US20130059851A1 - Methods of Diagnosing and Treating Cancer in Patients Having or Developing Resistance to a First Cancer Therapy - Google Patents

Methods of Diagnosing and Treating Cancer in Patients Having or Developing Resistance to a First Cancer Therapy Download PDF

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US20130059851A1
US20130059851A1 US13/583,056 US201113583056A US2013059851A1 US 20130059851 A1 US20130059851 A1 US 20130059851A1 US 201113583056 A US201113583056 A US 201113583056A US 2013059851 A1 US2013059851 A1 US 2013059851A1
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kinase
protein kinase
inhibitor
raf
protein
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Levi A. Garraway
Cory M. Johannessen
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Dana Farber Cancer Institute Inc
Broad Institute Inc
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Definitions

  • Oncogenic mutations in the serine/threonine kinase B-RAF are found in 50-70% of malignant melanomas. (Davies, H. et al., Nature 417, 949-954 (2002).)
  • BRAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma (Hoeflich, K. P. et al., Cancer Res. 69, 3042-3051 (2009); McDermott, U. et al., Proc. Natl. Acad. Sci. USA 104, 19936-19941 (2007); Solit, D.
  • MAPK mitogen-activated protein kinase
  • the present invention relates to the development of resistance to therapeutic agents in the treatment of cancer and identification of targets that confer resistance to treatment of cancer.
  • the present invention also relates to identification of parallel drug targets for facilitating an effective long-term treatment strategy and to identifying patients that would benefit from such treatment.
  • a method of identifying a subject having cancer who is likely to benefit from treatment with a combination therapy with a RAF inhibitor and a second inhibitor includes assaying a gene copy number, a mRNA or a protein level or phosphorylation of one or more kinase targets selected from the group consisting of MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3) in cancer cells obtained from the subject.
  • MAP3K8 TPL2/COT
  • RAF1 CRAF
  • CrkL CrkL
  • FGR Fgr
  • PRKCE PRKCE
  • Prkch PRKCH
  • ErbB2 ErbB2
  • AXL Axl
  • PAK3 PAK3
  • the method further includes comparing the gene copy number, the mRNA or the protein level or the phoshorylation with a gene copy number, a mRNA or a protein level or phosphorylation of the target kinase in cells obtained from a subject without the cancer and correlating increased gene copy number or an alteration in mRNA expression or protein overexpression or phosphorylation of the target kinase in the cancer cells relative to the cells from the subject without the cancer with the subject having the cancer who is likely to benefit from treatment with the combination therapy.
  • a method of treating cancer in a subject in need thereof includes administering to the subject an effective amount of a RAF inhibitor and an effective amount of a second inhibitor, wherein the second inhibitor is a MEK inhibitor or a TPL2/COT inhibitor.
  • a method of identifying a kinase target that confers resistance to a first inhibitor includes culturing cells having sensitivity to the first inhibitor and expressing a plurality of kinase ORF clones in the cell cultures, each cell culture expressing a different kinase ORF clone. The method further includes exposing each cell culture to the inhibitor and identifying cell cultures having greater viability than a control cell culture after exposure to the inhibitor to identify the kinase ORF clone that confers resistance to the first inhibitor.
  • FIG. 1 illustrates an ORF-based functional screen which identified COT and C-RAF kinases as drivers of resistance to B-RAF inhibition.
  • ORFs Nine ORFs (circles) scored 2 standard deviations (dashed line, 58.64%) from the mean of all ORFs (dashed line, 44.26%);
  • Secondary screen in A375 and SKMEL28 prioritizes the top 9 candidate ORFs across a multipoint PLX4720 concentration scale.
  • FIG. 2 illustrates resistance to B-RAF inhibition via MAPK pathway activation.
  • Indicated ORFs were expressed in A375. Levels of phosphorylated MEK and ERK were assayed following 18 h. treatment with DMSO ( ⁇ ) or PLX4720 (concentration noted);
  • Proliferation of A375 expressing indicated ORFs. Error bars represent standard deviation between replicates (n 6).
  • COT mRNA has an internal start codon (30M) resulting in two protein products of different lengths; amino acids 1-467 or 30-467, noted with arrows.
  • FIG. 3 illustrates COT expression predicts resistance to B-RAF inhibition in cancer cell lines.
  • OUMS-23 and M307 represent cell lines with COT expression/amplification and all others represent cell lines with undetectable/unaltered COT;
  • FIG. 4 illustrates COT-expressing B-RAFV600E cell lines exhibit resistance to allosteric MEK inhibitors.
  • PLX4720 positions C-RAF in a signaling-competent complex (upper right panel) that is activated by oncogenic events upstream of C-RAF (lower right panel), subsequently driving resistance.
  • COT/RAF-containing complexes are sufficient to activate the MAPK pathway and mediate resistance (lower left panel).
  • FIG. 5 illustrates a schematic outline of an ORF-based functional screen for kinases that drive resistance to B-RAF inhibition.
  • the B-RAF V600E cell line A375 was lentivirally transduced with the 597 kinases in the CCSB/Broad Institute Kinase ORF Collection.
  • ORFs having a positive or negative effect on proliferation in control-treated A375 were identified and removed from final analysis.
  • Resistance-promoting ORFs were identified by generating a differential viability ratio between B-RAF inhibited (PLX4720-treated) and control-treated cells. Differential viability was normalized to a constitutively active MEK1 allele, MEK1 DD ; an assay specific positive control.
  • FIG. 6 illustrates that the CCSB/Broad Institute Kinase ORF Collection is well expressed via high titer lentivirus.
  • (a) schematic of the pLX-BLAST-V5 lentiviral expression vector used for all ORF-screens and subsequent validation.
  • (b) GFP-tagged ORFs representing a broad size range were lentivirally expressed in Jurkat cells and the percentage of GFP-expressing cells/ORF (e.g., infected cells) quantified, demonstrating high viral titer across a range of ORF sizes.
  • FIG. 7 illustrates the expression of candidate resistance ORFs.
  • 293T were transiently transfected with pLX-BLAST-V5-ORF (indicated) and expression detected using an anti-V5-HRP antibody.
  • the AXL clone is ‘closed’ and has a stop codon preceding the V5 tag. See FIG. 12 for verification of expression; (*) on dark-exposure indicate the expression of three ORFs not visible in the lighter exposure.
  • FIG. 8 illustrates that a secondary screen prioritizes the top 9 candidate B-RAF inhibitor resistance ORFs.
  • the top nine ORFs scoring in the primary screen were expressed in A375 or SKMEL28 and a GI 50 from an 8-point PLX4720 concentration range.
  • FIG. 9 illustrates the effects of ORF expression on proliferation in B-RAF V600E cell lines. Proliferation, relative to MEK1, in (a) A375 or (b) SKMEL28 expressing indicated ORFs after 7 days of growth.
  • FIG. 10 illustrates that ectopic expression of constitutively active MEK1 (MEK1 DD ) and COT lead to increased pMEK/pERK in A375, whereas C-RAF reduces pMEK/pERK levels.
  • Lysates from A375 ectopically expressing GFP, MEK1, MEK DD , COT or C-RAF were analyzed via immunoblot for levels of pERK and pMEK.
  • GFP and MEK1 (lanes 1-3) were separated from COT/C-RAF (lanes 4-5) to prevent residual V5-MEK1 signal from overwhelming that of COT and C-RAF, which are expressed at much lower levels.
  • FIG. 11 illustrates that the kinase activity of COT and C-RAF is required for sustained ERK phosphorylation in the context of PLX4720 treatment.
  • Immunoblot analysis of A375 expressing ectopic (a) MEK1, wild type COT or kinase inactive COT (COT K167R ) or (b) MEK1, wild type C-RAF or kinase inactive C-RAF (C-RAF K375M ) treated with 1 ⁇ M PLX4720 for 18 h.
  • FIG. 12 illustrates the effects of ORF expression on MAPK signaling in the context of the B-RAF inhibitor PLX4720.
  • MAPK pathway activation was assessed by immunoblot analysis of pERK and pMEK in A375 expressing the indicated ORFs in the presence of PLX4720 (18 h., concentration indicated).
  • (*) indicates the use of an antibody directed against the expressed ORF, not the V5 epitope.
  • AXL was cloned without the V5 tag.
  • FIG. 13 illustrates that B-RAF associates with immunoprecipitated C-RAF in A375 following 18 hr. treatment with 1 ⁇ M PLX4720 (+) or DMSO ( ⁇ ), (a). WCE; whole cell extract controls. Ectopically expressed C-RAF constitutively associates with B-RAF and is phosphorylated at S338, consistent with membrane localization and activation. MEK1, MEK DD and COT-expressing A375 show no evidence of C-RAF activation, (b).
  • FIG. 14 illustrates that Retroviral expression of a wild-type C-RAF or a high-activity truncation mutant of C-RAF (C-RAF(22W)) renders A375 resistant to the B-inhibitor PLX4720 (a) and leads to sustained pERK levels in the context of PLX4720 treatment (1 ⁇ M, 18 h.), (b).
  • C-RAF expression levels achieved with retroviruses are significantly lower than with lentiviral-based systems, resulting in a lower GI 50 than that achieved with lentiviral C-RAF.
  • FIG. 15 illustrates the effects of B-RAF V600E on COT mRNA
  • a Quantitative RT/PCR of COT mRNA expression relative to GAPDH mRNA expression in transformed primary melanocytes expressing wild-type B-RAF (vector) or B-RAF V600E COT expression was normalized to that of vector-expressing primary melanocytes.
  • ** Significant, p 0.05 (Student's two-tailed, paired T-Test). Endogenous COT mRNA is undetectable in PLX4720-sensitive A375 and ectopically expressed COT mRNA levels are unaffected by 1 ⁇ M PLX4720 treatment. A375 expressing GFP or COT were treated for 18 h.
  • FIG. 16 illustrates that B- and C-RAF protein levels are not required for COT-mediated ERK phosphorylation.
  • A375 expressing ectopic MEK1 (control) or COT were sequentially infected with lentivirus expressing shRNAs targeting B-RAF, C-RAF or control shRNA (shLuc) and assayed for expression of the indicated proteins in the presence (+) or absence ( ⁇ ) of 1 ⁇ M PLX4720, 18 h.
  • FIG. 17 illustrates SNP analysis of 752 cell lines reveals copy number alterations in MAP3K8/COT.
  • 752 cell lines hat had undergone copy number analysis, 534 had also undergone mutation profiling.
  • Thirty-eight (7.1%) of mutation-profiled cells harbor the B-RAF V600E mutation.
  • Two cell lines (OUMS-23, RPMI-7951, indicated) harbor the B-RAF V600E mutation along with copy number gain in MAP3K8/COT.
  • FIG. 18 illustrates MAP3K8/COT alterations in the cancer cell line OUMS-23.
  • OUMS-23 is one of the top 2% (of 765 cell lines) expressing COT mRNA.
  • FIG. 19 illustrates that COT mRNA and protein are expressed in B RAF-inhibitor resistant cell lines and tissue.
  • FIG. 20 illustrates that depletion of COT affects viability in the COT amplified cell line RPMI-7951.
  • FIG. 21 illustrates effects of ORF expression on the GI 50 of a panel of MAPK pathway inhibitors in SKMEL28.
  • the half-maximal growth-inhibitory concentration (GI 50 ) of SKMEL28 ectopically expressing MEK1, MEK1 DD or COT was determined for the RAF inhibitors PLX4720 and RAF265 and the MEK1/2 inhibitors CI-1040 and AZD6244.
  • the change in GI 50 for MEK1 DD and COT was determined for each compound.
  • FIG. 22 illustrates that COT can activate ERK via MEK-independent and MEK-dependent mechanisms.
  • FIG. 23 illustrates that combinatorial MAPK pathway inhibition effectively suppresses proliferation in SKMEL28.
  • Error bars represent the standard deviation between replicates.
  • FIG. 24 illustrates that COT over expression is sufficient to render melanoma cancer cells with the B-RAF V600E mutation resistant to B-RAF inhibition.
  • FIG. 25 illustrates the top nine ORFs scoring in the primary screen were expressed in (a) SKEL28 of (b) A375 and a GI50 is shown for 4 MAPK pathway inhibitors (PLX4720, RAF265, CI-1040, AZD-6244).
  • FIG. 26 illustrates that CRKL expression modifies sensitivity to the selective B-RAF inhibitor PLX4720 in a panel of B-RAFv600E cell lines.
  • FIG. 27 illustrates the MAP3K8/COT-amplified/B-RAF V600E -mutant cancer cell line OUMS-23 shows constitutive phosphorylation of ERK/MEK across a dose range of PLX4720.
  • FIG. 28 illustrates the insensitivity to MAPK pathway inhibition corresponds with MAP3K8/COT copy number gains in a subset of skin cancer cell lines.
  • the present invention relates to the development of resistance to therapeutic agents in the treatment of cancer and identification of targets that confer resistance to treatment of cancer.
  • the present invention also relates to identification of parallel drug targets for facilitating an effective long-term treatment strategy and to identifying patients that would benefit from such treatment.
  • the present invention relates to kinases and in particular to MAP kinase pathway components.
  • the mitogen-activated protein kinase (MAPK) cascade is a critical intracellular signaling pathway that regulates signal transduction in response to diverse extracellular stimuli, including growth factors, cytokines, and proto-oncogenes. Activation of this pathway results in transcription factor activation and alterations in gene expression, which ultimately lead to changes in cellular functions including cell proliferation, cell cycle regulation, cell survival, angiogenesis and cell migration.
  • Classical MAPK signaling is initiated by receptor tyrosine kinases at the cell surface, however many other cell surface molecules are capable of activating the MAPK cascade, including integrins, heterotrimeric G-proteins, and cytokine receptors.
  • Ligand binding to a cell surface receptor typically results in phosphorylation of the receptor.
  • the adaptor protein Grb2 associates with the phosphorylated intracellular domain of the activated receptor, and this association recruits guanine nucleotide exchange factors including SOS-I and CDC25 to the cell membrane. These guanine nucleotide exchange factors interact with and activate the GTPase Ras.
  • Ras isoforms include K-Ras, N-Ras, H-Ras and others.
  • Raf serine/threonine kinase Raf (e.g., A-Raf, B-Raf or Raf-1) is recruited to the cell membrane through interaction with Ras. Raf is then phosphorylated. Raf directly activates MEKI and MEK2 by phosphorylation of two serine residues at positions 217 and 221. Following activation, MEKI and MEK2 phosphorylate tyrosine (Tyr-185) and threonine (Thr-183) residues in serine/threonine kinases Erkl and Erk2, resulting in Erk activation.
  • Tyr-185 tyrosine
  • Thr-183 threonine residues in serine/threonine kinases Erkl and Erk2, resulting in Erk activation.
  • Erk Activated Erk regulates many targets in the cytosol and also translocates to the nucleus, where it phosphorylates a number of transcription factors regulating gene expression.
  • Erk kinase has numerous targets, including Elk-I, c-Etsl, c-Ets2, p90RSKI, MNKI, MNK2, MSKI, MSK2 and TOB. While the foregoing pathway is a classical representation of MAPK signaling, there is considerable cross talk between the MAPK pathway and other signaling cascades.
  • Ras signaling Aberrations in MAPK signaling have a significant role in cancer biology. Altered expression of Ras is common in many cancers, and activating mutations in Ras have also been identified. Such mutations are found in up to 30% of all cancers, and are especially common in pancreatic (90%) and colon (50%) carcinomas. In addition, activating Raf mutations have been identified in melanoma and ovarian cancer. The most common mutation, BRAF V600E , results in constitutive activation of the downstream MAP kinase pathway and is required for melanoma cell proliferation, soft agar growth, and tumor xenograft formation. Based on the defined role of MAPK over-activation in human cancers, targeting components of the MAPK pathway with specific inhibitors is a promising approach to cancer therapy. However, patients may have innate resistance or acquire resistance to these promising therapies. Identification of target kinases, diagnostic and/or prognostic markers and treatment therapies for these patients with innate or acquired resistance are described below.
  • the present invention relates to methods of identifying targets capable of driving resistance to clinically efficacious therapies using a high throughput screening assay.
  • the method may include an open reading frame (ORF)-based functional screen for kinases that drive resistance to therapeutic agents.
  • the method may include providing a cell line with a kinase known to have an oncogenic mutation.
  • a library of kinase ORFs may be individually expressed in the cell line so that a plurality of clones each expressing a different ORF from the library may be further evaluated.
  • Each clone may be (1) exposed to an inhibitor of the known kinase in the cell line and (2) monitored for growth changes based on the expression of the ORF in the cell line without the inhibitor.
  • the remaining clones each expressing a different kinase are then compared for viability between a control and a treated clone and normalized to a positive control.
  • Increased cell viability after treatment with the inhibitor identifies ORFs that confer resistance and therefore identifies kinase targets for treatment with an additional inhibitor.
  • clones scoring above two standard deviations from the normalized mean may be target kinases indicating treatment with an additional inhibitor is beneficial to the subject.
  • FIG. 5 a schematic of a high throughput functional screening assay for kinases that drive resistance to B-RAF inhibition is shown in FIG. 5 .
  • CCSB Center for Cancer Systems Biology
  • FIGS. 1 a , 1 b Table 3
  • This publically available collection can be rapidly transferred into a variety of expression vectors for various end-applications. Any type of expression vector known to one skilled in the art may be used to express the Kinase ORF collection.
  • a selectable, epitope-tagged, lentiviral expression vector capable of producing high titer virus and robust ORF expression in mammalian cells may be created to express the kinase collection, (pLX-BLAST-V5, FIG. 6 a ).
  • the arrayed kinase ORF collection may be stably expressed in A375, a B-RAF V600E malignant melanoma cell line that is sensitive to the RAF kinase inhibitor PLX4720 ( FIGS. 1 a , 1 b and 6 c , Table 3).
  • Clones of ORF expressing cells treated with 1 ⁇ M PLX4720 are screened for viability relative to untreated cells and normalized to an assay-specific positive control, MEK1 S218/222D (MEK1 DD ) (Table 4). ORFs that affected baseline viability or proliferation are removed from the analysis.
  • the gene encoding the target kinase may be MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3).
  • the gene encoding the target kinase may be a MAPK pathway activator.
  • the gene encoding the target kinase may be a MAP3 kinase that directly phosphorylates and activates MEK. In some embodiments, the gene encoding the target kinase may encode an adapter protein that is amplified and phosphorylated in melanoma.
  • the ORF collection may be stably expressed in a cell line having a different mutation in B-RAF, for example, another mutation at about amino acid position 600 such as V600K, V600D, and V600R. Additional B-RAF mutations include the mutations described in Davies et al. Nature, 417, 949-954, 2002, Table 1. Cell lines may be used that are sensitive to other RAF kinase inhibitors including, but not limited to, PLX4032; GDC-0879; RAF265; sorafenib; SB590855 and/or ZM 336372. In some embodiments, the ORF collection may be stably expressed in a cell line having a sensitivity to a MEK inhibitor.
  • Non-limiting examples of MEK inhibitors include, AZD6244; CI-1040; PD184352; PD318088, PD98059, PD334581, RDEA119, 6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrile and 4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile. Additional RAF and MEK inhibitors are described below. By way of non-limiting example, exemplary RAF inhibitors are shown in Table 1 and exemplary MEK inhibitors are shown in Table 2.
  • RAF Inhibitors Name CAS No. Structure 1 RAF265 927880- 90- 2 Sorafenib Tosylate Nexavar Bay 43-9006 475207- 59-1 Sorafenib 4-[4-[[4-chloro-3- (trifluoromethyl)phenyl]carbamoylamino] phenoxy]-N-methyl-pyridine-2- carboxamide 284461- 73-0 3 SB590885 4 PLX4720 918505- 84-7 5 PLX4032 1029872- 54-5 6 GDC-0879 905281- 76-7 7 ZM 336372 208260- 29-1
  • the present invention relates to methods of detecting the presence of one or more diagnostic or prognostic markers in a sample (e.g. a biological sample from a cancer patient).
  • a sample e.g. a biological sample from a cancer patient.
  • a variety of screening methods known to one of skill in the art may be used to detect the presence of the marker in the sample including DNA, RNA and protein detection.
  • the techniques described below can be used to determine the presence or absence of a kinase target in a sample obtained from a patient.
  • the patient may have innate or acquired resistance to kinase targeted therapies, including B-RAF inhibitors or MEK inhibitors.
  • the patient may have an innate or acquired resistance to B-RAF inhibitors PLX4720 and/or PLX4032.
  • the patient may have innate or acquired resistance to MEK inhibitor AZD6244.
  • Identification of one or more kinase targets markers in a patient assists the physician in determining a treatment protocol for the patient. For example, in a patient having one or more kinase target markers, the physician may treat the patient with a combination therapy as described in more detail below.
  • the kinase target may include, but is not limited to, MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3).
  • the marker may be an increase in the gene copy number, an increase in protein expression, phosphorylation of one or more MAP kinase pathway members, a change in mRNA expression and the like, for the kinase target.
  • identification of an activated target kinase can be useful for characterizing a treatment protocol for the patient.
  • treatment with a RAF inhibitor alone may indicate that the patient is at relatively high risk of acquiring resistance to the treatment after a period of time.
  • identification of an activated kinase target in that patient may indicate inclusion of a second inhibitor in the treatment protocol.
  • Identification of an activated kinase target may include an analysis of a gene copy number and identification of an increase in copy number of a target kinase.
  • a copy number gain in MAP3K8 is indicative of a patient having innate resistance or developing acquired resistance, in particular if the patient also has a B-RAF V600E mutation.
  • identification of an activated kinase target may include an analysis of phosphorylation of a kinase target and/or a member of the MAP kinase pathway.
  • phosphorylation of C-RAF at S338 is indicative of a patient having innate resistance or developing acquired resistance, in particular if the patient also has a B-RAF V600E mutation.
  • identification of an increase in MEK/ERK phosphorylation may be indicative of a patient having innate resistance or developing acquired resistance. Increased COT protein expression in patients having a B-RAF V600E mutation may predict resistance to RAF inhibition and MEK inhibition.
  • Identification of an activated kinase target may include an analysis of mRNA expression of a kinase target. For example, an increase in COT mRNA expression following initial treatment with a first kinase inhibitor is indicative of a patient having or developing resistance.
  • the first kinase inhibitor may be a RAF inhibitor or a MEK inhibitor.
  • the invention provides methods for treatment of a patient having cancer.
  • the methods generally comprise administration of a first inhibitor and a second inhibitor.
  • One inhibitor may be a RAF inhibitor.
  • the RAF inhibitor may be a pan-RAF inhibitor or a selective RAF inhibitor.
  • Pan-RAF inhibitors include but are not limited to RAF265, sorafenib, or SB590885.
  • the RAF inhibitor is a B-RAF inhibitor.
  • the selective RAF inhibitor is PLX4720, PLX4032, or GDC-0879-A.
  • One inhibitor may be a MEK inhibitor (see Table 2 illustrating exemplary MEK inhibitors).
  • One inhibitor may be a COT inhibitor.
  • the COT inhibitor may be a shRNA inhibitor as described below or a small molecule COT inhibitor, 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine (EMD; TPL2 inhibitor I; catalogue number 616373, PubChem ID: 9549300)
  • Inhibitors of the present invention inhibit one or more of the kinase targets including MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3) or other MAP kinase pathway targets.
  • MAP3K8 TPL2/COT
  • RAF1 CRAF
  • CrkL CrkL
  • FGR Fgr
  • a combination therapy for cancer comprising an effective amount of a RAF inhibitor and an effective amount of a MAP3K8 (TPL2/COT) inhibitor.
  • a combination therapy for cancer comprising an effective amount of a RAF inhibitor and an effective amount of a MEK inhibitor.
  • combination therapies include an effective amount of a RAF inhibitor and an effective amount of a second inhibitor targeting the gene, mRNA or protein encoded by one or more of the following: MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3).
  • the combination therapy is suitable for treatment of a patient wherein the cancer contains B-RAF mutant cells and in particular, B-RAF V600E mutant cells.
  • the present invention further provides a combination therapy for cancer, comprising an effective amount of a RAF inhibitor and an effective amount of a MEK inhibitor, wherein the subject with the cancer contains cells with altered MAP3K8 (TPL2/COT) expression or gene copy number.
  • the MEK inhibitor is CI-1040/PD184352 or AZD6244.
  • the MEK inhibitor provided herein can be CI-1040, AZD6244, PD318088, PD98059, PD334581, RDEA119, 6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrile or 4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile, Roche compound RG7420, or combinations thereof. Additional MEK inhibitors known in the art may also be used.
  • the RAF inhibitor provided herein is PLX4720, PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881, LBT-613, or CJS352 (NVP-AAL881-NX (hereafter referred to as AAL881) and NVP-LBT613-AG-8 (LBT613) are isoquinoline compounds (Novartis, Cambridge, Mass.).
  • Additional exemplary RAF inhibitors useful for combination therapy include pan-RAF inhibitors, inhibitors of B-RAF, inhibitors of A-RAF, and inhibitors of RAF-1.
  • RAF inhibitors useful for combination therapy include PLX4720, PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881, LBT-613, and CJS352.
  • Exemplary RAF inhibitors further include the compounds set forth in PCT Publication No. WO/2008/028141, the entire contents of which are incorporated herein by reference.
  • Exemplary RAF inhibitors additionally include the quinazolinone derivatives described in PCT Publication No. WO/2006/024836, and the pyridinylquinazolinamine derivatives described in PCT Publication No. WO/2008/020203, the entire contents of which are incorporated herein by reference.
  • Administration of the combination includes administration of the combination in a single formulation or unit dosage form, administration of the individual agents of the combination concurrently but separately, or administration of the individual agents of the combination sequentially by any suitable route.
  • the dosage of the individual agents of the combination may require more frequent administration of one of the agents as compared to the other agent in the combination. Therefore, to permit appropriate dosing, packaged pharmaceutical products may contain one or more dosage forms that contain the combination of agents, and one or more dosage forms that contain one of the combinations of agents, but not the other agent(s) of the combination.
  • Agents may contain one or more asymmetric elements such as stereogenic centers or stereogenic axes, e.g., asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms.
  • asymmetric elements such as stereogenic centers or stereogenic axes, e.g., asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms.
  • These compounds can be, for example, racemates or optically active forms.
  • these compounds with two or more asymmetric elements these compounds can additionally be mixtures of diastereomers.
  • compounds having asymmetric centers it should be understood that all of the optical isomers and mixtures thereof are encompassed.
  • compounds with carbon-carbon double bonds may occur in Z- and E-forms; all isomeric forms of the compounds are included in the present invention.
  • the single enantiomers can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column.
  • reference to compounds useful in the combination therapy of the invention includes both the free base of the compounds, and all pharmaceutically acceptable salts of the compounds.
  • a preferred salt is the hydrochloride salt.
  • pharmaceutically acceptable salts includes derivatives of the disclosed compounds, wherein the parent compound is modified by making non-toxic acid or base addition salts thereof, and further refers to pharmaceutically acceptable solvates, including hydrates, of such compounds and such salts.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid addition salts of basic residues such as amines; alkali or organic addition salts of acidic residues such as carboxylic acids; and the like, and combinations comprising one or more of the foregoing salts.
  • the pharmaceutically acceptable salts include non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, and cesium salt; and alkaline earth metal salts, such as calcium salt and magnesium salt; and combinations comprising one or more of the foregoing salts.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric
  • other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, and cesium salt
  • alkaline earth metal salts such as calcium salt and magnesium salt
  • organic salts include salts prepared from organic acids such as acetic, trifluoroacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC(CH 2 ) n COOH where n is 0-4; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N′-dibenzylethylenediamine salt; and amino acid salts such as arginate, as
  • an “effective amount” of a combination of agents e.g., MEK and RAF inhibitors, or RAF and COT inhibitors, or RAF and an inhibitor targeting MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3)
  • RAF1 CRAF
  • CrkL CrkL
  • FGR Fgr
  • PRKCE PRKCE
  • Prkch PRKCH
  • ErbB2 ErbB2
  • AXL Axl
  • PAK3 PAK3
  • oral dosage form is meant to include a unit dosage form prescribed or intended for oral administration.
  • An oral dosage form may or may not comprise a plurality of subunits such as, for example, microcapsules or microtablets, packaged for administration in a single dose.
  • the pharmaceutical products can be released in various forms. “Releasable form” is meant to include instant release, immediate-release, controlled-release, and sustained-release forms.
  • “Instant-release” is meant to include a dosage form designed to ensure rapid dissolution of the active agent by modifying the normal crystal form of the active agent to obtain a more rapid dissolution.
  • “Immediate-release” is meant to include a conventional or non-modified release form in which greater than or equal to about 50% or more preferably about 75% of the active agents is released within two hours of administration, preferably within one hour of administration.
  • “Sustained-release” or “extended-release” includes the release of active agents at such a rate that blood (e.g., plasma) levels are maintained within a therapeutic range but below toxic levels for at least about 8 hours, preferably at least about 12 hours, more preferably about 24 hours after administration at steady-state.
  • the term “steady-state” means that a plasma level for a given active agent or combination of active agents, has been achieved and which is maintained with subsequent doses of the active agent(s) at a level which is at or above the minimum effective therapeutic level and is below the minimum toxic plasma level for a given active agent(s).
  • treat is used herein to mean to relieve, reduce or alleviate at least one symptom of a disease in a subject.
  • treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder, such as cancer.
  • the term “treat” also denote to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.
  • the term “protect” is used herein to mean prevent delay or treat, or all, as appropriate, development or continuance or aggravation of a disease in a subject.
  • the disease is associated with a cancer.
  • subject or “patient” is intended to include animals, which are capable of suffering from or afflicted with a cancer or any disorder involving, directly or indirectly, a cancer.
  • subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from cancers.
  • the term “about” or “approximately” usually means within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems, the term “about” means within about a log (i.e., an order of magnitude) preferably within a factor of two of a given value.
  • the instant invention provides a drug combination useful for treating, preventing, arresting, delaying the onset of and/or reducing the risk of developing, or reversing at least one symptom of cancer, in a subject comprising administering to the subject a combination therapy, comprising an effective amount of a RAF inhibitor and an effective amount of a MAP3K8 (TPL2/COT) inhibitor, or an effective amount of a RAF inhibitor and an effective amount of MEK inhibitor or an effective amount of a RAF inhibitor and an effective amount of a second inhibitor targeting MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL (CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3).
  • these inhibitors are administered at therapeutically effective dosages which, when combined, provide a beneficial effect.
  • the administration may be simultaneous or sequential
  • cancer is used herein to mean a broad spectrum of tumors, including all solid tumors and hematological malignancies.
  • tumors include but are not limited to leukemias, lymphomas, myelomas, carcinomas, metastatic carcinomas, sarcomas, adenomas, nervous system cancers and geritourinary cancers.
  • the foregoing methods are useful in treating adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, anal cancer, cancer of the appendix, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brain stem glioma, cerebellar astrocytoma, malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoid tumor, carcinoma of unknown origin, central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic lymphocytic
  • the cancer may be associated with a mutation in the B-RAF gene.
  • These cancers include melanoma, breast cancer, colorectal cancers, glioma, lung cancer, ovarian cancer, sarcoma and thyroid cancer.
  • the therapeutic combination provided herein is effective for the treatment of moderate to severe cancer in a subject.
  • the optimal dose of the combination of agents for treatment of cancer can be determined empirically for each subject using known methods and will depend upon a variety of factors, including the activity of the agents; the age, body weight, general health, gender and diet of the subject; the time and route of administration; and other medications the subject is taking. Optimal dosages may be established using routine testing and procedures that are well known in the art.
  • the amount of combination of agents that may be combined with the carrier materials to produce a single dosage form will vary depending upon the individual treated and the particular mode of administration.
  • the unit dosage forms containing the combination of agents as described herein will contain the amounts of each agent of the combination that are typically administered when the agents are administered alone.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above and is readily determined by one having skill in the art.
  • therapeutically effective doses of the compounds of this invention for a patient when used for the indicated analgesic effects, will range from about 0.0001 to about 1000 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • compositions comprising a combination of agents for the treatment of cancer, e.g., melanoma.
  • the pharmaceutical formulations may additionally comprise a carrier or excipient, stabilizer, flavoring agent, and/or coloring agent.
  • compositions comprising combination of agents which can be, for example, a combination of two types of agents: (1) a RAF inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the inhibitor and (2) a MAP3K8 (TPL2/COT) inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the COT inhibitor.
  • agents which can be, for example, a combination of two types of agents: (1) a RAF inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the inhibitor and (2) a MAP3K8 (TPL2/COT) inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the COT inhibitor.
  • the combination of agents may be provided for a subject comprising BRAF mutant cells or comprising cells over expressing MAP3K8 (TPL2/COT) and include: (1) a RAF inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the inhibitor and (2) a MEK inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the MEK inhibitor.
  • the combination of agents may be administered using a variety of routes of administration known to those skilled in the art.
  • the combination of agents may be administered to humans and other animals orally, parenterally, sublingually, by aerosolization or inhalation spray, rectally, intracisternally, intravaginally, intraperitoneally, bucally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
  • Topical administration may also involve the use of transdermal administration such as transdermal patches or ionophoresis devices.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.
  • compositions for use in the present invention can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art.
  • sterile injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 propanediol or 1,3 butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono or di glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations may also be prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissues.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, acetyl alcohol and
  • compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
  • the active compounds can also be in micro-encapsulated form with one or more excipients as noted above.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • buffering agents include polymeric substances and waxes.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending
  • Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulations, ear drops, and the like are also contemplated as being within the scope of this invention.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • compositions of the invention may also be formulated for delivery as a liquid aerosol or inhalable dry powder.
  • Liquid aerosol formulations may be nebulized predominantly into particle sizes that can be delivered to the terminal and respiratory bronchioles.
  • Aerosolized formulations of the invention may be delivered using an aerosol forming device, such as a jet, vibrating porous plate or ultrasonic nebulizer, preferably selected to allow the formation of an aerosol particles having with a mass medium average diameter predominantly between 1 to 5 m.
  • the formulation preferably has balanced osmolarity ionic strength and chloride concentration, and the smallest aerosolizable volume able to deliver effective dose of the compounds of the invention to the site of the infection.
  • the aerosolized formulation preferably does not impair negatively the functionality of the airways and does not cause undesirable side effects.
  • Aerosolization devices suitable for administration of aerosol formulations of the invention include, for example, jet, vibrating porous plate, ultrasonic nebulizers and energized dry powder inhalers, that are able to nebulize the formulation of the invention into aerosol particle size predominantly in the size range from 1 5 m. Predominantly in this application means that at least 70% but preferably more than 90% of all generated aerosol particles are within 1 5 m range.
  • a jet nebulizer works by air pressure to break a liquid solution into aerosol droplets. Vibrating porous plate nebulizers work by using a sonic vacuum produced by a rapidly vibrating porous plate to extrude a solvent droplet through a porous plate.
  • An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets.
  • a variety of suitable devices are available, including, for example, AERONEB and AERODOSE vibrating porous plate nebulizers (AeroGen, Inc., Sunnyvale, Calif.), SIDESTREAM nebulizers (Medic Aid Ltd., West Wales, England), PARI LC and PARI LC STAR jet nebulizers (Pari Respiratory Equipment, Inc., Richmond, Va.), and AEROSONIC (DeVilbiss Medizinische Kunststoffische Kunststoffische Kunststoffische Kunststoffische Kunststoffische Kunststoffo Kunststoffotechnik (Deutschland) GmbH, Heiden, Germany) and ULTRAAIRE (Omron Healthcare, Inc., Vernon Hills, Ill.) ultrasonic nebulizers.
  • Compounds of the invention may also be formulated for use as topical powders and sprays that can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound to the body.
  • dosage forms can be made by dissolving or dispensing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin.
  • the rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
  • the compounds of the present invention can also be administered in the form of liposomes.
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono or multi lamellar hydrated liquid crystals that are dispersed in an aqueous medium.
  • any non toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like.
  • the preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott (ed.), “Methods in Cell Biology,” Volume XIV, Academic Press, New York, 1976, p. 33 et seq.
  • ORF expressing cells treated with 1 ⁇ M PLX4720 were screened for viability relative to untreated cells and normalized to an assay-specific positive control, MEK1 S218/222D (MEK1 DD ) (Emery, C. M. et al. Proc. Natl. Acad. Sci. USA 106, 20411-20416 (2009).) (Table 4 and summarized in FIG. 5 ).
  • MEK1 S218/222D MEK1 DD
  • FIG. 5 Nine ORFs conferred resistance at levels exceeding two standard deviations from the mean ( FIG. 1 b and Table 4) and were selected for follow-up analysis ( FIG. 7 ).
  • Three of nine candidate ORFs were receptor tyrosine kinases, underscoring the potential of this class of kinases to engage resistance pathways.
  • tyrosine kinases such as BCR-ABL (Birge, R. B. et al., Cell Commun Signal 7, 13 (2009)), but lacks intrinsic kinase activity.
  • COT and C-RAF reduced sensitivity to PLX4720 in multiple B-RAF V600E cell lines ( FIG. 1 c ) confirming the ability of these kinases to mediate resistance to RAF inhibition.
  • a secondary screen in A375 and SKMEL28 prioritizes the top 9 candidate ORFs across a multipoint PLX4720 concentration scale ( FIG. 1 d ).
  • MAP3Ks Ser/Thr MAP kinase kinase kinases
  • C-RAF is a MAP3K in the canonical MAPK cascade (McKay, M. M. and Morrison, D. K. Oncogene 26, 3113-3121 (2007)) that was previously implicated in resistance associated with stepwise selection in vitro using a pan-RAF inhibitor (Montagut, C. et al. Cancer Res 68, 4853-4861 (2008)).
  • COT the protein product of the human MAP3K8 gene
  • MAP3K Basalmeron, A.
  • C-RAF activation and heterodimerization with B-RAF constitute critical components of the cellular response to B-RAF inhibition.
  • endogenous C-RAF B-RAF heterodimers were measurable and inducible following treatment with PLX4720 ( FIG. 13 ).
  • ectopically expressed C-RAF was phosphorylated on S338 ( FIG. 13 ) and its PLX4720 resistance phenotype was associated with sustained MEK/ERK activation ( FIGS. 2 a and 13 ).
  • C-RAF(W22) a high-activity C-RAF truncation mutant
  • C-RAF(W22) a high-activity C-RAF truncation mutant
  • FIG. 14 ectopic expression of a high-activity C-RAF truncation mutant
  • C-RAF(W22) was more effective than wild-type C-RAF in mediating PLX4720 resistance and ERK activation
  • elevated C-RAF activity directs resistance to this agent.
  • oncogenic alleles of NRAS and KRAS conferred PLX4720 resistance in A375 cells ( FIG. 2 b ) and yielded sustained C-RAF(S338) and ERK phosphorylation in the context of drug treatment ( FIG. 2 c ).
  • a panel of melanoma short-term cultures was also screened for COT protein expression.
  • One of these lines expressed COT M307, a short-term culture derived from a B-RAF V600E tumor that developed resistance to allosteric MEK inhibition following initial disease stabilization ( FIG. 3 c ).
  • All three cell lines were refractory to PLX4720 treatment, exhibiting GI 50 values in the range of 8-10 ⁇ M ( FIG. 3 d ) and showing sustained ERK phosphorylation in the context of B-RAF inhibition ( FIGS. 3 e , 3 f ).
  • OUMS-23 and RPMI-7951 are MAPK pathway inhibitor-na ⁇ ve cell lines; thus, these results demonstrate that COT confers de novo resistance to RAF inhibition (a phenomenon observed in ⁇ 10% of B-RAF V600E melanomas).
  • COT expression in the context of resistance to the clinical RAF inhibitor PLX4032 was examined by obtaining biopsy material from 3 patients with metastatic, B-RAF V600E melanoma. Each case consisted of frozen, lesion-matched biopsy material obtained prior to and during treatment (“pre-treatment” and “on-treatment”; FIG. 3 g , Table 6); additionally, one sample contained two independent biopsy specimens from the same relapsing tumor site (“post-relapse”; FIG. 3 g ). Consistent with the experimental models presented above, quantitative real-time RT-PCR (qRT/PCR) analysis revealed increased COT mRNA expression concurrent with PLX4032 treatment in 2 of 3 cases.
  • qRT/PCR quantitative real-time RT-PCR
  • COT mRNA levels were further increased in a relapsing specimen relative to its pre-treatment and on-treatment counterparts ( FIG. 3 g , Patient #1).
  • An additional, unmatched relapsed malignant melanoma biopsy showed elevated COT mRNA expression comparable to levels observed in RAF inhibitor-resistant, COT-amplified cell lines ( FIG. 19 ).
  • This specimen also exhibited robust MAPK pathway activation and elevated expression of B-RAF, C-RAF and COT relative to matched normal skin or B-RAF V600E cell lines ( FIG. 19 ). Sequencing studies of this tumor revealed no additional mutations in BRAF, NRAS or KRAS (data not shown). These analyses provided clinical evidence that COT-dependent mechanisms are operant in PLX4032-resistant malignant melanomas.
  • COT-expressing cancer cells remain sensitive to MAPK pathway inhibition at a target downstream of COT or RAF was analyzed.
  • the OUMS-23 and RPMI-7951 cell lines were queried for sensitivity to the MEK1/2 inhibitor CI-1040. Both cell lines were refractory to MEK inhibition ( FIG. 4 a ) and displayed sustained ERK phosphorylation even at 1 ⁇ M CI-1040 ( FIG. 4 b ).
  • Ectopic COT expression in A375 and SKMEL28 cells also conferred decreased sensitivity to the MEK inhibitors CI-1040 and AZD6244, suggesting that COT expression alone was sufficient to induce this phenotype ( FIGS. 4 c , 4 d and 21 ).
  • RAF and MEK inhibitors in combination can override resistance to single-agents as shown in FIG. 23 . It was tested whether the combined RAF/MEK inhibition might circumvent COT-driven resistance. In the setting of ectopic COT expression, exposure to AZD6244 or CI-1040 in combination with PLX470 (1 ⁇ M each) reduced cell growth and pERK expression more effectively than did single-agent PLX4720, even at concentrations of 10 ⁇ M ( FIGS. 4 e , 4 f and 23 ). These data underscore the importance of this pathway in B-RAF V600E tumor cells and demonstrate that dual B-RAF/MEK inhibition helps circumvent resistance to RAF inhibitors.
  • CCSB Center for Cancer Systems Biology (CCSB)/Broad Institute Kinase Open Reading Frame Collection
  • kinases ORFs were assembled from multiple sources; 337 kinases were isolated as single clones from the ORFeome 5.1 collection (http://horfdb.dfci.harvard.edu), 183 kinases were cloned from normal human tissue RNA (Ambion) by reverse transcription and subsequent PCR amplification to add Gateway sequences (Invitrogen), 64 kinases were cloned from templates provided by the Harvard Institute of Proteomics (HIP), and 13 kinases were cloned into the Gateway system from templates obtained from collaborating laboratories.
  • the Gateway-compatible lentiviral vector pLX-Blast-V5 was created from the pLKO.1 backbone.
  • LR Clonase enzymatic recombination reactions were performed to introduce the 597 kinases into pLX-Blast-V5 according to the manufacturer's protocol (Invitrogen).
  • A375 melanoma cells were plated in 384-well microtiter plates (500 cells per well). The following day, cells were spin-infected with the lentivirally-packaged kinase ORF library in the presence of 8 ug/ml polybrene. 48 hours post-infection, media was replaced with standard growth media (2 replicates), media containing 1 ⁇ M PLX4720 (2 replicates, 2 time points) or media containing 10 ug/ml blasticidin (2 replicates). After four days and 6 days, cell growth was assayed using Cell Titer-Glo (Promega) according to manufacturer instructions. The entire experiment was performed twice.
  • MEK1 DD normalized differential proliferation for each individual ORF was averaged across two duplicate experiments, with two time points for each experiment (day 4 and day 6). A z-score was then generated, as described above for average MEK1 DD normalized differential proliferation. ORFs with a z-score of >2 were considered hits and were followed up in the secondary screen.
  • ORFs were expressed from pLX-Blast-V5 (lentiviral) or pWZL-Blast, pBABE-Puro or pBABE-zeocin (retroviral) expression plasmids.
  • lentiviral transduction 293T cells were transfected with 1 ⁇ g of pLX-Blast-V5-ORF or pLKO.1-shRNA, 900 ng ⁇ 8.9 (gag, pol) and 100 ng VSV-G using 6 ⁇ l Fugene6 transfection reagent (Roche). Viral supernatant was harvested 72 h post-transfection.
  • Mammalian cells were infected at a 1:10-1:20 dilution of virus in 6-well plates in the presence of 5 ⁇ g/ml polybrene and centrifuged at 2250 RPM for 1 h at 37° C. Twenty-four hours after infection blasticidin (pLX-Blast-V5, 10 ⁇ g/ml) or puro (pLKO.1, 0.75 ⁇ g/ml) was added and cells were selected for 48 hrs.
  • 293T were transfected with 1 ⁇ g of retroviral plasmid-ORF, 1 ⁇ g pCL-AMPHO and 100 ng VSV-G, as described above. Cells were infected with retrovirus containing supernatant at a 1:2 dilution in 5 ⁇ g/ml polybrene overnight, followed by media change to growth medium. Infection was repeated once more (twice total), followed by selection, above.
  • A375 (1.5 ⁇ 10 3 ) and SKMEL28 cells (3 ⁇ 10 3 ) were seeded in 96-well plates for 18 h.
  • ORF-expressing lentivirus was added at a 1:10 dilution in the presence of 8 ⁇ g/ml polybrene, and centrifuged at 2250 RPM and 37° C. for 1 h. Following centrifugation, virus-containing media was changed to normal growth media and allowed to incubate for 18 h. Twenty-four hours after infection, DMSO (1:1000) or 10 ⁇ PLX4720 (in DMSO) was added to a final concentration of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 or 0.00001 ⁇ M. Cell viability was assayed using WST-1 (Roche), per manufacturer recommendation, 4 days after the addition of PLX4720.
  • A375, SKMEL28, SKMEL30, COLO-679, WM451lu, SKMEL5, Malme 3M, SKMEL30, WM3627, WM1976, WM3163, WM3130, WM3629, WM3453, WM3682 and WM3702 were all grown in RPMI (Cellgro), 10% FBS and 1% penicillin/streptomycin.
  • M307 was grown in RPMI (Cellgro), 10% FBS and 1% penicillin/streptomycin supplemented with 1 mM sodium pyruvate. 293T and OUMS-23 were grown in DMEM (Cellgro), 10% FBS and 1% penicillin/streptomycin.
  • RPMI-7951 cells were grown in MEM (Cellgro), 10% FBS and 1% penicillin/streptomycin. Wild-type primary melanocytes were grown in HAM's F10 (Cellgro), 10% FBS and 1% penicillin/streptomycin.
  • B-RAF V600E -expressing primary melanocytes were grown in TIVA media [Ham's F-10 (Cellgro), 7% FBS, 1% penicillin/streptomycin, 2 mM glutamine (Cellgro), 100 uM IBMX, 50 ng/ml TPA, 1 mM dbcAMP (Sigma) and 1 ⁇ M sodium vanadate].
  • CI-1040 (PubChem ID: 6918454) was purchased from Shanghai Lechen International Trading Co., AZD6244 (PubChem ID: 10127622) from Selleck Chemicals, and PLX4720 (PubChem ID: 24180719) from Symansis.
  • RAF265 (PubChem ID: 11656518) was a generous gift from Novartis Pharma AG. Unless otherwise indicated, all drug treatments were for 16 h. Activated alleles of NRAS and KRAS have been previously described. (Boehm, J. S. et al. Cell 129, 1065-1079 (2007); Lundberg, A. S. et al. Oncogene 21, 4577-4586 (2002)).
  • Cultured cells were seeded into 96-well plates (3,000 cells per well) for all melanoma cell lines; 1,500 cells were seeded for A375. Twenty-four hours after seeding, serial dilutions of the relevant compound were prepared in DMSO added to cells, yielding final drug concentrations ranging from 100 ⁇ M to 1 ⁇ 105 ⁇ M, with the final volume of DMSO not exceeding 1%. Cells were incubated for 96 h following addition of drug. Cell viability was measured using the WST1 viability assay (Roche). Viability was calculated as a percentage of control (untreated cells) after background subtraction. A minimum of six replicates were performed for each cell line and drug combination.
  • NP-40 buffer 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF and 1% NP-40] containing 2 ⁇ protease inhibitors (Roche) and 1 ⁇ Phosphatase Inhibitor Cocktails I and II (CalBioChem). Lysates were quantified (Bradford assay), normalized, reduced, denatured (95° C.) and resolved by SDS gel electrophoresis on 10% Tris/Glycine gels (Invitrogen).
  • Protein was transferred to PVDF membranes and probed with primary antibodies recognizing pERK1/2 (T202/Y204), pMEK1/2 (S217/221), MEK1/2, MEK1, MEK2, C-RAF (rabbit host), pC-RAF (pS338) (Cell Signaling Technology; 1:1,000), V5-HRP (Invitrogen; (1:5,000), COT (1:500), B-RAF (1:2,000), Actin (1:1,000), Actin-HRP (1:1,000; Santa Cruz)), C-RAF (mouse host; 1:1,000; BD Transduction Labs), Vinculin (Sigma; 1:20,000), AXL (1:500; R&D Systems).
  • Lysates from tumor and matched normal skin were generated by mechanical homogenization of tissue in RIPA [50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1.0% NaDOC, 1.0% Triton X-100, 25 mM NaF, 1 mM NA3VO4] containing protease and phosphatase inhibitors, as above. Subsequent normalization and immunoblots were performed as above.
  • Biopsied tumor material consisted of discarded and de-identified tissue that was obtained with informed consent and characterized under protocol 02-017 (paired samples, Massachusetts General Hospital) and 07-087 (unpaired sample, Dana-Farber Cancer Institute). For paired specimens, ‘on-treatment’ samples were collected 10-14 days after initiation of PLX4032 treatment (Table 6).
  • Adherent RPMI-7951 cells were washed twice with 1 ⁇ PBS and incubated overnight in serum-free growth media. Subsequently, 4-(3-Chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine (EMD; TPL2 inhibitor I; Cat#: 616373, PubChem ID: 9549300), suspended in DMSO at the indicated concentration, was added to cells for 1 hour, after which protein extracts were made as described above.
  • EMD 3-Chloro-4-fluorophenylamino-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine
  • Adherent RPMI-7951 cells were infected with virus expressing shRNAs against COT or Luciferase as described above. Following selection, cells were plated (1.5 ⁇ 10 5 cells/well) onto a 24-well plate in quadruplicate. Viable cells were counted via trypan blue exclusion using a VI-CELL Cell Viability Analyzer, per manufacturer's specifications. Quadruplicate cell counts were averaged and normalized relative to that of the control shRNA.
  • the Cancer Cell Line Encyclopedia (COLE) project is a collaboration between the Broad Institute, the Novartis Institutes for Biomedical Research (NIBR) and the Genomics Institute of the Novartis Research Foundation (GNF) to conduct a detailed genetic and pharmacologic characterization of a large panel of human cancer models, to develop integrated computational analyses that link distinct pharmacologic vulnerabilities to genomic patterns and to translate cell line integrative genomics into cancer patient stratification. Chromosomal copy number and gene expression data used for this study are available online at http://www.broadinstitute.org/cgi-bin/cancer/datasets.cgi.
  • Oligonucleotide microarray analysis was carried out using the GeneChip Human Genome U133 Plus 2.0 Affymetrix expression array (Affymetrix). Samples were converted to labeled, fragmented, cRNA following the Affymetrix protocol for use on the expression microarray.
  • NRTK protein kinase
  • NRTK nucleophilicity factor-1
  • NRS/TK viral oncogene homolog 1 protein kinase
  • YSK4 80122 hypothetical protein FLJ23074 protein kinase ZAK 51776 sterile alpha motif and leucine zipper containing kinase AZK protein kinase (NRS/TK) ZAP70 7535 zeta-chain (TCR) associated protein kinase 70 kDa protein kinase (NRTK)
  • RS/TK Receptor Serine/Threonine Kinase
  • RTK Receptor Tyrosine Kinase
  • NRTK Non-Receptor Tyrosine Kinase

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