790M mutation (Bivona et al, 2011; Sos et al, 2009). Interestingly, EGFR knockdown by two specific short hairpin RNAs, shRNA-El and -E2, almost completely inhibited cell growth (FIG. 1A), whereas inhibition of EGFR by treating with TKI, gefitinib or erlotinib, had virtually no effect on cell growth (FIG. 1A and 7A), which was expected.
  • TKIresistant cells sensitized to EGFR depletion suggested an oncogenic addiction via unknown roles of mutant EGFR independently of TKI responsiveness and that roles of EGFR may maintain cancer cell survival by activating downstream signaling, including Akt, Erk, and RelA phosphorylation, in TKI-resistant H1650 cells.
  • endogenous EGFR (del 19, kinase activated) was knocked down by shRNA-El targeting 3 -UTR of EGFR in H1650 cells and then re-expressed it or its corresponding kinase-dead (dell9-kd) form in these cells.
  • HCC827 cells were used as control cells, which require EGFR kinase activity for cell survival, and sensitive to both TKI and EGFR depletion, and thus cannot be rescued by re-expression of the kinase-dead mutant (EGFR-dell9-kd; FIG. 7C, right).
  • EGFR knockdown and rescue experiments in H1650, H1975, and HCC827 cells validated the specificity of EGFR shRNA-El (FIG. 1C and 7C). Together, these results demonstrated a previously undiscovered role of EGFR in the survival of TKI-resistant cells.
  • acquired TKI-resistant clones were generated from TKI-sensitive HCC827 cells by exposing them to 1 mM of gefitinib for 6 months.
  • ICso > 1 mM median inhibitory concentration
  • IC50 -0.006 pM IC50 -0.006 pM
  • Each of the GR clones was also resistant to afatinib, a clinically used irreversible TKI (FIG. 7D, right).
  • Sequence analysis of EGFR in each GR clone revealed that none harbored the resistant T790M mutation.
  • PKC8 serves as a common mediator in TKI-insensitive EGFR pathways and a contributor to TKI resistance: Because EGFR depletion, but not kinase inhibition, sensitized TKI-resistant cells, such as H1650 and all fifteen HCC827-derived GR cell lines, harboring different resistant mechanisms to TKI, it was hypothesized that the TKI- insensitive pathways of EGFR may confer TKI resistance through a common mediator. If so, therapeutic targeting of the common mediator may provide an effective strategy to overcome recurrent disease in patients with TKI-resistant EGFR mutant NSCLC.
  • EDR EGFR-depleted resistant subclone
  • EDR cells After culturing for an additional 7 days, most of the EGFR-depleted cells had died; the few cells that survived were further cultured for more than 3 months and isolated as EDR cells (III, FIG. 2A). EGFR depletion was subsequently validated in EDR cells by immunoblotting (FIG. 2B, bottom, lane 4). Analysis of EGFR knockdown efficiency by flow cytometric analysis also supported the EGFR depletion as only about 10.5% EDR cells expressed EGFR compared with the control shRNA group, which had > 92% EGFR-positive cells (FIG. 2A, bottom).
  • EDR cells maintained similar EGFR depletion level during EDR cell establishment, and suggested the EDR cells could be used to search for survival signaling pathways that are present in the parental H1650 but significantly reduced by EGFR depletion and restored in the EDR clones.
  • the identified pathways may represent survival signaling against the lethality induced by EGFR depletion in EDR cells and serve as ideal therapeutic target to overcome TKI-resistance for the EGFR-mutant NSCLC.
  • NSCLCs with resistance to TKI are NSCLCs with resistance to TKI.
  • M male
  • F female
  • Gef gefitinib
  • Erl erlotinib
  • PR partial response
  • SD stable disease
  • N/A not available.
  • Table 2 27 potential mediators in the TKI-insensitive EGFR pathways identified by antibody array. ratio of shCtrl shCtrl+TKI shEGFR EDR
  • CD 5 (Ab-453) 1.00 1.15 0.14 1.11 8.10 c-Jun (Ab-73) 1.00 1.20 0.05 1.27 26.78
  • Cytokeratin 8 (Ab-431) 1.00 1.16 0.08 0.55 7.12
  • Ephrin B (Ab-330) 1.00 0.99 0.07 1.88 28.43
  • Estrogen Receptor-alpha (Ab-106) 1.00 0.81 0.03 0.93 29.80 FAK (Ab-576)* 1.00 0.85 0.11 0.91 8.00
  • HDAC5 (Ab-498) 1.00 1.04 0.05 0.98 21.25
  • MAP3K7/TAK1 (Ab-439) 1.00 1.13 0.05 0.83 16.26 NFkB-pl05/p50 (Ab-337)* 1.00 0.92 0.03 0.72 24.17
  • PAK1 (Ab-212) 1.00 1.10 0.08 0.76 9.03
  • PKC beta/PKCB Phospho-Ser661
  • PKC delta Phospho-Thr505
  • PLCg2 Ab-1217
  • VASP (Ab-238) 1.00 1.13 0.06 0.64 11.51
  • IL-8 Signaling 9.84E00 F AK,B -Raf, Raf 1 ,c- Jun,PKC delta, NF-kB- pl05/p50, VASP,PKC beta
  • Table 4 Synthetic lethal screen of gefitinib with inhibitors targeting potential mediators or their impacted pathways in H 1650 cells.
  • NSC668036 0.5-50 uM >1.1 IkB kinase BMS-345541 1-30 uM 0.4-1.1
  • Sotrastaurin (AEB071) ** Phase 2 0.5-20 uM *0.1-0.8
  • IWP4 (Porcupine inactivator) 0.5-50 uM 0.5-0.9
  • LGK-974 (Porcupine 0.01-1 uM >1.1 inhibitor)
  • Non-steroidal anti-inflammatoiy drug NSAID
  • combination index Cl
  • PKC8 is required for TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in TKI resistance: To validate the role of PKC5 in
  • PKC5 is known to elicit several survival signaling pathways in cancer cells (Basu and Pal, 2010), and interestingly, Erk/MAPK, PI3K, and NF-kB signaling were among those identified in the first screen (Tables 2 and 3).
  • Akt activation is associated with TKI resistance in PTEN-loss H1650 cells (Sos et al, 2009). Therefore, Akt, Erk, and NF-kB phosphorylation was also examined in tumors treated with the gefitinib-sotra combination. Single treatment of gefitinib or sotra reduced phosphorylation of AKT, RelA, and ERK (FIG. 2G and 8E), suggesting either EGFR or PKC5 are potential upstream molecules of these survival signaling.
  • the effective doses of the gefitinib-sotra combination did not significantly affect mouse body weight or the values of the indicators of liver and kidney functions (FIG. 9C and D). These results suggested that the gefitinib-sotra combination at the doses administered may be a safe and effective therapeutic strategy to treat EGFR-mutant NSCLC with TKI resistance. Since gefitinib has received regulatory approval in NSCLC patients, and PKCi sotra is available for clinical studies, the combination could be readily tested in clinical trials, especially for patients whose tumor has developed resistance to TKIs.
  • PKC8 is sufficient to induce TKI resistance: Next, to determine whether ectopic expression of PKC5 in TKI-sensitive NSCLC cells is sufficient to induce gefitinib resistance, PKC5-ectopic expressing stable clones were established from two TKI sensitive H3255 and HCC827 cells. Ectopic expression of PKC5 significantly induced resistance to gefitinib in vitro (FIG. 3D and 3E) and in vivo (FIG. 3F and 3G). Notably, TKI- induced cleaved PARP (cPARP, a marker for apoptosis) was abolished by ectopic PKC5 expression (FIG. 3H, lane 4 vs. 2). Thus, enhanced expression of PKC5 may protect TKI- sensitive cells from TKI-induced apoptosis in EGFR-mutant NSCLC and is sufficient to cause TKI resistance.
  • cPARP a marker for apoptosis
  • PKC5 nuclear localization of PKC8 is present in multiple TKI-resistant NSCLC cells and contributes to TKI resistance: PKC5 is activated in specific subcellular compartments, such as the nucleus (Mochly-Rosen et al , 2012). To determine the molecular mechanism underlying the contribution of PKC5 to TKI resistance, the expression and subcellular distribution of PKC5 was compared between GR and parental (gefitinib-sensitive) HCC827 cells. Western blot analysis indicated that the total expression of PKC5 did not change significantly in both GR and parental HCC827 cells (FIG. 10A).
  • nPKCd PKC5 nuclear localization
  • NLS sequence was first at the C-terminal of human PKC5 using in silico analysis of NLS (FIG. 10H, top) and showed that the NLS sequence was highly conserved among different species.
  • T505A phosphorylation-defective
  • T505D 17 and T505E phosphorylation-mimic mutant PKC5
  • FIG. 10F immunofluorescence staining
  • TKI-insensitive EGFR pathways contribute to the heterogeneity of TKI resistance mechanisms via nPKCb upregulation:
  • EGFR was knocked down in H1650 cells and compared the effects on nPKCd to TKI treatment. Immunofluorescence staining showed that EGFR depletion, but not kinase inhibition, reduced nPKCd (FIG. 4C, lane 3 vs. 2), suggesting an unknown TKI-insensitive role of EGFR in promoting nPKCd in TKIresistant NSCFC.
  • EGFR is a membrane-bound receptor that can interact with other RTKs, such as Her-2 and Axl, which limits the sensitivity to anti -EGFR therapies (Hirsch et al , 2009; Meyer et al, 2013).
  • RTKs such as Her-2 and Axl
  • these RTKs have been implicated in PKC5 activation (Allen-Petersen et al., 2014; Elkabets et al , 2015).
  • FIG. 4C indicated that EGFR knockdown, which eliminated all of EGFR pathways, but not TKI, which only reduced kinase-dependent activity, suppressed nPKCd levels.
  • EGFR phosphorylation status was examined by a Human EGFR Phosphorylation Antibody Array in GR4 cells treated with TKI (gefitinib) and Axl inhibitor (R428).
  • EGFR Yl 173 when phosphorylated, functions as a docking site for phospholipase Cy (PLCy) (Chattopadhyay et ai , 1999) which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2), resulting in the production of the secondary messengers diacyl glycerol (DAG) and inositol 1,4, 5 -triphosphate (IP3). DAG activates isozymes of the PKC family, including PKC5 (Rosse el al.. 2010).
  • EGFR-Y1173- PFOy2-nPKC5 is a common axis of TKI-insensitive EGFR pathways that contributes to the heterogeneity of TKI resistance.
  • nPKCd is upregulated in human EGFR-mutant NSCLCs with acquired TKI resistance and correlates with poor survival in EGFR-mutant NSCLC patients treated with first-line single agent TKI: To strengthen the findings that nPKCd is upregulated in TKI-resistant cells and confers TKI resistance, nPKCd status was examined by immunohistochemistry (IHC) in matched pretreatment and TKI-resistant EGFR-activating mutation-harboring NSCFC specimens from 41 patients (Tables 1, and FIG. 12A, 12B, 12C). All of these patients were treated with erlotinib or gefitinib and had met the established clinical definition of acquired resistance to TKI.
  • IHC immunohistochemistry
  • nPKCd was present in more than 40% of their resistant tumors (17/41, 41.5%). Twelve among the resistant tumors (29.3%) had higher nPKCd expression levels than their matched pretreatment tumors (FIG. 6A), suggesting acquired TKI resistance in these tumors. The other five nPKCd-positive tumors (-12.2%, 3 high, 1 medium, and 1 low level of nPKCd) had similar nPKCd levels compared with their matched pretreatment tumors (FIG. 6A), suggesting the role of nPKCd in intrinsic TKI- resistance.
  • nPKCd was examined in a larger cohort including 166 naive tumors from patients with EGFR-mutant NSCLC treated with single-agent gefitinib, erlotinib, or afatinib as first-line therapy. Analysis of nPKCd expression in these TKI- naive tumor samples showed that nPKCd was highly expressed in 19 (11.4%) of the 166 patients (FIG. 6B and Table 5), well consistent with the -12% from the previous cohort (FIG. 6A). Furthermore, high expression of nPKCd in these 19 patients was associated with worse progression-free survival following TKI treatment (FIG. 6C).
  • nPKCd may contribute to both acquired (-29.3%, FIG. 6A) and intrinsic (-11.4%; FIG. 6B) resistance to TKI and that it may be a common mechanism underlying TKI resistance in human EGFR-mutant NSCLC.
  • Table 5 Objective response and PFS of 19 patients with high nPKCd tumors.
  • Gef gefitinib
  • Erl erlotinib
  • SD stable disease
  • PR partial response
  • PD progression disease
  • NA data not available
  • nPKCd induces resistance to third-generation TKI in T790M+ tumors:
  • Third-generation TKIs are currently the most potent anti-cancer drugs against TKI- resistant EGFR-mutant NSCLC with T790M mutation (Rotow and Bivona, 2017). From the results above, it was noticed that T790M-positive (T790M+) patients (case 6 and 9 in Table 1), who may be considered for third-generation TKI treatment, concurrently harbored increased nPKCd in their resistant tumors (Table 1). Co-occurrence of EGFR T790M mutation and reactivation of other resistant RTKs, such as Axl, was reported in TKI-treated NSCLC (Zhang el al , 2012).
  • mice with H1975 tumor as well as the T790M+PDX (TM0204) tumor harboring the EGFR dell9/T790M mutation and PKC5-resistant feature positive nPKCd staining; FIG. 8F, right.
  • the AZD929l-sotra combination effectively led to tumor regression in H1975 and TM0204 PDX models but not sotra or AZD9291 (partially delayed tumor growth) alone (FIG. 6D and 6E).
  • nPKCb is upregulated in TKI-resistant tumors
  • nPKCb expression levels were detected by IHC staining in two untreated control tumors and five lst generation TKI erlotinib resistant tumors from genetically engineered EGFR dell 9-mutant mice as well as in four untreated control tumors and two 3rd generation TKI AZD9291 -resistant tumors from EGFR L858R T790M mutant mice (Ji et al. , 2006; Li el al.
  • nPKCb renders NSCLC tumors resistant to 3rd generation TKI and that sotra and AZD9291 prevent tumor growth in heterogenous T790M+ tumor models with AZD9291 resistance in a cooperative manner.
  • nPKCd immunoreactivity was ranked as previously described (Lo et al, 2005; Lo et al, 2007; Xia et al, 2004). Briefly, nPKCd immunoreactivity was categorized into four groups (score 0, 1, 2, and 3) according to a well- established system in which H score was generated by the percentage of positive tumor cells. The scores with their indicating percentage of positive cells are score 0 (0%), 1 (less than 50%), 2 (51-75%), 3 (more than 75%).
  • DNA fragment containing EGFR exons 20 were amplified with intron-based primers EGFR-20F (5 - GTCCCTGTGCTAGGTCTTTT-3 ' (SEQ ID NO: l)) and EGFR-20R (5 - ATCTCCCTTCCCTGATTAC-3 ' (SEQ ID NO:2)). PCR reaction was performed at 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s, 56 °C for 30 s, and 72 °C for 30 s, then by 10 min extension at 72 °C.
  • PCR products were bidirectional sequenced on ABI 3730 XL sequencers (Applied Biosystems) with ABI BigDye Terminator v3.1 Cycle Sequencing Kits and analyzed by Chromas Sequence Scanner Software.
  • GenBank NM_005228 was used as the reference DNA for nucleotide positions.
  • Hl650/luc and GR6/luc cells were injected directly into the right chest of B ALB/c nude mice (six week-old, female). Tumor volume as indicated by luciferase intensity was measured by an IVIS system on the days shown. H1975 cells were inoculated subcutaneously into nude mice. TM0204 PDX bearing mice were purchased from Jackson Laboratory. Tumor-bearing mice were randomized and drugs administered according to treatment group. Gefitinib (5 mg/kg/day), AZD9291 (1 mg/kg/day, ⁇ 6% clinically equivalent dose), and sotrastaurin (30 mg/kg/day, 30-50% clinically equivalent dose) were administered orally five times per week (1 week equaled one treatment cycle) and continued for indicated cycles.
  • sotrastaurin Treatment of sotrastaurin (AEB071) in patients has been shown to be well tolerated (Martin-Liberal et al, 2014). For instance, previous studies suggested that the clinical activity of sotrastaurin without toxicity in uveal melanoma patients treated with multiple concentrations of sotrastaurin (800 mg/day as maximum tolerable dose) (Pipemo-Neumann et al, 2014).
  • HCC827-vector and HCC827-PKC5 cells were inoculated subcutaneously into the hind limbs of NSG mice. Tumor-bearing mice were randomized and treated with gefitinib (50 mg/kg/day). Data represent mean ⁇ SEM. Immunohistochemical staining was performed as previously described (Shen et al, 2013). All tumors after drug treatment for 5 days were collected for immunohistochemical staining. All animal procedures were conducted under the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at MD Anderson Cancer Center (Protocol Number 06-87-06139).
  • IACUC Institutional Animal Care and Use Committee
  • Human NSCLC cell lines (H1650, HCC827, H1975, and H820) were obtained from ATCC. H1650, HCC827, H1975, and H820 and the corresponding subclones were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS).
  • Human NSCLC cell line H3255 was a gift from Dr. Zhen Fan and were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS). All cell lines have been tested for mycoplasma contamination and were validated by short tandem repeat (STR) DNA fingerprinting using the AmpFLSTR® Identifiler® PCR Amplification Kit (Life Technologies Grand Island, NY).
  • STR short tandem repeat
  • HCC827 gefitinib-resistant cells were generated by continuous (> 2 months) culture in standard RPMI medium in the presence of 1 mM gefitinib, followed by single-cell cloning.
  • EDR cells were generated from H1650 cells depleted of EGFRby lentiviral infection.
  • H1650 cells were infected with viruses overnight in the presence of polybrene (10 pg/mL), then cultured in fresh medium for 24 h and subsequently selected by puromycin (2 pg/mL) for 2 days. The cells were then subcultured and maintained in 1 pg/mL puromycin. After 7 days, most of the cells had died; the few that survived were then cultured with 1 mg/mL puromycin for 3 more months to obtain the EDR clone.
  • EGFR antibody (ab-l2) was obtained from Thermo Scientific; phospho-EGFR (#2234), cleaved PARP (#9541), phospho-p44/42 MAPK (Erkl/2) (Thr202/Tyr204) (D13.14.4E, #4370p), phospho-Akt (ser473) (#927ls), Akt (#9272s), phospho-NF-kB p65 (Ser 536) (7F1, #3036s), IkBa (44D4, #48 l2s), Ki67 (#9027s), gH2AC (97l8s) antibodies from Cell Signaling Technology; PKC5 antibody (EPR17075, abl82l26) from Abeam.
  • Gefitinib, erlotinib, and SP600125 were purchased from LC Laboratories; Edelfosine from R & D Systems; FIPI, NFAT inhibitor, IWP-2, and IWP-4 from Cayman Chemical; VU0359595, aspirin, sulindac, PNU-74654, resveratrol, and NSC 668036 from Sigma-Aldrich; LGK-974 from Xcess Biosciences.
  • Dvl-PDZ Domain Inhibitor II was obtained from EMD Millipore; Go6983, U73122, afatinib, BMS-345541, KN-62, ICG-001, and AZD9291 from Selleck Chemicals; Sotrastaurin from Chemscene.
  • both WT and K721A mutant were used as template to further prepare the exon 19 deletion mutant lacking amino acids 722- 726 (ELREA) using the same kit to generate the EGFR-dell9 and EGFR-dell9-KD constructs, respectively.
  • PKC5-NLSml and NLSm3 was generated from human wide-type PKC5 vector. Each construct was verified by sequencing before use. Human EGFR shRNAs and scrambled control shRNA were constructed and described previously ⁇ . Human PKC5 shRNAs were obtained from Sigma-Aldrich.
  • Cell counting and cell viability assays Cellular responses to the treatments were estimated by cell counting or cell viability assay. To count the cells with a hemocytometer, cells were seeded on six-well plates (5 c 104 cells/well) and cultured for the indicated period. For the synthetic lethal screen, H1650 cells were seeded in 24-well plates in RPMI 1640 medium containing 10% FBS overnight, then treated with the respective agent(s) for 3 days. Viable cells were identified by the Cell Counting Kit-8 (Donjindo) according to the manufacturer's protocol. For the validated cell viability assays, cells were seeded in 24-well plates in RPMI 1640 medium containing 10% FBS overnight, then treated with the respective agent(s).
  • IP Immunoprecipitation
  • WB Western blot analysis
  • cells were washed twice with phosphate-buffered saline solution (PBS), lysed in lysis buffer, briefly sonicated, and then subjected to IP-WB.
  • PBS phosphate-buffered saline solution
  • proteins were separated by sodium dodecyl sulfate electrophoresis on a 10% or 12% polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Invitrogen). After overnight incubation with primary antibody, washing, and incubation with secondary antibodies, blots were developed with a chemiluminescence system (Pierce).
  • PKC8 Protein Kinase C8 (PKC8) kinase activity assay: PKC5 was immunoprecipitated (IP) from HCC827 cells expressing WT PKC5 or NLS mutant (NLSml and NLSm3) and immunoprecipitates were then subjected to Western blot (WB) analysis and PKC kinase activity assay using a PKC kinase activity kit (Enzo Life Sciences, ADI-EKS50 420A). The PKC activities measured were normalized to the quantitated levels of PKC5 protein expression from IP-WB.
  • Antibody array The Phospho-Explorer Antibody Microarray was purchased from Full Moon Biosystems. Microarray images were analyzed with the GenePixTM Pro 4.0 image analysis software. Fluorescence intensity measurements were normalized against local background, and cytoskeletal antibodies (b-actin and GAPDH) were used for normalization of total protein quantity between samples.
  • Biological network and pathway analysis Biological networks and pathways related to the 27 mediators were analyzed with Ingenuity Pathway Analysis (IP A) software (Qiagen). All mediators identified by the antibody array analysis were uploaded into the IPA software. For the analysis of networks and pathways, the cutoff values were set as p ⁇ 10 8
  • Confocal microscopy analysis was performed as described previously. Briefly, drug-treated cells were washed with PBS and fixed in 100% methanol for 20 min at -20 °C. Cells were then subjected to permeabilization with 0.5% Triton X-100 with 3% bovine serum albumin overnight at 4 °C. After that, cells were incubated with primary antibodies overnight at 4 °C, washed with PBS and further incubated with the appropriate secondary antibody. Nuclei were counterstained with 4,6-diamidino-2- phenylindole (DAPI) before mounting. Confocal fluorescence images were captured using a Zeiss LSM710 laser microscope. The relative intensity of PKC5 in nuclei to that in the whole cell was determined by Image J version 1.49 software.

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Abstract

Provided herein are methods of treating EGFR-resistant cancer by administering a protein kinase C delta (PKC5) inhibitor and/or a phospholipase C gamma (PLCγ) inhibitor in combination with an epidermal growth factor (EGFR) tyrosine kinase inhibitor (TKI). Further provided herein are methods of identifying a subject as EGFR TKI resistant.

Description

DESCRIPTION
COMBINATION THERAPY FOR THE TREATMENT OF EGFR TYROSINE
KINASE INHIBITOR RESISTANT CANCER
[0001] This application claims the benefit of United States Provisional Application No. 62/754,889, filed November 2, 2018, the entirety of which is incorporated herein by reference.
INCORPORATION OF SEQUENCE FISTING
[0002] The sequence listing that is contained in the file named “UTFCPl404WO_ST25.txt”, which is 1 KB (as measured in Microsoft Windows®) and was created on November 1, 2019, is filed herewith by electronic submission and is incorporated by reference herein.
BACKGROUND
1. Field
[0003] The present invention relates generally to the field of medicine. More particularly, it concerns the combination therapy for the treatment of epidermal growth factor (EGFR) tyrosine kinase inhibitor resistant cancer.
2. Description of Related Art
[0004] EGFR-activating mutant non-small cell lung cancer (NSCLC) often initially responds well to EGFR tyrosine kinase inhibitors (TKIs) (Haber et al, 2011); however, the disease almost always recurs about 10-33 months of therapy. Analysis of clinical specimens indicated that TKI-resistant NSCLCs harbor multiple acquired-resistance mechanisms, including amplification or upregulation of Axl, Her-2, c-Met, Akt, Erk, and NF-kB signaling, and EGFR second-site mutation T790M (Rotow and Bivona, 2017). To overcome T790M- mediated resistance, third-generation TKIs, e.g., AZD9291 (osimertinib), were developed and showed promising results (Janne et al, 2015), but virtually all tumors eventually develop resistance after about 10 months of treatment (Minari et al, 2016). Likewise, tumors from patients who failed AZD9291 treatment also harbor similar mechanisms underlying disease progression, e.g., EGFR C797S mutation, activation of Akt and MAPK, and amplification of HER-2, MET, or EGFR, which are highly heterogeneous even within an individual patient (Minari et al, 2016; Piotrowska et al, 2015). [0005] In addition to acquired resistance, intrinsic resistance, which by definition is a lack of response or an initial response with tumor re-progression < 4 months (Park et al. , 2014), may also be attributed to heterogeneous resistant mechanisms. Previous in vitro studies indicated that EGFR amplification in EGFR-mutant NSCFC cells causes resistance to an irreversible TKI (Ercan et al, 2010). Those findings raise the interesting question of whether and how the expression level of EGFR per se plays a role in resistance to EGFR kinase inhibition. The heterogeneity of TKI resistance remains a challenge in treating TKI-intrinsic or acquired resistant EGFR mutant non-small cell lung cancer (NSCFC). Thus, identification of a common and targetable mediator involved in the TKI-insensitive EGFR pathways may provide a treatment strategy to overcome disease recurrence.
SUMMARY
[0006] In one embodiment, the present disclosure provides a method for treating cancer in a subject comprising administering an effective amount of a protein kinase C delta (PKC5) inhibitor and/or a phospholipase C gamma (PFCy) inhibitor in combination with an epidermal growth factor (EGFR) tyrosine kinase inhibitor (TKI) to the subject. In some aspects, the cancer is lung cancer, such as non-small cell lung cancer (NSCFC). In some aspects, the subject is human.
[0007] In some aspects, the subject is administered the PKC5 inhibitor and EGFR TKI. In certain aspects, the subject is administered the PFCy inhibitor and EGFR TKI. In some aspects, the subject is administered the PKC5 inhibitor, PFCy inhibitor and EGFR TKI.
[0008] In certain aspects, the cancer is an EGFR-mutant cancer. In particular aspects, the cancer is an EGFR TKI -resistant cancer. In certain aspects, the EGFR-TKI resistant cancer comprises amplification or upregulation of Axl, Her-2, c-Met, Akt, Erk, and/or NF-KB signaling. The EGFR-TKI resistant cancer may comprise an EGFR second-site mutation, such as T790M and/or C797S.
[0009] In certain aspects, the PKC5 inhibitor is a pan-PKC inhibitor. For example, the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893. The sotrastaurin may be administered at a dose of 300-500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day. In particular aspects, the PKC5 inhibitor is not enzastaurin. In particular aspects, the PFCy inhibitor is U73122. [0010] In some aspects, the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib. In particular aspects, the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day. For example, The EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
[0011] In some aspects, the PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI are administered orally, intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. The PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI may be administered intravenously. The PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI may be administered more than once, such as multiple times a day, once daily, once every 2 days, once every 3 days, or once weekly. In certain the PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI are administered concurrently. The PKC5 inhibitor may be administered before or after the EGFR TKI.
[0012] In additional aspects, the method further comprises the step of administering at least one additional therapeutic agent to the subject. In some aspects, the at least one additional therapeutic agent is selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, and immunotherapy. In some apsects, the at least one additional therapeutic agent is an immunomodulator, growth factor, or cytokine.
[0013] In a further embodiment, there is provided a pharmaceutical composition comprising a PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI. In some aspects, the composition comprises the PKC5 inhibitor and EGFR TKI. In certain aspects, the composition comprises the PFCy inhibitor and EGFR TKI. In some aspects, the composition comprises the PKC5 inhibitor, PFCy inhibitor and EGFR TKI. In certain aspects, the PKC5 inhibitor is a pan- PKC inhibitor. For example, the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893. The sotrastaurin may be administered at a dose of 300-500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day. In particular aspects, the PKC5 inhibitor is not enzastaurin. In particular aspects, the PFCy inhibitor is U73122. In some aspects, the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib. In particular aspects, the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day. For example, The EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib. Further provided herein is a pharmaceutical composition of the embodiments for use in the treatment of EGFR- resistant cancer.
[0014] Another embodiment provides the use of a therapeutically effective amount of a PKC5 inhibitor, PFOy inhibitor, and/or EGFR TKI for the treatment of EGFR-resistant cancer. In some aspects, the use comprises the PKC5 inhibitor, PFCy inhibitor and EGFR TKI. In certain aspects, the PKC5 inhibitor is a pan-PKC inhibitor. For example, the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893. The sotrastaurin may be administered at a dose of 300- 500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day. In particular aspects, the PKC5 inhibitor is not enzastaurin. In particular aspects, the PFOy inhibitor is U73122. In some aspects, the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib. In particular aspects, the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day. For example, The EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
[0015] Further provided herein is a composition comprising a therapeutically effective amount of a PKC5 inhibitor, PFCy inhibitor, and/or EGFR TKI for the treatment of EGFR- resistant cancer in a subject. In some aspects, the composition comprises the PKC5 inhibitor and EGFR TKI. In certain aspects, the composition comprises the PFCy inhibitor and EGFR TKI. In some aspects, the composition comprises the PKC5 inhibitor, PFCy inhibitor and EGFR TKI. In certain aspects, the PKC5 inhibitor is a pan-PKC inhibitor. For example, the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893. The sotrastaurin may be administered at a dose of 300-500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day. In particular aspects, the PKC5 inhibitor is not enzastaurin. In particular aspects, the PFOy inhibitor is U73122. In some aspects, the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib. In particular aspects, the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day. For example, The EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
[0016] In another embodiment, there is provided a method of treating cancer a subject comprising administering an effective amount of a PKC5 inhibitor to the subject, wherein the subject has been identified to have PKC5 activation. In particular aspects, the EGFR-resistant cancer is NSCFC. [0017] In some aspects, PKC5 activation is detected by increased nuclear PKC5 expression as compared to a control. In certain aspects, nuclear PKC5 is detected by immunohistochemistry, immunofluorescence, or western blot. In some aspects, the method comprises administering the PKC5 inhibitor and EGFR TKI. In certain aspects, the composition comprises the PFCy inhibitor and EGFR TKI. In some aspects, the method comprises administering the PKC5 inhibitor, PFCy inhibitor and EGFR TKI. In certain aspects, the PKC5 inhibitor is a pan-PKC inhibitor. For example, the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893. The sotrastaurin may be administered at a dose of 300-500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day. In particular aspects, the PKC5 inhibitor is not enzastaurin. In particular aspects, the PFOy inhibitor is U73122. In some aspects, the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib. In particular aspects, the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day. For example, The EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
[0018] A further embodiment provides a method of predicting response to an EGFR TKI comprising detecting the level of nuclear PKC5 in a sample, wherein an increased nuclear PKC5 as compared to a control indicates a subject is resistant to the EGFR TKI. In some aspects, nuclear PKC5 is detected by immunohistochemistry, immunofluorescence, or western blot.
[0019] In additional aspects, the method further comprises administering a PKC5 inhibitor and EGFR TKI to the subject identified to be resistant to the EGFR TKI.
[0020] In some aspects, the method comprises administering the PKC5 inhibitor and EGFR TKI. In certain aspects, the composition comprises the PLCy inhibitor and EGFR TKI. In some aspects, the method comprises administering the PKC5 inhibitor, PLCy inhibitor and EGFR TKI. In certain aspects, the PKC5 inhibitor is a pan-PKC inhibitor. For example, the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893. The sotrastaurin may be administered at a dose of 300-500 mg/day, such as 325, 350, 375, 400, 425, 450, 475, or 500 mg/day. In particular aspects, the PKC5 inhibitor is not enzastaurin. In particular aspects, the PFCy inhibitor is U73122. In some aspects, the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib. In particular aspects, the EGFR TKI is administered at a dose of 50-300 mg/day, such as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/day. For example, The EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib), 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
[0021] In another embodiment, there is provided an in vitro method of identifying an EGFR TKI resistant sample comprising: (a) obtaining a cancer sample; and (b) detecting a level of nuclear PKC5 in the sample, wherein an elevated level of nuclear PKC5 indicates the sample is EGFR TKI resistant. In some aspects, nuclear PKC5 is detected by immunohistochemistry, immunofluorescence, or western blot. In additional aspects, the method further comprises detecting the level of PLCy. In some aspects, an elevated level of PLCy further indicates the sample is EGFR TKI resistant.
[0022] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0024] FIGS. 1A-1E: A TKI-insensitive role of activating-mutant EGFR maintains survival of NSCLC resistant to EGFR TKIs. (A) Comparison of responses to EGFR depletion and to EGFR kinase inhibition in H1650 cells. Cells were counted after treatment with 1 mM gefitinib (Gef), 0.1 mM erlotinib (Erl), or an EGFR shRNA (El or E2) for the indicated time. Error bars are based on assays that were repeated at least in triplicate and are present for each time point, but nominal in some cases. (B) Effects of protein knockdown and kinase inhibition of EGFR on survival signaling in TKI-resistant H1650 cells. Western blots showing phosphorylation of Akt, Erk, and RelA in H1650 cells treated with gefitinib, control shRNA, or EGFR shRNA-El. (C) Re-expression of either endogenous EGFR (del 19) or kinase-dead (dell9-kd) EGFR reversed EGFR depletion-induced H1650 cell death. H1650 cells infected with EGFR shRNA and re-expressed shRNA-resistant EGFR (rEGFR) variants were counted on day 7. Phosphorylated and total EGFR status was determined by Western blot in the cells 2 days after lentiviral infection. (D) Validation of the effects of EGFR depletion in NSCLC cell lines with various EGFR mutations and TKI responses. Cells were counted, and phosphorylated and total EGFR status was determined by Western blot in HCC827, H1650, H1975, and H820 cells after treatments as in (A). S, sensitive; R, resistant. (E) Responses to EGFR depletion in 15 clones with acquired gefitinib resistance (GR). Previously established resistant features are detected by WB (FIG. 7E) and indicated at the bottom. Each 35 GR cell group was counted as described in (A). Data are represented as mean ± SD.
[0025] FIGS. 2A-2H: is involved in TKI-insensitive pathways of mutant EGFR and confers resistance to EGFR TKIs. (A) Top, flow diagram of strategies used for establishing (I) scrambled shRNA control (shCtrl), (II) EGFR-depleted, and (III) EGFR- depletion resistant (EDR) stable cells. Bottom, flow cytometric analysis of EGFR expression in shCtrl and EDR cells at the end of treatment. (B) Schematic of antibody array analysis identifying potential mediators. Stable shCtrl cells (I) were treated with or without 1 mM gefitinib for 24 h (1+) and subjected to antibody array analysis for comparison with EGFR- depleted cells (II) and EDR cells (III). Spots of interest were identified by using the following three criteria: (1) the spot was expressed similar levels (between 0.8- 1.2 fold) in control (I) and TKI-treated groups (I+); (2) the difference in expression level of the spots between the EGFR-depleted group (II) and the control group (I) was < 0. l5-fold; (3) the observed difference in (2) changed in the opposite direction in the EDR group (III) (> 4-fold from the EGFR-depleted group). A total of 27 candidates were thus identified. Phosphorylated and total EGFR status in the indicated groups was determined by Western blot. (C) Gefitinib dose response in H 1650 cells expressing scrambled shRNA (control), two PKC5 shRNAs, and/or re-expression of shRNA-resistant PKC5 (rPKCd). Each stable cell was treated with gefitinib 36 for 10 days. PKC5 levels in each cell were determined by Western blot. (D) Sensitivity to sotra in H 1650 cells harboring active (del 19) or inactive (dell9-kd) EGFR. The cells generated for the experiment shown in FIG. 1B were treated with sotra for 10 days and cell viability was assayed. Data are represented as mean ± SD. (E) Synergistic effects of gefitinib with PKC inhibitor (PKCi) in H1650 cells. Cells were treated with PKCi, sotra (Sotra) or Go-6983 (Go) in combination with gefitinib at the indicated concentrations for 10 days. Cell viability was assayed and combination indexes (Cl) were then calculated, where Cl < 0.3 indicates strong synergy; Cl = 0.3-0.9, synergy; Cl = 0.9-1.1, additive; and Cl > 1.1, antagonism. (F) Quantification of tumor growth (as represented by luciferase intensity) in intrinsically TKI- resistant xenografts treated as indicated. Mice with orthotopic H1650 tumors were imaged and treated with 5 mg/kg gefitinib alone (-10% clinically equivalent dose, n = 9) per day, 30 mg/kg sotra alone (-30-50% clinically equivalent dose, n = 9) per day, or the combination (n = 9). **p < 0.01, for the gefitinib-sotra combination versus untreated control (n = 9), gefitinib alone, and sotra alone. (G) The H-score of phosphorylation of Akt, RelA, and, Erk and levels of proliferation marker (Ki67), nuclear and cytosolic PKC5, and phosphorylation of EGFR in Hl650-derived xenograft tumors from mice treated as in (F). (H) Dose response of drug treatment. Data are represented as mean ± SEM.
[0026] FIGS. 3A-3H: PKC8 is required and sufficient for EGFR TKI-resistance.
(A) The IC50 of gefitinib in GR cells expressing scrambled control shRNA (shCtrl), PKC5 shRNA (shPKCd) or re-expressed shRNA-resistant PKC5 (shPKC5-rPKC5) were measured after treatment with gefitinib for 10 days. PKC5 expression in indicated cells was determined by Western blot. (B) The IC50 of gefitinib in GR cells was measured after 10 days of treatment with vehicle (control) or sotra. Data are represented as mean ± SD. (C) Quantification of tumor growth (represented by luciferase intensity) in lung orthotopic xenografts treated as indicated. Mice with established GR6 tumors were imaged every 7 days after oral treatment with gefitinib alone (n = 9), sotra alone (n = 9), or the combination (n = 9) as in FIG. 2F. Data are represented as mean ± SEM. ** p < 0.01, for the gefitinib-sotra combination versus untreated control (n = 9), gefitinib alone, and sotra alone. (D, E) Gefitinib dose response in TKI-sensitive H3255 (D) and HCC827 (E) cells ectopically expressing PKC5 in vitro. Vector control and PKC5- expressing H3255 and HCC827 cells were treated with gefitinib at the doses shown and cell viability was then assayed. Top, expression of PKC5 in cells expressing vector control or PKC5. (F) Effects of ectopic expression of PKC5 on gefitinib sensitivity in 38 HCC827 xenograft tumors compared to vector control tumors. Established tumors (250-300 mm3, n = 7 per treatment group) were randomized and treated for 3 days with 50 mg/kg gefitinib per day. (G) Left, the percentage of proliferation (Ki67) and nuclear PKC5-positive cells in HCC827- derived xenograft tumors from mice treated with or without gefitinib. Right, representative IHC staining of tumors from the experiment in F. Bar, 50 pm. (H) Expression of indicated proteins in HCC827 expressing control vector or PKC5 in the presence or absence of gefitinib was determined by Western blot.
[0027] FIGS. 4A-4F: Nuclear localization of PKC8 is required for TKI-resistance.
(A) Confocal microscopy analysis showed significant PKC5 nuclear localization in GR cells compared to HCC827 parental cells. PKC5 was determined in HCC827 and GR cells by immunofluorescence staining. Nuclei were counterstained with DAPI (blue). Bar, 10 pm. (B) PKC5 expression in nuclear extracts (NE) of parental and GR cells was determined by Western blot analysis. (C) Effects of gefitinib, EGFR shRNA, and sotra and Go6983 (Go), on subcellular localization of PKC5 in H1650 cells. Cells were treated with gefitinib, EGFR shRNA, sotra and Go6983. Top, at the end of treatment, PKC5 was identified by immunofluorescence staining as in (A). Botom, the percentage of H1650 cells with high, medium, and low levels of nuclear PKC5 (nPKCd). Relative intensity of nPKCd was determined by fluorescence microscopy. Bar, 10 pm. (D) PKC5 expression in nuclei and cytosol of H1650 cells after sotra treatment. The levels of PKC5 in nuclear extract (NE) and cytosol extract (CE) were determined by Western blot analysis. (E) Top, the ICso of gefitinib in cells expressing vector control, wide-type (WT) PKC5, or NLS-mutant PKC5 (NLSml and NLSm3). Data are represented as mean ± SD. Botom, nPKCd was identified by immunofluorescence staining as in (A). Bar, 50 pm. (F) Western blots showing pAkt, pErk, pRelA, pT505 PKC5, and total PKC5 expression in HCC827 expressing vector control, WT PKC5, NLSml, or NLSm3 PKC5 mutants in present of gefitinib.
[0028] FIGS. 5A-5D: Nuclear localization of PKC8 is induced by EGFR heterodimers in TKI-resistant cells. (A) Gefitinib induced EGFR interactions with Axl and Her-2. Untreated or gefitinib-treated GR4 and GR10 cell lysates were subject to immunoprecipitation (IP) with EGFR antibody. The IP (left) or cell lysates (right) were then bloted with the indicated antibodies. (B) Western blot showing PKC5 expression in nuclear extracts of GR4 and GR10 cells treated with 1 pM gefitinib in combination with 2.5 pM R428 and 5 pM lapatinib (Lapa). Lapa is known to target both Her-2 and EGFR. (C) Western blot showing phospho-PLCy2 in cells treated as indicated. SE, short exposure; LE, long exposure. (D) Western blot showing PKC5 expression in NE and phospho40 PLCy2, ERK in whole cell extracts (WCE) of GR4 and GR10 cells treated with U73122 (5 pM).
[0029] FIGS. 6A-6G: nPKCd reduces progression-free survival in patients with naive EGFR-mutant NSCLC treated with a first-line EGFR TKI, and confers resistance of EGFR T790M+ NSCLCs to 3rd generation EGFR TKIs. (A) Among the 41 matched EGFR-mutant NSCLC specimens from patients before single-agent TKI treatment(pretreatment) and after development of resistance to these drugs (resistance) as in Table 1, 17 patients whose resistance (R) tumor samples had detectable nPKCd (nPKCd scored 3 as high level, 2 as medium and 1 as low) presented a higher level of nPKC5 (12 of 41 cases, 29.3%) or a similar level of nPKCd (5 of 41, 12.2%) as their matched pretreatment (Pre) tumors. (B) Median progression-free survival (PFS) and 95% confidence intervals of low nPKCd (score = 0 and 1) and high nPKCd (score = 2 and 3) groups and the entire cohort. (C) Effects of nPKCd on PFS in patients with EGFR-mutantNSCLC treated with a first-line single agent TKI. (D, E) H1975 (D) and TM0204 PDX (E) tumor growth. Mice bearing tumor were treated with AZD9291, sotra, or the combination. Data are represented as mean ± SEM. (F) The H-score of nuclear PKC5, in untreated and TKI-resistant tumors from genetically engineered EGFR del 19 (left) and EGFR L858R T790M (right) mice. (G) Model for the development of heterogeneous resistances to TKI and the proposed therapeutic strategies against resistant tumors in EGFR-mutant NSCLC. Patients with resistant tumors C and D harboring additional EGFR mutations, such as T790M and C797S, respectively, could be treated with next generation TKI, whereas for resistant tumor A and B, multiple combination treatments of the same TKI with specific kinase inhibitors targeting single resistant pathway were unsuccessful in clinical due to tumor heterogeneity of resistance to TKI. Inhibition of PKC5, a common mediator involved in multiple resistant mechanisms, such as Axl and Her-2 upregulation, has potential to overcome such heterogeneous resistance through combination with the same TKI.
[0030] FIGS. 7A-7E: related to FIG. 1. (A) Western blot showing Y1068 and Y1086 phosphorylation of EGFR in H 1650 cells treated with gefitinib (gef) or erlotinib (erl) for 1, 5, and 7 days. (B) KY of gefitinib in HCC827 sensitive and H1650, H1975, and H820 resistant cells. (C) Re-expression of endogenous EGFR reversed EGFR depletion-induced cell death. H1975 and HCC827 cells were infected with EGFR shRNA and re-expressed shRNA-resistant EGFR (rEGFR) variants, L858R+T790M (H1975) and del 19 (HCC827) in presence or absence of gefitinib and AZD9291, respectively. The cells were counted after treatments for 7 days. (D) Cells were treated with gefitinib and afatinib for 3 days. Cell number were counted and expressed as percent of control cells and mean ± SD of three independent experiments. ICY of gefitinib and afatinib in HCC827 parental and 15 GR cells were calculated and showed in right. (E) Parental and GR cell lysates were subjected to Western blots analysis with the indicated antibodies. Antibodies used correspond to previously reported features of known TKI resistance. [0031] FIGS. 8A-8F: (A) The numbers of canonical pathways involving the 27 candidates identified in FIG. 2B. Ingenuity pathway analysis identified a total 32 canonical pathways involving the 27 candidates (Table 3). The canonical pathways involving each individual candidate were counted. (B) The effects of sotrastaurin treatment on T505 phosphorylation of PKC5 in H1650 cells. Western blot showing T505 phosphorylation of PKC5 in H1650 cells treated with sotrastaurin. (C) Images of mice with H1650 tumors at day 14. (D) Mice survival in combination group compared to control, gefitinib (Get) and sotrastaurin (Sotra) alone groups. (E) Related to FIG. 2G. Representative IHC images of pAkt, pRelA, pErk, Ki67, PKC5, and pEGFR in H1650 xenografts from mice treated as indicated. Arrows denote representative nuclear PKC5-positive cells. Bar, 50 pm. (F) Representative IHC images of nPKCd positive PDX tumors. Arrow denotes representative nuclear PKC5-positive cells. Bar, 10 pm.
[0032] FIGS. 9A-9D: (A) Western blot showing pAkt, pErk, pRelA in GR cells treated with the indicated inhibitor(s). (B) Images of mice with GR6 tumors at day 28. (C, D) Body weight changes (C) as well as indicators for liver and kidney functions (D) in each treatment group before and after drug treatment for 3 weeks. The normal range of aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), blood urea nitrogen (BUN), and creatinine are 63-253 U/L, 35-90 U/L, 17-38 mg/dl, and 0.3-0.5 mg/dl, respectively.
[0033] FIGS. 10A-10J: (A) Western blots showed total PKC5 expression in HCC827 parental (P) and GR cells. (B) Confocal microscopy analysis of PKC5 localization in H1650 cells in the presence or absence of sotrastaurin treatment with the LS-C 199448 antibody that recognizes the N-terminal region of PKC5 * Bar, 10 pm. (C) Sotrastaurin reduced nuclear PKC5 expression in GR4 and GR6 cells. (D) Sotrastaurin reduced total PKC5 expression in whole cell extracts (WCE) of H 1650, GR4, and GR6 cells. (E) Real-time PCR analysis of PKC5 messenger RNA levels in H1650 cells in the presence of sotrastaurin followed by the treatment with or without proteasome inhibitor MG132. (F, G) Western blots showing PKC5 protein (F) and ubiquitination (G) levels in H 1650 cells in presence of sotrastaurin followed by the treatment with or without MG132. (H) Top, sequence alignment of PKC5 nuclear localization signal (NLS) domain across species. Bottom, NLS of human wide-type (WT) PKC5 and NLS mutants. Red letters indicate the mutated amino acid residues within the NLS. (I, J) Analysis of PKC5 kinase activity in HCC827 cells expressing endogenous PKC5 (Vector), exogenous WT PKC5, PKC5 mutant NLSml, or PKC5 mutant NLSm3. (I) PKC5 protein expression levels (top) and the raw PKC5 activities (bottom) normalized to PKC5 activity by protein expression level (J). (K) Western blot analysis of total PKC5 and pT505 PKC5 expression in HCC827 cells expressing vector, WT, T505A, T505D, or T505E mutant PKC5. (L) Confocal microscopy analysis of PKC5 localization in HCC827 expressing WT, T505A, T505D, or T505E mutant PKC5. Bar, 20 pm. (M) Western blot analysis of pT505 PKC5 expression in ectopic WT PKC5 expressing HCC827 cells treated with gefitinib. * Previous study reported a cleaved form of PKC5, 5CF, which is proteolytically cleaved by caspase 3 at the hinge region, in response to apoptotic stimulus (Reyland, 2007). The cleavage of PKC5 separates the regulatory domain (N-terminal) from the catalytic domain (C-terminal) and releases a constitutively active catalytic C-terminal fragment (PKC5 catalytic fragment, 5CF). This 5CF, containing an NLS sequence, accumulates in the nucleus and plays a pro- apoptotic role (Reyland, 2007). To confirm full-length PKC5 in our study, two antibodies, AM82126 (FIG. 4A, 4C, and 4D) and FS-C199448 were used which recognize the C-terminal (aa 500 to C-terminus) and N-terminal (aa 18-67) domain of PKC5, respectively, for immunofluorescence staining.
[0034] FIGS. 11A-11C: (A) The median inhibitory concentrations (ICso) of gefitinib in Axl -positive GR4 and Her-2 -positive GR10 cells measured after 10 days of treatment of R428 (Axli) and Fapatinib (Fapa, Her2i) as well as sotrastaurin and Go6983 (PKCi) in GR4 and GR10 cells with gefitinib. (B) Western blot showing phosphorylation of EGFR Y1173, Y845, Y1068, and Y1086 in cells treated as indicated. SE, short exposure; LE, long exposure. (C) Western blot showing phosphorylation of PLCyl in cells treated as indicated.
[0035] FIGS. 12A-12G: (A and B) Western blot (A) and IHC staining (B) in H1650 cells showing the specificity of PKC5 antibody (abeam ab 182126) used for human IHC staining. The samples were H1650 shRNA control (shControl), PKC5-depleted (shPKCd), and re-expressing shRNA-resistant PKC5 cells from left to right in that order. PKC5-depleted samples were used as negative control. Bar, 30 pm. (C) Representative images of PKC5 by IHC staining in paired pretreatment and resistance specimens of cases 4, 5, 7, and 8 in Table 1. Bar, 50 pm. (D) H1975 cells were treated with AZD9291 for 24 h. The cell extracts were subjected to Western bloting. (E) Synergistic effects of sotrastaurin (Sotra) with mutant- selective AZD9291 (AZD) in T790M-mutant H1975 cells. Cells were treated with AZD9291 and/or sotrastaurin at the concentrations shown for 15 days. Cell viability* was assayed and the value of combination index (Cl) calculated as shown in FIG. 2E. Data are expressed as percentage of control cells and mean ± s.d. of three independent experiments. (F) Confocal microscopy analysis showing PKC5 localization in H1975 cells in absence or presence of sotrastaurin treatment. Bar, 10 pm. (G) H1975 cells were treated with sotrastaurin for 24 h. The cell extracts were subjected to Western bloting. (H) Left, the H-score of Erk, RelA, and Akt phosphorylation, proliferation marker Ki67, nuclear and cytosolic PKC5, apoptosis (TUNEL), and gH2AC in El 1975 -derived xenograft tumors from mice treated as indicated. Right, representative IHC images of pErk, pRelA, pAkt, Ki67, PKC5, TUNEL, and gH2AC in H1975 xenografts from mice treated as indicated. Yellow arrows denote representative nuclear PKC5-positive cells. Bar, 50 pm.
[0036] FIGS. 13A-13E: (A) Western blot showing Y1068 and Y1086 phosphorylation of EGFR in H1650 cells treated with gefrtinib (gef) or erlotinib (erl) for 1, 5, and 7 days. (B) IC50 of gefrtinib in HCC827 sensitive and H1650, H1975, and H820 resistant cells. (C) Re expression of endogenous EGFR reversed EGFR depletion-induced cell death. H1975 and HCC827 cells were infected with EGFR shRNA and re-expressed shRNA-resistant EGFR (rEGFR) variants, L858R+T790M (H1975) and dell9 (HCC827) in presence or absence of gefrtinib and AZD9291, respectively. The cells were counted after treatments for 7 days. (D) Cells were treated with gefitinib and afatinib for 3 days. Cell number were counted and expressed as percent of control cells and mean ± SD of three independent experiments. IC50 of gefitinib and afatinib in HCC827 parental and 15 GR cells were calculated and showed in right. (E) Parental and GR cell lysates were subjected to Western blots analysis with the indicated antibodies. Antibodies used correspond to previously reported features of known TKI resistance.
[0037] FIGS. 14A-14F: (A) The numbers of canonical pathways involving the 27 candidates identified in FIG. 2B. Ingenuity pathway analysis identified a total 32 canonical pathways involving the 27 candidates (Table 3). The canonical pathways involving each individual candidate were counted. (B) The effects of sotrastaurin treatment on T505 phosphorylation of PKC5 in H1650 cells. Western blot showing T505 phosphorylation of PKC5 in H1650 cells treated with sotrastaurin. (C) Images of mice with H1650 tumors at day 14. (D) Mice survival in combination group compared to control, gefitinib (Gef) and sotrastaurin (Sotra) alone groups. (E) Related to FIG. 2G. Representative IHC images of pAkt, pRelA, pErk, Ki67, PKC5, and pEGFR in H1650 xenografts from mice treated as indicated. Arrows denote representative nuclear PKC5-positive cells. Bar, 50 pm. (F) Representative IHC images of nPKCd positive PDX tumors. Arrow denotes representative nuclear PKC5-positive cells. Bar, 10 pm.
[0038] FIGS. 15A-15D: (A) Western blot showing pAkt, pErk, pRelA in GR cells treated with the indicated inhibitor(s). (B) Images of mice with GR6 tumors at day 28. (C, D) Body weight changes (C) as well as indicators for liver and kidney functions (D) in each treatment group before and after drug treatment for 3 weeks. The normal range of aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), blood urea nitrogen (BUN), and creatinine are 63-253 U/L, 35-90 U/L, 17-38 mg/dl, and 0.3-0.5 mg/dl, respectively.
[0039] FIGS. 16A-16J: (A) Western blots showed total PKC5 expression in HCC827 parental (P) and GR cells. (B) Confocal microscopy analysis of PKC5 localization in H1650 cells in the presence or absence of sotrastaurin treatment with the LS-C 199448 antibody that recognizes the N-terminal region of PKC5 * Bar, 10 pm. (C) Sotrastaurin reduced nuclear PKC5 expression in GR4 and GR6 cells. (D) Sotrastaurin reduced total PKC5 expression in whole cell extracts (WCE) of H 1650, GR4, and GR6 cells. (E) Real-time PCR analysis of PKC5 messenger RNA levels in H1650 cells in the presence of sotrastaurin followed by the treatment with or without proteasome inhibitor MG132. (F, G) Western blots showing PKC5 protein (F) and ubiquitination (G) levels in H 1650 cells in presence of sotrastaurin followed by the treatment with or without MG132. (H) Top, sequence alignment of PKC5 nuclear localization signal (NLS) domain across species. Bottom, NLS of human wide-type (WT) PKC5 and NLS mutants. Red letters indicate the mutated amino acid residues within the NLS. (I, J) Analysis of PKC5 kinase activity in HCC827 cells expressing endogenous PKC5 (Vector), exogenous WT PKC5, PKC5 mutant NLSml, or PKC5 mutant NLSm3. (I) PKC5 protein expression levels (top) and the raw PKC5 activities (bottom) normalized to PKC5 activity by protein expression level (J). (K) Western blot analysis of total PKC5 and pT505 PKC5 expression in HCC827 cells expressing vector, WT, T505A, T505D, or T505E mutant PKC5. (L) Confocal microscopy analysis of PKC5 localization in HCC827 expressing WT, T505A, T505D, or T505E mutant PKC5. Bar, 20 pm. (M) Western blot analysis of pT505 PKC5 expression in ectopic WT PKC5 expressing HCC827 cells treated with gefitinib.* Previous study reported a cleaved form of PKC5, 5CF, which is proteolytically cleaved by caspase 3 at the hinge region, in response to apoptotic stimulus (Reyland, 2007). The cleavage of PKC5 separates the regulatory domain (N-terminal) from the catalytic domain (C-terminal) and releases a constitutively active catalytic C-terminal fragment (PKC5 catalytic fragment, 5CF). This 5CF, containing an NLS sequence, accumulates in the nucleus and plays a pro-apoptotic role (Reyland, 2007). To confirm full- length PKCo in the study, two antibodies were used, AM82126 (FIG. 4A, 4C, and 4D) andLS-C199448, which recognize the C-terminal (aa 500 to C-terminus) and N-terminal (aa 18-67) domain of PKC5, respectively, for immunofluorescence staining.
[0040] FIGS. 17A-17C: (A) The median inhibitory concentrations (IC50) of gefitinib in Axl-positive GR4 and Her-2-positive GR10 cells measured after 10 days of treatment of R428 (Axli) and Lapatinib (Lapa, Her2i) as well as sotrastaurin and Go6983 (PKCi) in GR4 and GR10 cells with gefitinib. (B) Western blot showing phosphorylation of EGFR Y1173, Y845, Y1068, and Y1086 in cells treated as indicated. SE, short exposure; LE, long exposure. (C) Western blot showing phosphorylation of PLCyl in cells treated as indicated.
[0041] FIGS. 18A-18H: (A and B) Western blot (A) and IHC staining (B) in H1650 cells showing the specificity of PKC5 antibody (abeam ab 182126) used for human IHC staining. The samples were H1650 shRNA control (shControl), PKC5-depleted (shPKCo). and re-expressing shRNA-resistant PKC5 cells from left to right in that order. PKC5-depleted samples were used as negative control. Bar, 30 mih. (C) Representative images of PKC5 by IHC staining in paired pretreatment and resistance specimens of cases 4, 5, 7, and 8 in Table 1. Bar, 50 mih. (D) H1975 cells were treated with AZD9291 for 24 h. The cell extracts were subjected to Western blotting. (E) Synergistic effects of sotrastaurin (Sotra) with mutant- selective AZD9291 (AZD) in T790M-mutant H1975 cells. Cells were treated with AZD9291 and/or sotrastaurin at the concentrations shown for 15 days. Cell viability* was assayed and the value of combination index (Cl) calculated as shown in FIG. 2E. Data are expressed as percentage of control cells and mean ± s.d. of three independent experiments. (F) Confocal microscopy analysis showing PKC5 localization in H1975 cells in absence or presence of sotrastaurin treatment. Bar, 10 mih. (G) H1975 cells were treated with sotrastaurin for 24 h. The cell extracts were subjected to Western blotting. (H) Left, the H-score of Erk, RelA, and Akt phosphorylation, proliferation marker Ki67, nuclear and cytosolic PKC5, apoptosis (TUNEL), and gH2AC in Hl975-derived xenograft tumors from mice treated as indicated. Right, representative IHC images of pErk, pRelA, pAkt, Ki67, PKC5, TUNEL, and gH2AC in H1975 xenografts from mice treated as indicated. Yellow arrows denote representative nuclear PKC5-positive cells. Bar, 50 mih. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0042] Lung cancer is the leading cancer killer in both men and women in the United States. Nearly 40% of lung cancers are adenocarcinoma, in which EGFR is one of the addicted oncogenes. Lung adenocarcinoma with activating mutations in EGFR often responds to treatment with EGFR tyrosine kinase inhibitors (TKls), but the degrees of tumor regressions are variable and the therapeutic outcomes are invariably limited by the emergence of drug resistance. Although distinct resistant mechanisms were reported in a portion of patients, there is no effective therapy for individuals who develop such resistance.
[0043] Multiple mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKIs) have been identified in EGFR-mutant non-small cell lung cancer (NSCLC); however, recurrent resistance to EGFR TKIs due to the heterogeneity of the mechanisms underlying resistance within a single patient remains a major challenge in the clinic. It was hypothesized that EGFR- mutant NSCLC is addicted to EGFR via the well-known kinase-mediated downstream signaling (TKI sensitive) and additional unknown roles of EGFR (TKI insensitive), and that the TKI-insensitive EGFR pathways, including multiple known resistant mechanisms, contribute to the heterogeneity of TKI resistance.
[0044] The present studies discovered that depletion of total EGFR, as opposed to using an EGFR kinase inhibitor, is an effective therapy to avoid TKI resistance in EGFR-mutant lung adenocarcinoma, since both kinase -dependent and -independent roles of EGFR are essential for maintaining the tumor growth. In addition, phospholipase C gamma (PLCy) and protein kinase C delta (PKC5) are common targetable modifiers for initial tumor response and acquired drug -resistance to TKI. It was found that inhibition of PLCy and PKC5 using small molecule inhibitors enhanced the killing effects of TKI treatment in the tumor cells with acquired resistance to TKI in vitro.
[0045] A clinically-used PKC5 inhibitor, such as sotrastaurin, in combination with TKI in xenograft mice induced tumor regression in tumors that were TKI resistant. Moreover, PKC5 activation, which was determined by nuclear translocation of PKC5, as well as PLCy overexpression were observed in most TKI-acquired resistant cells, but not in parental cells. In addition, it was found that there was a higher level of nuclear PKC5 in human tumors with acquired resistance to TKI in comparison with their baseline tumors. Moreover, the nuclear PKC5 in naive tumors was negatively correlated with the tumor response and progression-free survival in patients treated with first-line TKls. Together, PKC5 activation and PLCy overexpression in human lung adenocarcinoma with activating EGFR mutation may serve as markers for resistance to TKIs and be able to stratify patients who will benefit most from combination therapy of the PKC inhibitor with TKI.
[0046] Specifically, the present studies demonstrated that TKI-inactivated EGFR induces its dimerization with other membrane receptors implicated in TKI resistance to promote PKC5 nuclear translocation. Moreover, the level of nuclear PKC5 is associated with TKI response in patients. The combined inhibition of PKC5 and EGFR was shown to induce marked regression of resistant tumors with EGFR mutation.
[0047] Thus, in certain embodiments, the present disclosure provides methods for treating cancer by administering a PKC5 inhibitor and/or PLCy inhibitor to the subject in combination with an EGFR TKI, such as a third generation EGFR TKI. Further provided herein are methods for identifying a subject that is sensitive to EGFR TKI therapy by detecting the level of nuclear PKC5 and/or PFCy expression, wherein the subject is identified to be sensitive if the level of nuclear PKC5 and/or PFCy expression is elevated.
I. Definitions
[0048] As used herein the specification,“a” or“an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word“comprising,” the words“a” or “an” may mean one or more than one.
[0049] The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.” As used herein “another” may mean at least a second or more. The terms “about”, “substantially” and “approximately” mean, in general, the stated value plus or minus 5%.
[0050] As used herein, the term“patient” or“subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non- limiting examples of human patients are adults, juveniles, infants and fetuses. [0051] “Treating” or treatment of a disease or condition refers to executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease . Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition,“treating” or“treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.
[0052] The term“effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating or preventing a disease, is an amount sufficient to effect such treatment or prevention of the disease.
[0053]‘‘Prevention” or“preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.
[0054] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
[0055]“Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as l,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalene sulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene- l-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and dicarboxybc acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methane sulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, />-chlorobenzene sulfonic acid, phenyl-substituted alkanoic acids, propionic acid, /Molucncsulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, L'-mcthylgl ucam i nc and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).
[0056] A“pharmaceutically acceptable carrier,”“drug carrier,” or simply“carrier” is a pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and/or transporting a chemical agent. Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and/or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery to the specific target sites. Examples of carriers include: liposomes, microspheres (e.g., made of poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.
[0057] The term “determining an expression level” as used herein means the application of a gene specific reagent such as a probe, primer or antibody and/or a method to a sample, for example a sample of the subject and/or a control sample, for ascertaining or measuring quantitatively, semi-quantitatively or qualitatively the amount of a gene or genes, for example the amount of mRNA. For example, a level of a gene can be determined by a number of methods including for example immunoassays including for example immunohistochemistry, ELISA, Western blot, immunoprecipitation and the like, where a biomarker detection agent such as an antibody for example, a labeled antibody, specifically binds the biomarker and permits for example relative or absolute ascertaining of the amount of polypeptide biomarker, hybridization and PCR protocols where a probe or primer or primer set are used to ascertain the amount of nucleic acid biomarker, including for example probe based and amplification based methods including for example microarray analysis, RT-PCR such as quantitative RT-PCR, serial analysis of gene expression (SAGE), Northern Blot, digital molecular barcoding technology, for example Nanostring mCounter™ Analysis, and TaqMan quantitative PCR assays. Other methods of mRNA detection and quantification can be applied, such as mRNA in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples or cells. This technology is currently offered by the QuantiGene®ViewRNA (Affymetrix), which uses probe sets for each mRNA that bind specifically to an amplification system to amplify the hybridization signals; these amplified signals can be visualized using a standard fluorescence microscope or imaging system. This system for example can detect and measure transcript levels in heterogeneous samples; for example, if a sample has normal and tumor cells present in the same tissue section. As mentioned, TaqMan probe-based gene expression analysis (PCR-based) can also be used for measuring gene expression levels in tissue samples, and for example for measuring mRNA levels in FFPE samples. In brief, TaqMan probe-based assays utilize a probe that hybridizes specifically to the mRNA target. This probe contains a quencher dye and a reporter dye (fluorescent molecule) attached to each end, and fluorescence is emitted only when specific hybridization to the mRNA target occurs. During the amplification step, the exonuclease activity of the polymerase enzyme causes the quencher and the reporter dyes to be detached from the probe, and fluorescence emission can occur. This fluorescence emission is recorded and signals are measured by a detection system; these signal intensities are used to calculate the abundance of a given transcript (gene expression) in a sample.
[0058] The term“sample” as used herein includes any biological specimen obtained from a patient. Samples include, without limitation, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), ductal lavage fluid, nipple aspirate, lymph (e.g., disseminated tumor cells of the lymph node), bone marrow aspirate, saliva, urine, stool (i.e., feces), sputum, bronchial lavage fluid, tears, fine needle aspirate (e.g., harvested by fine needle aspiration that is directed to a target, such as a tumor, or is random sampling of normal cells, such as periareolar), any other bodily fluid, a tissue (e.g., tumor tissue) such as a biopsy of a tumor (e.g., needle biopsy) or a lymph node (e.g., sentinel lymph node biopsy), and cellular extracts thereof. In some embodiments, the sample is whole blood or a fractional component thereof such as plasma, serum, or a cell pellet.
[0059] As used herein, a“fixed” sample refers to a sample which has undergone preservation. The fixation can terminate any biochemical reactions and increase the tissue’ s stability. Chemical fixation methods can include subjecting the sample to aldehydes, such as formaldehyde or glutaraldehyde, or alcohols, such as methanol or ethanol.
[0060] The terms “increased”, “elevated”, “overexpress”, “overexpression”, “overexpressed”,“up-regulate”, or“up-regulated” interchangeably refer to a biomarker that is present at a detectably greater level in a biological sample, e.g. plasma, from a patient with cancer, in comparison to a biological sample from a patient without cancer. The term includes overexpression in a sample from a patient with cancer due to transcription, post-transcriptional processing, translation, post-translational processing, cellular localization (e.g, organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a sample from a patient without cancer. Overexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins (i.e., ELISA, immunohistochemical techniques, mass spectroscopy, Luminex® xMAP technology). Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a sample from a patient without cancer. In certain instances, overexpression is l-fold, 2-fold, 3-fold, 4-fold 5, 6, 7, 8, 9, 10, or l5-fold or higher levels of transcription or translation in comparison to a sample from a patient without cancer.
[0061] As used herein, the term“detecting” refers to observing a signal from a label moiety to indicate the presence of a biomarker in the sample. Any method known in the art for detecting a particular detectable moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. II. EGFR Tyrosine Kinase Inhibitor Resistance
[0062] In certain embodiments, the present disclosure concerns methods for detecting PKC5 and/or PLCy expression to determine if a subject is resistant to EGFR TKIs. In particular, the methods concern the detection of PKC5 activation by measuring the level of nuclear PKC5. Nuclear PKC5 may be detected by methods known in the art, such as immunohistochemistry, immunofluorescence, or western blot. An elevated level of nuclear PKC5 and/or PLCy expression can indicate EGFR TKI-resistance, which may be overcome by the administration of a PKC5 and/or PLCy inhibitor.
[0063] Any antibody-based method of detection is contemplated for use with the present methods. For example, the present methods could be used for the detection of immune checkpoint molecules such as by immunoblotting, quantitative EFISA, immunofluorescence (IF) imaging, and IHC staining. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g. , Nakamura et al. (1987), incorporated herein by reference.
[0064] As used herein, sample may refer to a whole organism or a subset of its tissues, cells or component parts. A sample may also refer to a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. Non-limiting examples of samples include urine, blood, cerebrospinal fluid (CSF), pleural fluid, sputum, and peritoneal fluid, bladder washings, secretions, oral washings, tissue samples, touch preps, or fine-needle aspirates. In some embodiments, a sample may be a cell line, cell culture or cell suspension. Preferably, a sample corresponds to the amount and type of expression products present in a parent cell from which the sample was derived. A sample can be from a human or non-human subject. In some embodiments, the sample used for performing antibody-based detection is a formalin fixed paraffin embedded (FFPE) specimen.
[0065] The sample may comprise body fluids and tissue samples that include but are not limited to blood, tissue biopsies, spinal fluid, meningeal fluid, urine, alveolar fluid. For those tissue samples in which the cells do not naturally exist in a monolayer, the cells can be dissociated by standard techniques known to those skilled in the art. These techniques include but are not limited to trypsin, collagenase or dispase treatment of the tissue.
[0066] The present methods may be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al, 1990; Abbondanzo et al, 1990; Allred et al, 1990).
[0067] Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized" tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections.
[0068] Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.
[0069] In one exemplary IHC method, the slides may be dried at 40-45°C in an oven overnight and then incubated at 58-65 C for 1-3 hours. The slides can then be deparaffmized with xylene and ethanol and hydrated in distilled H20. Antigen retrieval can be performed in 10 mM citric acid (pH 6.0) in a microwave for 10 min (2 min 1000W, 8 min 200W), cooled down at room temperature for 60 min, and washed with PBS twice. The slides can then be blocked in 3% H 202/methanol for 10 min at room temperature and washed with PBS three times. Normal horse serum or goat serum (10% normal serum in PBS) is applied for 30 min in a humid chamber at room temperature and normal serum is wiped off. The primary antibody in applied in a humid chamber at 4°C overnight and then washed with PBS three times. The secondary antibody is applied in a humid chamber for 1 hour at room temperature and then washed with PBS three times. Peroxidase conjugated avidin biotin complex is applied in a humid chamber for 1 hour at room temperature and then washed with PBS three times. AEC chromogen substrate is applied for 5-10 min and washed with distilled H20 three times. The sample is then counterstained with Mayer’ s hematoxylin for 30 seconds and washed with distilled H20 three times. Finally, the slides are mounted with aqua-mount (Lemer Laboratories Inc). [0070] In one exemplary immunofluorescence staining followed by confocal microscopy analysis method, drug-treated cells are washed with PBS and fixed in 100% methanol for 20 min at 20 °C. Cells are then subjected to permeabilization with 0.5% Triton X-100 with 3% bovine serum albumin overnight at 4 °C. After that, cells are incubated with primary antibodies overnight at 4 °C, washed with PBS and further incubated with the appropriate secondary antibody. Nuclei are counterstained with 4,6-diamidino-2-phenylindole (DAPI) before mounting. Confocal fluorescence images are captured using a Zeiss LSM710 laser microscope. The relative intensity of PKC5 in nuclei to that in the whole cell was determined by ImageJ version 1.49 software.
III. Methods of Treatment
[0071] In certain embodiments, the present disclosure provides methods for the treatment of cancer by the combination of an EGRF TKI and a PKC5 inhibitor, such as sotrastaurin. The combination treatment may comprise the combination of an EGFR TKI and a PLCy inhibitor. Also provided herein are methods of identifying a subject that is resistant to EGFR TKIs and may respond to a PKC5 inhibitor and/or a PLCy inhibitor in combination with the EGFR TKI. This method may comprise the detection of an elevated expression of nuclear PKC5 and/or PLCy. The sotrastaurin may be administered at a dose of 300-500 mg per day, with a dose of 800 mg/day as the maximum tolerable dose. The EGFR TKI may be administered at a dose of 50-300 mg/day, such as 250 mg/day of gefitinib, 150 mg/day of erlotinib, and 80 mg/day of osimertinib.
[0072] The EGFR TKI for use in the present methods may be selected from the group consisting of: erlotinib, afatinib, Cetuximab, panitumumab, Erlotinib HC1, Gefitinib, Lapatinib, Neratinib, Lifirafenib, HER2-nhibitor-l, Nazartinib, Naquotinib, Canertinib, Lapatinib, AG-490, CP- 724714, Dacomitinib, WZ4002, Sapitinib, CUDC-101, AG-1478, PD153035 HCL, pelitinib, AC480, AEE788, AP26113-analog, OSI-420, WZ3146, WZ8040, AST-1306, Rociletinib, Genisten, Varlitinib, Icotinib, TAK-285, WHI-P154, Daphnetin, PD168393, Tyrphostin9, CNX-2006, AG- 18, AZ5104, Osimertinib, CL-387785, Olmutinib, AZD3759, Poziotinib, vandetanib, and necitumumab.The PKC5 inhibitor for use in the present methods may be sotrastaurin (AEB071) or Go-6893. The PLCy inhibitor for use in the present methods may be U73122.
[0073] Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastemas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.
[0074] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fimgoides; other specified non-hodgkin's lymphomas; B-cell lymphoma; low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia. [0075] The combination therapy can be administered once, for a limited time period, or is administered as maintenance therapy (for a longer period of time until the condition is improved, cure, or continuous lifetime subject). Finite periods of time may be 1 week, 2 weeks, 3 weeks, 4 weeks and up to one year, contained in any time period between these values, inclusive of the endpoints included. In some embodiments, the combination therapy may be administered about 1 day, about 3 days, about 1 week, about 10 days, about 2 weeks, about 18 days, about 3 weeks, or any range between any one of these values, inclusive of the endpoints included.
[0076] In one embodiment, the composition is administered once a day to a subject in need thereof. In another embodiment, every other day, one week or once every three days composition. In another embodiment, the composition is administered twice a day. In yet another embodiment, four times a day or three times a day administration of the composition. In another embodiment, the composition is administered least once a day, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In yet another embodiment, the composition is administered least once a day for a long time, such as at least 4, 6, 8, 10, 12 or 24 months. In some embodiments, administration including but not limited to, at least daily frequency is 2, 3 or 4 times the dose of the composition administered l0-50mg. In some embodiments, once a week, once a month, once every other week, or is administered the composition.
[0077] The PKC5 or PLCy inhibitor and EGFR TKI may be administered orally, intravenously, intraperitoneally, directly by injection to a tumor, topically, or a combination thereof. In some embodiments, the PKC5 or PLCy inhibitor and EGFR TKI are administered as a combination formulation. In certain embodiments, the PKC5 or PLCy inhibitor and EGFR TKI are administered as individual formulations. In some embodiments, the PKC5 or PLCy inhibitor and EGFR TKI r are administered sequentially. In other embodiments, the PKC5 or PLCy inhibitor and EGFR TKI are administered simultaneously.
A. Combination Therapies
[0078] In certain embodiments, the methods provided herein further comprise a step of administering at least one additional therapeutic agent to the subject. All additional therapeutic agents disclosed herein will be administered to a subject according to good clinical practice for each specific composition or therapy, taking into account any potential toxicity, likely side effects, and any other relevant factors. [0079] In certain embodiments, the additional therapy may be immunotherapy, radiation therapy, surgery (e.g., surgical resection of a tumor), chemotherapy, bone marrow transplantation, or a combination of the foregoing. The additional therapy may be targeted therapy. In certain embodiments, the additional therapy is administered before the primary treatment (i.e.. as adjuvant therapy). In certain embodiments, the additional therapy is administered after the primary treatment (i.e., as neoadjuvant therapy.
[0080] In certain embodiments, the additional therapy comprises an immunotherapy. In certain embodiments, the immunotherapy comprises an immune checkpoint inhibitor.
[0081] The PKC5 or PLCy inhibitor and EGFR TKI may be administered before, during, after, or in various combinations relative to an additional cancer therapy. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the PKC5 or PLCy inhibitor and EGFR TKI is provided to a patient separately from an additional therapeutic agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the PKC5 or PLCy inhibitor and EGFR TKI and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6- 12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.
[0082] Various combinations may be employed. For the example below the PKC5 or PFCy inhibitor and EGFR TKI is“A” and an anti-cancer therapy is“B”:
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0083] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. 1. Chemotherapy
[0084] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti -adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpoly saccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-l l); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine ,plicomycin, gemcitabien, navelbine, famesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above^
2. Radiotherapy
[0085] Other factors that cause DNA damage and have been used extensively include what are commonly known as g-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation, and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
3. Immunotherapy
[0086] The skilled artisan will understand that immunotherapies may be used in combination or in conjunction with methods of the embodiments. In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells
[0087] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs and may be used in combination therapies. This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in“armed” MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index. Exemplary ADC drugs inlcude ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).
[0088] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e.. is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, erb b2 and pl55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-l, MCP-l, IL-8, and growth factors, such as FLT3 ligand.
[0089] Examples of immunotherapies include immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds); cytokine therapy, e.g., interferons a, b, and g, IL-l, GM-CSF, and TNF; gene therapy, e.g., TNF, IL-l, IL-2, and p53; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti- p 185. It is contemplated that one or more anti -cancer therapies may be employed with the antibody therapies described herein. [0090] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3 -dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-l), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-l axis and/or CTLA- 4.
[0091] The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies. Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used. As the skilled person will know, alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and/or equivalent names are interchangeable in the context of the present disclosure. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.
[0092] In some embodiments, the PD-l binding antagonist is a molecule that inhibits the binding of PD-l to its ligand binding partners. In a specific aspect, the PD-l ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-l and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD- 1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
[0093] In some embodiments, the PD-l binding antagonist is an anti -PD-l antibody (e. g. , a human antibody, a humanized antibody, or a chimeric antibody) . In some embodiments, the anti-PD-l antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-l binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-l binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-l binding antagonist is AMP-224. Nivolumab, also known as MDX- 1106-04, MDX-l 106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-l antibody that may be used. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab,
KEYTRUDA , and SCH-900475, is an exemplary anti-PD-l antibody. CT-011, also known as hBAT or hBAT-l, is also an anti-PD-l antibody. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.
[0094] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an“off” switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA- 4, an inhibitory receptor for B7 molecules.
[0095] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
[0096] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. An exemplary anti-CTLA- 4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).
4. Surgery
[0097] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).
[0098] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti -cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
5. Other Agents
[0099] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. B. Pharmaceutical Compositions
[00100] In another aspect, provided herein are pharmaceutical compositions and formulations comprising a PKC5 or PLCy inhibitor and EGFR TKI and a pharmaceutically acceptable carrier.
[00101] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012), in the form of aqueous solutions, such as normal saline (e.g., 0.9%)and human serum albumin ( e.g 10%). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zinc-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
IV. Articles of Manufacture or Kits
[00102] An article of manufacture or a kit is provided comprising a method of detecting nuclear PKC5 and/or PLCy expression is also provided herein. The kit may further comprise PKC5 and/or PLCy inhibitors. The article of manufacture or kit can further comprise a package insert comprising instructions for using the inhibitors to treat or delay progression of cancer in an individual. Any of the inhibitors described herein may be included in the article of manufacture or kits. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti -neoplastic agent). Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
V. Examples
[00103] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1 - Mechanisms of EGFR TKI Resistance
[00104] A TKI-insensitive role of EGFR maintains cell survival of EGFR- mutant NSCLC with TKI resistance: To corroborate the TKI-insensitive role of EGFR in TKI resistance, EGFR was depleted to compare treatment with TKIs in H1650 cells, which harbor EGFR-activating mutation and are resistant to TKIs via mechanisms unrelated to T790M mutation (Bivona et al, 2011; Sos et al, 2009). Interestingly, EGFR knockdown by two specific short hairpin RNAs, shRNA-El and -E2, almost completely inhibited cell growth (FIG. 1A), whereas inhibition of EGFR by treating with TKI, gefitinib or erlotinib, had virtually no effect on cell growth (FIG. 1A and 7A), which was expected.
[00105] PTEN loss and subsequent Akt activation as well as NF-kB pathway in H1650 have been shown to contribute to cell survival and resistance to TKIs (Bivona et al, 2011; Sos et al, 2009). Therefore, the phosphorylation status of Akt and NF-kB was assessed as well as EGFR downstream survival signaling, Erk in EGFR-depleted H1650 cells (FIG. 1B). EGFR depletion attenuated all Akt, Erk, and RelA phosphorylation compared with EGFR kinase inhibition, which did not affect Akt or RelA and moderately reduced Erk phosphorylation (FIG. 1B). The results showing TKIresistant cells sensitized to EGFR depletion suggested an oncogenic addiction via unknown roles of mutant EGFR independently of TKI responsiveness and that roles of EGFR may maintain cancer cell survival by activating downstream signaling, including Akt, Erk, and RelA phosphorylation, in TKI-resistant H1650 cells. To validate these potential roles of EGFR that maintain survival of TKI-resistant cancer cells, endogenous EGFR (del 19, kinase activated) was knocked down by shRNA-El targeting 3 -UTR of EGFR in H1650 cells and then re-expressed it or its corresponding kinase-dead (dell9-kd) form in these cells. Re-expression of either EGFR-dell9 or kinase-dead mutant (EGFR-dell9-kd) rescued EGFR depletion-induced lethality (FIG. 1C), indicating that the lethal activity requires EGFR depletion but is independent of EGFR kinase activity.
[00106] Similar effects by EGFR depletion were observed in two other NSCLC cell lines, H1975 carrying the EGFR L858R/T790M mutation and H820 cells carrying the EGFR dell9/T790M mutation and Met amplification (FIG. 1D and FIG. 7B). Re-expression of endogenous EGFR in H1975 cells rescued EGFR depletion induced lethality (FIG. 7C, left, lanes 5 and 6 vs. 3 and 4). HCC827 cells were used as control cells, which require EGFR kinase activity for cell survival, and sensitive to both TKI and EGFR depletion, and thus cannot be rescued by re-expression of the kinase-dead mutant (EGFR-dell9-kd; FIG. 7C, right). EGFR knockdown and rescue experiments in H1650, H1975, and HCC827 cells validated the specificity of EGFR shRNA-El (FIG. 1C and 7C). Together, these results demonstrated a previously undiscovered role of EGFR in the survival of TKI-resistant cells.
[00107] To systematically interrogate the newly discovered role of EGFR in acquired TKI resistance, acquired TKI-resistant clones were generated from TKI-sensitive HCC827 cells by exposing them to 1 mM of gefitinib for 6 months. Fifteen HCC827 gefitinib- resistant (GR) clones isolated from a single cell demonstrated resistance to gefitinib as indicated by their much greater median inhibitory concentration (ICso > 1 mM) than the parental cells (IC50 -0.006 pM) (FIG. 7D, left). Each of the GR clones was also resistant to afatinib, a clinically used irreversible TKI (FIG. 7D, right). Sequence analysis of EGFR in each GR clone revealed that none harbored the resistant T790M mutation.
[00108] Next, it was investigated whether features of known resistant mechanisms exist in GR clones and whether they are sensitive to EGFR depletion. Consistent with prior studies (Bivona et al, 2011; Rotow and Bivona, 2017), these GR clones showed distinctive features of resistance, such as upregulation of Her-2, Axl, Erk, Akt, or NF-kB, on Western blot (FIG. 1E, bottom, and FIG. 7E), suggesting the heterogeneity of TKI resistance mechanisms in these GR cells. Nevertheless, knocking down endogenous EGFR by lentiviral shRNA-El significantly reduced the growth of all GR clones (FIG. 1E, top). Together with the data from other three well characterized TKI-resistant cell lines (FIG. 1D), these results suggested that in addition to the well-known kinase activity of EGFR, there exists an unknown role of activating -mutant EGFR that is insensitive to TKI but is required for survival of TKI- resistant EGFR mutant NSCLC.
[00109] PKC8 serves as a common mediator in TKI-insensitive EGFR pathways and a contributor to TKI resistance: Because EGFR depletion, but not kinase inhibition, sensitized TKI-resistant cells, such as H1650 and all fifteen HCC827-derived GR cell lines, harboring different resistant mechanisms to TKI, it was hypothesized that the TKI- insensitive pathways of EGFR may confer TKI resistance through a common mediator. If so, therapeutic targeting of the common mediator may provide an effective strategy to overcome recurrent disease in patients with TKI-resistant EGFR mutant NSCLC. To identify this mediator, an EGFR-depleted resistant subclone (EDR) was generated from H1650 cells by first knocking down EGFR with lentiviral shRNA-El and then selecting survived cells as EDR clone (FIG. 2A). EGFR depletion was first validated two days after lentiviral shRNA infection by comparison to scrambled control (II, FIG. 2A, and lane 3 vs. 1, FIG. 2B bottom).
[00110] After culturing for an additional 7 days, most of the EGFR-depleted cells had died; the few cells that survived were further cultured for more than 3 months and isolated as EDR cells (III, FIG. 2A). EGFR depletion was subsequently validated in EDR cells by immunoblotting (FIG. 2B, bottom, lane 4). Analysis of EGFR knockdown efficiency by flow cytometric analysis also supported the EGFR depletion as only about 10.5% EDR cells expressed EGFR compared with the control shRNA group, which had > 92% EGFR-positive cells (FIG. 2A, bottom). These data indicated that the cells maintained similar EGFR depletion level during EDR cell establishment, and suggested the EDR cells could be used to search for survival signaling pathways that are present in the parental H1650 but significantly reduced by EGFR depletion and restored in the EDR clones. The identified pathways may represent survival signaling against the lethality induced by EGFR depletion in EDR cells and serve as ideal therapeutic target to overcome TKI-resistance for the EGFR-mutant NSCLC.
[00111] It was then aimed to identify protein kinase candidates that can be targeted by drugs currently available for clinical use. To this end, phosphorylation profiling of broad-scope proteins was performed in these cells (FIG. 2B, top) to identify common mediators that may be involved in the TKI-insensitive EGFR pathways and maintaining EDR cell survival by using a Phospho-Explorer Antibody Microarray (Table 2). A wide range of protein phosphorylation was detected and compared among the four different groups (FIG. 2B, top): (I) shRNA control, (1+) shRNA control treated with TKI, (II) EGFR depleted (day 2 population), and (III) EDR. To identify the potential mediators of the TKI-insensitive EGFR pathways, three criteria (see FIG. 2 legend) were used which identified 27 candidates that were expressed at relatively lower levels in group II than in groups I, and recovered in group III, with no difference between groups (I) and (I+). [00112] Table 1. Nuclear PKC5 is upregulated in human EGFR-mutant
NSCLCs with resistance to TKI.
Figure imgf000040_0001
77 F No 4 L858R PR 14.1 Gef 81 M Yes 4 19Del PR 28.5 Gef 81 M No 4 19Del SD 22.2 Gef 53 M No 4 19Del PR 26.8 Gef 73 F No 3b L858R PR 10.8 Gef 62 F No 4 19Del PR 22.4 Gef
8 44 F No 4 19Del PR 8.6 Gef
9 72 F No 4 19Del PR 16.0 Gef
10 47 F No 4 L858R PD 2.2 Gef
11 60 M No 4 19Del SD 8.4 Gef
12 78 M No 4 19Del SD 6.1 Gef
13 56 F No 4 19Del PR 4.9 Erl
14 66 F No 4 L858R PD 1.6 Gef
15 66 F No 4 L858R PD 2.7 Gef
16 60 M No 4 19Del SD 9.2 Gef
17 87 F No 4 19Del PD 2.4 Gef
18 61 M No 4 L858R N/A PR 9.3 Gef
19 50 M No 4 L858R SD 3.5 Gef 20 58 M Yes 4 L858R PR 3.7 Gef
21 49 F No 4 L858R PD 1.6 Gef
22 57 F No 3b L861Q SD 8.7 Gef
23 81 F No 4 L858R PR 3.9 Gef
24 45 F No 4 19Del PD 3.0 Gef
25 67 F No 4 19Del SD 6.0 Gef
26 54 M Yes 4 L858R PR 34.0 Gef
27 61 M No 4 L861Q PR 14.4 Gef
28 89 F No 4 L858R PR 10.5 Gef
29 63 M Yes 4 L858R PR 11.9 Gef
30 37 F No 4 19Del PR 8.2 Gef
31 41 F No 4 L858R PR 7.2 Gef
32 67 F No 4 19Del PR 6.4 Gef
33 50 F No N/A 19Del SD 6.8 Erl
34 52 F No 4 19Del PR 6.7 Gef
35 75 F No 4 L858R PR 9.3 Gef
36 72 F No 4 L858R SD 11.9 Gef
37 54 M Yes 4 19Del PR 6.0 Gef
38 37 F No 4 19Del PR 7.7 Gef
39 56 F No 4 19Del SD 5.8 Gef
40 71 F No 4 L858R N/A PR 7.0 Gef
41 55 M Yes 4 L858R SD 6.0 Gef
M, male; F, female; Gef, gefitinib; Erl, erlotinib; PR, partial response; SD, stable disease; N/A, not available.
[00113] Table 2 : 27 potential mediators in the TKI-insensitive EGFR pathways identified by antibody array. ratio of shCtrl shCtrl+TKI shEGFR EDR
Modifier (C) /
(A) (A+) (B) (C)
(B)
B-RAF (Ab-446)* 1.00 0.97 0.04 1.20 30.51
CaMK2 (Phospho-Thr305) 1.00 0.86 0.02 1.03 49.32
CaMK4 (Phospho-Thrl96/200) 1.00 1.17 0.06 0.95 16.63 Cateninbeta (CTNNB) (Phospho-
1.00 0.91 0.10 1.15 11.58 Tyr489)*
Catenin delta- 1 (Ab-228) 1.00 1.14 0.06 1.13 18.41
CD 5 (Ab-453) 1.00 1.15 0.14 1.11 8.10 c-Jun (Ab-73) 1.00 1.20 0.05 1.27 26.78
CK2-b (Phospho-Ser209) 1.00 0.85 0.13 0.63 4.93 c-met (Ab-1003)* 1.00 0.95 0.08 2.05 26.95 cofilin (Ab-3) 1.00 1.13 0.12 1.06 9.00
Cytokeratin 8 (Ab-431) 1.00 1.16 0.08 0.55 7.12
Ephrin B (Ab-330) 1.00 0.99 0.07 1.88 28.43
Estrogen Receptor-alpha (Ab-106) 1.00 0.81 0.03 0.93 29.80 FAK (Ab-576)* 1.00 0.85 0.11 0.91 8.00
FKHR (Phospho-Ser319)* 1.00 0.96 0.09 1.79 19.37
HDAC5 (Ab-498) 1.00 1.04 0.05 0.98 21.25
LAT (Ab-191) 1.00 0.84 0.09 0.61 6.65
MAP3K7/TAK1 (Ab-439) 1.00 1.13 0.05 0.83 16.26 NFkB-pl05/p50 (Ab-337)* 1.00 0.92 0.03 0.72 24.17
PAK1 (Ab-212) 1.00 1.10 0.08 0.76 9.03
PKC beta/PKCB (Phospho-Ser661) 1.00 0.90 0.12 0.96 8.32 PKC delta (Phospho-Thr505) 1.00 0.99 0.14 0.68 4.74 PLCg2 (Ab-1217) 1.00 0.88 0.12 0.86 7.29
Rail (Ab-338)* 1.00 0.91 0.14 1.39 9.91
Rb (Ab-795) 1.00 1.08 0.10 1.07 10.53
SRF (Phospho-Ser77) 1.00 0.99 0.11 2.22 19.65
VASP (Ab-238) 1.00 1.13 0.06 0.64 11.51
* indicates that the candidates have previously been implicated with resistance to EGFR inhibitors. [00114] Interestingly, among these mediators (Table 2), several, including B- RAF, b-catenin, c-Met, FAK, FKHR, NF- KB, and Rafl, have previously been implicated in resistance to EGFR inhibition (Rotow and Bivona, 2017; Tomasello et al, 2018). Ingenuity pathways analysis (IPA) identified 32 canonical signaling pathways involving these 27 mediators (Table 3). Interestingly, PKC5 was involved in 22 of the 32 pathways (> 68%; FIG. 8A) suggesting that PKC5 may be a common mediator in the TKI-insensitive EGFR pathways involved in TKI resistance.
[00115] To further identify which of these mediators and their related pathways are critical in TKI resistance, a synthetic lethality screen was performed by counting the viable TKI-resistant H1650 cells treated with gefitinib in combination of a commercially available inhibitor targeting these potential mediators or their corresponding pathways for 3 days; the combination index (Cl) was calculated for each combination (Table 4), with results indicating that Go-6983 (pan-PKC 10 inhibitor, PKCi), sotra (sotra), a PKCi currently in phase I/II clinical trials (Mochly-Rosen et al, 2012), and U73122 (phospholipase C [PLC] inhibitor) demonstrated the strongest synergy (Cl < 0.3) with gefitinib (Table 4). Both RKO'b and PKC5 were identified in the first screen (Table 2). However, a comparison of each of the three PKC inhibitors (Table 4) indicated that only Go-6983 and sotra, which are able to inhibit both RKEb and PKC5, but not enzastaurin (inhibiting RKEb, but not PKC5, Cl value > 1; Table 4), exhibited synergy with gefitinib, suggesting that inhibition of PKC5 sensitized the TKI- resistant H1650 cells to gefitinib. Although the results indicated that both PKC5 and PLCy2 are involved in the TKI-insensitive EGFR pathways and TKI resistance, only the PKC5 inhibitor (sotra in this study) is currently available in clinical trials. Thus, the further studies focused on PKC5 as a clinically targetable mediator that could rapidly benefit patients with EGFR-mutant NSCLC. [00116] Table 3: Top 32 impacted pathways by ingenuity pathway analysis based on 27 potential mediators.
Ingenuity Canonical Pathways -log Molecules
(p-value)
Molecular Mechanisms of Cancer 1.69E01 Rafl,NF-kB-pl05/p50,B-
Raf,FAK,RB,PAKl,
c-Jun, FKHR, PKC delta, MAP3K7,Catenin beta, PKC beta, catenin delta, CaMK2 B Cell Receptor Signaling 1.55E01 FAK,Rafl,CaMK4,c- Inn FKI Ik Cofilin Pl .CiO MAP3K7, NF- kB-pl05/p50, CaMK2,PKC beta
HGF Signaling 1.38E01 F AK,c-Met,Raf 1 ,P AK 1 ,c- Jun,PKC
delta, PLCg2, MAP3K7,PKC beta
Chemokine Signaling 1.34E01 FAK,Rafl,CaMK4,c-Jun, Cofilin, PLCg2,
CaMK2, PKC beta
LPS-stimulated MAPK Signaling 1.31E01 Raf 1 ,P AK 1 ,c- Jun,PKC delta, SRF,MAP3K7,
NF-kB-pl05/p50,PKC beta
GNRH Signaling 1.3E01 FAK, Rafl,PAKl,c-Jun, PKC delta, MAP3K7,
NF-kB-pl05/p50,CaMK2,PKC beta
Protein Kinase A Signaling 1.18E01 FAK, B -Raf , Raf 1 , CaMK4 ,PKC delta, PLCg2,
NF-kB-pl05/p50,Cateninbeta,
VASP,CaMK2, PKC beta
Renin- Angiotensin S ignaling 1.17E01 FAK, Raf 1 ,P AK 1 ,c- Jun,PKC
delta, PLCg2,NF-kB-pl05/p50, PKC beta
ERK/MAPK Signaling 1.15E01 F AK,B-Raf,Raf 1 ,P AK 1 ,PKC
delta, PLCg2,SRF,
ER alpha, PKC beta
Role of Macrophages, Fibroblasts 1.14E01 Raf 1 , CaMK4,c-Jun,PKC
and Endothelial Cells in delta, PLCg2,MAP3K7,
Rheumatoid Arthritis NF-kB-p 105/p50,Catenin beta, CaMK2, PKC beta
Melatonin Signaling 1.13E01 B -Raf, Raf 1 , CaMK4 ,PKC
delta, PLCg2,CaMK2,
PKC beta
ErbB Signaling 1.06E01 Raf 1 ,P AK 1 ,c-Jun,FKHR,PKC
delta, PLCg2, PKC beta
Glioma Signaling 1.03E01 Rafl,RB,CaMK4,PKC delta, PLCg2,CaMK2,
PKC beta
Role of NFAT in Cardiac 9.98E00 Rafl,CaMK4,PKC delta, PLCg2, MAP3K7,
Hypertrophy HDAC5, CaMK2, PKC beta
Corticotropin Releasing Hormone 9.86E00 B -Raf, Raf 1 , CaMK4,c- Jun,PKC delta, PLCg2, Signaling PKC beta
IL-8 Signaling 9.84E00 F AK,B -Raf, Raf 1 ,c- Jun,PKC delta, NF-kB- pl05/p50, VASP,PKC beta
Thrombin Signaling 9.77E00 F AK,Raf 1 , CaMK4 ,PKC delta, PLCg2,NF- kB-pl05/p50, CaMK2, PKC beta
Axonal Guidance Signaling 9.67E00 F AK,c-Met,Raf 1 ,Ephrin
B,RAKI, Cofilin, PKC delta,
PLCg2,VASP, PKC beta
PI3K Signaling in B Lymphocytes 9.43E00 Raf 1 , CaMK4 ,c- Jun,PL Cg2 ,NF -kB -pl05/p50,
CaMK2, PKC beta
Erythropoietin Signaling 9.31E00 Rafl,c-Jun,PKC delta, PLCg2,NF-kB- pl05/p50,
PKC beta IL-3 Signaling 9.16E00 Raf 1 ,P AK 1 ,c-Jun,FKHR,PKC delta, PKC
beta
Phospholipase C Signaling 9.04E00 Raf 1 , CaMK4,PKC delta, PLCg2,LAT,NF- kB-pl05/p50, HDAC5,PKC beta
PDGF Signaling 8.94E00 Raf 1 ,c-Jun,PLCg2, SRF, CK2-b ,PKC beta
Regulation of IL-2 Expression in 8.91E00 Rafl,CaMK4,c-Jun,PLCg2,LAT,NF-kB-
Activated and Anergic T pl05/p50
Lymphocytes
Xenobiotic Metabolism Signaling 8.69E00 Rafl,CaMK4,PKC delta, MAP3K7,NF -kB - pl05/p50,HDAC5,CaMK2,PKC beta
NF-kB Signaling 8.46E00 B-Raf,Rafl,PLCg2,MAP3K7,CK2-b,NF-kB- pl05/p50, PKC beta
UVC-Induced MAPK Signaling 8.45E00 B -Raf,Raf 1 ,c- Jun,PKC delta, PKC beta
IGF-1 Signaling 8.33E00 FAK,Rafl,c-Jun,FKHR,SRF,CK2-b
Cholecy stokinin/ Gastrin-mediated 8.22E00 FAK,Rafl,c-Jun,PKC delta, SRF, PKC beta
Signaling
Rac Signaling 8.17E00 F AK,Raf 1 ,P AK 1 ,c- Jun, Cofilin,NF-kB- pl05/p50
Leukocyte Extravasation Signaling 8.06E00 FAK,PKC delta, PLCg2,Catenin beta, VASP,
catenin delta, PKC beta
Natural Killer Cell Signaling 8.05E00 Rafl,PAKl,PKC delta, PLCg2,LAT, PKC
beta
[00117] Table 4: Synthetic lethal screen of gefitinib with inhibitors targeting potential mediators or their impacted pathways in H 1650 cells.
Target Inhibitor name Clinical Dose range Synergy with
status gefitinib (Cl) b-catenin ICG-001 1-15 uM >1.1 nuclear
complex PNU-74654 1-15 uM >1.1
Resveratrol (non-selective) 1-50 uM >1.1 b-catenin Aspirin FDA 0.5-8 uM 0.5-0.9
(non-selective, NS AID) approved
Sulindac FDA 1-20 uM 0.5-0.9
(non-selective, NS AID) approved
CaMKII KN-62 0.1-10 uM 0.5-0.9
FZD-Dvl Dvl-PDZ (3289-8625) 0.5-25 uM 0.9-1.1
NSC668036 0.5-50 uM >1.1 IkB kinase BMS-345541 1-30 uM 0.4-1.1
JNK SP600125 1-20 uM 0.9-1.1
MEK Trametinib FDA 2.5-20 nM 0.4-0.5
approved
NFAT NFAT inhibitor 1-20 uM >1.1
PKC Enzastaurin** Phase 3 0.5-20 uM 1.0-1.4
PKC-pan Go6983** 1-40 uM *0.2-0.8
Sotrastaurin (AEB071) ** Phase 2 0.5-20 uM *0.1-0.8
PLC U-73122 (Stroidamine) 1-5 uM *0.3-0.7
PI-PLC Edelfosine 1.5-30 uM >1.1
PLD1, PLD2 FIPI 0.1-10 uM 0.5-0.9
PLD VU0359595 0.1-10 uM 0.5-0.9
Wnt# IWP2 (Porcupine inactivator) 0.05-10 uM 0.5-0.9
IWP4 (Porcupine inactivator) 0.5-50 uM 0.5-0.9
LGK-974 (Porcupine 0.01-1 uM >1.1 inhibitor)
Non-steroidal anti-inflammatoiy drug (NSAID); combination index (Cl).
*Strong synergy (Cl value < 0.3); shown in red.
**Go6983 inhibited PKCa, b, g, d, z, and m isoforms, and sotrastaurin inhibited a, b, d, e, h, and q. Both inhibitors synergized with EGFR TKI and shared the a, b, and d isoforms as targets. PKCa and b were subsequently excluded because enzastaurin (so-call RKEb inhibitor), which did not synergize with EGFR TKI, inhibited PKCa, b, g, e. Together, PKC6 is the only isoform as a therapeutic target that is shared by both Go6983 and sotrastaurin and excluded by enzastaurin.
#Mediators involved in canonical and non-canonical Wnt signaling although the Wnt receptor was not identified. PKC inhibitors are shown in bold.
[00118] PKC8 is required for TKI resistance: To validate the role of PKC5 in
TKI resistance, PKC5 expression was first knocked down by two different shRNAs, #1 and #
2, targeting CDS and 3 '-UTRof PKC5, respectively, in H1650 cells; PKC5 depletion sensitized cells to gefitinib whereas re-expression of shRNA-resistant PKC5 reversed this sensitization effect (FIG. 2C), suggesting that PKC5 is required for TKI resistance.
[00119] To further investigate the role of PKC5 in EGFR-mutant NSCLC, two TKI-resistant H1650 cell lines expressing EGFR-dell9 (activated) or EGFR-dell9-kd (inactivated), as shown in FIG. 1C, were treated with PKCi. Interestingly, H1650 cells expressing EGFR-dell9-kd were more sensitive to sotra than those expressing EGFR-dell9 (FIG. 2D). These results suggested that EGFR kinase -inactive cells are more dependent on PKC5 activity than cells with active EGFR such that EGFR kinase activity rendered cells resistant to PKCi.
[00120] The synergistic effects induced by inhibition of both EGFR kinase activity and PKC5 pathways were further validated by examining colony formation of H 1650 cells treated with gefitinib and PKCi for a longer time point of 10 days (FIG. 2E and 8B). Remarkably, in almost all doses used, the Cl values of the gefitinib-sotra and gefitinib-Go- 6983 combinations were < 0.3 (FIG. 2E), further supporting the strong synergy initially observed in the synthetic lethality screen.
[00121] On the basis of the in vitro synergies of the TKI-PKCi combination in the intrinsically TKI-resistant H1650 cells (FIG. 2C), the combination of gefitinib with sotra was further evaluated in an H1650 orthotopic xenograft tumor model. The gefitinib-sotra combination, but not each agent alone, induced significant tumor regression and extended mice survival (FIG. 2F and FIG. 8C, 8D). All mice in the control, gefitinib, and sotra groups died within 55 days but three of the mice in the gefitinib-sotra combination group (n = 9) survived longer than 99 days, with one surviving beyond one year.
[00122] PKC5 is known to elicit several survival signaling pathways in cancer cells (Basu and Pal, 2010), and interestingly, Erk/MAPK, PI3K, and NF-kB signaling were among those identified in the first screen (Tables 2 and 3). In addition, Akt activation is associated with TKI resistance in PTEN-loss H1650 cells (Sos et al, 2009). Therefore, Akt, Erk, and NF-kB phosphorylation was also examined in tumors treated with the gefitinib-sotra combination. Single treatment of gefitinib or sotra reduced phosphorylation of AKT, RelA, and ERK (FIG. 2G and 8E), suggesting either EGFR or PKC5 are potential upstream molecules of these survival signaling.
[00123] The combination treatment suppressed more pAkt, pRelA, and Ki67 levels than single drug treatment (FIG. 2G), implying AKT and NF-kB are mechanisms for the synergy of EGFR and PKC inhibitors. These results supported the therapeutic potential of TKI- PKCi combination in TKI-resistant NSCLC and suggested that Akt and NF-kB survival signaling may be involved in the TKI-PKCi synergy in H 1650 cells.
[00124] To further validate the role of PKC5 as a common mediator in acquired TKI resistance, genetic and pharmacological inhibition of PKC5 was performed in GR cells. Knockdown of PKC5 by shRNA in multiple GR cell lines rendered them sensitive to gefitinib (FIG. 3 A), whereas reexpression of PKC5 rescued the gefitinib-resistant phenotype (FIG. 3A).
[00125] Consistent with those findings, inhibition of PKC5 by sotra significantly enhanced suppression of both cell viability and survival signaling in GR cells treated with gefitinib in vitro (FIG. 3B and 9A). The combination of gefitinib and sotra was further evaluated in mice with NSCLC tumors (GR6) harboring acquired gefitinib resistance. The gefitinib-sotra combination effectively induced tumor regression (-88.5% regression after a one-week treatment and -98.3% regression after a 4-week treatment) whereas gefitinib or sotra alone only modestly delayed tumor growth (FIG. 3C).
[00126] Furthermore, the effective doses of the gefitinib-sotra combination did not significantly affect mouse body weight or the values of the indicators of liver and kidney functions (FIG. 9C and D). These results suggested that the gefitinib-sotra combination at the doses administered may be a safe and effective therapeutic strategy to treat EGFR-mutant NSCLC with TKI resistance. Since gefitinib has received regulatory approval in NSCLC patients, and PKCi sotra is available for clinical studies, the combination could be readily tested in clinical trials, especially for patients whose tumor has developed resistance to TKIs.
[00127] PKC8 is sufficient to induce TKI resistance: Next, to determine whether ectopic expression of PKC5 in TKI-sensitive NSCLC cells is sufficient to induce gefitinib resistance, PKC5-ectopic expressing stable clones were established from two TKI sensitive H3255 and HCC827 cells. Ectopic expression of PKC5 significantly induced resistance to gefitinib in vitro (FIG. 3D and 3E) and in vivo (FIG. 3F and 3G). Notably, TKI- induced cleaved PARP (cPARP, a marker for apoptosis) was abolished by ectopic PKC5 expression (FIG. 3H, lane 4 vs. 2). Thus, enhanced expression of PKC5 may protect TKI- sensitive cells from TKI-induced apoptosis in EGFR-mutant NSCLC and is sufficient to cause TKI resistance.
[00128] Nuclear localization of PKC8 is present in multiple TKI-resistant NSCLC cells and contributes to TKI resistance: PKC5 is activated in specific subcellular compartments, such as the nucleus (Mochly-Rosen et al , 2012). To determine the molecular mechanism underlying the contribution of PKC5 to TKI resistance, the expression and subcellular distribution of PKC5 was compared between GR and parental (gefitinib-sensitive) HCC827 cells. Western blot analysis indicated that the total expression of PKC5 did not change significantly in both GR and parental HCC827 cells (FIG. 10A).
[00129] Interestingly, however, immunofluorescence staining and Western blot analysis indicated greater PKC5 nuclear localization (nPKCd) in the GR cells than in the parental HCC827 cells (FIG. 4A and 4B). Furthermore, nPKCd was readily detectable in a TKI-resistant EGFR-mutant H1650 cell model (FIG. 4C, lane 1 and 10B, left), suggesting an association between nPKCd and TKI resistance.
[00130] To further characterize the role of nPKCd in TKI resistance, the effects of PKC5 inhibition on its nuclear localization were examined. Treatment of H1650 and GR cells with sotra, attenuated nPKCb (FIG. 4C, lanes 4 and 5, 10B, and 4D) and reduced its overall expression (FIG. 10D). Moreover, sotra led to downregulation of PKC5 protein but not mRNA levels, and the reduction of PKC5 was attributed to the ubiquitination and proteasome degradation pathway (FIG. 10E, 10F, and 10G). To determine whether nuclear localization of PKC5 is required to induce TKI resistance, identified a potent NLS sequence was first at the C-terminal of human PKC5 using in silico analysis of NLS (FIG. 10H, top) and showed that the NLS sequence was highly conserved among different species.
[00131] Two mutations were then constructed in the NLS region (NLSml and NLSm3) of human wild-type PKC5 (PKC5-WT; FIG. 10H, bottom). Two TKI-sensitive cells, H3255 and HCC827, were transduced with empty vector, PKC5-WT, PKC5-NLSml, or PKC5-NLSm3. Results from immunofluorescence analysis indicated that PKC5 increased in both nuclei and cytoplasm of PKC5-WT cells compared with vector control cells whereas increased PKC5 was detected only in cytoplasm of both NLSml - and NLSm3 -expressing cells, confirming that these two NLS PKC5 mutants effectively blocked PKC5 nuclear translocation (FIG. 4E).
[00132] These stable transfectant cells were then treated with gefitinib and it was found that ectopic expression of PKC5-WT, which increased nuclear PKC5 expression, but not PKC5-NLSml or PKC5-NLSm3 mutants, induced pAkt, pErk, and pRelA (FIG. 4F) and rendered cells resistant to gefitinib (FIG. 4E). In addition, WT and NLS mutant PKC5 expressed similar kinase activity in HCC827 cells (FIG. 101 and 10J), indicating that the differences in TKI sensitivity between WT PKC5 and the NLS mutants were attributed to their localization, but not kinase activity. [00133] Collectively, these findings indicated that nuclear PKC5 is required for TKI resistance. Phosphorylation on the activation loop of PKC serves as a priming step allowing catalytic maturation of PKC5 (Dutil et al, 1998), which involves T505 phosphorylation (pT505) (Parekh et al. 1999), a phosphorylation site that was identified in our first screen (Table 2). However, pT505 does not seem to be necessary for the catalytic activity of PKC5 (Stempka et al, 1997) but may be associated with its localization (Leitges et al, 2002). To determine whether pT505 is required for PKC5 nuclear translocation in EGFR- mutant lung cancer model, three stable cells were established by ectopically expressing phosphorylation-defective (T505A) and phosphorylation-mimic (T505D 17 and T505E) mutant PKC5 (FIG. 10K). These three T505 mutants, similar to WT PKC5, were detected in both the nucleus and cytoplasm of the cells (FIG. 10F) by immunofluorescence staining. These results indicated that all three T505 mutants were still localized in nucleus unlike the NFS mutants, and suggested that PKC5 pT505 is not required for controlling its nuclear translocation.
[00134] To determine whether PKC5 pT505 is only accompanied by its nuclear localization, the levels of pT505 were examined in the WT and NFS mutants expressing cells treated with gefitinib (FIG. 4F and 10M). It was found that pT505 PKC5 was enhanced in WT PKC5 cells compared to vector control cells but was not affected by TKI gefitinib (gef; FIG. 10M). These data supported the results from initial screening (Table 2) that TKI is not able to suppress pT505 in TKI-resistant cells. Interestingly, cells expressing NFS mutants (ml and m3), which do not contain nuclear PKC5 (FIG. 4E), exhibited substantially lower levels of pT505 PKC5 compared with WT cells in the presence of gefitinib (FIG. 4F). These results suggested that pT505 of PKC5 does not control but may accompany its nuclear translocation, which promotes survival signaling pErk, pRelA, and pAkt, and confers TKI resistance.
[00135] TKI-insensitive EGFR pathways contribute to the heterogeneity of TKI resistance mechanisms via nPKCb upregulation: To further validate the role of EGFR on nPKCd in EGFR-mutant NSCFC, EGFR was knocked down in H1650 cells and compared the effects on nPKCd to TKI treatment. Immunofluorescence staining showed that EGFR depletion, but not kinase inhibition, reduced nPKCd (FIG. 4C, lane 3 vs. 2), suggesting an unknown TKI-insensitive role of EGFR in promoting nPKCd in TKIresistant NSCFC. EGFR is a membrane-bound receptor that can interact with other RTKs, such as Her-2 and Axl, which limits the sensitivity to anti -EGFR therapies (Hirsch et al , 2009; Meyer et al, 2013). [00136] Interestingly, these RTKs have been implicated in PKC5 activation (Allen-Petersen et al., 2014; Elkabets et al , 2015). The above results (FIG. 4C) indicated that EGFR knockdown, which eliminated all of EGFR pathways, but not TKI, which only reduced kinase-dependent activity, suppressed nPKCd levels. Thus, it was asked whether the RTKs that are overexpressed in acquired resistant cells upregulate nPKCd and whether TKI-inactivated EGFR promotes nPKCd and TKI resistance through heterodimer formation with those RTKs. Because previous studies have reported the roles of Axl and Her-2 in TKI resistance (Rotow and Bivona, 2017), it was first validated that inhibition of Axl and Her-2 enhances TKI sensitivity in our GR4 (Axl overexpression) and GR10 (Her-2 overexpression) cells, respectively (FIG. 11A). These results also confirmed that these resistant features led to TKI- resistance in the GR cells. Immunoprecipitation followed by Western blot analysis to assess EGFR interactions in the two GR cells treated with TKI indicated that TKI induced the interaction between EGFR with Axl in GR4 and with Her-2 in GR10 cells (FIG. 5 A), suggesting the existence of EGFR heterodimers upon the addition of TKI. Interestingly, GR4 or GR10 cells treated with R428 (Axli) or lapatinib (Her-2i) alone or with gefitinib alone did not significantly reduce nPKCd. In contrast, only coinhibition of EGFR and Axl or Her-2 in GR4 or GR10 cells, respectively, attenuated the nPKCd levels (FIG. 5B). Fapatinib treatment alone slightly reduced nPKCd in the G10 cells, which may be attributed to the dual inhibitory effects on both Her-2 and EGFR (FIG. 5B, bottom). These data suggested that the EGFR kinase activity in TKI-resistant cells remains important for upregulation of nPKCd. The TKI-induced interaction between EGFR and other resistance-mediating RTKs (heterodimers) may upregulate nPKCd and lead to TKI resistance in EGFR-mutant NSCFC.
[00137] To investigate the mechanisms of how EGFR heterodimers induce nuclear PKC5 and to elucidate the effects of Axl and Her-2 upregulation on EGFR phosphorylation, EGFR phosphorylation status was examined by a Human EGFR Phosphorylation Antibody Array in GR4 cells treated with TKI (gefitinib) and Axl inhibitor (R428). Although the array data suggested that co-inhibition of EGFR and Axl blocked more EGFR Yl 173 and Y845 phosphorylation than TKI alone, Western blot analysis indicated that only EGFR Yl 173 phosphorylation (pYH73) but not Y845 was suppressed by co-inhibition compared with gefitinib alone (FIG. 11B, lane 6 vs. lane 4). Similar results were also observed in GR10 cells treated with TKI and Her-2 inhibitor (FIG. 11B, right, lane 6 vs. lane 4). [00138] In addition to EGFR Y1173 and Y845, EGFR Y1068 and Y1086 phosphorylation were also detected (FIG. 11B). pYH73 was only partially inhibited by gefitinib treatment (FIG. 5C, lane 4) whereas Y1068 and Y1086 EGFR phosphorylation were almost completely suppressed (FIG. 11B, lane 4). The sustained phosphorylation on EGFR Y 1173 was further suppressed by co-treating GR4 and GR10 cells with Axl or Her-2 inhibitor, respectively (FIG. 11B, lane 6 vs. 4). These results suggested that the interaction of EGFR and Axl or Her-2 may sustain EGFR pYH73 in resistant cells treated with TKI, and raised an interesting question of whether the sustained EGFR pYH73 may contribute to the TKI- insensitive EGFR pathway. EGFR Yl 173, when phosphorylated, functions as a docking site for phospholipase Cy (PLCy) (Chattopadhyay et ai , 1999) which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2), resulting in the production of the secondary messengers diacyl glycerol (DAG) and inositol 1,4, 5 -triphosphate (IP3). DAG activates isozymes of the PKC family, including PKC5 (Rosse el al.. 2010).
[00139] Consistently, EGFR pY 1173 observed in GR cells treated with gefitinib and lapatinib (FIG. 11B) was associated with the phosphorylation of PFOy2 (FIG.5C) but not PFOyl (FIG. 11C). Interestingly, PFOy2 was identified in our first screen (Table 2), and the second synthetic lethality screen indicated synergistic effects of gefitinib and PFCy inhibitor (U73122) (Table 4). To further investigate the role of PFCy2 in nuclear PKC5, GR4 and GR10 cells with were treated U73122 and a significant suppression of nuclear PKC5 was observed (FIG. 5D). These data suggested that the interaction of EGFR with Axl or Her-2 sustains EGFR pYH73 and PFCy2 activity in the presence of EGFR inhibitor. Such activation enhanced nuclear PKC5 and conferred TKI resistance. Therefore, EGFR-Y1173- PFOy2-nPKC5 is a common axis of TKI-insensitive EGFR pathways that contributes to the heterogeneity of TKI resistance.
[00140] nPKCd is upregulated in human EGFR-mutant NSCLCs with acquired TKI resistance and correlates with poor survival in EGFR-mutant NSCLC patients treated with first-line single agent TKI: To strengthen the findings that nPKCd is upregulated in TKI-resistant cells and confers TKI resistance, nPKCd status was examined by immunohistochemistry (IHC) in matched pretreatment and TKI-resistant EGFR-activating mutation-harboring NSCFC specimens from 41 patients (Tables 1, and FIG. 12A, 12B, 12C). All of these patients were treated with erlotinib or gefitinib and had met the established clinical definition of acquired resistance to TKI. [00141] Surprisingly, it was found that nPKCd was present in more than 40% of their resistant tumors (17/41, 41.5%). Twelve among the resistant tumors (29.3%) had higher nPKCd expression levels than their matched pretreatment tumors (FIG. 6A), suggesting acquired TKI resistance in these tumors. The other five nPKCd-positive tumors (-12.2%, 3 high, 1 medium, and 1 low level of nPKCd) had similar nPKCd levels compared with their matched pretreatment tumors (FIG. 6A), suggesting the role of nPKCd in intrinsic TKI- resistance. To further validate the clinical importance of the findings in TKI -intrinsic resistance, the status of nPKCd was examined in a larger cohort including 166 naive tumors from patients with EGFR-mutant NSCLC treated with single-agent gefitinib, erlotinib, or afatinib as first-line therapy. Analysis of nPKCd expression in these TKI- naive tumor samples showed that nPKCd was highly expressed in 19 (11.4%) of the 166 patients (FIG. 6B and Table 5), well consistent with the -12% from the previous cohort (FIG. 6A). Furthermore, high expression of nPKCd in these 19 patients was associated with worse progression-free survival following TKI treatment (FIG. 6C). These findings suggested that nPKCd may contribute to both acquired (-29.3%, FIG. 6A) and intrinsic (-11.4%; FIG. 6B) resistance to TKI and that it may be a common mechanism underlying TKI resistance in human EGFR-mutant NSCLC.
[00142] Table 5: Objective response and PFS of 19 patients with high nPKCd tumors.
ID smoking EGFR alteration TKI Objective response PFS (m)
1 no 19DEL Gef SD 9.2
2 no 19DEL Erl PR 4.9
no L858R Gef PD 1.6 no L858R Gef PD 2.7 no 19DEL Gef SD 6.1 no 19DEL Gef SD 13.0 no 19DEL Gef SD 8.4 no G719A Gef SD 7.9 no V769_D770inASV Erl/Gef PD 1.9 no 19DEL Gef PR 15.9 no L858R Gef PR 3.2
12 no 19DEL Gef NA 2.1
13 no 19DEL Erl PD 1.5 14 no T790M, L858R Gef PD 0.7
15 no S768I, G719A Erl SD 6.8
16 no 19DEL Gef SD 6
17 yes 19DEL Gef PD 0.5
18 no L858R Gef PR 3.9
19 no 19DEL Gef/Erl PD 3
Gef, gefitinib; Erl, erlotinib; SD, stable disease; PR, partial response; PD, progression disease; NA, data not available
[00143] nPKCd induces resistance to third-generation TKI in T790M+ tumors: Third-generation TKIs are currently the most potent anti-cancer drugs against TKI- resistant EGFR-mutant NSCLC with T790M mutation (Rotow and Bivona, 2017). From the results above, it was noticed that T790M-positive (T790M+) patients (case 6 and 9 in Table 1), who may be considered for third-generation TKI treatment, concurrently harbored increased nPKCd in their resistant tumors (Table 1). Co-occurrence of EGFR T790M mutation and reactivation of other resistant RTKs, such as Axl, was reported in TKI-treated NSCLC (Zhang el al , 2012). These findings prompted the question whether PKC5 plays a direct role in resistance to third-generation TKI and whether the combination of third-generation TKI with PKCi may also benefit T790M+ patients with a PKC5-resistant feature. Using the H1975 cell line model system, which harbors the T790M resistant mutation but sensitive to EGFR depletion as shown in an earlier experiment (FIG. 1D and 7C), H1975 cells were treated with third-generation TKI, AZD9291 (0.1-0.2 mM). which nearly abolished all EGFR phosphorylation (FIG. 12D) but only partially inhibited cell growth (FIG. 12E). nPKCd was readily observed in untreated H1975 cells and was reduced by treatment of sotra (FIG. 12F). These findings together suggested that nPKCd may cause resistance to third generation TKI in T790M+ cells.
[00144] To assess the role of PKC5 in these cells, H1975 tumors were treated with AZD9291 in combination with sotra in vitro (FIG. 12E and 12G) and in vivo (FIG. 6D). A strong synergy (Cl < 0.3) between AZD9291 and sotra in H1975 cells in vitro (FIG. 12E) suggested that inhibition of PKC5 may provide additional benefit when combined with a third generation TKI to treat T790M+NSCLC patients with a PKC5-resistant marker. This concept was further validated in mice with H1975 tumor as well as the T790M+PDX (TM0204) tumor harboring the EGFR dell9/T790M mutation and PKC5-resistant feature (positive nPKCd staining; FIG. 8F, right). The AZD929l-sotra combination effectively led to tumor regression in H1975 and TM0204 PDX models but not sotra or AZD9291 (partially delayed tumor growth) alone (FIG. 6D and 6E). IHC staining of drug -treated tumors demonstrated that sotra at the dose used effectively reduced nPKCb.
[00145] Moreover, the combination of sotra and AZD9291 suppressed survival signaling pErk and pRelA, and enhance apoptosis (TUNEL) (FIG. 12H). To strengthen the conclusion that nPKCb is upregulated in TKI-resistant tumors, nPKCb expression levels were detected by IHC staining in two untreated control tumors and five lst generation TKI erlotinib resistant tumors from genetically engineered EGFR dell 9-mutant mice as well as in four untreated control tumors and two 3rd generation TKI AZD9291 -resistant tumors from EGFR L858R T790M mutant mice (Ji et al. , 2006; Li el al. , 2007). Encouragingly, all tumors resistant to TKI expressed higher levels of nPKCb than untreated control tumors, particularly in AZD9291 -resistant tumors (FIG. 6F). Together, these results supported the conclusion that nPKCb renders NSCLC tumors resistant to 3rd generation TKI and that sotra and AZD9291 prevent tumor growth in heterogenous T790M+ tumor models with AZD9291 resistance in a cooperative manner.
[00146] These findings offer new insights into the addiction of TKI-resistant EGFR-mutant NSCLC via TKI-insensitive EGFR survival pathways, and may partially explain the differences in the response to EGFR kinase inhibition versus EGFR protein reduction in patients with TKI-resistant EGFR mutant NSCLC. Moreover, targetable PKC5 was identified as a common mediator of the EGFR pathway that plays a role in multiple TKI-resistant mechanisms.
Example 2 - Materials and Methods
[00147] Analysis of human NSCLC clinical specimens. All specimens were acquired from patients under the auspices of clinical protocols approved by the respective ethics review board at each hospital; informed consent was obtained in all cases. The tumor tissues were obtained as pleural effusion or biopsy specimen from patients with stage IIIB or IV NSCLC that had been screened for EGFR-activating mutations. The tissues were then fixed in formalin and embedded in paraffin. Pretreated EGFR-mutant NSCLC specimens (n = 127) were retrieved from the archive of Department of Pathology at Lin-Kou Chang -Gung Memorial Hospital, Taiwan. Forty-one of these have matched TKI resistant specimens. Another 39 specimens of pretreated EGFR-mutant NSCLC were obtained from China Medical University Hospital, Taiwan. From all tissue blocks, 4- pm tissue slides were prepared for immunohistochemical staining. A rabbit monoclonal PKC5 antibody (clone ERR17075, ab 182126, Abeam, diluted at 1:2000) raised against PKC5 was used according to the product instructions.
[00148] IHC staining was performed automatically with a Leica Bone-MAX (Leica Microsystems GmbH) according to the manufacturer’s standard protocol. The slides were counterstained with hematoxylin. The immunoreactivity of nPKCd was ranked as previously described (Lo et al, 2005; Lo et al, 2007; Xia et al, 2004). Briefly, nPKCd immunoreactivity was categorized into four groups (score 0, 1, 2, and 3) according to a well- established system in which H score was generated by the percentage of positive tumor cells. The scores with their indicating percentage of positive cells are score 0 (0%), 1 (less than 50%), 2 (51-75%), 3 (more than 75%). All slides were independently viewed and scored by two pathologists. Slides in which there was a scoring discrepancy were reevaluated and reconciled by a two-headed microscope. The probability of cancer recurrence in low (score = 0 and 1) and high (score = 2 and 3) nPKCd populations was determined by Kaplan Meier analysis. The Mantel-Cox (log rank) p is reported (with p < 0.05 considered significant). For T790M mutation analysis, exons 20 of the EGFR gene in all cases were sequenced by the Sanger method. Briefly, DNA was isolated from formalin-fixed paraffin embedded (FFPE) tumor by using a QuickExtract™ FFPE DNA Extraction Kit (Epicentre). DNA fragment containing EGFR exons 20 were amplified with intron-based primers EGFR-20F (5 - GTCCCTGTGCTAGGTCTTTT-3 ' (SEQ ID NO: l)) and EGFR-20R (5 - ATCTCCCTTCCCTGATTAC-3 ' (SEQ ID NO:2)). PCR reaction was performed at 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s, 56 °C for 30 s, and 72 °C for 30 s, then by 10 min extension at 72 °C. The PCR products were bidirectional sequenced on ABI 3730 XL sequencers (Applied Biosystems) with ABI BigDye Terminator v3.1 Cycle Sequencing Kits and analyzed by Chromas Sequence Scanner Software. GenBank NM_005228 was used as the reference DNA for nucleotide positions.
[00149] Tumorigenesis assays and Immunohistochemical Staining:
Hl650/luc and GR6/luc cells were injected directly into the right chest of B ALB/c nude mice (six week-old, female). Tumor volume as indicated by luciferase intensity was measured by an IVIS system on the days shown. H1975 cells were inoculated subcutaneously into nude mice. TM0204 PDX bearing mice were purchased from Jackson Laboratory. Tumor-bearing mice were randomized and drugs administered according to treatment group. Gefitinib (5 mg/kg/day), AZD9291 (1 mg/kg/day, ~6% clinically equivalent dose), and sotrastaurin (30 mg/kg/day, 30-50% clinically equivalent dose) were administered orally five times per week (1 week equaled one treatment cycle) and continued for indicated cycles. Treatment of sotrastaurin (AEB071) in patients has been shown to be well tolerated (Martin-Liberal et al, 2014). For instance, previous studies suggested that the clinical activity of sotrastaurin without toxicity in uveal melanoma patients treated with multiple concentrations of sotrastaurin (800 mg/day as maximum tolerable dose) (Pipemo-Neumann et al, 2014).
[00150] In other clinical studies, sotrastaurin at a dose of 300-500 mg/day was safe to treat patients for organ transplantation, psoriasis, and malignant diseases (Wu-Zhang and Newton, 2013). HCC827-vector and HCC827-PKC5 cells were inoculated subcutaneously into the hind limbs of NSG mice. Tumor-bearing mice were randomized and treated with gefitinib (50 mg/kg/day). Data represent mean ± SEM. Immunohistochemical staining was performed as previously described (Shen et al, 2013). All tumors after drug treatment for 5 days were collected for immunohistochemical staining. All animal procedures were conducted under the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at MD Anderson Cancer Center (Protocol Number 06-87-06139).
[00151] Cell culture and generation of GR and EDR clones: Human NSCLC cell lines (H1650, HCC827, H1975, and H820) were obtained from ATCC. H1650, HCC827, H1975, and H820 and the corresponding subclones were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS). Human NSCLC cell line H3255 was a gift from Dr. Zhen Fan and were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS). All cell lines have been tested for mycoplasma contamination and were validated by short tandem repeat (STR) DNA fingerprinting using the AmpFLSTR® Identifiler® PCR Amplification Kit (Life Technologies Grand Island, NY). The STR profiles were compared with ATCC fingerprints and the Cell Line Integrated Molecular Authentication database. HCC827 gefitinib-resistant cells (GR cells) were generated by continuous (> 2 months) culture in standard RPMI medium in the presence of 1 mM gefitinib, followed by single-cell cloning. EDR cells were generated from H1650 cells depleted of EGFRby lentiviral infection. H1650 cells were infected with viruses overnight in the presence of polybrene (10 pg/mL), then cultured in fresh medium for 24 h and subsequently selected by puromycin (2 pg/mL) for 2 days. The cells were then subcultured and maintained in 1 pg/mL puromycin. After 7 days, most of the cells had died; the few that survived were then cultured with 1 mg/mL puromycin for 3 more months to obtain the EDR clone.
[00152] Antibodies and compounds. EGFR antibody (ab-l2) was obtained from Thermo Scientific; phospho-EGFR (#2234), cleaved PARP (#9541), phospho-p44/42 MAPK (Erkl/2) (Thr202/Tyr204) (D13.14.4E, #4370p), phospho-Akt (ser473) (#927ls), Akt (#9272s), phospho-NF-kB p65 (Ser 536) (7F1, #3036s), IkBa (44D4, #48 l2s), Ki67 (#9027s), gH2AC (97l8s) antibodies from Cell Signaling Technology; PKC5 antibody (EPR17075, abl82l26) from Abeam. Her2 (E2 4001, AHO1011) from ThermoFisher Scientific; Axl (GTX108560) from GeneTex; Anti-MAP Kinase 1/2 (Erkl/2) antibody (06-182) from EMD Millipore. TUNEF-based TumorTACS™ In Situ Apoptosis Detection Kit from Trevigen. Gefitinib, erlotinib, and SP600125 were purchased from LC Laboratories; Edelfosine from R & D Systems; FIPI, NFAT inhibitor, IWP-2, and IWP-4 from Cayman Chemical; VU0359595, aspirin, sulindac, PNU-74654, resveratrol, and NSC 668036 from Sigma-Aldrich; LGK-974 from Xcess Biosciences. Dvl-PDZ Domain Inhibitor II was obtained from EMD Millipore; Go6983, U73122, afatinib, BMS-345541, KN-62, ICG-001, and AZD9291 from Selleck Chemicals; Sotrastaurin from Chemscene.
[00153] Constructs and shRNAs: Human EGFR-dell9 (purchased from Addgene) and human PKC5 (OriGene) were subcloned into the pCDH-CMV-MCS vector (System Biosciences). Kinase-dead EGFR del 19 was constructed by site-directed mutagenesis. In brief, WT EGFR ORF with a C-terminus Flag-tag was amplified and subcloned it into the pCDH-CMV vector. The K721A mutation (kinase dead) was generated using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer’s protocol. After verification of the K721A mutation by DNA sequencing, both WT and K721A mutant were used as template to further prepare the exon 19 deletion mutant lacking amino acids 722- 726 (ELREA) using the same kit to generate the EGFR-dell9 and EGFR-dell9-KD constructs, respectively. PKC5-NLSml and NLSm3 was generated from human wide-type PKC5 vector. Each construct was verified by sequencing before use. Human EGFR shRNAs and scrambled control shRNA were constructed and described previously^. Human PKC5 shRNAs were obtained from Sigma-Aldrich.
[00154] Cell counting and cell viability assays: Cellular responses to the treatments were estimated by cell counting or cell viability assay. To count the cells with a hemocytometer, cells were seeded on six-well plates (5 c 104 cells/well) and cultured for the indicated period. For the synthetic lethal screen, H1650 cells were seeded in 24-well plates in RPMI 1640 medium containing 10% FBS overnight, then treated with the respective agent(s) for 3 days. Viable cells were identified by the Cell Counting Kit-8 (Donjindo) according to the manufacturer's protocol. For the validated cell viability assays, cells were seeded in 24-well plates in RPMI 1640 medium containing 10% FBS overnight, then treated with the respective agent(s). After the indicated days, cells were washed with PBS, fixed with ice-cold methanol, and stained with 0.5% crystal violet. Crystal violet was dissolved in acetic acid and optical density of each well measured at 570 nm (OD570) using an ELISA plate reader. The average OD570 of untreated cells was set to 100%. The percentage of treated cells that were viable were then calculated accordingly. The median inhibitory concentration (IC50) for each drug was determined from the dose-effect relationship at four or five concentrations of each drug using the CompuSyn software (version 1.0.1; CompuSyn, Inc.) by the method of Chou and Martin based on the medianeffect principle and plot. Data are expressed as percentage of control cells and mean ± s.d. of three independent experiments. The interactions of two drug treatments were evaluated by the Chou-Talalay combination indices (Chou, 2006; Chou, 2010).
[00155] Immunoprecipitation and Western blot analysis:
Immunoprecipitation (IP) and Western blot (WB) analysis were performed as previously described. Briefly, cells were washed twice with phosphate-buffered saline solution (PBS), lysed in lysis buffer, briefly sonicated, and then subjected to IP-WB. For Western blot analysis, proteins were separated by sodium dodecyl sulfate electrophoresis on a 10% or 12% polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Invitrogen). After overnight incubation with primary antibody, washing, and incubation with secondary antibodies, blots were developed with a chemiluminescence system (Pierce).
[00156] Protein Kinase C8 (PKC8) kinase activity assay: PKC5 was immunoprecipitated (IP) from HCC827 cells expressing WT PKC5 or NLS mutant (NLSml and NLSm3) and immunoprecipitates were then subjected to Western blot (WB) analysis and PKC kinase activity assay using a PKC kinase activity kit (Enzo Life Sciences, ADI-EKS50 420A). The PKC activities measured were normalized to the quantitated levels of PKC5 protein expression from IP-WB.
[00157] Antibody array: The Phospho-Explorer Antibody Microarray was purchased from Full Moon Biosystems. Microarray images were analyzed with the GenePixTM Pro 4.0 image analysis software. Fluorescence intensity measurements were normalized against local background, and cytoskeletal antibodies (b-actin and GAPDH) were used for normalization of total protein quantity between samples.
[00158] Biological network and pathway analysis: Biological networks and pathways related to the 27 mediators were analyzed with Ingenuity Pathway Analysis (IP A) software (Qiagen). All mediators identified by the antibody array analysis were uploaded into the IPA software. For the analysis of networks and pathways, the cutoff values were set as p < 10 8
[00159] Confocal microscopy analysis: Confocal microscopy analysis was performed as described previously. Briefly, drug-treated cells were washed with PBS and fixed in 100% methanol for 20 min at -20 °C. Cells were then subjected to permeabilization with 0.5% Triton X-100 with 3% bovine serum albumin overnight at 4 °C. After that, cells were incubated with primary antibodies overnight at 4 °C, washed with PBS and further incubated with the appropriate secondary antibody. Nuclei were counterstained with 4,6-diamidino-2- phenylindole (DAPI) before mounting. Confocal fluorescence images were captured using a Zeiss LSM710 laser microscope. The relative intensity of PKC5 in nuclei to that in the whole cell was determined by Image J version 1.49 software.
[00160] Quantification and Statistical Analysis: All experiments were repeated at least three times unless otherwise indicated. Error bars represent standard deviation (SD). Student's t test was used to compare two groups of independent samples. Correlations were analyzed using the Pearson chi-square test. A p value < 0.05 was considered statistically significant.
[00161] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
Allen-Petersen, B. L., Carter, C. J., Ohm, A. M., and Reyland, M. E. (2014). Oncogene 33, 1306-1315.
Basu, A., and Pal, D. (2010). Scientific World Journal 10, 2272-2284.
Bivona, T. G., Hieronymus, H., Parker, J., Chang, K., Taron, M., Rosell, R., Moonsamy, P., Dahlman, K., Miller, V. A., Costa, C., et al. (2011). Nature 471, 523-526.
Chattopadhyay, A., Vecchi, M., Ji, Q., Memaugh, R., and Carpenter, G. (1999). J Biol Chem 274, 26091-26097.
Dutil, E. M., Toker, A., and Newton, A. C. (1998). Curr Biol 8, 1366-1375.
Elkabets, M., Pazarentzos, E., Juric, D., Sheng, Q., Pelossof, R. A., Brook, S., Benzaken, A.
O .,
Rodon, J., Morse, N., Yan, J. J., et al. (2015). Cancer Cell 27, 533-546.
Ercan, D., Zejnullahu, K., Yonesaka, K., Xiao, Y., Capelletti, M., Rogers, A., Lifshits, E., Brown, A., Lee, C., Christensen, J. G., et al. (2010). Oncogene 29, 2346-2356.
Haber, D. A., Gray, N. S., and Baselga, J. (2011). Cell 145, 19-24.
Hirsch, F. R., Varella-Garcia, M., and Cappuzzo, F. (2009). Oncogene 28 Suppl 1 , S32-37.
Janne, P. A., Yang, J. C., Kim, D. W., Planchard, D., Ohe, Y., Ramalingam, S. S., Ahn, M. J., Kim, S. W., Su, W. C., Horn, L„ et al. (2015). N Engl J Med 372, 1689-1699.
Ji, H., Li, D., Chen, L., Shimamura, T., Kobayashi, S., McNamara, K., Mahmood, U., Mitchell,
A., Sun, Y., Al-Hashem, R., et al. (2006). Cancer Cell 9, 485-495.
Leitges, M., Gimbom, K., Elis, W., Kalesnikoff, J., Hughes, M. R., Krystal, G., and Huber,
M.
(2002). Mol Cell Biol 22, 3970-3980.
Li, D., Shimamura, T., Ji, H., Chen, L., Haringsma, H. J., McNamara, K., Liang, M. C., Perera,
S. A., Zaghlul, S., Borgman, C. L., et al. (2007). Cancer Cell 12, 81-93.
Martin-Liberal, J., Cameron, A. J., Claus, J., Judson, I. R., Parker, P. J., and Linch, M.
(2014).
Biochim Biophys Acta 1846, 547-559. Meyer, A. S., Miller, M. A., Gertler, F. B., and Lauffenburger, D. A. (2013). Sci Signal 6, ra66.
Minari, R., Bordi, P., and Tiseo, M. (2016). Transl Lung Cancer Res 5, 695-708.
Mochly-Rosen, D., Das, K., and Grimes, K. V. (2012). Nat Rev Drug Discov 11, 937-957.
Parekh, D., Ziegler, W., Yonezawa, K., Hara, K., and Parker, P. J. (1999). J Biol Chem 274, 34758-34764.
Park, K. S., Raffeld, M., Moon, Y. W., Xi, L., Bianco, C., Pham, T., Lee, L. C., Mitsudomi, T.,
Yatabe, Y., Okamoto, L, et al. (2014). J Clin Invest 124, 3003-3015.
Piotrowska, Z., Niederst, M. J., Karlovich, C. A., Wakelee, H. A., Neal, J. W., Mino- Kenudson,
M., Fulton, L., Hata, A. N., Lockerman, E. L., Kalsy, A., et al. (2015). Cancer Discov 5, 713- 722.
Pipemo-Neumann, S., Kapiteijn, E., Larkin, J. M. G., Carvajal, R. D., Luke, J. J., Seifert, H., Roozen, L, Zoubir, M., Yang, L., Choudhury, S., et al. (2014). Journal of Clinical Oncology 32.
Reyland, M. E. (2007). Biochem Soc Trans 35, 1001-1004.
Rosse, C., Linch, M., Kermorgant, S., Cameron, A. J., Boeckeler, K., and Parker, P. J.
(2010) .
Nat Rev Mol Cell Biol 11, 103-112.
Rotow, J., and Bivona, T. G. (2017). Nat Rev Cancer 77, 637-658.
Sos, M. L., Koker, M., Weir, B. A., Heynck, S., Rabinovsky, R., Zander, T., Seeger, J. M., Weiss, J., Fischer, F., Frommolt, P., et al. (2009). Cancer Res 69, 3256-3261.
Stempka, L., Girod, A., Muller, H. J., Rincke, G., Marks, F., Gschwendt, M., and
Bossemeyer, D. (1997). J Biol Chem 272, 6805-6811.
Wu-Zhang, A. X., and Newton, A. C. (2013). Biochem J 452, 195-209.
Zhang, Z., Lee, J. C., Lin, L., Olivas, V., Au, V., LaFramboise, T., Abdel-Rahman, M., Wang, X., Levine, A. D., Rho, J. K., et al. (2012). Nat Genet 44, 852-860.

Claims

1. A method for treating cancer in a subject comprising administering an effective
amount of a protein kinase C delta (PKC5) inhibitor and/or a phospholipase C gamma (PLCy) inhibitor, in combination with an epidermal growth factor (EGFR) tyrosine kinase inhibitor (TKI), to the subject.
2. The method of claim 1, wherein the subject is administered the PKC5 inhibitor and EGFR TKI.
3. The method of claim 1, wherein the subject is administered the PLCy inhibitor and EGFR TKI.
4. The method of claim 1, wherein the subject is administered the PKC5 inhibitor, PLCy inhibitor and EGFR TKI.
5. The method of any of claims 1-4, wherein the cancer is an EGFR-mutant cancer.
6. The method of any of claims 1-4, wherein cancer is an EGFR TKI-resistant cancer.
7. The method of claim 6, wherein the EGFR-TKI resistant cancer comprises
amplification or upregulation of Axl, Her-2, c-Met, Akt, Erk, and/or NF-kB signaling.
8. The method of claim 6, wherein the EGFR-TKI resistant cancer comprises an EGFR second-site mutation.
9. The method of claim 6 or 7, wherein the EGFR second-site mutation is T790M and/or C797S.
10. The method of any of claims 1-9, wherein the cancer is lung cancer.
11. The method of claim 10, wherein the lung cancer is non-small cell lung cancer
(NSCLC).
12. The method of any of claims 1-11, wherein the PKC5 inhibitor is a pan-PKC
inhibitor.
13. The method of any of claims 1-12, wherein the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893.
14. The method of claim 13, wherein the sotrastaurin is administered at a dose of 300-500 mg/day.
15. The method of any of claims 1-14, wherein the PKC5 inhibitor is not enzastaurin.
16. The method of any of claims 1-15, wherein the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib.
17. The method of any of claims 1-16, wherein the EGFR TKI is administered at a dose of 50-300 mg/day.
18. The method of any of claims 1-17, wherein the PLCy inhibitor is U73122.
19. The method of any of claims 1-18, wherein PKC5 inhibitor, PLCy inhibitor, and/or EGFR TKI are administered orally, intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.
20. The method of any of claims 1-19, wherein PKC5 inhibitor, PLCy inhibitor, and/or EGFR TKI are administered intravenously.
21 The method of any of claims 1-19, wherein PKC5 inhibitor, PLCy inhibitor, and/or EGFR TKI are administered more than once.
22 The method of any of claims 1-19, wherein the PKC5 inhibitor, PLCy inhibitor, and/or EGFR TKI are administered daily.
23. The method of any of claims 1-21, wherein the PKC5 inhibitor, PLCy inhibitor, and/or EGFR TKI are administered concurrently.
24. The method of any of claims 1-22, wherein the PKC5 inhibitor is administered before the EGFR TKI.
25. The method of any of claims 1-22, wherein the PKC5 inhibitor is administered before the EGFR TKI.
26. The method of any of claims 1-25, wherein the subject is human.
27. The method of any of claims 1-26, further comprising the step of administering at least one additional therapeutic agent to the subject.
28. The method of claim 27, wherein the at least one additional therapeutic agent is selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, and immunotherapy.
29. The method of claim 27 or 28, wherein the at least one additional therapeutic agent is an immunomodulator, growth factor, or cytokine.
30. A pharmaceutical composition comprising a PKC5 inhibitor, PLCy inhibitor, and/or EGFR TKI.
31. The composition of claim 30, wherein the composition comprises the PKC5 inhibitor and EGFR TKI.
32. The composition of claim 30, wherein the composition comprises the PLCy inhibitor and EGFR TKI.
33. The composition of claim 30, wherein the composition comprises the PKC5 inhibitor, PLCy inhibitor and EGFR TKI.
34. The composition of any of claims 30-33, wherein the PKC5 inhibitor is sotrastaurin (AEB071) or Go-6893.
35. The composition of any of claims 30-34, wherein the EGFR TKI is AZD9291
(osimertinib), gefitinib, or erlotinib.
36. The composition of any of claims 30-35, wherein the PLCy inhibitor is U73122.
37. The pharmaceutical composition of any of claims 30-36 for use in the treatment of
EGFR-resistant cancer.
38. The use of a therapeutically effective amount of a PKC5 inhibitor, PLCy inhibitor, and/or EGFR TKI for the treatment of EGFR-resistant cancer.
39. The use of claim 38, wherein the PKC5 inhibitor is sotrastaurin (AEB071) or Go- 6893.
40. The use of claim 38 or 39, wherein the EGFR TKI is AZD9291 (osimertinib),
gefitinib, or erlotinib.
41. The use of any of claims 38-40, wherein the PLCy inhibitor is U73122.
42. A composition comprising a therapeutically effective amount of a PKC5 inhibitor, PLCy inhibitor, and/or EGFR TKI for the treatment of EGFR-resistant cancer in a subject.
43. A method of treating cancer a subject comprising administering an effective amount of a PKC5 inhibitor to the subject, wherein the subject has been identified to have PKC5 activation.
44. The method of claim 43, wherein PKC5 activation is detected by increased nuclear PKC5 expression as compared to a control.
45. The method of claim 43 or 44, wherein nuclear PKC5 is detected by
immunohistochemistry, immunofluorescence, or western blot.
46. The method of any of claims 43-45, wherein the PKC5 inhibitor is sotrastaurin
(AEB071) or Go-6893.
47. The method of any of claims 43-46, further comprising administering an EGFR TKI.
48. The method of claim 47, wherein the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib.
49. The method of any of claims 44-48, wherein the EGFR-resistant cancer is NSCLC.
50. A method of predicting response to an EGFR TKI comprising detecting the level of nuclear PKC5 in a sample, wherein an increased nuclear PKC5 as compared to a control indicates a subject is resistant to the EGFR TKI.
51. The method of claim 50, wherein nuclear PKC5 is detected by
immunohistochemistry, immunofluorescence, or western blot.
52. The method of claim 50 or 51, further comprising administering a PKC5 inhibitor and EGFR TKI to the subject identified to be resistant to the EGFR TKI.
53. The method of claim 52, wherein the PKC5 inhibitor is sotrastaurin (AEB071) or Go- 6893.
54. The method of claim 52 or 53, wherein the EGFR TKI is AZD9291 (osimertinib), gefitinib, or erlotinib.
55. An in vitro method of identifying an EGFR TKI resistant sample comprising:
(a) obtaining a cancer sample; and
(b) detecting a level of nuclear PKC5 in the sample, wherein an elevated level of nuclear PKC5 indicates the sample is EGFR TKI resistant.
56. The method of claim 55, wherein nuclear PKC5 is detected by
immunohistochemistry, immunofluorescence, or western blot.
57. The method of claim 55 or 56, further comprising detecting the level of PLCy.
58. The method of claim 57, wherein an elevated level of PLCy further indicates the sample is EGFR TKI resistant.
PCT/US2019/059424 2018-11-02 2019-11-01 Combination therapy for the treatment of egfr tyrosine kinase inhibitor resistant cancer WO2020092924A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022042681A1 (en) * 2020-08-28 2022-03-03 Shanghai Junshi Biosciences Co., Ltd. Use of an anti-pd-1 antibody and a cytotoxic anticancer drug in treatment of non-small cell lung cancer
WO2024051679A1 (en) * 2022-09-05 2024-03-14 应世生物科技(南京)有限公司 Pharmaceutical combination of fak inhibitor and egfr-tki, and use

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110245256A1 (en) * 2010-03-30 2011-10-06 Novartis Ag Use of a pkc inhibitor
US20170107577A1 (en) * 2014-03-11 2017-04-20 The Council Of The Queensland Institute Of Medical Research Determining Cancer Aggressiveness, Prognosis and Responsiveness to Treatment
WO2017100642A1 (en) * 2015-12-11 2017-06-15 Regeneron Pharmaceuticals, Inc. Methods for reducing or preventing growth of tumors resistant to egfr and/or erbb3 blockade
US20170252322A1 (en) * 2012-09-19 2017-09-07 Faller & Williams Technology, Llc PKC Delta Inhibitors for use as Therapeutics
US20180179181A1 (en) * 2014-08-06 2018-06-28 Novartis Ag Protein kinase c inhibitors and methods of their use

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110245256A1 (en) * 2010-03-30 2011-10-06 Novartis Ag Use of a pkc inhibitor
US20170252322A1 (en) * 2012-09-19 2017-09-07 Faller & Williams Technology, Llc PKC Delta Inhibitors for use as Therapeutics
US20170107577A1 (en) * 2014-03-11 2017-04-20 The Council Of The Queensland Institute Of Medical Research Determining Cancer Aggressiveness, Prognosis and Responsiveness to Treatment
US20180179181A1 (en) * 2014-08-06 2018-06-28 Novartis Ag Protein kinase c inhibitors and methods of their use
WO2017100642A1 (en) * 2015-12-11 2017-06-15 Regeneron Pharmaceuticals, Inc. Methods for reducing or preventing growth of tumors resistant to egfr and/or erbb3 blockade

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DEVRIES ET AL.: "Nuclear import of PKC6 is required for apoptosis: identification of a novel nuclear import sequence", EMBO J, vol. 21, 15 November 2002 (2002-11-15), pages 6050 - 6060, XP055705893 *
LEE ET AL.: "Targeting PKC5 as a Therapeutic Strategy against Heterogeneous Mechanisms of EGFR Inhibitor Resistance in EGFR-Mutant Lung Cancer", CANCER CELL, vol. 34, 10 December 2018 (2018-12-10), pages 954 - 969, XP085555160 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022042681A1 (en) * 2020-08-28 2022-03-03 Shanghai Junshi Biosciences Co., Ltd. Use of an anti-pd-1 antibody and a cytotoxic anticancer drug in treatment of non-small cell lung cancer
WO2024051679A1 (en) * 2022-09-05 2024-03-14 应世生物科技(南京)有限公司 Pharmaceutical combination of fak inhibitor and egfr-tki, and use

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