WO2011060380A1 - L'état de mutation de pik3ca et l'expression de sash1 prédisent la synergie entre le lapatinib et un inhibiteur d'akt dans le cancer du sein her2 positif - Google Patents

L'état de mutation de pik3ca et l'expression de sash1 prédisent la synergie entre le lapatinib et un inhibiteur d'akt dans le cancer du sein her2 positif Download PDF

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WO2011060380A1
WO2011060380A1 PCT/US2010/056743 US2010056743W WO2011060380A1 WO 2011060380 A1 WO2011060380 A1 WO 2011060380A1 US 2010056743 W US2010056743 W US 2010056743W WO 2011060380 A1 WO2011060380 A1 WO 2011060380A1
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patient
expression level
gene
inhibitor
akt inhibitor
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James E. Korkola
Joe W. Gray
Nora Bayani
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The Regents Of The University Of California
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • 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

  • This invention relates generally to genetic markers involved in the diagnosis and prognosis of ERBB2/HER2 -positive cancer (HER2 positive), predicting patient response to specific therapeutic compounds and providing such therapty to patients predicted to benefit from such therapy.
  • HER2 amplification occurs in approximately 20% of all breast cancer patients.
  • Therapeutic agents such as trastuzumab and lapatinib have been developed that target this alteration.
  • many patients with HER2 amplification are non-responsive to these drugs, or develop resistance to the drugs.
  • patients who are non-responsive or develop resistance to Lapatinib are put onto experimental trials with other chemotherapeutic agents for treatment of advanced breast cancer.
  • Lapatinib is currently given to patients with metastatic HER2 positive breast cancer in combination with traditional chemotherapeutic agents such as paclitaxel.
  • the invention provides for a method for identifying a HER2 -positive cancer patient suitable for treatment with a 4-anilinoquinazoline kinase inhibitor Lapatinib and an AKT inhibitor, comprising: (a) obtaining the sequence of the PIK3CA gene, mRNA, or protein from a sample from a patient; and (b) identifying any mutations in the PIK3CA gene or protein in a sample from the patient as compared to the wild-type sequence found in SEQ ID NOs:3, 4, or 11, wherein a mutation in the PIK3CA gene, mRNA, or protein indicates the patient is suitable for treatment with the 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.
  • the method further comprising (c) measuring the genomic copy number or expression level of a gene encoding HER2 in a sample from the patient, and (d) comparing the HER2 genomic copy number in the patient to normal copy number or expression level of the gene encoding HER2, the expression level of the gene encoding HER2 in a normal tissue sample or a reference expression level, or the average expression level of HER2 in a panel of normal cell lines or cancer cell lines, wherein an increase in the expression level of HER2 indicates the patient is suitable for treatment with the 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.
  • the method in another embodiment, (c) measuring the HER2 protein levels in a sample from the patient, and (d) comparing the HER2 protein levels from the sample to normal HER2 protein levels in a normal tissue sample or a reference protein level, or the average protein level of HER2 in a panel of normal cell lines or cancer cell lines, wherein an increase in the protein levels of HER2 indicates the patient is suitable for treatment with the 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.
  • Patients identified by the present invention will respond to the synergistic treatment of cancer with the 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.
  • a method for identifying a HER2 -positive cancer patient suitable for treatment with a 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor comprising: (a) measuring the expression level of the SASH1 gene in a sample from the patient; and (b) comparing the expression level of said gene from the patient with the expression level of the gene in a normal tissue sample or a reference expression level (such as the average expression level of the gene in a cell line panel or a cancer cell or tumor panel, or the like), wherein an increase in the expression level of SASH1 indicates the patient is suitable for treatment with the 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.
  • the patient identified as suitable for treatment with a combination of 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor are selected from the group consisting of 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.
  • the 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor are selected from the group consisting of 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.
  • a method for identifying a HER2 -positive cancer patient suitable for treatment with a 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor comprising: (a) obtaining the sequence of the PIK3CA gene from a sample from a patient; (b) identifying any mutations in the PIK3CA gene in a sample from the patient as compared to the wild-type sequence found in SEQ ID NOs:3, 4 or 1 1, (c) measuring the expression level of the SASH1 gene in the sample from the patient; and (d) comparing the expression level of said gene from the patient with the expression level of the gene in a normal tissue sample or a reference expression level (such as the average expression level of the gene in a cell line panel or a cancer cell or tumor panel, or the like), wherein both a mutation in the PIK3CA gene and an increase in the expression level of SASH1 indicates the patient is suitable for treatment with the 4-anilinoquinazoline kinase inhibitor
  • the invention provides for a method of treating a cancer patient comprising (a) identifying a cancer patient who is suitable for treatment with a 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor using a method of the present invention, and (b) administering a therapeutically effective amount of the 4-anilinoquinazoline kinase inhibitor and the AKT inhibitor to the patient.
  • a combination of the 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor and in other embodiments, the 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor are administered concurrently or sequentially.
  • the cancer is breast cancer and the cancer patient is a breast cancer patient.
  • the breast cancer patient is an ERBB2-positive breast cancer patient.
  • Figure 1 shows representative examples of HER2 positive breast cancer cell lines subtypes and their response to an AKT inhibitor (triangle), lapatinib (square), or a combination of the two (diamond).
  • Y-axis represents % growth inhibition,
  • X-axis represents dose.
  • Each graph is labeled with the cell line name and the PIK3CA mutation found.
  • Figure 2 shows a heatmap showing synergy across nine different doses of a 1 : 1 combinations of Lapatinib + AKT inhibitor (light gray indicates significant synergy, black indicates additivity/no significant interaction, and dark gray indicates antagonism). Note synergistic interactions between the inhibitors in the cell lines with PI3K pathway mutations, while those without PI3K mutations show additivity or antagonism.
  • FIG 3 shows relative SASHl expression levels in breast cancer cell lines that show synergy with lapatinib + AKT ((HCC202; MDAMB361; HCC1954; SUM190PT;
  • BT474 does not cluster with either group.
  • Figure 4 shows a heat map which demonstrates synergy found between Lapatinib and the AKT inhibitor GSK690693 is dependent upon PI3K pathway status. Combination index values were calculated across all nine doses of drug and transformed to take into account significance (upper 95% confidence interval less than 1). Significant synergy is denoted as light gray indicates significant synergy, black indicates additivity/no significant interaction, and dark gray indicates antagonismin the heat map. Clustering of the data revealed that all the lines with similar synergistic responses had PI3K pathway mutations (cells with mutations in the PI3K pathway are denoted in red or blue). In contrast, all but two (BT474, MDAMB453) of the non-synergistic lines were wild-type for PI3K.
  • Figure 5 shows a heatmap which demonstrates synergy found between Lapatinib and the AKT inhibitor GSK2141795 is dependent upon PI3K pathway status. Combination index values were calculated across all nine doses of drug and transformed to take into account significance (upper 95% confidence interval less than 1). Significant synergy is denoted as light gray indicates significant synergy, black indicates additivity/no significant interaction, and dark gray indicates antagonism in the heat map. Clustering of the data revealed that all the lines with similar synergistic responses had PI3K pathway mutations except one (cells with mutations in the PI3K pathway are denoted in red or blue). In contrast, all but two (BT474, HCC1569) of the non-synergistic lines were wild-type for PI3K.
  • FIG. 6 shows graphs showing that introduction of a PIK3CA mutation results in synergy in previously non-synergistic HER2+ cell line.
  • Cell lines highlighted in bold HCC202; MDAMB361 ; HCC1954; SUM190PT show strongest synergistic interaction between Lapatinib and AKT.
  • the present invention provides a new combination of targeted therapeutic agents that should have clinical efficacy for the treatment of HER2 positive breast cancer.
  • the present invention also provides as genetic markers mutant PIK3CA and SASH1 which are herein shown to be predictive of patients most likely to respond to this combination.
  • the cancer patient is either patient who is known to be ERBB2-positive, i.e., HER2- positive, and overexpresses the ERBB2 protein also known as the HER2 protein, or it is not known whether patient is ERBB2-positive or not.
  • the ERBB2 status of the patient is to be determined.
  • the present methods describe the measurement and detection of expression levels of a gene as measured from a sample from a patient that comprises essentially a cancer cell or cancer tissue of a cancer tumor. Such methods for obtaining such samples are well known to those skilled in the art.
  • the expression level of a gene is measured from a sample from the patient that comprises essentially a breast cancer cell or breast cancer tissue of a breast cancer tumor.
  • Methods for detection of expression levels of a gene can be carried out using known methods in the art including but not limited to, fluorescent in situ hybridization (FISH), immunohistochemical analysis, comparative genomic hybridization, PCR methods including real-time and quantitative PCR, and other sequencing and analysis methods.
  • the expression level of the gene in question can be measured by measuring the amount or number of molecules of mRNA or transcript in a cell.
  • the measuring can comprise directly measuring the mRNA or transcript obtained from a cell, or measuring the cDNA obtained from an mRNA preparation thereof.
  • Such methods of extracting the mRNA or transcript from a cell, or preparing the cDNA thereof are well known to those skilled in the art.
  • the expression level of a gene can be measured by measuring or detecting the amount of protein or polypeptide expressed, such as measuring the amount of antibody that specifically binds to the protein in a dot blot or Western blot.
  • the proteins described in the present invention can be overexpressed and purified or isolated to homogeneity and antibodies raised that specifically bind to each protein. Such methods are well known to those skilled in the art.
  • Comparison of the detected expression level of a gene in a patient sample is often compared to the expression levels detected in a normal tissue sample or a reference expression level.
  • the reference expression level can be the average or normalized expression level of the gene in a panel of normal cell lines or cancer cell lines.
  • the method comprises measuring the expression level of ERBB2 of the patients in order to determine whether the patient is an ERBB2-positive patient.
  • the expression level of a gene encoding ERBB2 can be measured using an oligonucleotide derived from the mouse v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (Erbb2), mR A sequence of GenBank Accession No. NM_001003817.1 GL54873609, hereby incorporated by reference and shown as SEQ ID NO: 1 :
  • the expression level of a gene encoding ERBB2 can also be measured using an oligonucleotide derived from the human nucleotide sequence of GenBank Accession No. NM 004448.2 GL54792095, Homo sapiens v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), transcript variant 1, mRNA, hereby incorporated by reference and shown as SEQ ID NO: 2: 1 ggaggaggtg gaggaggagg gctgcttgag gaagtataag aatgaagttg tgaagctgag 61 attccctcc attgggaccg gagaaaccag gggagcccccc cgggcagccg cgcgcccttt 121 cccacgg
  • Methods of assaying for ERBB2 or HER2 protein overexpression include methods that utilize immunohistochemistry (IHC) and methods that utilize fluorescence in situ hybridization (FISH).
  • IHC immunohistochemistry
  • FISH fluorescence in situ hybridization
  • a commercially available IHC test is DAKO HercepTest® (DAKO Corp., Carpinteria, Calif). Patient samples having an IHC staining score of 0 -1,2 is normal, and scores of 2+ may be borerderline, while results of 2,3+ are scored as positive for multiple copies of HER2 (HER2 positive).
  • a commercially available FISH test is PathVysion® (Vysis Inc., Downers Grove, 111.).
  • the HER2 genomic copy number of a patient sample is determined using FISH.
  • HER2 -positive patients suffer from metastatic breast cancer
  • a patient's HER2 status can also be determined in relation to other types of cancers including but not limited to epithelial cancers such as pancreatic, lung, cervical, ovarian, prostate, non-small cell lung carcinomas, melanomas, squamous cell cancers, etc. It is contemplated that the present methods described herein may find use in prognosis and predicting patient response to certain combination therapies that may be used in various cancer treatments for multiple types of cancers so long as the patient criteria described herein is present as identifying a patient suitable for such combination therapy.
  • a method for identifying a HER2 -positive cancer patient suitable for treatment with a 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor comprising: (a) obtaining the sequence of the PIK3CA gene from a sample from a patient; and (b) identifying any mutations in the PIK3CA gene in a sample from the patient as compared to the wild-type sequence found in SEQ ID NO: l, wherein a mutation in the PIK3CA gene indicates the patient is suitable for treatment with the 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.
  • the PIK3CA gene is identified as Homo sapiens phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) on chromosome 3, and the wild-type PIK3CA gene sequence is found at GenBankAccession No. NG_012113.1 GL237858742, hereby incorporated by reference and identified as SEQ ID NO: 11.
  • PIK3CA mRNA sequence is found at NM_006218.2 GL54792081, hereby incorporated by reference, and shown here as SEQ ID NO:3.
  • the PIK3CA mRNA is expressed as the phosphatidylinositol-4,5-bisphosphate 3- kinase catalytic subunit alpha isoform [Homo sapiens] (PI3 K or PIK3CA) protein having GenBank Accession No. NP 006209.2 GL54792082, hereby incorporated by reference, and shown here as SEQ ID NO:4.
  • Missense mutations are most commonly found in two hotspot locations in PIK3CA, occurring in either the helical domain (amino acids 542 545) or the kinase domain (amino acids 1047 and 1049). See Saal et al. Cancer Research 65, 2554-2559, April 1, 2005, "PIK3CA Mutations Correlate with Hormone Receptors, Node Metastasis, and ERBB2, and Are Mutually Exclusive with PTEN Loss in Human Breast Carcinoma," hereby incorporated by reference. PIK3CA missense mutations detected by Saal et al. and others have found the following substitutions: E542K, E545K, H1047R, H1047L, H1047Y and G1049R.
  • tumors with both PIK3CA copy number gain and mutation had moderate levels of gain and were enriched for E545K and other non-H1047R mutations.
  • Table 1 shows the various mutations found in the cell lines tested and include:
  • E545K, H1047R, Kl 1 IN, E307K Cell lines having these mutations were found to have synergistic response. Either mutation is thought to be sufficient to activate the PI3K pathway, and both of these mutations have been shown to be transforming in vitro. Mutation of the PIK3CA gene is one of the most common mutations found in breast cancer, with mutation rates exceeding 20%. While cell lines from patients with the 542/545 mutations appear to be most resistant and those with 1047 mutation appear to be most sensitive to lapatinib as a monotherapy, both types appear to have comparable synergistic responses to the combination of lapatinib plus an AKT inhibitor.
  • a biopsy, tissue or fluid sample is obtained from a patient and the mutation status of a patient at the PIK3CA gene is obtained by methods known and used the in the art.
  • the two main PIK3CA mutation sites: aa 542-545 (E542K, E545K) and aa 1047 (H1047R, H1047L) correspond to PIK3CA niRNA sequence sites of 1624 bp, 1633, 1634 and 3140 bp.
  • the patient's PIK3CA gene or mRNA is amplified and sequenced to identify any mutations in the PIK3CA gene. It may be preferable to use primers within the PIK3CA gene or mRNA sequence that are near the two hotspot locations for PIK3CA mutation to use to amplify those regions and then the resulting PCR products sequenced.
  • the PIK3CA mutation in the PIK3CA protein expressed in the patient's tissue is identified. For example, immunohistochemical analysis by utilizing an antibody that specifically identifies an E545K or an H1047R mutation can be performed.
  • MDAMB453 features a E307Kmutation in PTEN and showed synergistic response to both lapatinib and the AKT inhibitor.
  • Another cell line HCC1569 with a PTEN mutation showed antagonistic response to the combination therapy. It may be useful to identify the mutation status of a patient at the PTEN gene.
  • the PTEN gene was identified as a tumor suppressor that is mutated in a large number of cancers at high frequency.
  • the protein encoded this gene is a phosphatidylinositol- 3,4,5-trisphosphate 3-phosphatase. It contains a tensin like domain as well as a catalytic domain similar to that of the dual specificity protein tyrosine phosphatases. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates
  • phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating AKT/PKB signaling pathway.
  • the expression level or the mutation status can be evaluated using primers or oligonucleotides derived from PTEN mRNA sequence found at GenBank Accession No. NM 000314.4 GI: 110224474, hereby incorporated by reference and shown here as SEQ ID NO: 5 :
  • a method for identifying a HER2 -positive cancer patient suitable for treatment with a 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor comprising: (a) measuring the expression level of the SASH1 gene in a sample from the patient; and (b) comparing the expression level of said gene from the patient with the expression level of the gene in a normal tissue sample or a reference expression level (such as the average expression level of the gene in a cell line panel or a cancer cell or tumor panel, or the like), wherein an increase in the expression level of SASH1 indicates the patient is suitable for treatment with the 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor.
  • a method for identifying a HER2 -positive cancer patient suitable for treatment with a 4-anilinoquinazoline kinase inhibitor and an AKT inhibitor comprising: (a) obtaining the sequence of the PIK3CA gene from a sample from a patient; (b) identifying any mutations in the PIK3CA gene in a sample from the patient as compared to the wild-type sequence found in SEQ ID NO: 3, 4, or 11, (c) measuring the expression level of the SASH1 gene in the sample from the patient; and (d) comparing the expression level of said gene from the patient with the expression level of the gene in a normal tissue sample or a reference expression level (such as the average expression level of the gene in a cell line panel or a cancer cell or tumor panel, or the like), wherein both a mutation in the PIK3CA gene and an increase in the expression level of SASH1 indicates the patient is suitable for treatment with the 4-anilinoquinazoline kinase inhibitor.
  • the expression level of the SASH1 gene is measured by measuring the amount or number of molecules of mRNA or transcript in a cell.
  • the measuring can comprise directly measuring the mRNA or transcript obtained from a cell, or measuring the cDNA obtained from an mRNA preparation thereof. Such methods of extracting the mRNA or transcript from a cell, or preparing the cDNA thereof are well known to those skilled in the art.
  • the expression level of a gene can be measured by measuring or detecting the amount of protein or polypeptide expressed, such as measuring the amount of antibody that specifically binds to the protein in a dot blot or Western blot.
  • the proteins described in the present invention can be overexpressed and purified or isolated to homogeneity and antibodies raised that specifically bind to each protein. Such methods are well known to those skilled in the art.
  • the SASHl protein (also known as KIAA0790, 094885, PEPEl, Q8TAI0, Q9H7R7, and SASHl) is a poorly described protein so named because of SAM and SH3 domains contained in the protein [GenBank Accession Number NM_015278.3 GL45935384, GenBank mRNA accession number CCDS5212.1, and protein sequence 094885.3
  • SASHl has been implicated as a putative tumor suppressor gene in both breast and lung cancer. It is thought to be involved in signal transduction, and recent evidence suggests it may be involved in PI3K pathway signaling.
  • the expression level of a gene encoding SASHl can be measured using an oligonucleotide derived from the nucleotide sequence of GenBank Accession No. NM_015278.3 GL45935384, hereby incorporated by reference, and shown here as SEQ ID NO: 7:
  • SASHl protein levels can be detected and compared to normal or reference levels.
  • the SASHl protein sequence is found at protein sequence GenBank
  • the method further comprises administering a therapeutically effective amount of the 4-anilinoquinazoline kinase inhibitor to the patient.
  • 4-anilinoquinazoline kinase inhibitors suitable for use in the present invention, and the dosages and methods of administration thereof, are taught in U.S. Patent Nos. 6,391,874; 6,713,485; 6,727,256; 6,828,320; and 7,157,466, and International Patent Application Nos. PCT/EP97/03672, PCT/EP99/00048, and PCT/US01/20706 (which are incorporated in their entireties by reference).
  • the lapatinib is lapatinib ditosylate
  • Lapatinib ditosylate monohydrate is commercially available under the brand name TYKERB® (GlaxoSmithKline; Research Triangle Park, NC). The prescription information of TYKERB® (Full Prescribing Information, revised March 2007,
  • GlaxoSmithKline which is incorporated in its entirety by reference, teaches one method of administration of the lapatinib to the patient.
  • the AKT gene is Homo sapiens v-akt murine thymoma viral oncogene homolog 1 (AKTl) on chromosome 14.
  • the genomic sequence for AKT is found at GenBank Accession No. NG_012188.1 GL237874257, hereby incorporated by reference and identified as SEQ ID NO: 12 .
  • the serine -threonine protein kinase encoded by the AKTl gene is catalytically inactive in serum-starved primary and immortalized fibroblasts.
  • AKTl and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKTl .
  • AKT is a critical mediator of growth factor-induced neuronal survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKTl, which then phosphorylates and inactivates components of the apoptotic machinery. Multiple alternatively spliced transcript variants have been found for this gene and are collectively referred to here as AKT. Homo sapiens v-akt murine thymoma viral oncogene homo log 1 (AKT1), transcript variant 2, m NA at GenBank Accession No:
  • AKT protein found as GenBank Accession No. NP 001014432.1 GL62241015, and hereby incorporated by reference is shown here at SEQ ID NO: 10
  • the AKT inhibitor can target AKT in various ways including targeting the AKT gene (SEQ ID NO: 12), AKT mRNA (SEQ ID NO: 9) expression or expression levels, or the AKT protein (SEQ ID NO: 10) or protein levels,
  • the AKT inhibitor is a small molecule kinase inhibitor, more specifically a serine/threonine kinase inhibitor.
  • compounds and formulations of an AKT kinase inhibitor include small molecule protein kinase inhibitors such as an aminofuruzan.
  • An aminofuruzan suitable for use in the present invention, and the dosages and methods of administration thereof include GSK690693 and other compounds taught in U.S. Patent Nos. 7,157,476; 7,348,339; and 7,547,779, and International Patent Application No. PCT/US2004/027182, hereby
  • the AKT kinase inhibitor suitable for use in the present invention, and the dosages and methods of administration thereof include heterocyclic carboxamide compounds such as GSK2141795 and other compounds taught in WO2008/098.104 and WO/2010/093885, both of which are hereby incorporated by reference.
  • These heterocyclic carboxamide compounds are inhibitors of the activity of one or more of the isoforms of the serine/threonine kinase, Akt.
  • Some of the compounds of described are inhibitors of the activity of all three isoforms of the serine/threonine kinase, Akt.
  • Akt kinase inhibitor developed by Glaxo SmithKline (GSK690693), which is an ATP-competitive, low-nanomolar pan- Akt kinase inhibitor. It is selective for the three Akt isoforms, although it does inhibit other members of the AGC kinase family.
  • the AKT inhibitor can be a therapeutic including but not limited to, antisense or inhibitory oligonucleotides, RNA interference, siRNAs, aptamers, monoclonal antibodies, and small molecules.
  • Antibodies In one embodiment, a method of treatment using a humanized monoclonal AKT antibody to down-regulate AKT.
  • Specific antibodies can be made by general methods known in the art. A preferred method of generating these antibodies is by first synthesizing peptide fragments. These peptide fragments should likely cover the coding gene region. Since synthesized peptides are not always immunogenic by their own, the peptides should be conjugated to a carrier protein before use. Appropriate carrier proteins include but are not limited to Keyhole limpet hemacyanin (KLH).
  • KLH Keyhole limpet hemacyanin
  • the conjugated peptides should then be mixed with adjuvant and injected into a mammal, preferably a rabbit through intradermal injection, to elicit an immunogenic response.
  • a mammal preferably a rabbit through intradermal injection
  • Samples of serum can be collected and tested by ELISA assay to determine the titer of the antibodies and then harvested.
  • Polyclonal (e.g., anti-AKT) antibodies can be purified by passing the harvested antibodies through an affinity column.
  • Monoclonal antibodies are preferred over polyclonal antibodies and can be generated according to standard methods known in the art of creating an immortal cell line which expresses the antibody.
  • Nonhuman antibodies are highly immunogenic in human and that limits their therapeutic potential. In order to reduce their immunogenicity, nonhuman antibodies need to be humanized for therapeutic application. Through the years, many researchers have developed different strategies to humanize the nonhuman antibodies. One such example is using "HuMAb-Mouse” technology available from MEDAREX, Inc. and disclosed by van de Winkel, in U.S. Pat. No. 6,111,166 and hereby incorporated by reference in its entirety. "HuMAb-Mouse” is a strain of transgenic mice which harbor the entire human
  • immunoglobin (Ig) loci and thus can be used to produce fully human monoclonal antibodies.
  • RNAi RNA interference
  • the approaches to be taken will depend on the detailed characteristics of the genes, but in some embodiments, will begin with strategies to inhibit RNA transcription since they can, in principal, be used to attack over expressed genes independent of their biochemical composition.
  • Work in the past two decades on transcriptional inhibitors focused on oligodeoxynucleotides and ribozymes. These approaches have had some clinical success but delivery issues limited their clinical utility.
  • siRNA short interfering RNA
  • Promising therapeutic approaches include siRNAs complexed with cationic liposomes (Liao, Y., et al, Enhanced paclitaxel cytotoxicity and prolonged animal survival rate by a nonviral-mediated systemic delivery of El A gene in orthotopic xenograft human breast cancer. Cancer Gene Ther 11, 594-602 (2004); Yano, J. et al. Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer. Clin Cancer Res 10, 7721-6 (2004)), virus vector-mediated RNAi (Zhao, N. et al. Knockdown of Mouse Adult beta-Globin Gene Expression in MEL Cells by Retrovirus Vector-Mediated RNA Interference.
  • siRNAs against the high priority targets complexed with cationic liposomes and small molecule approaches to inhibit the over expressed candidate genes will allow rapid development of this line of attack.
  • the expression of AKT is manipulated.
  • such manipulation can be made using optimized siRNAs. See Hannon, G. J. RNA
  • amplification/expression levels and pro-apoptotic effects of siRNAs will indicate that copy number/expression levels determine the extent of apoptotic responses to target gene inhibitors. Spearman rank test correlations between amplification detected and the level of induced apoptosis will indicate response to the targeted therapeutics.
  • High throughput methods can be used to identify AKT inhibitors such as siRNA and/or small molecular inhibitor formulations to deliver AKT (and other) inhibitors efficiently to cultured cells and xenografts.
  • AKT (and other) inhibitory formulations will be preferentially effective against xenografts that are amplified at the target loci and that these will enhance response to 4-anilinoquinazoline kinase inhibitor compounds. Effective formulations using such methods as described herein can be developed for clinical application.
  • RNA interference is used to generate small double-stranded RNA (small interference RNA or siRNA) inhibitors to affect the expression of a candidate gene generally through cleaving and destroying its cognate RNA.
  • small interference RNA small interference RNA
  • siRNA is typically 19-22 nt double-stranded RNA.
  • siRNA can be obtained by chemical synthesis or by DNA-vector based RNAi technology. Using DNA vector based siRNA technology, a small DNA insert (about 70 bp) encoding a short hairpin RNA targeting the gene of interest is cloned into a commercially available vector. The insert-containing vector can be transfected into the cell, and expressing the short hairpin RNA. The hairpin RNA is rapidly processed by the cellular machinery into 19-22 nt double stranded RNA (siRNA).
  • the siRNA is inserted into a suitable RNAi vector because siRNA made synthetically tends to be less stable and not as effective in transfection.
  • siRNA can be made using methods and algorithms such as those described by Wang L, Mu FY. (2004) A Web-based Design Center for Vector-based siRNA and siRNA cassette. Bioinformatics . (In press); Khvorova A, Reynolds A, Jayasena SD. (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell. 115(2):209-16; Harborth J, Elbashir SM, Vandenburgh K, Manninga H, Scaringe SA, Weber K, Tuschl T. (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev.
  • siRNA Target Finder and Construct Builder available from GenScript (http://www.genscript.com), Oligo Design and Analysis Tools from Integrated DNA Technologies
  • siRNA are suggested to built using the ORF (open reading frame) as the target selecting region, preferably 50-100 nt downstream of the start codon. Because siR As function at the mRNA level, not at the protein level, to design an siRNA, the precise target mRNA nucleotide sequence may be required. Due to the degenerate nature of the genetic code and codon bias, it is difficult to accurately predict the correct nucleotide sequence from the peptide sequence.
  • siRNAs since the function of siRNAs is to cleave mRNA sequences, it is important to use the mRNA nucleotide sequence and not the genomic sequence for siRNA design, although as noted in the Examples, the genomic sequence can be successfully used for siRNA design. However, designs using genomic information might inadvertently target introns and as a result the siRNA would not be functional for silencing the corresponding mRNA.
  • Rational siRNA design should also minimize off-target effects which often arise from partial complementarity of the sense or antisense strands to an unintended target. These effects are known to have a concentration dependence and one way to minimize off-target effects is often by reducing siRNA concentrations. Another way to minimize such off-target effects is to screen the siRNA for target specificity.
  • the siRNA can be modified on the 5 '-end of the sense strand to present compounds such as fluorescent dyes, chemical groups, or polar groups. Modification at the 5 '-end of the antisense strand has been shown to interfere with siRNA silencing activity and therefore this position is not recommended for modification. Modifications at the other three termini have been shown to have minimal to no effect on silencing activity.
  • primers be designed to bracket one of the siRNA cleavage sites as this will help eliminate possible bias in the data (i.e., one of the primers should be upstream of the cleavage site, the other should be downstream of the cleavage site). Bias may be introduced into the experiment if the PCR amplifies either 5' or 3' of a cleavage site, in part because it is difficult to anticipate how long the cleaved mRNA product may persist prior to being degraded. If the amplified region contains the cleavage site, then no amplification can occur if the siRNA has performed its function.
  • siRNA is designed based upon the mRNA sequence of AKT, SEQ ID NO: 9.
  • antisense oligonucleotides can be designed to inhibit AKT and other candidate gene function.
  • Antisense oligonucleotides are short single - stranded nucleic acids, which function by selectively hybridizing to their target mRNA, thereby blocking translation. Translation is inhibited by either RNase H nuclease activity at the DNA:RNA duplex, or by inhibiting ribosome progression, thereby inhibiting protein synthesis. This results in discontinued synthesis and subsequent loss of function of the protein for which the target mRNA encodes.
  • antisense oligos are phosphorothioated upon synthesis and purification, and are usually 18-22 bases in length. It is contemplated that the AKT gene antisense oligos may have other modifications such as 2'-0-Methyl RNA,
  • methylphosphonates methylphosphonates, chimeric oligos, modified bases and many others modifications, including fluorescent oligos.
  • active antisense oligos should be compared against control oligos that have the same general chemistry, base composition, and length as the antisense oligo. These can include inverse sequences, scrambled sequences, and sense sequences. The inverse and scrambled are recommended because they have the same base composition, thus same molecular weight and Tm as the active antisense oligonucleotides. Rational antisense oligo design should consider, for example, that the antisense oligos do not anneal to an unintended mRNA or do not contain motifs known to invoke immunostimulatory responses such as four contiguous G residues, palindromes of 6 or more bases and CG motifs.
  • Antisense oligonucleotides can be used in vitro in most cell types with good results. However, some cell types require the use of transfection reagents to effect efficient transport into cellular interiors. It is recommended that optimization experiments be performed by using differing final oligonucleotide concentrations in the 1-5 ⁇ range with in most cases the addition of transfection reagents.
  • the window of opportunity i.e., that concentration where you will obtain a reproducible antisense effect, may be quite narrow, where above that range you may experience confusing non-specific, non-antisense effects, and below that range you may not see any results at all.
  • down regulation of the targeted mRNA e.g.
  • AKT mRNA SEQ ID NO: 9 will be demonstrated by use of techniques such as northern blot, real-time PCR, cDNA/oligo array or western blot. The same endpoints can be made for in vivo experiments, while also assessing behavioral endpoints.
  • antisense oligonucleotides should be re-suspended in sterile nuclease- free water (the use of DEPC-treated water is not recommended). Antisense oligonucleotides can be purified, lyophilized, and ready for use upon re-suspension. Upon suspension, antisense oligonucleotide stock solutions may be frozen at -20 °C and stable for several weeks.
  • aptamer sequences which bind to specific R A or DNA sequences can be made.
  • Aptamer sequences can be isolated through methods such as those disclosed in U.S. Patent Nos. 5,756,291; 5,843,653; and 7,329,742, which are hereby incorporated by reference.
  • sequences described herein may be varied to result in substantially homologous sequences which retain the same function as the original.
  • a polynucleotide or fragment thereof is “substantially homologous” (or “substantially similar”) to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other polynucleotide (or its complementary strand), using an alignment program such as BLASTN (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool.” J. Mol. Biol. 215:403-410), and there is nucleotide sequence identity in at least about 80%, preferably at least about 90%, and more preferably at least about 95-98%> of the nucleotide bases.
  • the AKT therapeutics of the present invention such as the small molecule AKT inhibitor, also can be used to treat or prevent a variety of disorders associated with reduced survival rate, especially as related to cancers.
  • the AKT therapeutics are administered to a patient in an amount sufficient to elicit a therapeutic response in the patient ⁇ e.g. , reduction of tumor size and growth rate, prolonged survival rate, reduction in concurrent cancer therapeutics administered to patient). An amount adequate to accomplish this is defined as "therapeutically effective dose or amount.”
  • the AKT inhibitors of the invention can be administered directly to a mammalian subject using any route known in the art, including e.g., by injection ⁇ e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, or intradermal), inhalation, transdermal application, rectal administration, or oral administration.
  • injection e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, or intradermal
  • inhalation e.g., transdermal application, rectal administration, or oral administration.
  • compositions of the invention may comprise a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • solvents dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • Administration of the AKT inhibitors of the invention can be in any convenient manner, e.g., by injection, intratumoral injection, intravenous and arterial stents (including eluting stents), cather, oral administration, inhalation, transdermal application, or rectal administration.
  • the peptides and nucleic acids are formulated with a pharmaceutically acceptable carrier prior to administration.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered (e.g., nucleic acid or polypeptide), as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical
  • compositions of the present invention see, e.g., Remington 's Pharmaceutical Sciences, 17 m ed., 1989).
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the dose will be determined by the efficacy of the particular vector (e.g. peptide or nucleic acid) employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular peptide or nucleic acid in a particular patient.
  • the physician evaluates circulating plasma levels of the polypeptide or nucleic acid, polypeptide or nucleic acid toxicities, progression of the disease (e.g., breast cancer), and the production of antibodies that specifically bind to the peptide.
  • the dose equivalent of a polypeptide is from about 0.1 to about 50 mg per kg, preferably from about 1 to about 25 mg per kg, most preferably from about 1 to about 20 mg per kg body weight.
  • the dose equivalent of a naked c acid is from about 1 ⁇ g to about 100 ⁇ g for a typical 70 kilogram patient, and doses of vectors which include a viral particle are calculated to yield an equivalent amount of therapeutic nucleic acid.
  • theAKT inhibitor of the present invention can be administered at a rate determined by the LD-50 of the polypeptide or nucleic acid, and the side-effects of the polypeptide or nucleic acid at various concentrations, as applied to the mass and overall health of the patient.
  • Administration can be accomplished via single or divided doses, e.g. , doses administered on a regular basis (e.g., daily) for a period of time (e.g., 2, 3, 4, 5, 6, days or 1-3 weeks or more).
  • compositions comprising the AKT inhibitor therapies disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally as described in U. S. Patent
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U. S. Patent 5,466,468).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • aqueous solution for parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington 's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions disclosed herein may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
  • oligonucleotides and standard or siSTABLE siRNAs are readily soluble in aqueous solution and can be resuspended at concentrations as high as 2.0 mM. However, viscosity of the resultant solutions can sometimes affect the handling of such concentrated solutions.
  • lipid formulations have been used extensively for cell culture experiments, the attributes for optimal uptake in cell culture do not match those useful in animals.
  • the principle issue is that the cationic nature of the lipids used in cell culture leads to aggregation when used in animals and results in serum clearance and lung accumulation.
  • Polyethylene glycol complexed-liposome formulations are currently under investigation for delivery of siRNA by several academic and industrial investigators, including Dharmacon, but typically require complex formulation knowledge. There are a few reports that cite limited success using lipid-mediated delivery of plasmids or oligonucleotides in animals.
  • Oligonucleotides can also be administered via bolus or continuous administration using an ALZET mini-pump (DURECT Corporation). Caution should be observed with bolus administration as studies of antisense oligonucleotides demonstrated certain dosing-related toxicities including hind limb paralysis and death when the molecules were given at high doses and rates of bolus administration. Studies with antisense and ribozymes have shown that the molecules distribute in a related manner whether the dosing is through intravenous (IV), subcutaneous (sub-Q), or intraperitoneal (IP) administration. For most published studies, dosing has been conducted by IV bolus administration through the tail vein. Less is known about the other methods of delivery, although they may be suitable for various studies. Any method of administration will require optimization to ensure optimal delivery and animal health.
  • IV intravenous
  • subcutaneous subcutaneous
  • IP intraperitoneal
  • dosing can occur once or twice per day.
  • the clearance of oligonucleotides appears to be biphasic and a fairly large amount of the initial dose is cleared from the urine in the first pass.
  • Dosing should be conducted for a fairly long term, with a one to two week course of administration being preferred. This is somewhat dependent on the model being examined, but several metabolic disorder studies in rodents that have been conducted using antisense oligonucleotides have required this course of dosing to
  • Liposomes In certain embodiments, the inventors contemplate the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the administration of the AKT inhibitory peptides and nucleic acids of the present invention.
  • the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • liposomes are generally known to those of skill in the art (see for example, Couvreur et al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy for intracellular bacterial infections and diseases).
  • liposomes were developed with improved serum stability and circulation half-times (Gabizon & Papahadjopoulos, 1988; Allen and Choun, 1987; U. S. Patent 5,741,516).
  • various methods of liposome and liposome like preparations as potential drug carriers have been reviewed (Takakura, 1998; Chandran et al., 1997; Margalit, 1995; U. S. Patent 5,567,434; U.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 m.
  • Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the peptide compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e. in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug-bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation.
  • Targeting is generally not a limitation in terms of the present invention.
  • antibodies may be used to bind to the liposome surface and to direct the liposomes and its contents to particular cell types.
  • Carbohydrate determinants may also be used as recognition sites as they have potential in directing liposomes to particular cell types.
  • the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987).
  • ultrafme particles sized around 0.1 m
  • Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention.
  • Such particles may be are easily made, as described (Couvreur et al., 1980; 1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry et al., 1995 and U. S. Patent 5,145,684).
  • the AKT inhibitor is administered in combination with a 4-anilinoquinazoline kinase inhibitor for treating a HER2+ cancer, including breast cancer.
  • a 4-anilinoquinazoline kinase inhibitor for treating a HER2+ cancer, including breast cancer.
  • an inhibitory AKT siR A may be administered in conjunction with lapatinib.
  • the AKT inhibitor and the 4-anilinoquinazoline kinase inhibitor may be administered simultaneously or sequentially.
  • the AKT inhibitor may be administered first, followed by lapatinib.
  • the 4-anilinoquinazoline kinase inhibitor may be administered first, followed by the AKT inhibitor.
  • the AKT inhibitor and 4-anilinoquinazoline kinase inhibitor are administered in the same formulation.
  • the AKT inhibitor and the 4-anilinoquinazoline kinase inhibitor are
  • AKT inhibitor and the 4-anilinoquinazoline kinase inhibitor are administered in different formulations, their administration may be simultaneous or sequential.
  • combination index (upper 95% less than 1) at multiple drug concentrations, while one additional line showed significant synergy at a single concentration. Synergy was observed when cells were co-treated (i.e. simultaneously) with Lapatinib and the AKT inhibitor.
  • Kits The present invention further provides kits for use within any of the above diagnostic methods.
  • Such kits typically comprise two or more components necessary for performing a diagnostic assay.
  • Components may be compounds, reagents, containers and/or equipment.
  • one container within a kit may contain lyophilized primers to amplify and sequence PIK3CA.
  • One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.
  • Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.
  • Kits can also be supplied for therapeutic uses.
  • the subject composition of the present invention may be provided, usually in a lyophilized form, in a container.
  • the 4-anilinoquinazoline kinase inhibitor and AKT inhibitors described herein are included in the kits with instructions for use, and optionally with buffers, stabilizers, biocides, or inert proteins. It may be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% weight of the total composition.
  • EXAMPLE 1 EXAMPLE 1
  • Lapatinib is a dual inhibitor of EGFR/HER2. Recent evidence suggests that resistance to HER2 inhibition by lapatinib may be in part due to re-activation of PI3K-AKT signaling mediated by HER3. The purpose of this study was to screen lapatinib in combination with a pan-AKT inhibitor in a panel of HER2 amplified breast cancer cell lines to determine if this dual inhibition had synergistic effects in preventing cell line growth.
  • SASH1 has previously been implicated as a tumor suppressor gene in breast, although at this point, it is unclear whether it plays any functional role or is simply a marker for synergistic response to lapatinib and AKTi.
  • Our work demonstrates that a combination of lapatinib plus and AKT inhibitor may be beneficial in HER2 positive patients who also have PI3K pathway mutations. Care should be taken in screening patients prior to treatment, as the combination was found to be antagonistic in some cell lines that did not have PIK3CA mutations.
  • SASH1 may be a useful screening tool in identifying such patients.
  • PIK3CA and introduced an H1047R PIK3CA mutant by lentiviral transduction.
  • the cell line EFM192A was transduced with a mutant H1047R PIK3CA lentiviral vector using standard techniques. Briefly, the lentiviral vector containing mutant PIK3CA was transfected into the cell line 293 along with plasmids containing the viral gag and env genes for packaging. Supernatant containing virus was collected at 48 hours post- transfection and filtered to remove cell debris. Supernatant was then used to transducer EFM192A cells grown to 70% confluence. After 8 hours of treatment of the cells with supernatant, the medium was replaced. Following one day of growth, a selective agent (blasticidin, 10 ug/ml) was added to select for transduced cells.
  • a selective agent blasticidin, 10 ug/ml
  • Lapatinib is currently approved for use in patients with advanced HER2 positive breast cancer in conjunction with capecitabine.
  • Patients eligible for Lapatinib + AKT inhibitor therapy would be those HER2 positive patients who also harbor either a PI3K pathway mutation (PIK3CA mutation and possibly PTEN mutation/deletion) or high levels of expression of SASH1.
  • Paraffin embedded tumor blocks could be assessed for mutations in PIK3CA or PTEN using standard sequencing approaches.
  • SASH1 levels could be assessed by RT-PCR approaches from paraffin embedded tissue using primers spanning a short amplicon or by immunohistochemical staining of tissue sections if suitable antibodies can be identified.
  • Lapatinib is an orally bioavailable small molecule inhibitor, while an AKT inhibitor such as GSK690693 or GSK2141795 may be delivered orally or intravenously to the patient.

Abstract

Méthode d'identification d'un cancer chez un patient, par exemple d'un cancer du sein, adaptée au traitement par un inhibiteur de 4-anilinoquinazoline kinase, tel que le lapatinib, et un inhibiteur d'AKT, ladite méthode comprenant la détection de l'expression modulée de HER2 (ERBB2) et de SASH1 ou de la protéine pour laquelle il code, et la détection de l'état de mutation de PIK3CA. Des teneurs élevées d'expression de HER2 et des teneurs élevées de SASH1 et/ou un état de mutation positif de PIK3CA indiquent que le traitement par un inhibiteur de 4-anilinoquinazoline kinase, tel que le lapatinib, et un inhibiteur d'AKT est adapté au patient.
PCT/US2010/056743 2009-11-14 2010-11-15 L'état de mutation de pik3ca et l'expression de sash1 prédisent la synergie entre le lapatinib et un inhibiteur d'akt dans le cancer du sein her2 positif WO2011060380A1 (fr)

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EP2572199A4 (fr) * 2010-05-21 2013-10-16 Glaxosmithkline Llc Combinaison
WO2013015833A3 (fr) * 2011-07-28 2013-09-26 Genentech, Inc Modèle de cancer du sein d'animal non humain knock-in pik3ca h1047r
WO2013083810A1 (fr) 2011-12-09 2013-06-13 F. Hoffmann-La Roche Ag Identification de non-répondeurs aux inhibiteurs de her2
WO2013113796A1 (fr) * 2012-01-31 2013-08-08 Smithkline Beecham (Cork) Limited Méthode de traitement du cancer
CN102994627A (zh) * 2012-06-21 2013-03-27 海尔施生物医药股份有限公司 一种肿瘤用药个体化指导的检测方法
CN102994627B (zh) * 2012-06-21 2015-04-15 宁波海尔施基因科技有限公司 一种肿瘤用药个体化指导的检测方法
CN104902899A (zh) * 2012-10-22 2015-09-09 葛兰素史克公司 组合
EP2908815A4 (fr) * 2012-10-22 2016-06-22 Novartis Ag Combinaison
WO2019213689A1 (fr) * 2018-05-08 2019-11-14 Queensland University Of Technology Détermination de la réactivité d'un cancer à un traitement
JP2021523146A (ja) * 2018-05-08 2021-09-02 クイーンズランド ユニバーシティ オブ テクノロジー 治療に対するがんの反応性を明らかにする

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