WO2024249331A1

WO2024249331A1 - Combined rtk and alk inhibition in rtk driven cancers

Info

Publication number: WO2024249331A1
Application number: PCT/US2024/031075
Authority: WO
Inventors: Amyn HABIB; Xiaoyao YANG
Original assignee: University of Texas System; US Department of Veterans Affairs; University of Texas at Austin
Current assignee: University of Texas System; US Department of Veterans Affairs; University of Texas at Austin
Priority date: 2023-05-26
Filing date: 2024-05-24
Publication date: 2024-12-05
Anticipated expiration: 2025-11-26

Abstract

The present disclosure generally relates to compositions and methods for treating cancer, particularly certain treatment resistant cancers. Aspects include providing a combination of a receptor tyrosine kinase (RTK) inhibitor and an Anaplastic Lymphoma Kinase (ALK) inhibitor for the treatment of cancer. Aspects provide for treatment of cancers resistant or at risk of becoming resistant to RTK inhibition, including those that do would not normally be treated with ALK inhibition.

Description

TITLE

COMBINED RTK AND ALK INHIBITION IN RTK DRIVEN CANCERS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/504,683, filed May 26, 2023, and titled “COMBINED RTK AND ALK INHIBITION IN RTK DRIVEN CANCERS,” which is incorporated by reference herein in its entirety.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Nos. CA244212 and NS119225 awarded by the National Institutes of Health. The government has certain rights in this invention.

INCORPORATION OF SEQUENCE LISTING

[0003] The present application contains a Sequence Listing which has been submitted in .XML format via Patent Center and is hereby incorporated by reference in its entirety. Said WIPO Sequence Listing was created on May 20, 2024, is named 106546-793650_Sequence Listing, and is 5 kilobytes in size.

BACKGROUND

[0004] 1. Field

[0005] The present disclosure is directed to compositions and methods of treating delayed RTK inhibitor resistance in certain cancers.

[0006] 2. Discussion of Related Art

[0007] Receptor tyrosine kinase (RTK) inhibition is a widely used treatment in cancer and is assumed to render the RTK inactive. However, ultimately many targeted treatments using tyrosine kinase inhibitors (TKIs) (including RTKs) ultimately fail because of secondary resistance. Mechanisms of secondary resistance include mutations in RTKs such as the C797S mutation in EGFR in NSCLC or RTK amplification, detected months after initial treatment. RTK inhibition also results in a rapid adaptive reprogramming of cellular signaling networks as the cancer cell tries to restore homeostasis. This rapid adaptive response may lead to the survival of drug resistant persister clones and secondary resistance. For example, it has been shown that activation of inflammatory signals such as TNF-NF-KB can mediate resistance to EGFR inhibition in NSCLC. However, the ultimate underlying cause behind RTK drug resistance is unknown. New methods and compositions are needed to treat and reverse RTK inhibitor resistance in patients. SUMMARY

[0008] The present disclosure is based, at least in part, on the discovery that ALK activation contributes to RTK inhibitor resistance. Therefore, in accord with certain aspects of the present disclosure, a method is of treating cancer in a subject is provided, the method comprising administering to the subject in need thereof at least one receptor tyrosine kinase (RTK) inhibitor that does not inhibit ALK, and at least one ALK inhibitor and wherein the subject has or is suspected of having a malignant tumor.

[0009] In various aspects, the malignant tumor can comprise comprises a carcinoma, a sarcoma, a hematological malignancy or any combination thereof. For example in some aspects, the malignant tumor comprises a lung tumor, a breast tumor, a pancreatic tumor, a colorectal tumor, an esophageal tumor, a head-and-neck tumor, a hematological malignancy, a brain tumor, an ovarian tumor, a stomach tumor, a gastric tumor, a gastrointestinal stromal tumor, a prostate tumor, a thyroid tumor, bladder tumor, dermatofibrosarcoma protuberans, a giant cell tumor, a lymphoma, melanoma, a myeloma, a kidney tumor, a soft tissue sarcoma, chronic eosinophilic leukemia (CEL), chronic lympocytic leukemia (CLL), urothelial carcinoma, a cervical tumor, liver or bile duct tumor, endometrial tumor or any combination thereof.

[0010] In various aspects, the malignant tumor is derived from a non-small-cell lung cancer (NSCLC), HER-2 positive breast cancer, glioblastoma, gastrointestinal stromal tumor, esophageal tumor, liver or bile duct tumor, bladder tumor or any combination thereof.

[0011] In various aspects, the malignant tumor has one or more somatic mutations, translocations, amplifications, and/or other disruptions in at least one gene encoding a receptor tyrosine kinase that is not ALK. In some aspects, the at least one gene encoding a RTK that is not ALK comprises an EGFR gene, a HER2 gene, a HER3 gene, a HER4 gene, a c-Met gene, a ROS1 gene, a IGF-1R gene, a TRKA gene, a TRKB gene, a TRKC gene, a PDGFR gene, a VEGFR gene, a FGFR gene, a FLT3 gene, a NTRK gene, a RET gene or any combination thereof.

[0012] In various aspects, the malignant tumor expresses one or more hyperactive or overactive receptor tyrosine kinases (RTKs) that are not ALK. In various aspects, the the one or more receptor tyrosine kinases are selected from EGFR, HER2, HER3, HER4, c-Met, ROS1 , IGF-1 R, TRKA, TRKB, TRKC, PDGFR, VEGFR, FGFR, FLT3, NTRK, and RET.

[0013] In various aspects, the malignant tumor does not have any somatic mutations and/or translocations in an ALK gene.

[0014] In any of the above or related aspects, the at least one ALK inhibitor comprises at least one of a peptide, an antibody, a small molecule pharmaceutical compound, an oligo, a nucleic acid molecule, or a combination thereof. [0015] In various aspects, the at least one ALK inhibitor comprises a small molecule pharmaceutical compound. For example, in some aspects the at least one ALK inhibitor comprises Alectinib, Lorlatinib, Brigatinib, Ceritinib, TAE684 (NVP-TAE684), AP26113 analog (ALK-IN-1), GSK1838705A, AZD3463, ASP3026, Entrectinib, Crizotinib, or any combination thereof.

[0016] In any of the aspects herein, the at least one RTK inhibitor inhibits an epidermal growth factor receptor (EGFR), a platelet-derived growth factor (PDGF)/Kit receptor, a vascular endothelial growth factor receptor (VEGFR), a fibroblast growth factor (FGF) receptor, a hepatocyte growth factor (HGF receptor), a mesenchymal-epithelial transition factor receptor (MET), an insulin or insulin-like growth factor (IGF) receptor or any combination thereof. For example, in some aspects, the at least one RTK inhibitor comprises an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, a pan-HER inhibitor, a MET inhibitor, an insulin or insulin-like growth factor (IGF) receptor inhibitor, an FGF receptor inhibitor, a PDGFR receptor inhibitor or any combination thereof. In various aspects, the the at least one RTK inhibitor is an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, or a pan-HER inhibitor.

[0017] In any of the foregoing or related aspects, the at least one RTK inhibitor comprises least one of a peptide, an antibody, small molecule pharmaceutical compound, an oligo, a nucleic acid molecule, or a combination thereof.

[0018] In some aspects, the at least one RTK inhibitor comprises a small molecule pharmaceutical compound or a monoclonal antibody. For example, the at least one RTK inhibitor can comprise Osimertinib, Imatinib, Capmatinib, Linsitinib, Infigratinib, Avapritinib, Pemigatinib, Ripretinib, Selpercatinib, Tucatinib, Entrectinib, Erdafitinib, Pexidartinib, Dacomitinib, Gilteritinib, Acalabrutinib, Midostaurin, Neratinib, Cobimetinib, Lenvatinib, Nintedanib, Afatinib, Trametinib, Axitinib, Cabozantinib, Ponatinib, Regorafenib, Tofacitinib, Vandetanib, Pazopanib, Lapatinib, Nilotinib, Dasatinib, Sunitinib, Sorafenib, Erlotinib, Gefitinib, Imatinib, Varlinitib, Infigratinib, Mobocertinib, Pralsetinib, Tepotinib, Tivozanib, Trastuzumab, Aflibercept, Larotrectinib, Trastuzumab, Pertuzumab, Amivantamab, Bevacizumab, Margetuximab, Necitumumab, Ramucirumab, Panitumumab, Cetuximab or any combination thereof.

[0019] In various aspects, the at least one RTK inhibitor comprises Osimertinib, Imatinib, Capmatinib, Linsitinib, Infigratinib, Erlotinib, Gefitinib, Afatinib, Dacomitinib, Lapatinib, Neratinib, Varlitinib, Tucatinib, Trastuzumab, Pertuzumab, Cetuximab or any combination thereof.

[0020] In various aspects, the at least one RTK inhibitor comprises Osimertinib, Erlotinib, Gefitinib, Afatinib, Dacomitinib, Lapatinib, Neratinib, Varlitinib, Trastuzumab, Pertuzumab, Cetuximab, or any combination thereof. [0021] In any of the foregoing or related aspects, the malignant tumor cells in the malignant tumor have a higher rate of cell death in the subject compared to an untreated subject with identical disease condition and predicted outcome.

[0022] In any of the foreging or related aspects, the incidence of treatment resistance to the RTK therapy may be decreased in the subject compared to a subject treated with the RTK inhibitor that does not inhibit ALK alone, wherein the subject has identical disease condition and predicted outcome.

[0023] Further aspects of the present disclosure are related to a method of treating a tumor in a subject in need thereof, the method comprising administering to the subject at least one ALK inhibitor, wherein the subject has undergone, is undergoing or will undergo an anti-cancer therapy comprising one or more targeted receptor tyrosine kinase (RTK) inhibitors that do not target ALK.

[0024] In various aspects, the tumor can be resistant to the anti-cancer therapy comprising one or more targeted RTK inhibitors. In various aspects, the tumor can comprise a lung tumor, a breast tumor, a pancreatic tumor, a colorectal tumor, an esophageal tumor, a head-and- neck tumor, a hematological malignancy, a brain tumor, an ovarian tumor, a stomach tumor, a gastric tumor, a gastrointestinal stromal tumor, a prostate tumor, a thyroid tumor, bladder tumor, dermatofibrosarcoma protuberans, a giant cell tumor, a lymphoma, melanoma, a myeloma, a kidney tumor, a soft tissue sarcoma, chronic eosinophilic leukemia (CEL), chronic lympocytic leukemia (CLL), urothelial carcinoma, cervical tumor, a liver or bile duct tumor, endometrial tumor, or any combination thereof.

[0025] In various aspects, the tumor can be non-small cell lung cancer (NSCLC), HER-2 positive breast cancer, glioblastoma, gastrointestinal stromal tumor, esophageal tumor, liver or bile duct tumor, bladder tumor or any combination thereof.

[0026] In various aspects, the tumor can have one or more somatic mutations, translocations, amplifications, and/or other disruptions in at least one gene encoding a receptor tyrosine kinase that is not ALK. In some aspects, the at least one gene encoding a RTK that is not ALK is selected from EGFR, HER2, HER3, HER4, c-Met, ROS1, IGF-1R, TRKA, TRKB, TRKC, PDGFR, VEGFR, FGFR, FLT3, NTRK, RET, and any combination thereof. In various aspects, the tumor does not have any somatic mutations and/or translocations in an ALK gene.

[0027] In various aspects, the at least one ALK inhibitor comprises at least one of a peptide, an antibody, a small molecule pharmaceutical compound, an oligo, a nucleic acid molecule, or a combination thereof.

[0028] In various aspects, the at least one ALK inhibitor comprises a small molecule pharmaceutical compound. For example, in some aspects, the at least one ALK inhibitor is selected from the group consisting of Lorlatinib, Brigatinib, Alectinib, Ceritinib, TAE684 (NVP- TAE684), AP26113 analog (ALK-IN-1), GSK1838705A, AZD3463, ASP3026, Entrectinib, and Crizotinib.

[0029] In various aspects, the targeted RTK inhibitor targets an EGFR/ERBB receptor, a platelet-derived growth factor (PDGF)/Kit receptor, a vascular endothelial growth factor receptor (VEGFR), a fibroblast growth factor (FGF) receptor, a hepatocyte growth factor (HGF receptor), a mesenchymal-epithelial transition factor receptor (MET), or an insulin or insulinlike growth factor (IGF) receptor. For example, in some aspects, the targeted RTK inhibitor is an EGFR/ERBB inhibitor. In still other aspects, the targeted RTK inhibitor is an EGFR inhibitor, a (PDGF)/Kit receptor inhibitor, an FGF receptor inhibitor, an IGF receptor inhibitor, a MET inhibitor or any combination thereof.

[0030] In various aspects, the targeted RTK inhibitor comprises least one of a peptide, an antibody, a small molecule pharmaceutical compound, an oligo, a nucleic acid molecule, or a combination thereof.

[0031] In various aspects, the targeted RTK inhibitor comprises a small molecule pharmaceutical compound or a monoclonal antibody. For example, in some aspects, the targeted RTK inhibitor can be selected from Osimertinib, Lapatinib, Avapritinib, Capmatinib, Pemigatinib, Ripretinib, Selpercatinib, Selumetinib, Tucatinib, Entrectinib, Erdafitinib, Fedratinib, Pexidartinib, Upadacitinib, Zanubrutinib, Baricitinib, Binimetinib, Dacomitinib, Fostamatinib, Gilteritinib, Larotrectinib, Acalabrutinib, Midostaurin, Neratinib, Cobimetinib, Lenvatinib, Nintedanib, Afatinib, Ibrutinib, Trametinib, Axitinib, Bosutinib, Cabozantinib, Ponatinib, Regorafenib, Tofacitinib, Ruxolitinib, Vandetanib, Pazopanib, Nilotinib, Dasatinib, Sunitinib, Sorafenib, Erlotinib, Gefitinib, Imatinib, Varlinitib Trastuzumab, Pertuzumab, Cetuximab Imatinib, Capmatinib, Linsitinib, and Infratinib. In various aspects, the targeted RTK inhibitor is selected from Osimertinib, Lapatinib, Erlotinib, Gefitinib, Afatinib, Dacomitinib, Neratinib, Varlitinib Trastuzumab, Pertuzumab, Cetuximab, Imatinib, Capmatinib, Linsitinib, and Infratinib. In still other aspects, the targeted RTK inhibitor is selected from the group consisting of Osimertinib, Lapatinib, Erlotinib, Gefitinib, Afatinib, Dacomitinib, Neratinib, Varlitinib Trastuzumab, Pertuzumab, and Cetuximab.

[0032] Further aspects of the present disclosure are directed to methods of preventing sensitization and/or resistance to an anti-cancer therapy comprising a receptor tyrosine kinase (RTK) inhibitor, the method comprising administering to a subject in need thereof, an ALK inhibitor prior to or concurrent with the anti-cancer therapy comprising the RTK inhibitor.

[0033] In various aspects, the RTK inhibitor is an EGFR inhibitor, a (PDGF)/Kit receptor inhibitor, an FGF receptor inhibitor, an IGF receptor inhibitor, a MET inhibitor or any combination thereof. For example, in some aspects the RTK inhibitor is an EGFR inhibitor.

[0034] In various aspects, the subject responds to the RTK inhibitor over a longer time period than a subject that did not receive the ALK inhibitor. [0035] In any of the foregoing or related aspects, the subject can be a human.

[0036] Further aspects of the present disclosure are directed to a method of increasing sensitivity of a cancer cell to a receptor tyrosine kinase (RTK) inhibitor comprising: contacting a cancer cell with at least one ALK inhibitor, where the cancer cell is sensitized and/or resistant to a receptor tyrosine kinase (RTK) inhibitor, with at least one ALK inhibitor increases the cell’s sensitivity to an RTK inhibitor as compared to a cell not contacted with the ALK inhibitor.

[0037] In various aspects, cell survival of the cancer cell is decreased after contacting the cancer cell with at least one ALK inhibitor. In various aspects, cell proliferation of the cancer cell is decreased after contacting the cancer cell with at least one ALK inhibitor.

[0038] In various aspects, the cancer cell can be located in or be obtained from a lung tumor, a breast tumor, a pancreatic tumor, a colorectal tumor, an esophageal tumor, a head-and- neck tumor, a hematological malignancy, a brain tumor, an ovarian tumor, a stomach tumor, a gastric tumor, a prostate tumor, a thyroid tumor, bladder tumor, a lymphoma, melanoma, a kidney tumor, a soft tissue sarcoma, chronic eosinophilic leukemia (CEL), chronic lympocytic leukemia (CLL), urothelial carcinoma, cervical tumor, a liver or bile duct tumor, endometrial tumor, or any combination thereof. In various aspects, the cancer cell can be located in or be obtained from a non-small cell lung cancer (NSCLC) tumor, a HER-2 positive breast cancer tumor, a glioblastoma, a gastrointestinal stromal tumor, an esophageal tumor, a liver or bile duct tumor, a bladder tumor or any combination thereof.

[0039] The foregoing is intended to be illustrative and is not meant in a limiting sense. Many features and subcombinations of the present disclosure may be made and will be readily evident upon a study of the following specification and accompanying drawings comprising a part thereof. These features and subcombinations may be employed without reference to other features and subcombinations.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Aspects of the present disclosure are illustrated by way of example in which like reference numerals indicate similar elements and in which:

[0041] FIG. 1A is a representative heatmap indicating proteins that bind to EGFR in cells after osimertinib treatment.

[0042] FIG. 1 B-1 D are representative immunoblots showing association of SUMO3 and EGFR in HCC827 cells (FIG. 1 B), PC9 cells (FIG. 1C), and H3255 cells (FIG. 1 D) after treatment with Osimertinib and immunoprecipitation with EGFR antibody.

[0043] FIG. 1 E-1F are representative immunoblots showing association of SUMO3 and EGFR in HCC827 xenograft tumor tissue (FIG. 1 E) and HCC4190 PDX xenograft tumor tissue (FIG. 1 F) after treatment with Osimertinib and immunoprecipitation with EGFR antibody. [0044] FIG. 1G-1I are representative immunoblots showing lack of association of SLIMO3 and EGFR in HCC827 cells (FIG. 1G), PC9 cells (FIG. 1 H) and H3255 cells (FIG. 11) that were transfected with SLIMO3 siRNA, treated with osimertinib, and immunoprecipitated with EGFR antibody.

[0045] FIG. 1 J-1K are representative immunoblots showing lack of association of SLIMO3 and EGFR in HCC827 cells (FIG. 1 J) and PC9 cells (FIG. 1 K) that were transfected with Flag- SENP2 and then treated with 100 nM and 50 nM Osimertinib, respectively, followed by immunoprecipitation with EGFR antibody.

[0046] FIG. 1L-1 M are representative immunoblots of BT474 cells (FIG. 1 L) and OE19 cells (FIG. 1M) following 1 pM lapatinib treatment and immunoprecipitation with HER2 antibody.

[0047] FIG. 1 N-1O are representative immunoblots of H1703 cells (FIG. 1 N) and H661 cells (FIG. 10) following 1 pM Imatinib treatment and immunoprecipitation with PDGFRa antibody.

[0048] FIG. 1P-1Q are representative immunoblots of H1993 cells (FIG. 1 P) and EBC-1 cells (FIG. 1Q) following 1 pM capmatinib treatment and immunoprecipitation with MET antibody.

[0049] FIG. 1R-1S are representative immunoblots of HepG2 cells (FIG. 1 R) and Hep3B cells (FIG. 1S) following 1 pM linsitinib treatment and immunoprecipitation with IGF1-R antibody.

[0050] FIG. 1T-1U are representative immunoblots of RT112 cells (FIG. 1T) and RT4 cells (FIG. 11I) following 1 pM infigratinib treatment and immunoprecipitation with FGFR3 antibody. [0051] FIG. 2A-2C are representative bar graphs indicating fold-change in TNF mRNA transcript levels as measured by qPCR in HCC827 cells (FIG. 2A), PC9 cells (FIG. 2B), and H3255 cells (FIG. 2C) that were transfected with control or SLIMO3 siRNA for 48 hours followed by osimertinib treatment for 48 hours. Insets show illustrative immunoblot of SLIMO3 expression in siRNA treated cells.

[0052] FIG. 2D-2F are representative plots indicating fold-change in TNF protein levels in supernatant as measured by ELISA in HCC827 cells (FIG. 2D), PC9 cells (FIG. 2E), and H3255 cells (FIG. 2F) that were transfected with control or SLIMO3 siRNA for 48 hours followed by osimertinib treatment for 48 hours. Insets show illustrative immunoblot of SLIMO3 expression in siRNA treated cells.

[0053] FIG. 2G-2I are representative plots indicating relative NF-KB activity as measured by dual-luciferase assay in HCC827 cells (FIG. 2G), PC9 cells (FIG. 2H), and H3255 cells (FIG. 2I) that were transfected with control or SLIMO3 siRNA for 48 hours followed by osimertinib treatment for 48 hours. Insets show illustrative immunoblot of SLIMO3 expression in siRNA treated cells. [0054] FIG. 2J-2K are representative bar graphs indicating fold-change in TNF mRNA transcript levels as measured by qPCR in HCC827 cells (FIG. 2J) and PC9 cells (FIG. 2K) that were transfected with control or EGFR siRNA for 48 hours followed by osimertinib treatment for 48 hours. Insets show illustrative immunoblot of EGFR expression in siRNA treated cells.

[0055] FIG. 2L-2M are representative plots indicating fold-change in TNF protein levels in supernatant as measured by ELISA in HCC827 cells (FIG. 2L) and PC9 cells (FIG. 2M), that were transfected with control or EGFR siRNA for 48 hours followed by osimertinib treatment for 48 hours. Insets show illustrative immunoblot of EGFR expression in siRNA treated cells. [0056] FIG. 2N-2O are representative plots indicating relative NF-KB activity as measured by dual-luciferase assay in HCC827 cells (FIG. 2N) and PC9 cells (FIG. 20) that were transfected with control or EGFR siRNA for 48 hours followed by osimertinib treatment for 48 hours. Insets show illustrative immunoblot of EGFR expression in siRNA treated cells.

[0057] FIG. 3A-3B are representative immunoblots of HCC827 cells (FIG. 3A) and PC9 cells (FIG. 3B) following osimertinib treatment and immunoprecipitation with TRIM28 antibody. [0058] FIG. 3C-3D are representative immunoblots of HCC827 cells (FIG. 3C) and PC9 cells (FIG. 3D) that were transfected with control or TRIM28 siRNA followed by osimertinib treatment and immunoprecipitation with EGFR antibody.

[0059] FIG. 3E-3F are representative immunoblots of HCC827 cells (FIG. 3E) and PC9 cells (FIG. 3F) that were transfected with control or LIBC9 siRNA followed by osimertinib treatment and immunoprecipitation with EGFR antibody.

[0060] FIG. 4A-4B are representative immunoblots showing EGFR expression in H661 cells (FIG. 4A) and H1975_EGFRKO cells (FIG. 4B) stably transfected with vectors encoding Del, Del_K37R, L858R, L858_K37R mutant EGFRs.

[0061] FIG. 4C-4D are representative immunoblots showing EGFR activity (pEGFR levels) in Del and Del_K37R expressing H661 cells (FIG. 4G) and Del and Del_K37R expressing EGFRKO H1975 cells (FIG. 4D) following treatment with EGF for 5 min.

[0062] FIG. 4E-4F are representative immunoblots of Del and Del_K37R expressing H661 cells (FIG. 4E) and Del and Del_K37R expressing EGFRKO H1975 cells (FIG. 4F) following Osimertinib treatment and immunoprecipitation with EGFR antibody.

[0063] FIG 4G is a representative bar graph indicating fold-change in TNF mRNA transcript levels as measured by qPCR in Del and Del_K37R expressing H661 cells following Osimertinib treatment.

[0064] FIG. 4H is a representative plot indicating relative NF-KB activity as measured by dual-luciferase assay in Del and Del_K37R expressing H661 cells following Osimertinib treatment. [0065] FIG. 41 is a representative bar graph indicating fold-change in TNF mRNA transcript levels as measured by qPCR in Del and Del_K37R expressing EGFRKO H1975 cells following Osimertinib treatment.

[0066] FIG. 4J is a representative plot indicating relative NF-KB activity as measured by dual-luciferase assay in Del and Del_K37R expressing EGFRKO H1975 cells following Osimertinib treatment.

[0067] FIG. 4K-4L are representative immunoblots of HCC827 cells (FIG. 4K) and PC9 cells (FIG. 4L) that were transfected with HA-Del and HA-Del-K37R, followed by osimertinib treatment and immunoprecipitation with HA antibody.

[0068] FIG. 5A-5B are representative immunoblots of HCC827 cells (FIG. 5A) and PC9 cells (FIG. 5B) following osimertinib treatment and immunoprecipitation with TFG antibody.

[0069] FIG. 5C-5D are representative immunoblots of HCC827 cells (FIG. 5C) and PC9 cells (FIG. 5D) following osimertinib treatment and immunoprecipitation with EML4 antibody.

[0070] FIG. 5E-5F are representative immunoblots of HCC827 cells (FIG. 5E) and PC9 cells (FIG. 5F) following osimertinib treatment and immunoprecipitation with TPM4 antibody.

[0071] FIG. 5G-5H are representative immunoblots showing levels of activated ALK (p- ALK), activated MET (p-MET), and activated HERT2 (p-HER2) in HCC827 cells (FIG. 5G) and PC9 cells (FIG. 5H) following Osimertinib treatment.

[0072] FIG. 5I is a representative immunoblot showing p-ALK levels in HCC4190 PDX xenograft tumor tissue collected 1 or 30 days after in vivo treatment with Osimertinib.

[0073] FIG. 5J-5K are representative immunoblots showing pALK levels in HCC827 cells (FIG. 5J) and PC9 cells (FIG. 5K) that were transfected with control or SLIMO3 siRNA followed by osimertinib treatment.

[0074] FIG. 5L-5N are representative immunoblots of Del and Del_K37R expressing H661 cells that were treated with Osimertinib and immunoprecipitated with TFG antibody (FIG. 5L), EML4 antibody (FIG. 5M), or TPM4 antibody (FIG. 5N).

[0075] FIG. 50 is a representative immunoblot of Del and Del_K37R expressing H661 cells following treatment with 100 nM Osimertinib.

[0076] FIG. 5P-5R are representative immunoblots of Del and Del_K37R expressing H1975_EGFRKO cells that were treated with Osimertinib and immunoprecipitated with TFG antibody (FIG. 5P), EML4 antibody (FIG. 5Q), or TPM4 antibody (FIG. 5R).

[0077] FIG. 5S is a representative immunoblot of Del and Del_K37R expressing H1975_EGFRKO cells following treatment with 100 nM Osimertinib.

[0078] FIG. 5T-5V are representative immunoblots showing p-ALK levels after Osimertinib treatment in HCC827 cells transfected with control siRNA or TFG siRNA (FIG. 5T), control siRNA or EML4 siRNA (FIG. 5U), and control siRNA or TPM4 siRNA (FIG. 5V). [0079] FIG. 5W-5Y are representative immunoblots showing p-ALK levels after Osimertinib treatment in PC9 cells transfected with control siRNA or TFG siRNA (FIG. 5W), control siRNA or EML4 siRNA (FIG. 5X), and control siRNA or TPM4 siRNA (FIG. 5Y).

[0080] FIG. 6A-6B are representative immunoblots showing activated ALK (p-ALK) levels after Lapatinib treatment in BT474 cells (FIG. 6A) and OE19 cells (FIG. 6B) that were transfected with control or SLIMO3 siRNA.

[0081] FIG. 6C-6D are representative immunoblots showing activated ALK (p-ALK) levels after Imatinib treatment in H1703 cells (FIG. 6C) and H661 cells (FIG. 6D) that were transfected with control or SUMO3 siRNA.

[0082] FIG. 6E-6F are representative immunoblots showing activated ALK (p-ALK) levels after Capmatinib treatment in H1993 cells (FIG. 6E) and EBC-1 cells (FIG. 6F) that were transfected with control or SUMO3 siRNA.

[0083] FIG. 6G-6H are representative immunoblots showing activated ALK (p-ALK) levels after Infigratinib treatment in RT112 cells (FIG. 6G) and RT4 cells (FIG. 6H) that were transfected with control or SUMO3 siRNA.

[0084] FIG. 6I-6J are representative immunoblots showing activated ALK (p-ALK) levels after Linsitinib treatment in HepG2 cells (FIG. 6I) and Hep3B cells (FIG. 6J) that were transfected with control or SUMO3 siRNA.

[0085] FIG. 6K-6L are representative immunoblots of BT474 cells that were treated with Lapatinib and immunoprecipitated with TFG antibody (FIG. 6K) or TPM4 antibody (FIG. 6L).

[0086] FIG. 6M-6N are representative immunoblots of EBC-1 cells that were treated with Capmatinib and immunoprecipitated with TFG antibody (FIG. 6M) or TPM4 antibody (FIG. 6N).

[0087] FIG. 7A is a representative immunoblot of HCC827 (parental) and HCC827 erlotinib-resistant (ER3, ER4A and ER4B) cells following immunoprecipitation with EGFR antibody.

[0088] FIG. 7B is a representative immunoblot of H1975 (parental) and H1975 osimertinib-resistant (OR5 and OR16) cells following immunoprecipitation with EGFR antibody.

[0089] FIG. 7C is a representative immunoblot of HCC827 (parental) and HCC827 erlotinib-resistant cells (ER3, ER4A and ER4B) following immunoprecipitation with ALK antibody.

[0090] FIG. 7D is a representative immunoblot of H1975 (parental) and H1975 osimertinib-resistant cells (OR5 and OR16) following immunoprecipitation with ALK antibody. [0091] FIG. 7E is a representative immunoblot of HCC4190 PDX (Parental) and HSCC4190 osimertinib-resistant PDX tumor tissue. [0092] FIG. 7F-7G are representative plots showing cell viability (via AlamarBlue) following Osimertinib treatment of H1975 osimertinib-resistant OR5 cells (FIG. 7F) and H1975 osimertinib-resistant OR16 cells (FIG. 7G) transfected with control and SLIMO3 siRNA.

[0093] FIG. 8A-8B are representative plots showing cell viability after Osimertinib treatment as measured by AlamarBlue assay of HCC827 cells (FIG. 8A) and PC9 cells (FIG. 8B) transfected with control or SLIMO3 siRNA. Insets are representative immunoblots of SLIMO3 expression in siRNA treated cells.

[0094] FIG. 8C-8D are representative plots showing cell viability after Osimertinib treatment as measured by AlamarBlue assay of H661 cells (FIG. 8C) and H1975_EGFR_KO cells (FIG. 8D) stably expressing Del or Del_K37R EGFR.

[0095] FIG. 8E-8F are representative plots showing cell viability after Osimertinib treatment as measured by AlamarBlue assay of H661 cells (FIG. 8E) and H1975_EGFR_KO cells (FIG. 8F) stably expressing L858R and L858R_K37R EGFR.

[0096] FIG. 8G depicts a summary plot and images showing tumor growth in athymic mice injected with EGFR_del or EGFR_del K37R H1975_EGFR KO cells with or without Osimertinib treatment.

[0097] FIG. 8H depicts representative plots and images showing tumor growth in athymic mice injected with EGFR_del or EGFR_del K37R H661 cells with or without Osimertinib treatment.

[0098] FIG. 8I depicts representative plots and images showing tumor growth in athymic mice injected with HCC4190 PDX xenograft cells after treatment with or without Osimertinib and Alectinib, alone or in combination.

[0099] FIG. 8J depicts representative plots and images showing tumor growth in athymic mice injected with PC9 xenograft cells after treatment with or without Osimertinib and Alectinib, alone or in combination.

[0100] FIG. 8K-8L depict representative plots and images showing tumor growth in the presence or absence of Osimertinib in athymic mice subcutaneously injected with HCC827 xenograft cells (FIG. 8K) or PC9 xenograft cells (FIG. 8L) stably transfected with lentiviral control shRNA or ALK shRNA (shALK) and showing stable silencing of ALK (insets).

[0101] FIG. 8M depicts a representative plot and images showing tumor growth in NOD/SCID mice subcutaneously injected with HCC4190 EGFR_L858R Osimertinib resistant PDX cells and treated with Osimertinib and/or Alectinib.

[0102] FIG. 9A-9B are representative images of PLA analysis of EGFR and SLIMO3 in HCC827 cells (FIG. 9A) or paraffin-embedded HCC827 tumor slides (FIG. 9B).

[0103] FIG. 9C-9D depict representative images of PLA analysis of EGFR and SLIMO3 in paraffin-embedded human tumor slides from three patients that are EGFR-TKI naive (FIG. 9C) or treated with EGFR- TKI (FIG. 9D). [0104] FIG. 9E depict representative PLA analysis of pre and post-TKI treated tissue from the same patient.

[0105] FIG. 9F is a plot quantifying PLA positive staining in TKI naive and TKI treated tissue from 27 patients.

[0106] FIG. 9G-9H are representative images showing p-ALK immunostaining (FIG. 9G) and quantification of p-ALK positive cells (FIG. 9H) in HCC827 xenograft tumor with or without Osimertinib treatment.

[0107] FIG. 9I-9J are representative images showing p-ALK immunostaining (FIG. 9I) and quantification of p-ALK positive cells (FIG. 9J) in HCC4190 xenograft tumor with or without Osimertinib treatment.

[0108] FIG. 9K is a representative immunohistochemistry (IHC) image for p-ALK in a representative TKI naive and TKI treated resected tissue from patients.

[0109] FIG. 9L is a representative immunohistochemistry (IHC) image for p-ALK in a representative TKI naive and TKI treated resected tissue from the same patient.

[0110] FIG. 10A-10B are representative immunoblots showing lack of association of SUMO1 and EGFR upon osimertinib treatment in HCC827 cells (FIG.10A) and PC9 cells (FIG. 10B) assessed by immunoprecipitation with an EGFR antibody.

[0111] FIG. 10C-10D are representative immunoblots showing lack of association of SUMO2 and EGFR upon osimertinib treatment in HCC827 cells (FIG.10C) and PC9 cells (FIG. 10D) assessed by immunoprecipitation with an EGFR antibody.

[0112] FIG. 11A-11C are representative immunoblots showing EGFR expression in HCC827 cells (FIG. 11A), PC9 cells (FIG. 11 B) and H3255 cells (FIG. 11C) after treatment with Osimertinib.

[0113] FIG. 11D-11 E are representative immunoblots showing EGFR expression in HCC827 cells (FIG. 11 D) and PC9 cells (FIG. 11 E) after treatment with Osimertinib and actinomycin D.

[0114] FIG. 12A-12B are representative immunoblots showing activated EGFR (p-EGFR) and ERK (pERK) in H661 cells (FIG. 12A) or H1975_EGFRKO cells (FIG. 12B) stably expressing L858R or L858R_K37R_EGFR and after exposure to a vehicle (Mock) or EGF.

[0115] FIG. 12C-12D are representative immunoblots showing association or lack of association between SUMO3 and EGFR in H661 cells (FIG. 12C) or H1975_EGFRKO cells (FIG. 12D) stably expressing L858R or L858R_K37R EGFR after treatment with Osimertinib and immunoprecipitation with an EGFR antibody.

[0116] FIG. 12E-12F are representative plots showing fold change in TNF mRNA levels as measured by qPCR (FIG. 12E) or relative NF-KB activity as measured by a dual-luciferase screen (FIG. 12F) in H661 cells stably expressing L858R_EGFR or L858R_K37R_EGFR. [0117] FIG. 12G-12H are representative plots showing fold change in TNF mRNA levels as measured by qPCR (FIG. 12G) or relative NF-KB activity as measured by a dual-luciferase screen (FIG. 12H) in H1975_EGFRKO cells stably expressing L858R_EGFR or L858R_K37R_EGFR.

[0118] FIG. 13A-13B are representative immunoblots showing protein levels in the cytosol, membrane or nucleus of HCC827 cells (FIG. 13A) or PC9 cells (FIG. 13B) after treatment with Osimertinib.

[0119] FIG. 14A-14B are representative immunoblots showing ALK expression in HCC827 cells (FIG. 14A) and PC9 cells (FIG. 14B) after transfection with control (siCtr) or ALK siRNA (siALK_1 and siALK_2).

[0120] FIG. 14C-14D are representative immunoblots showing ALK activation (p-ALK) and EGFR activation (p-EGFR) in HCC827 cells (FIG. 14C) and PC9 cells (FIG. 14D) treated with Osimertinib or siRNA to EGFR.

[0121] FIG. 15 depicts representative images of immunohistochemistry (IHC) for p-ALK from another patient with or without TKI treatment.

[0122] FIG. 16A-16B are representative plots indicating relative NF-KB activity as measured by dual-luciferase assay at baseline in parent HCC827 or HCC827 erlatanib resistant clones (FIG. 16A) or in parent H1975 and H1975 osimertinib resistant clones (FIG. 16B).

[0123] FIG. 17A-17B depict representative cell viability plots as measured by AlamarBlue assay of HCC827 cells (FIG. 17A) or PC9 cells (FIG. 17B) treated with vehicle, osimertinib, lorlatinib, or lorlatanib and osimertinib for 72 hours.

[0124] FIG. 17C-17D depict representative cell viability plots as measured by AlamarBlue of HCC827 cells (FIG. 17C) or PC9 cells (FIG. 17D) treated with siRNA to silence ALK expression (siALK) and then treated with osimertinib or vehicle for 72 hours.

[0125] FIG. 17E depicts a representative plot tracking tumor size in NOD/SCID mice implanted with HCC4190 EGFR+L858R NSCLC PDX cells and treated with 5 mg/kg Osimertinib, 10 mg/kg of Lorlatinib or a combination thereof.

[0126] The drawing figures do not limit the present disclosure to the specific aspects disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain aspects of the present disclosure.

DETAILED DESCRIPTION

[0127] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects and aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0128] The instant disclosure is based, in part, on the surprising discovery that RTKs do not become inert after TKI treatment. Instead, RTKs undergo a rapid SUMOylation following tyrosine kinase inhibition which causes the kinase inhibited RTK to signal in an ALK-mediated adaptor platform, ultimately triggering activation of key survival signals that promote resistance. Surprisingly, it has been shown that ALK inhibitors can reverse this effect and restore sensitivity to RTKs, even in patient populations that would not normally be treated with ALK inhibitors.

[0129] Accordingly, provided herein are compositions and methods for treating cancer and, in some aspects, a malignant tumor. In some aspects, the compositions herein comprise an RTK inhibitor. In some aspects, the compositions herein comprise an ALK inhibitor. In various aspects, the methods herein comprise administering the ALK inhibitor to a subject in need thereof concurrently with or after a treatment with the RTK inhibitor (RTKi). In various aspects, the methods herein ultimately prevent treatment resistance and improve responsiveness of a cancer to the RTKi treatment.

I. Terminology

[0130] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred aspects and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0131] As used in the specification, articles “a” and “an” are used herein to refer to one or to more than one (i.e. , at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0132] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The term “about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.

[0133] Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.

[0134] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0135] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0136] Moreover, the present disclosure also contemplates that in some aspects, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0137] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise- Indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1 % to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0138] As used herein, "treatment," "therapy" and/or "therapy regimen" refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.

[0139] As used herein, “prevent” or “prevention” refers to eliminating or delaying the onset of a particular disease, disorder or physiological condition, or to the reduction of the degree of severity of a particular disease, disorder or physiological condition, relative to the time and/or degree of onset or severity in the absence of intervention.

[0140] The term “effective amount" or "therapeutically effective amount" refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.

[0141] As used herein, “individual”, “subject”, “host”, and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis or therapy is desired, for example, humans, pets, livestock, horses or other animals. As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In some aspects, the subject can be a human. In other aspects, the subject can be a human in need of treating a cancer (e.g., a melanoma).

[0142] As used interchangeably herein, “treatment of cancer” and “cancer treatment” encompass any of increased inhibition of cancer progression and/or metastases, inhibition of an increase in tumor volume, reduction in tumor volume and/or growth, reduction in tumor growth rate, eradication or killing of a tumor and/or cancer cell, or any combination thereof. In some aspects, the treatment can also prolong the survival of a subject, improve the prognosis and/or improve the quality of life of the subject.

II. Compositions

[0143] In certain aspects, compositions disclosed herein may comprise at least one receptor tyrosine kinase inhibitor. In certain aspects, compositions herein comprise at least one ALK inhibitor. In yet another aspect, compositions disclosed herein may comprise a combination of at least one RTK inhibitor and at least one ALK inhibitor, wherein the RTK inhibitor does not also target or inhibit ALK. In further aspects, compositions disclosed herein may be pharmaceutical compositions. In various aspects, compositions as disclosed herein exclude a SUMOylation inhibitor.

(a) Receptor Tyrosine Kinases (RTK) Inhibitors

[0144] Receptor tyrosine kinases encompass a large family of receptors that are localized to a cell’s plasma membrane, dimerize when bound to a ligand, and have an active tyrosine kinase intracellular domain. Many tumors are characterized by disrupted RTK signaling and RTK inhibition is often a first line targeted therapy in many cancers. In various aspects, the methods herein comprise administering an RTK inhibitor.

[0145] In certain aspects, compositions disclosed herein may include at least one RTK inhibitor. As used herein, an “RTK inhibitor” (RTKi) can include any biomolecule(s) that can inhibit RTK direct activity, inhibit RTK indirect activity, inhibit formation of an RTK receptor dimer, decrease expression of a RTK gene, decrease expression of a RTK protein, or a combination thereof. In some apects, compositions having a RTK inhibitor can include any biomolecule(s) that are modulators and/or inhibitors of targets upstream or downstream of a RTK signaling cascade that would effectively inhibit the physiological outcome of RTK inhibition. In another aspect, biomolecule(s) capable of inhibiting one or more RTKs can be a peptide, an antibody, a chemical, a compound, an oligo, a nucleic acid molecule, or a combination thereof.

[0146] In various aspects, the RTK inhibitor herein may inhibit epidermal growth factor receptor (EGFR), a platelet-derived growth factor (PDGF)/Kit receptor, a vascular endothelial growth factor receptor (VEGFR), a fibroblast growth factor (FGF) receptor, a hepatocyte growth factor (HGF receptor), a mesenchymal-epithelial transition factor receptor (MET), an insulin or insulin-like growth factor (IGF) receptor, or a combination of any thereof. For example, in some aspects, the RTK inhibitor may be an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, or a pan-HER inhibitor.

[0147] In certain aspects, RTK inhibitors for use in compositions herein can include a nucleic acid molecule. The term “nucleic acid molecule” as used herein refers to a molecule having nucleotides. The nucleic acid can be single, double, or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. In some aspects, a nucleic acid molecule for use herein can be a double-stranded RNA. In some other aspects a double stranded RNA suitable for use herein can be small temporal RNA, small nuclear RNA, small nucleolar RNA, short hairpin RNA, microRNA, or the like.

[0148] In certain aspects, RTK inhibitors for use in compositions herein may be a polypeptide, a protein, a peptide, a protein fragment, an antibody, or any combination thereof. As used herein, the terms “peptide,” “polypeptide,” and “protein,” refer to polymers comprised of amino acid monomers linked by amide bonds. Peptides may include the standard 20 a- amino acids that are used in protein synthesis by cells (i.e., natural amino acids), as well as non-natural amino acids (non-natural amino acids may be found in nature, but not used in protein synthesis by cells, e.g., ornithine, citrulline, and sarcosine, or may be chemically synthesized), amino acid analogs, and peptidomimetics. In some aspects, a peptide inhibitor of a receptor tyrosine kinase may be fused at its C-terminus, its N-terminus, or both with at least one other peptide and/or polypeptide. In some aspects, the at least one other peptide or polypeptide may be a carrier peptide, allowing cell penetration of the resulting fusion peptide. As an example, a peptide inhibitor of RTK may be fused to a cell penetrating peptide (GPP). GPPs are short peptides that facilitate cellular uptake of various molecular cargo, e.g. via endocytosis. Non-limiting examples of GPP that may be suitable for use herein include Antennapedia Penetratin, HIV-1 TAT protein, pVEC Cadherin, Transportan Galanine/Mastoparan, MPG HIV-gp41/SV40 T-antigen, Pep-1 HIV-reverse transcriptase/SV40 T-antigen, Polyarginines, MAP, R6W3, NLS, 8-lysines, ARF (1-22), Azurin-p28, and the like. As another example, a peptide inhibitor of RTK may be fused to celltargeting peptides (CTPs). CTPs are ideal carrier molecules as that bind with high affinity to overexpressed receptors on the tumor cell surface, effectively targeting a peptide inhibitor of RTK to the target tumor. CTPs can target, for example integrin receptors, epidermal growth factor receptors (EGFR), neuropeptide Y (NPY) receptors, gastrin-releasing peptide receptors (GRPR), somatostatin receptors (e.g., SSTR2), gonadotropin-releasing hormone receptors (GnRHR), vasoactive intestinal peptide (VIP) receptors, melanocortin 1 receptors (MC1 R), neurotensin receptors (e.g., NTSR1), and the like. In certain aspects, the CTP can bind to the target RTK, so that the RTK inhibiting peptide is near the receptor.

[0149] In certain aspects, RTK inhibitors for use in compositions herein may be an RTK antibody. As used herein, the terms “RTK antibody” can refer to an antibody capable of binding to and/or blocking activity of a receptor tyrosine kinase (RTK). In some aspects, blocking activity may comprise blocking dimerization and/or kinase activity of the receptor. Blocking activity may also comprise blocking association of various target proteins (those that are usually substrates of the RTK) from binding to or associating with the RTK. In some aspects, antibodies acting as an inhibitor of RTK may be full-length antibodies, antigen binding fragments of full-length antibodies, Fab fragments, single chain antibodies (scFv), diabodies, triabodies, minibodies, nanobodies, single-domain antibodies, camelids, or any combination thereof. In some aspects, septin antibodies disclosed herein may be of a suitable origin, for example, murine, rat, or human. Such antibodies are non-naturally occurring, i.e. , would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof or isolated from antibody libraries). Any of the antibodies described herein, e.g., RTK antibodies, can be either monoclonal or polyclonal. A “monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made. Exemplary RTK inhibitors that are antibodies comprise Trastuzumab, Pertuzumab, Amivantamab, Bevacizumab, Margetuximab, Necitumumab, Ramucirumab, Panitumumab, and Cetuximab.

[0150] In certain aspects, RTK inhibitors for use in compositions herein may be a compound (also referred to herein as “small molecule”). In some aspects, a RTK inhibitor compound may comprise Osimertinib, Imatinib, Capmatinib, Linsitinib, Infigratinib, Avapritinib, Pemigatinib, Ripretinib, Selpercatinib, Tucatinib, Entrectinib, Erdafitinib, Pexidartinib, Dacomitinib, Gilteritinib, Acalabrutinib, Midostaurin, Neratinib, Cobimetinib, Lenvatinib, Nintedanib, Afatinib, Trametinib, Axitinib, Cabozantinib, Ponatinib, Regorafenib, Tofacitinib, Vandetanib, Pazopanib, Lapatinib, Nilotinib, Dasatinib, Sunitinib, Sorafenib, Erlotinib, Gefitinib, Imatinib, Varlinitib, Infigratinib, Mobocertinib, Pralsetinib, Tepotinib, Tivozanib, Aflibercept, Larotrectinib, any analog thereof, or any combination thereof.

[0151] In any of the above and foregoing examples, the RTK inhibitor may comprise Osimertinib, Imatinib, Capmatinib, Linsitinib, Infigratinib, Erlotinib, Gefitinib, Afatinib, Dacomitinib, Lapatinib, Neratinib, Varlitinib, Tucatinib, Trastuzumab, Pertuzumab, Amivantamab, Bevacizumab, Margetuximab, Necitumumab, Ramucirumab, Panitumumab, and Cetuximab.

Targeted Cancer Therapy Compositions comprising RTK Inhibitors

[0152] In various aspects, compositions provided herein comprise a targeted RTK inhibitor designed to treat a certain cancer. In many aspects, the RTK inhibitor is a standard line of treatment for the given cancer. In further aspects, the RTK inhibitor may be used for treatment of a cancer selected from: bladder cancer, breast cancer, cervical cancer, colorectal cancer, dermatofibrosarcoma protuberans, endometrial cancer, esophageal cancer, head and neck cancer, gastrointestinal stromal tumor, giant cell tumor, kidney cancer, leukemia, liver and bile duct cancer, lung cancer, lymphoma, multiple myeloma, myelodysplastic/myeloproliferative disorders, ovarian epithelial/fallopian tube/primary peritoneal cancers, pancreatic cancer, soft tissue sarcoma, solid tumors with an NTRK gene fusion, stomach (gastric) cancer, systemic mastocytosis, and thyroid cancer. In various aspects, the RTK inhibitor is f used or treatment of a lung cancer such as non-small cell lung carcinoma.

[0153] Exemplary RTK inhibitors approved for various cancers listed above are provided below. It is noted that this list is non-exhaustive and other RTK inhibitors may be contemplated. [0154] In some aspects, the RTK inhibitor may be used for treating bladder cancer and may comprise erdafitinib (Balversa).

[0155] In some aspects, the RTK inhibitor may be used for treating bladder cancer and may comprise Bevacizumab (Avastin®).

[0156] In some aspects, the RTK inhibitor may be used for treating breast cancer and be selected from trastuzumab (Herceptin®), lapatinib (Tykerb®), pertuzumab (Perjeta®), ado- trastuzumab emtansine (Kadcyla®), neratinib maleate (Nerlynx®), fam-trastuzumab deruxtecan-nxki (Enhertu®), tucatinib (Tukysa®), and margetuximab-cmkb (Margenza®).

[0157] In some aspects, the RTK inhibitor may be used for treating cervical cancer and comprises bevacizumab (Avastin®).

[0158] In some aspects, the RTK inhibitor may be used for treating colorectal cancer and be selected from Cetuximab (Erbitux®), panitumumab (Vectibix®), bevacizumab (Avastin®), ziv-aflibercept (Zaltrap®), regorafenib (Stivarga®), and ramucirumab (Cyramza®). In some aspects, the RTK inhibitor may be approved for treating colorectal cancer and be selected from Cetuximab (Erbitux®), panitumumab (Vectibix®), bevacizumab (Avastin®), and ramucirumab (Cyramza®).

[0159] In some aspects, the RTK inhibitor may be used for treating dermatofibrosarcoma protuberans and comprise imatinib mesylate (Gleevec®).

[0160] In still further aspects, the RTK inhibitor may be used for treating endometrial cancer and comprise lenvatinib mesylate (Lenvima®). [0161] In some aspects, the RTK inhibitor may be used for treating esophageal cancer: and be selected from Trastuzumab (Herceptin), ramucirumab (Cyramza®), fam-trastuzumab deruxtecan-nxki (Enhertu®).

[0162] In some aspects, the RTK inhibitor may be used for treating head and neck cancer and comprise Cetuximab (Erbitux®).

[0163] In some aspects, the RTK inhibitor may be used for treating gastrointestinal stromal tumor and be selected from imatinib mesylate (Gleevec®), sunitinib (Sutent®), regorafenib (Stivarga®), avapritinib (Ayvakit), and ripretinib (Qinlock®).

[0164] In some aspects, the RTK inhibitor may be used for treating giant cell tumor and comprise Pexidartinib (Turalio®).

[0165] In some aspects, the RTK inhibitor may be used for treating kidney cancer and is selected from bevacizumab (Avastin®), sorafenib (Nexavar®), sunitinib (Sutent), pazopanib (Votrient®), axitinib (Inlyta®), cabozantinib (Cabometyx®), lenvatinib mesylate (Lenvima®), and tivozanib hydrochloride (Fotivda®). In some aspects, the RTK inhibitor may be used for treating kidney cancer and comprise Bevacizumab (Avastin®).

[0166] In some aspects, the RTK inhibitor may be used for treating leukemia and be selected from imatinib mesylate (Gleevec®), dasatinib (Sprycel®), midostaurin (Rydapt®), rituximab and hyaluronidase human (Rituxan Hycela®), gilteritinib (Xospata®), avapritinib (Ayvakit®).

[0167] In still further aspects, the RTK inhibitor may be used for treating liver and bile duct cancer and be selected from sorafenib (Nexavar®), regorafenib (Stivarga®), lenvatinib mesylate (Lenvima®), cabozantinib (Cabometyx®), ramucirumab (Cyramza®), pemigatinib (Pemazyre®), bevacizumab (Avastin®), and infigratinib phosphate (Truseltiq®). In still further aspects, the RTK inhibitor may be used for treating liver and bile duct cancer and be selected from ramucirumab (Cyramza®), and bevacizumab (Avastin®).

[0168] In still further aspects, the RTK inhibitor may be used for treating lung cancer and be selected from afatinib dimaleate (Gilotrif®), Nintedanib (Ofev®), Varlinitib (ASLAN001), Bevacizumab (Avastin®), erlotinib (Tarceva®), gefitinib (Iressa®), ramucirumab (Cyramza®), osimertinib (Tagrisso®), necitumumab (Portrazza®), dacomitinib (Vizimpro®), selpercatinib (Retevmo®), pralsetinib (Gavreto®), tepotinib hydrochloride (Tepmetko®), amivantamab-vmjw (Rybrevant®), and mobocertinib succinate (Exkivity®). For example, in some aspects the RTK inhibitor may be used for treating lung cancer and be selected from afatinib dimaleate (Gilotrif®), Nintedanib (Ofev®), Varlinitib (ASLAN001), Bevacizumab (Avastin), erlotinib (Tarceva®), gefitinib (Iressa), ramucirumab (Cyramza®), osimertinib (Tagrisso®), necitumumab (Portrazza®), dacomitinib (Vizimpro®), amivantamab-vmjw (Rybrevant®), and mobocertinib succinate (Exkivity®). In some aspects, the RTK inhibitor may be used for treating lung cancer and comprise osimertinib (Tagrisso®). [0169] In still further aspects, the RTK inhibitor may be used for treating lymphoma and comprise rituximab and hyaluronidase human (Rituxan Hycela®).

[0170] In still further aspects, the RTK inhibitor may be used for treating multiple myeloma and comprise daratumumab and hyaluronidase-fihj (Darzalex Faspro®).

[0171] In still further aspects, the RTK inhibitor may be used for treating myelodysplastic/myeloproliferative disorders and comprise imatinib mesylate (Gleevec®).

[0172] In still further aspects, the RTK inhibitor may be used for treating ovarian epithelial/fallopian tube/primary peritoneal cancers and comprise bevacizumab (Avastin®).

[0173] In still further aspects, the RTK inhibitor may be used for treating pancreatic cancer and be selected from erlotinib (Tarceva®) and sunitinib (Sutent®). For example, the RTK inhibitor may be approved for treating pancreatic cancer and comprise erlotinib (Tarceva®).

[0174] In still further aspects, the RTK inhibitor may be used for treating soft tissue sarcoma and comprise pazopanib (Votrient®).

[0175] In still further aspects, the RTK inhibitor may be used for treating solid tumors with an NTRK gene fusion and comprise larotrectinib sulfate (Vitrakvi®).

[0176] In still further aspects, the RTK inhibitor may be used for treating stomach (gastric) cancer and be selected from trastuzumab (Herceptin), ramucirumab (Cyramza®), and famtrastuzumab deruxtecan-nxki (Enhertu®).

[0177] In still further aspects, the RTK inhibitor may be used for treating systemic mastocytosis and be selected from imatinib mesylate (Gleevec®), midostaurin (Rydapt®), and avapritinib (Ayvakit®).

[0178] In still further aspects, the RTK inhibitor may be used for treating thyroid cancer and be selected from cabozantinib (Cometriq®), vandetanib (Caprelsa®), sorafenib (Nexavar®), lenvatinib mesylate (Lenvima®), selpercatinib (Retevmo®), and pralsetinib (Gavreto®).

[0179] In certain aspects, inhibitors of RTK(s) disclosed herein can be used to treat, attenuate, or prevent tumor growth and/or progression. In certain aspects, inhibitors of RTK(s) disclosed herein can be used to treat, attenuate, or prevent tumor metastasis. In certain aspects inhibitors of RTK(s) disclosed herein can be used to treat, attenuate, or prevent treatment-resistant tumor growth and/or metastasis. In certain aspects, inhibitors of RTK(s) disclosed herein can be used to increase the therapeutic effect of one or more cancer treatments.

(b) Anaplastic Lymphoma Kinase (ALK) Receptor Tyrosine Kinases (ALK-RTK)

[0180] A specific receptor tyrosine kinase that may be active in tumor cells is Anaplastic Lymphoma Kinase (ALK). As described further herein, it has been surprisingly discovered that tumor cells treated with many receptor tyrosine kinase inhibitors targeting non-ALK receptors display SUMOylation at the kinase site which, in turn, recruits ALK and ALK associated molecules to the inhibited receptor. This association triggers excessive ALK activity which can contribute to delayed resistance to the RTK therapy.

[0181] Accordingly, in various aspects, the compositions herein comprise or further comprise an ALK inhibitor. As used herein, an “ALK inhibitor” can include any biomolecule(s) that can inhibit ALK direct activity, inhibit ALK indirect activity, inhibit formation of an ALK receptor dimer, decrease expression of a ALK gene, decrease expression of a ALK protein, or a combination thereof. In some aspects, compositions having a ALK inhibitor can include any biomolecule(s) that are modulators and/or inhibitors of targets upstream or downstream of a ALK signaling cascade that would effectively inhibit the physiological outcome of ALK inhibition. In some aspects, biomolecule(s) capable of inhibiting one or more ALK can be a peptide, an antibody, a chemical, a compound, an oligo, a nucleic acid molecule, or a combination thereof.

[0182] In certain aspects, ALK inhibitors for use in compositions herein can include a nucleic acid molecule. The term “nucleic acid molecule” as used herein refers to a molecule having nucleotides. The nucleic acid can be single, double, or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. In some aspects, a nucleic acid molecule for use herein can be a double-stranded RNA. In some other aspects a double stranded RNA suitable for use herein can be small temporal RNA, small nuclear RNA, small nucleolar RNA, short hairpin RNA, microRNA, or the like.

[0183] In certain aspects, ALK inhibitors for use in compositions herein may be a polypeptide, a protein, a peptide, a protein fragment, an antibody, or any combination thereof. As used herein, the terms “peptide,” “polypeptide,” and “protein,” refer to polymers comprised of amino acid monomers linked by amide bonds. Peptides may include the standard 20 a- amino acids that are used in protein synthesis by cells (i.e., natural amino acids), as well as non-natural amino acids (non-natural amino acids may be found in nature, but not used in protein synthesis by cells, e.g., ornithine, citrulline, and sarcosine, or may be chemically synthesized), amino acid analogs, and peptidomimetics. In some aspects, a peptide inhibitor of a receptor tyrosine kinase may be fused at its C-terminus, its N-terminus, or both with at least one other peptide and/or polypeptide. In some aspects, the at least one other peptide or polypeptide may be a carrier peptide, allowing cell penetration of the resulting fusion peptide. As an example, a peptide inhibitor of ALK may be fused to a cell penetrating peptide (CPP). CPPs are short peptides that facilitate cellular uptake of various molecular cargo, e.g. via endocytosis. Non-limiting examples of CPP that may be suitable for use herein include Antennapedia Penetratin, HIV-1 TAT protein, pVEC Cadherin, Transportan Galanine/Mastoparan, MPG HIV-gp41/SV40 T-antigen, Pep-1 HIV-reverse transcriptase/SV40 T-antigen, Polyarginines, MAP, R6W3, NLS, 8-lysines, ARF (1-22), Azurin-p28, and the like. As another example, a peptide inhibitor of ALK may be fused to celltargeting peptides (CTPs). CTPs are ideal carrier molecules as that bind with high affinity to overexpressed receptors on the tumor cell surface, effectively targeting a peptide inhibitor of ALK to the target tumor. CTPs can target, for example integrin receptors, epidermal growth factor receptors (EGFR), neuropeptide Y (NPY) receptors, gastrin-releasing peptide receptors (GRPR), somatostatin receptors (e.g., SSTR2), gonadotropin-releasing hormone receptors (GnRHR), vasoactive intestinal peptide (VIP) receptors, melanocortin 1 receptors (MC1 R), neurotensin receptors (e.g., NTSR1), and the like. In certain aspects, the CTP can bind to the target ALK, so that the ALK inhibiting peptide is near the receptor.

[0184] In certain aspects, ALK inhibitors for use in compositions herein may be an ALK antibody. As used herein, the terms “ALK antibody” can refer to an antibody capable of binding to and/or blocking activity of a receptor tyrosine kinase (ALK). In some aspects, blocking activity may comprise blocking dimerization and/or kinase activity of the receptor. Blocking activity may also comprise blocking association of various target proteins (those that are usually substrates of the ALK) from binding to or associating with the ALK. In some aspects, antibodies acting as an inhibitor of ALK may be full-length antibodies, antigen binding fragments of full-length antibodies, Fab fragments, single chain antibodies (scFv), diabodies, triabodies, minibodies, nanobodies, single-domain antibodies, camelids, or any combination thereof. In some aspects, septin antibodies disclosed herein may be of a suitable origin, for example, murine, rat, or human. Such antibodies are non-naturally occurring, i.e. , would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof or isolated from antibody libraries). Any of the antibodies described herein, e.g., ALK antibodies, can be either monoclonal or polyclonal. A “monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

[0185] In certain aspects ALK inhibitors for use in compositions herein may be a compound (also referred to herein as “small molecule”). In some aspects, a ALK inhibitor compound may comprise Lorlatinib, Brigatinib, Alectinib, Ceritinib, TAE684 (NVP-TAE684), AP26113 analog (ALK-IN-1), GSK1838705A, AZD3463, ASP3026, Entrectinib, Crizotinib, any analog thereof, or any combination thereof.

[0186] In some aspects, an ALK inhibitor compound may be a lortinib analog. In accordance with these aspects, an analog of lortinib suitable for use herein may be TAE684 (NVP-TAE684), AP26113 analog (ALK-IN-1), GSK1838705A, AZD3463, ASP3026, or any combination thereof. [0187] In certain aspects, inhibitors of ALK disclosed herein can be used to treat, attenuate, or prevent tumor growth and/or progression. In certain aspects, inhibitors of ALK disclosed herein can be used to treat, attenuate, or prevent tumor metastasis. In certain aspects inhibitors of ALK disclosed herein can be used to treat, attenuate, or prevent treatment-resistant tumor growth and/or metastasis. In certain aspects, inhibitors of ALK disclosed herein can be used to increase the therapeutic effect of one or more cancer treatments.

[0188] In various aspects, inhibitors of ALK disclosed herein can be used to increase the therapeutic effect of one or more RTK inhibitor cancer treatment. In particular aspects, inhibitors of ALK disclosed herein may be used to increase the therapeutic effect of one or more EGFR inhibitor. For example, the ALK inhibitor provided herein may increase the therapeutic effect of an EGFR inhibitor selected from Erlotinib, Gefitinib, Afatinib, Osimertinib, Dacomitinib, Lapatinib, Neratinib, Varlitinib, Trastuzumab, Pertuzumab, Tucatinib, and Cetuximab. In some aspects, the ALK inhibitor increases the therapeutic effect of Osimertinib. As described further below, “increasing a therapeutic effect” comprises increasing sensitivity or reversing resistance to the given RTK inhibitor in a tumor, tumor cell, cancer, or cancer cell in a patient.

(c) Additional Targeted Therapies

[0189] The RTK and ALK inhibitors described above are types of targeted therapies - drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and/or spread of cancer. Targeted cancer therapies are sometimes called "molecularly targeted drugs," "molecularly targeted therapies," "precision medicines," or similar names. Non-limiting examples of targeted therapies include hormone therapies, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapies, toxin delivery molecules, and the like. In some aspects, additional targeted therapies may be combined with the RTK and ALK inhibitors above. In some aspects, targeted therapies disclosed herein may be a small molecule, an antibody, or the like. One of skill in the art will appreciate that the appropriate targeted therapies for use herein will depend on, for example, the type of cancer, the stage of cancer, if the cancer is associated with a gene mutation, and the like. The following targeted therapies associated with one or more cancer types are nonlimiting examples of suitable targeted therapies for use in the compositions and methods of the present disclosure: 1) Bladder cancer: Atezolizumab (Tecentriq®), nivolumab (Opdivo®), avelumab (Bavencio®), pembrolizumab (Keytruda®), enfortumab vedotin-ejfv (Fadeev®), sacituzumab govitecan-hziy (Trodelvy®); 2) Brain cancer: everolimus (Afinitor®), belzutifan (Welireg®); 3) Breast cancer: Everolimus (Afinitor®), tamoxifen (Nolvadex®), toremifene (Fareston®), fulvestrant (Faslodex®), anastrozole (Arimidex®), exemestane (Aromasin®), letrozole (Femara®), palbociclib (Ibrance®), ribociclib (Kisqali®), abemaciclib (Verzenio®), olaparib (Lynparza®), talazoparib tosylate (Talzenna®), sacituzumab govitecan-hziy (Trodelvy®), pembrolizumab (Keytruda®), 4) Cervical cancer: pembrolizumab (Keytruda®), tisotumab vedotin-tftv (Tivdak®); 5) Colorectal cancer: nivolumab (Opdivo®), ipilimumab (Yervoy®), encorafenib (Braftovi®), pembrolizumab (Keytruda®);6) Endometrial cancer: Pembrolizumab (Keytruda®), dostarlimab-gxly (Jemperli®); 7) Esophageal cancer: pembrolizumab (Keytruda®), nivolumab (Opdivo®), 8) Head and neck cancer: pembrolizumab (Keytruda®), nivolumab (Opdivo®);9) Giant cell tumor: Denosumab (Xgeva®), pexidartinib hydrochloride (Turalio®); 10) Kidney cancer: temsirolimus (Torisel®), everolimus (Afinitor®), nivolumab (Opdivo®), ipilimumab (Yervoy®), pembrolizumab (Keytruda®), avelumab (Bavencio®), belzutifan (Welireg®); 11) Leukemia: Tretinoin (Vesanoid®), nilotinib (Tasigna®), bosutinib (Bosulif®), rituximab (Rituxan®), alemtuzumab (Campath®), ofatumumab (Arzerra®), obinutuzumab (Gazyva®), ibrutinib (Imbruvica®), idelalisib (Zydelig®), blinatumomab (Blincyto®), venetoclax (Venclexta®), ponatinib hydrochloride (Iclusig®), enasidenib mesylate (Idhifa®), inotuzumab ozogamicin (Besponsa®), tisagenlecleucel (Kymriah®), gemtuzumab ozogamicin (Mylotarg®), ivosidenib (Tibsovo®), duvelisib (Copiktra®), moxetumomab pasudotox-tdfk (Lumoxiti®), glasdegib maleate (Daurismo®), tagraxofusp-erzs (Elzonris®), acalabrutinib (Calquence®), brexucabtagene autoleucel (Tecartus®), asciminib hydrochloride (Scemblix®); 12) Liver and bile duct cancer: nivolumab (Opdivo®), pembrolizumab (Keytruda®), ipilimumab (Yervoy®), atezolizumab (Tecentriq®), ivosidenib (Tibsovo®); 13) Lung cancer: nivolumab (Opdivo®), pembrolizumab (Keytruda®), atezolizumab (Tecentriq®), trametinib (Mekinist®), dabrafenib (Tafinlar®), durvalumab (Imfinzi®), capmatinib hydrochloride (Tabrecta®), ipilimumab (Yervoy®), cemiplimab-rwlc (Libtayo®), sotorasib (Lumakras®), 14) Lymphoma: Ibritumomab tiuxetan (Zevalin®), denileukin diftitox (Ontak®), brentuximab vedotin (Adcetris®), rituximab (Rituxan®), vorinostat (Zolinza®), romidepsin (Istodax®), bexarotene (Targretin®), bortezomib (Velcade®), pralatrexate (Folotyn®), ibrutinib (Imbruvica®), siltuximab (Sylvant®), belinostat (Beleodaq®), obinutuzumab (Gazyva®), nivolumab (Opdivo®), pembrolizumab (Keytruda®), copanlisib hydrochloride (Aliqopa®), axicabtagene ciloleucel (Yescarta®), acalabrutinib (Calquence®), tisagenlecleucel (Kymriah®), venetoclax (Venclexta®), mogamulizumab-kpkc (Poteligeo®), duvelisib (Copiktra®), polatuzumab vedotin-piiq (Polivy®), zanubrutinib (Brukinsa®), tazemetostat hydrobromide (Tazverik®), selinexor (Xpovio®), tafasitamab-cxix (Monjuvi®), brexucabtagene autoleucel (Tecartus®), umbralisib tosylate (Ukoniq®), lisocabtagene maraleucel (Breyanzi®), loncastuximab tesirine-lpyl (Zynlonta®); 15) Multiple myeloma: Bortezomib (Velcade®), carfilzomib (Kyprolis®), daratumumab (Darzalex®), ixazomib citrate (Ninlaro®), elotuzumab (Empliciti®), selinexor (Xpovio®), isatuximab-irfc (Sarclisa®), belantamab mafodotin-blmf (Blenrep®), idecabtagene vicleucel (Abecma®), ciltacabtagene autoleucel (Carvykti®); 16) Myelodysplastic/myeloproliferative disorders: ruxolitinib phosphate (Jakafi®), fedratinib hydrochloride (Inrebic®), pacritinib citrate (Vonjo®); 17) Ovarian epithelial/fallopian tube/primary peritoneal cancers: olaparib (Lynparza®), rucaparib camsylate (Rubraca®), niraparib tosylate monohydrate (Zejula®); 18) Pancreatic cancer: everolimus (Afinitor®), olaparib (Lynparza®), belzutifan (Welireg®); 19) Soft tissue sarcoma: alitretinoin (Panretin®), tazemetostat hydrobromide (Tazverik®), sirolimus protein-bound particles (Fyarro®); 20) Stomach (gastric) cancer: Pembrolizumab (Keytruda®), nivolumab (Opdivo®); 21) Thyroid cancer: trametinib (Mekinist®), dabrafenib (Tafinlar®).

[0190] The present disclosure can provide for use of one or more anticancer therapies in combination with any of the compositions disclosed herein. In accordance with some aspects of the disclosure, the one or more anticancer therapies may be prescribed to a subject with increased likelihood of RTK inhibitor resistance, or may be used and/or administered to treat a cancer, a solid tumor, a metastasis, or any other cancerous condition characterized by RTK inhibitor resistance. In some aspects, one or more anticancer therapies may be any one or more of surgery, radiation, chemotherapy, immunotherapy, vaccine or combinations thereof. [0191] In some aspects, the one or more anticancer therapies may be chemotherapy. Chemotherapeutic agents may be selected from any one or more of cytotoxic antibiotics, antimetabolities, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: doxorubicin, epirubicin, etoposide, camptothecin, topotecan, irinotecan, teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy- 5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halichondrin, colchicine, and rhizoxin. Compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. In another aspect, PARP (e.g., PARP-1 and/or PARP-2) inhibitors are used and such inhibitors are well known in the art (e.g., Olaparib, ABT- 888, BSI-201 , BGP-15, INO-1001 , PJ34, 3-aminobenzamide, 4-amino-1 ,8-naphthalimide, 6(5H)-phenanthridinone, benzamide, NU1025).

[0192] In some aspects, the one or more anticancer therapies may be radiation therapy. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy. In some aspects, the radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. In other aspects, the radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A and 2BA-2-DMHA.

[0193] In some aspects, the one or more anticancer therapies may be immunotherapy. Immunotherapy may comprise, for example, use of cancer vaccines and/or sensitized antigen presenting cells. The immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of a pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines.

[0194] In some aspects, the one or more anticancer therapies may be hormonal therapy, Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LLIPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0195] In certain aspects, the duration and/or dose of treatment with anticancer therapies may vary according to the particular anti-cancer agent or combination thereof. An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan. In some aspects, the continued assessment of optimal treatment schedules for each cancer therapeutic agent is contemplated, where the genetic signature of the cancer of the subject as determined by the methods of the disclosure is a factor in determining optimal treatment doses and schedules.

(d) Pharmaceutical Formulations and Treatment Regimens

[0196] In certain aspects, any one or more active agents disclosed herein (i.e RTK inhibitor, ALK inhibitor, or any combination thereof) may be provided per se or as part of a pharmaceutical composition, where the active agent(s) can be mixed with suitable carriers or excipients.

[0197] As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

(i) Pharmaceutically acceptable carriers and excipients

[0198] Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” are interchangeably used herein to refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0199] In certain aspects, compositions disclosed herein may further compromise one or more pharmaceutically acceptable diluent(s), excipient(s), and/or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer’s solution, phosphate solution or buffer, buffered saline, and other carriers known in the art.

[0200] In some aspects, pharmaceutical compositions herein may also include stabilizers, anti-oxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, or any combination thereof. Herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0201] In certain aspects, pharmaceutical compositions described herein may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used pharmaceutically. In some aspects, any of the well-known techniques, carriers, and excipients may be used as suitable and/or as understood in the art. [0202] In certain aspects, pharmaceutical compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, polymers that may comprise pharmaceutical compositions described herein include: water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water-insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% total amount of polymers as suspending agent(s) by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of polymers as suspending agent(s) by total weight of the composition.

[0203] In certain aspects, pharmaceutical compositions disclosed herein may comprise a viscous formulation. In some aspects, viscosity of composition herein may be increased by the addition of one or more gelling or thickening agents. In some aspects, compositions disclosed herein may comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% total amount of gelling or thickening agent(s) by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of gelling or thickening agent(s) by total weight of the composition. In some aspects, suitable thickening agents for use herein can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethylcellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), or any combination thereof.

[0204] In certain aspects, pharmaceutical compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% total amount of one or more agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more agents by total weight of the composition. In some aspects, one or more of these agents may be added to improve the performance, efficacy, safety, shelf-life and/or other property of the muscarinic antagonist composition of the present disclosure. In some aspects, additives may be biocompatible, without being harsh, abrasive, and/or allergenic.

[0205] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid may be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more acidifying agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more acidifying agents by total weight of the composition.

[0206] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more alkalizing agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more alkalizing agents by total weight of the composition.

[0207] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite and other materials known to one of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more antioxidants by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more antioxidants by total weight of the composition.

[0208] In certain aspects, pharmaceutical compositions disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more buffering agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more buffering agents by total weight of the composition.

[0209] In some aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some aspects, pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In some aspects, pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0210] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more preservatives by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more preservatives by total weight of the composition.

[0211] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more surface-acting reagents or detergents. In some aspects, surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic. In some aspects, compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more surface-acting reagents or detergents by total weight of the composition.

[0212] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more stabilizers by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more stabilizers by total weight of the composition.

[0213] In some aspects, pharmaceutical compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose and others known to those or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm/L). Osmolarity may be measured using methods commonly known in the art. In some aspects, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein may result in a composition osmolarity of about 150 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about 280 mOsm/L to about 370 mOsm/L or about 250 mOsm/L to about 320 mOsm/L. In some aspects, a composition herein may have an osmolality ranging from about 100 mOsm/kg to about 1000 mOsm/kg, from about 200 mOsm/kg to about 800 mOsm/kg, from about 250 mOsm/kg to about 500 mOsm/kg, or from about 250 mOsm/kg to about 320 mOsm/kg, or from about 250 mOsm/kg to about 350 mOsm/kg or from about 280 mOsm/kg to about 320 mOsm/kg. In some aspects, a pharmaceutical composition described herein may have an osmolarity of about 100 mOsm/L to about 1000 mOsm/L, about 200 mOsm/L to about 800 mOsm/L, about 250 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about 250 mOsm/L to about 320 mOsm/L, or about 280 mOsm/L to about 320 mOsm/L. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more tonicity modifiers by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more tonicity modifiers by total weight of the composition.

(ii) Dosage formulations

[0214] In certain aspects, the present disclosure provides compositions formulated for one or more routes of administration. Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal, and/or parenteral delivery. In some aspects, compositions herein formulated can be formulated for parenteral delivery. In some aspects, compositions herein formulated can be formulated intramuscular, subcutaneous, intramedullary, intravenous, intraperitoneal, and/or intranasal injections. [0215] In certain aspects, one may administer a composition herein in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some aspects, a pharmaceutical composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra-cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some aspects, a pharmaceutical composition disclosed herein can administered to subject as disclosed herein. In some aspects, a pharmaceutical composition disclosed herein can administered to human patient. In some aspects, a pharmaceutical composition disclosed herein can administered to a human patient via at least two administration routes. In some aspects, the combination of administration routes by be intracerebroventricular injection and intravenous injection; intrathecal injection and intravenous injection; intra-cisterna magna injection and intravenous injection; and/or intra-parenchymal injection and intravenous injection.

[0216] In certain aspects, pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0217] In certain aspects, pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of a pharmaceutical composition herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, physiological salt buffer, or any combination thereof.

[0218] In certain aspects, pharmaceutical compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection herein may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some aspects, compositions herein may be suspensions, solutions or emulsions in oily or aqueous vehicles, and/or may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0219] In certain aspects, pharmaceutical compositions herein formulated for parenteral administration may include aqueous solutions of the active preparation (e.g., septin inhibitors, bleb inhibitors, targeted therapies, or any combination thereof) in water-soluble form. In some aspects, compositions herein comprising suspensions of the active preparation may be prepared as oily or water-based injection suspensions. Suitable lipophilic solvents and/or vehicles for use herein may include, but are not limited to, fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. In some aspects, compositions herein comprising aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, and/or dextran. In some aspects, compositions herein comprising a suspension may also contain one or more suitable stabilizers and/or agents which increase the solubility of the active ingredients (e.g., septin inhibitors, bleb inhibitors, targeted therapies, or any combination thereof) to allow for the preparation of highly concentrated solutions.

[0220] In some aspects, compositions herein may comprise the active ingredient in a powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0221] Pharmaceutical compositions suitable for use in context of the present disclosure may include compositions wherein the active ingredients can be contained in an amount effective to achieve the intended purpose. In some aspects, a therapeutically effective amount means an amount of active ingredients (e.g., septin inhibitors, bleb inhibitors, targeted therapies, or any combination thereof) effective to prevent, slow, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

[0222] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0223] For any preparation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays and or screening platforms disclosed herein. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0224] In some aspects, toxicity and therapeutic efficacy of the active ingredients disclsoed herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In some aspects, data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in a human subject. In some aspects, a dosage for use herein may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1).

[0225] In certain aspects, dosage amounts and/or dosing intervals may be adjusted individually to brain or blood levels of the active ingredient that are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). In some aspects, the MEC for an active ingredient (e.g.,RTK inhibitor or ALK inhibitor) may vary for each preparation but can be estimated from in vitro data. In some aspects, dosages necessary to achieve the MEC herein may depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0226] In certain aspects, depending on the severity and responsiveness of the condition to be treated, dosing with compositions herein can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0227] In certain aspects, amounts of a composition herein to be administered will be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like. In some aspects, effective doses may be extrapolated from dose-responsive curves derived from in vitro or in vivo test systems.

III. Methods of Use

[0228] The present disclosure provides for methods for improving sensitivity and/or preventing resistance of cancer cells to an RTK inhibitor, either in vitro or in vivo.

[0229] Accordingly, in one aspect, the disclosure provides for a method of increasing sensitivity of a cancer cell to a receptor tyrosine kinase inhibitor (RTK inhibitor) - including but not limited to an RTK inhibitor described above. In various aspects, the methods comprise contacting a cancer cell with at least one ALK inhibitor (e.g., an ALK inhibitor described above) wherein contacting the cell with the at least one ALK inhibitor increases the cell’s sensitivity to an RTK inhibitor as compared to a cell not contacted with the ALK inhibitor. In various aspects, the cell survival of the cancer cell is decreased after contacting the cancer cell with at least one ALK inhibitor. In further aspects, the cell proliferation of the cancer cell is decreased after contacting the cancer cell with at least one ALK inhibitor. In any of these aspects, the cancer cell may be located in or obtained from a lung tumor, a breast tumor, a pancreatic tumor, a colorectal tumor, an esophageal tumor, a head-and-neck tumor, a hematological malignancy, a brain tumor, an ovarian tumor, a stomach tumor, a gastric tumor, a prostate tumor, a thyroid tumor, bladder tumor, a lymphoma, melanoma, or a kidney tumor. In various aspects, the cell is in vitro or is in vivo. When the cell is in vivo the method may have therapeutic benefits and may be applied to treat, attenuate, and/or prevent cancer in a subject. In various aspects, methods of increasing sensitivity of a cancer cell as disclosed herein exclude contacting the cell with a SUMOylation inhibitor.

[0230] Therefore, in various aspects, the disclosure provides for methods of treating, attenuating, and preventing cancer in a subject in need thereof. The present disclosure also provides for methods of impairing tumor growth compared to tumor growth in an untreated subject with identical disease condition and predicted outcome. In certain aspects, a method for treating, attenuating, or preventing tumor growth or a method for treating, attenuating, or preventing a cancer and/or metastasis in a subject can generally comprise increasing sensitivity of a cancer cell in the subject to a targeted therapy targeting receptor tyrosine kinase signaling (e.g., sensitivity to an RTK inhibitor).

[0231] In some aspects, a method for treating, attenuating and/or preventing cancer in a subject in need thereof may comprise administering to the subject in need thereof (a) at least one receptor tyrosine kinase (RTK) inhibitor as provided herein, wherein the RTK inhibitor does not inhibit ALK; and (b) at least one ALK inhibitor as provided herein. In various aspects, methods of treating, attenuating and/or preventing cancer in a subject in need thereof as disclosed herein exclude administering a SUMOylation inhibitor.

[0232] In various aspects, a method for treating a tumor in a subject in need thereof is provided, the method comprising: administering to the subject at least one ALK inhibitor as provided herein, wherein the subject has undergone, is undergoing or will undergo an anticancer therapy comprising one or more targeted receptor tyrosine kinase (RTK) inhibitors that do not target ALK, as provided herein.

[0233] Still other aspects are directed to methods of preventing sensitization and/or resistance to an anti-cancer therapy comprising a receptor tyrosine kinase (RTK) inhibitor, the methods comprising administering to a subject in need thereof, an ALK inhibitor prior to or concurrent with the anti-cancer therapy comprising the RTK inhibitor. In some aspects disclosed herein, compositions and methods disclosed herein are designed to re-sensitize or sensitize a tumor in a subject to RTK inhibition to reduce costs, improve outcome, and reduce or eliminate patient exposure to an anticancer therapy without significant effect. As noted, suitable RTK inhibitors and ALK inhibitors that may be used in the methods herein are described above.

(a) Malignant Tumors

[0234] In any of these aspects, the compositions and methods herein are directed to treating a malignant tumor in a subject. In various aspects, the malignant tumor may be resistant to an RTK inhibitor (RTKi) or be at risk of developing resistance to the RTKi. In some aspects the malignant tumor comprises a solid tumor or a non-solid tumor. In some aspects, the malignant tumor comprises a carcinoma, a sarcoma, a hematological malignancy, or any combination thereof.

[0235] In some aspects, the malignant tumor is a solid tumor. In some aspects, a solid tumor can be an abnormal mass of tissue that is devoid of cysts or liquid regions within the tumor. In some aspects, a solid tumor herein can be a malignant cancer that has metastasized. In other aspects, solid tumors contemplated herein can include, but are not limited to, sarcomas, carcinomas, lymphomas, gliomas or a combinational thereof. In accordance with some aspects herein, malignant tumors can include, but are not limited to, lung tumor, a breast tumor, a pancreatic tumor, a colorectal tumor, an esophageal tumor, a head-and-neck tumor, a brain tumor, an ovarian tumor, a stomach tumor, a gastric tumor, a prostate tumor, a thyroid tumor, bladder tumor, melanoma, a kidney tumor, a soft tissue sarcoma, a urothelial carcinoma, a cervical tumor, a liver tumor, an endometrial tumor, a solid tumor with an NTRK gene fusion, systemic mastocytosis, or a combination thereof. In some aspects, a targeted tumor contemplated herein can include a solid tumor such as non-small- cell lung cancer (NSCLC), HER-2 positive breast cancer, glioblastoma, gastrointestinal stromal tumor, esophageal tumor, liver tumor, bladder tumor or any combination thereof.

[0236] In some aspects, the malignant tumor (e.g., that is resistant or suspected of becoming resistant to RTKi) is a hematological malignancy. As used herein a “hematological malignancy” refer to a tumor that begin in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. There are three main types of hematologic malignancies: leukemia, lymphoma and multiple myeloma. The hematological malignancy may be a solid tumor or a non-solid tumor. In some aspects, the hematological malignancy is a leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lympocytic leukemia (CLL) or hairy cell leukemia). In some aspects, the hematological malignancy is a lymphoma (e.g., a Hodgkin’s lymphoma such as nodular sclerosis class HL (NSHL), a mixed cellularity classic HL (MCHL), a lymphocyte rich classic HL (LRHL) or a lymphocyte depleted classic HL (LDHL) or a nonHodgkin’s lymphoma such as a B-cell neoplasm or a T-cell or putative NK-cell neoplasm). In other aspects, the hematological malignancy is multiple myeloma.

[0237] Accordingly, in various aspects, the malignant tumor may comprise a lung tumor, a breast tumor, a pancreatic tumor, a colorectal tumor, an esophageal tumor, a head-and- neck tumor, a hematological malignancy, a brain tumor, an ovarian tumor, a stomach tumor, a gastric tumor, a gastrointestinal stromal tumor, a prostate tumor, a thyroid tumor, bladder tumor, dermatofibrosarcoma protuberans, a giant cell tumor, a lymphoma, melanoma, a myeloma, a kidney tumor, a soft tissue sarcoma, chronic eosinophilic leukemia (CEL), chronic lympocytic leukemia (CLL), urothelial carcinoma, a cervical tumor, liver or bile duct tumor, endometrial tumor or any combination thereof. In certain aspects, the malignant tumor can be derived from a non-small-cell lung cancer (NSCLC), HER-2 positive breast cancer, glioblastoma, gastrointestinal stromal tumor, esophageal tumor, liver or bile duct tumor, bladder tumor or any combination thereof.

[0238] In various aspects, the malignant tumor is a candidate for a targeted therapy targeting aberrant receptor tyrosine kinase signaling. For example, the malignant tumor may comprise one or more somatic mutations, translocations, amplifications and/or other disruptions in a gene encoding a receptor tyrosine kinase (RTK). In aspects, the somatic mutations, translocations, amplifications and/or other disruptions occur in a gene for an RTK that is not ALK. In various aspects, the at least one gene encoding a RTK that is not ALK comprises an EGFR gene, HER2 gene, HER3 gene, HER4 gene, c-Met gene, R0S1 gene, IGF-1R gene, TRKA gene, TRKB gene, TRKC gene, a PDGFR gene, a VEGFR gene, a FGFR gene, a FLT3 gene, a NTRK gene, a RET gene or any combination thereof. In various aspects, the malignant tumor has excessive RTK signaling that may be caused, for example, by the one or more somatic mutations, translocations, amplifications and/or other disruptions in a gene encoding the RTK. For example, the tumor may express one or more hyperactive or overactive RTKs that are not ALK. In some aspects, the malignant tumor has aberrant (i.e., excessive) RTK signaling caused by a different pathway (e.g., not caused by a direct mutation in an RTK gene) but is still considered a candidate for RTK inhibition. In any of these aspects, the receptor tyrosine kinase that is hyperactive, overactive, or that is involved in excessive RTK signaling comprises EGFR, HER2, HER3, HER4, c-Met, ROS1 , IGF-1 R, TRKA, TRKB, TRKC, PDGFR, VEGFR, FGFR, FLT3, NTRK, or RET.

[0239] In still further aspects, the malignant tumor is not a candidate for ALK inhibition - that is, the malignant tumor has no genetic or molecular characteristics or biomarkers that would identify it as a candidate for ALK inhibitor therapy. In aspects, the malignant tumor does not express an ALK fusion protein or any other mutant or overactive ALK.

[0240] In still further aspects, the malignant tumor is at high risk for resistance to RTK inhibition. In some aspects, the tumor is refractory. As used herein, “refractory” refers to the tumor that does not respond to or becomes resistant to a treatment. In some aspects, the subject herein may have a tumor that is resistant to at least one targeted therapy. In some aspects, the subject herein may have a tumor that is resistant or is at high risk of becoming resistant to RTK inhibition. In some aspects, the subject may be a human patient having a relapsed disease. As used herein, “relapsed” or “relapses” refers to a tumor that returns or progresses following a period of improvement (e.g., a partial or complete response) with treatment. In certain aspects, the “treatment” administered before the relapse may be an RTK inhibitor.

[0241] A subject having a malignant tumor as disclosed herein, for example, non-small- cell carcinoma can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, genetic tests, interventional procedure (biopsy, surgery) any and all relevant imaging modalities. In some aspects, a subject to be treated by the methods described herein may have one or more cancers or one or more tumors having at least one somatic mutation. In some aspects, a subject herein may have a cancer and/or tumor with one or more somatic mutations in an RTK gene that is not an ALK gene (e.g., in a an EGFR gene, a HER2 gene, a HER3 gene, a HER4 gene, a c-Met gene, a ROS1 gene, a IGF-1R gene, a TRKA gene, a TRKB gene, a TRKC gene, a PDGFR gene, a VEGFR gene, a FGFR gene, a FLT3 gene, a NTRK gene, a RET gene or any combination thereof. In some aspects, the subject to be treated by the methods described herein is a human cancer patient who has undergone or is subjecting to an anti-cancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, or surgery. In some aspects, a subject shows disease progression through the treatment. In other aspects, a subject is resistant to the treatment (either de novo or acquired). In some aspects, such a subject is demonstrated as having advanced malignancies (e.g., inoperable or metastatic). Alternatively, or in addition, in some aspects, the subject has no standard therapeutic options available or ineligible for standard treatment options, which refer to therapies commonly used in clinical settings for treating the corresponding solid tumor.

(b) Therapeutic Effects

[0242] In certain aspects, compositions and methods disclosed herein can treat and/or prevent cancer in a subject in need. In some aspects, compositions and methods disclosed herein can impair tumor growth compared to tumor growth in an untreated subject with identical disease condition and predicted outcome. In some aspects, tumor growth can be stopped following treatment with compositions disclosed herein. In other aspects, tumor growth can be impaired at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject with identical disease condition and predicted outcome. In other words, tumors in subject treated using a composition of the disclosure have tumors that grow at least 5% less (or more as described above) when compared to an untreated subject with identical disease condition and predicted outcome. In some aspects, tumor growth can be impaired at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease condition and predicted outcome. In some aspects, tumor growth can be impaired at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated subject with identical disease condition and predicted outcome.

[0243] In some aspects, treatment of tumors with compositions and methods disclosed herein can result in a shrinking of a tumor in comparison to the starting size of the tumor. In some aspects, tumor shrinking is at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% (meaning that the tumor is completely gone after treatment) compared to the starting size of the tumor. [0244] In certain aspects, compositions disclosed herein can improve patient life expectancy compared to the cancer life expectancy of an untreated subject with identical disease condition and predicted outcome. As used herein, “patient life expectancy” is defined as the time at which 50 percent of subjects are alive and 50 percent have passed away. In some aspects, patient life expectancy can be indefinite following treatment with a composition disclosed herein. In other aspects, patient life expectancy can be increased at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject with identical disease condition and predicted outcome. In some aspects, patient life expectancy can be increased at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease condition and predicted outcome. In some aspects, patient life expectancy can be increased at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated patient with identical disease condition and predicted outcome.

[0245] In some aspects, the methods of the present disclosure increase anti-tumor activity (e.g., reduce cell proliferation, tumor growth, tumor volume, and/or tumor burden or load or reduce the number of metastatic lesions over time) by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels prior to treatment or in a control subject. In some aspects, reduction is measured by comparing cell proliferation, tumor growth, and/or tumor volume in a subject before and after administration of the pharmaceutical composition. In some aspects, the method of treating or ameliorating a cancer in a subject allows one or more symptoms of the cancer to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some aspects, methods disclosed herein may include administration of the compositions herein to reduce tumor volume, size, load or burden in a subject to an undetectable size, or to less than about 1 %, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the subject's tumor volume, size, load or burden prior to treatment. In other aspects, methods disclosed herein may include administration of the compositions herein to reduce the cell proliferation rate or tumor growth rate in a subject to an undetectable rate, or to less than about 1 %, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate prior to treatment.

[0246] In some aspects, a subject to be treated by any of the methods and/or compositions herein can present with one or more malignant tumors, metastatic nodes, or a combination thereof. In some aspects, a subject herein may have a cancerous tumor cell source that can be less than about 0.2 cm³ to at least about 20 cm³ or greater, at least about 2 cm³ to at least about 18 cm³ or greater, at least about 3 cm³ to at least about 15 cm³ or greater, at least about 4 cm³ to at least about 12 cm³ or greater, at least about 5 cm³ to at least about 10 cm³ or greater, or at least about 6 cm³ to at least about 8 cm³ or greater.

[0247] In certain aspects, the compositions disclosed herein can be effective for treating at least one tumor cell in a solid tumor from a subject in need. In some aspects, the amount of viable tumor cells may be reduced by at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease condition and predicted outcome.

(c) Methods of administration

[0248] In some aspects, compositions of use herein can include at least one ALK inhibitor as disclosed herein. In some other aspects, a combination therapy herein may include administering to a subject in need at least one RTK inhibitor and at least one ALK inhibitor disclosed herein. In certain aspects, ALK inhibitors disclosed herein can be administered to a subject alone or in combination with a RTK inhibitor, daily, every other day, twice weekly, every other day, every other week, weekly, monthly, or any other suitable dosing regimen.

[0249] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the compositions disclosed herein to a subject, depending upon the type of disease to be treated or the site of the disease. In some aspects, compositions herein can be administered to a subject by intravenous infusion, by subcutaneous administration, by inhalation, by intranasal administration or other mode of administration. In some aspects, compositions herein can be administered to a subject orally.

[0250] In some aspects, one or more disclosed active agents (e.g., ALK inhibitor) can be administered concurrently with the one or more RTK inhibitor by the same or different modes of administration. In some aspects, one or more disclosed active agents (e.g., ALK inhibitor) can be administered before, during or after the one or more RTK inhibitors. In some aspects, the one or more RTK inhibitors and/or the one or more ALK inhibitors can be administered systemically. In certain aspects, the one or more RTK inhibitors and/or the one or more ALK inhibitors can be administered locally directly to one or more tumors in the subject. In some aspects, the one or more RTK inhibitors and/or the one or more ALK inhibitors can be administered by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intra-articular, intrasynovial, intrathecal, intratumoral, oral, inhalation or topical routes. In other aspects, the one or more RTK inhibitors and/or the one or more ALK inhibitors can be administered to the subject by intravenous infusion.

[0251] An effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, systemically or locally. In some aspects, one or more disclosed active agents (e.g., septin inhibitor and/or bleb inhibitor) can be administered by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intra-articular, intrasynovial, intrathecal, intratumoral, oral, inhalation or topical routes. In some aspects, one or more disclosed active agents (e.g., septin inhibitor and/or bleb inhibitor) can be administered orally.

[0252] In some aspects, methods herein of treating a cancer with one or more ALK inhibitors and/or RTK inhibitors as disclosed herein can further include treating a subject with at least one additional therapeutic regimen, for example, chemotherapy, radiotherapy, immunotherapy, or surgery. In some aspects, a subject treated with any of the methods herein can have completed an additional therapeutic regimen, be receiving an additional therapeutic regimen, or can receive an additional therapeutic regimen following treatment according to the methods herein.

[0253] In some aspects, any of the methods disclosed herein can further include monitoring occurrence of one or more adverse effects in the subject. Exemplary adverse effects include, but are not limited to, hepatic impairment, hematologic toxicity, neurologic toxicity, cutaneous toxicity, gastrointestinal toxicity, or a combination thereof. When one or more adverse effects are observed, the method disclosed herein can further include reducing or increasing the dose of one or more of the disclosed active agents (e.g., ALK inhibitor), the dose of one or more RTK inhibitor, or both depending on the adverse effect or effects in the subject. For example, when a moderate to severe hepatic impairment is observed in a subject after treatment, one or more compositions can be reduced in concentration, frequency of dosing, or a combination thereof.

IV. Kits

[0254] The present disclosure provides kits for use in treating or alleviating cancer and/or a malignant tumor described herein. Such kits can include one or more containers including one or more disclosed agents (e.g., ALK inhibitor). In some aspects, kits can include one or more containers including one or more one or more disclosed agents (e.g., ALK inhibitor) and one or RTK inhibitors described herein. [0255] In some aspects, the kits herein can include instructions for use in accordance with any of the methods described herein. The included instructions can have a description of administration of the one or more disclosed active agents (e.g., ALK inhibitor), and/or the one or more RTK inhibitors described herein, to treat, delay the onset, or alleviate a target disease as those described herein, or a combination thereof. In some aspects, the kit can further include a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease, e.g., applying a diagnostic method as described herein. In still other aspects, the instructions can have a description of administering any one of the compositions described herein to an individual at risk of the target disease.

[0256] In some aspects, kit instructions relating to the use of one or more disclosed active agents (e.g., ALK inhibitor), one or more RTK inhibitors described herein, or a combination thereof can generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers can be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0257] The label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating the solid tumor. In some aspects, instructions are provided for practicing any of the methods described herein.

[0258] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. In some aspects, a kit has a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some aspects, the container also has a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0259] In some aspects, kits herein can optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some aspects, the disclosure provides articles of manufacture comprising contents of the kits described above.

******* [0260] Having described several aspects, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present disclosure. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present disclosure. Accordingly, this description should not be taken as limiting the scope of the present disclosure.

[0261] Those skilled in the art will appreciate that the presently disclosed aspects teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.

EXAMPLES

[0262] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

Example 1 - Introduction to Examples

[0263] Molecularly targeted cancer therapy generally involves inhibition of specific oncogenic pathways, and has been most successful in the presence of activating mutations in receptor tyrosine kinase (RTK) pathways that result in accentuated signaling. However, secondary resistance to RTK inhibition remains a pervasive problem. In non-small cell lung cancer (NSCLC), activating EGFR mutations represent an important target. EGFR inhibition using tyrosine kinase inhibitors (TKIs) such as osimertinib is highly effective initially in the subset of patients with activating EGFR mutations. However, secondary resistance inevitably develops in initially responsive EGFR mutant tumors. The emergence of secondary resistance implies the survival of persister clones that are not eliminated during the initial treatment and is a major therapeutic hurdle.

[0264] In addition to NSCLC, a number of other cancers harbor activated receptor tyrosine kinases that serve as oncogenic drivers and respond to targeted therapies. For example, lapatinib is used in metastatic HER2+ breast cancer, and multiple RTK inhibitors such as sunitinib or caboxantinib have been used effectively in renal cell carcinoma (RCC). Multi kinase RTK inhibitors are used for hepatocellular carcinoma (HCC) and include sorafenib which targets PDGFR and VEGFR. Gastrointestinal tumors expressing KIT are responsive to Imatinib, and lenvatinib and sorafenib are effective for thyroid cancers.

[0265] The mechanisms of resistance to EGFR inhibition in NSCLC have been extensively investigated. Both mutational and non-mutational mechanisms of resistance have been described. For example, major mutational mechanisms of resistance to EGFR inhibition in NSCLC include secondary EGFR mutations such as the T790M mutation, and amplification of other RTKs such as MET. These genetic changes are detected after months of exposure to TKIs. In addition, inhibition of RTK signaling pathways in cancer cells leads to a rapid reprogramming of signaling pathways as the cancer cell seeks to restore homeostasis. This adaptive response to RTK inhibition may play a key role in early drug resistance and the formation of persister clones. It may also protect cells from a loss of RTK signals and could play an important role in mediating therapeutic resistance, although this has been difficult to prove in patients because of difficulty in obtaining tumor tissue at early time point after treatment. However, there is substantial evidence from experimental studies that EGFR inhibition triggers a rapid adaptive response in NSCLC that likely contributes to secondary resistance. A substantial component of this adaptive response includes activation of inflammatory signaling pathways. For example, upregulation of TNF and activation of NF-KB may play a central role in mediating survival of persister clones and secondary resistance to EGFR inhibition in NSCLC. For example, a rapid TNF-driven adaptive response may play a key role in resistance to EGFR inhibition in NSCLC and in glioblastoma (GBM). Bypass RTK signaling is also an important component of the RTK inhibition induced adaptive response. While rapid signaling changes following RTK inhibition are well documented, the mechanisms that trigger these signaling events remain unknown.

[0266] Oncogenic anaplastic lymphoma kinase (ALK) fusion proteins are found in multiple cancer types including NSCLC, renal cell carcinoma (RCC), breast cancer, colon carcinoma, serous ovarian carcinoma (SOC) and esophageal squamous cell carcinoma (ESCC). ALK gene fusions are found in 3-5% of NSCLC and result in oncogenic activation of ALK, which has emerged as an important therapeutic target in NCSLC. EGFR mutations and ALK fusions are generally mutually exclusive in NSCLC. In one large study, only 1.3% of tumors had concomitant EGFR mutation and ALK rearrangements.

[0267] Protein modification by small ubiquitin-like modifier, termed SUMOylation plays an important role in multiple biological process in health and disease. SUMO regulated processes are found in multiple cancer hallmark functions. Enhanced SUMOylation can be oncogenic and correlate with poor prognosis. Expression of SUMO pathway enzymes is frequently upregulated in cancer including lung adenocarcinoma. SUMO enzymes are enriched in the nucleus but are also detected in the cytoplasm, and proteins in multiple cellular compartments including the plasma membrane can become SUMOylated. SUMOylation of proteins may have multiple molecular consequences and one outcome is that the attached SUMO acts as a platform to recruit new interacting proteins to the substrate.

[0268] Receptor tyrosine kinase inhibition is a commonly used treatment for various cancers, leads to a loss of the tyrosine kinase activity of the RTK and is considered to render the receptor inactive. The following examples explore and define the surprising finding that kinase inhibited RTKs continue to function by becoming SUMOylated and converting to signaling platforms that drive adaptive resistance to RTK inhibition. In the examples, an EGFR mutant NSCLC was used as an exemplary model to examine this signaling function of kinase inhibited receptor tyrosine kinases and its role in therapeutic resistance.

Example 2 - Tyrosine kinase inhibition causes SUMOylation of EGFR

[0269] This example demonstrates that receptor tyrosine kinases are SUMOylated in response to tyrosine kinase inhibition (TKI).

[0270] In an initial experiment, a mass spectrometry analysis was performed to identify proteins that associate/dissociate with an epidermal growth factor receptor (EGFR) in EGFR mutant HCC827 NSCLC cell lines following inhibition of the EGFR with osimertinib. Specifically, HCC827 cells were treated with 100 nM Osimertinib for 5 min, 30 min, 2 hours, 6 hours or 24 hours and then lysed. Cell lysates were then immunoprecipitated by EGFR antibody followed by Mass Spectrometry (see Materials and Methods in Example 14). Despite an expectation that proteins would dissociate from a kinase inhibited EGFR, it was instead surprisingly found that many proteins associate with the EGFR at various time points after osimertinib treatment. FIG. 1A shows a heat map indicating proteins that bind to EGFR after Osimertinib treatment with an affinity increase of over twofold and show association of SUMPO3 and ALK fusion proteins (TFG, EML4, and TPM4). Indeed, upon tyrosine kinase inhibition, it was found that numerous proteins are rapidly recruited to the EGFR in temporal waves. The association of SUMO3 was of particular interest because it indicated that the EGFR may become SUMOylated, a post-translation modification that in implicated in key biological processes.

[0271] In another experiment to confirm association of SUMO3 to the EGFR following Osimertinib treatment, an immunoprecipitation/western blot experiment was performed. Specifically, HCC827, PC9 and H3255 cells were treated with 100 nM, 50 nM and 50 nM Osimertinib, respectively, and then lysed and immunoprecipitated with an EGFR antibody. Resulting complexes were analyzed with Western Blot using anti-EGFR, anti-phospho- specific EGFR (Y1068), and anti-SUMO3 antibodies (P-acting was used as a loading control). As shown in FIG. 1B-1D, all three cell lines showed association between SLIMO3 and EGFR following Osimertinib treatment. The experiment was then repeated in NOD-SCID mice inoculated with HCC827 or HCC4190 PDX tumor cells derived from an EGFR mutant NSCLC patient. Specifically, HCC827 and HCC4190 PDX xenograft tumor bearing mice were treated with 0.5 mg/kg and 5 mg/kg Osimertinib, respectively, followed by collection of tumor 0, 2 or 6 hours after treatment. Tumor cells were isolated, lysed, immunoprecipitated with anti-EGFR antibody and examined using western blot. FIG. 1E-1 F depict representative immunoblots showing that EGFR receptors associated with SLIMO3 within a day of exposure to Osimertinib in both cell lines.

[0272] To confirm that the post-translational EGFR modification detected is indeed SUMOylation, two experiments were performed to knockdown expression of SLIMO3. In a first experiment, siRNA knockdown of SLIMO3 resulted in a loss of Osimertinib-induced EGFR SUMOylation (FIG. 1G-1I). Specifically, HCC827, PC9 and H3255 cells were transfected with siRNA control and siRNA SUMO3, then treated by 100 nM and 50 nM osimertinib, respectively, followed by immunoprecipitation with EGFR antibody and Western blot (FIG. 1G- 11). In a second experiment, SUMO specific protease 2 (SENP2) was overexpressed in HCC827 cells before treatment with Osimertinib (FIG. 1J-1 K). SENP Sumo specific proteases (SENPs) are cysteine proteases with isopeptidase activity facilitating the de-conjugation of SUMO proteins. In this experiment, HCC827 and PC9 cells were transfected with FLAGSEN P2 and then treated with 100 nM and 50 nM Osimertinib, respectively, followed by immunoprecipitation with anti-EGFR antibody and western blot. As shown in FIG. 1J-1K, SENP2 overexpression resulted in a loss of Osimertinib-induced EGFR SUMOylation. Additional control experiments were performed to determine whether association was specific to SUMO3. Briefly, HCC827 and PC9 cells were treated with 100 nM and 50 nM osimertinib, respectively, followed by immunoprecipitation with EGFR antibody and Western blot with anti- SUMO1 or anti-SUMO2 antibodies. No association of SUMO1 and SUMO2 with EGFR was detected after Osimertinib treatment (FIG. 10A-10D). Further, to test whether Osimertinib could affect EGFR stability, HCC827, PC9 and H3255 cells were treated by osimertinib followed by collection and western blot analysis of EGFR expression in lysates 24, 48 or 72 hours later. Further, HCC827 and PC9 cells were also treated with Osimertinib and actinomycin D for 0 to 24 hours and examined for EGFR expression via western blot. These experiments confirmed that Osimertinib had no detectible effect on EGFR stability (FIG. 11A- 11E). [0273] Next, experiments were performed to determine whether RTK SUMOylation was specific to EGFR kinase inhibition or a general feature of kinase inhibition of RTKs. It was found that RTK SUMOylation can be detected upon kinase inhibition of other RTKs as well. Specifically, BT474 and OE19 cells were treated with 1 pM lapatinib, H1703 and H661 cells were treated with 1 pM Imatinib, H1993 and EBC-1 cells were treated with 1 pM capmatinib, HepG2 and Hep3B cells were treated with 1 pM linsitinib, and RT112 and RT4 cells were treated with 1 pM infigratinib for 2 hours, 6 hours, 12 hours or 24 hours before cells were collected, lysed, immunoprecipitated with an anti-HER2 antibody, anti- PDGFRa antibody, anti- MET antibody, anti-IGF1-R antibody or anti-FGFR3 antibody, respectfully, and analyzed by Western Blot. FIGs. 1L-1U show that these alternative kinase inhibitors also induced SUMOylation at their target receptors. For example, kinase inhibition of HER2 with lapatinib, leads to HER2 SUMOylation in the HER2 amplified cell lines OE19 and BT474 (Fig. 1 L-M). Inhibition of PDGFRA with imatinib in the PDGFR amplified cell lines H1703 and H661 leads to PDGFR SUMOylation (Fig. 1N-O). Inhibition of MET with capmatinib in the Met amplified lines H1993 and EBC1 results in MET SUMOylation (Fig. 1 P-Q), while inhibition of IGF-1 R in HepG2 and Hep3B cells with linsitinib resulted in IGF1-R SUMOylation (Fig. 1R-S). Finally, inhibition of FGFR3 in RT112 and RT4 cell lines with infigratinib resulted in SUMOylation of FGFR3 (Fig. 1T-U). These data indicated that multiple RTKs rapidly become SUMOylated following tyrosine kinase inhibition.

Example 3 - EGFR SUMOylation is required for osimertinib-induced upregulation of TNF and activation of NF-KB

[0274] This example presents data showing that there are functional consequences of TKI-induced SUMOylation in various cell lines.

[0275] Previous studies have reported that TNF upregulation and NF-KB activation is a universal response to EGFR inhibition in EGFR mutant NSCLC. In fact, it was previously reported that TNF upregulation is a biologically significant and widespread outcome of EGFR inhibition in NSCLC and GBM. In these examples, EGFR mutant NSCLC and its response to osimertinib is used as a representative model to examine of the mechanisms and biological significance of TKI induced RTK SUMOylation.

[0276] In a first set of experiments, it was found that siRNA knockdown of SUMO3 abolished osimertinib-induced TNF upregulation and NF-KB activation in multiple cell lines. Specifically, HCC827, PC9 and H3255 cells were transfected with siRNA SUMO3 or control siRNA for 48 h, followed by doses of osimertinib for 48 hr and then qPCR was performed to detect TNF mRNA. FIG. 2A-2C showed that SUMO3 siRNA suppressed TNF upregulation following Osimertinib treatment. Next, HCC827, PC9 and H3255 cells were transfected with SLIM03 or control siRNA for 48 hr, followed by doses of Osimertinib (50nM for PC9 and H3255, 100nM for HCC827) for 48 hr and then the concentration of TNF was measured in supernatants by ELISA. FIG. 2D-2F show that SLIMO3 siRNA suppressed the increase in TNF protein levels observed after Osimertinib treatment. Finally, HCC827, PC9 and H3255 cells were transfected with SLIMO3 or control siRNA for 48 hr, followed by indicated doses of osimertinib for 24 hr and then NF-KB activity was detected by a dual luciferase reporter assay. Renilla luciferase was used as an internal control. FIG. 2G-2I show that siSUMO3 treatment again suppressed the increase in NF-KB activity observed following Osimertinib treatment.

[0277] In a second set of experiments, neither TNF upregulation nor NF-KB activation were observed upon siRNA knockdown of EGFR. Specifically, HCC827 and PC9 cells were treated with Osimertinib or transfected with EGFR siRNA for 48 hr before a qPCR analysis or ELISA was performed to detect TNF mRNA regulation and expression levels respectively. Again, GAPDH was used as an endogenous control. FIGs. 2J and FIG. 2K show that siRNA knockdown of EGFR did not result in an upregulation of TNF that was observed with Osimertinib treatment, a result that was corroborated by TNF protein levels in cell supernatant (FIG. 2K and FIG. 2M). Finally, HCC827 and PC9 cells treated with Osimertinib or transfected with EGFR siRNA were tested for NF-KB activity using a dual-luciferase reporter assay using renilla luciferase as internal control. FIG. 2N and FIG. 20 show that EGFR knockdown (via treatment with EGFR siRNA for 48 hours) again does not result in the same increase in NF- KB activity observed following Osimertinib treatment over the same time frame. Collectively, these data suggest that this adaptive response to EGFR TKI inhibition requires the presence of the kinase-inhibited EGFR.

[0278] In summary, the above data shows that siRNA knockdown of SUMO3 abolished osimertinib-induced EGFR SUMOylation and blocked TNF upregulation and NF-KB activation in multiple cell lines. Importantly, TNF upregulation and NF-KB activation were not detected upon siRNA knockdown of the EGFR, suggesting that the adaptive response to EGFR TKI inhibition requires the presence of the kinase inhibited EGFR. These data indicated that the cellular response to EGFR TKIs is distinct from siRNA knockdown of the EGFR.

Example 4 - TRIM28 is the E3 ligase for EGFR SUMOylation in response to osimertinib

[0279] A number of proteins have been reported to have E3 ligase activity in the SUMO pathway. As shown above in FIG. 1 A, it was found that TRIM28 becomes associated with the EGFR in response to osimertinib in EGFR mutant lung adenocarcinoma HCC827 cells. TRIM28 is known to have E3 SUMO ligase activity so a series of experiments were performed to evaluate its role in EGFR SUMOylation in response to osimertinib. First, it was confirmed that TRIM28 associated with the EGFR upon exposure to osimertinib by treating HCC827 and PC9 cells (both EGFR mutant NSCLC cell lines) with 100 nM and 50 nM Osimertinib, respectively, followed by immunoprecipitation with TRIM28 antibody and Western blot (FIG.

3A-3B).

[0280] Next, HCC827 and PC9 cells were transfected with siRNA control and siRNA TRIM28, then treated with 100 nM and 50 nM osimertinib, respectively, followed by immunoprecipitation with EGFR antibody and Western blot. It was found that TRIM28 was knockdown inhibited that osimertinib-induced EGFR SUMOylation (FIG. 3C-3D). These data support an essential role for TRIM28 in osimertinib-induced EGFR SUMOylation. In another experiment, HCC827 and PC9 cells were transfected with siRNA control and siRNA UBC9, then treated by 100 nM and 50 nM osimertinib, respectively, followed by immunoprecipitation with EGFR antibody and Western blot. This experiment showed that siRNA knockdown of Ubc9, the known SUMO-conjugating enzyme also blocked osimertinib-induced EGFR SUMOylation (FIG. 3E-3F).

Example 5 - Identification of the site of osimertinib-induced EGFR SUMOylation

[0281] As noted, the previous examples establish that SUMO3, but not SUMO1 and SUMO2, becomes associated with the EGFR following treatment with osimertinib (FIG. 1A- 1F, FIG. 10A-10D). This Example describes experiments performed to identify the site of Osimertinib-induced SUMOylation on the EGF Receptor.

[0282] K37 has been identified as a major site of SUMOylation of the wild type EGFR under basal conditions. Specifically, it was shown that EGFR was associated with SUMO-1 in the nucleus and with SUMO-2/3 in the cytoplasm. Therefore, using the most common EGFR mutations found in NSCLC (EGFR del 19 and L858R) as a template, the K37 residue was mutated. To examine the effects of the EGFR K37 mutant without the confounding effect of endogenous EGFR, this mutant receptor was introduced into H661 cells which do not express endogenous EGFR. CRISPR mediated gene editing was also used to knockout endogenous EGFR in H1975 cells (which normally express the L858R/T790M EGFR mutant). The H661 and H1975 EGFR knockout lines were then transfected to stably express EGFR del mutant, EGFR del 19 mutant with K37R mutation, EGFR L858R mutant, or EGFR L858R mutation with K37R mutation. Stable expression of EFFR in all transfected cell lines was confirmed with western blot (FIG. 4A-4B).

[0283] First, it was confirmed that mutants are functional by measuring EGF induced ERK activation via western blot. Specifically, EGFR_del and EGFR_delK37R expressing H661 cells, EGFR_del and EGFR_delK37R expressing H1975_EGFRKO cells, L858R and L858R_K37R expressing H661 cells, and L858R and L858R_K37R expressing EGFRKO H1975 cells were treated with EGF (50 ng/mL for 5 minutes) or control (Mock) followed by western blot and other antibodies. EGF induced robust levels of pERK in all treated cells (FIG.

4A-4C and FIG. 12A-12B) .

[0284] Next, it was found that the K37R mutation in the EGFR del 19 mutant or the L858R mutant resulted in a substantial inhibition of osimertinib induced SUMOylation in H661 and H1975_EGFRKO cells (Fig. 4E-4F, FIG. 12C-12D). In this experiment, EGFR_del, EGFR_del K37R, L758R and L858R_K37R expressing H661 cells and H1975_EGFRKO cells were treated with 100 nM osimertinib followed by IP with EGFR antibody, then Western blot with SLIMO3 antibody, as described previously. As shown in FIG. 4E, FIG. 4F and FIG. 12C-12D ., mutating the K37 SUMOylation site resulted in a loss of EGFR SUMOylation in response to osimertinib, confirming that the K37 site is the major site of EGFR sumoylation in response to osimertinib.

[0285] Next, it was found that osimertinib (100 nM) induced upregulation of TNF (measured by qPCR detection of TNF mRNA in treated cells as described above) and activation of NF-KB (measured by a NF-KB dual luciferase assay as described above) are blocked in H661 cells and H1975_EGFRKO cells expressing EGFR_del, EGFR_del K37R, L758R and L858R_K37R (FIG. 4G-4J and FIG. 12E-12H). This data provided strong evidence that osimertinib induces TNF upregulation and NF-KB activation requires SUMOylation of the kinase inactive EGFR.

[0286] Next to examine the effect of the K37R mutation on EGFR SUMOylation in oncogene addicted lines expressing endogenous mutant EGFR, HA tagged EGFR del 19 mutant or del 19 plus K37R double mutant were transfected into HCC827 and PC9 cells before treatment with Osimertinib (100 nM and 50 nM, respectively). This was followed by immunoprecipitation with HA antibodies and immunoblot with SUMO3 antibodies (FIG. 4K- 4L). The results again demonstrated that the major site of osimertinib induced EGFR SUMOylation is K37 and that EGFR SUMOylation is required for osimertinib induced TNF upregulation and NF-KB activation.

[0287] K37 is an extracellular residue which raises the question of how EGFR becomes

SUMOylated on this residue. Importantly, previous studies support a cytoplasmic localization of NSCLC EGFR tyrosine kinase mutants including a constitutive endocytosis and cytoplasmic localization, and a rapid internalization of membrane-associated EGFR with EGFR TKIs. Therefore, localization of EGFR in HCC827 and PC9 cells was examined by treating HCC827 and PC9 cells with 100 nM and 50 nM Osimertinib, respectively, for 0, 2 or 6 hours before cell fractionation and Western blot. It was found that EGFR is found in multiple cellular compartments including plasma membrane and the cytoplasm, (FIG. 13A-13B). Thus, without wishing to be bound by theory, it is likely that EGFR SUMOylation in response to osimertinib occurs in the cytosol.

Example 6: EGFR inhibition leads to a SUMOylation dependent activation of ALK

[0288] EGFR mutations and ALK fusions are generally mutually exclusive in NSCLC. Thus, it was quite surprising that multiple proteins that form oncogenic fusions with ALK in NSCLC became rapidly associated with the kinase inhibited EGFR with osimertinib treatment (FIG. 1A). First, it was confirmed that the ALK fusion proteins TFG, EML-4 and TPM4, which in certain tumors can form genetic fusions with ALK (and are thus referred to here as “ALK fusion proteins”) bound to the EGFR in EGFR mutant NSCLC lines following osimertinib treatment by coimmunoprecipitation studies (FIG. 5A-5F). In these experiments, HCC827 and PC9 cells were treated with 100 nM and 50 nM Osimertinib, respectfully, followed by immunoprecipitation with TFG, EM4 or TPM4 antibody and then western blot.

[0289] The association of these proteins suggest that ALK is activated in response to EGFR kinase inhibition. In support of this, it was found that ALK is expressed in EGFR mutant NSCLC cell lines and activated in response to osimertinib (FIG. 5G-5H, FIG. 14A-14B). At the same time, no concomitant activation of other RTKs such as MET or ERBB2 was detected in this experiment, although a reduction in activation of both RTKs can be detected suggesting that mutant EGFR drives activation of these RTKs in these oncogene addicted cell lines (FIG. 5G-5H). In these experiments, phosphorylated ALK (p-ALK), MET (p-MET) and ERBB2 (p- ERBB2) were measured by western blot of cell lysates after 2 to 24 hours of Osimertinib treatment as a measure of ALK, MET and ERBB2 activity. Further, it was found that this ALK activation persisted and could be detected after 30 days of osimertinib treatment in the HCC4190 EGFR mutant PDX (FIG. 5I).

[0290] Interestingly, the osimertinib-induced pALK activation is lost with siRNA knockdown of SUMO3, suggesting that EGFR SUMOylation is required for ALK activation (FIG. 5J-5K). To confirm the requirement for EGFR SUMOylation in recruitment of ALK fusion proteins, H661 cells expressing EGFR del19 or EGFR del 19plus K37 mutants were examined. It was found that osimertinib readily induced an association of TFG, EML4 and TPM4 with the EGFR in H661 cells expressing the EGFR deletion mutant but not in the del 19K37R mutant (FIG. 5L-5N). Consistent with this result, osimertinib-induced ALK activation was detected in H661 cells expressing EGFR dell 9 mutant but not the EGFR del19/K37R mutant (FIG. 50). A similar result was obtained in 1975 EGFR knockout cells reconstituted with EGFR dell 9 or EGFR del19/K37R mutant (FIG. 5P-S). Additionally, siRNA knockdown of any of the ALK-fusion partners in EGFR mutant NSCLC lines (e.g., TFG, EML-4 and TPM4) resulted in a loss of ALK activation after treatment with 100 nM osimertinib (FIG. 5T-5Y). Importantly, siRNA knockdown of the EGFR failed to activate ALK (FIG. 14A-14D). suggesting that presence of the kinase inactive EGFR is required for ALK activation.

[0291] These data suggest that osimertinib-induced EGFR SUMOylation at K37 is required for binding of ALK fusion proteins and ALK activation.

Example 7: RTK inhibition of multiple RTKs results in ALK activation

[0292] This example presents data demonstrating that activation of ALK occurs in response to TKIs directed at other RTKs. Remarkably, ALK activation was detected upon inhibition of HER2 with lapatinib in BT474 and in OE-19 cells (FIG. 6A-6B). Lapatinib-induced (1 pM) ALK activation induced in breast and esophageal cancer cell lines can be inhibited by siRNA knockdown of SUMO3 supporting a requirement for HER2 SUMOylation in ALK activation in these cell lines (FIG. 6A-6B). Similarly, PDGFR inhibition with imatinib (1 pM) in H1703 and H661 lung cancer cell lines resulted in ALK activation that could be inhibited by siRNA knockdown of SUMO3 (FIG. 6C-6D). Also, MET inhibition with capmatinib (1 pM) resulted in ALK activation in EBC-1 or H1993 lung cancer cell lines which could be inhibited by siRNA knockdown of SUMO3 (FIG. 6E-6F). Similarly, FGFR3 inhibition with infligratinib (1 pM) and IGFR1 R inhibition with linsitinib (1 pM) resulted in ALK activation (FIG. 6G-6J). The mechanism of ALK activation with inhibition of these RTKs appears to be similar to EGFR and representative experiments demonstrated a rapid association of ALK fusion proteins with the kinase-inhibited RTK (Fig. 6K-6N).

Example 8: EGFR SUMOylation and ALK activation in secondary resistant cell lines

[0293] Cell line models of secondary resistance to EGFR TKIs are useful tools to study mechanisms of resistance. Therefore, a series of experiments were performed to examine EGFR mutant cell lines that have rendered experimentally resistant to EGFR TKIs. Two types of secondary resistant lines were used - specifically, HCC827 ER (erlotinib resistant) cell lines and H1975 OR (osimertinib resistant) cell lines. It was found that all EGFR TKI resistant lines tested had a striking increase in EGFR SUMOylation, as measured by immunoprecipitation with EGFR antibody and immunoblotting with SUMO3 antibody (FIG. 7A-7B). We have previously reported that TNF is upregulated in the ER resistant lines. Additionally, these lines have increased ALK activation compared to the parental lines, as measured by western blot (FIG. 7C-7D) and showed increased NF-KB activity as measured using a dual-luciferase assay, as compared to parental lines (FIG. 16A-16B). ALK activation was also increased in the HCC4190 PDX rendered experimentally resistant to osimertinib (FIG. 7E).

[0294] To determine the effect of SUMOylaton on resistance to TKI, SUMO3 was silenced by siRNA for 48 hours before administration of osimertinib. Treated cells were then monitored with AlamarBlue cell viability assay. Importantly, siRNA knockdown of SLIMO3 enhances sensitivity to osimertinib in secondary resistant cell lines (cell viability curves), demonstrating that EGFR SUMOylation plays an important role in resistance to osimertinib treatment (FIG. 7F-7G). These data support a model in which EGFR SUMOylation and ALK activation play a key role in resistance to EGFR TKIs.

Example 9 - EGFR SUMOylation and ALK activation induce resistance to osimertinib treatment in EGFR mutant NSCLC models.

[0295] Cell survival assays in vitro and animal xenograft tumor growth studies in vivo were performed to evaluate the biological significance EGFR SUMOylation and ALK activation following Osimertinib treatment.

[0296] In a first experiment, HCC827 and PC9 cells were transfected with siRNA SUMO3 or control siRNA for 48 hours followed by increasing doses of osimertinib for 72 hours before quantification of cell viability by alamarBlue assay. SUMO3 siRNA expression was confirmed by western blot. It was found that cells treated with SUMO3 siRNA and osimertinib had lower viability than those treated with osimertinib alone, demonstrating that siRNA to SUMO3 sensitized these cells to osimertinib (FIG. 8A- 8B).

[0297] In a second experiment, EGFR_del EGFR_del K37R, EGFR_L858R, and EGFR_L858R K37R expressing H661 and H1975_EGFR cells were treated with increasing doses of osimertinib for 72 hours (one dose per group) and cell viability was measured by alamarBlue assay. Data from this experiment shows that expression of the SUMOylation defective K37 del 19 mutant EGFR in cell lines that do not express endogenous EGFR resulted in enhanced sensitivity to osimertinib compared to expression of del 19 mutant alone (FIG. 8C-8D) suggesting EGFR SUMOylation confers resistance to EGFR TKIs. Similar results were found with H661 or H1975 EGFR knockout cells expressing the EGFR L858R mutant or EGFR L858R plus K37 mutant (FIG. 8E-8F).

[0298] In a third experiment, whether EGFR SUMOylation influences the biological response to osimertinib in vivo was tested using mouse models of EGFR mutant NSCLC. Athymic mice were injected s.c with 2x10⁶ EGFR_del or EGFR_del K37R stably expressing H1975_EGFR KO cells. When tumors formed, mice were randomly divided into 4 groups (Del_Ctrl vehicle, EGFR_del osimertinib, Del_K37R_Ctrl vehicle, and Del_K37R osimertinib, n=6). The indicated mice were treated with 1 mg/kg osimertinib by oral gavage for 30 consecutive days. It was found that tumors generated from H1975 EGFR knockout cells stably expressing the EGFR del19/K37R double mutant were more responsive to osimertinib treatment compared to H1975 EGFR knockout tumors expressing the single del 19 mutant (FIG. 8G). A similar result was found in H661 cells expressing the double mutant (EGFR del19/K37R) compared to the single del 19 mutant (FIG. 8H). These experiments suggest that EGFR SUMOylation confers resistance to osimertinib treatment.

Example 10: Combining RTK inhibitor and ALK inhibitor has synergistic anti-tumor effect compared to effect of either inhibitor receptor alone.

[0299] Next, it was examined if osimertinib-induced ALK activation was biologically significant. First, a combination of osimertinib and alectinib, an ALK inhibitor used clinically in current practice, was tested in an EGFR mutant expressing PDX model. Specifically, HCC4190 EGFR_L858R NSCLC PDX cells were implanted subcutaneously into NOD/SCID mice. When tumors formed, mice were divided into 4 groups (n = 6) and treated with(a) 5 mg/kg Osimertinib, (b) 10 mg/ kg Alectinib, or (c) 5 mg/kg Osimertinib and 10 mg/ kg Alectinib for 30 days. Tumor growth was measured over the course of the treatment. It was found that the combination treatment was significantly more effective than osimertinib alone in this PDX model (FIG. 8I). Similar results were found in PC9 tumors in athymic mice (formed by injection with 5 x10⁶ PC9 cells) treated with (a) 0.25 mg/kg Osimertinib, (b) 10 mg/kg Alectinib, or (c) 0.25 mg/kg Osimertinib and 10 mg/kg Alectinib via oral gavage for 30 consecutive days. As in the PDX model, a combination of EGFR plus ALK inhibition was more effective than osimertinib alone used at low concentrations (FIG. 8J). The role of ALK in enhancing sensitivity to Osimertinib was tested in a mouse model. Specifically, in these experiments PC9 and HCC827cells were stably infected with lentivirus control shRNA (Ctrl) or shRNA for ALK lentivirus and silencing was confirmed by western blot. Cells with stable silencing of ALK or control were subcutaneously injected into 6 athymic mice per group. Osimertinib was administered orally at 0.1 mg/kg and 0.25 mg/kg, respectively. Stable silencing of ALK in HCC827 or PC9 cells also resulted in enhanced sensitivity to osimertinib in a mouse model (FIG. 8K-8L) and in vitro (FIG. 17C-17D).

[0300] Additionally, a HCC4190 PDX model that is completely resistant to osimertinib was generated by implanting HCC4190 EGFR_L858R osimertinib-resistant PDX cells subcutaneously into NOD/SCID mice. When tumors formed, mice were divided into 4 groups (n = 6) and treated with 5 mg/kg osimertinib or/with 10 mg/ kg Alectinib for 30 days. It was found that a combination of osimertinib and alectinib resulted in effective suppression of tumor growth in this Osimertinib resistant model (FIG. 8M).

[0301] Additional synergistic studies were conducted with Lorlatinib and Osimertinib. In a first set of experiments, in vitro, cell viability of cell lines treated with EGFR inhibitors with and without ALK inhibitor was evaluated. Specifically, in a first experiment, HCC827 and PC9 cells were treated with Osimertinib alone or with Lorlatinib before evaluation with an AlamarBlue assay after 72 hours. FIG. 17A-17B show that cells treated with a combination of Osimertinib and Lorlatinib had significantly reduced viability compared to cells treated with either inhibitor alone. The combination of Lorlatinib and Osimertinib in vivo was also tested. Specifically, HCC4190 EGFR_L858R NSCLC PDX was implanted subcutaneously into NOD/SCID mice. When tumors formed mice were divided into 4 groups (n=6) and treated with 5 mg/kg Osimertinib or 10 mg/kg lorlatinib or both for 40 days. It was found that there was a significant reduction in tumor growth over the treatment period in mice treated with both Osimertinib and lorlatinib (FIG. 17E).

[0302] Thus, data from multiple mouse models and cell culture studies confirmed that TKI- induced EGFR SUMOylation is biologically significant. Furthermore, silencing ALK results in enhanced sensitivity to osimertinib and a combination of EGFR plus ALK inhibition can overcome osimertinib-resistance in mouse models.

Example 11 - EGFR SUMOylation in TKI treated NSCLC patient tissues

[0303] In another experiment, a proximity ligation assay (PLA) was used to detect SUMOylation of EGFR in cultured cells (see Materials and Methods in Example 14 for protocol). It was found that EGFR and SUMO-3 interact in response to osimertinib (FIG. 9A). EGFR-SUMO3 interaction was also detected in HCC827 mouse tumors treated with osimertinib (FIG. 9B). More importantly, EGFR SUMOylation was detected in resected tissue from post TKI treated patient tissue. Both TKI naive and - TKI treated tissue (e.g., from patients treated with erlotinib or Osimertinib) were examined. Evidence of EGFR SUMOylation was consistently found in TKI treated tumor tissue but not in TKI naive tissue as shown in representative sections from 3 TKI naive and 3 TKI treated patients and also paired pre- and post-TKI tissues from the same patient (FIG. 9C-9F).

Example 12 - Additional Evidence of ALK activation in EGFR TKI treated tumor tissue

[0304] EGFR mutations and ALK fusions are generally mutually exclusive. Patients with EGFR mutations are treated with EGFR TKIs such as osimertinib. Patients with ALK fusion are treated with ALK TKIs such as alectinib. The data shown in these examples indicate that osimertinib induced inhibition of mutant EGFR in NSCLC leads to a rapid ALK activation. It was shown in earlier examples that ALK is persistently activated in EGFR mutant PDX HCC4190 derived mouse tumors by Western blot (FIG. 5K). In this example, ALK activation was detected in osimertinib treated HCC827 and HCC4190 PDX tumors by IHC (FIG. 9G-9J). Additionally, increased ALK activation was detected in TKI-treated resected patient tissues compared to TKI-naTve samples (n=27), including in one pair of tissues from the same patient (FIG. 9K-9L, FIG. 15). Table 1 below summarizes quantification of p-ALK IHC staining in human paraffin sections visually depicted in FIG. 9L. Table 1 : Quantification of p-ALK IHC staining in human paraffin sections visually depicted in FIG. 9L

Example 13 - Discussion of Examples 1 to 12

[0305] Targeted therapy using tyrosine kinase inhibitors represents a major advance in the treatment of multiple cancers. By inhibiting kinase activity, TKIs shut down receptor signal transduction. The major finding of this study is that, in contrast to prior assumptions, receptor tyrosine kinases do not become inert upon TKI treatment. Instead, they undergo a rapid SUMOylation, which in turn, leads to an alternate signal transduction in which the kinase- inhibited RTK now functions as a hub to continue to signal as an adaptor platform. Thus, the term “escape RTK signaling” is provided as a term for this continued signal transduction by kinase-inhibited receptors. Escape RTK signaling is distinct from the well documented concept of bypass RTK signaling in which a different RTK becomes activated when a given RTK is inhibited by its specific TKI. An important outcome of protein SUMOylation is that SUMO acts as a platform that recruits new interacting proteins to the SUMOylated protein. The findings herein indicate that SUMOylation of the kinase inhibited RTK triggers activation of specific adaptive signals that promote resistance. The SUMOylation of RTKs in response to TKI inhibition is detected with multiple receptors and TKIs. in multiple cancer cell lines with RTK amplfications. Examples include, EGFR/osimertinib, MET/capmanitnib, HER2/lapatinib, and PDGFR/imatinib. These data suggested that RTK SUMOylation may be a general outcome of RTK inhibition by TKIs. However, since TKIs are clearly effective in multiple oncogene- addicted cancers by suppression of RTK kinase activity, the escape RTK signaling transduced by the kinase inactive SUMOylated RTK cannot be identical to signaling generated by the noninhibited receptor and may play role in secondary resistance which emerges later. Additionally, continued signal transduction by kinase inhibited SUMOylated RTKs could be an important factor underlying the limited effectiveness of TKIs in general, and could also play a role in intrinsic or primary resistance.

[0306] These examples focused on signal transduction by the kinase-inhibited EGFR in EGFR mutant NSCLC as a representative disease model. Surprisingly, osimertinib treatment induces a recruitment of multiple proteins to the kinase inhibited EGFR in lung cancer cells. SLIMO3 is among these proteins, and it was found that the mutant EGFR becomes rapidly SUMOylated in response to osimertinib. Further experiments identified the major site of osimertinib-induced EGFR SUMOylation as K37 and found that mutation of this EGFR SUMOylation site or siRNA knockdown of SLIMO3 results in a loss of key adaptive signals triggered by osimertinib such as TNF-NF-KB and ALK and enhances the sensitivity of EGFR mutant tumors to osimertinib in mouse models. A study of cell lines rendered secondarily resistant to EGFR TKIs showed a large increase in EGFR SUMOylation in every line tested and a reversal of TKI resistance upon siRNA knockdown of SUMO3. Further, a proximity ligation assay (PLA demonstrated that EGFR and SUMO3 associate in post-TKI treated mouse tumors and human NSCLC tissue. SUMOylation has no effect on EGFR stability.

[0307] It has been shown that inhibition of RTKs leads to a rapid activation of multiple intracellular signaling pathways, such as TNF- NF-KB, ERK, or STAT3 activation, which may promote the survival of persister clones. What triggers these early compensatory signals is a fundamentally important and unanswered question. The data in these examples indicate that the kinase-inactive EGFR itself serves as the nidus to trigger the adaptive response. Thus, TNF upregulation and NF-KB activation and ALK activation are readily detected following treatment with EGFR TKIs but, importantly, not upon siRNA knockdown of the EGFR. These findings suggested that the cellular response to EGFR kinase inhibition is distinct from loss of EGFR expression.

[0308] Data in these examples also show that ALK activation is an important preferential outcome of TKI inhibition of multiple receptors. Although bypass RTK signaling in response to RTK inhibition is well described, the ALK activation trigged by the kinase inhibited SUMOylated RTK has unique features. First, ALK activation is remarkably widespread having been detected with TKI treatment of all receptors tested. Second, ALK activation occurs early, generally detected within 2-6 hours of RTK treatment initiation. Tellingly, no activation of other RTKs such as MET or HER2 was detected at this early time point. The mechanism of ALK activation is also rather distinct. Upon treatment with TKIs, ALK and ALK fusion proteins become associated with multiple RTKs using the SUMOylated RTK as a platform and leading to ALK activation. The recruitment of multiple ALK fusion proteins and ALK activation also appears to be a general outcome of RTK inhibition using TKIs. This ALK activation is biologically significant and knockdown of ALK from NSCLC cancer cell lines enhances sensitivity to osimertinib, and a combined EGFR plus ALK inhibition results in more effective treatment in mouse NSCLC models. Further, siRNA knockdown of ALK fusion partners inhibits ALK activation in response to EGFR inhibition. Mechanistically, the ALK activation is likely triggered not by genetic fusion but by oligomerization with its fusion partners since siRNA knockdown of ALK fusion partners inhibits TKI-induced ALK activation. siRNA knockdown of the RTK does not result in ALK activation, supporting the idea that the kinase inactive RTK itself serves as the nidus for ALK activation. Furthermore, RTK SUMOylation is required for TKI-induced ALK activation. ALK activation can trigger many canonical downstream signaling pathways that could mediate the resistance to EGFR inhibition. ALK activation can also be detected in osimertinib treated tumor tissue, in both experimental xenografted tumor tissue as well as post-TKI treated human NSCLC tissue. These data suggest that ALK activation is a bonafide and widespread outcome of TKI treatment in multiple cancers. Therefore, a combined treatment targeting EGFR and ALK should be effective in EGFR mutant NSCLC and other cancers. Since the initial treatment of EGFR mutant NSCLC with osimertinib is quite effective and well tolerated, and because of concerns of combined toxicity and cost, pragmatically the best time to add alectinib could be when osimertinib resistance emerges. Importantly, our data indicate that ALK activation is a general outcome of RTK inhibition with TKIs and may also be useful in other cancers that are treated with TKIs.

[0309] TKIs have been widely used for cancer treatment for decades, but their effectiveness is limited due to therapeutic resistance. The findings disclosed herein suggested that tyrosine kinase inhibition does not abrogate signaling by the kinase-inhibited RTK. While the kinase activity was suppressed by TKIs, the kinase inhibited RTK becamee SUMOylated and continued to signal. Thus, the SUMOylated RTK escapes the constraints of kinase inhibition by transforming into an adaptor signaling platform. It is proposed that this post-TKI SUMOylation of RTKs serves a key mechanism underlying the rapid compensatory signaling changes that have been extensively documented following TKI exposure. Our study also raises a more fundamental question about the best approach to inhibiting oncogenic RTKs in cancer with the finding that tyrosine kinase inhibition does not shut down RTK signaling, and instead continues to signal as an adaptor that generating survival signals. Thus, combination therapies such as those combining RTK inhibitor with an ALK inhibitor may be more effective. Regardless, the identification of key mechanisms driving resistance to TKIs, such as RTK SUMOylation and ALK activation, may continue to enhance effectiveness of targeted treatment using TKIs

Example 14 - Materials and Methods

[0310] Cell lines and drugs. OE19, RT4, EBC-1 , HepG2 and Hep 3B was purchased from Millipore-Sigma. RT112 was purchased from CLS Cell Lines Service GmbH. BT474 was purchased from the American Type Culture Collection. HCC827/ER3, HCC827/ER4(A) have been described previously (Gong, K., et al., J Clin Invest (2018) 128, 2500-2518, which is incorporated herein by reference in its entirety). H1975/OR5 and H1975 OR16 were provided by Dr. John V. Heymach (MD Anderson Cancer Center, Houston), and the method of generating these lines has been described previously Le, X Clin Cancer Res (2018) 24, 6195- 6203, which is incorporated herein by reference in its entirety). All other NSCLC cell lines including HCC827, PC9, H3255, H661 , H1975, H1993 and H1703 were from the Hamon Center for Therapeutic Oncology Research at UT Southwestern Medical Center. HepG2 and Hep 3B were cultured in DM EM containing 10% FBS. OE19 and RT112 were cultured in RPMI-1640 containing 10% FBS. RT4 was cultured in McCoy’s 5A containing 10% FBS. All other cell lines were cultured in RPMI-1640 containing 5% FBS. Cell lines were authenticated by DNA fingerprints for cell-line individualization using Promega Stem Elite ID system, a short tandem repeat (STR)-based assay, at UT Southwestern Medical Center Genomics Core. Cells were tested for Mycoplasma contamination using an e-Myco kit (Boca Scientific).

[0311] Osimertinib (16237), Lapatinib (11493), Imatinib (mesylate) (13139), Capmatinib (20056), Linsitinib (17708), Infigratinib (19157) Alectinib (18516), Lorlatanib (18371), Crizotinib (31114), were obtained from Cayman Chemical.

[0312] siRNA and shRNA lentiviral particles. Human EGFR (L-003114-00-0005), TRIM28 (L-005046-00-0005), SUMO3 (L-019730-00-0005), ALK_1 (L-003103-00-0005), EML4 (L-008398-01-0005), TFG (L-016366-00-0005), TPM4 (L-019753-00-0005) siRNA were obtained from Dharmacon. siRNA pool from Dharmacon contain four sequences for each target. Human ALK_2 (sc-40083), UBC9 (sc-36773) and scrambled siRNA (sc-37007) were purchased from Santa Cruz Biotechnology. siRNA pool from Santa Cruz Biotechnology contain three sequences for each target. Cells were transfected with the siRNA pool using Lipofectamine RNAiMAX (Invitrogen). Experiments were conducted 48 h after siRNA transfection.

[0313] Human ALK shRNA lentiviral particles (sc-40083- V) and control lentiviral particles (sc-437282) were obtained from Santa Cruz Biotechnology. To generate HCC827 and PC9 cells with stable shRNA-mediated ALK knockdown, the cells were infected with ALK shRNA or control lentiviral particles. Clones were selected in culture containing 0.8 .g/ml and 1.5 .g/ml puromycin, knockdown efficiency was determined by western blot.

[0314] Construction of lentiviral vectors and cell lines. DNA fragments encoding proteins of interest were amplified by PCR with Q5 Hot Start High-Fidelity 2 x Master Mix. The amplified DNA fragments were purified and cloned into pLX317 vector (Nhel/Mlul sites) using NEBuilder HiFi DNA Assembly Master Mix. Inserted sequences were further validated by Sanger sequencing. EGFR^K37R was generated by replacing K37 with R using PCR.

[0315] To package lentivirus, HEK293T cells were seeded in a 6-well plate at a confluency of ~ 90% were transfected with 1.2 ug pLX137 plasmid carrying the gene of interest, together with 0.5 ug envelope plasmid pMD2.G and 0.8 ug packaging plasmid psPAX2 using Effectene transfection reagent according to manufacturer’s manual. Culture medium were collected 48 hr post transfection and filtered through 0.45 mm sterilized Millex-HV Syringe Filters (Millipore) as lentivirus preparation. Lentivirus infection was carried out by adding lentivirus into cell culture medium supplemented with 2 mg/mL protamine sulfate. 48 hr post infection, lentivirus was removed by changing with fresh culture medium. In all experiments, lentivirus amount was titrated to achieve expression level comparable to that of endogenous proteins, as confirmed by western blotting or quantitative real-time PCR.

[0316] CRISPR-Cas9 genomic editing for gene deletion was performed as previously described. For deletion of the gene encoding EGFR, two TrueGuide Mod EGFR-sgRNA (ThermoFisher, A35533) and TrueCut Cas9 Proteins (ThermoFisher, A36498) were transfected by Neo Tranfection Kit (ThermoFisher, MPK10025K).

[0317] Western blot and antibodies. Western blot was performed according to standard protocols. Western blot results were representative of at least three independent experiments. EGFR (06-847) antibody was from EMD Millipore; p-EGFR(Tyr1068) (2236), p-ERBB2 (Tyr1221/1222) (2243), ERBB2 (4290), HA-Tag (2367), Phospho-p44/42 MAPK (Erk1/2) (4370), p44/42 MAPK (Erk1/2) (4696), TIF1 p (KAP1/TRIM28) (4123), UBC9 (4786) , p-MET (Tyr1234/1235) (3077), MET (8198), p-PDGF Receptor a (Tyr754) (2992), PDGF Receptor a (5241), Lamin A/C (4777), E-Cadherin (3195), EML4 (12548), p-ALK (Tyr 1604) (3341), ALK (3633), p-IGF-l Receptor p (Tyr1135/1136)/lnsulin Receptor p (Tyr1150/1151) (3024) and IGF-I Receptor p (9750) antibodies were from Cell Signaling Technology; ALK (ab275964), TPM4 (ab181085) were from Abeam; p-actin (sc-47778), SUMO-3 (sc-130884), SUMO-1 (sc- 5308), FGFR-3 (sc-390423) and GAPDH (sc-47724) were from Santa Cruz Biotechnology. SUMO-2 (VMA00340) was from Bio-Rad. TFG (A302-341A) was from Bethyl Laboratories. Phospho-FGFR3 (Y724) (AP1274) was from ABclonal Technology.

[0318] Immunoprecipitation and Immunoblot Analysis of SUMOylation. Cells were lysed with 100 ul of denaturing lysis solution (0.15 M Tris-HCI, pH 6.7, 5% SDS, and 30% glycerol), which was then diluted 1 :10 in PBS/0.5% NP40 plus complete protease inhibitor (Roche). If the lysate was highly viscous, sonication was performed until viscosity was reduced. Lysates were centrifuged at 16,000g for 10 min at 4 °C to remove cellular debris. Immunoprecipitation was performed with anti-EGFR beads (CST, #5735), HA-Tag beads (3956), MET beads (SantCruz, sc-8057 AC), FGFR-3 beads (SantCruz, sc-390423 AC), ERBB2 beads (SantCruz, sc-33684), PDGF Receptor a beads (SantCruz, sc-398206) and IGF-1 R (SantCruz, sc-462) for overnight at 4 °C. Thereafter, the precipitants were washed four times with washing buffer (PBS/0.5% NP40 plus complete protease inhibitor), and the immune complexes were 2 x SDS sample buffer and separated on a SDS-PAGE, followed by Immunoblot analysis. [0319] Real-time PCR and primers. cDNA synthesis and real-time PCR Total RNA was isolated by TRIzol Reagent (Fisher Scientific). cDNA reverse transcription was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Each PCR reaction was carried out in triplicate in a 20- l volume using SYBR Green Master Mix (Applied Biosystems). Values for each gene were normalized to expression levels of GAPDH mRNA GAPDH. Primer sequences were as follows. GAPDH, 5’-GTGAAGGTCGGAGTCAACGG-3’ (SEQ ID NO: 1 , forward), 5’-TGATGACAAGCTTCCCGTTCTC-3’ (SEQ ID NO: 2, reverse); TNF, 5’-CCCAGGGACCTCTCTCTAATCA-3’ (SEQ ID NO: 3, forward), 5’- GCTACAGGCTTGTCACTCGG-3’ (SEQ ID NO: 4, reverse). These primers were synthesized by Millipore-Sigma.

[0320] Cell viability assay. Cell viability assays were conducted with alamarBlue cell viability reagent from Thermo Fisher Scientific, following the manufacturer’s protocol. Cells were cultured in Corning 96-well black plates with clear bottom and detected by the POLARstar Omega Microplate Reader (BMG LABTECH) (excitation at 544 nm and emission at 590 nm). At least three independent experiments were performed.

[0321] Luciferase assays. Cells were transfected with NF-KB and Renilla luciferase plasmid by Lipofectamine 2000. Renilla luciferase was co-transfected as an internal control. A dual-luciferase reporter assay system (Promega) was used according to the manufacturer’s protocol. Firefly luciferase activity was measured in the POLARstar Omega Microplate Reader (BMG LABTECH) and normalized based on Renilla luciferase activity. NF-KB luciferase plasmid was provided by Ezra Burstein (UT Southwestern).

[0322] ELISA. To detect TNF levels in medium, cells were cultured in serum-free medium and treated with indicated drugs for 48 h. Supernatant was then collected and concentrated using a Pierce protein concentrator (Thermo Fisher Scientific). Then, the levels of TNF protein were measured by ELISA using human TNF ELISA kit (BD Bioscience, 550610) according to the manufacturer’s protocol.

[0323] Immunohistochemistry and DuoLink in Situ Proximity Ligase Assay (PLA). Formalin-Fixed Paraffin-Embedded (FFPE) tissue slides were dewaxing and antigen retrieval as described previously. Immunohistochemistry analysis was performed using the phospho- ALK antibody (Invitrogen, PA5-40168) and ABC streptavidin-biotin method with a Vectastain ABC kit (Vector Laboratories) as described previously. The PLA assay was conducted according to the manufacturer’s instructions. Two primary antibodies (SUMO3, santa cruz sc- 130884 and EGFR, invitrogen PA1-1110) raised in different species are used to detect two unique protein targets. A pair of oligonucleotide-labeled secondary antibodies (PLA probes) then binds to the primary antibodies. Next, hybridizing connector oligos join the PLA probes only if they are in close proximity to each other and ligase forms a closed, circle DNA template that is required for rolling-circle amplification (RCA). The PLA probe then acts as a primer for a DNA polymerase, which generates concatemeric sequences during RCA. This allows up to 1000-fold amplified signal that is still tethered to the PLA probe, allowing localization of the signal. Lastly, labeled oligos hybridize to the complementary sequences within the amplicon, which are then visualized and quantified as discrete spots (PLA signals). Analysis was conducted with a Laser scanning confocal Zeiss LSM780 microscope, at 40* magnification.

[0324] Animal study. Cell lines. Athymic mice (088) at 4-6 weeks old were purchased from Charles River Laboratories. Mice were injected with 100 pl Matrigel containing 5 x 10⁶ H661 (stable cell lines derived) and PC9 (including stable cell lines derived) cells in the right flank. Two million (2 x 10⁶) HCC827 (including stable cell lines derived) and H1975 (stable cell lines derived) were injected subcutaneously into the flanks of Athymic mice. About 2 weeks later, mice developed subcutaneous tumors. Mice were randomly divided into the indicated groups. Mice were treated with drugs using doses described in figure legends. For combination treatment, both drugs were given concurrently for the indicated period. Tumor dimensions were measured every 4 d and tumor volumes were calculated by the formula: volume = 0.5 x length x width x width. Mice were euthanized when tumors reached >2000 mm³ in length or after the indicated number of days.

[0325] PDX. NSCLC specimens (P0) for HCC4190 PDXs were surgically resected from a patient diagnosed with NSCLC at UT Southwestern Medical Center, after obtaining Institutional Review Board (IRB) approval and informed consent. HCC4190 harbors EGFR L858R mutation identified by exome sequencing. The 4-6-week-old female NOD-SCID mice (001303) were purchased from Jackson Laboratory. PDX tumor tissues were cut into small pieces (~20 mm³) and subcutaneously implanted into NOD-SCID mice of serial generations (P1 , P2 and so on). P4 tumor-bearing SCID mice were used in this study. To generate resistant PDX isogenic models, HCC4190 PDX models were continuously treated with osimertinib for 3 in vivo passages (weekly treated with increasing doses of osimertinib: 5, 10, 20 mg/kg). The corresponding untreated PDX was passaged in parallel as corresponding sensitive model.

[0326] Patient data. Patient formalin-fixed, paraffin-embedded (FFPE) tissues were obtained from UT Southwestern Medical Center (n = 29), including two paired patients slides. Twenty slides were from patients treated with TKI prior to the tissue collection (15 slides are from erlotinib treated patients and 5 slides are from osimertinib treated patients), while nine were never treated with TKI before. [0327] Study approval. All animal studies were performed under Institutional Animal Care and Use Committee-approved protocols at UT Southwestern Medical Patient tissues and medical records were obtained from UT Southwestern Medical Center with IRB approval.

[0328] Statistics. Error bars represent the mean ± s.e.m. of three independent experiments unless indicated otherwise. Combination effects in vivo were analyzed by two- way ANOVA with Bonferroni’s correction to adjust significance level for multiple comparisons. ELISA experiments were analyzed by two-tailed one-sample Student’s t-test, two-tailed two- sample Student’s t-test. IHC staining experiments were analyzed by Mann-Whitney U test. The family-wise error rate was set at 0.05. All analyses above were performed using GraphPad Prism 9 software. P value, or an adjusted P value for multiple comparisons, <0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, p > 0.05, not statistically significant). Data from cell survival assay, qPCR assay and luciferase assay were presented as mean lines with three dots from three cell cultures within one.

Claims

CLAIMS What is claimed is:

1. A method of treating a cancer in a subject, the method comprising administering to the subject in need thereof at least one receptor tyrosine kinase (RTK) inhibitor that does not inhibit ALK, and at least one ALK inhibitor and wherein the subject has or is suspected of having a malignant tumor.

2. The method of claim 1 , wherein the malignant tumor comprises a carcinoma, a sarcoma, a hematological malignancy or any combination thereof.

3. The method of either claim 1 or claim 2, wherein the malignant tumor comprises a lung tumor, a breast tumor, a pancreatic tumor, a colorectal tumor, an esophageal tumor, a head- and-neck tumor, a hematological malignancy, a brain tumor, an ovarian tumor, a stomach tumor, a gastric tumor, a gastrointestinal stromal tumor, a prostate tumor, a thyroid tumor, bladder tumor, dermatofibrosarcoma protuberans, a giant cell tumor, a lymphoma, melanoma, a myeloma, a kidney tumor, a soft tissue sarcoma, chronic eosinophilic leukemia (CEL), chronic lympocytic leukemia (CLL), urothelial carcinoma, a cervical tumor, liver or bile duct tumor, endometrial tumor or any combination thereof.

4. The method of any one of claims 1 to 3, wherein the malignant tumor is derived from a non-small-cell lung cancer (NSCLC), HER-2 positive breast cancer, glioblastoma, gastrointestinal stromal tumor, esophageal tumor, liver or bile duct tumor, bladder tumor or any combination thereof.

5. The method of any one of claims 1 to 3, wherein the malignant tumor has one or more somatic mutations, translocations, amplifications, and/or other disruptions in at least one gene encoding a receptor tyrosine kinase that is not ALK.

6. The method of claim 5, wherein the at least one gene encoding a RTK that is not ALK comprises an EGFR gene, a HER2 gene, a HER3 gene, a HER4 gene, a c-Met gene, a R0S1 gene, a IGF-1R gene, a TRKA gene, a TRKB gene, a TRKC gene, a PDGFR gene, a VEGFR gene, a FGFR gene, a FLT3 gene, a NTRK gene, a RET gene or any combination thereof.

7. The method of any one of claims 1 to 6, wherein the malignant tumor expresses one or more hyperactive or overactive receptor tyrosine kinases (RTKs) that are not ALK.

8. The method of claim 7, wherein the one or more receptor tyrosine kinases are selected from EGFR, HER2, HER3, HER4, c-Met, ROS1 , IGF-1 R, TRKA, TRKB, TRKC, PDGFR, VEGFR, FGFR, FLT3, NTRK, and RET.

7. The method of any one of claims 1 to 6, wherein the malignant tumor does not have any somatic mutations and/or translocations in an ALK gene.

8. The method of any one of claims 1 to 7, wherein the at least one ALK inhibitor comprises at least one of a peptide, an antibody, a small molecule pharmaceutical compound, an oligo, a nucleic acid molecule, or a combination thereof.

9. The method of any one of claims 1 to 8, wherein the at least one ALK inhibitor comprises a small molecule pharmaceutical compound.

10. The method of any one of claims 1 to 9, wherein the at least one ALK inhibitor comprises Alectinib, Lorlatinib, Brigatinib, Ceritinib, TAE684 (NVP-TAE684), AP26113 analog (ALK-IN-1), GSK1838705A, AZD3463, ASP3026, Entrectinib, Crizotinib, or any combination thereof.

11 . The method of any one of claims 1 to 10, wherein the at least one RTK inhibitor inhibits an epidermal growth factor receptor (EGFR), a platelet-derived growth factor (PDGF)/Kit receptor, a vascular endothelial growth factor receptor (VEGFR), a fibroblast growth factor (FGF) receptor, a hepatocyte growth factor (HGF receptor), a mesenchymal-epithelial transition factor receptor (MET), an insulin or insulin-like growth factor (IGF) receptor or any combination thereof.

12. The method of any one of claims 1 to 11 , wherein the at least one RTK inhibitor comprises an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, a pan- HER inhibitor, a MET inhibitor, an insulin or insulin-like growth factor (IGF) receptor inhibitor, an FGF receptor inhibitor, a PDGFR receptor inhibitor or any combination thereof.

13. The method of any one of claims 1 to 12, wherein the at least one RTK inhibitor is an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, or a pan-HER inhibitor.

14. The method of any one of claims 1 to 13, wherein the at least one RTK inhibitor comprises least one of a peptide, an antibody, small molecule pharmaceutical compound, an oligo, a nucleic acid molecule, or a combination thereof.

15. The method of any one of claims 1 to 14, wherein the at least one RTK inhibitor comprises a small molecule pharmaceutical compound or a monoclonal antibody.

16. The method of any one of claims 1 to 15, wherein the at least one RTK inhibitor comprises Osimertinib, Imatinib, Capmatinib, Linsitinib, Infigratinib, Avapritinib, Pemigatinib, Ripretinib, Selpercatinib, Tucatinib, Entrectinib, Erdafitinib, Pexidartinib, Dacomitinib, Gilteritinib, Acalabrutinib, Midostaurin, Neratinib, Cobimetinib, Lenvatinib, Nintedanib, Afatinib, Trametinib, Axitinib, Cabozantinib, Ponatinib, Regorafenib, Tofacitinib, Vandetanib, Pazopanib, Lapatinib, Nilotinib, Dasatinib, Sunitinib, Sorafenib, Erlotinib, Gefitinib, Imatinib, Varlinitib, Infigratinib, Mobocertinib, Pralsetinib, Tepotinib, Tivozanib, Trastuzumab, Aflibercept, Larotrectinib, Trastuzumab, Pertuzumab, Amivantamab, Bevacizumab, Margetuximab, Necitumumab, Ramucirumab, Panitumumab, Cetuximab or any combination thereof.

17. The method of any one of claims 1 to 16, wherein the at least one RTK inhibitor comprises Osimertinib, Imatinib, Capmatinib, Linsitinib, Infigratinib, Erlotinib, Gefitinib, Afatinib, Dacomitinib, Lapatinib, Neratinib, Varlitinib, Tucatinib, Trastuzumab, Pertuzumab, Cetuximab or any combination thereof.

18. The method of any one of claims 1 to 17, wherein the at least one RTK inhibitor comprises Osimertinib, Erlotinib, Gefitinib, Afatinib, Dacomitinib, Lapatinib, Neratinib, Varlitinib, Trastuzumab, Pertuzumab, Cetuximab, or any combination thereof.

19. The method of any one of claims 1 to 18, wherein malignant tumor cells in the malignant tumor have a higher rate of cell death in the subject compared to an untreated subject with identical disease condition and predicted outcome.

20. The method of any one of claims 1 to 19, wherein the incidence of treatment resistance to the RTK therapy is decreased in the subject compared to a subject treated with the RTK inhibitor that does not inhibit ALK alone, wherein the subject has identical disease condition and predicted outcome.

21 . A method of treating a tumor in a subject in need thereof comprising administering to the subject at least one ALK inhibitor, wherein the subject has undergone, is undergoing or will undergo an anti-cancer therapy comprising one or more targeted receptor tyrosine kinase (RTK) inhibitors that do not target ALK.

22. The method of claim 21 , wherein the tumor is resistant to the anti-cancer therapy comprising one or more targeted RTK inhibitors.

23. The method of claim 21 or 22, wherein the tumor comprises lung tumor, a breast tumor, a pancreatic tumor, a colorectal tumor, an esophageal tumor, a head-and-neck tumor, a hematological malignancy, a brain tumor, an ovarian tumor, a stomach tumor, a gastric tumor, a gastrointestinal stromal tumor, a prostate tumor, a thyroid tumor, bladder tumor, dermatofibrosarcoma protuberans, a giant cell tumor, a lymphoma, melanoma, a myeloma, a kidney tumor, a soft tissue sarcoma, chronic eosinophilic leukemia (CEL), chronic lympocytic leukemia (CLL), urothelial carcinoma, cervical tumor, a liver or bile duct tumor, endometrial tumor, or any combination thereof.

24. The method of any one of claims 21 to 23, wherein the tumor is non-small cell lung cancer (NSCLC), HER-2 positive breast cancer, glioblastoma, gastrointestinal stromal tumor, esophageal tumor, liver or bile duct tumor, bladder tumor or any combination thereof.

25. The method of any one of claims 21 to 24, wherein the tumor has one or more somatic mutations, translocations, amplifications, and/or other disruptions in at least one gene encoding a receptor tyrosine kinase that is not ALK.

26. The method of claim 25, wherein the at least one gene encoding a RTK that is not ALK is selected from EGFR, HER2, HER3, HER4, c-Met, ROS1, IGF-1R, TRKA, TRKB, TRKC, PDGFR, VEGFR, FGFR, FLT3, NTRK, RET, and any combination thereof.

27. The method of any one of claims 21 to 26, wherein the tumor does not have any somatic mutations and/or translocations in an ALK gene.

28. The method of any one of claims 21 to 27, wherein the at least one ALK inhibitor comprises at least one of a peptide, an antibody, a small molecule pharmaceutical compound, an oligo, a nucleic acid molecule, or a combination thereof.

29. The method of any one of claims 21 to 28, wherein the at least one ALK inhibitor comprises a small molecule pharmaceutical compound.

30. The method of any one of claims 21 to 29, wherein the at least one ALK inhibitor is selected from the group consisting of Lorlatinib, Brigatinib, Alectinib, Ceritinib, TAE684 (NVP- TAE684), AP26113 analog (ALK-IN-1), GSK1838705A, AZD3463, ASP3026, Entrectinib, and Crizotinib

31. The method of any one of claims 21 to 30, wherein the targeted RTK inhibitor targets an EGFR/ERBB receptor, a platelet-derived growth factor (PDGF)/Kit receptor, a vascular endothelial growth factor receptor (VEGFR), a fibroblast growth factor (FGF) receptor, a hepatocyte growth factor (HGF receptor), a mesenchymal-epithelial transition factor receptor (MET), or an insulin or insulin-like growth factor (IGF) receptor.

32. The method of any one of claims 21 to 31 , wherein the targeted RTK inhibitor is an EGFR/ERBB inhibitor.

33. The method of any one of claims 21 to 31 , wherein the targeted RTK inhibitor is an EGFR inhibitor, a (PDGF)/Kit receptor inhibitor, an FGF receptor inhibitor, an IGF receptor inhibitor, a MET inhibitor or any combination thereof.

34. The method of any one of claims 21 to 33, wherein the targeted RTK inhibitor comprises least one of a peptide, an antibody, a small molecule pharmaceutical compound, an oligo, a nucleic acid molecule, or a combination thereof.

35. The method of any one of claims 21 to 34, wherein the targeted RTK inhibitor comprises a small molecule pharmaceutical compound or a monoclonal antibody.

36. The method of any one of claims 21 to 35, wherein the targeted RTK inhibitor is selected from Osimertinib, Lapatinib, Avapritinib, Capmatinib, Pemigatinib, Ripretinib, Selpercatinib, Selumetinib, Tucatinib, Entrectinib, Erdafitinib, Fedratinib, Pexidartinib, Upadacitinib, Zanubrutinib, Baricitinib, Binimetinib, Dacomitinib, Fostamatinib, Gilteritinib, Larotrectinib, Acalabrutinib, Midostaurin, Neratinib, Cobimetinib, Lenvatinib, Nintedanib, Afatinib, Ibrutinib, Trametinib, Axitinib, Bosutinib, Cabozantinib, Ponatinib, Regorafenib, Tofacitinib, Ruxolitinib, Vandetanib, Pazopanib, Nilotinib, Dasatinib, Sunitinib, Sorafenib, Erlotinib, Gefitinib, Imatinib, Varlinitib Trastuzumab, Pertuzumab, Cetuximab Imatinib, Capmatinib, Linsitinib, and Infratinib.

37. The method of any one of claims 21 to 36, wherein the targeted RTK inhibitor is selected from Osimertinib, Lapatinib, Erlotinib, Gefitinib, Afatinib, Dacomitinib, Neratinib, Varlitinib Trastuzumab, Pertuzumab, Cetuximab, Imatinib, Capmatinib, Linsitinib, and Infratinib.

38. The method of any one of claims 21 to 37, wherein the targeted RTK inhibitor is selected from the group consisting of Osimertinib, Lapatinib, Erlotinib, Gefitinib, Afatinib, Dacomitinib, Neratinib, Varlitinib Trastuzumab, Pertuzumab, and Cetuximab.

39. A method of preventing sensitization and/or resistance to an anti-cancer therapy comprising a receptor tyrosine kinase (RTK) inhibitor, the method comprising administering to a subject in need thereof, an ALK inhibitor prior to or concurrent with the anti-cancer therapy comprising the RTK inhibitor.

40. The method of claim 39, wherein the RTK inhibitor is an EGFR inhibitor, a (PDGF)/Kit receptor inhibitor, an FGF receptor inhibitor, an IGF receptor inhibitor, a MET inhibitor or any combination thereof

41. The method of claim 40, wherein the RTK inhibitor is an EGFR inhibitor.

42. The method of any one of claims 39 to 41, wherein the subject responds to the RTK inhibitor over a longer time period than a subject that did not receive the ALK inhibitor.

43. The method of any one of claims 1 to 42, wherein the subject is a human.

44. A method of increasing sensitivity of a cancer cell to a receptor tyrosine kinase (RTK) inhibitor comprising: contacting a cancer cell with at least one ALK inhibitor, where the cancer cell is sensitized and/or resistant to a receptor tyrosine kinase (RTK) inhibitor, with at least one ALK inhibitor increases the cell’s sensitivity to an RTK inhibitor as compared to a cell not contacted with the ALK inhibitor.

45. The method of claim 44, wherein cell survival of the cancer cell is decreased after contacting the cancer cell with at least one ALK inhibitor.

46. The method of claim 44 or 45, wherein cell proliferation of the cancer cell is decreased after contacting the cancer cell with at least one ALK inhibitor.

47. The method of any one of claims 44 to 46, wherein the cancer cell is located in or is obtained from a lung tumor, a breast tumor, a pancreatic tumor, a colorectal tumor, an esophageal tumor, a head-and-neck tumor, a hematological malignancy, a brain tumor, an ovarian tumor, a stomach tumor, a gastric tumor, a prostate tumor, a thyroid tumor, bladder tumor, a lymphoma, melanoma, a kidney tumor, a soft tissue sarcoma, chronic eosinophilic leukemia (CEL), chronic lympocytic leukemia (CLL), urothelial carcinoma, cervical tumor, a liver or bile duct tumor, endometrial tumor, or any combination thereof.

48. The method of any one of claims 44 to 47, wherein the cancer cell is located in or is obtained from a non-small cell lung cancer (NSCLC) tumor, a HER-2 positive breast cancer tumor, a glioblastoma, a gastrointestinal stromal tumor, an esophageal tumor, a liver or bile duct tumor, a bladder tumor or any combination thereof.