WO2023059801A1 - Biomarqueurs de résistance à médiation egfr dans des cancers provoqués par un oncogène et méthodes de traitement, de prévention et/ou d'atténuation de cancers provoqués par un oncogène - Google Patents

Biomarqueurs de résistance à médiation egfr dans des cancers provoqués par un oncogène et méthodes de traitement, de prévention et/ou d'atténuation de cancers provoqués par un oncogène Download PDF

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WO2023059801A1
WO2023059801A1 PCT/US2022/045903 US2022045903W WO2023059801A1 WO 2023059801 A1 WO2023059801 A1 WO 2023059801A1 US 2022045903 W US2022045903 W US 2022045903W WO 2023059801 A1 WO2023059801 A1 WO 2023059801A1
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mig6
inhibitor
alk
level
ros1
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PCT/US2022/045903
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English (en)
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Robert DOEBELE
Nan Chen
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The Regents Of The University Of Colorado, A Body Corporate
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    • 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
    • G01N33/53—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation
    • 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

  • NSCLC non-small cell lung cancer
  • EGFR epidermal growth factor receptor
  • ALK or ROS1 inhibitors improved survival for these advanced or metastatic cancer patients, few achieve complete response and all patients ultimately experience disease progression and ultimately succumb to the disease due to drug resistance.
  • cancer cells Following the treatment with inhibitors specific to ALK or ROS1, cancer cells typically become resistant via three broad mechanisms: 1) on target mutations such as gate keeper or solvent front mutations within the kinase domain of the ALK or ROS1 gene, 2) histologic transformation or epithelial to mesenchymal transition, or finally 3) bypass signaling.
  • target mutations such as gate keeper or solvent front mutations within the kinase domain of the ALK or ROS1 gene
  • histologic transformation or epithelial to mesenchymal transition or finally 3) bypass signaling.
  • EGFR epidermal growth factor receptor
  • the invention relates broadly to drug resistance mechanisms in cancer cells, such as non-small cell lung cancer cells.
  • the invention provides Mitogen-inducible gene 6 (MIG6, also known as ERBB Receptor Feedback Inhibitor 1 (ERRFI1), RALT or Gene 33) as a novel regulator for ErbB signaling-mediated adaptive and acquired resistance to ALK/RO SI -targeting TKI’s.
  • MIG6 Mitogen-inducible gene 6
  • ERRFI1 ERBB Receptor Feedback Inhibitor 1
  • RALT Gene 33
  • the phosphorylation, transcriptional and/or protein level of MIG6 as well as the mutation status of Mig6 can guide the decision to combine ErbB inhibitors with ALK or ROS1 inhibitors following drug resistance to prior ALK or ROS1 inhibitors to improve therapeutic responses and overcome resistance emergence in certain cancers.
  • the present invention is directed to the following non-limiting embodiments:
  • the present invention is directed to a method of treating, ameliorating, and/or preventing a cancer in a subject in need thereof with a combination therapy.
  • the method includes administering to the subject an effective amount of an inhibitor for anaplastic lymphoma kinase (ALK) and/or an inhibitor for ROS proto-oncogene 1 (ROS1).
  • ALK anaplastic lymphoma kinase
  • ROS1 ROS proto-oncogene 1
  • the method includes administering to the subject an effective amount of an inhibitor for epidermal growth factor receptor (EGFR) and/or an inhibitor for Erb-B2 receptor tyrosine kinase 2 (ERBB2).
  • EGFR epidermal growth factor receptor
  • ERBB2 Erb-B2 receptor tyrosine kinase 2
  • the method includes administering to the subject an effective amount of an inhibitor for anaplastic lymphoma kinase (ALK) and/or an inhibitor for ROS proto-oncogene 1 (ROS1) and administering to the subject an effective amount of an inhibitor for epidermal growth factor receptor (EGFR) and/or an inhibitor for Erb-B2 receptor tyrosine kinase 2 (ERBB2).
  • ALK anaplastic lymphoma kinase
  • ROS1 ROS proto-oncogene 1
  • EGFR epidermal growth factor receptor
  • ERBB2 Erb-B2 receptor tyrosine kinase 2
  • the subject had not previously been treated with an inhibitor for anaplastic lymphoma kinase (ALK) and/or an inhibitor for ROS proto-oncogene 1 (ROS1).
  • ALK anaplastic lymphoma kinase
  • ROS ROS proto-oncogene 1
  • the subject had not developed resistance to an inhibitor for anaplastic lymphoma kinase (ALK) and/or an inhibitor for ROS proto-oncogene 1 (ROS1).
  • ALK anaplastic lymphoma kinase
  • ROS1 ROS proto-oncogene 1
  • the cancer is an ALK positive cancer and/or a ROS1 positive cancer.
  • the cancer is a non-small cell lung cancer.
  • the cancer is resistant to a treatment of the RTK inhibitor alone.
  • the inhibitor for ALK includes alectinib, alkotinib, AP26113, ASP3026, AZD3463, belizatinib, brigatinib, CEP -28122, CEP-37440, ceritinib, crizotinib, ensartinib, entrectinib, foritinib, HG-14-10-04, Lorlatinib, NVL-655, PF-06463922, PLB1003, repotrectinib, TAE684, TPX-0131, TQ-B3139, TSR-011, X-376, or derivatives thereof.
  • the inhibitor for ROS1 includes ceritinib, crizotinib, entrectinib, lorlatinib, NUVL-520, repotrectinib, or derivatives thereof.
  • the inhibitor for EGFR includes afatinib, amivantamab, cetuximab, dacomitinib, erlotinib, gefitinib, mobocertinib, necitumumab, neratinib, osimertinib, panitumumab, poziotinib, vandetanib, or derivatives thereof.
  • the inhibitor for ERBB2 includes dacomitinib, famtrastuzumab deruxtecan-nxki, lapatinib, margetuximab, neratinib, pertuzumab, poziotinib, trastuzumab, tucatinib, or derivatives thereof.
  • the present invention is directed to a method of treating, ameliorating and/or preventing cancer in a subject in need thereof, employing MIG6 as a predictive biomarker.
  • the method includes providing a sample of the subject. In some embodiments, the method includes testing the sample for a MIG6 level and/or a MIG6 activity. In some embodiments, if the MIG6 level and/or the MIG6 activity are below a threshold level, administering to the subject an effective amount of an EGFR inhibitor and/or an ERBB2 inhibitor.
  • the cancer is an ALK positive cancer and/or a ROS1 positive cancer.
  • the cancer is a non-small cell lung cancer.
  • the sample includes at least one selected from a blood sample and a cancer biopsy sample.
  • the sample is tested for the MIG6 level, and the MIG6 level includes a MIG6 mRNA level or a MIG6 protein level.
  • the sample is tested for the MIG6 activity, and the MIG6 activity is determined based on a binding strength between MIG6 and EGFR and/or ERBB2, or based on an inhibition strength by MIG6 on EGFR and/or ERBB2.
  • the sample is tested for the MIG6 mRNA level, and the MIG6 mRNA level is determined by reverse transcription PCR, real time PCR, RNA sequencing, or in situ hybridization.
  • the sample is tested for the Mig6 protein level, and the Mig6 protein level is determined by western blotting, ELISA, flow cytometry, immunocytochemistry, mass spectrometry, or quantitative proteomics.
  • the Mig6 activity is determined by a post-translational modification on the MIG6 6 protein, such as a phosphorylation of the MIG6 protein, such as phosphorylations on residues tyrosine 394 and/or tyrosine 395 of the Mig6 protein.
  • a post-translational modification on the MIG6 6 protein such as a phosphorylation of the MIG6 protein, such as phosphorylations on residues tyrosine 394 and/or tyrosine 395 of the Mig6 protein.
  • the Mig6 activity is determined by a presence or absence of a genetic mutation in the MIG6 gene, such as a genetic mutation affecting an interaction between MIG6 and EGFR and/or an interaction between MIG and ERBB2.
  • the MIG6 activity is determined by the phosphorylation of the MIG6 protein, and the phosphorylation is measured by western blotting, ELISA, flow cytometry, immunocytochemistry, mass spectrometry, or phosphoproteomics.
  • the MIG6 activity is determined by the presence or absence of a genetic mutation, and the presence or absence of a genetic mutation in the MIG6 gene is determined by PCR, RNA sequencing, targeted next-generation sequencing (NGS), whole exome sequencing, or whole genome sequencing.
  • NGS next-generation sequencing
  • a reduced level of phosphorylation of MIG6 corresponds to a decreased MIG6 activity.
  • the phosphorylation level is expressed as a ratio of a level of an unphosphorylated amino acid to a level of the phosphorylated counterpart.
  • the method further includes: if the MIG6 level and/or the MIG6 activity are the same as or higher than the threshold level, administering to the subject an effective amount of an ALK inhibitor and/or an ROS1 inhibitor.
  • the subject is administered with: the effective amount of the EGFR inhibitor and/or the ERBB2 inhibitor; and an effective amount of an ALK inhibitor and/or an RO SI inhibitor.
  • the EGFR inhibitor includes afatinib, amivantamab, cetuximab, dacomitinib, erlotinib, gefitinib, mobocertinib, necitumumab, neratinib, osimertinib, panitumumab, poziotinib, vandetanib, or derivatives thereof,
  • the ERBB2 inhibitor includes dacomitinib, fam-trastuzumab deruxtecan-nxki, lapatinib, margetuximab, neratinib, pertuzumab, poziotinib, trastuzumab, tucatinib, or derivatives thereof.
  • the ALK inhibitor includes alectinib, alkotinib, AP26113, ASP3026, AZD3463, belizatinib, brigatinib, CEP -28122, CEP-37440, ceritinib, crizotinib, ensartinib, entrectinib, foritinib, HG-14-10-04, Lorlatinib, NVL-655, PF-06463922, PLB1003, repotrectinib, TAE684, TPX-0131, TQ-B3139, TSR-011, X-376, or derivatives thereof,
  • the ROS1 inhibitor includes ceritinib, crizotinib, entrectinib, lorlatinib, NUVL-520, repotrectinib, or derivatives thereof.
  • the present invention is directed to one or more methods below:
  • the present invention is directed to a method of delaying and/or preventing resistance development to an ALK inhibitor and/or a ROS1 inhibitor in a patient suffering from non-small cell lung cancer.
  • the method includes providing a sample of the patient after the patient has received treatment with the ALK inhibitor and/or the ROS1 inhibitor. In some embodiments, the method includes testing the sample for a MIG6 level or a MIG6 activity. In some embodiments, if the MIG6 level and/or the MIG6 activity are below a threshold level, administering to the patient: an effective amount of an ALK inhibitor and/or a ROS1 inhibitor; and an effective amount of an EGFR inhibitor and/or an ERBB2 inhibitor, thereby delaying or preventing resistance development to the at least one ROS1 and ALK inhibitor.
  • the present invention is directed to a method of adapting treatment in a patient with non-small cell lung cancer that is being treated with an ALK inhibitor and/or a RO SI inhibitor.
  • the method includes providing a sample of a patient. In some embodiments, the method includes testing sample for a MIG6 level and/or a MIG6 activity. In some embodiments, if the MIG6 level and/or the MIG6 activity are equal to or above a threshold level, continuing treating the patient with the ALK inhibitor and/or the ROS1 inhibitor. In some embodiments, if the MIG6 level and/or the MIG6 activity are below the threshold level: treating the patient with the ALK inhibitor and/or the ROS1 inhibitor; and treating the patient with an EGFR inhibitor and/or an ERBB2 inhibitor, thereby adapting the treatment.
  • the present invention is directed to a method of determining whether new treatment should be adapted in a patient with non-small cell lung cancer and has been treated or is being treated with a ROS1 and/or ALK inhibitor.
  • the method includes providing a sample of the patient. In some embodiments, the method includes testing sample for a MIG6 level or a MIG6 activity. In some embodiments, if the MIG6 level or the MIG6 activity are below a threshold level, adapting new treatment.
  • the present invention is directed to a method of predicting resistance development to treatment of an ALK inhibitor and/or a ROS1 inhibitor in a patient suffering from non-small cell lung cancer.
  • the method includes providing a first sample of patient prior to initiation of treatment. In some embodiments, the method includes testing the first sample for MIG6 level and/or MIG6 activity to establish a first value. In some embodiments, the method includes treating the patient with the ALK inhibitor or the ROS1 inhibitor for a time period. In some embodiments, the method includes providing a second sample of the patient after the time period. In some embodiments, the method includes testing the second sample for MIG6 level and/or MIG6 activity to establish a second value. In some embodiments, if the second level is smaller than the first level for a value higher a threshold level, predicting that the patient will develop resistance to ALK and/or ROS1 inhibitors. In some embodiments, if the second level is smaller than the first level for a value equal to or smaller than the threshold level, predicting that the patient will not develop resistance to ALK and/or ROS1 inhibitors.
  • the non-small cell lung cancer is an ALK positive non-small cell lung cancer or a ROS1 positive non-small cell lung cancer.
  • the sample includes at least one selected from a blood sample and a cancer biopsy sample.
  • the sample is tested for the MIG6 level, and the MIG6 level includes a MIG6 mRNA level or a MIG6 protein level, or
  • the sample is tested for the MIG6 activity, and the MIG6 activity is determined based on a binding strength between MIG6 and EGFR and/or ERBB2, or based on an inhibition strength by MIG6 on EGFR and/or ERBB2.
  • the sample is tested for the MIG6 activity, and the MIG6 activity is determined by a post-translational modification on the Mig 6 protein, such as a phosphorylation of the MIG6 protein, such as phosphorylations on residues tyrosine 394 and/or tyrosine 395 of the MIG6 protein.
  • a post-translational modification on the Mig 6 protein such as a phosphorylation of the MIG6 protein, such as phosphorylations on residues tyrosine 394 and/or tyrosine 395 of the MIG6 protein.
  • the sample is tested for the MIG6 activity, and the MIG6 activity is determined by a presence or absence of a genetic mutation in the MIG6 gene, such as a genetic mutation affecting an interaction between MIG6 and EGFR and/or an interaction between MIG6 and ERBB2.
  • the MIG6 activity is determined by a phosphorylation of the MIG6 protein, and wherein the phosphorylation is measured by western blotting, ELISA, flow cytometry, immunocytochemistry, mass spectrometry, or phosphoproteomics,
  • the MIG6 activity is determined by the presence or absence of a genetic mutation, and the presence or absence of a genetic mutation in the MIG6 gene is determined by PCR, RNA sequencing, targeted next-generation sequencing (NGS), whole exome sequencing, or whole genome sequencing.
  • NGS next-generation sequencing
  • a reduced level of phosphorylation of MIG6 corresponds to a decreased MIG6 activity.
  • the phosphorylation level is expressed as a ratio of a level of an unphosphorylated amino acid to a level of the phosphorylated counterpart.
  • the ALK inhibitor includes alectinib, alkotinib, AP26113, ASP3026, AZD3463, belizatinib, brigatinib, CEP -28122, CEP-37440, ceritinib, crizotinib, ensartinib, entrectinib, foritinib, HG-14-10-04, Lorlatinib, NVL-655, PF-06463922, PLB1003, repotrectinib, TAE684, TPX-0131, TQ-B3139, TSR-011, X-376, or derivatives thereof,,
  • the ROS1 inhibitor includes ceritinib, crizotinib, entrectinib, lorlatinib, NUVL-520, repotrectinib, or derivatives thereof,
  • the EGFR inhibitor includes afatinib, amivantamab, cetuximab, dacomitinib, erlotinib, gefitinib, mobocertinib, necitumumab, neratinib, osimertinib, panitumumab, poziotinib, vandetanib, or derivatives thereof,
  • the ERBB2 inhibitor includes dacomitinib, fam-trastuzumab deruxtecan-nxki, lapatinib, margetuximab, neratinib, pertuzumab, poziotinib, trastuzumab, tucatinib, or derivatives thereof.
  • the present invention is directed to method of overcoming resistance of a non-small cell lung cancer in a patient to a receptor tyrosine kinase inhibitor that inhibits at least one of ALK and ROS 1.
  • the method includes administering to the patient a compound or construct that increases the expression level and/or phosphorylation level of MIG6 in the cancer cell.
  • FIG. 1 is a schematic demonstration of a timeline of resistance to ALK and RO1 inhibitors over time.
  • FIG. 2 is a diagram of the phosphoproteomics approach to explore early adaptive signaling changes under ALK/ROS1 oncogene inhibition.
  • FIGs. 3 A-3D depict Volcano plots demonstrating that phosphorylation sites and phosphoproteins are significantly regulated following crizotinib treatment.
  • Control DMSO treatment. Red: p value ⁇ 0.05, FC (fold change) >1.5.
  • FIG. 3 A and 3B H3122 cells (ALK cells);
  • FIG. 3C and 3D CUTO28 cells (ROS1 cells).
  • FIG. 3 A H3122pY; FIG. 3B, H3122pSTY, FIG. 3C, CUTO28pY, FIG. 3D, CUTO28pSTY.
  • 1252 S/T/Y phosphorylation sites and 798 phosphoproteins are significantly regulated under crizotinib treatment in H3122.
  • In total 2201 S/T/Y phosphorylation sites and 1095 phosphoproteins are significantly regulated under crizotinib treatment in CUTO28.
  • FIGs. 4A-4C depict bar graphs and tables relating to certain crizoinib-regulated phosphoproteins.
  • FIG. 4A top enriched GO terms in crizotinib vs DMSO treated H3133 cells
  • FIG. 4B top 20 enriched GO terms in crizotinib vs DMSO treated CUTO28 cells.
  • FIG. 4C 27 regulated genes in the GO term “regulation of ERBB signaling pathway”.
  • FIGs. 5A-5B depict Volcano plots showing that phosphorylation of MIG6 at Y394/Y395 is decreased following crizotinib treatment in H3122 (FIG. 5A) and CUTO28 cells (FIG. 5B).
  • FIG. 6 depicts a representation of the interaction of MIG6 with EGFR under conditions where MIG6 is not or less phosphorylated (EGFR active, left) and when MIG6 is phosphorylated (EGFR inactive, right).
  • FIGs. 7A-7D depict western blots and graphs showing that MIG6 mRNA and protein levels are decreased following ALK/ROS1 inhibition.
  • FIG. 7A western blot of H3122 cells incubated with crizotinib for 0, 6, 24 or 48h
  • FIG. 7B relative MIG6 expression in H3122
  • FIG. 7C western blot of CUTO28 cells incubated with crizotinib for 0, 6, 24 or 48h
  • FIG. 7D relative MIG6 expression in CUTO28 cells.
  • FIG. 8 depicts a western blot showing that MEK inhibition also decreases MIG6 protein levels, mediated by the MAPK pathway.
  • FIGs. 9A-9E depict a series of graphs showing that MIG6 knockdown promotes cell survival under ALK/ROS1 inhibition via EGFR signaling in H3122 cells.
  • FIG. 9A cells incubated with various concentrations of crizotinib
  • FIG. 9B cells incubated with various concentrations of alectinib
  • FIG. 9C cells incubated with various concentrations of crizotinib plus afatinib
  • FIG. 9D cells incubated with various concentrations of alectinib plus afatinib.
  • FIG. 9E ICso values derived from the inhibition curves.
  • FIGs. 10A-10E depict a series of graphs showing that MIG6 knockdown promotes cell survival under ALK/ROS1 inhibition via EGFR signaling in CUTO28 cells.
  • FIG. 10A cells incubated with various concentrations of crizotinib
  • FIG. 10B cells incubated with various concentrations of entrectinib
  • FIG. 10C cells incubated with various concentrations of crizotinib plus afatinib
  • FIG. 10D cells incubated with various concentrations of entrectinib plus afatinib.
  • FIG. 10E IC50 values derived from the inhibition curves.
  • FIGs. 11 A-l IB are western blots showing that MIG6 knockdown rescues MAPK signaling under ALK/ROS1 inhibition.
  • FIG. 11 A results obtained with H3122 cells;
  • FIG. 1 IB results obtained with CUTO28 cells.
  • FIGs. 12A-12B depict a schematic representation and a table showing that MIG6 and Shcl compete for the same substrate binding cleft on EGFR.
  • FIG. 12A depicts a schematic representation showing that Shcl is a signaling adapter shared by ALK, ROS1, and EGFR.
  • FIG. 12B depicts a table listing certain candidate proteins to be phosphorylated by EGFR in the substrate binding cleft (adapted from Begley et al., Nature Structural & Molecular Biology 2015. PubMed ID: 26551075).
  • FIGs. 13A-13B depict two western blots showing that MIG6 knockdown rescues phosphorylation of Shcl Y239/Y240 in EGFR under ALK/ROS1 inhibition.
  • FIG. 13A results obtained with H3122 cells;
  • FIG. 13B results obtained with CUTO28 cells.
  • FIGs. 14A-14D depict experimental results showing the loss of MIG6 mediates acquired resistance to ALK inhibition.
  • FIG. 14A is a graph showing the viability of cells in the presence of crizotinib;
  • FIG. 14B is a graph showing the viability of cells in the presence of crizotinib plus afatinib;
  • FIG. 14C is a table listing the IC50 values;
  • FIG. 14D is a western blot showing MIG6 loss and upregulation of EGFR activity in crizotinib acquired resistance in H3122 cells.
  • FIGs. 15A-15C depict experimental results showing that MIG6 overexpression resensitizes resistant H3122 cells to ALK inhibition.
  • FIG. 15A is a western blot
  • FIG 15B shows H3122 (EV and MIG6 OE) cells incubated with various concentrations of crizotinib
  • FIG. 15C is a Table with the ICso values for crizotinib in cells transformed with empty vector (EV) or MIG6-0E.
  • FIGs. 16A-16B depict photographs of 2-week clonogenic assay for cells incubated with combinations of inhibitors showing that upfront inhibition of EGFR eliminates residual resistant colonies in response to ALK/ROS1 inhibitors.
  • FIG. 16A H3122 cells incubated with DMSO (left), crizotinib (middle) or alectinib (right), without afatinib (DMSO, top panels) or with afatinib (lower panels).
  • FIG. 16B CUTO28 cells incubated with DMSO (left), crizotinib (middle) or entrectinib (right), without afatinib (DMSO, top panels) or with afatinib (lower panels).
  • FIG. 17 is a schematic representation showing that MIG6 is suppressed under ALK/ROS1 inhibition, driving adaptive resistance by releasing its inhibition on EGFR.
  • FIGs.l8A-18E demonstrate that phosphorylation of MIG6 Y394 and Y395 is decreased in ALK and ROS1 fusion driven cells following crizotinib treatment, in accordance with some embodiments.
  • FIG. 18A Schematic workflow of phosphoproteomics for H3122 and CUTO28 cells under 2-hour crizotinib treatment to explore the early adaptive signaling reprograming under ALK/ROS1 inhibition.
  • FIG. 18B-18C Proteins with phosphorylation sites significantly regulated by crizotinib (
  • FIG. 18D EGFR interactome plot for significantly regulated phosphorproteins in CUTO28 pY phosphoproteomics.
  • FIG. 18E Volcano plot showing decreased phosphorylation of MIG6 Y394 and Y395 under crizotinib treatment in H3122 and CUTO28 pY phosphoproteomics. Significantly regulated phosphoproteins (
  • FIGs. 19A-19B demonstrate that MIG6 protein level is sustained in 2-hr crizotinib treatment, in accordance with some embodiments.
  • FIGs. l9A-19B H3122 and CUTO28 cells were treated with 250 nM and 100 nM crizotinib respectively for 2 hours. Lysates were probed with indicated antibodies.
  • FIGs. 20A-20E demonstrate that the upfront inhibition of ERBB signaling enhances responses to ALK and ROS1 inhibitors and MIG6 protein level is decreased following ALK and ROS1 inhibition, in accordance with some embodiments.
  • FIG. 20A H3122 and CUTO28 were treated with various ALK and RO SI inhibitors alone or in combination of 100 nM afatinib for 72 hours and then assayed with MTS for cell viability. Data represent the mean ⁇ SEM for three biological replicates.
  • FIG. 20A H3122 and CUTO28 were treated with various ALK and RO SI inhibitors alone or in combination of 100 nM afatinib for 72 hours and then assayed with MTS for cell viability. Data represent the mean ⁇ SEM for three biological replicates.
  • H3122 and CUTO28 cells were treated with DMSO, ALK and ROS1 inhibitors (500 nM crizotinib or 100 nM alectinib for H3122, 100 nM crizotinib or 100 nM entrectinib for CUTO28) alone or in combination of 100 nM afatinib for 6 hours.
  • Cell lysates were probed with indicated antibodies.
  • H3122 and CUTO28 cells were seeded 2000 cells/well in a 12-well plate and treated with DMSO, ALK/ROS1 inhibitors (125 nM crizotinib or 25 nM alectinib for H3122, 25 nM crizotinib or 12.5 nM entrectinib for CUTO28) alone or in combination of 100 nM afatinib for two weeks. Colonies were stained by crystal violet.
  • H3122 cells (FIG. 20D) were treated with 250 nM crizotinib or 50 nM alectinib
  • CUTO28 cells FIG. 20E were treated with 250 nM crizotinib or 100 nm entrectinib for indicated time points. Lysates were probed with indicated antibodies.
  • FIGs. 21 A-21H demonstrate that the MIG6 protein reduction correlates with the responsiveness to ALK/ROS1 inhibitors, in accordance with some embodiments.
  • FIG. 21A CUTO29.1 cells harboring EML4-ALK (E6;A19) with ALK p.Cl 156Y mutation
  • FIG. 21B CUTO29.2 cells harboring EML4-ALK (E6;A19) with ALK p.L1996F;Il 171T;C1156Y mutations were treated with various ALK inhibitors alone or in combination of 100 nM of afatinib for 72 hours. Cell viability was assayed by MTS.
  • FIG. 21A CUTO29.1 cells harboring EML4-ALK (E6;A19) with ALK p.Cl 156Y mutation
  • FIG. 21B CUTO29.2 cells harboring EML4-ALK (E6;A19) with ALK p.L1996F;Il 171T
  • FIG. 21C CUTO29.1 and CUTO29.2 cells were treated with 10 nM lorlatinib
  • FIG. 21D CUTO29.2 cells were treated with 250 nM crizotinib with indicated times. Lysates were probed with indicated antibodies.
  • FIG. 2 IE CUTO52 cells harboring EML4-ALK (E13:A20) with ALK p.Ll 196M and D1203N within the kinase domain
  • FIG. 21F CUTO23 cells harboring CD74-ROS1 (C6;R34) were treated with various ALK or ROS1 inhibitors alone or in combination of 100 nM afatinib for 72 hours. Cell viability was assayed by MTS.
  • 21G and 21H CUTO52 and CUTO23 cells were treated with 250nM crizotinib with indicated times and lysates were probed with indicated antibodies.
  • Data in FIGs. 21 A, 21B, 21E and 21F represent the mean ⁇ SEM for three biological replicates.
  • FIGs. 22A-22E demonstrate that MIG6 expression is decreased by ALK/ROS1 inhibition via MAPK pathway, in accordance with some embodiments.
  • FIGs.22A and 22B H3122 and CUTO28 were treated with crizotinib with indicated times. RNA was extracted and expression levels of ERRFI1 (encodes MIG6) were measured by qRT-PCR. Data were shown as the mean ⁇ SEM for three biological replicates.
  • FIGs. 22C and 22D H3122 and CUTO28 were treated with or without 10 nM trametinib for 6hrs. RNA was extracted and expression levels of ERRFI1 were measured by qRT-PCR. Data were shown as the mean ⁇ SEM for three biological replicates.
  • FIG. 22E H3122 and CUTO28 were treated with 10 nM trametinib for indicated times and lysates were probed with indicated antibodies.
  • FIGs. 23A-23E demonstrate that MIG6 knockdown induces resistance to ALK/ROS1 inhibition via EGFR- She 1 -MAPK pathway, in accordance with some embodiments.
  • FIGs. 23 A and 23B H3122 and CUTO28 were transduced with two different shRNA targeting MIG6 or non-targeting control (NTC) shRNA.
  • NTC non-targeting control
  • H3122 and CUTO28 NTC and MIG6 knockdown cells were treated with crizotinib alone (500 nM for H3122 and 100 nM for CUTO28) or in combination of 100 nM afatinib. Lysates were probed with indicated antibodies.
  • FIG. 23E H3122 NTC and MIG6 knockdown cells were stimulated with 10 ng/ml EGF for 15 mins. Cell lysates were subjected to immunoprecipitation (IP) using EGFR antibody and were immunoblotted for SHC1. In parallel, whole cell lysates (WCL) were immunoblotted with indicated antibodies.
  • IP immunoprecipitation
  • WCL whole cell lysates
  • FIGs. 24A-24B demonstrate that MIG6 siRNA knockdown rescues of She 1 -MAPK signaling under crizotinib treatments, in accordance withs some embodiments.
  • FIGs. 24A and 24B, H3122 and CUTO28 were transfected with siRNA targeting MIG6 or control siRNA for 48 hours, and then treated with DMSO, crizotinib (500 nM for H3122 and 100 nM for CUTO28) alone or in combination of 100 nM afatinib for 2 hours. Lysates were probed with indicated antibodies.
  • FIGs. 25 A-25B demonstrate that MIG6 overexpression enhances sensitivity of H3122 to ALK inhibition, in accordance with some embodiments.
  • FIG. 25 A H3122 transduced with an empty vector (EV) or stably overexpressing Myc-tagged MIG6 (MIG6 OE) were treated with crizotinib for 72 hours and then assayed by MTS. Data represent the mean ⁇ SEM for three biological replicates.
  • FIG. 25B H3122 EV and MIG6 OE cells were treated with DMSO or 500 nM crizotinib for 2 hours. Lysates were probed with indicated antibodies. Overexpressed Myc-tagged MIG6 was detected by MIG6 antibody.
  • FIG. 26A-26E Loss of MIG6 observed in EGFR mediated acquired resistance to ALK and ROS1 inhibition, in accordance with some embodiments.
  • FIG. 26A H3122 and crizotinib-resistant (CR1-CR3) cell lysates were immunoblotted with indicated antibodies.
  • FIG. 26B H3122 and CR1-CR3 cells were treated with crizotinib alone or in combination of 100 nM afatinib for 72 hours and assayed with MTS for cell viability. Data represent the mean ⁇ SEM for three biological replicates.
  • FIG. 26A H3122 and crizotinib-resistant (CR1-CR3) cell lysates were immunoblotted with indicated antibodies.
  • FIG. 26B H3122 and CR1-CR3 cells were treated with crizotinib alone or in combination of 100 nM afatinib for 72 hours and assayed with MTS for cell viability. Data represent the mean ⁇ SEM for three biological
  • 26C Indicated cells were treated with DMSO, 250 nM crizotinib, 100 nM afatinib alone, or a combination of crizotinib and afatinib for 2 hours. Lysates were probed with indicated antibodies.
  • FIG. 26D CUTO37 and entrectinib-resistant CUTO37-ER cells were treated with entrectinib or afatinib alone for 72 hours and assayed with MTS for cell viability. Data represent the mean ⁇ SEM for three biological replicates.
  • 26E CUTO37 and ER cells were treated with DMSO, 100 nM entrectinib, 100 nM afatinib alone, or a combination of crizotinib and afatinib for 2 hours. Lysates were probed with indicated antibodies. All the resistant cells were seeded free of TKI for 24 hours prior to drug treatments for immunoblots.
  • FIGs. 27A-27B demonstrate that MIG6 protein is reduced in HCC78-TR and PR2 cells compared to their parental counterparts, in accordance with some embodiments.
  • HCC78 SLC34A2-ROS1
  • LC-2/ad CCDC6-RET
  • FIGs 27A and 27B Lysates from HCC78 and LC-2 and their resistant counterparts were analyzed by immunoblots with indicated antibodies.
  • FIGs. 28A-28E demonstrate that phosphorylation of MIG6 Y394/395 is critical for suppressing EGFR-mediated acquired resistance to ALK/ROS1 inhibition, in accordance with some embodiments.
  • FIG. 28A H3122 CR1 transduced with empty vector (EV), wildtype (WT) MIG6 or Y394F/Y395F mutant MIG6 were treated with crizotinib for 72 hours and assayed with MTS for cell viability. Data represent the mean ⁇ SEM for three biological replicates.
  • FIG. 28B Cells were treated with DMSO or 250 nM crizotinib for 2 hours and lysates were probed with indicated antibodies.
  • FIG. 28C CUTO37 ER cells were transduced with empty vector (EV), wild-type (WT) MIG6 or Y394F/Y395F mutant MIG6. After one week of puromycin selection, cells were cultured for another week free of entrectinib incubation. Colonies were stained with crystal violet and quantified in FIG. 28D. Data represent the mean ⁇ SEM for three biological replicates. Unpaired Student
  • CUTO37 ER cells transduced with empty vector (EV), wild-type (WT) MIG6 or Y394F/Y395F mutant MIG6 lentivirus were treated with DMSO or 100 nM entrectinib for 2 hours. Lysates were probed with indicated antibodies.
  • FIGs. 29A-29C demonstrate that ERRFI1 mutations resulting in a truncation of MIG6 EGFR-binding domain predicts ROS1 TKI resistance and EGFR TKI sensitivity in a patient- derived ROS1 line, in accordance with some embodiments.
  • FIG. 29A Schematic diagram of MIG6 truncation mutation 24 IQ* in CUTO63 cells harboring SLC34A2-ROS1 (S13:R32, S13:R34). This mutation results in a complete deletion of EGFR binding domain on MIG6.
  • FIG. 29A Schematic diagram of MIG6 truncation mutation 24 IQ* in CUTO63 cells harboring SLC34A2-ROS1 (S13:R32, S13:R34). This mutation results in a complete deletion of EGFR binding domain on MIG6.
  • FIG. 29A Schematic diagram of MIG6 truncation mutation 24 IQ* in CUTO63 cells harboring SLC34A2-ROS1
  • FIG. 29B Loss of full-length MIG6 ( ⁇ 55 kDa) and a truncated MIG6 ( ⁇ 26 kDa) were detected in CUTO63 cells. MIG6 is probed by an antibody raised against MIG6 amino acids 111-221 (Abnova).
  • FIG. 29C CUTO63 cells were treated with indicated inhibitors for 72 hours and assayed with MTS. Data represent the mean ⁇ SEM for three biological replicates.
  • FIG. 30 IGV view of MIG6 c.721G>A:p.Q241* truncation mutation (23% variant frequency) revealed by CUTO63 RNA-seq, in accordance with some embodiments.
  • FIG. 31 Model depicting MIG6 role in regulating EGFR-mediated adaptive and acquired resistance to ALK/ROS1 inhibition, in accordance with some embodiments.
  • ALK/ROS1 as the primary driver signals through Shcl-MAPK pathway to promote cancer cell survival and proliferation.
  • EGFR activity is inhibited by its endogenous protein inhibitor, MIG6.
  • MIG6 endogenous protein inhibitor
  • MIG6 starts to disassociate from EGFR with a decrease of Y394/Y395 phosphorylation.
  • MIG6 expression is also suppressed due to the initial downregulation of MAPK pathway under ALK/ROS1 inhibition.
  • EGFR is then released from MIG6 inhibition with an increased Shcl adaptor binding to maintain MAPK pathway to support the residual disease and the following tumor progression.
  • Shcl adaptor binding to maintain MAPK pathway to support the residual disease and the following tumor progression.
  • Upfront inhibition of EGFR together with primary ALK/ROS1 oncogene inhibition will minimize or eliminate residual disease, therefore delaying or preventing resistance emergence.
  • afatinib refers to N-[4-[(3-Chloro-4-fluorophenyl)amino]- 7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, or a salt or solvate thereof.
  • a disease or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
  • co-administered and “co-administration” as relating to a subject refer to administering to the subject a compound contemplated herein or salt thereof along with a compound that may also treat the disorders or diseases contemplated herein.
  • the co-administered compounds are administered separately, or in any kind of combination as part of a single therapeutic approach.
  • the co-administered compound may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.
  • composition refers to a mixture of at least one compound contemplated herein with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, nasal, pulmonary and topical administration.
  • determining generally refers to any form of measurement, and includes detecting the presence of a mutation, including, for example, an EGFR exon 20 insertion mutation in the tumor cells, as disclosed herein.
  • the term “determining” includes both quantitative and/or qualitative determination.
  • the mutation e.g., EGFR exon 20 insertion mutation
  • a “disease” as used herein is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a “disorder” as used herein in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • the terms “effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of a compound or agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • a therapeutic benefit or improvement need not be complete ablation of any one, most or all symptoms, complications, consequences or underlying causes associated with the disorder or disease.
  • a satisfactory endpoint is achieved when there is a transient, medium or long term, incremental improvement in a subject’s condition, or a partial reduction in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal, of one or more associated adverse symptoms or complications or consequences or underlying causes, worsening or progression (e.g., stabilizing one or more symptoms or complications of the condition, disorder or disease), of the disorder or disease, over a duration of time (hours, days, weeks, months, and so forth).
  • EGFR epidermal growth factor receptor
  • “likelihood”, “likely to”, and similar generally refers to an increase in the probability of an event.
  • “likelihood”, “likely to”, and similar, when used in reference to responsiveness to cancer therapy generally contemplates an increased probability that the individual will exhibit a reduction in the severity of cancer or the symptoms of cancer or the retardation or slowing of the cancer progression.
  • the term “likelihood”, “likely to”, and similar, when used in reference to responsiveness to cancer therapy, can also generally mean the increase of indicators that may evidence an increase in cancer treatment.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient is a human.
  • the subject is a subject in need of treatment thereof.
  • the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • phosphorylation refers to a biological process where a phosphoryl group is added to an amino acid.
  • the amino acid is serine, although phosphorylation also occurs on threonine and tyrosine in eukaryotes.
  • Phosphorylation is an esterification reaction where a phosphate group reacts with the hydroxyl (-OH) group of a serine, threonine, or tyrosine side chain.
  • the enzyme protein kinase covalently binds a phosphate group to the amino acid.
  • the best-studied forms of phosphorylation are posttranslational modifications (PTM), in which the proteins are phosphorylated after their translation from an RNA template.
  • PTM posttranslational modifications
  • dephosphorylation is catalyzed by protein phosphatases.
  • protein phosphorylation refers to a widespread post-translational modification of proteins that regulates a broad range of cellular processes. Several diseases are linked with defects or altered states of phosphorylation. Phosphoproteomics is a technology to identify which amino acids in a protein are phosphorylated. Candidate amino acids that are most commonly phosphorylated in a protein are serine, threonine and tyrosine. Additionally, arginine, lysine, aspartic acid, glutamic acid and cysteine may be phosphorylated. Phosphoproteomics can identify if an amino acid is phosphorylated as well as the ratio of amino acid in its phosphorylated/unphosphorylated state. For instance, the ratio of phosphorylated serine to unphoshorylated serine in a protein.
  • the term “predict” can mean to determine or tell in advance.
  • the term “predict” can mean that the likelihood of the outcome of the cancer treatment can be determined at the outset, before the treatment has begun, or before the treatment period has progressed substantially.
  • a predictive method may also be described as a prognostic method.
  • prevent means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences.
  • a "proliferative disease” is defined as a tumor disease (including benign or cancerous) and/or any metastases, wherever the tumor or the metastasis are located, more especially a tumor selected from the group comprising one or more of (and in some variations selected from the group consisting of) breast cancer, genitourinary cancer, lung cancer, gastrointestinal cancer, epidermoid cancer, melanoma, ovarian cancer, pancreatic cancer, neuroblastoma, colorectal cancer, head and neck cancer.
  • the proliferative disease is cancer.
  • the proliferative disease is a non-cancerous disease.
  • the proliferative disease is a benign or malignant tumor.
  • a tumor a tumor disease, a carcinoma or a cancer
  • metastasis in the original organ or tissue and/or in any other location are implied alternatively or in addition, whatever the location of the tumor and/or metastasis is.
  • the phrase “providing tumor cells” refers to the step of obtaining cells of the individual (e.g. by way of biopsy or otherwise), and/or refers to the step of receiving a sample of tumor cells that has previously been obtained from the individual.
  • responsiveness refers to the degree of effectiveness of the treatment in lessening or decreasing the symptoms of a disease, disorder, or condition being treated.
  • increased responsiveness when used in reference to a treatment of a cell or a subject, refers to an increase in the effectiveness in lessening or decreasing the symptoms of the disease when measured using any methods known in the art.
  • the increase in the effectiveness is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.
  • tumor cells comprise a “sample.”
  • the sample comprises a biological sample and can be, for instance, a cell, a cell culture, a tissue, and/or a biological fluid.
  • the biological sample can comprise a tumor cell biopsy, a plurality of samples from a clinical trial, or the like.
  • the sample can be a crude sample, or can be purified to various degrees prior to storage, processing, or measurement.
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
  • threshold value is a value is a value beyond which a biological system, i.e. a protein in a cell, changes the way it operates.
  • a threshold value in phosphorylation of a protein is a value beyond which a protein may or may not interact with another protein.
  • the value can be an absolute value, or a ratio.
  • the threshold value in phosphorylation is established by phosphoproteomics.
  • the ratio refers to the quotient of the amount of a phorphorylated protein to the amount of the same protein in non-phosphorylated state.
  • a phosphorylated counterpart is a phosphorylated amino acid residue that is at the same position in the protein as the amino acid residue that is non-phosphorylated.
  • treatment is defined as the application or administration of a therapeutic agent, i.e., a compound disclosed herein (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein and/or a symptom of a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein and/or the symptoms of a condition contemplated herein.
  • Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • the term “treatment” or “treating” of a cancer refers to an action that occurs while an individual is suffering from the specified cancer, which reduces the severity of the cancer or the symptoms of the cancer, and/or retards or slows the progression of the cancer.
  • “treatment” or “treat” refers to a 5%, 10%, 25%, 50%, or 100% decrease in the rate of progress of a tumor.
  • “treatment” refers to a 5%, 10%, 25%, 50%, or 100% decrease in determined tumor burden (i.e., number of cancerous cells present in the individual, and/or the size of the tumor).
  • treatment refers to a 5%, 10%, 25%, 50%, or 100% decrease in any physical symptom(s) of a cancer. In yet other embodiments, “treatment” refers to a 5%, 10%, 25%, 50%, or 100% increase in the general health of the individual, as determined by any suitable means, such as cell counts, assay results, or other suitable means.
  • the cancer can be any cancer, including those contemplated herein, including, for example, a HER-driven drug-resistant cancer.
  • reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so forth.
  • Reference to a range of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, and so forth.
  • a series of ranges are disclosed throughout this document.
  • ranges include combinations of the upper and lower ranges to provide another range. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document.
  • reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100, 20-150, and so forth. This applies regardless of the breadth of the range.
  • protein kinase is a protein that transfers phosphate groups from ATP to serine, threonine, or tyrosine residues on protein peptide substrates, directly affecting the activity and function of the target. Approximately 30% of proteins in a eukaryotic cell may be phosphorylated. This crucial post-translational modification regulates a broad range of cellular activities including the cell cycle, differentiation, metabolism, and neuronal communication. Abnormal phosphorylation events in cells are implicated in many disease states. Protein kinases are often common elements in multiple signaling networks and can influence numerous downstream effectors responsible for a biological response. Assessing the activity of a single specific kinase often provides valuable information on parallel pathways.
  • tyrosine kinase inhibitor refers to a pharmaceutical drug that inhibits at least one tyrosine kinase.
  • Tyrosine kinases are enzymes responsible for activation of proteins involved in signal transduction cascades. Such proteins are activated by the addition of a phosphate group to the protein, a step which is catalyzed by kinase enzymes.
  • TKI inhibit the kinases involved in the phosphorylation step. TKIs are used as anticancer drugs.
  • the TKI’s are inhibitors for ALK, inhibitors for In some embodiments, TKI’s are drugs that have been approved for anti-cancer treatment, such as but not limited to crizotinib, ceritinib, alectinib, brigatinib, or lorlatinib. In some embodiments, the TKI’s inhibit ROS1. In some embodiments, approved drugs for R0S1 inhibition comprise crizotinib and entrectinib.
  • RTK receptor tyrosine kinase
  • the present invention is directed to a method of treating, ameliorating and/or preventing cancer in a subject in need thereof.
  • the method includes administering to the subject an effective amount of an RTK inhibitor that inhibits at least one of ALK and ROS1.
  • the method includes administering to the subject an effective amount of an EGFR inhibitor and/or an ERBB2 inhibitor.
  • the method includes administering to the subject an effective amount of an RTK inhibitor that inhibits at least one of ALK and ROS1 and an effective amount of an EGFR inhibitor and/or an ERBB2 inhibitor.
  • the subject had not previously been treated with an inhibitor for anaplastic lymphoma kinase (ALK) and/or an inhibitor for ROS proto-oncogene 1 (ROS1).
  • ALK anaplastic lymphoma kinase
  • ROS ROS proto-oncogene 1
  • the subject had not developed resistance to an inhibitor for anaplastic lymphoma kinase (ALK) and/or an inhibitor for ROS proto-oncogene 1 (ROS1).
  • ALK anaplastic lymphoma kinase
  • ROS ROS proto-oncogene 1
  • the cancer in the subject includes a cancer cell including a mutant ALK and/or ROS1, such as a hyperactive mutant ALK and/or ROS1, such as a hyperactive ALK fusion protein and/or a hyperactive fusion ROS1 protein.
  • a mutant ALK and/or ROS1 such as a hyperactive mutant ALK and/or ROS1, such as a hyperactive ALK fusion protein and/or a hyperactive fusion ROS1 protein.
  • the cancer is an oncogene-driven cancer.
  • the cancer is an ALK-driven cancer, a BRAF driven cancer, an MAPK-driven cancer, an NTRK driven cancer, a RET driven cancer, a ROS1 driven cancer, or the like.
  • the cancer is a lung cancer, such as a non-small cell lung cancer (NSCLC, such as advanced NSCLC), or an advanced solid tumor malignancy.
  • the cancer is a lung cancer, a sarcoma (such as an inflammatory myofibroblastic tumor), or another solid tumor driven by ALK or ROS1.
  • the cancer includes a cancer cell including a hyperactive receptor tyrosine kinase (RTK).
  • RTK is a fusion RTK, such as a fusion RTK produced by gene arrangements.
  • the hyperactive RTK includes a hyperactive ROS1 or a hyperactive ALK. Hyperactive ROS1 and/or hyperactive ALK that result from fusion with other proteins are often found in NSCLC’s.
  • ROS1 refers to c-ros oncogene and ALK refers to “anaplastic lymphoma kinase,” hyperactive fusion proteins of both of which are found in cancers, such as lung cancers (e.g., NSCLC’s).
  • the cancer has been treated with the RTK inhibitor previously. In some embodiments, the cancer is resistant to the treatment of the RTK inhibitor alone.
  • Non-limiting examples of inhibitors for ALK include alectinib, alkotinib (also known as ZG-0418), AP26113, ASP3026, AZD3463, belizatinib (also known as TSR-011), brigatinib, CEP -28122, CEP-37440, ceritinib, crizotinib, ensartinib (also known as X-396), entrectinib (also known as NMS-E628 and RXDX-101), foritinib (SAF-189), HG-14-10-04, Lorlatinib, NVL-655, PF-06463922, PLB1003, repotrectinib (also known as TPX-0005), TAE684, TPX-0131, TQ-B3139, TSR-011, X-376, or derivatives thereof.
  • alectinib also known as ZG-0418
  • AP26113 also known as ZG-0418
  • Non-limiting examples of inhibitors for ROS 1 include ceritinib, crizotinib, entrectinib, lorlatinib, NUVL-520, repotrectinib, or derivatives thereof.
  • Non-limiting examples of inhibitors for EGFR include afatinib, amivantamab, cetuximab, dacomitinib, erlotinib, gefitinib, mobocertinib, necitumumab, neratinib, osimertinib, panitumumab, poziotinib, vandetanib, or derivatives thereof.
  • Non-limiting examples of inhibitors for ERBB2 include dacomitinib, famtrastuzumab deruxtecan-nxki, lapatinib, margetuximab, neratinib, pertuzumab, poziotinib, trastuzumab, tucatinib, or derivatives thereof.
  • the present study employed a phosphoproteomics approach to explore the early signaling reprogramming in hyperactive ALK/RO SI -driven cancer cell lines following the treatment of crizotinib, an ALK/ROS1 inhibitor.
  • MIG6 as regulator for EGFR-mediated adaptive and acquired resistance to ALK/ROS1 inhibitors.
  • MIG6 level such as mRNA/protein level or mutation status
  • activity such as post-translational modification or mutation status
  • the present study proposes methods of using loss of MIG6 expression/activity as a predictive biomarker to select patients with oncogene-driven cancers including, but not limited to, ALK or ROS1+ NSCLC for combination therapies including EGFR inhibitors.
  • the present invention is directed to a method of treating, ameliorating and/or preventing cancer in a subject in need thereof.
  • the present invention is directed to a method of identifying a subject suitable for receiving a treatment with an EGFR/ERBB2 inhibitor. In some embodiments, the present invention is directed to a method of identifying a subject suitable for receiving a combination treatment including an RTK inhibitor that inhibits ALK and/or ROS1, as well as an EGFR/ERBB2 inhibitor.
  • the method includes obtaining a sample from the subject.
  • the method includes testing the sample for an alteration of MIG6 level and/or for an alteration of MIG6 activity.
  • the MIG6 level is affected by, for example, the gene copy number (which may be altered by genetic mutations, chromosomal abnormalities such as deletion, duplication or translocation, and etc.), the transcriptional efficiencies, the translational efficiencies, protein degradation rate, and so on.
  • the MIG6 activity is affected by, for example, post-translation modification (such as phosphorylation) and genetic mutations that affect MIG6 functions (such as the ability of MIG6 to bind to and/or inhibit EGFR and ERBB2). Methods of measuring these parameters are known in the art and described briefly below.
  • identifying the subject as not benefiting from a treatment including an EGFR/ERBB2 inhibitor if the MIG6 levels and/or the MIG6 activity are above a threshold level, identifying the subject as not benefiting from a treatment including an EGFR/ERBB2 inhibitor.
  • identifying the subject as benefiting from treatment including an EGFR/ERBB2 inhibitor if the MIG6 levels and/or the MIG6 activity are below a threshold level, identifying the subject as benefiting from treatment including an EGFR/ERBB2 inhibitor.
  • the method further includes: if the MIG6 levels and/or the MIG6 activity are below the threshold level, administering to the subject an effective amount of the EGFR/ERBB2 inhibitor.
  • the method further includes: if the MIG6 levels and/or the MIG6 activity are below the threshold level, identifying the subject as benefiting from treatment of an RTK inhibitor that inhibits at least one of ALK and ROS1 and the EGFR/ERBB2 inhibitor. In some embodiments, the method further includes: if the MIG6 levels and/or the MIG6 activity are below a threshold level, administering to the subject an effective amount of the RTK inhibitor that inhibits the at least one of ALK and ROS1 and an effective amount of the EGFR/ERBB2 inhibitor.
  • the ALK inhibitor, the ROS1 inhibitor, the EGFR inhibitor and/or the ERBB2 inhibitor are the same as or similar to those described elsewhere herein, such as in the “Treating, Ameliorating and/or Preventing Cancer with Combination of ALK/ROS1 Inhibitor and EGFR/ERBB2 Inhibitor” section.
  • the cancer is an oncogene-driven cancer.
  • the cancer is an ALK-driven cancer, a BRAF driven cancer, an MAPK-driven cancer, an NTRK driven cancer, a RET driven cancer, a ROS1 driven cancer, or the like.
  • the cancer is a lung cancer, such as a non-small cell lung cancer (NSCLC, such as advanced NSCLC), or an advanced solid tumor malignancy.
  • NSCLC non-small cell lung cancer
  • the cancer includes a cancer cell including a hyperactive receptor tyrosine kinase (RTK).
  • RTK is a fusion RTK, such as a fusion RTK produced by gene arrangements, such as a chromosomal rearrangement.
  • the hyperactive RTK includes a hyperactive ROS1 or a hyperactive ALK. Hyperactive ROS1 and/or hyperactive ALK that result from fusion with other proteins are often found in NSCLC’ s.
  • ROS1 refers to c-ros oncogene and ALK refers to “anaplastic lymphoma kinase,” hyperactive fusion proteins of both of which are found in cancers, such as lung cancers (e.g., NSCLC’ s).
  • the cancer has been treated with the RTK inhibitor. In some embodiments, the cancer is resistant to a treatment of the RTK inhibitor alone.
  • the cancer with hyperactive receptor tyrosine kinase has previously been treated with a tyrosine kinase inhibitor, such as an inhibitor for ALK and/or an inhibitor for ROS 1.
  • a tyrosine kinase inhibitor such as an inhibitor for ALK and/or an inhibitor for ROS 1.
  • the sample includes a blood sample, a cancer sample (such as a biopsy sample from the cancer tissue), or the like.
  • the mechanism of resistance of cells to ALK or ROS1 inhibitors can be determined by measuring in a cancer cell or a blood cell alteration (such as, but not limited to, absence) of MIG6 mRNA or protein expression and/or an alteration in MIG6 that impairs MIG6 function or expression.
  • the cell is a cancer cell from a patient.
  • the cancer cell is obtained via a tissue biopsy.
  • the cell is a blood cell from a patient, i.e., a lymphocyte.
  • the alteration of MIG6 level includes an alteration of MIG6 mRNA level and/or an alteration of MIG6 protein level.
  • Methods of detecting and/or measuring mRNA levels or protein levels are known in the art and one of ordinary skill in the art would know how to choose the proper detection method and/or measurement method.
  • Non-limiting examples of method of detecting/measuring mRNA levels include northern blot analysis, nuclease protection assays (NPA), in situ hybridization, RNA sequencing, real time PCR, reverse transcription-PCR, or the like.
  • Non-limiting examples of methods of detecting/measuring protein levels include western blotting, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunocytochemistry, mass spectrometry, quantitative proteomics, or the like.
  • ELISA enzyme-linked immunosorbent assay
  • the alteration of MIG6 activity includes a post-translational modification, or a mutation in the MIG6 gene. Since the MIG6 functions by binding to and inhibiting EGFR and ERBB2, stronger binding to and/or stronger inhibition of EGFR/ERBB2 correspond to higher activity, and vice versa.
  • the post-translational modification includes a phosphorylation.
  • the MIG6 activity is altered by a change of phosphorylation levels in residues Y394 and/or Y395. Since the phosphorylation of these 2 residues is involved in MIG6 binding and subsequent inhibition of EGFR, increased phosphorylation levels of residues Y394 and/or Y395 corresponds to higher MIG6 activity, and decreased phosphorylation levels of residues Y394 and/or Y395 corresponds to lower MIG6 activity.
  • the alteration of MIG6 activity includes a mutation to the MIG6 gene.
  • the mutation of the MIG6 gene results in a MIG6 protein having weaker binding to and/or weaker inhibition of EGFR and/or ERBB2, which results in a lower MIG6 activity.
  • the mutation of the MIG6 gene results in a MIG6 protein having stronger binding to and/or stronger inhibition of EGFR and/or ERBB2, which results in a higher MIG6 activity.
  • the mutation of the MIG6 gene results in reduced or loss of expression of the Mig6 protein, which cause the alteration of MIG6 level with or without affecting MIG6 activity.
  • Non-limiting examples of method of detecting MIG6 mutation status include PCR, targeted NGS sequencing, whole exome sequencing and whole genome sequencing, RNA sequencing, or the like.
  • MIG6 mRNA and protein levels were also decreased following ALK/ROS1 inhibition.
  • the MEK inhibitor trametinib decreased MIG6 transcripts and protein levels with a similar magnitude as ALK/ROS1 inhibition.
  • the regulation of MIG6 expression by ALK/ROS1 inhibition is mediated by the MAPK pathway.
  • MIG6 knock-down rescues cell survival and pErk suppression from ALK/ROS1 inhibition.
  • NSCLC in a subject is treated with a combination of a tyrosine kinase inhibitor that inhibits at least one of ALK and ROS1, and an inhibitor of EGFR/ERBB2.
  • a subject is identified that would benefit from a combination treatment of a tyrosine kinase inhibitor that inhibits at least one of ALK and ROS1, and an inhibitor of EGFR/ERBB2.
  • resistance to ALK or ROS1 inhibitors can be minimized, reversed, and/or prevented by treating the subject with a combination of a tyrosinke kinase inhibitor that inhibits at least one of ALK and ROS1, and an inhibitor of EGFR/ERBB2.
  • treatment can be adapted to include treatment with a combination of a tyrosine kinase inhibitor that inhibits at least one of ALK and ROS1, and an inhibitor of EGFR/ERBB2.
  • methods are provided to determine if treatment should be adapted to include a combination treatment contemplated herein. In some embodiments, it can be predicted if resistance to ALK or ROS1 inhibitors will develop by measuring, in a cancer cell or a blood cell, absence of MIG6 mRNA or protein expression and/or an alteration in MIG6 that impairs MIG6 function or expression.
  • crizotinib-resistant ALK cell lines loss of MIG6 and resistance can be reversed by MIG6 overexpression.
  • afatinib which is an ERBB family pan inhibitor, was combined with ALK/ROS1 TKIs.
  • the combination therapy of afatinib and ALK/ROS1 TKIs eliminated residual colony formation for 2 weeks.
  • the present study discovered that low MIG6 level and/or activity predict favorable results from a combined treatment with an RTK inhibitor that inhibits at least one of ALK and ROS1, and an EGFR/ERBB2 inhibitor.
  • the present invention is directed to a method of identifying a subject with non-small cell lung cancer or other cancers that would benefit from a combination treatment with a RTK inhibitor that inhibits at least one of ALK and ROS1 and an EGFR/ERBB2 inhibitor.
  • the method includes providing sample of a patient treated with at least one of ROS 1 and ALK inhibitor.
  • the method includes testing the sample for an alteration of MIG6 level and/or for an alteration of MIG6 activity.
  • the MIG6 level is affected by, for example, the gene copy number (which may be altered by genetic mutations, chromosomal abnormalities such as deletion, duplication or translocation, and etc.), the transcriptional efficiencies, the translational efficiencies, protein degradation rate, and so on.
  • the MIG6 activity is affected by, for example, post-translation modification (such as phosphorylation) and genetic mutations that affect MIG6 functions (such as the ability of MIG6 to bind to and/or inhibit EGFR and ERBB2). The methods of detecting/measuring the MIG6 level and/or activity are detailed elsewhere herein.
  • identifying the patient as not benefiting from a combination treatment of a ROS1 or ALK inhibitor and an EGFR/ERBB2 inhibitor if the MIG6 level and/or MIG6 activity are above a threshold level, identifying the patient as not benefiting from a combination treatment of a ROS1 or ALK inhibitor and an EGFR/ERBB2 inhibitor.
  • identifying the patient at benefiting from a combination treatment of a ROS1 or ALK inhibitor, and an EGFR/ERBB2 inhibitor if the MIG6 level and/or MIG6 activity are below a threshold level, identifying the patient at benefiting from a combination treatment of a ROS1 or ALK inhibitor, and an EGFR/ERBB2 inhibitor.
  • the present study discovered that low MIG6 level and/or activity predict resistance development to RTK inhibitor that inhibits at least one of ALK and ROS1 in non-small cell lung cancer.
  • the present study further discovered that, under such situations, the additional administration of EGFR/ERBB2 inhibitor was able to delay and/or prevent the development of resistance.
  • the present invention is directed to a method of delaying and/or preventing resistance development to a RTK inhibitor that inhibits at least one of ALK and ROS1 in a patient suffering from non-small cell lung cancer.
  • the method includes providing a sample of a patient treated with at least one of ALK and ROS1 inhibitor.
  • the method includes testing the sample for alteration of MIG6 level and/or MIG6 activity.
  • MIG6 level and/or MIG6 activity are below a threshold level, treating the patient with ROS1 or ALK inhibitor, and further treating the patient with an EGFR/ERBB2 inhibitor, thereby delaying or preventing resistance development to the at least one ROS1 and ALK inhibitor.
  • the present invention is directed to a method of adapting treatment in a patient with non-small cell lung cancer that is being treated with an inhibitor that inhibits at least one of ROS 1 and ALK.
  • the method includes providing a sample of a patient.
  • the method includes testing the sample for alteration of MIG6 level and/or MIG6 activity.
  • MIG6 level and/or MIG6 activity are above a threshold level, continuing treating the patient with an inhibitor that inhibits at least one of ROS 1 and ALK.
  • MIG6 level and/or MIG6 activity are below a threshold level, treating the patient with a ROS1 or ALK inhibitor and further treating the patient with an EGFR/ERBB2 inhibitor, thereby adapting the treatment.
  • the present invention is directed to a method of determining whether treatment should be adapted in a patient with non-small cell lung cancer and treated with a ROS1 and/or ALK inhibitor.
  • the method includes providing a sample of the patient.
  • the method includes testing the sample for a MIG6 protein level and/or a MIG6 protein activity.
  • the MIG6 protein level and/or the MIG6 protein activity are lower than a threshold level, adapting the treatment.
  • the present study discovered that lowered MIG6 level and/or MIG6 activity in response to a treatment by a receptor tyrosine kinase inhibitor that inhibits at least one of ALK and ROS1 in non-small cell lung cancer correspond with the development of resistance against the treatment.
  • the present invention is directed to a method of predicting resistance development to a receptor tyrosine kinase inhibitor that inhibits at least one of ALK and ROS1 in a patient suffering from non-small cell lung cancer.
  • the method includes providing a first sample of a patient prior to initiation of treatment.
  • the method includes testing the first sample for MIG6 level and/or MIG6 activity to establish a first value.
  • the method includes treating the patient with an ALK and/or ROS1 inhibitors for a time period.
  • the method includes providing a second sample of the patient after the time period.
  • the method includes testing the second sample for MIG6 level and/or MIG6 activity to establish a second value.
  • the second value is smaller than the first value for a value the same as or higher than a threshold level, predicting that the patient will develop resistance to ALK and/or ROS1 inhibitors.
  • the second value is not smaller than the first value for a value the same as or higher than the threshold level, predicting that the patient will not develop resistance to ALK and/or ROS1 inhibitors.
  • the nature of the cancer(s), the nature of the sample(s), the nature of the MIG6 level/activity, the method of determining the MIG6 level/activity, the ALK inhibitor, the RO SI inhibitor, the EGFR inhibitor, the ERBB2 inhibitor, and so on, are the same as or similar to those as described elsewhere herein, such as in the “Treating, Ameliorating and/or Preventing Cancer with Combination of ALK/ROS1 Inhibitor and EGFR/ERBB2 Inhibitor” and the “Treating, Ameliorating and/or Preventing Cancer Employing MIG6 as Predictive Biomarker” sections.
  • the subject is further administered at least one additional agent that treats, ameliorates, and/or prevents a disease and/or disorder contemplated herein.
  • the compound and the at least one additional agent are co-administered to the subject, either together, or sequentially, one after the other.
  • the compound and the at least one additional agent are co-formulated.
  • the compounds contemplated within the disclosure are intended to be useful in combination with one or more additional compounds.
  • additional compounds may comprise compounds of the present disclosure and/or at least one additional agent for treating cancer, and/or at least one additional agent that treats one or more diseases or disorders contemplated herein.
  • a synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55).
  • Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination.
  • the corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a disease and/or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions contemplated within the disclosure may be carried out using known procedures, at dosages and for periods of time effective to treat a disease and/or disorder contemplated herein in the patient.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the disclosure to treat a disease and/or disorder contemplated herein in the patient.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a nonlimiting example of an effective dose range for a therapeutic compound contemplated within the disclosure is from about 1 and 5,000 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within the disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • physician or veterinarian could start doses of the compounds contemplated within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms contemplated within the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease and/or disorder contemplated herein.
  • compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more.
  • the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.
  • Compounds of the disclosure for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
  • the dose of a compound of the disclosure is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the present disclosure is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of cancer in a patient.
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranial, intrathecal, oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • routes of administration of any of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.
  • the compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate).
  • the tablets may be coated using suitable methods and coating materials such as OP ADR YTM film coating systems available from Colorcon, West Point, Pa.
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxy benzoates or sorbic acid
  • the present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the disclosure, and a further layer providing for the immediate release of another medication.
  • a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the disclosure, and a further layer providing for the immediate release of another medication.
  • a wax/pH-sensitive polymer mix Using a wax/pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.
  • the compounds of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
  • Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.
  • Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos.
  • the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • the therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the cancer in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
  • a suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
  • the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday").
  • the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the patient's condition, to a level at which the improved disease is retained.
  • patients require intermittent treatment on a longterm basis upon any recurrence of symptoms and/or infection.
  • the compounds for use in the method of the disclosure may be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50.
  • Capsid assembly modulators exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
  • the dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity.
  • the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
  • the compound contemplated herein can be more efficiently delivered to the cell nucleus by coupling the compound with the monoclonal anti-DNA antibody 3E10, which penetrates living cells and localizes in the nucleus without causing any apparent harm to the cell (Hansen JE, et al., Intranuclear protein transduction through a nucleoside salvage pathway. J Biol Chem 2007;282:20790-3; see also WO 2020/047353 and WO 2021/042060, all of which are incorporated herein in their entireties by reference).
  • 3E10 and its single-chain variable fragment (3E10 scFv) have been developed as an intracellular delivery system for macromolecules. After localizing in the cell nucleus, 3E10 scFv is largely degraded within 4 hours, thus further minimizing any potential toxicity.
  • the compounds contemplated herein can be more efficiently delivered to the cancer using certain lipid nanoparticle formulations known in the art, such as but not limited to those described in Cullis, P. R. et al., Molecular Therapy Vol. 25 No 7 July 2017. See also US20150165039 and WO 2014/008334, all of which are incorporated herein in their entireties by reference.
  • the compounds contemplated herein can be more efficiently delivered to tissue by coupling with certain protein fragments, called “pHLIP” (pH (Low) Insertion Peptide), which allow for the cargo to accumulate in acidic environments within the body.
  • pHLIP protein fragments
  • a polypeptide with a predominantly hydrophobic sequence long enough to span a membrane lipid bilayer as a transmembrane helix (TM) and comprising one or more dissociable groups inserts across a membrane spontaneously in a pH- dependent fashion placing one terminus inside cell.
  • the polypeptide conjugated with various functional moieties delivers and accumulates them at cell membrane with low extracellular pH.
  • the functional moiety conjugated with polypeptide terminus placed inside cell are translocated through the cell membrane in cytosol.
  • the peptide and its variants or nonpeptide analogs can be used to deliver therapeutic, prophylactic, diagnostic, imaging, gene regulation, cell regulation, or immunologic agents to or inside of cells in vitro or in vivo in tissue at low extracellular pH. See also US20080233107, WO2012/021790, US20120039990, US20120142042, US20150051153, US20150086617, and US20150191508, all of which are incorporated herein in their entireties by reference.
  • the present study comprises two studies: the first study described in FIGs. 1-17 and Example 1, and the second study described in FIGs. 18A-31 and Example 2.
  • Phosphoproteomics, LC/MS-MS and data analysis p-Tyr enriched phosphoproteomics, global (pSTY) phosphoproteomics and following peptide identification, normalization and quantification were carried out as described in Solanki et al., 2021. Differential expression analysis was performed for DMSO versus crizotinib-treated conditions. GO enrichment analysis was conducted for all the significantly regulated phosphoproteins (
  • H3122 was obtained from Dr. Paul A Bunn at University of Colorado. CUTO28 was derived in the Doebele laboratory from the pleural effusion of a patient harboring TPM3- ROS1 fusion under an IRB-approved consent (#11-1621). Three biological triplicates of H3122 crizotinib-resistant cells (H3122 CR1, CR2 and CR3) were generated by chronic exposures to a fixed dose of 500 nM crizotinib for approximately 3 months. Crizotinib, alectinib and entrectinib were purchased from Selleck Chemicals. EGF was purchased from R&D Systems.
  • Antibodies were used as follows: total ERK (4696), pERKl/2 T202/Y204 (4370), total AKT (2920), pAKT S473 (4060), total ALK (3791), pALK Y1604 (3341), pEGFR Y1068 (3777), total ROS1 (3266), pROSl Y2274 (3678), pSHCl Y239Y240 (2434) from Cell Signaling Technology; total EGFR (610017) from BD; total SHC1 (H00006464- M01) from Abnova; total MIG6 (sc-137154) from Santa Cruz Biotechnology.
  • Cells were seeded at a density of 1000 cells/well in 96 well plates 24 hours prior to treatments. After 72 hours of drug treatments, cell viability was assayed by MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega).
  • Lentivirus was produced by co-transfecting pCMV-VSV-G and pCMVDR8.2 into 293T cells along with non-targeting control (SHC002) or two MIG6 shRNA (Functional Genomics Facility, University of Colorado, Aurora, CO) or Myc-DDK-tagged MIG6 expressing plasmid (Origene, CAT#: RC206883L3), using Minis TransIT-293 reagent. Viral supernatants were collected 48 hours after transfection and applied to target cells for 24 hours. Transduced cells were then selected by puromycin (10 ug/ml for H3122 and 2 ug/ml for CUTO28) for one week.
  • Example 2 MIG6 Mediates Adaptive and Acquired Resistance to ALK and ROS1 Fusion Kinase Inhibition through Feedback Activation of EGFR
  • ALK and ROS1 fusions defines subsets of lung adenocarcinoma.
  • ALK/ROS1 inhibitors improved therapeutic outcome of patients harboring those oncogenic fusions, complete responses were rare, and resistance eventually develops from the residual tumor.
  • the present study performed phosphoproteomics to explore the signaling adaption shortly after ALK/ROS1 inhibition.
  • the present study found the phosphorylation of MIG6, a potent inhibitor for EGFR, was decreased following ALK/ROS1 inhibition, impairing MIG6 binding and inhibition on EGFR. Furthermore, MIG6 mRNA and protein levels were decreased rapidly by ALK/ROS1 inhibitors, potentiating EGFR activity to support cell survival.
  • the present study also uncovered a novel mechanism mediated by MIG6 to regulate EGFR activity without impacting EGFR phosphorylation, but rather altering signaling adaptor SHC1 binding to EGFR. MIG6 expression was also lost following long-term exposure to ALK/ROS1 inhibitors to support EGFR-mediated acquired resistance. Finally, a MIG6 EGFR-binding domain truncation mutation was identified in a patient-derived ROS1 cell line, rendering its resistance to ROS1 inhibitors but sensitivity to HER family inhibitors. The work established a rationale to evaluate combinations of ALK/ROS1 and EGFR inhibitors to limit residual tumor formation, therefore preventing or delaying subsequent resistance emergence.
  • the present study identified MIG6 as a novel regulator for EGFR-mediated adaptive and acquired resistance to ALK/ROS1 inhibitors.
  • the present study also suggests MIG6 mutation status as a novel biomarker to predict the responsiveness to EGFR inhibitor in ALK/ROS1 fusion-driven lung adenocarcinoma.
  • Drug resistance is a major challenge for targeted therapeutics including tyrosine kinase inhibitors (TKI) targeting oncogenic ALK and ROS1 fusions, which drive cancer progression in 3-9% of non-small cell lung cancer patients.
  • TKI tyrosine kinase inhibitors
  • ALK and ROS1 TKIs significantly improve patient outcomes compared to chemotherapies, however, complete responses are rare, and the therapeutic resistance always develops.
  • Several mechanisms that could contribute to ALK/ROS1 TKI resistance have been identified including kinase mutations hindering inhibitor binding, and off-target signaling bypass. To cope with resistance mutations, next generations of ALK/ROS1 inhibitors have been employed but resistance to those inhibitors is still inevitable.
  • drug combinations have been employed to target bypassing signaling in anecdotal cases, this strategy has not yet led to approved combinations to overcome resistance.
  • ALK/ROS1 inhibitors Another hindrance to studying the resistance mechanism to ALK/ROS1 inhibitors is the lack of cell line models harboring those fusion kinase oncogenes, as opposed to the relatively abundant EGFR and KRAS mutation-driven cell models in the non-small cell lung cancer field. In the past decade, multiple patient-derived ALK and RO SI cell lines have been generated, which represent a unique and valuable resource for studying the resistance mechanisms to ALK/ROS1 inhibitors.
  • HER family activation as a bypassing signaling mechanism has been shown to support cell survival under months of chronic exposure to ALK and ROS1 inhibitors.
  • EGFR could mediate cellular responses following ALK/ROS1 inhibition as an early adaptive resistance mechanism.
  • MIG6 an endogenous EGFR inhibitor encoded by the ERRFI1 gene, was identified as a novel regulator for EGFR-mediated adaptive and acquired resistance to ALK/ROS1 inhibitors.
  • the sphosphorylation and mRNA of MIG6 are rapidly regulated following ALK and R0S1 inhibition, potentiating EGFR activity and thereby supporting cell survival in response to primary oncogene inhibition.
  • This study provides a strong rationale for combing EGFR and ALK/ROS1 TKIs as the first-line therapy to reduce the residual disease burden and therefore delay or eliminate resistance development in ALK and ROS1 fusion-positive lung cancer patients.
  • Example 2-2 Material and Methods
  • EGFR interactome analysis significantly regulated phosphoproteins by crizotinib in CUTO28 p-Tyr phosphoproteomics were analyzed by STRING (string-db dot org/, version 11.0b) to plot protein-protein interaction (PPI) network.
  • STRING string-db dot org/, version 11.0b
  • PPI protein-protein interaction
  • H3122 was obtained from Dr. Paul A Bunn at University of Colorado (Aurora, CO).
  • CUTO23, 28, 29.1, 29.2, 37, 52 and 63 cells were derived from pleural effusion or tumor biopsy from the ALK and ROS1 fusion-positive patients.
  • the cell line derivation process was performed as described in McCoach et al. (Clin Cancer Res 2018;24:3334-47).
  • the fusions and associated mutations in all derived cells were verified by Archer Fusionplex and Variantplex assays in the Colorado Molecular Correlates Laboratory (CMOCO).
  • CMOCO Colorado Molecular Correlates Laboratory
  • H3122 and patient-derived cells were cultured in RPMI1640 supplemented with 10% FBS.
  • Crizotinib, alectinib, entrectinib, afatinib, lorlatinib and trametinib were purchased from Selleck Chemicals.
  • EGF was purchased from R&D Systems.
  • Cells were seeded 1000 or 2000 cells per well in 96 well plates 24 hours prior to treatments. After 72 hours of drug treatments, cell viability was assayed by MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega). Dose-responsive curves were generated in GraphPad Prism software. For clonogenic assay, cells were seeded 2000 cells per well in 24 well plates and treated with drugs for 2 weeks with media changed every 3 or 4 days. Colonies were stained with crystal violet and the total colony area per well was quantified by MetaMorph software.
  • Mission non-target shRNA control #SHC002 and two MIG6 shRNAs, shMIG6#l (TRC Clone ID TRCN0000291921) and shMIG6#2 (TRC Clone ID TRCN0000118128) were ordered from Functional Genomics Facility at University of Colorado (Aurora, CO).
  • WT MIG6 lentiviral plasmid #RC206883L3 and the empty vector (#PS 100092) were purchased from Origene.
  • To generate MIG6 mutant lentiviral plasmid a short sequence containing Y394FY395F mutations from a MIG6 Y394FY395F pBabe plasmid (a gift from Dr.
  • Lentivirus was produced by transfecting shRNA or protein-expressing plasmids along with pCMV-VSV-G and pCMVAR8.2 into 293T cells using Minis TransIT- 293 reagent. Viral supernatants were collected 48 hours after transfection and applied to target cells for 24 hours. Transduced cells were selected by puromycin for one week (5 ug/ml for H3122 and H3122-CR cells, 2 ug/ml for CUTO28 cells, and 1 ug/ml for CUTO37 and CUTO37-ER cells). H3122-CR and CUTO37-ER cells were maintained free of TKI after transduction to minimize cell death for the clones highly expressing WT MIG6.
  • EDTA 1% Triton X-100
  • 800 pg protein was incubated with 10 pg EGFR rabbit antibody (CST #4267) in 500 ml lysis buffer at 4°C overnight.
  • the protein-antibody complex was captured by PureProteome Protein A/G Mix Magnetic Beads (Millipore) for 1 hr at 4°C and washed by cold co-IP buffer. Buffer for lysing, incubation and washing were all supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Proteins were eluted from beads by boiling in Protein Sample Loading Buffer (LI-COR) supplemented with lOOmM DTT for 5 mins.
  • LI-COR Protein Sample Loading Buffer
  • RNA for H3122, CUTO37 and their resistant counterparts were extracted using RNeasy Plus Kits (Qiagen) and sent to Novogene for sequencing. RNA-seq raw counts were normalized and the following differentially expression analyses were performed using DESeq2 R package.
  • Example 2-3 Phosphoproteomics Reveals that Crizotinib-Regulated Phosphoproteins are Enriched in ERBB Signaling Pathways
  • the present study conducted quantitative phosphoproteomics on H3122 (EML4- ALK) and CUTO28 (TPM3-ROS1) cells following a 2-hour crizotinib treatment (FIG. 18A). Due to the limitation of global (pSTY) phosphoproteomics in detecting phosphotyrosine (pY), the present study also conducted pY phosphoproteomics in parallel using pY antibody to pull down and enrich pY proteome. All significantly regulated phosphoproteins (
  • the present study investigated phosphorylation changes of EGFR physical interactors under crizotinib treatment in the EGFR interactome constructed with CUTO28 pY data (FIG. 18D).
  • the present study identified MIG6, an endogenous EGFR protein inhibitor.
  • MIG6 Y394 phosphorylation in H3122 cells (Y395 phosphorylation undetected) and both Y394 and Y395 phosphorylation in CUTO28 cells were decreased by crizotinib (FIG. 18E). Phosphorylation of Y394 and Y395 is important for MIG6 to bind and inhibit EGFR.
  • Example 2-4 Upfront Inhibition of EGFR Enhances Responses to ALK and ROS1 Inhibition
  • MIG6 protein levels were evaluated in an extended ALK/ROS1 inhibition across 48 hours. Strikingly, MIG6 protein level starts to decrease after 6-hour treatment of various ALK/ROS1 inhibitors in H3122 and CUTO28 cells (FIGs. 20A and 20E).
  • the present study also examined MIG6 protein level under ALK/ROS1 inhibition across other patient-derived ALK and ROS1 lines available to the present study.
  • CUTO29.1 and CUTO29.2 are EML4-ALK variant 3-driven cells derived from the same patient in a treatment course.
  • CUTO29.1 was derived during brigatinib treatment; the patient had been previously treated with and progressed on crizotinib and alectinib.
  • CUTO29.2 was derived later under the treatment of the next-generation ALK inhibitor, lorlatinib.
  • CUTO29.1 harbors the crizoinib resistant mutation ALK p.Cl 156Y but was still sensitive to loratinib (FIG. 21 A).
  • CUTO29.2 harbored ALK p.L1998F; Il 171T; Cl 156Y mutations, which rendered loratinib resistance but induces re-sensitization to crizotinib (FIG. 2 IB).
  • MIG6 protein was decreased by lorlatinib in CUTO29.1 cells but not in lorlatinib-resistant CUTO29.2 cells (FIG. 21C). However, MIG6 could be decreased by crizotinib in CUTO29.2 cells, in line with its sensitivity to crizotinib (FIG. 2 ID).
  • CUTO52 cell harbors EML4-ALK (vl) and the ALK p.Ll 196M and D1203N resistance mutations, derived from a patient following treatment with crizotinib, alectinib and brigatinib.
  • CUTO52 is resistant to crizotinib and moderately sensitive to alectinib (FIG. 21E) and its MIG6 protein level could not be reduced by crizotinib treatment (FIG. 21G).
  • CUTO23 harboring WT CD74-ROS1 fusion is sensitive to crizotinib and demonstrated a reduction of MIG6 following crizotinib treatment (FIGs. 2 IF and 21H).
  • MIG6 downregulation correlates with cellular sensitivity to ALK/ROS1 inhibition
  • MAPK pathway is a critical downstream pathway shared by ALK and R0S1 kinases and has also been reported to regulate MIG6 mRNA expression.
  • ALK/ROS1 inhibition reduced mRNA levels of ERRFI1 (MIG6) (FIGs. 22A-22B).
  • Trametinib a MEKl/2 inhibitor, also significantly suppressed ERRFI1 mRNA expression in ALK and ROS1 cells (FIGs. 22C-22D).
  • Example 2-7 MIG 6 Modulates Responses to ALK/ROS1 Inhibition via EGFR-SHC1- MAPK Pathway
  • MIG6 expression was depleted by shRNA knockdown in H3122 and CUTO28 cells. It was found that MIG6 knockdown induced resistance to various ALK/ROS1 inhibitors (FIGs. 23A-23B). However, addition of pan-HER inhibitor restored ALK/ROS1 inhibition sensitivity in absence of MIG6 expression, suggesting MIG6 knockdown supported cell survival via ERBB signaling pathway in responses to ALK/ROS1 inhibition. Consistent with the responses in cell viability, ERK signaling was also rescued by MIG6 knockdown under ALK/ROS1 inhibition while this rescue could be abrogated by addition of afatinib (FIGs.
  • MIG6 binds and blocks the EGFR substrate-binding cleft where protein substrates, including signaling adaptors, bind to EGFR to be phosphorylated for following signaling transduction.
  • SHC1 is one of these protein substrates competing with MIG6 for this EGFR binding site and is phosphorylated at Tyr239 by EGFR after binding.
  • SHC1 is also known as a critical signaling adaptor shared by EGFR, ALK, and presumably, ROS1 to drive MAPK signaling. It was therefore hypothesized that MIG6 depletion increases SHC1 binding to EGFR to maintain cell survival following ALK/ROS1 fusion kinase inhibition. Indeed, SHC1 Y239/240 phosphorylation decrease under ALK/ROS1 inhibition could be partially rescued by MIG6 knockdown (FIGs. 23C-23D). However, when cotreated with afatinib, SHC1 Y239/240 phosphorylation was further inhibited regardless of MIG6 knockdown, in line with a more profound suppression of ERK signaling.
  • MIG6 overexpression in H3122 further enhanced cellular responses to ALK inhibition with more SHC1 and ERK phosphorylation suppression (FIGs. 25A-25B).
  • MIG6 knockdown increases SHC1 binding to EGFR (FIG. 23E).
  • Example 2-8 MIG6 Downregulation Associated with EGFR-Mediated Acquired Resistance to ALK/ROS1 Inhibition
  • An entrectinib- resistant ROS1 cell line (CUTO37-ER) was also developed by exposing CUTO37, a patient- derived line harboring CD74-ROS1 (C6;R34) fusion, to an escalating dose of entrectinib for ⁇ 4 months and ultimately maintaining in 500 nM entrectinib.
  • RNA-seq data revealed the mRNA of two EGFR ligands HB-EGF and EREG (Epiregulin), had a ⁇ 1.7-fold and ⁇ 2.5-fold increase, respectively, in H3122-CR lines versus H3122 cells (data not shown).
  • Afatinib was able to partially re-sensitize those resistant cells to crizotinib treatment (FIG. 26B).
  • crizotinb and afatinib combination were able to suppress ERK phosphorylation in H3122 CR1-3 cells, indicating a dual dependency of ALK and EGFR in H3122 crizotinib resistant cells (FIG. 26C).
  • CUTO37-ER cells demonstrated a complete signaling switch from ROS1 to EGFR for survival (FIG. 26D).
  • RNA-seq and immunoblots revealed MIG6 mRNA and protein were depleted in CUTO37-ER cells accompanied by a robust upregulation of EGFR transcripts, protein and phosphorylation (FIG. 26E).
  • Afatinib alone was able to eliminate ERK phosphorylation in CUTO37-ER cells (FIG. 26E).
  • the present study also determined MIG6 protein level in other fusion kinase inhibitorresistant cells available to the present study.
  • HCC78 a ROS 1 -fusion driven cell line and LC- 2/ad, a RET-fusion driven cell line, were continuously exposed to their oncogene-targeted TKIs for months to derive HCC78-TR and PR2 resistant cells.
  • EGFR bypass signaling was reported as the resistance mechanism for those cells.
  • MIG6 was attenuated in HCC78-TR and PR2 cells as well compared to their parental counterparts (FIGs. 27A-27B), supporting the correlation between MIG6 downregulation and EGFR- mediated acquired resistance to fusion kinase inhibitors.
  • Example 2-9 Phosphorylation of MIG6 Y394/395 is Critical for Suppressing EGFR- Mediated Acquired Resistance to ALK/RO SI Inhibition
  • the present study investigated whether MIG6 reconstitution would re-sensitize resistant cells to ALK/ROS1 inhibition. Furthermore, because the phosphoproteomics identified a decrease of phosphorylation of Y394/Y395 of MIG6 under crizotinib treatment (FIG. 18D), the present study interrogated the biological relevance of these phosphorylation in regulating the cell survival under ALK/ROS1 inhibition.
  • tyrosine residues on MIG6 394 and 395 were mutated to phenylalanine, which mimics the structure of tyrosine but lacks the hydroxyl to be phosphorylated, resulting in an EGFR-binding deficient MIG6 mutant.
  • WT MIG6 overexpression was able to re-sensitize H3122 CR1 cells to crizotinib treatment by suppressing SHC1 and ERK activation (FIGs. 28A-28B).
  • this ability to overcome crizotinib resistance by MIG6 was diminished, but not completely abrogated, by introduction of the Y394F/Y395F mutation.
  • WT MIG6 overexpression alone did not decrease SHC1 and ERK phosphorylation, consistent with the finding showing single-agent afatinib treatment did not impact downstream signaling of H3122-CR1 cells (FIG. 26C).
  • CUTO37-ER survival and colony formation could be suppressed by WT MIG6 overexpression alone but not by the MIG6 Y394FY395F mutant (FIGs. 28C-28D), in line with its complete signaling switch from ROS1 to EGFR to survive (FIGs. 26D-26E).
  • MIG6 Y394FY395F has a much higher expression than MIG6 WT, suggesting MIG6 WT expression alone is cytotoxic to CUTO37-ER cells.
  • Example 2-10 MIG6 EGFR-binding Domain Truncation Mutation Predicts Resistance to ROS1 Inhibition and Sensitivity to EGFR Inhibition
  • ERRFI1 mutation status through RNA-seq conducted in several patient-derived ALK and ROS1 cells in-house and identified a Q241* nonsense mutation on ERRFI1 from CUTO63 cell line (FIG. 29A). This line harbors SLC34A2-ROS1 (S13:R32, S13:R34) fusion and was derived from a patient’s pleural effusion while progressing on crizotinib treatment.
  • the cell line was derived in the absence of ROS 1 TKI selective pressure.
  • An ERRFIl Q241* mutation results in a complete deletion of EGFR-binding domain on MIG6, potentially releasing EGFR from MIG6 inhibition.
  • the mutant variant frequency was only -23% (FIG. 30)
  • the full-length MIG6 (-55 kDa) was absent in CUTO63, but a truncated MIG6 protein with a predicted size ( ⁇ 26kDa) could be detected by an antibody raised against an MIG6 peptide generated before the truncation site (FIG. 29B).
  • EGFR could be preferably selected by ALK/ROS1 cancer cells to maintain the critical downstream signaling when the primary oncogene is inhibited.
  • Upfront inhibition of EGFR with fusion kinase inhibitors indeed eliminated the residual colony formation (FIG. 20C). It was also demonstrated the co-inhibition of EGFR and ALK in vivo further suppressed H3122 xenograft tumor growth compared to ALK inhibitor monotherapy.
  • Another notable finding in this study is that the present study discovered a novel machinery that regulates EGFR signaling activity without altering EGFR phosphorylation itself.
  • MIG6 and SHC1 bind to the same substrate-binding site of EGFR, it was demonstrated for the first time that MIG6 competes with SHC1 for EGFR binding in ALK/RO SI -driven cancer cells and this competition has biological and functional significance in regulating responses to ALK/ROS1 TKIs.
  • SHC1 serves as a critical signaling adaptor for ALK/ROS1 primary oncogene under treatment-naive condition.
  • MIG6 and ACK1 which contains a homology region of MIG6 proline-rich and EGFR-binding domains, physically interacted with ALK via GRB2 but not with kinase-inactive ALK. It was speculated that MIG6 could be phosphorylated by ALK/ROS1 fusion kinases directly or their downstream effectors indirectly. Further study is required to understand the phosphorylation mechanism of MIG6 in ALK/RO SI -driven cancer cells.
  • ERRFI1 mutation resulting in MIG6 EGFR- binding domain truncation can be considered as a biomarker to predict a potential response to this drug combination and/or to an EGFR inhibitor monotherapy despite an absence of oncogenic EGFR mutations.
  • the data provided a strong mechanistic basis for evaluating the combination of an EGFR inhibitor with ALK/ROS1 inhibition in the first-line setting to minimize residual disease and potentially delay the development of resistance.
  • the present invention is directed to the following non-limiting embodiments:
  • Embodiment 1 A method of treating, ameliorating, and/or preventing a cancer in a subject in need thereof, comprising: administering to the subject an effective amount of an inhibitor for anaplastic lymphoma kinase (ALK) and/or an inhibitor for ROS proto-oncogene 1 (ROS1); and administering to the subject an effective amount of an inhibitor for epidermal growth factor receptor (EGFR) and/or an inhibitor for Erb-B2 receptor tyrosine kinase 2 (ERBB2).
  • ALK anaplastic lymphoma kinase
  • ROS1 ROS proto-oncogene 1
  • EGFR epidermal growth factor receptor
  • ERBB2 Erb-B2 receptor tyrosine kinase 2
  • Embodiment 2 The method of Embodiment 1, wherein the cancer is an ALK positive cancer or a ROS1 positive cancer.
  • Embodiment 3 The method of any one of Embodiments 1-2, wherein the cancer is a non-small cell lung cancer.
  • Embodiment 4 The method of any one of Embodiments 1-3, wherein the cancer is resistant to a treatment of the RTK inhibitor alone.
  • Embodiment 5 The method of any one of Embodiments 1-4, wherein at least one of the following applies:
  • the inhibitor for ALK comprises alectinib, alkotinib, AP26113, ASP3026, AZD3463, belizatinib, brigatinib, CEP-28122, CEP-37440, ceritinib, crizotinib, ensartinib, entrectinib, foritinib, HG-14-10-04, Lorlatinib, NVL-655, PF-06463922, PLB1003, repotrectinib, TAE684, TPX-0131, TQ-B3139, TSR-011, X-376, or derivatives thereof,,
  • the inhibitor for RO SI comprises ceritinib, crizotinib, entrectinib, lorlatinib, NUVL-520, repotrectinib, or derivatives thereof
  • the inhibitor for EGFR comprises afatinib, amivantamab, cetuximab, dacomitinib, erlotinib, gefitinib, mobocertinib, necitumumab, neratinib, osimertinib, panitumumab, poziotinib, vandetanib, or derivatives thereof,
  • the inhibitor for ERBB2 comprises dacomitinib, fam-trastuzumab deruxtecan-nxki, lapatinib, margetuximab, neratinib, pertuzumab, poziotinib, trastuzumab, tucatinib, or derivatives thereof.
  • Embodiment 6 A method of treating, ameliorating and/or preventing cancer in a subject in need thereof, the method comprising: providing a sample of the subject; testing the sample for a MIG6 level and/or a MIG6 activity; if the MIG6 level and/or the MIG6 activity are below a threshold level, administering to the subject an effective amount of an EGFR inhibitor and/or an ERBB2 inhibitor.
  • Embodiment 7 The method of Embodiment 6, wherein the cancer is an ALK positive cancer or a ROS1 positive cancer.
  • Embodiment 8 The method of any one of Embodiments 6-7, wherein the cancer is a non-small cell lung cancer.
  • Embodiment 9 The method of any one of Embodiment 6-8, wherein the sample comprises at least one selected from a blood sample and a cancer biopsy sample.
  • Embodiment 10 The method of any one of Embodiments 6-9, wherein
  • the sample is tested for the MIG6 level, and the MIG6 level comprises a MIG6 mRNA level or a MIG6 protein level, or
  • the sample is tested for the MIG6 activity, and the MIG6 activity is determined based on a binding strength between MIG6 and EGFR and/or ERBB2, or based on an inhibition strength by MIG6 on EGFR and/or ERBB2.
  • Embodiment 11 The method of Embodiment 10, wherein
  • the sample is tested for the MIG6 mRNA level, and the MIG6 mRNA level is determined by reverse transcription PCR, real time PCR, RNA sequencing, or in situ hybridization,
  • the sample is tested for the Mig6 protein level, and the Mig6 protein level is determined by western blotting, ELISA, flow cytometry, immunocytochemistry, mass spectrometry, or quantitative proteomics.
  • Embodiment 12 The method of Embodiment 10, wherein the sample is tested for the
  • Mig6 activity is determined by at least one of the following:
  • a post-translational modification on the MIG6 6 protein such as a phosphorylation of the MIG6 protein, such as phosphorylations on residues tyrosine 394 and/or tyrosine 395 of the Mig6 protein,
  • a presence or absence of a genetic mutation in the MIG6 gene such as a genetic mutation affecting an interaction between MIG6 and EGFR and/or an interaction between MIG and ERBB2.
  • Embodiment 13 The method of Embodiment 12, wherein at least one of the following applies:
  • the MIG6 activity is determined by the phosphorylation of the MIG6 protein, and the phosphorylation is measured by western blotting, ELISA, flow cytometry, immunocytochemistry, mass spectrometry, or phosphoproteomics,
  • the MIG6 activity is determined by the presence or absence of a genetic mutation, and the presence or absence of a genetic mutation in the MIG6 gene is determined by PCR, RNA sequencing, targeted next-generation sequencing (NGS), whole exome sequencing, or whole genome sequencing.
  • NGS next-generation sequencing
  • Embodiment 14 The method of any one of Embodiments 12-13, wherein a reduced level of phosphorylation of MIG6 corresponds to a decreased MIG6 activity.
  • Embodiment 15 The method of Embodiment 14, wherein the phosphorylation level is expressed as a ratio of a level of an unphosphorylated amino acid to a level of the phosphorylated counterpart.
  • Embodiment 16 The method of any one of Embodiments 6-15, wherein the method further comprises: if the MIG6 level and/or the MIG6 activity are the same as or higher than the threshold level, administering to the subject an effective amount of an ALK inhibitor and/or an ROS1 inhibitor.
  • Embodiment 17 The method of any one of Embodiments 6-15, wherein, if the MIG6 level and/or the MIG6 activity are below the threshold level, the subject is administered with: the effective amount of the EGFR inhibitor and/or the ERBB2 inhibitor; and an effective amount of an ALK inhibitor and/or an RO SI inhibitor.
  • Embodiment 18 The method of any one of Embodiments 6-17, wherein at least one of the following applies:
  • the EGFR inhibitor comprises afatinib, amivantamab, cetuximab, dacomitinib, erlotinib, gefitinib, mobocertinib, necitumumab, neratinib, osimertinib, panitumumab, poziotinib, vandetanib, or derivatives thereof,
  • the ERBB2 inhibitor comprises dacomitinib, fam-trastuzumab deruxtecan-nxki, lapatinib, margetuximab, neratinib, pertuzumab, poziotinib, trastuzumab, tucatinib, or derivatives thereof.
  • Embodiment 19 The method of Embodiments 16-17, wherein at least one of the following applies:
  • the ALK inhibitor comprises alectinib, alkotinib, AP26113, ASP3026, AZD3463, belizatinib, brigatinib, CEP-28122, CEP-37440, ceritinib, crizotinib, ensartinib, entrectinib, foritinib, HG-14-10-04, Lorlatinib, NVL-655, PF-06463922, PLB1003, repotrectinib, TAE684, TPX-0131, TQ-B3139, TSR-011, X-376, or derivatives thereof,
  • the ROS1 inhibitor comprises ceritinib, crizotinib, entrectinib, lorlatinib, NUVL-520, repotrectinib, or derivatives thereof.
  • Embodiment 20 A method of delaying and/or preventing resistance development to an ALK inhibitor and/or a ROS1 inhibitor in a patient suffering from non-small cell lung cancer, the method comprising: providing a sample of the patient after the patient has received treatment with the ALK inhibitor and/or the RO SI inhibitor; testing the sample for a MIG6 level or a MIG6 activity; if the MIG6 level and/or the MIG6 activity are below a threshold level, administering to the patient: an effective amount of an ALK inhibitor and/or a ROS1 inhibitor; and an effective amount of an EGFR inhibitor and/or an ERBB2 inhibitor, thereby delaying or preventing resistance development to the at least one ROS1 and ALK inhibitor.
  • Embodiment 21 A method of adapting treatment in a patient with non-small cell lung cancer that is being treated with an ALK inhibitor and/or a ROS1 inhibitor, the method comprising: providing a sample of a patient; testing sample for a MIG6 level and/or a MIG6 activity; if the MIG6 level and/or the MIG6 activity are equal to or above a threshold level, continuing treating the patient with the ALK inhibitor and/or the ROS1 inhibitor, if the MIG6 level and/or the MIG6 activity are below the threshold level: treating the patient with the ALK inhibitor and/or the ROS1 inhibitor; and treating the patient with an EGFR inhibitor and/or an ERBB2 inhibitor, thereby adapting the treatment.
  • Embodiment 22 A method of determining whether new treatment should be adapted in a patient with non-small cell lung cancer and has been treated or is being treated with a ROS1 and/or ALK inhibitor, the method comprising: providing a sample of the patient; testing sample for a MIG6 level or a MIG6 activity; if the MIG6 level or the MIG6 activity are below a threshold level, adapting new treatment.
  • Embodiment 23 A method of predicting resistance development to treatment of an ALK inhibitor and/or a ROS1 inhibitor in a patient suffering from non-small cell lung cancer, the method comprising: providing a first sample of patient prior to initiation of treatment; testing the first sample for MIG6 level and/or MIG6 activity to establish a first value; treating the patient with the ALK inhibitor or the RO SI inhibitor for a time period; providing a second sample of the patient after the time period; testing the second sample for MIG6 level and/or MIG6 activity to establish a second value; if the second level is smaller than the first level for a value higher a threshold level, predicting that the patient will develop resistance to ALK and/or ROS1 inhibitors; if the second level is smaller than the first level for a value equal to or smaller than the threshold level, predicting that the patient will not develop resistance to ALK and/or ROS1 inhibitors.
  • Embodiment 24 The method of any one of Embodiments 20-23, wherein the non- small cell lung cancer is an ALK positive non-small cell lung cancer or a ROS1 positive non- small cell lung cancer.
  • Embodiment 25 The method of any one of Embodiments 20-24, wherein the sample comprises at least one selected from a blood sample and a cancer biopsy sample.
  • Embodiment 26 The method of any one of Embodiments 20-25, wherein
  • the sample is tested for the MIG6 level, and the MIG6 level comprises a MIG6 mRNA level or a MIG6 protein level, or
  • the sample is tested for the MIG6 activity, and the MIG6 activity is determined based on a binding strength between MIG6 and EGFR and/or ERBB2, or based on an inhibition strength by MIG6 on EGFR and/or ERBB2.
  • Embodiment 27 The method of any one of Embodiments 20-26, wherein the sample is tested for the MIG6 activity, and the MIG6 activity is determined by at least one of the following:
  • a post-translational modification on the Mig 6 protein such as a phosphorylation of the MIG6 protein, such as phosphorylations on residues tyrosine 394 and/or tyrosine 395 of the MIG6 protein,
  • a presence or absence of a genetic mutation in the MIG6 gene such as a genetic mutation affecting an interaction between MIG6 and EGFR and/or an interaction between MIG6 and ERBB2.
  • Embodiment 28 The method of Embodiment 27, wherein at least one of the following applies:
  • the MIG6 activity is determined by a phosphorylation of the MIG6 protein, and wherein the phosphorylation is measured by western blotting, ELISA, flow cytometry, immunocytochemistry, mass spectrometry, or phosphoproteomics,
  • the MIG6 activity is determined by the presence or absence of a genetic mutation, and the presence or absence of a genetic mutation in the MIG6 gene is determined by PCR, RNA sequencing, targeted next-generation sequencing (NGS), whole exome sequencing, or whole genome sequencing.
  • NGS next-generation sequencing
  • Embodiment 29 The method of any one of Embodiments 27-28, wherein a reduced level of phosphorylation of MIG6 corresponds to a decreased MIG6 activity.
  • Embodiment 30 The method of Embodiment 29, wherein the phosphorylation level is expressed as a ratio of a level of an unphosphorylated amino acid to a level of the phosphorylated counterpart.
  • Embodiment 31 The method of any one of Embodiments 20-31, wherein at least one of the following applies:
  • the ALK inhibitor comprises alectinib, alkotinib, AP26113, ASP3026, AZD3463, belizatinib, brigatinib, CEP-28122, CEP-37440, ceritinib, crizotinib, ensartinib, entrectinib, foritinib, HG-14-10-04, Lorlatinib, NVL-655, PF-06463922, PLB1003, repotrectinib, TAE684, TPX-0131, TQ-B3139, TSR-011, X-376, or derivatives thereof,,
  • the ROS1 inhibitor comprises ceritinib, crizotinib, entrectinib, lorlatinib, NUVL-520, repotrectinib, or derivatives thereof,
  • the EGFR inhibitor comprises afatinib, amivantamab, cetuximab, dacomitinib, erlotinib, gefitinib, mobocertinib, necitumumab, neratinib, osimertinib, panitumumab, poziotinib, vandetanib, or derivatives thereof,
  • the ERBB2 inhibitor comprises dacomitinib, fam-trastuzumab deruxtecan-nxki, lapatinib, margetuximab, neratinib, pertuzumab, poziotinib, trastuzumab, tucatinib, or derivatives thereof.
  • Embodiment 32 A method of overcoming resistance of a non-small cell lung cancer in a patient to a receptor tyrosine kinase inhibitor that inhibits at least one of ALK and ROS1, the method comprising administering to the patient a compound or construct that increases the expression level and/or phosphorylation level of MIG6 in the cancer cell.

Abstract

Actuellement, il n'existe pas de biomarqueurs prédictifs ou de thérapies approuvées pour répondre à une résistance aux médicaments médiée par une signalisation de dérivation dans ALK ou ROS1 + NSCLS ou d'autres cancers après une thérapie par TKI classique. La présente invention identifie MIG6 en tant que nouveau régulateur de la résistance adaptative et acquise médiée par la signalisation d'ErbB aux TKI ciblant ALK/ROS1. Les niveaux et/ou l'activité de MIG6, telle que déterminée en fonction des niveaux de phosphorylation, de transcription et/ou de protéine, ainsi que l'état de mutation de MIG6, sont utilisés pour guider la décision de combiner des inhibiteurs d'ErbB avec des inhibiteurs d'ALK/ROS1 après le développement de la progression d'une maladie après un traitement par un inhibiteur d'ALK ou de ROSl pour surmonter la résistance thérapeutique.
PCT/US2022/045903 2021-10-06 2022-10-06 Biomarqueurs de résistance à médiation egfr dans des cancers provoqués par un oncogène et méthodes de traitement, de prévention et/ou d'atténuation de cancers provoqués par un oncogène WO2023059801A1 (fr)

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NAN CHEN; ANH T. LE; ERIC A. WELSH; BIN FANG; ERIC B. HAURA; ROBERT C. DOEBELE: "Abstract P071: Phosphoproteomics identifies Mig6 as a keymediator of adaptive resistance to ALK/ROS1 oncogeneinhibition", MOLECULAR CANCER THERAPEUTICS, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 20, no. 12, Suppl., 1 December 2021 (2021-12-01) - 10 October 2021 (2021-10-10), US , pages P071, XP009545787, ISSN: 1535-7163, DOI: 10.1158/1535-7163.TARG-21-P071 *

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CN116763792B (zh) * 2023-06-19 2024-03-12 西安国际医学中心医院 Hg-14-10-04在制备治疗食管鳞癌的药物中的应用

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