WO2020092924A1 - Polythérapie pour le traitement du cancer résistant aux inhibiteurs de la tyrosine kinase egfr - Google Patents

Polythérapie pour le traitement du cancer résistant aux inhibiteurs de la tyrosine kinase egfr Download PDF

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

Definitions

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

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Abstract

La présente invention concerne des méthodes de traitement du cancer résistant à l'EGFR par l'administration d'un inhibiteur de protéine kinase C delta (PKC5) et/ou d'un inhibiteur de phospholipase C gamma (PLCγ) en association avec un inhibiteur de la tyrosine kinase (TKI) facteur de croissance épidermique (EGFR). L'invention concerne en outre des procédés d'identification d'un sujet comme étant résistant à la TKI EGFR.
PCT/US2019/059424 2018-11-02 2019-11-01 Polythérapie pour le traitement du cancer résistant aux inhibiteurs de la tyrosine kinase egfr WO2020092924A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2022042681A1 (fr) * 2020-08-28 2022-03-03 Shanghai Junshi Biosciences Co., Ltd. Utilisation d'un anticorps anti-pd-1 et d'un médicament anticancéreux cytotoxique dans le traitement du cancer du poumon non a petites cellules
WO2024051679A1 (fr) * 2022-09-05 2024-03-14 应世生物科技(南京)有限公司 Association pharmaceutique d'inhibiteur de fak et d'egfr-tki et utilisation

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US20170107577A1 (en) * 2014-03-11 2017-04-20 The Council Of The Queensland Institute Of Medical Research Determining Cancer Aggressiveness, Prognosis and Responsiveness to Treatment
WO2017100642A1 (fr) * 2015-12-11 2017-06-15 Regeneron Pharmaceuticals, Inc. Méthodes pour ralentir ou empêcher la croissance de tumeurs résistantes au blocage de l'egfr et/ou d'erbb3
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US20180179181A1 (en) * 2014-08-06 2018-06-28 Novartis Ag Protein kinase c inhibitors and methods of their use

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US20110245256A1 (en) * 2010-03-30 2011-10-06 Novartis Ag Use of a pkc inhibitor
US20170252322A1 (en) * 2012-09-19 2017-09-07 Faller & Williams Technology, Llc PKC Delta Inhibitors for use as Therapeutics
US20170107577A1 (en) * 2014-03-11 2017-04-20 The Council Of The Queensland Institute Of Medical Research Determining Cancer Aggressiveness, Prognosis and Responsiveness to Treatment
US20180179181A1 (en) * 2014-08-06 2018-06-28 Novartis Ag Protein kinase c inhibitors and methods of their use
WO2017100642A1 (fr) * 2015-12-11 2017-06-15 Regeneron Pharmaceuticals, Inc. Méthodes pour ralentir ou empêcher la croissance de tumeurs résistantes au blocage de l'egfr et/ou d'erbb3

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022042681A1 (fr) * 2020-08-28 2022-03-03 Shanghai Junshi Biosciences Co., Ltd. Utilisation d'un anticorps anti-pd-1 et d'un médicament anticancéreux cytotoxique dans le traitement du cancer du poumon non a petites cellules
WO2024051679A1 (fr) * 2022-09-05 2024-03-14 应世生物科技(南京)有限公司 Association pharmaceutique d'inhibiteur de fak et d'egfr-tki et utilisation

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