Methods of treating TKI resistance and SDH-deficient GIST

Abstract

Provided herein are methods of treating diseases, disorders, or conditions associated with succinate dehydrogenase (SDH) deficiency using a multi-target tyrosine kinase inhibitor (TKI). Also provided herein are methods of treating diseases using a multi-target TKI in combination with a Bcl-2 inhibitor.

Description

Technical Field

[0001] This invention relates to methods for treating diseases, disorders, or conditions associated with succinate dehydrogenase (SDH) deficiency using multi-target tyrosine kinase inhibitors (TKIs). This invention also relates to methods for treating diseases using a combination of multi-target TKIs and BCL-2 inhibitors. Background Technology

[0002] Gastrointestinal stromal tumors (GISTs) are the most common malignant tumors of the gastrointestinal mesenchyme (Unc, M. et al., Cancers (Basel) 15,1498 (2023)). Approximately 85% to 90% of the pathogenesis was coded. KIT Or platelet-derived growth factor receptor α (PGF receptor α) PDGFRA Oncogenic mutations in the gene for tyrosine kinases drive the process. Other mutations are not present. KIT or PDGFRA Mutations were found in wild-type (WT) GISTs, with succinate dehydrogenase (SDH) deficient GISTs being the most common, accounting for 5% to 7.5% of all GISTs (Unc, M. et al.). Cancers (Basel) 15,1498 (2023)). SDH-deficient GIST typically results from a germline and / or somatic loss-of-function mutation in any of the four SDH subunits (A, B, C, or D) (Pasini, B. et al., Eur. J. Hum. Genet., 16, 79–88 (2008)), or due to SDHC promoter methylation in the tumor, which leads to silencing of SDH expression. Clinically, compared with KIT / PDGFRA mutant GIST, SDH-deficient GIST tends to be multifocal and presents an indolent clinical course, even in advanced or metastatic stages (Nannini, M. et al., Ther. Adv. Med. Oncol. 13, 17588359211023278 (2021)).

[0003] For patients with SDH-deficient GIST, surgery is the only potentially curative treatment option (Unc, M. et al.). Cancers (Basel) 15,1498 (2023)). Other treatment alternatives include systemic or targeted therapies, such as tyrosine kinase inhibitors (TKIs). The introduction and use of TKIs have improved the treatment prospects for GIST, especially imatinib as... KIT or PDGFRAFirst-line treatment for patients with imatinib mutations. Other TKIs such as sunitinib and regorafenib have been approved as second- and third-line treatment options for patients developing imatinib resistance. However, the role of TKIs in SDH-deficient GIST patients remains controversial, with limited published results. Imatinib has no proven efficacy for this type (Unc, M. et al., Cancers (Basel) 15,1498 (2023)). Other approved TKIs have limited efficacy, and studies of these drugs in metastatic SDH-deficient GIST have shown median progression-free survival (PFS) of less than 10 months (Nannini, M. et al., Ther. Adv. Med. Oncol. 13, 17588359211023278 (2021)). These factors highlight the unmet clinical need of SDH-deficient GIST patients, especially those who are insensitive to previous PKI treatments. Summary of the Invention

[0004] Throughout this disclosure, the articles “a,” “a kind,” and “the” are used herein to refer to one or more of the grammatical objects of the article (i.e., at least one). For example, “method” means one or more methods.

[0005] Throughout this disclosure, all references cited herein are incorporated herein by reference in their entirety.

[0006] The inventors of this application have now discovered that administration of a multi-target TKI (e.g., olabatinib) is particularly effective in treating diseases, disorders, or conditions associated with SDH deficiency (e.g., GIST), especially in subjects who are insensitive to at least one prior TKI treatment. The inventors of this application have also discovered that the combined administration of a multi-target TKI (e.g., olabatinib) with a BCL-2 inhibitor (e.g., levofloxacin) is particularly effective in treating cancer (e.g., GIST).

[0007] In one aspect, this disclosure provides a method for treating a subject with a disease, disorder, or condition associated with succinate dehydrogenase (SDH) deficiency, the method comprising administering to the subject a therapeutically effective amount of a multitarget tyrosine kinase inhibitor (TKI) thereby treating the subject with the disease, disorder, or condition associated with SDH deficiency.

[0008] On the other hand, this disclosure provides a method for identifying subjects suffering from diseases, disorders, or conditions associated with succinate dehydrogenase (SDH) deficiency and for selecting treatment for the identified subjects, the method comprising: a) Determine whether the biological sample from the subject contains an SDH deficiency; b) Based on the SDH deficiency identified in step a), identify the subject as having a disease, disorder, or condition related to the SDH deficiency, and c) It is recommended that the subject receive treatment with a multi-target TKI.

[0009] In some implementations, the multi-target TKI includes olabatrinib.

[0010] In some implementations, the disease, disorder, or condition associated with SDH deficiency is cancer (e.g., SDH-deficient cancer).

[0011] In some implementations, the cancer is selected from: gastrointestinal cancer (e.g., gastrointestinal stromal tumor (GIST)), cholangiocarcinoma, lung cancer, melanoma, breast cancer, colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), bladder cancer, pancreatic cancer, kidney cancer, esophageal cancer, head and neck cancer, thyroid cancer, squamous cell carcinoma of the skin, glioblastoma, neuroblastoma, urethral bladder cancer, hysterocarcinoma, melanoma, osteosarcoma, lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma), leukemia (e.g., T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia (ALL)), multiple myeloma, uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma. Squamous cell carcinoma of the lung, cervical cancer, esophageal cancer, sarcoma, chromophobe carcinoma, renal cell carcinoma, clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low-grade glioma (LGG), mesothelioma, pheochromocytoma, paraganglioma, thymoma, and adenoid cystic carcinoma (ACC).

[0012] In some implementations, the cancer is SDH-deficient GIST.

[0013] In some implementations, the SDH-deficient cancer is selected from SDH-deficient ALL, renal cell carcinoma, colon cancer, pheochromocytoma, thyroid adenoma (e.g., papillary thyroid adenoma, multifocal papillary thyroid adenoma, differentiated thyroid carcinoma, papillary (follicular variant) thyroid adenoma, follicular thyroid adenoma), pancreatic neuroendocrine tumor / pancreatic paraganglioma, breast cancer (e.g., triple-negative breast cancer), neuroblastoma, follicular lymphoma, B-cell lymphoma, Hodgkin lymphoma, complex paraganglioma / neuroblastoma, rectal adenocarcinoma, Burkitt lymphoma, thyroid paraganglioma, adrenocortical carcinoma, adrenal adenoma-like tumor, neuroblastoma, melanoma, synovial sarcoma, squamous cell carcinoma of the lung, embryonal rhabdomyosarcoma, lung adenocarcinoma, prostate adenocarcinoma, endometrial cancer, poorly differentiated gastric cancer, bladder urothelial carcinoma, colon adenocarcinoma, sigmoid colon adenocarcinoma, testicular seminoma, and pituitary adenoma.

[0014] In some implementations, the subject has been identified as having an SDH deficiency.

[0015] In some implementations, SDH deficiency is identified in biological samples from the subject.

[0016] In some implementations, the biological sample is a tissue (e.g., tumor tissue), a fluid (e.g., whole blood, plasma, PBMC), or any biological sample that can be used to detect germline / somatic mutations in the SDH gene.

[0017] In some implementations, SDH deficiency is determined by immunohistochemistry, Western blotting, amplification-based assays, hybridization-based assays, or sequencing.

[0018] In some implementations, SDH deficiency is indicated by the presence of one or more loss-of-function mutations in SDHA, SDHB, SDHC, and SDHD or any combination thereof, or by SDHC promoter methylation, which leads to reduced SDH enzyme activity, instability of the SDH protein complex, or reduced SDH expression.

[0019] In some implementations, the one or more loss-of-function mutations include mutations selected from missense substitutions, deletions, insertions, translocations, and premature stop codons, or any combination thereof.

[0020] In some implementations, the one or more loss-of-function mutations in SDHB include one or more mutations selected from p.A15T, p.W218Ter, and SDHB KO exon 2+3 (heterozygote).

[0021] In some implementations, the one or more loss-of-function mutations in the SDHB include one or more mutations selected from c.17_42dup, c.71_72insA, c.136C>T, c.137G>A, c.274T>A, c.301delT, c.380T>G, c.423+1G>A, c.423+20T>A, c.566G>T, c.600G>T, c.643-1G>C, c.725G>A, and c.778_779insG, or any combination thereof.

[0022] In some implementations, the one or more loss-of-function mutations in SDHA include one or more mutations selected from p.G184R and p.P279S.

[0023] In some implementations, the one or more loss-of-function mutations in SDHA include: c.91C>T, c.113A>T, c.160C>T, c.511C>T, c.533C>T, c.457–3_457–1delCAG, c.562C>T, c.628C>T, c.666del, c.688delG, c.767C>T, c.800C>T, c.818C>T, c.923C>T, c.985C>T, c.1043–1055del, c.1046_1047delTG, c.1054C>T, c.1151C>G, c.1215_1216insCC, c.1255G>A, c.1334C>T, c.1352G>A, c.1360G>A, c.1361C>A, c.1367C>T, c.1468delG, c.1513delA, c.1532T>C, c.1534C>T, c.1753C>T, c.1765C>T, c.1766G>A, c.1690G>A, c.1794G>C, c.1795–1G>T, c.1798C>T, and c.1799G>A, or any combination thereof.

[0024] In some implementations, the one or more loss-of-function mutations in the SDHC comprise one or more mutations selected from the following: c.1A>G, c.6delT, c.65G>A, c.160C>A, c.221A>G, c.224G>A, c.238C>T, c.301delT, c.380A>G, c.397C>T, and c.405+1G>A or any combination thereof.

[0025] In some implementations, the one or more loss-of-function mutations in SDHD comprise one or more mutations selected from the following: c.34G>A, c.352delG, c.416T>C, and c.455G>A, or any combination thereof.

[0026] In some implementations, the subject (e.g., the subject's biological sample) has been further identified as having wild-type KIT, wild-type PDGFR, or a combination thereof.

[0027] In some implementations, the subject (e.g., the subject's biological sample) has been identified as having one or more mutations in the NF1 / BRAF / RAS pathway gene, PIK3CA, or a fusion affecting NTRK3, or any combination thereof.

[0028] In some implementations, the subject (e.g., a biological sample of the subject) has been identified as overexpressing VEGFR or FGF ligands.

[0029] In some implementations, the subject has been identified as insensitive to at least one existing TKI.

[0030] In some implementation schemes, subjects do not achieve complete remission (CR), partial remission (PR), or stable disease (SD) within 6 months of receiving at least one existing TKI treatment; or achieve initial remission or stable disease after ≥6 months of receiving at least one existing TKI treatment, but subsequently experience disease progression.

[0031] In some implementations, the subject has been identified as insensitive to at least one of the following existing TKIs: imatinib, riprinib, avatinib, crenolanib, masitinib, ponatinib, dasatinib, nilotinib, sunitinib, regorafenib, sorafenib, anlotinib, surufatinib, pazopanib, rogatinib, infelgratinib, erdatinib, pemitinib, fisogatinib, and famitinib, or any combination thereof.

[0032] In some implementations, the subject has been identified as insensitive to at least one existing TKI selected from the following: ripretinib, dasatinib, sunitinib, regorafenib, anlotinib, and famitinib, or any combination thereof.

[0033] In some embodiments, the multi-target TKI is capable of inhibiting at least two tyrosine kinases selected from the following: KIT, PDGFR, SRC, VEGFR (e.g., any one of VEGFR1, VEGFR2, and VEGFR3 or any combination thereof) and FGFR (e.g., any one of FGFR1, FGFR2, FGFR3, and FGFR4 or any combination thereof), or any combination thereof.

[0034] In some implementations, the multi-target TKI is capable of inhibiting at least two tyrosine kinases selected from the following: KIT, PDGFR, SRC, VEGFR2, and FGFR1, or any combination thereof.

[0035] In some implementations, this multi-target TKI can inhibit the proliferation of SDH-deficient tumor cells.

[0036] In some implementations, this multi-target TKI has an IC50 effect in inhibiting the proliferation of SDH-deficient tumor cells. 50 Not exceeding 10 μM (e.g., not exceeding 5 μM, 1 μM, 500 nM, 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM or 10 nM), as determined by CCK8 assay or CTG assay.

[0037] In some implementations, SDH-deficient tumor cells are Jurkat clones E6-1, OS-RC-2, RKO, and PC12#5F7, or any combination thereof.

[0038] In some embodiments, the multi-target TKI comprises a heterocyclic acetylenoid compound having the following formula (I): Z is independently selected from CH or N; L 1 Independently selected from NH, -N=, CH; L 2 Independently selected from -CONH- or -NHCO-; R1 is independently selected from: 1) H; 2) C1-C6 alkyl groups; 3) C3-C6 cycloalkyl groups; 4) C1-C5 alkyl groups substituted with one or two hydroxyl groups; 5) Phenyl; 6) It can pass through L at the Z atomic site. 1 Fused A rings to form a ring containing 1-3 N atoms and L 1 Fused five-membered heterocyclic groups, such as , Among them, X, Y, and Z are independently selected from N and CH; ring D is an aromatic heterocycle containing 1-3 N atoms; R2 is independently selected 1) H; 2) Halogens; 3) C1-C5 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C5 alkyl groups; R3 is selected independently 1) H; 2) Halogens; 3) C1-C4 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C4 alkyl groups; When R5 is H, R4 is independently selected. 1) H; 2) (CH2) n NR6R7; 3) (CH2) n -Het 1 ; Alternatively, when R4 is H, R5 is independently selected. 1) H; 2) Het 2 ; n is independently selected from 0 or 1; Het 1 Defined as a non-aromatic heterocycle containing 1-3 N atoms; Het 2 Defined as an aromatic five-membered heterocycle containing heteroatoms such as N, O and / or S; alkyl, cycloalkyl or NR6R7 may be incorporated into any C or N position of substituted non-aromatic heterocycles and aromatic heterocycles; R6 or R7 is selected independently from: 1) H; 2) C1-C3 alkyl groups; 3) F-containing C1-C3 alkyl groups; 4) C3-C6 cycloalkyl groups; Alternatively, R6 and R7 can be further formed into five-, six-, seven-, or eight-membered ring structures through C, O, N, and S atoms; Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0039] In some embodiments, the multi-target TKI is a compound having the following formula (II): (II) Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0040] In some embodiments, the multi-target TKI is a compound selected from the following: olabatinib, ripretinib, avatinib, cristatinib, masatitinib, ponatinib, dasatinib, nilotinib, sunitinib, regorafenib, sorafenib, anlotinib, surufatinib, infuratinib, erdatinib, pemitinib, rogatinib, and lincitinib, or a pharmaceutically acceptable salt or stereoisomer or prodrug of any of the foregoing.

[0041] In some implementations, the multi-target TKI is administered at doses of approximately 10 mg to 200 mg, 10 mg to 150 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 20 mg to 50 mg.

[0042] In some implementations, the multitarget TKI is administered at a dose of approximately 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg.

[0043] In some implementations, the multi-target TKI is administered once daily, every other day, once weekly, once every other week, once monthly, or once every other month.

[0044] In some implementations, the treatment includes one or more 28-day treatment cycles.

[0045] In some implementations, the treatment includes at least 1, 2, 3, 6, 14, 16, or 36 treatment cycles.

[0046] In some implementations, the multi-target TKI is administered via oral or enteral routes (e.g., intravenous, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, or intraocular injection).

[0047] In some implementations, the multi-target TKI is administered orally once every other day during a 28-day treatment cycle.

[0048] In some implementations, the method further includes administering a therapeutically effective amount of one or more additional therapies.

[0049] In some implementations, the multi-target TKI is administered in combination with one or more other therapies for treating diseases, disorders, or conditions.

[0050] In some implementations, the one or more additional therapies include radiation therapy, chemotherapy, targeted cancer therapy, therapy using immune checkpoint molecule modulators, or therapy using BCL-2 inhibitors.

[0051] In some implementations, after treatment with a compound having formula (II), the subject experiences partial remission or disease stabilization.

[0052] In some implementations, after a 28-day treatment cycle with a compound having formula (II), the objective response rate (complete response and partial response) is approximately or greater than 20%, and / or the clinical benefit rate (complete response, partial response and stable disease) is approximately or greater than 90%.

[0053] On the other hand, this disclosure provides a method for treating cancer in a subject, comprising administering to the subject: a) A therapeutically effective dose of a multi-target tyrosine kinase inhibitor (TKI); and b) A therapeutically effective dose of Bcl-2 inhibitors.

[0054] In some implementations, the cancer is selected from: gastrointestinal cancer (e.g., gastrointestinal stromal tumor (GIST)), cholangiocarcinoma, lung cancer, melanoma, breast cancer, colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), bladder cancer, pancreatic cancer, kidney cancer, esophageal cancer, head and neck cancer, thyroid cancer, squamous cell carcinoma of the skin, glioblastoma, neuroblastoma, urethral and bladder cancer, endometrial cancer, melanoma, osteosarcoma, lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma), leukemia (e.g. T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia (ALL), multiple myeloma, uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophageal cancer, sarcoma, chromophobe carcinoma, renal cell carcinoma, clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low-grade glioma (LGG), mesothelioma, pheochromocytoma, paraganglioma, thymoma, and adenoid cystic carcinoma (ACC).

[0055] In some implementations, the cancer is GIST.

[0056] In some implementations, the subjects have been identified as including KIT, PDGFR (For example, PDGFRA and PDGFRB ), SDH (For example, SDHA, SDHB, SDHC and SDHD ), NF1, BRAF, EGFR, HRAS, NRAS, KRAS, ZNF217, MEN1, PIK3CA, ARID1A, ARID1B, FGFR1, FGFR2, FGFR3, FGFR4, SDHAF2, FGFR1-HOOK3, FGFR1-TACC1, NTRK1 (For example, NTRK1 Fusion NTRK2 (For example, NTRK2 Fusion NTRK3 (For example, NTRK3 Fusion KIT- PDGFRA and PRKAR1B-BRAF Or one or more mutations in any combination of them.

[0057] In some implementations, the subject has been identified as insensitive to at least one existing TKI.

[0058] In some implementation schemes, subjects do not achieve complete remission (CR), partial remission (PR), or stable disease (SD) within 6 months of receiving at least one existing TKI treatment; or achieve initial remission or stable disease after ≥6 months of receiving at least one existing TKI treatment, but subsequently experience disease progression.

[0059] In some implementations, the subject has been identified as insensitive to at least one existing TKI selected from the following: imatinib, riprinib, avatinib, cristatinib, masitinib, ponatinib, dasatinib, nilotinib, sunitinib, regorafenib, sorafenib, anlotinib, surufatinib, pazopanib, rogatinib, infelgratinib, erdatinib, pemitinib, fexotinib, and famitinib, or any combination thereof.

[0060] In some implementations, the subject has been identified as insensitive to at least one existing TKI selected from the following: ripretinib, dasatinib, sunitinib, regorafenib, anlotinib, and famitinib, or any combination thereof.

[0061] In some embodiments, the multi-target TKI is capable of inhibiting at least two tyrosine kinases selected from the following: KIT, PDGFR, SRC, VEGFR (e.g., any one of VEGFR1, VEGFR2, and VEGFR3 or any combination thereof) and FGFR (e.g., any one of FGFR1, FGFR2, FGFR3, and FGFR4 or any combination thereof), or any combination thereof.

[0062] In some implementations, the multi-target TKI is capable of inhibiting at least two tyrosine kinases selected from the following: KIT, PDGFR, SRC, VEGFR2, and FGFR1, or any combination thereof.

[0063] In some embodiments, the multi-target TKI comprises a heterocyclic acetylenoid compound having the following formula (I): Z is independently selected from CH or N; L 1 Independently selected from NH, -N=, CH; L 2 Independently selected from -CONH- or -NHCO-; R1 is independently selected from: 1) H; 2) C1-C6 alkyl groups; 3) C3-C6 cycloalkyl groups; 4) C1-C5 alkyl groups substituted with one or two hydroxyl groups; 5) Phenyl; 6) It can pass through L at the Z atomic site. 1 Fused A rings to form a ring containing 1-3 N atoms and L 1 Fused five-membered heterocyclic groups, such as , Among them, X, Y, and Z are independently selected from N and CH; ring D is an aromatic heterocycle containing 1-3 N atoms; R2 is independently selected 1) H; 2) Halogens; 3) C1-C5 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C5 alkyl groups; R3 is selected independently 1) H; 2) Halogens; 3) C1-C4 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C4 alkyl groups; When R5 is H, R4 is independently selected. 1) H; 2) (CH2) n NR6R7; 3) (CH2) n -Het 1 ; Alternatively, when R4 is H, R5 is independently selected. 1) H; 2) Het 2 ; n is independently selected from 0 or 1; Het 1 Defined as a non-aromatic heterocycle containing 1-3 N atoms; Het 2 Defined as an aromatic five-membered heterocycle containing heteroatoms such as N, O and / or S; alkyl, cycloalkyl or NR6R7 may be incorporated into any C or N position of substituted non-aromatic heterocycles and aromatic heterocycles; R6 or R7 is selected independently from: 1) H; 2) C1-C3 alkyl groups; 3) F-containing C1-C3 alkyl groups; 4) C3-C6 cycloalkyl groups; Alternatively, R6 and R7 can be further formed into five-, six-, seven-, or eight-membered ring structures through C, O, N, and S atoms; Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0064] In some embodiments, the multi-target TKI is a compound having the following formula (II): (II) Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0065] In some embodiments, the multi-target TKI is a compound selected from the following: olabatinib, ripretinib, avatinib, cristatinib, masatitinib, ponatinib, dasatinib, nilotinib, sunitinib, regorafenib, sorafenib, anlotinib, surufatinib, infuratinib, erdatinib, pemitinib, rogatinib, and lincitinib, or a pharmaceutically acceptable salt or stereoisomer or prodrug of any of the foregoing.

[0066] In some implementations, the multi-target TKI is administered at doses of approximately 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg.

[0067] In some implementations, the multi-target TKI is administered once daily, every other day, once weekly, once every other week, once monthly, or once every other month.

[0068] In some implementations, the multi-target TKI is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 4 months.

[0069] In some implementations, the multi-target TKI is administered via oral or enteral routes (e.g., intravenous, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, or intraocular injection).

[0070] In some implementations, the multi-target TKI is administered every other day at a dose of approximately 15 mg / kg for 3 weeks.

[0071] In some embodiments, the Bcl-2 inhibitor is a compound having the following formula (III). (III), Or its pharmaceutically acceptable salt or stereoisomer or prodrug, wherein: A3 is selected from ; E3 is nitrogen and It is a single bond; X 31 X 32 and X33 Each independently selected from -CR 38 = and -N=; R 31a and R 31b Together with the carbon atoms they are attached to, they form 3-, 4-, or 5-membered cycloalkyl groups; R 32 Selected from -NO2, -SO2CH3 and -SO2CF3; R 32a Selected from hydrogen and halogens; R 33 Selected from -N(R) 34a (R) 34b ); R 34a Selected from C (optional substitution) 1-6 Alkyl, optionally substituted C 3-6 Cycloalkyl, heterocyclic, heteroalkyl, (cycloalkyl)alkyl, and (heterocyclic)alkyl; R 34b Selected from hydrogen and C 1-4 alkyl; R 38 Selected from hydrogen and halogens; In some implementations, the Bcl-2 inhibitor is selected from: , Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0072] In some embodiments, the Bcl-2 inhibitor is a compound having the following formula (IV). (IV) Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0073] In some implementations, the Bcl-2 inhibitor is administered at doses of approximately 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg.

[0074] In some implementations, the Bcl-2 inhibitor is administered twice daily, once daily, every other day, once weekly, every other week, once monthly, or once every month.

[0075] In some implementations, the Bcl-2 inhibitor is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 4 months.

[0076] In some implementations, the Bcl-2 inhibitor is administered via oral or non-enteric routes (e.g., intravenous, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, or intraocular injection).

[0077] In some implementations, the Bcl-2 inhibitor is administered once daily at a dose of approximately 50 mg / kg for 3 weeks.

[0078] In some implementations, the multi-target TKI and the Bcl-2 inhibitor are administered simultaneously.

[0079] In some implementations, the multi-target TKI and the Bcl-2 inhibitor are administered sequentially.

[0080] In some implementations, the multi-target TKI is administered every other day at a dose of approximately 15 mg / kg for 3 weeks, and the Bcl-2 inhibitor is administered once daily at a dose of approximately 50 mg / kg for 3 weeks.

[0081] In some implementations, the combination of the multi-target TKI and the Bcl-2 inhibitor treats the subject's cancer in an additive or synergistic manner.

[0082] In some implementations, after treatment with a compound having formula (II) in combination with a compound having formula (IV), the subject has a tumor growth inhibition (TGI) rate of at least 75%.

[0083] In some implementations, the combination of this multi-target TKI with the Bcl-2 inhibitor can inhibit the proliferation of imatinib-resistant GIST cells.

[0084] In some implementations, the combination of the multi-target TKI and the Bcl-2 inhibitor can inhibit the proliferation of imatinib-resistant GIST cells in an additive or synergistic manner.

[0085] In some implementations, imatinib-resistant GIST cells are GIST430, GIST48, and GIST48B, or any combination thereof.

[0086] On the other hand, this disclosure provides a method for treating a subject with a disease, disorder, or condition associated with the upregulation of hypoxia-inducible factors (HIF-1α, HIF-2α), vascular endothelial growth factor-A (VEGFA), fibroblast growth factor receptor-1 (FGFR1), phosphorylated FGFR1, insulin-like growth factor-1 receptor (IGF-1R), phosphorylated IGF-1R, STAT3, phosphorylated STAT3, and / or phosphorylated Src, including administering a therapeutically effective amount of a multitarget tyrosine kinase inhibitor (TKI) to the subject, thereby treating the subject with a lipid metabolism-related disease, disorder, or condition.

[0087] In some implementations, the disease, disorder, or condition can be cancer.

[0088] In some implementations, the cancer is selected from: gastrointestinal cancer (e.g., gastrointestinal stromal tumor (GIST)), cholangiocarcinoma, lung cancer, melanoma, breast cancer, colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), bladder cancer, pancreatic cancer, kidney cancer, esophageal cancer, head and neck cancer, thyroid cancer, squamous cell carcinoma of the skin, glioblastoma, neuroblastoma, urethral and bladder cancer, endometrial cancer, melanoma, osteosarcoma, lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma), leukemia (e.g. T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia (ALL), multiple myeloma, uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophageal cancer, sarcoma, chromophobe carcinoma, renal cell carcinoma, clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low-grade glioma (LGG), mesothelioma, pheochromocytoma, paraganglioma, thymoma, and adenoid cystic carcinoma (ACC). Attached Figure Description

[0089] Figures 1A to 1B This study demonstrated the antitumor efficacy of olabatinib in patients with SDH-deficient GIST. Figure 1A This displays treatment duration and best response (RECIST v1.1). Each row represents the treatment duration and best response for a patient. The two columns on the left show each patient's treatment history and gene mutations. (Im, imatinib; Sun, sunitinib; Das, dasatinib; Reg, regorafenib; Rep, ripretinib; Anlo, anlotinib; Fam, famitinib; TMZ, temozolomide. PD, disease progression; PR, partial response; SD, stable disease). Figure 1BThe Kaplan–Meier estimate of progression-free survival (PFS) based on RECIST v1.1 is shown. The median PFS was 25.7 months (range 12.9 – not reached).

[0090] Figures 2A to 2C The efficacy of olaribatitinib in SDH-deficient or knockdown (KD) tumor cells and the molecular pathways regulated by olaribatitinib were demonstrated. Figure 2A This study demonstrated the antiproliferative activity of TKIs in SDH-deficient primary GIST cells. Cells were extracted from tumor tissue of SDH-deficient patients, seeded into 96-well plates, and incubated in GIST medium for 24 hours. Cells were then exposed to olabrutinib and other TKIs for 72 hours, followed by CellTiter 96... ® Aqueous non-radioactive cell proliferation assays are used to detect cell viability. Figure 2B The images show: Left panel, cell death assay in rat pheochromocytoma cells PC12 or PC12#5F7 (SDHBKD). Cells were seeded in 24-well plates, incubated overnight in complete medium, exposed to olaribinib for 24 hours, and then collected for propidium iodide (PI) staining. Right panel, colony formation assay in renal cell carcinoma cells 786O (SDHB-wild type) and OS-RC-2 (SDHB-deficient type). Cells were seeded in 6-well plates and incubated overnight in complete medium. 786O cells were exposed to olaribinib for 7 days (and OS-RC-2 cells for 10 days), and then stained with crystal violet. Figure 2C This study demonstrates the regulation of multiple oncogenic signaling proteins by olabrutinib in SDHB KD PC12#5F7 cells. Cell lysates were collected for Western blot analysis 24 hours after treatment with olabrutinib at the indicated concentration. p, phosphorylation; C, control. Detailed Implementation

[0091] I. Definition In accordance with this disclosure and as used herein, unless otherwise expressly stated, the following terms are defined as follows. Unless otherwise defined, all technical terms, symbols, and other scientific or medical terms used herein are intended to have the meanings commonly understood by those skilled in the art of chemistry and medicine.

[0092] As used in this article, "TKI" is an abbreviation for tyrosine kinase inhibitor, which is an agent that inhibits one or more tyrosine kinases. Tyrosine kinases are important mediators of signaling cascades and play a crucial role in various biological processes such as growth, differentiation, metabolism, and apoptosis in response to external and internal stimuli. The role of tyrosine kinases in the pathophysiology of diseases such as cancer has been discovered and investigated (Manash K. Paul et al., Int J Med Sci., 2004; 1(2): 101–115). Therefore, TKIs are commonly used as anticancer agents. For example, TKIs have shown significant improvement in chronic myeloid leukemia. Tyrosine kinases are mainly classified into receptor tyrosine kinases (RTKs) such as EGFR, PDGFR, FGFR, and IR, and non-receptor tyrosine kinases (NRTKs) such as SRC, ABL, FAK, and Janus kinase. As used herein, “multi-target TKI” refers to a tyrosine kinase inhibitor that can inhibit at least two tyrosine kinases.

[0093] As used herein, the term "biomarker" refers to a biomolecule that serves as a measurable indicator of a biological state or condition. The term "biomarker" as used herein is intended to encompass a target polynucleotide or polypeptide (e.g., a polypeptide encoded by a target polynucleotide). Examples of biomarkers provided herein may be genes (e.g., genomic DNA, cDNA), or the products of a gene such as mRNA transcribed from that gene, and proteins encoded by that gene. Specific examples of biomarkers provided herein include SDHs (including SDHA, SDHB, SDHC, and SDHD).

[0094] The term "level" for biomarkers refers to the amount or quantity of a target biomarker present in a sample. This amount or quantity can be expressed as an absolute measure, i.e., the total amount of the biomarker in the sample, or as a relative measure, i.e., the concentration or percentage of the biomarker in the sample. Biomarker levels can be measured at the DNA level (e.g., as the amount or quantity or copy number of genes in chromosomal regions), at the RNA level (e.g., as the amount or quantity of mRNA), or at the protein level (e.g., as the amount or quantity of proteins or protein complexes).

[0095] The term “activity” in relation to biomarkers refers to the biological activity (e.g., catalytic or regulatory capacity) of a protein as described herein.

[0096] As used herein, “likelihood” and “probability” regarding a subject’s response to treatment are measures of the likelihood that a treatment response will occur in a subject. It is used interchangeably with “probability.” Likelihood refers to a probability greater than speculation but less than certainty. Therefore, a treatment response is likely if a rational person, using common sense, training, or experience, concludes that a treatment response is possible under given circumstances. In one implementation, the terms “likelihood” and “probability” represent the percentage probability of a treatment response occurring. In some implementations, a subject with cancer identified as “likely to respond” means a subject with cancer has a greater than 30% chance, greater than 40% chance, greater than 50% chance, greater than 60% chance, greater than 70% chance, greater than 80% chance, or greater than 90% chance of responding to treatment.

[0097] The terms "responsive" or "reactive" used in the context of a subject's response to cancer therapy are interchangeable and refer to a beneficial response to treatment, not an adverse response, i.e., an adverse event. In subjects, a beneficial response can be represented by many clinical parameters, including detectable disappearance of the tumor (complete remission), a reduction in tumor size and / or the number of cancer cells (partial remission), tumor growth arrest (disease stabilization), an enhanced anti-tumor immune response that may lead to tumor regression or rejection; some relief of one or more tumor-related symptoms; prolonged survival after treatment; and / or a reduced mortality rate at a given time point after treatment. A sustained increase in tumor size and / or the number of cancer cells and / or tumor metastasis indicates a lack of beneficial response to treatment, and therefore reduced responsiveness.

[0098] As used herein, “cancer” or “tumor” are used interchangeably; they are generic terms for a broad range of malignant cellular tumors characterized by unregulated growth, lack of differentiation, and the potential or ability to invade local tissues and metastasize. These malignancies affect every tissue and organ of the body at varying degrees of incidence and can be benign or malignant. Cancer involves the presence of cells with typical characteristics of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain characteristic morphological features. Cancer cells typically exist in the form of solid tumors, but such cells can also exist alone or circulate in the bloodstream as independent cells such as leukemia cells.

[0099] As used in this article, a “pharmaceuticalally acceptable” component is one that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions) and has a reasonable benefit / risk ratio.

[0100] The terms “determination,” “measurement,” and “detection” are used interchangeably and refer to quantitative and semi-quantitative determinations or qualitative determinations.

[0101] The term "hybridization" refers to the binding, double-stranding, or pairing of at least some complementary strands of a nucleic acid molecule. Nucleic acid strands can specifically hybridize with target nucleic acid strands when sufficient complementarity exists to avoid non-specific binding to non-target nucleic acid sequences.

[0102] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to any length of polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or their analogues). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0103] The term "complementarity" refers to the ability of one nucleic acid sequence to pair with another nucleic acid sequence via traditional Watson-Crick or other non-traditional types. Complementarity can be partial or complete. Partial complementarity occurs when one or more nucleic acid bases do not match according to the base pairing rules. The complementarity percentage indicates the percentage of nucleic acid bases in the nucleic acid molecule that can form base pairs with a second nucleic acid sequence (e.g., Watson-Crick base pairing) (e.g., 50%, 60%, 70%, 80%, 90%, and 100% complementarity for 5, 6, 7, 8, 9, and 10 base pairs out of 10).

[0104] As used in this article, the term "prognosis" refers to the prediction or forecast of the future course or outcome of a disease or condition.

[0105] Generally, a "protein" is a polypeptide (i.e., a string of at least two amino acids linked together by peptide bonds). Proteins may include portions other than amino acids (e.g., glycoproteins) and / or may be otherwise processed or modified. Those skilled in the art will understand that a "protein" can be a complete polypeptide chain produced by a cell (with or without a signaling sequence), or it can be its functional portion. Those skilled in the art will also understand that proteins may sometimes comprise more than one polypeptide chain, for example, linked by one or more disulfide bonds or otherwise bound together.

[0106] As used herein, the term "treatment" for a condition includes preventing or alleviating the condition, slowing the onset or rate of occurrence of the condition, reducing the risk of developing the condition, preventing or delaying the occurrence of symptoms associated with the condition, reducing or ending symptoms associated with the condition, producing complete or partial remission of the condition, curing the condition, or some combination thereof. In particular, for cancer, unless otherwise stated, the term "treatment" for cancer as used herein means partially or completely reversing, alleviating, inhibiting, or preventing the progression of or growth of a tumor, metastasis, or other cancerous or tumor-causing cells in a subject.

[0107] As used herein, “combination therapy” is understood to mean the administration of two or more active agents, using a single formulation or a single drug formulation, or in any sequential order, such that the two (or all) active agents exert their biological activity simultaneously over a period of time. This document considers an active agent (e.g., a multi-target TKI) that enhances the activity of a second agent, for example, by sensitizing target cells (e.g., cancer cells) to the activity of the second agent. Combination therapy does not require the agents to be administered at the same time, at the same frequency, or via the same route of administration.

[0108] The term "administration" includes any method of delivering a pharmaceutical composition or agent to a specific area within or on the body of a subject. Administration can be local or systemic. In some embodiments, administration is via an oral or enteral route (e.g., intravenous, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, or intraocular injection; direct injection into a tumor; or intravenous infusion). In some embodiments, routes of administration include pulmonary administration, suppositories, and transdermal or percutaneous application. Administration of a pharmaceutical agent can be performed by a group of people working together. Administration of a pharmaceutical agent includes, for example, prescribing a pharmaceutical agent to be administered to a subject, and / or providing instructions directly or through another person for self-administration (e.g., via oral administration, subcutaneous administration, administration via a central venous catheter, etc.); or administration by a trained professional, such as intravenous administration, intramuscular administration, intratumoral administration, continuous infusion, etc.

[0109] As used herein, the term "subject" refers to a human or any non-human animal or mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). In many embodiments, the subject is a human. A subject can be a patient, meaning a person who goes to a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably with "individual" or "patient" herein. A subject may have or be susceptible to a disease or disorder, but may or may not show symptoms of the disease or disorder.

[0110] The term "therapeutic effective amount" or "effective amount" refers to the amount of a compound that produces some desired local or systemic therapeutic effect with a reasonable benefit / risk ratio suitable for any treatment. When administered for disease prevention, this amount is sufficient to prevent or delay the onset of the disease. A therapeutic effective amount or effective amount is not necessarily curative or preventative of the occurrence of the disease or condition. In some embodiments, the therapeutic effective amount of a compound will depend on its therapeutic index, solubility, etc.

[0111] In all instances where a series of numerical values ​​are listed in this application, it should be understood that any of the listed values ​​can serve as an upper or lower limit of the numerical range. It should also be understood that this invention covers all such numerical ranges, i.e., ranges having a combination of upper and lower numerical limits, where each upper and lower limit can be any of the values ​​listed herein. The ranges provided herein should be understood to include all values ​​within that range. For example, 1-10 should be understood to include all values ​​1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, as well as appropriate fractional values. Similarly, ranges defined by “at least” should be understood to include the provided lower limit value and all higher numbers.

[0112] As used herein, the term "about" for a value or parameter includes (and describes) the implementation of that value or parameter itself. For example, a description referring to "about X" includes a description of "X". Numerical ranges include the numbers that define the range. Generally, the term "about" refers to the indicated value of a variable, and all variable values ​​within the experimental error range of the indicated value (e.g., within the 95% confidence interval of the mean) or within 10% of the indicated value, whichever is greater. When the term "about" is used in the context of a time period (year, month, week, day, etc.), the term "about" refers to that time period plus or minus a quantity of the next subordinate time period (e.g., about 1 year means 11–13 months; about 6 months means 6 months plus or minus 1 week; about 1 week means 6–8 days; etc.), or within 10% of the specified value, whichever is greater.

[0113] The term “including” in this document means the phrase “including but not limited to” and is used interchangeably with it. Similarly, “such as” in this document means the phrase “such as but not limited to” and is used interchangeably with it.

[0114] Unless the context clearly indicates otherwise, the term “or” is used inclusively in this document to mean the term “and / or” and is used interchangeably with it.

[0115] II. Methods for treating diseases, disorders, or conditions associated with SDH deficiency. This article describes methods for treating subjects with diseases, disorders, or conditions associated with SDH deficiency, and methods for predicting the responsiveness of subjects in need to multitarget TKI therapy.

[0116] The inventors of these compounds have generated numerous TKIs targeting various tyrosine kinases and demonstrated their efficacy as antitumor and antileukemic agents (e.g., Anafi M et al., Blood, 1993, 82(12): 3524–9). Commercial TKIs have been developed, including, for example, imatinib, gefitinib, erlotinib, dasatinib, etc., to combat cancers such as GIST, the most common mesenchymal tumor of the gastrointestinal tract. Prior to the advent of TKIs, treatment options for patients with metastatic GIST were limited. However, treatment resistance is a significant challenge in managing locally advanced or metastatic GIST. SDH-deficient GIST patients have been reported to be insensitive to imatinib and other TKIs. The inventors have unexpectedly discovered that subjects with TKI-resistant and SDH-deficient GIST may respond to treatment with multi-target TKIs such as those presented herein (e.g., olaribatinib).

[0117] In one aspect, this disclosure provides a method for treating a subject with an SDH deficiency-related disease, disorder, or condition, the method comprising administering a therapeutically effective amount of TKI to the subject thereby treating the subject's SDH deficiency-related disease, disorder, or condition.

[0118] In another aspect, this disclosure provides a method for identifying a subject suffering from a disease, disorder, or condition associated with succinate dehydrogenase (SDH) deficiency and for selecting treatment for the identified subject, the method comprising: a) determining whether an SDH deficiency is present in a biological sample from the subject; b) identifying the subject as suffering from a disease, disorder, or condition associated with SDH deficiency based on the SDH deficiency determined in step a); and c) recommending that the subject receive treatment with a multitarget TKI.

[0119] 2.1 SDH Defects In some implementations, such as those described herein, the subjects are identified as or have been identified as having SDH deficiency.

[0120] As used herein, “SDH” is an abbreviation for succinate dehydrogenase, also known as succinate-coenzyme Q reductase (SQR) or respiratory complex II. The term “SDH” is intended to include both the SDH gene (e.g., genomic DNA, cDNA) and the SDH gene products (e.g., mRNA transcribed from the gene, proteins encoded by the gene). SDH is an enzyme complex found in the inner mitochondrial membrane of many bacterial cells and eukaryotes. It participates in both the citric acid cycle and the electron transport chain by oxidizing succinate to fumarate and reducing ubiquinone to ubiquinol in the electron transport chain (James MacFarlane et al.). Clinical Endocrinology (2020)93:528–538).

[0121] Mitochondrial and many bacterial SDH are composed of four structurally distinct subunits: two hydrophilic and two hydrophobic. The first two subunits, flavoprotein (SDHA) and iron-sulfur protein (SDHB), form the hydrophilic head where the enzymatic activity of the complex occurs. The latter two subunits are the hydrophobic membrane-anchoring subunits SDHC and SDHD.

[0122] Exemplary sequences of human SDHA are available in the UniProtKB database under accession number P31040 (SDHA_HUMAN) and in the GenBank database under gene ID 6389. Exemplary sequences of human SDHB are available in the UniProtKB database under accession number P21912 (SDHB_HUMAN) and in the GenBank database under gene ID 6390. Exemplary sequences of human SDHC are available in the UniProtKB database under accession number Q99643 (C560_HUMAN) and in the GenBank database under gene ID 6391. Exemplary sequences of human SDHB are available in the UniProtKB database under accession number O14521 (DHSD_HUMAN) and in the GenBank database under gene ID 6392.

[0123] As used herein, “deficiency” or “deficiency” of a biomarker (such as SDH) means an insufficient activity or level of the biomarker, and may include, for example, below normal activity or level, or the absence or zero activity or level of the biomarker. The terms “SDH deficiency” or “SDH deficiency” as used herein refer to an insufficient level or activity of SDH in a biological sample. In some embodiments, “SDH deficiency” or “SDH deficiency” results in reduced SDH enzyme activity, reduced SDH expression, or instability of the SDH protein complex.

[0124] In some implementations, such as the methods provided herein, it includes determining whether an SDH defect is present in a biological sample.

[0125] In some implementations, SDH deficiency is indicated by the presence of one or more loss-of-function mutations in SDHA, SDHB, SDHC, SDHD, or any combination thereof. Therefore, methods as provided herein may include the step of detecting the presence of one or more loss-of-function mutations in a biological sample in which SDHA, SDHB, SDHC, SDHD, or any combination thereof are present.

[0126] As used in this paper regarding biomarkers, the term "loss-of-function mutation" refers to a mutation that results in at least a partial (or complete) loss of function or activity of the gene or gene product of the biomarker, or a mutation that results in a non-functional gene or gene product of the biomarker, or a mutation that results in reduced gene expression (or complete absence of gene expression) (e.g., reduced mRNA or protein abundance in SDHA, SDHB, SDHC, or SDHD). The activity and / or level of the affected gene or gene product of the biomarker will be significantly lower than that of the wild-type counterpart or even eliminated. Loss of function in the SDH enzyme complex is most commonly caused by genetic mutations in one of the four SDHx genes (SDHA, SDHB, SDHC, and SDHD).

[0127] In some implementations, loss-of-function mutations can be missense substitutions, deletions, insertions, translocations, and premature termination codons, or any combination thereof.

[0128] Loss-of-function mutations can be caused by germline oncogenic missense mutations in SDHB, which affect the acquisition of important amino acid residues (L(I)YR motif amino acid residues) or the connection of important amino acid residues (cysteine) to the Fe-S cluster (Neetu Saxena et al.). JNCI J Natl Cancer Institute , (2016) 108(1):djv287).

[0129] As used in this article, a "missense substitution" is a mutation that replaces one nucleobase in a polynucleotide sequence with another, or one amino acid residue in a polypeptide sequence with another. Such substitutions in polynucleotide sequences can change a codon to one that encodes a different amino acid residue, thus causing a change in the amino acid sequence of the resulting protein.

[0130] As used in this article, a “deletion” is a mutation in which one or more nucleobase pairs are lost or omitted from a polynucleotide sequence, or one or more amino acid residues are omitted from a polypeptide sequence.

[0131] As used in this article, "insertion" is the insertion of one or more additional nucleobase pairs into a position in a polynucleotide sequence, or the insertion of one or more amino acid residues into a polypeptide sequence.

[0132] In some implementations, insertions or deletions in a polynucleotide sequence can cause a frameshift, altering the reading frame of a codon and resulting in a translated gene product that is entirely different from the original gene product. This typically produces a truncated protein, leading to loss of function.

[0133] As used in this article, "translocation" is a genetic change in which a segment of one chromosome breaks off and attaches to another chromosome. Sometimes, segments from two different chromosomes exchange positions.

[0134] As used in this article, a "premature stop codon" is a mutation in the mRNA that precedes the normal position of the gene. This means that the information for producing the target protein is incomplete; therefore, only an incomplete (often called a truncated) protein is formed.

[0135] In some embodiments, the one or more loss-of-function mutations include mutations as shown in the expressed protein. Exemplary mutations known in the art include, but are not limited to, the following mutations.

[0136] SDHA: p.G184R and p.P279S.

[0137] SDHB: p.A15T, p.W218Ter, SDHB KO exon 2+3 (heterozygote), p.I44N, p.R46G, p.R46Q, p.C93R, p.C93Y, p.C98R, p.C98Y, p.C101Y, p.C113Y, p.C186Y, p.C189R, p.C189F, p.C191Y, p.C192R, p.C192Y, p.C196Y, p.C196W, p.R242S, p.R242C, p.R242H, p.C243S, p.C249G, p.C249Y, and p.C253Y (see, for example, Neetu Saxena et al., JNCI J Natl Cancer Institute (2016) 108(1): djv287).

[0138] In some implementations, the one or more loss-of-function mutations include mutations as shown in the nucleic acid sequence (see, for example, Ahmad Ibrahim and Shefali Chopra, Arch Pathol Lab Med (2020) 144 (5):655–660). Exemplary mutations known in the art include, but are not limited to, the mutations listed in Table 1.

[0139] Table 1. Mutations in SDH It should be understood that this disclosure is not limited to any specific SDH mutation. Any loss-of-function mutation in SDH can be used in this disclosure. Mutations as provided herein are represented according to general rules in the art (see, for example, Ahmad Ibrahim and Shefali Chopra, Arch Pathol Lab Med (2020) 144 (5): 655–660), and this will be clearly understood by one of ordinary skill in the art.

[0140] The following is a simplified explanation for reference, not for any purpose of limitation.

[0141] Missense substitution can be represented as p.AnB, where "n" is the number indicating the nth amino acid residue in the polypeptide sequence, "A" is the amino acid residue at the nth residue in the wild-type polypeptide sequence, and "B" is the mutated amino acid residue at the nth residue. For example, "p.A15T" means that the 15th amino acid residue Ala (A) is changed to threonine (T).

[0142] Premature termination codons can be represented as "Ter" following the terminal amino acid residue of an incomplete protein. For example, "p.W218Ter" indicates that translation terminates aberrantly after the 218th amino acid residue, tryptophan (W), which is the C-terminal amino acid residue of the resulting incomplete protein.

[0143] As shown in Table 1, substitutions in a nucleic acid sequence can be represented as c.nA>B, where "n" is the number indicating the nth nucleotide in the nucleic acid sequence, "A" is the nucleotide at the nth residue in the wild-type nucleic acid sequence, and "B" is the mutant nucleotide at the nth nucleotide. For example, "c.91C>T" means that the 91st nucleotide C is changed to T.

[0144] As shown in Table 1, deletions in a nucleic acid sequence can be indicated by “del” after the nucleotide number indicating the deletion site. For example, “c.1043–1055del” indicates the deletion of nucleotides 1043–1055. Optionally, “del” is followed by one or more nucleotides indicating the nucleotides missing from the given deletion site.

[0145] As shown in Table 1, the repetition of one or more nucleotides in a nucleic acid sequence can be indicated by "dup" after the nucleotide number, indicating the repeating nucleotide. For example, "c.17_42dup" indicates a repetition of 26 nucleotides at positions 17 to 42.

[0146] As shown in Table 1, insertions in nucleic acid sequences can be indicated as "ins" after the nucleotide number flanking the insertion site, followed by the inserted nucleotide. For example, "c.1215_1216insCC" indicates that CC is inserted between nucleotides 1215 and 1216.

[0147] In some implementations, loss-of-function mutations can be single-copy or double-copy mutations. Therefore, such mutations can result in loss of function, even if they only lead to haploinsufficiency. Thus, loss-of-function mutations can be homozygous deletion mutations of SDH, point mutations in one allele of SDH and the other allele, or heterozygous mutations of SDH.

[0148] In some implementations, SDH deficiency is indicated by SDHC promoter methylation. Therefore, methods as provided herein may include steps for detecting the presence of SDHC promoter methylation in biological samples.

[0149] To determine the presence of an SDH deficiency, methods such as those provided herein may include the step of determining whether the SDHC promoter is negatively altered in a manner that leads to decreased SDHC expression. In some embodiments, the methods of this disclosure include determining whether the SDHC promoter in a biological sample is hypermethylated, wherein hypermethylation of the promoter indicates an SDH deficiency.

[0150] It has been reported that approximately 75% to 80% of GIST patients have mutations in the KIT gene, while 5% to 10% of GIST patients have mutations in the platelet-derived growth factor receptor α (PDGFRA) gene (Vallilas C et al., Int J Mol Sci , 2021;22(2):493). Mutations in 707 driver genes have been identified, including oncogenes (OG) and tumor suppressor genes (TSG) and fusion genes (F) (Keiichi Ohshima et al., Cancer Sci. Dec 2019;110(12):3821–3833). Subjects with SDH adequacy as described herein may also carry other gene mutations. In some embodiments, subjects with SDH adequacy (e.g., subject biosamples) also contain one or more of wild-type KIT, wild-type PDGFR, or combinations thereof. In some embodiments, subjects with SDH adequacy also contain wild-type KIT. In some embodiments, subjects with SDH adequacy (e.g., subject biosamples) also contain one or more mutations in NF1 / BRAF / RAS pathway genes, PIK3CA, or fusions affecting NTRK3, or any combination thereof. In some embodiments, subjects (e.g., subject biosamples) have been identified as overexpressing VEGFR or FGF ligands.

[0151] In some implementations, SDH deficiency is identified in biological samples from the subject.

[0152] Any biological sample suitable for performing the methods provided herein can be obtained from the subject. As used herein, "biological sample" means a biological specimen obtained by sampling from the subject, optionally subject to additional processing. In some embodiments, the biological sample can be any biological sample that can be used to detect germline mutations in the SDH gene.

[0153] In some implementations, the biological sample can be any fresh or archived sample containing cancerous or non-cancerous cells, obtained, for example, from tumor tissue via tumor biopsy or fine-needle aspiration. In some implementations, the sample can be any biological fluid containing cancerous or non-cancerous cells. The sample is collected from the subject according to standard protocols typically followed by the hospital or clinic, such as during a biopsy. Examples of biological samples include, but are not limited to, tissues such as biopsy tissue (e.g., biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tissue, lung tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, kidney tissue, endometrial tissue, diffuse cervical tissue, lymph node tissue, muscle tissue, or skin tissue), frozen tissue, paraffin-embedded tissue; and fluids such as blood, plasma, serum, peripheral blood mononuclear cells (PBMCs), urine, vaginal fluid, uterine or vaginal lavage fluid, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, etc. In another embodiment, the biological sample includes cells, tissues (e.g., tumor tissue), whole blood, plasma, serum, PBMCs, urine, mouthwash, feces, saliva, and any combination thereof. In another embodiment, the biological sample is tissue (e.g., tumor tissue), whole blood, plasma, or PBMCs. In a preferred embodiment, the biological sample is tumor tissue.

[0154] In some implementations, the sample may be further processed by desired methods for determining the genetic state, activity, or level of at least one biomarker, such as SDH.

[0155] In some implementations, the method also includes isolating or extracting cancer cells (such as circulating tumor cells) from biological tissue samples or fluid samples (such as PBMCs) obtained from the subject. Cancer cells can be isolated using immunomagnetic separation techniques, such as those available from Immunicon (Huntingdon Valley, PA).

[0156] In some implementations, tissue samples may be processed for in situ hybridization. For example, tissue samples may be embedded in paraffin before being fixed onto a microscope slide and then dewaxed with a solvent (typically xylene).

[0157] In some implementations, if the goal is to measure the RNA or DNA levels of a biomarker, the method further includes isolating nucleic acids from the sample. Various extraction methods are suitable for isolating DNA or RNA from cells or tissues, such as phenol and chloroform extraction, as well as various other methods, such as those employed by Ausubel et al. Current Protocols of Molecular Biology (1997) John Wiley & Sons, and Sambrook and Russell, Molecular Cloning: A Laboratory Manual As described in the 3rd edition (2001).

[0158] Commercially available kits can also be used to isolate RNA, including, for example, the NucliSens extraction kit (Biomerieux, Marcy l'Etoile, France) and QIAamp. TM mini blood test kit, AgencourtGenfind TM Rneasy® mini columns (Qiagen), PureLink® RNA mini extraction kit (Thermo Fisher Scientific), and Eppendorf Phase Lock Gels TM Technicians can easily extract or separate RNA or DNA according to the manufacturer's instructions.

[0159] In some implementations, SDH deficiency is determined by immunohistochemistry, Western blotting, amplification-based assays, hybridization-based assays, and sequencing.

[0160] The mutational status of biomarkers (e.g., SDH as provided herein) at the DNA or RNA level can be measured by any method known in the art, such as, but not limited to, amplification assays, hybridization assays, or sequencing assays. The mutational status at the protein level can be measured by any method known in the art, such as, but not limited to, immunoassays. In some embodiments, immunohistochemical detection of absent SDHB staining can be used to diagnose pathogenic germline variants in the SDHx gene and to rapidly assess the pathogenicity of novel SDHx variants of uncertain significance (Papathomas TG et al.). Mod Pathol , 2015; 28(6):807-821).

[0161] The expression level of a biomarker (e.g., SDH as provided herein) at the DNA or RNA level can be measured by any method known in the art, such as, but not limited to, amplification assays, hybridization assays, or sequencing assays. The expression level of a biomarker (e.g., SDH as provided herein) at the protein level can be measured by any method known in the art, such as, but not limited to, immunoassays.

[0162] The activity level of a biomarker can be measured by appropriate functional assays known in the art, such as, but not limited to, phosphorylation assays.

[0163] These methods are well known in the art and are described in detail below as exemplary embodiments.

[0164] (a) Amplification assay Nucleic acid amplification assays involve replicating target nucleic acids (e.g., DNA or RNA), thereby increasing the copy number of the amplified nucleic acid sequence. Amplification can be exponential or linear. Exemplary nucleic acid amplification methods include, but are not limited to, the use of polymerase chain reaction (“PCR”, see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide To Methods And Applications (edited by Innis et al., 1990)), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR); quantitative PCR, such as TaqMan®, nested PCR, ligase chain reaction (Abravaya, K. et al., Nucleic Acids Research, 23:675-682, (1995)), branched DNA signal amplification (Urdea, MS et al., AIDS, 7 (suppl 2):S11-S14, (1993)), amplifiable RNA reporter, Q-β replication (Lizardi et al., Biotechnology (1988) 6: 1197), and transcription-based amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA (1989)). 86:1173-1177), boomerang DNA amplification, strand displacement activation, circular probe technology, self-persistent sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA (1990) 87:1874-1878), rolling circle replication (US Patent No. 5,854,033), isothermal nucleic acid sequence-based amplification (NASBA), and serial analysis of gene expression (SAGE).

[0165] In some implementations, to measure the mRNA level of a biomarker, the target RNA of the biomarker is reverse transcribed into cDNA prior to amplification. Various reverse transcriptases can be used, including but not limited to MMLV RT, RNase H mutants of MMLV RT such as Superscript and Superscript II (Life Technologies, GIBCO BRL, Gaithersburg, MD), AMV RT, and thermostable reverse transcriptases from thermophilic bacteria. For example, one method that can be used to convert RNA into cDNA is a protocol adapted from the Superscript II pre-amplification system (Life Technologies, GIBCO BRL, Gaithersburg, MD; catalog number 18089-011), as described in Rashtchian, A., PCR Methods Applic., 4:S83-S91, (1994).

[0166] In some implementations, the expression level of the biomarker RNA (e.g., mRNA) or the copy number variation of DNA is quantified after nucleic acid amplification assay. For example, the amplification products can be separated on an agarose gel, stained with ethidium bromide, and then detected and quantified using standard gel electrophoresis. Alternatively, the amplification products can be integrally labeled with a suitable detectable marker (e.g., radioactive or fluorescent nucleotides) and then visualized using X-ray film or under a suitable stimulus spectrum.

[0167] In some embodiments, the expression level of a biomarker's RNA (e.g., mRNA) or copy number variation of DNA is quantified during a nucleic acid amplification assay, also known as real-time amplification or quantitative amplification. Methods for quantitative amplification are disclosed, for example, in U.S. Patent Nos. 6,180,349, 6,033,854, and 5,972,602, and, for example, in Gibson et al. Genome Research (1996) 6:995-1001; DeGraves et al., Biotechniques (2003) 34(1): 106-10,112-5; Deiman B et al., Mol Biotechnol (2002) 20(2): 163-79. Quantification is typically based on monitoring a detectable signal representing template copies in an amplification (e.g., PCR) reaction cycle. The detectable signal can be generated by intercalating agents (e.g., SYBR GREEN™ and SYBR GOLD™) or labeled primers or labeled probes used during amplification.

[0168] In some embodiments, the labeled primer or labeled probe contains a detectable label containing a fluorophore. In some embodiments, the labeled primer or labeled probe may further contain a quenching substance. The presence of both a fluorophore and a quenching substance (“dual-labeled”) in a primer or probe helps to provide self-quenching probes, such as TaqMan (US Patent Nos. 5,210,015 and 5,538,848) or molecular beacon probes (US Patent Nos. 5,118,801 and 5,312,728), or other stemless or linear beacon probes (Livak et al., 1995, PCR Method Appl., 4:357-362; Tyagi et al., 1996, Nature Biotechnology, 14:303-308; Nazarenko et al., 1997, Nucl. Acids Res., 25:2516-2521; US ​​Patent Nos. 5,866,336 and 6,117,635). In a complete primer or probe, the quencher and the fluorophore are very close together, so that when the fluorophore is excited by radiation, it transfers energy to the quencher in the same probe via fluorescence resonance energy transfer (FRET), thus not emitting a signal.

[0169] In quantitative amplification assays (such as real-time PCR), the expression level of RNA (e.g., mRNA) or copy number variation of DNA of a biomarker can be quantified using methods known in the art. For example, during amplification, the fluorescence signal can be monitored and calculated during each PCR cycle. A threshold period, or Ct value, can be further calculated. The Ct value is the period at which fluorescence intersects a predetermined value. A standard curve can be used to correlate Ct with the initial amount of nucleic acid or the number of starting cells. A standard curve is constructed to correlate the difference between the Ct value and the logarithmic level of the measured biomarker.

[0170] As a quality control measure, the expression level or copy number variation of an internal control biomarker may be measured. Those skilled in the art will understand that internal control biomarkers may inherently be present in the sample, and their expression level or copy number variation can be used to standardize the expression level or copy number variation of the target biomarker being measured to offset any differences in the absolute amount of the sample.

[0171] (b) Hybridization determination Nucleic acid hybridization assays use probes to hybridize with target nucleic acids, thereby allowing the detection of target nucleic acids. Non-limiting examples of hybridization assays include RNA blotting, DNA blotting, in situ hybridization, microarray analysis, and multiplex hybridization-based assays.

[0172] In some embodiments, the probes used for hybridization assays are detectably labeled. In other embodiments, the nucleic acid-based probes used for hybridization assays are unlabeled. Such unlabeled probes can be immobilized on a solid support such as a microarray and can hybridize with detectably labeled target nucleic acid molecules.

[0173] In some implementations, hybridization assays can be performed by the following steps: isolating nucleic acids (e.g., RNA or DNA), isolating nucleic acids (e.g., by gel electrophoresis), and then transferring the isolated nucleic acids to a suitable membrane filter (e.g., a nitrocellulose membrane), where the probe hybridizes with the target nucleic acid and allows detection. See, for example, *Molecular Cloning: A Laboratory Manual*, edited by J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, 1989, Chapter 7. Hybridization of the probe and target nucleic acid can be detected or measured by methods known in the art. For example, autoradiographic detection of hybridization can be performed by exposing the hybridized filter membrane to photographic film. A precise measurement of the target nucleic acid level is provided by scanning the optical density of the photographic film exposed to the hybridized filter membrane. Computer imaging systems can also be used to quantify the levels of biomarkers.

[0174] In some implementations, hybridization assays can be performed on a microarray. Microarrays provide a method for simultaneously measuring the levels of a large number of target nucleic acid molecules. Target nucleic acids can be RNA, DNA, cDNA reverse-transcribed from mRNA, or chromosomal DNA. The target nucleic acids can be hybridized to a microarray comprising a substrate with multiple immobilized nucleic acid probes arranged at a density of up to several million probes per square centimeter of substrate surface. RNA or DNA in a sample hybridizes with complementary probes on the array and is then detected by laser scanning. The hybridization intensity of each probe on the array is determined and converted into a quantitative value representing the relative level of RNA or DNA. See U.S. Patent Nos. 6,040,138, 5,800,992, 6,020,135, 6,033,860, and 6,344,316.

[0175] Techniques for synthesizing these arrays using mechanosynthetic methods are described, for example, in U.S. Patent No. 5,384,261. While planar array surfaces are typically employed, the array can be fabricated on surfaces of virtually any shape, or even multiple surfaces. Arrays can be peptides or nucleic acids on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate, see U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193, and 5,800,992. Arrays can be encapsulated in specific ways to support diagnostic or other operations of integrated devices. Useful microarrays are also commercially available, such as those from Affymetrix, Nano String Technologies, and Panomics' QuantiGene 2.0 Multiplex Assay.

[0176] In some embodiments, the hybridization assay may be an in situ hybridization assay. An in situ hybridization assay can be used to detect the presence of copy number variations (e.g., increases or amplifications) at a locus of a target biomarker (e.g., SDH). Probes used for in situ hybridization assays may be locus-specific probes that hybridize with a specific locus on a chromosome to detect the presence or absence of a specific target locus (e.g., SDH). Other types of probes may also be useful, such as chromosome counting probes (e.g., those that hybridize with repetitive sequence regions in a target chromosome to indicate the presence or absence of an entire chromosome) and chromosome arm probes (e.g., those that hybridize with a chromosomal region and indicate the presence or absence of an arm of a specific chromosome). Methods for in situ hybridization using unique sequence probes are described in U.S. Patent No. 5,447,841, which is incorporated herein by reference. Probes can be observed using a fluorescence microscope and filters appropriate for each fluorophore, or by observing multiple fluorophores using a set of dual or triple bandpass filters. See, for example, U.S. Patent No. 5,776,688 to Bittner et al., which is incorporated herein by reference. Any suitable microscopic imaging method can be used to visualize hybridization probes, including automated digital imaging systems. Alternatively, techniques such as flow cytometry can be used to examine the hybridization pattern of the probes.

[0177] (c) Sequencing methods Sequencing methods allow for the determination of the nucleic acid sequence of a target nucleic acid, and also allow for the counting of sequenced target nucleic acids, thereby measuring the level of the target nucleic acid. Examples of sequencing methods include, but are not limited to, RNA sequencing, pyrosequencing, and high-throughput sequencing.

[0178] High-throughput sequencing encompasses synthetic sequencing, ligation sequencing, and ultra-deep sequencing (as described in Marguiles et al., Nature 437(7057): 376-80 (2005)). Synthetic sequencing involves synthesizing the complementary strand of a target nucleic acid by incorporating a labeled nucleotide or nucleotide analog into polymerase amplification. Immediately after or following successful incorporation of the labeled nucleotide, the label signal is measured and the identity of the nucleotide is recorded. The detectable label on the incorporated nucleotide is removed before repeating the incorporation, detection, and identification steps. Examples of synthetic sequencing methods are known in the art and are described, for example, in U.S. Patent Nos. 7,056,676, 8,802,368, and 7,169,560, the contents of which are incorporated herein by reference. Synthetic sequencing can be performed on a solid surface (or microarray or chip) using foldback PCR and anchored primers. The target nucleic acid fragment can be attached to the solid surface by hybridization with the anchored primer and amplified by bridging. This technique is used in, for example, Illumina… ® Sequencing platform.

[0179] Pyrosequencing involves hybridizing the target nucleic acid region with primers and extending the chain in the presence of polymerase by sequentially incorporating deoxyribonucleotide triphosphates corresponding to bases A, C, G, and T(U). Each base incorporation is accompanied by the release of pyrophosphate, which is converted to ATP by a sulfatase, driving the synthesis of oxidative luciferin and the release of visible light. Since the amount of pyrophosphate released is equimolar to the number of bases incorporated, the emitted light is proportional to the number of nucleotides added in any step. This process is repeated until the entire sequence is determined.

[0180] In some implementations, the detection of the mutation and / or wild-type status described herein and the measurement of the levels of the target biomarker are performed by whole transcriptome sequencing or RNA sequencing (e.g., RNA-Seq). RNA sequencing methods have been described (see Wang Z, Gerstein M, and Snyder M, Nature Review Genetics (2009) 10:57-63; MaherCA et al., Nature (2009) 458:97-101; Kukuba K and Montgomery SB, Cold Spring Harbor Protocols (2015) 2015(11): 951-969). In short, mRNA extracted from the sample is reverse transcribed into cDNA and cut into fragments. Fragments within an appropriate length range are selected and ligated with sequencing adapters, and then the reads are amplified, sequenced, and mapped to a reference genome.

[0181] In some implementations, whole-exome sequencing (WES) is used to determine the CNV of biomarkers. WES involves sequencing DNA exons (i.e., protein-coding regions) using high-throughput sequencing technologies. More details about WES can be found in, for example, Ng SB et al., Nature. 461 (7261): 272–276 (2009), and Bao R et al., Cancer Inform. 2014; 13(Suppl 2): ​​67–82, which are incorporated herein by reference in full.

[0182] (d) Immunoassay Immunoassays typically involve the detection or measurement of the presence or level of a target peptide or protein using an antibody that specifically binds to a biomarker peptide or protein (e.g., the SDH protein described herein). Such antibodies can be obtained using methods known in the art (see, for example, Huse et al.). Science (1989) 246:1275-1281; Ward et al., Nature (1989) 341:544-546), or examples of immunoassays available from commercial sources, including but not limited to Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), sandwich assay, competitive assay, immunofluorescence staining and imaging, immunohistochemistry (IHC), and fluorescence activated cell sorting (FACS). These methods are all routine assays in the field. For a review of immunology and immunoassay procedures, see Basic and Clinical Immunology (edited by Stites and Terr, 7th edition, 1991). Furthermore, immunoassays can be performed using any of a variety of configurations, which are extensively reviewed in Enzyme Immunoassay (edited by Maggio, 1980); and Harlow and Lane, see above. For a review of general immunoassays, see also [link to relevant documentation]. Methods in Cell Biology: Antibodies in Cell Biology Volume 37 (edited by Asai, 1993); Basic and Clinical Immunology (Edited by Stites and Terr, 7th edition, 1991).

[0183] In some implementations, the antibody is detectably labeled, or alternatively unlabeled but capable of reacting with a detectably labeled second molecule (e.g., a detectably labeled secondary antibody). Other detection systems such as time-resolved fluorescence, internal reflection fluorescence, amplification (e.g., polymerase chain reaction), and Raman spectroscopy are also useful.

[0184] In some embodiments, antibodies may be immobilized on a solid matrix. Immobilization can be performed via covalent or non-covalent linkage (e.g., coating). Examples of solid matrices include porous and non-porous materials, latex particles, magnetic particles, microparticles, strips, beads, membranes, microburette wells, and plastic tubing. The selection of the solid material and the method for detectably labeling antigens or antibodies is determined based on the desired performance characteristics of the assay.

[0185] (e) Activity assay The biological activity of proteins can be measured using bioassays. For example, SDH activity in a biological sample can be determined by contacting the sample with a substrate kinase (e.g., succinic acid) under suitable conditions and monitoring the production of fumarate, where fumarate production indicates SDH activity. Because proteins have the ability to perform a variety of biological activities, several acceptable bioassays may exist for a particular protein. Exemplary functional assays for measuring SDH activity can be found, for example, by Andrew JY Jones et al. Anal. Biochem , (2013) 442: 19–23.

[0186] 2.2 Diseases, disorders, or conditions related to SDH deficiencies The methods described in this article can be used to treat diseases, disorders, or conditions associated with SDH deficiency.

[0187] As used herein, “sickness, disorder, or condition associated with SDH deficiency” means a sickness, disorder, or condition that may be wholly or partially caused or aggravated by SDH deficiency (e.g., causing or accelerating the onset or development of a condition or symptoms associated with that condition, increasing the risk of developing a condition or symptoms associated with that condition, causing a condition or symptoms that are unresponsive or less responsive to existing therapies). In some embodiments, “sickness, disorder, or condition associated with SDH deficiency” can be identified by the loss of immunohistochemical expression of SDHB (Anthony J Gill, Histopathology , (2018) 72: 106–116).

[0188] In some implementations, the disease, impairment, or condition associated with SDH deficiency can be cancer (e.g., SDH-deficient cancer). In some implementations, the disease, impairment, or condition associated with SDH deficiency can be gastrointestinal cancer (e.g., GIST), bile duct cancer, lung cancer, melanoma, breast cancer, colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), bladder cancer, pancreatic cancer, kidney cancer, esophageal cancer, head and neck cancer, thyroid cancer, squamous cell carcinoma of the skin, glioblastoma, neuroblastoma, urethral and bladder cancer, endometrial cancer, melanoma, osteosarcoma, lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma), or leukemia. (For example, T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia (ALL)), multiple myeloma, uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophageal cancer, sarcoma, chromophobe carcinoma, renal cell carcinoma, clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low-grade glioma (LGG), mesothelioma, pheochromocytoma, paraganglioma, thymoma, or adenoid cystic carcinoma (ACC).)

[0189] In some implementations, the disease, disorder, or condition associated with SDH deficiency can be an SDH-deficient GIST.

[0190] In some implementations, the disease, disorder, or condition associated with SDH deficiency can be SDH-deficient ALL, renal cell carcinoma, colon cancer, pheochromocytoma, thyroid adenoma (e.g., papillary thyroid adenoma, multifocal papillary thyroid adenoma, differentiated thyroid carcinoma, papillary (follicular variant) thyroid adenoma, follicular thyroid adenoma), pancreatic neuroendocrine tumor / pancreatic paraganglioma, breast cancer (e.g., triple-negative breast cancer), neuroblastoma, follicular lymphoma, B-cell lymphoma, Hodgkin lymphoma, complex paraganglioma / neuroblastoma, rectal adenocarcinoma, Burkitt lymphoma, thyroid paraganglioma, adrenocortical carcinoma, adrenal adenoma-like tumor, neuroblastoma, melanoma, synovial sarcoma, squamous cell carcinoma of the lung, embryonal rhabdomyosarcoma, lung adenocarcinoma, prostate adenocarcinoma, endometrial cancer, poorly differentiated gastric cancer, bladder urothelial carcinoma, colon adenocarcinoma, sigmoid colon adenocarcinoma, testicular seminoma, or pituitary adenoma.

[0191] 2.3 TKI resistance In some implementations, such as those described herein, the subject is identified or has been identified as insensitive to at least one existing TKI.

[0192] As used herein, “insensitive to,” “unresponsive to,” or “resistant to” means that a patient has not received clinical benefit or has little or no clinical benefit from prior treatment with at least one multi-target TKI. In some embodiments, a subject is defined as having acquired primary resistance to prior treatment with an existing TKI if the subject has not achieved a complete response (CR), partial response (PR), or stable disease (SD) within 6 months of receiving treatment with at least one existing TKI. In some embodiments, a subject is defined as having acquired resistance to prior treatment with an existing TKI if the subject has achieved initial response or stable disease after ≥6 months of receiving treatment with at least one existing TKI, but subsequently experiences disease progression.

[0193] In some implementation schemes, the existing TKI for prior treatment may be imatinib (Novartis), repretinib (Zai Lab in combination with Deciphera), avatinib (CStone Pharmaceuticals in combination with Blueprint), cristatinib (Pfizer), masitinib (AB Science), ponatinib (Takeda Pharmaceuticals), dasatinib (Bristol-Myers Squibb), nilotinib (Novartis), sunitinib (CPPI CV), regorafenib (Bayer), sorafenib (Bayer), anlotinib (Chia Tai Tianqing Pharmaceutical Group), surufatinib (Hutchison Whampoa), pazopanib (GSK), rogatinib (Bayer), bridgeBio, erdatinib (Johnson & Johnson), pemitinib (Incyte), fexotinib (Blueprint), famitinib (Hengrui), or any combination thereof. In a further implementation scheme, the existing TKI for prior treatment may be repretinib (Zai Lab in combination with Deciphera), dasatinib (Bristol-Myers Squibb), sunitinib (CPPICV), regorafenib (Bayer), anlotinib (Chia Tai Tianqing), famitinib (Hengrui) or any combination thereof.

[0194] 2.4 Multi-target TKIs The multi-target TKI disclosed in this invention can inhibit at least two tyrosine kinases. Tyrosine kinases are enzymes responsible for activating many proteins through signal transduction cascades. Protein activation occurs by adding a phosphate group to the protein (phosphorylation), a step that TKIs inhibit.

[0195] In some embodiments, the multi-target TKI provided herein can inhibit at least two tyrosine kinases selected from the following: KIT, PDGFR, SRC, VEGFR (e.g., any one of VEGFR1, VEGFR2, VEGFR3, or any combination thereof), FGFR (e.g., any one of FGFR1, FGFR2, FGFR3, FGFR4, or any combination thereof), or any combination thereof. In a further embodiment, the multi-target TKI can inhibit at least two tyrosine kinases selected from the following: KIT, PDGFR, SRC, VEGFR2, FGFR1, or any combination thereof.

[0196] In some embodiments, this multi-target TKI is able to inhibit the proliferation of SDH-deficient tumor cells. "SDH-deficient tumor cells" refers to any tumor cell line that is SDH-deficient, or any tumor cells primarily isolated from SDH-deficient subjects. The SDH-deficient tumor cells used herein may be Jurkat clone E6-1, OS-RC-2, RKO, PC12#5F7, or any combination thereof. In some embodiments, this multi-target TKI has an IC50 value in inhibiting the proliferation of SDH-deficient tumor cells. 50 Not exceeding 10 μM, as determined by CCK8 assay or CTG assay. In a further embodiment, this multi-target TKI has an IC50 value in inhibiting the proliferation of SDH-deficient tumor cells. 50 Not exceeding 5 μM, 1 μM, 500 nM, 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, or 10 nM, as determined by the CCK8 or CTG assay. The CCK8 or CTG assay can be performed by a person skilled in the art using conventional methods. Commercially available kits are also available for CCK8 or CTG assays (e.g., Shanghai Liji Medical Technology Co., Ltd., #G7573, Promega). Those skilled in the art can readily use these assays according to the manufacturer's protocol.

[0197] In some implementations, such as the multi-target TKIs provided herein, the TKIs comprise heterocyclic acetylenes having the following formula (I): Z is independently selected from CH or N; L 1 Independently selected from NH, -N=, CH; L 2 Independently selected from -CONH- or -NHCO-; R1 is independently selected from: 1) H; 2) C1-C6 alkyl groups; 3) C3-C6 cycloalkyl groups; 4) C1-C5 alkyl groups substituted with one or two hydroxyl groups; 5) Phenyl; 6) It can pass through L at the Z atomic site. 1 Fused A rings to form a ring containing 1-3 N atoms and L 1 Fused five-membered heterocyclic groups, such as , Among them, X, Y, and Z are independently selected from N and CH; ring D is an aromatic heterocycle containing 1-3 N atoms; R2 is independently selected 1) H; 2) Halogens; 3) C1-C5 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C5 alkyl groups; R3 is selected independently 1) H; 2) Halogens; 3) C1-C4 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C4 alkyl groups; When R5 is H, R4 is independently selected. 1) H; 2) (CH2) n NR6R7; 3) (CH2) n -Het 1 ; Alternatively, when R4 is H, R5 is independently selected. 1) H; 2) Het 2 ; n is independently selected from 0 or 1; Het 1 Defined as a non-aromatic heterocycle containing 1-3 N atoms; Het 2 Defined as an aromatic five-membered heterocycle containing heteroatoms such as N, O and / or S; alkyl, cycloalkyl or NR6R7 may be incorporated into any C or N position of substituted non-aromatic heterocycles and aromatic heterocycles; R6 or R7 is selected independently from: 1) H; 2) C1-C3 alkyl groups; 3) F-containing C1-C3 alkyl groups; 4) C3-C6 cycloalkyl groups; Alternatively, R6 and R7 can be further formed into five-, six-, seven-, or eight-membered ring structures through C, O, N, and S atoms; Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0198] In some implementations, the multi-target TKIs provided herein are compounds having the following formula (II) (also known as olabatinib, HQP1351): (II) Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0199] In some implementations, the multi-target TKIs provided herein are selected from the following compounds: olaribatinib, repretinib (Zai Lab & Deciphera), avatinib (CStone Pharmaceuticals & Blueprint), cristatinib (Pfizer), masitinib (AB Science), ponatinib (Takeda Pharmaceutical), dasatinib (Bristol-Myers Squibb), nilotinib (Novartis), sunitinib (CPPI CV), regorafenib (Bayer), sorafenib (Bayer), anlotinib (Chia Tai Tianqing Pharmaceutical Group), surufatinib (Hutchison Whampoa), infineratinib (BridgeBio), erdatinib (Johnson & Johnson), pemitinib (Incest), rogatinib, and lincitinib, or pharmaceutically acceptable salts or stereoisomers or prodrugs of any of the foregoing.

[0200] Further multi-target TKIs that can be used in this application are disclosed in WO2012000304A1, which is incorporated herein by reference.

[0201] Multi-target TKIs as described herein can exist as salts. Pharmaceutically acceptable salts of multi-target TKIs as described herein are generally preferred in the methods of the present invention. As used herein, the term "pharmaceutically acceptable salt" refers to a salt or zwitterionic form of a compound of formula (I) or (II). Salts of compounds of formula (I) or (II) can be prepared during the final separation and purification of the compound, or separately by reacting the compound with an acid having a suitable cation, such as, but not limited to, alkali metal and alkaline earth metal ions, for example, Na+. + K + Ca 2+ and Mg 2+ and organic cations, such as, but not limited to, ammonium and substituted ammonium ions, such as NH4+. + NHMe3 + NH2Me2 + NHMe3 + and NMe4 + Examples of pharmaceutically acceptable monovalent and divalent cations are discussed, for example, by Berge et al. J. Pharm. Sci., 66:1-19 (1997).

[0202] Pharmaceutically acceptable salts of compounds of structural formula (I) or (II) can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids that can be used to form pharmaceutically acceptable salts include inorganic acids such as nitric acid, boric acid, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. Non-limiting examples of salts of the compounds of the present invention include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, 2-hydroxyethanesulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, hydrogen sulfate, butyrate, camphorate, camphor sulfonate, diglucuronate, glyceryl phosphate, hemisulfate, heptanate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, hydroxyethyl sulfonate, salicylate, methanesulfonate, trimethylbenzenesulfonate, naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, picrate, neopentanoate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, undecanoate, lactate, citrate, tartrate, glucuronate, methanesulfonate, ethanedisulfonate, benzenesulfonate, and p-toluenesulfonate. Furthermore, the available amino groups present in the compounds of the present invention can be quaternized with: methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and dipentyl sulfate; decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. Based on the foregoing, any references to the compounds of the present invention appearing herein are intended to include compounds of structural formula (I) and their pharmaceutically acceptable salts.

[0203] Compounds having one or more chiral centers can exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in spatial arrangement. Stereoisomers include all diastereomers, enantiomers, and epimers, as well as their racemic mixtures and mixtures.

[0204] The term "geometric isomer" refers to a cyclic compound having at least two substituents, wherein both substituents are on the same side of the ring (cis) or wherein the substituents are on opposite sides of the ring (trans). When a disclosed compound is named or described by its structure without specifying its stereochemistry, it should be understood that the name or structure covers one or more possible stereoisomers or geometric isomers, or mixtures of such stereoisomers or geometric isomers.

[0205] When a geometric isomer is described by name or structure, it should be understood that the named or described isomer exists to a greater extent than another isomer, i.e., the geometric isomer purity of the named or described geometric isomer is greater than 50%, such as a purity of at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight. Geometric isomer purity is determined by dividing the weight of the named or described geometric isomer in the mixture by the total weight of all geometric isomers in the mixture.

[0206] A racemic mixture refers to a mixture where 50% is one enantiomer and 50% is the corresponding enantiomer. When a compound having one chiral center is named or described without specifying the stereochemistry of the chiral center, it should be understood that the name or structure covers both possible enantiomeric forms of the compound (e.g., enantiomerically pure, enantiomerically enriched, or racemic). When a compound having two or more chiral centers is named or described without specifying the stereochemistry of the chiral centers, it should be understood that the name or structure covers all possible diastereomeric forms of the compound (e.g., diastereomerically pure, diastereomerically enriched, and equimolar mixtures of one or more diastereomers (e.g., racemic mixtures)).

[0207] Mixtures of enantiomers and diastereomers can be resolved into their component enantiomers or stereoisomers by known methods such as chiral gas chromatography, chiral high-performance liquid chromatography, crystallization of the compound into a chiral salt complex, or crystallization of the compound in a chiral solvent. Enantiomers and diastereomers can also be obtained from diastereomerically pure or enantiomerically pure intermediates, reagents, and catalysts by known asymmetric synthetic methods.

[0208] When a compound is named with a name or structure representing a single enantiomer, unless otherwise stated, the compound is optically pure (also known as “enantiomer purity”) of at least 60%, 70%, 80%, 90%, 99%, or 99.9%. Optical purity is the weight of a mixture of named or described enantiomers divided by the total weight of the mixture of the two enantiomers.

[0209] When the stereochemistry of the disclosed compound is named or described using a structure, and the named or described structure covers more than one stereoisomer (e.g., as in a diastereomer pair), it should be understood that this includes one of the covered stereoisomers or any mixture of the covered stereoisomers. It should also be understood that the stereoisomer purity of the named or described stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight. In this case, the stereoisomer purity is determined by dividing the total weight of the mixture of stereoisomers covered by the name or structure by the total weight of all the stereoisomer mixtures.

[0210] “Prodrug” refers to a compound that can be converted into the bioactive compound described herein under physiological conditions or by solvent degradation. Therefore, the term “prodrug” refers to a precursor of a pharmaceutically acceptable bioactive compound. A prodrug may be inactive when administered to a subject, but is converted into the active compound in vivo, for example, by hydrolysis. Prodrug compounds typically offer advantages such as solubility, tissue compatibility, or delayed release in mammalian organisms (see, for example, Bundgard, H., Design of Prodrugs (1985), pp. 7–9, 21–24 (Elsevier, Amsterdam)). Discussions of prodrugs are provided in Higuchi, T. et al., ACS Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, edited by Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference in their entirety. The term “prodrug” is also intended to include any covalently bonded carrier that, when administered to a mammalian subject, releases the active compound in vivo. Prodrugs of the active compounds described herein can be prepared by modifying the functional groups present in the active compound, such that the modified compound is cleaved into the parent active compound in conventional procedures or in vivo. Prodrugs include compounds in which a hydroxyl, amino, or thiol group is bonded to any group that, when the prodrug of the active compound is administered to a mammalian subject, cleaved to form a free hydroxyl, free amino, or free thiol group, respectively.

[0211] 2.5 Dosage Regimen In some embodiments, such as the multi-target TKIs provided herein, the pharmaceutical composition may be administered as a pharmaceutical composition. This pharmaceutical composition may comprise a multi-target TKI and pharmaceutically acceptable excipients.

[0212] As used herein, a "pharmaceuticalally acceptable excipient" means a substance that contributes to the formulation and / or administration to and / or absorption by a subject of an active agent, and may be included in the compositions disclosed herein without causing significant adverse toxicological effects on the subject. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, aqueous saline solutions, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose), amylose or starch, hydroxymethyl cellulose, fatty acid esters, polyvinylpyrrolidone, and colorants. An excipient is "acceptable" in the sense that it is compatible with the other components of the pharmaceutical composition and is harmless to the recipient.

[0213] Those skilled in the art can determine, through routine experiments, the effective and non-toxic dose of the multi-target TKIs provided herein for treating diseases, disorders, or conditions associated with SDH deficiency as described herein. For example, the therapeutically active dose of a multi-target TKI can vary depending on a variety of factors, such as the subject's disease stage (e.g., stage I vs. stage IV), age, sex, medical complications (e.g., immunosuppressive conditions or diseases), and the subject's weight, as well as the multi-target TKI's ability to elicit the desired response in the subject. In some embodiments, the therapeutically active dose is a safe dose of the multi-target TKI that, when used in the manner disclosed herein, is sufficient to produce the desired therapeutic response without excessive adverse side effects (such as toxicity, irritation, and allergic reactions) and has a reasonable benefit / risk ratio.

[0214] Dosing regimens can be adjusted to provide optimal therapeutic response. For example, a number of fractions can be administered daily or via continuous dosing, or the dose can be reduced proportionally as indicated by an emergency in the treatment situation. In some embodiments, multi-target TKIs, such as those described herein, are administered at therapeutically effective amounts when delivered alone or in combination, i.e., the multi-target TKI is administered and / or as a therapeutic treatment for a disease, disorder, or condition associated with SDH deficiency, rather than primarily as an agent to alleviate the side effects of other treatments.

[0215] In some implementations, multi-target TKIs, as provided herein, are administered at a dose effective in treating diseases, disorders, or conditions associated with SDH deficiency. The dosages provided below can be used with any administration modality of multi-target TKIs as provided herein, including topical administration, inhalation administration, and intravenous administration (e.g., continuous infusion).

[0216] In some implementations, such as the multi-target TKIs provided herein, the TKIs can be administered orally, orally, via inhalation spray, sublingually, rectally, transdermally, via vaginal mucosa, via mucosal route, topically, via nasal route, or via enteral route; parenteral administration, such as intramuscular injection, subcutaneous injection, intramedullary injection, and intrathecal or direct administration to the brain, or in situ administration, subcutaneously, intraperitoneally, intravenously, intrasynovially, intrasternally, intrahepatically, intralesionally, intracranially, intraperitoneally, via nasal route, intraocularly, or by any other drug delivery method.

[0217] In some embodiments, the pharmaceutical composition comprises a multi-target TKI or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition is a solid dosage form. In some embodiments, the solid dosage form is a capsule. In some embodiments, the solid dosage form is a dry-filled capsule. In some embodiments, the capsule contains about 10 mg to 500 mg of the multi-target TKI as provided herein.

[0218] In some embodiments, the multi-target TKI is administered at doses of about 10 mg to 200 mg, 10 mg to 150 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 20 mg to 50 mg. In some embodiments, the multi-target TKI is administered at doses of about 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg. In some embodiments, olaribatinib is administered at doses of about 10 mg to 200 mg, 10 mg to 150 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 20 mg to 50 mg. In some embodiments, olaribatinib is administered at doses of about 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg.

[0219] In some implementations, the multi-target TKIs provided herein are administered once daily, every other day, weekly, once a week, monthly, or every other month. In some implementations, the multi-target TKIs provided herein are administered in one or more 7-day, 14-day, 28-day, 35-day, 42-day, two-month, or three-month treatment cycles. In some implementations, the treatment provided herein comprises at least 1, 2, 3, 6, 14, 16, or 36 treatment cycles. In some implementations, olaribatinib is administered once daily, every other day, weekly, once a week, monthly, or every other month. In some implementations, olaribatinib is administered in one or more 7-day, 14-day, 28-day, 35-day, 42-day, two-month, or three-month treatment cycles. In some implementations, the treatment provided herein comprises at least 1, 2, 3, 6, 14, 16, or 36 treatment cycles.

[0220] In a preferred embodiment, the multi-target TKI provided herein is administered orally once every other day during a 28-day treatment cycle. In a preferred embodiment, olaribatinib is administered orally once every other day during a 28-day treatment cycle.

[0221] In some implementations, after treatment with a compound having formula (II), subjects experience partial remission or disease stabilization. In some implementations, after a 28-day treatment cycle with a compound having formula (II), the objective response rate (complete remission and partial remission) is approximately or greater than 20%, and the clinical benefit rate (complete remission, partial remission, and disease stabilization) is approximately or greater than 90%.

[0222] In some embodiments, the methods provided herein further include administering a therapeutically effective amount of one or more additional therapies. In some embodiments, the multi-target TKI is administered in combination with one or more additional therapies for treating a disease, disorder, or condition as provided herein.

[0223] In some implementations, the one or more additional therapies include radiation therapy, chemotherapy, targeted cancer therapy, therapy using immune checkpoint molecule modulators, or therapy using BCL-2 inhibitors.

[0224] As used in this article, the term "radiotherapy" refers to the treatment of cancer with ionizing radiation. The term "chemotherapy" refers to the treatment of cancer with specific chemical agents. The term "targeted cancer therapy" refers to the treatment of cancer with agents (compounds or macromolecules) that selectively interact with selected biomolecules.

[0225] As used herein, "immune checkpoint" or "immune checkpoint molecule" refers to molecules in the immune system that regulate signaling. Immune checkpoint molecules can be co-stimulatory checkpoint molecules, i.e., those that enhance signaling, or inhibitory checkpoint molecules, i.e., those that weaken signaling. As used herein, "modulator of immune checkpoint molecules" is a drug that can alter the activity of immune checkpoints in a subject.

[0226] As used in this article, "Bcl-2 inhibitor" refers to a drug that can downregulate the level and / or activity of Bcl-2 in a subject.

[0227] III. Combination therapy for cancer using multi-target KTI and BCL-2 inhibitors This article describes a method for treating a subject’s cancer, comprising administering to the subject a) a therapeutically effective amount of a multitarget TKI; and b) a therapeutically effective amount of a Bcl-2 inhibitor.

[0228] 3.1 Cancer The combinations described herein can be used to treat cancer. In some implementations, the cancer may be gastrointestinal cancer (e.g., GIST), bile duct cancer, lung cancer, melanoma, breast cancer, colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), bladder cancer, pancreatic cancer, kidney cancer, esophageal cancer, head and neck cancer, thyroid cancer, squamous cell carcinoma of the skin, glioblastoma, neuroblastoma, urethral and bladder cancer, endometrial cancer, melanoma, osteosarcoma, lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma), or leukemia (e.g., T-cell lymphoma). The cancer is classified as follows: prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia (ALL), multiple myeloma, uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, squamous cell carcinoma of the lung, cervical cancer, esophageal cancer, sarcoma, chromophobe carcinoma, renal cell carcinoma, clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low-grade glioma (LGG), mesothelioma, pheochromocytoma, paraganglioma, thymoma, or adenoid cystic carcinoma (ACC). In a preferred embodiment, the cancer is GIST.

[0229] In some implementations, such as those described herein, the subjects are identified as or have been identified as including KIT, PDGFR (For example, PDGFRA and PDGFRB ), SDH (For example, SDHA、SDHB、SDHC and SDHD ), NF1、BRAF、EGFR、HRAS、NRAS、 KRAS、ZNF217、MEN1、PIK3CA、ARID1A、ARID1B、FGFR1、FGFR2、FGFR3、FGFR4、SDHAF2、FGFR1- HOOK3、FGFR1-TACC1、NTRK1 (For example, NTRK1 Fusion NTRK2 (For example, NTRK2 Fusion NTRK3 (For example, NTRK3 Fusion KIT-PDGFRA and PRKAR1B-BRAF Or one or more mutations in any combination of them (Lillian R. Klug et al., Nature Reviews Clinical Oncology. (2022) 19:328–341, JiHyun Lee et al. Cancers. 2022, 14(11), 2659).

[0230] In some implementations, such as those described herein, the subjects are identified as or have been identified as including ADGRB3、 AKT3、ALK、AMER1、APC、AR、ARAF、ASXL1、ATM、ATRX、BCL2、BCL9、BLM、BRIP1、BTG2、BTK、CBL、 CCDC6 / RET、CDK12、CDK8、CDKN2A、CDKN2B、CDKN2C、CHD2、CHEK2、CHIC2 / KDR、COL1A1-PDGFB、 CPEB3、CREB3L1、CREBBP、CSMD3、CTNNB1、DLL3、DPYD、DUSP2、EPAS1、EPCAM、ERBB2、ERBB3、 ERBB4、ERCC4、FANCD2、FANCF、FAT1、FAT3、FH、FLT3、FOXA1、GRIN2A、GSK3B、HDAC1、IGF1R、 IGF2、IGR / CDK8、IKBKE、IL7R、IQCJ-SCHIP1 / PRKCI、JAK3、KMT2A、KMT2B、KMT2C、LRP1B、 MAML3、MAP2K1、MAP3K1、MAX、MCL1、MDM4、MED12、MET、MGA、MLXIPL、MRE11、MSH2、MSH6、MTOR、 MUC16、MUC4、MUTYH、MYD88、NAT1、NCOR2、NF2、NFKBIE、NKX2-1、NOTCH1、NOTCH2、NOTCH3、 PAK3、PARP1、PDE11A、PGR、PIK3R1、PLK1、PMS2、POLD1、PRDM1、PRKN、PSIP1、PTCH1、PTEN、 PTPN11、PTPN14、RAD51D、RB1、RBM10、RECQL4、RICTOR、RNF43、SBDS、SETBP1、SETD1B、SETD2、 SF3B1、SGK1、SMAD4、SMARCB1、SMC1A、SMO、SRSF2、STAG2、STK11、TAP2、TCF7L1、TERC、TERT、TGFBR2, TNFAIP3, TNFRSF14, TP53, TRIM24, TSC1, TSC2, UGT1A1, USP44, USP8, VEGFA, WAS, WNK2, YAP1, ZEB1 and ZNF703 Or one or more mutations in any combination of them.

[0231] In some implementations, subjects, as described herein, are identified or have been identified as insensitive to at least one existing TKI. In some implementations, a subject is defined as having acquired primary resistance to prior treatment with an existing TKI if they do not achieve complete remission (CR), partial remission (PR), or stable disease (SD) within 6 months of receiving treatment with at least one existing TKI. In some implementations, a subject is defined as having acquired resistance to prior treatment with an existing TKI if they achieve initial remission or stable disease after ≥6 months of receiving treatment with at least one existing TKI, but subsequently experience disease progression.

[0232] In some implementation schemes, the existing TKI for prior treatment may be imatinib (Novartis), repretinib (Zai Lab in combination with Deciphera), avatinib (CStone Pharmaceuticals in combination with Blueprint), cristatinib (Pfizer), masitinib (AB Science), ponatinib (Takeda Pharmaceuticals), dasatinib (Bristol-Myers Squibb), nilotinib (Novartis), sunitinib (CPPI CV), regorafenib (Bayer), sorafenib (Bayer), anlotinib (Chia Tai Tianqing Pharmaceutical Group), surufatinib (Hutchison Whampoa), pazopanib (GSK), rogatinib (Bayer), bridgeBio, erdatinib (Johnson & Johnson), pemitinib (Incyte), fexotinib (Blueprint), famitinib (Hengrui), or any combination thereof.

[0233] 3.2 Multi-target TKIs and their dosing regimens In some embodiments, the multi-target TKI is capable of inhibiting at least two tyrosine kinases selected from the following: KIT, PDGFR, SRC, VEGFR (e.g., any one of VEGFR1, VEGFR2, VEGFR3 or any combination thereof), FGFR (e.g., any one of FGFR1, FGFR2, FGFR3, FGFR4 or any combination thereof), or any combination thereof.

[0234] In some implementations, the multi-target TKI is capable of inhibiting at least two tyrosine kinases selected from the following: KIT, PDGFR, SRC, VEGFR2, FGFR1, or any combination thereof.

[0235] In some embodiments, the multi-target TKI comprises a heterocyclic acetylenoid compound having the following formula (I): Z is independently selected from CH or N; L 1 Independently selected from NH, -N=, CH; L 2 Independently selected from -CONH- or -NHCO-; R1 is independently selected from: 1) H; 2) C1-C6 alkyl groups; 3) C3-C6 cycloalkyl groups; 4) C1-C5 alkyl groups substituted with one or two hydroxyl groups; 5) Phenyl; 6) It can pass through L at the Z atomic site. 1 Fused A rings to form a ring containing 1-3 N atoms and L 1 Fused five-membered heterocyclic groups, such as , Among them, X, Y, and Z are independently selected from N and CH; ring D is an aromatic heterocycle containing 1-3 N atoms; R2 is independently selected 1) H; 2) Halogens; 3) C1-C5 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C5 alkyl groups; R3 is selected independently 1) H; 2) Halogens; 3) C1-C4 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C4 alkyl groups; When R5 is H, R4 is independently selected. 1) H; 2) (CH2) n NR6R7; 3) (CH2) n -Het 1 ; Alternatively, when R4 is H, R5 is independently selected. 1) H; 2) Het 2 ; n is independently selected from 0 or 1; Het 1 Defined as a non-aromatic heterocycle containing 1-3 N atoms; Het2 Defined as an aromatic five-membered heterocycle containing heteroatoms such as N, O and / or S; alkyl, cycloalkyl or NR6R7 may be incorporated into any C or N position of substituted non-aromatic heterocycles and aromatic heterocycles; R6 or R7 is selected independently from: 1) H; 2) C1-C3 alkyl groups; 3) F-containing C1-C3 alkyl groups; 4) C3-C6 cycloalkyl groups; Alternatively, R6 and R7 can be further formed into five-, six-, seven-, or eight-membered ring structures through C, O, N, and S atoms; Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0236] In some embodiments, the multi-target TKI is a compound having the following formula (II): (II) Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0237] In some implementations, the multi-target TKIs provided herein are selected from the following compounds: olabatinib, ripretinib, avatinib, cristatinib, masitinib, ponatinib, dasatinib, nilotinib, sunitinib, regorafenib, sorafenib, anlotinib, surufatinib, infuratinib, erdatinib, pemitinib, rogatinib, and lincitinib, or pharmaceutically acceptable salts or stereoisomers or prodrugs of any of the foregoing.

[0238] In some embodiments, the multi-target TKIs provided herein are administered at doses of approximately 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg. In some embodiments, olaribatinib is administered at doses of approximately 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg.

[0239] In some implementations, the multi-target TKIs described herein are administered once daily, every other day, weekly, every other week, monthly, or every other month. In some implementations, olaribatitinib is administered once daily, every other day, weekly, every other week, monthly, or every other month.

[0240] In some implementations, the multi-target TKIs, as described herein, are administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 4 months. In some implementations, olaribatitinib is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 4 months.

[0241] In some implementations, multi-target TKIs such as olabrutinib (as described herein) are administered via oral or parenteral routes (e.g., intravenous, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, or intraocular injection).

[0242] In some implementations, the multi-target TKIs provided herein are administered at a dose of approximately 15 mg / kg every other day for 3 weeks. In some implementations, olabatinib is administered at a dose of approximately 15 mg / kg every other day for 3 weeks.

[0243] 3.3 Bcl-2 inhibitors and their dosing regimens Bcl-2 family proteins are important regulators of apoptosis. This protein family includes anti-apoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-1; and pro-apoptotic molecules including Bid, Bim, Bad, Bak, and Bax. Although normal cells express low levels of anti-apoptotic Bcl-2 and Bcl-xL proteins, these proteins have been found to be highly overexpressed in many different types of human tumors and are associated with tumor development, progression, and drug resistance. As used herein, “Bcl-2 inhibitor” refers to an agent that downregulates the level and / or activity of Bcl-2 in a subject.

[0244] In some implementations, such as the Bcl-2 inhibitors provided herein, the compounds are compounds having the following formula (III). (III), Or its pharmaceutically acceptable salt or stereoisomer or prodrug, wherein: A3 is selected from ; E3 is nitrogen and It is a single bond; X 31 X 32 and X 33 Each independently selected from -CR 38 = and -N=; R 31a and R 31b Together with the carbon atoms they are attached to, they form 3-, 4-, or 5-membered cycloalkyl groups; R 32 Selected from -NO2, -SO2CH3 and -SO2CF3; R 32aSelected from hydrogen and halogens; R 33 Selected from -N(R) 34a (R) 34b ); R 34a Selected from C (optional substitution) 1-6 Alkyl, optionally substituted C 3-6 Cycloalkyl, heterocyclic, heteroalkyl, (cycloalkyl)alkyl, and (heterocyclic)alkyl; R 34b Selected from hydrogen and C 1-4 alkyl; R 38 Selected from hydrogen and halogens; In some implementations, the Bcl-2 inhibitor is selected from: , Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0245] In a preferred embodiment, the Bcl-2 inhibitor is a compound having the following formula (IV) (also known as Lishenkel, APG-2575). (IV) Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

[0246] Further multi-target TKIs that can be used in this application are disclosed in WO2018027097A1, which is incorporated herein by reference.

[0247] Those skilled in the art can determine, through routine experiments, the effective and non-toxic dose of the Bcl-2 inhibitors described herein for treating cancers as described herein. For example, the therapeutically active dose of a Bcl-2 inhibitor can vary depending on a variety of factors, such as the subject's disease stage (e.g., stage I vs. stage IV), age, sex, medical complications (e.g., immunosuppressive conditions or diseases), and the subject's weight, as well as the ability of the Bcl-2 inhibitor to elicit the desired response in the subject. In some embodiments, the therapeutically active dose is a safe dose of the Bcl-2 inhibitor that, when used in the manner disclosed herein, is sufficient to produce the desired therapeutic response without excessive adverse side effects (such as toxicity, irritation, and allergic reactions) and has a reasonable benefit / risk ratio.

[0248] Dosing regimens can be adjusted to provide optimal therapeutic response. For example, a number of fractions can be administered daily or via continuous dosing, or the dose can be reduced proportionally as indicated by an emergency in the treatment situation. In some embodiments, Bcl-2 inhibitors, such as those described herein, are administered at a therapeutically effective amount when delivered alone or in combination; that is, multi-target TKIs are administered and / or as a therapeutic treatment for the desired cancer, rather than primarily as an agent to alleviate the side effects of other treatments.

[0249] In some implementations, Bcl-2 inhibitors, such as those provided herein, are administered at a dose effective in cancer treatment. The dosages provided below can be used in any administration modality of multi-target TKIs as provided herein, including topical administration, inhalation administration, and intravenous administration (e.g., continuous infusion).

[0250] In some implementations, such as the Bcl-2 inhibitors provided herein, the administration can be oral, oral, inhaled spray, sublingual, rectal, transdermal, vaginal mucosal, transmucosal, local, nasal, or enteric; parenteral, such as intramuscular, subcutaneous, intramedullary, intrathecal, intrathecal, intracranial, intrabrain, subcutaneous, intraperitoneal, intravenous, intrasynovial, intrasternal, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, intraocular, or any other drug delivery method.

[0251] In some embodiments, the pharmaceutical composition comprises a Bcl-2 inhibitor or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition is a solid dosage form. In some embodiments, the solid dosage form is a capsule. In some embodiments, the solid dosage form is a dry-filled capsule. In some embodiments, the capsule contains about 10 mg to 500 mg of a Bcl-2 inhibitor as provided herein.

[0252] In some embodiments, the Bcl-2 inhibitors provided herein are administered at doses of about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg. In some embodiments, lissex is administered at doses of about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg.

[0253] In some implementations, the Bcl-2 inhibitors provided herein are administered twice daily, once daily, every other day, once weekly, every other week, once monthly, or every other month. In some implementations, the Bcl-2 inhibitors provided herein are administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 4 months. In some implementations, lisqualane is administered twice daily, once daily, every other day, once weekly, every other week, once monthly, or every other month. In some implementations, lisqualane is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 4 months.

[0254] In a preferred embodiment, the Bcl-2 inhibitor, as described herein, is administered once daily at a dose of approximately 50 mg / kg for 3 weeks. In a preferred embodiment, lissaccharin is administered once daily at a dose of approximately 50 mg / kg for 3 weeks.

[0255] 3.4 Combination Therapy This article presents a method for treating cancer using a combination of multi-target KTI and Bcl-2 inhibitors, wherein the multi-target KTI and Bcl-2 inhibitors are as defined above. Cancer is also as defined above.

[0256] In some implementations, the multi-target TKI and the Bcl-2 inhibitor are each in separate formulations, or the multi-target TKI and the Bcl-2 inhibitor are in separate formulations.

[0257] In some implementations, the multi-target TKI and the Bcl-2 inhibitor are administered simultaneously, or the multi-target TKI and the Bcl-2 inhibitor are administered sequentially.

[0258] In some implementations, the multi-target TKI and the Bcl-2 inhibitor can be administered sequentially at time intervals of approximately 1 minute, approximately 5 minutes, approximately 10 minutes, approximately 15 minutes, approximately 30 minutes, approximately 45 minutes, approximately 1 hour, approximately 2 hours, approximately 4 hours, approximately 6 hours, approximately 12 hours, approximately 24 hours, approximately 48 hours, approximately 72 hours, approximately 96 hours, approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 8 weeks, or approximately 12 weeks.

[0259] In some embodiments, the combined product of the present invention comprising the multi-target TKI and the Bcl-2 inhibitor may be in the form of a pharmaceutical composition, wherein the multi-target TKI and the Bcl-2 inhibitor are each in separate dose units, or the multi-target TKI and the Bcl-2 inhibitor are in the form of a single dose unit.

[0260] In some implementations, the combination product can be administered in the following ways: orally, orally, via inhalation spray, sublingually, rectally, transdermally, via vaginal mucosa, via mucosa, topically, via the nose, or via the intestine; parenterally, such as intramuscularly, subcutaneously, intramedullary, and intrathecally or directly into the brain, or in situ, subcutaneously, intraperitoneally, intravenously, intrasynovially, intrasternally, intrahepatically, intralesionally, intracranially, intraperitoneally, via the nose, intraocularly, or by any other means of drug delivery.

[0261] In some embodiments, the multitarget TKI included in the combination therapy is administered at a dose of about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg; and the Bcl-2 inhibitor included in the combination therapy is administered at a dose of about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg. In some embodiments, olabrutinib, included in the combination therapy, is administered at a dose of about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg; and levofloxacin, included in the combination therapy, is administered at a dose of about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg.

[0262] In some embodiments, the multi-target TKI and the Bcl-2 inhibitor included in the combination therapy are administered alone or together, once daily, every other day, once weekly, once weekly, once monthly, or once monthly. In some embodiments, olaribatitinib and levofloxacin included in the combination therapy are administered alone or together, once daily, every other day, once weekly, once weekly, once monthly, or once monthly.

[0263] In some embodiments, the combination therapy comprising the multi-target TKI and the Bcl-2 inhibitor, as provided herein, is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 4 months. In some embodiments, the combination therapy comprising olaribatitinib and levofloxacin, as provided herein, is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 4 months.

[0264] In a preferred embodiment, the multi-target TKI as described herein is administered every other day at a dose of about 15 mg / kg for 3 weeks, and the Bcl-2 inhibitor as described herein is administered once daily at a dose of about 50 mg / kg for 3 weeks. In a preferred embodiment, olabatinib is administered every other day at a dose of about 15 mg / kg for 3 weeks, and levofloxacin is administered once daily at a dose of about 50 mg / kg for 3 weeks.

[0265] Combination therapies of multi-target TKIs (e.g., olabatinib) and Bcl-2 inhibitors (e.g., levofloxacin) as described herein may produce complementary, additive, or synergistic effects compared to monotherapy (i.e., multi-target TKIs or Bcl-2 inhibitors as described herein). A combination therapy producing a complementary effect is one where the overall clinical benefit of the combination therapy is greater than the clinical benefit of the monotherapy. A combination therapy producing an additive effect is one where the clinical benefit of the combination of agents reflects the sum of the individual monotherapies. A combination therapy producing a synergistic effect is one where the overall clinical benefit obtained from the combination is greater than the additive effect of each agent. Furthermore, in some embodiments, due to the additive or synergistic effect of the combination therapy, less frequent dosing and / or lower doses of one or more therapies may be used compared to monotherapy. In further embodiments, lower toxicity, resulting from fewer side effects from one or more therapies, may be obtained due to the lower doses and / or less frequent dosing of one or more therapies in the combination therapy. Therefore, in some embodiments, the pharmaceutically effective amount of the agent used as a component of the combination therapy to treat the subject's disease is less than the pharmaceutically effective amount of the same agent used as a monotherapy.

[0266] In some implementations, after treatment with a compound having formula (II) in combination with a compound having formula (IV), the subject has a tumor growth inhibition (TGI) rate of at least 75%.

[0267] In some embodiments, the combination of the multi-target TKI and the Bcl-2 inhibitor can inhibit the proliferation of imatinib-resistant GIST cells. In some embodiments, the combination of the multi-target TKI and the Bcl-2 inhibitor can inhibit the proliferation of imatinib-resistant GIST cells in an additive or synergistic manner. The imatinib-resistant GIST cells used herein can be GIST430, GIST48, GIST48B, or any combination thereof.

[0268] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below fall, in whole or in part, within the scope of this invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of this invention. Those skilled in the art can develop equivalent compositions, materials, and methods without inventive effort and without departing from the scope of this invention. It should be understood that many changes can be made in the processes described herein while still remaining within the scope of this invention. The inventors intend that such changes be included within the scope of this invention.

[0269] Example While we have described many embodiments of the invention, it will be apparent that our basic embodiments can be modified to provide other embodiments utilizing the compounds and methods of this disclosure. Therefore, it should be understood that the scope of this disclosure is defined by the appended claims, and not by the specific embodiments already shown by way of example.

[0270] All references cited throughout this application (including bibliographic references, granted patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly known to a person skilled in the art.

[0271] Example 1. Application of olabatrinib in late-stage SDH-deficient GIST: multicenter, open-label, phase 1 trial 1. Method Study design and patient population HQP1351-SJ-0003 is an ongoing, open-label, dose-escalation / dose-expansion phase 1 study being conducted at six sites in China to evaluate the safety and efficacy of olaribatinib in patients with refractory or relapsed advanced and / or metastatic GIST or other solid tumors (ClinicalTrials.gov identifier: NCT03594422). The dose-escalation portion of this study enrolled patients with advanced and / or metastatic GIST or other solid tumors that were refractory or relapsed after treatment with at least one TKI. In this group, oral olaribatinib QOD was administered in consecutive 28-day cycles at an initial dose of 20 mg, 30 mg, 40 mg, or 50 mg until the MTD was determined, with the aim of advancing to the dose-expansion portion of the study to determine the RP2D.

[0272] Eligible patients are adults (≥18 years) with advanced and / or metastatic GIST or other solid tumors confirmed by histology and / or cytology. SDH-deficient GIST is defined based on a pathological phenotype with negative immunohistochemical staining for SDHB. Study recruitment also requires an Eastern Cooperative Oncology Group Performance Status (ECOG PS) ≤2; and adequate renal, hepatic, cardiac, and bone marrow function. A complete list of inclusion and exclusion criteria is provided in the protocol. Recruitment trends, ongoing review of study data, and evolving knowledge about GIST treatment paradigms led the study sponsor's medical director and investigators to decide to analyze the SDH-deficient GIST patient subgroup separately.

[0273] Based on encouraging initial activity and good tolerability (Qiu, H. et al., J. Clin. Oncol.The study (40, 11513 (2022)) recruited additional SDH-deficient GIST patients to further investigate its efficacy, and allowed participation from SDH-deficient GIST patients who had not received TKI therapy and adolescent patients (12–18 years) with SDH-deficient GIST. Data from the dose-escalation portion of the study were reviewed during the principal investigator's meeting, and after recommending a post-marketing dose for the treatment of CML, all adult patients received 40 mg QOD (the determined RP2D dose), and adolescent patients received 20 mg to 40 mg QOD based on weight. Patients weighing at least 40 kg received 40 mg QOD; those in the 30–40 kg range received 30 mg QOD; and those in the 20–30 kg range received 20 mg QOD. Patients with SDH-deficient malignant paragangliomas who had failed at least one treatment and had no appropriate treatment (as determined by the investigator) were also recruited. Treatment can continue until exclusion due to toxicity, non-compliance, withdrawal of consent, physician decision, disease progression, death, or study termination. The trial protocol version released on May 27, 2022, identified all protocol modifications, including increases in patient and cohort numbers for dose expansion.

[0274] Tumor mutation status was determined through local detection and centrally confirmed using DNA from archived or new tumor biopsy samples. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and conformed to Good Clinical Practice as outlined in the International Conference on Harmonisation of Standards. The study was approved by the ethics review committee or independent ethics committee at each study site. Written informed consent was provided by all patients.

[0275] Research Evaluation Physical examinations and adverse events (AEs) were assessed at each follow-up visit (days –28 to –1 after informed consent); on days 1, 8, 15, and 22 during cycles 1 and 2; and on days 1 and 15 during cycle 3 and all subsequent cycles, up to 30 days after the final dose. All AEs were graded according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE; version 4.03). Routine blood tests and clinical laboratory studies of serum chemistry parameters were performed at each cycle (day 1); cycles 1 and 2 (days 8, 15, and 22); and 30 days after the last dose. Urinalysis, coagulation, cardiac troponin I / T, lipase, amylase, and electrocardiogram were assessed at baseline; in cycles 1 and 2 (days 1 and 15); in cycles 3, 5, 7, and 9; every 3 cycles after cycle 9 (day 1); and 14 days after the last dose. All patients underwent tumor imaging by computed tomography or magnetic resonance imaging at screening and every two cycles (8 weeks) to assess response until progression or discontinuation. Target and non-target lesions were assessed according to the Responsive Criteria for Solid Tumor Response, version 1.1 (RECIST v1.1).

[0276] A series of blood samples were collected to measure the plasma concentrations of olaribatinib. For adult patients, plasma samples were collected before administration on days 1 and 27 of cycle 1, and at 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours after administration, and before administration on days 15, 17, and 19. For adolescent patients, plasma samples were collected before administration on days 1 and 27 of cycle 1, and at 1, 2, 4, 6, 8, 24, and 48 hours after administration. Non-compartmental pharmacokinetic (PK) analysis was performed using Phoenix WinNonlin software (version 6.4). PK parameters for olaribatinib, including the area under the plasma concentration-time curve (AUC) and the maximum observed plasma concentration (C), were derived. max ), time to maximum observed plasma concentration (t) max ), apparent terminal elimination half-life (t) 1 / 2 Apparent total oral clearance (CL / F) and apparent volume of distribution at terminal elimination (Vz / F).

[0277] Research Results The primary endpoint was the determination of MTD / RP2D and the safety characteristics of olaribatib. Secondary endpoints included pharmacokinetic and pharmacodynamic characteristics, CBR, overall response rate (ORR), PFS, and overall survival (OS) according to RECIST v1.1.

[0278] CBR is defined as the proportion of patients with a proven CR or PR of any duration or SD of at least 16 weeks from the start of treatment (CBR = CR + PR + SD ≥ 16 weeks). ORR is defined as the proportion of patients with a proven CR or PR of any duration (ORR = CR + PR). PFS is defined as the time from the start of treatment to the date of first documented disease progression or death from any cause (whichever is earlier). OS is defined as the time from the start of treatment to the date of death.

[0279] Statistical analysis The data cutoff date for this analysis was December 27, 2023. The safety cohort included all patients who received at least one dose of olaribinib. The SDH-deficient GIST cohort (a pre-specified subgroup) included patients from the safety cohort. MTD was defined as the highest dose level at which no more than one dose-limiting toxicity occurred in six patients.

[0280] We evaluated antitumor activity in patients with SDH-deficient GIST who had undergone at least one post-baseline disease assessment. We used the Kaplan-Meier method to estimate PFS and OS; corresponding 95% CIs are provided. Treatment outcomes in patients with SDH-deficient GIST are reported separately. All statistical analyses were performed using SAS (version 9.3 or later; Cary SAS Institute, North Carolina).

[0281] 2. Results patient From July 11, 2018 to December 27, 2023, 66 patients were recruited into the safe population. The study population included 33 patients with non-SDH-deficient GIST, 26 patients with SDH-deficient GIST, 6 patients with paragangliomas, and 1 patient with pancreatic cancer. Baseline characteristics of patients in the non-SDH-deficient and SDH-deficient groups are shown in Table 2. In the non-SDH-deficient GIST group (n=33), the median age was 55 years (range 33–72); 26 (78.8%) patients were male; and the primary tumor sites were the stomach (18.2%) in 6 patients, the small intestine (54.5%) in 18 patients, and the large intestine (6.1%) in 2 patients. Most patients had previously received TKI therapy, with 15 patients (45.5%) and 13 patients (39.4%) having received at least two or three TKIs, respectively. In the SDH-deficient GIST group (n=26), the median age was 30 years (range 13–56); 7 patients (26.9%) were male; and the primary tumor site was the stomach in 24 patients (92.3%). Most patients had previously received TKI treatment, including 5 patients (19.2%) who had received at least 2, 9 patients (34.6%) who had received at least 3, and 2 patients (7.7%) who had received at least 4 or more TKIs. Data are median (range) or n (%). Due to rounding, the sum of percentages may not equal 100%. ECOG = Eastern Cooperative Oncology Group. *Small intestine, duodenum, jejunum, or ileum. † Colon and rectum. ‡ Unknown: One patient's initial assessment indicated multiple liver metastases; another patient had an abdominal mass.

[0282] Of the 33 patients with non-SDH-deficient GIST, 27 (81.8%) had KIT Mutation, 2 people (6.1%) had it PDGFRA The mutation was present in 2 patients (6.1%), and 2 patients (6.1%) had WT GIST. In the last subgroup, one patient had normal SDH function and neurofibromatosis type 1 (SDH). NF1 One patient tested positive, while the SDH status of another patient was unknown because tissue could not be obtained for immunohistochemical staining. Among the 26 patients with SDH-deficient GIST, germline... SDHB It is the most common SDH mutation (46.2% of 12 patients), and one patient has a germline mutation. SDHA and SDHC The remaining 12 (46.2%) individuals had no detectable SDH mutations.

[0283] The distribution of different GIST subtypes and the dosage of olaribatibin are summarized in Table 3. Most patients received olaribatibin 40 mg every other day (QOD). Based on the data from the dose escalation portion of this study and several studies on olaribatibin in CML, this regimen was selected as RP2D. This indication and the 40 mg QOD dosage have been approved for marketing by the China National Medical Products Administration. Security At the data cutoff, the median follow-up duration was 14.5 months (range, 0.9–57.5). Based on investigator judgment, all 66 subjects (100.0%) experienced treatment-related adverse events (TEAEs), with the majority being Grade 1 or 2. TEAEs (≥20%) included increased aspartate aminotransferase (AST) in 31 patients (47%), increased alanine aminotransferase (ALT) in 25 patients (37.9%), increased white blood cell count in 32 patients (48.5%), increased neutrophil count in 25 patients (37.9%), anemia in 31 patients (47.0%), fever in 28 patients (42.4%), constipation in 27 patients (40.9%), and abdominal pain in 15 patients (22.7%) (Table 4). 62 patients (93.9%) experienced treatment-related adverse events (TRAEs), the majority of which were Grade 1 or 2 (Table 5). Nine patients (13.6%) experienced ≥ grade 3 TRAEs, and five patients (7.6%) experienced treatment-related serious adverse events (Table 5). No dose-limiting toxicities were reported, and the maximum tolerated dose (MTD) was not reached. Of the total 66 patients, 21 (31.8%) required treatment discontinuation, the most common reasons being COVID-19 (9 patients), fever (4 patients), and abdominal pain and increased AST (2 patients each). Two patients required dose reduction due to increased AST or different patient selection. CRP, C-reactive protein; WBC, white blood cells.

[0284] therapeutic effect During a median follow-up period of 17.0 (range, 4.1–57.5) months, investigators confirmed that 6 out of 26 evaluable patients in the SDH-deficient GIST subgroup met the overall response criteria according to RECIST v1.1. No patients experienced complete response (CR), and 6 patients (23.1%) achieved partial response (PR). Clinical benefit was observed in 24 patients (92.3%), defined as the proportion of patients with confirmed CR, PR, or stable disease (SD) for ≥16 weeks. Figure 1A The median progression-free survival was 25.7 months (range, 12.9 - not reached); Figure 1B At the data cutoff, all patients in the SDH-deficient GIST cohort were alive, resulting in an overall survival of 100%.

[0285] Pharmacokinetic analysis Pharmacokinetics of olaribatinib (20 mg to 50 mg) was evaluated in patients with GIST after oral administration at QOD under food conditions. The pharmacokinetic results on day 27 are summarized in Table 6. Peak concentrations of olaribatinib occurred between 6 and 8 hours after oral administration at doses ranging from 20 mg to 50 mg. Mean terminal half-life (t...) 1 / 2 The effective duration of action ranged from 18.9 hours to 27 hours. Orabatinib showed the highest concentration (C) in the dose range of 20 mg to 50 mg. max ) and area under the concentration-time curve (AUC) last The approximate dose-proportion increase was observed. No significant accumulation of olaribinib was observed after multiple dose administrations.

[0286] Table 6. Summary of Pharmacokinetic Parameters of Orabatinib in GIST Patients at Steady State The value is T max The median (range) and the average (SD) of all other values.

[0287] Pharmacokinetic parameters, AUC, were obtained through non-compartmental analysis (NCA). last From time zero to the last measurable concentration (T) last Area under the curve at time ) ; CL / F, apparent total oral clearance; C max Maximum observed plasma drug concentration; T 1 / 2 Terminal elimination half-life; T max , the time to reach the maximum observed plasma drug concentration; Vz / F, apparent volume of distribution.

[0288] 3. Discussion Although the advent and use of imatinib has revolutionized care for most patients with advanced GIST, patients with SDH-deficient disease rarely respond to this agent, and other approved multi-kinase inhibitors have not been proven effective. Our study is the world's largest prospective clinical trial evaluating a TKI for the treatment of SDH-deficient GIST (to the best of our knowledge (Nannini, M. et al., Ther. Adv. Med. Oncol.13, 17588359211023278 (2021)) indicates that the new multi-kinase inhibitor olaribatib has clinical activity in patients with advanced SDH-deficient GIST, with durable response and manageable safety characteristics, including a median PFS of 25.7 months.

[0289] In this study, most patients with SDH-deficient GIST were women, younger, and had primary tumors located in the stomach. These factors, along with gender, [further context needed for accurate translation]. SDHB The expression and clinicopathological association with tumor site are consistent with the literature involving SDH-deficient GIST (Pasini, B. et al., Eur. J. Hum. Genet., 16, 79–88 (2008); Boikos, SA et al. JAMA Oncol. 7,922–928 (2016)). Loss of SDHB protein expression due to any SDH The biallelic inactivation of the gene occurs, and this inactivation is caused by... SDHC Mutations or hypermethylation in gene promoters cause this, leading to SDH deficiency (Unc, M. et al., Cancers (Basel) 15,1498 (2023); Pasini, B. et al. Eur. J. Hum. Genet., 16, 79–88 (2008); Killian, JK et al. Cancer Discov. 3,648–657 (2013)). SDHA Germplasm mutations occur in approximately 30% of SDH-deficient GISTs, while SDHB , SDHC and SDHD Germline mutations occur in 20% to 30% of cases (Pantaleo, MA et al.). J. Natl. Cancer Inst. 103, 983–987 (2011); Miettinen, M. and Lasota, J., Cell Biol. 53, 514–519 (2014)). The remaining 50% of SDH-deficient GISTs are lacking. SDH Mutation, but showing SDHC promoter hypermethylation (Unc, M. et al., Cancers (Basel) 15,1498 (2023); Killian, JK et al. Cancer Discov. 3,648–657 (2013). An observational study at the GIST clinic of the National Institutes of Health included 63 patients with SDH-mutant GIST, of whom 34 had SDHA Mutation, 16 people with SDHB Mutation, 12 people with SDHC Mutation, 1 person with SDHDMutations (Boikos, SA et al.) JAMA Oncol. 7, 922–928 (2016)). In our study, phylogenetic... SDHB It is the most common SDH Mutation (12 / 26 patients), while germline SDHA and SDHC There was only one case of each mutation. In our study, the germline... SDH The reason why the mutation frequency differs from previously reported values ​​is unclear, but one possible reason is that all patients in our study were Asian; further research is needed to explore potential racial differences in genotype. Consistent with previous reports, we did not detect it in 12 (46.2%) of the 26 patients with SDH-deficient GIST. SDH Mutation, this discovery may be related to SDHC It is related to hypermethylation of genes.

[0290] Because SDH-deficient GIST is difficult to treat and rare, clinical experience in its treatment is very limited. Complicating this issue is that most data in the literature reporting TKI activity in KIT / PDGFRA WT GIST lack characterization of SDH deficiency. In short, objective tumor response in this specific GIST subtype is uncommon, and previous experience with imatinib, sunitinib, and regorafenib (type 1 or 2 TKIs) has shown limited response or stable disease in SDH-deficient GIST (Nannini, M. et al.). Ther. Adv. Med. Oncol. 13, 17588359211023278 (2021); Boikos, SA et al. JAMA Oncol. 7, 922–928 (2016); Neppala, P. et al. Cancer Metastasis Rev. 38, 525–535 (2019). Imatinib is not effective in KIT / PDGFRA WT GIST (Boikos, SA et al., JAMA Oncol. 7, 922–928 (2016). In a US-led phase 3 trial, the effects of imatinib in advanced GIST were investigated. WT GIST patients had shorter time to tumor progression than those with more common exon 11 mutation-positive tumors, at 13 months vs. 25 months (Heinrich, MC et al.). J. Clin. Oncol. 33, 5360–5367 (2008)).

[0291] In adolescents with advanced GIST who failed imatinib treatment, sunitinib treatment resulted in disease stabilization (SD) for 6 months or longer in 56% of patients (Janeway, KA et al.). Pediatr. Blood Cancer52, 767–771 (2009)). In the aforementioned retrospective study conducted by Boikos et al. (Boikos, SA et al., JAMA Oncol. In 7,922–928 (2016), 7 out of 38 WT GIST patients treated with sunitinib achieved an objective response (18.4%), including partial response (n=3), mixed response (defined as progression in some sites and regression in others; n=3), and complete response (CR; n=1). In a phase 1 / 2 prospective trial of sunitinib in patients aged 6 to 18 years with advanced GIST, no tumor response was observed in six patients (five with SDH-deficient tumors and one untested), but three patients achieved SD, with a median PFS of only 5.8 months (Verschuur, AC et al.). Cancer Chemother. Pharmacol. 84, 41-50 (2019)).

[0292] In a phase 2 multicenter trial of regorafenib in patients with metastatic and / or unresectable GIST who had failed standard TKI therapy, the median progression-free survival (PFS) was 10 months in 6 patients with SDH-deficient GIST who received regorafenib, and all patients experienced clinical benefit for more than 16 weeks (Ben-Ami, E. et al.). Ann. Oncol. 27, 1794–1799 (2016)). In the treatment of metastatic and / or unresectable... KIT / PDGFR In a phase 2 single-arm trial of regorafenib as a single agent in WT GIST patients, 15 patients were enrolled. Although no CR was achieved, 13% of patients experienced PR, the 12-week disease control rate was 86.7%, and the median PFS was 11 months (Martin-Broto, J. et al.). Mol. Cancer 22,127 (2023)).

[0293] Because SDH-deficient GIST typically shows increased IGF-1R expression, von Mehren et al. prospectively tested the efficacy of the oral TKIIGF-1R inhibitor lincitinib in patients with WT GIST. Although no measurable response was observed, the 9-month clinical benefit rate was 40%, and the estimated 9-month PFS was 52% (von Mehren, M. et al.). Clin. Cancer Res. 26, 1837–1845 (2020)). In preclinical studies of patient-derived SDH-deficient GIST models, the DNA alkylating agent temozolomide (TMZ) has been shown to induce DNA damage and apoptosis (Yebra M. et al., Clin. Cancer Res.28, 187–200 (2022)). Preliminary results from a phase 2 clinical trial of TMZ in nine patients enrolled in advanced SDH-deficient GIST showed a 6-month ORR of 22.2% (Burgoyne, A. et al., Proceedings of the CTOS 2022 Annual Meeting, P096, Vancouver, BC, Canada (2022)).

[0294] Given that nearly half of the patients in our study had received extensive pretreatment with more than two TKIs, it is noteworthy that the clinical benefit rate (CBR) of patients receiving olaribatitinib exceeded 90%, with a median progression-free survival (mPFS) of 25.7 months; this level of disease control and durability of response appears to exceed those reported in all prospective clinical trials. Furthermore, olaribatitinib was well-tolerated in this group of patients, with a lower frequency and severity of adverse events compared to CML patients (Jiang, Q. et al.). J. Hematol. Oncol. 15,113 (2022)). Common drug-related toxicities were primarily Grade 1 or 2, and were usually manageable with standard supportive pharmacological treatment. No dose-limiting toxicities were observed, and no patients discontinued treatment due to adverse events.

[0295] In summary, olabrutinib was well tolerated in this prospective trial of patients with SDH-deficient GIST, with a CBR exceeding 90% and a significantly prolonged estimated median PFS, demonstrating the potential benefit of this treatment and providing a benchmark for future research in this rare subtype of GIST.

[0296] Example 2. Orabatinib for advanced SDH-deficient GIST: Preclinical Results 1. Method Cell lines and cell cultures Acute lymphoblastic leukemia (Jurkat, clone E6-1) and renal cell carcinoma (OS-RC-2) cell lines were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The colon cancer cell line (RKO) was generously donated by the laboratory of Professor Shaomeng Wang at the University of Michigan. The rat pheochromocytoma cell line (PC12) was obtained from Nanjing Kebai Biotechnology Co., Ltd. (Nanjing, China). The PC12 SDBH knockdown (KD) cell line (PC12#5F7) was obtained by Suzhou Genewiz Biotechnology Co., Ltd. (Anshengda Life Sciences; Suzhou, China) using the sgRNA-mediated clustered regularly spaced short palindromic repeat (CRISPR) / Cas9 system. All cell lines were identified by short tandem repeat (STR) analysis by Suzhou Genewiz Biotechnology Co., Ltd.; were negative for mycoplasma contamination; and were passaged in vitro less than 3 months after resuscitation. All cell lines were cultured in the recommended medium at 37°C and 5% CO2 atmosphere (Table 7).

[0297] Table 7. Cell lines and culture conditions HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; RPMI, Roswell Park Memorial Institute.

[0298] Human GlST tissue sample Frozen GIST tissues from 15 patients were used for RNA sequencing and LC-MS, and tissues from 3 patients with SDH-deficient GIST were used for primary cell culture. All samples were collected at Sun Yat-sen University Cancer Center, and informed consent was obtained from each participant. This study involving human subjects was approved by the Medical Ethics Committee of Sun Yat-sen University Cancer Center.

[0299] Tissue samples from GIST patients were maintained in DMEM / F12 medium containing 1% antibiotics and washed with phosphate-buffered saline (PBS). The tissues were then surgically fragmented using scissors. The tissues were digested with 1 mg / mL collagenase IV at 37°C for 30 minutes, and the suspension was then filtered into single cells by centrifugation using a single-cell filter. Finally, the cell suspension was collected in centrifuge tubes, centrifuged at 500×g for 5 minutes, and resuspended in GIST primary cell culture medium (DMEM) / F12 medium containing 10% FBS, 1% GlutaMAX™ (#35050061, ThermoFisher), 1% Anti-Anti (#15240062, GIBICO), 50 ng / ml SCF (#300-07-10, PPL), 125 ng / ml EGF (#PK0014-505, PPL), 20 ng / ml bFGF (#HY-P7004, MCE), 25 ng / ml hydrocortisone (#HY-N0583, MCE) and 5 ug / ml insulin (#HY-P0035, MCE). Cells were counted and seeded in 96-well plates (5,000 cells / well) and co-cultured with different concentrations of olaribatinib and other TKIs for 72 hours, then analyzed using CellTiter 96. ® Aqueous, non-radioactive cell proliferation assay (#G7570, Promega, Madison, WI, USA) was used to detect cell proliferation activity. The procedure was performed according to the manufacturer's instructions.

[0300] reagents Orabatinib was manufactured by Jiangsu Ascentage Pharmaceutical Co., Ltd. (Taizhou, China). Imatinib, avatinib, ponatinib, dasatinib, sunitinib, regorafenib, sorafenib, pazopanib, rogatinib, and pemitinib were all purchased from Jiangsu Aikon Biopharmaceutical R&D Co., Ltd. Ripretinib was purchased from Selleck Chem (Houston, TX, USA). Infiglioglatinib was purchased from MCE (MedChemExpress).

[0301] Cell proliferation assay The compound was prepared in a specific dose range by serial dilution and then added to 96-well plates, followed by cell seeding at the optimal density per well. Cells were incubated with the drug for 72 hours and counted using a cell counting kit-8 (Shanghai Liji Medical Technology Co., Ltd.) or CellTiter-Glo. ®Cell growth was quantified using a chemiluminescent cell viability assay kit (#G7573, Promega). For each condition, three replicates were measured for each concentration. After background subtraction, relative survival in the presence of the drug was normalized relative to untreated controls. Graphs were generated using Prism 9 (GraphPad software, La Jolla, CA, USA) based on the mean of the three replicates.

[0302] Using CellTiter-Glo ® Viability studies are performed using luminescence assays (Promega), where the luciferase-catalyzed luciferin / ATP reaction provides an indicator of cell number.

[0303] Cell death assay Rat pheochromocytoma cells (PC12 or PC12 SDHB KD cells PC12#5F7) were seeded into 24-well plates and incubated overnight in complete culture medium. Cells were exposed to olabrutinib for 24 hours, then collected and resuspended in culture medium. The cell suspension was then mixed with Viastain. TM AOPI staining solution (acridine orange / propidium iodide, #2210170205, Nexelcom Bioscience, Lawrence, MA, USA) was mixed in a 1:1 ratio. After staining, the solution was analyzed using a Cellometer. ® Counting dead cells on Auto 2000 Cell Counter (Nexelcom).

[0304] Cell colony formation assay For colony formation assays, 1,000 cells were seeded in each well of a 6-well plate, incubated for 7 to 14 days, stained with 0.1% crystal violet, and the number of colonies was counted.

[0305] Western blot analysis Harvested cells treated with dimethyl sulfoxide (DMSO) or a compound at indicated concentration were lysed in radioimmunoprecipitation (RIPA) buffer (#P0013, Beyotime, China) containing a mixture of protease inhibitors (#20104ES08, Yisheng Biotechnology Co., Ltd., Shanghai) and phosphatase inhibitors (#20109-A, #20109-B, Yisheng Biotechnology). Equal volumes of proteins were separated by 4%–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocking with 3% bovine serum, the membranes were incubated with primary antibodies and then with horseradish peroxidase (HRP)-conjugated secondary antibodies (#7074, anti-rabbit IgG, CST; #7076, anti-mouse IgG, CST). Finally, the binding of the secondary antibodies was observed using an ECL Western blot assay kit (Yisheng). The primary antibodies used included: SDHB (#AB14714, Abcam), phosphorylated FGFR1 (#52928, Cell Signaling Technology [CST]), FGFR1 (#9740, CST), FGF3 (#PA5-102906, Thermo Fisher Scientific, Waltham, MA, USA), FGF4 (#PA5-115228, Thermo Fisher Scientific), phosphorylated Src (#6943, CST), Src (#2109, CST), phosphorylated VEGFR2 (#3770, CST), VEGFR2 (#9698, CST), HIF-1α (#ab16066, Abcam), and HIF-2α (#... PA116510 (Invitrogen), VEGFA (#ab214424, Abcam), phosphorylated AKT (#4060, CST), AKT (#4685, CST), phosphorylated ERK (#4370, CST), ERK (#4695, CST), phosphorylated IGF-1Rβ (#3021, CST), IGF-1R (#ab182408, Abcam), C-MYC (#13987, CS) T), NFR2 (#20733, CST), phosphorylated STAT3 (#9145, CST), STAT3 (#9139, CST), phosphorylated STAT5 (#9359, CST), STAT5 (#94205, CST), PARP-1 (#9532, CST), caspase-3 (#9665, CST), cleaved caspase-3 (#9664, CST), and β-actin (#3700, CST).

[0306] 2. Orabatinib exhibits excellent antiproliferative activity in SDH-deficient tumor cell lines and primary SDH-deficient cells.

[0307] SDH is a key respiratory mitochondrial enzyme that connects the citrate cycle and the electron transport chain. The SDH complex (also known as "mitochondrial complex II") consists of four subunits: SDHA, SDHB, SDHC, and SDHD. Inactivation of the biallelic gene of one of the four SDH subunits, or... SDHC Promoter hypermethylation and epigenetic silencing of genes can lead to loss of function of the entire SDH complex. Subsequently, SDHB degrades and presents as an SDHB-negative immunohistochemical. No SDH-deficient GIST cell lines are available, and currently available lines contain mutants. KIT or PDFGR It cannot be used on GIST cell lines either, because olabatinib can effectively inhibit mutations. KIT / PDGFR This would mask its activity through other possible pathways. Therefore, other types of SDH-deficient tumor cell lines were used to test the in vitro antitumor activity of olaribatibin. These included Jurkat clone E6-1 (SDHB mutant [mut]), OS-RC-2 (SDHA and SDHB mut), RKO (SDHA mut), and rat pheochromocytoma PC12 cells (KD, PC12#5F7) with SDHB knockdown.

[0308] As shown in Table 8, olabatinib exhibited superior antiproliferative activity compared to other TKIs, including imatinib, sunitinib, and regorafenib (three other KIT inhibitors approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of GIST). Figure 2A As shown in Table 9, olabrutinib was the most effective inhibitor among all three primary cell lines derived from SDH-deficient GIST patients.

[0309] Table 8. Antiproliferative activity of TKIs in SDH-deficient cell lines and primary SDH-deficient cells Table 9. Antiproliferative activity of TKIs in SDH-deficient primary cells 3. Tumor cells with SDHB deficiency or knockdown (KD) are more sensitive to olabrutinib treatment than cells with normal SDHB function.

[0310] To detect the activity of olaribatibin in SDH-deficient cells, we knocked down olaribatibin in rat pheochromocytoma PC12 cells using CRISPR-Cas9. SDHBThe gene was extracted, and a clone 5F7 cell line (PC12#5F7) with significantly suppressed SDHB expression was generated. Orabatinib showed stronger dose-dependent activity in PC12#5F7 than in parental PC12 cells. Figure 2B (Left). When comparing the activity of olaribatitinib in naturally SDHB-functional and SDHB-deficient tumor cell lines, we found that colony formation in OS-RC-2 (SDHB-deficient renal cell carcinoma) was significantly inhibited by olaribatitinib compared to the 785-O (SDHB-functional renal adenocarcinoma) cell line. Figure 2B (See right image).

[0311] 4. Mechanism of action of olaribatibin in SDH-deficient tumors.

[0312] Western blot analysis showed that, compared with parental PC12 cells, SDHB Knockdown of these factors increased hypoxia-inducible factors (HIF-1α, HIF-2α), vascular endothelial growth factor-A (VEGFA), fibroblast growth factor receptor-1 (FGFR1), insulin-like growth factor-1 receptor (IGF-1R), and phosphorylated FGFR1 and IGF-1R. All of these factors have been reported to be involved in in vitro tumorigenesis due to SDH deficiency. Figure 2C In addition, in SDHB Knockdown also revealed upregulation of signal transducers and activator of transcription 3 (STAT3), phosphorylated STAT3, and Src, which had not been previously reported. Orabatinib treatment dose-dependently inhibited these proteins and their downstream proteins AKT (protein kinase B) and extracellular signal-regulated kinases 1 / 2 (ERK1 / 2). These signaling pathways are involved in hypoxia, angiogenesis, proliferation, and survival. Increased cleavage of caspase-3 and poly(ADP-ribose) polymerase 1 was also observed in tumor cell lines treated with olabatinib, indicating the induction of apoptosis.

[0313] 5. Discussion Although the exact oncogenic mechanisms of SDH deficiency in cancer are not yet fully elucidated, research into the complex biological events leading to SDH-deficient GIST tumors is ongoing. Most reported mechanisms of SDH-deficient cancers involve succinate-induced signaling pathways and upregulated FGFR pathways. SDH deficiency leads to the accumulation of succinate (a known oncogenic metabolite) (Eijkelenkamp, ​​K. et al., Clin. Genet., 97, 39–53 (2020); Kuo, C.-C. et al. J. Biomed. Sci. 29, 93 (2022); Selak, MA et al. Cancer Cell.7, 77–85 (2005)), which induces pseudohypoxia-associated proteins HIF-1α / 2α, IGF-1R, and angiogenic proteins (e.g., VEGF) in SDH-deficient GIST cell lines (Flavahan, WA et al., Nature. 575, 229–233 (2019)), paraganglioma (Gimenez-Roqueplo, AP et al., Am. J. Hum. Genet., 69, 1186–1197 (2001)) and pheochromocytoma (Gimenez-Roqueplo, AP et al., J. Clin. Endocrinol. Metab. 87, 4771–4774 (2002)). FGFR pathway signaling, including the receptor tyrosine kinase FGFR1 and two identified oncoproteins FGF3 and FGF4, is significantly highly expressed in SDH-deficient GISTs (Flavahan, WA et al., Nature. 575, 229–233 (2019)).

[0314] In our study, knockdown of SDHB in PC12 cells also revealed upregulation of proteins involved in the aforementioned signaling pathways, which in turn control cell angiogenesis, proliferation, survival, and apoptosis. These proteins, along with their downstream signaling molecules AKT, ERK, and STAT3, were inhibited by olaribinib in a dose-dependent manner; these effects partially explain the mechanism of action (MOA) of olaribinib in SDH-deficient GIST. Furthermore, our study also documented the inhibition of the proto-oncogene tyrosine protein kinase Src by olaribinib. Src interacts with IGF-1R to regulate tumorigenesis pathways, and its activity is increased in GIST (Rotert, JV et al.). J. Surg. Oncol. 109, 597–605 (2014)). Furthermore, increased cleavage of caspase-3 and poly(ADP-ribose) polymerase-1 after olaribinib treatment indicates its induction of apoptosis. In summary, olaribinib exerts its antitumor effects by modulating multiple signaling pathways involved in hypoxia, angiogenesis, proliferation, survival, and apoptosis.

[0315] Based on in vitro findings, we hypothesize that in SDH-deficient tumor cells, the MOA of olaribatinib involves the regulation of multiple signaling pathways involved in hypoxia, angiogenesis, apoptosis, proliferation, and survival. Further investigations are underway to validate these preliminary findings and elucidate the MOA of olaribatinib.

[0316] Example 3. The combination of olaribatitinib (HQP1351) and the BCL-2 inhibitor levofloxacin (APG-2575) overcame drug resistance in gastrointestinal stromal tumors (GIST). Olebatinib (HQP1351) is a new third-generation TKI that targets... BCR-ABL1 , KIT and PDGFRA It is currently being developed for relapsed or refractory chronic myeloid leukemia and GIST. APG-2575 is a selective BCL-2 inhibitor being developed for hematologic malignancies. The aim of this study was to evaluate whether the combination of APG-2 (a BCL-2 inhibitor) and olaribatibinib enhanced the treatment efficacy of imatinib-resistant GIST.

[0317] Increased BCL-2 protein expression was observed in imatinib-resistant GIST cells, particularly in the GIST430, GIST48, and GIST48B cell lines, compared to imatinib-sensitive GIST cell lines (GIST882 and GIST-T1). Cell-based antiproliferative studies demonstrated the additive activity of olaribatibin and limoncella in imatinib-resistant GIST cells. In vivo studies using an imatinib-resistant GIST430 cell-derived xenograft model further revealed enhanced antitumor activity with co-administration of olaribatibin at 15 mg / kg (every other day for 3 weeks) and limoncella at 50 mg / kg (daily for 3 weeks) compared to either agent alone. The tumor growth inhibition (TGI) rate of the olaribatibin and limoncella combination therapy reached 76.8%, superior to the TGI rates of either agent alone: ​​57.6% for olaribatibin and 31.2% for limoncella. Regarding the putative mechanism, olaribinib treatment showed downregulation of BCL-xL and MCL-1 protein expression, possibly via the KIT signaling pathway and inhibition of downstream signal transducers and activator of transcription 3 (STAT3). Resveratrol induces apoptosis by disrupting the BCL-2:BIM complex. Therefore, selective inhibition of BCL-2, through TKI-mediated MCL-1 and BCL-XL inhibition, significantly increases the cleavage of caspase-3 and poly(ADP-ribose) polymerase 1 (PARP-1), thereby inducing apoptosis and subsequently enhancing antitumor activity.

[0318] Our results demonstrate that olaribatitinib and the BCL-2 inhibitor levofloxacin have additive antitumor effects in imatinib-resistant GIST. This novel dual approach may have the potential to treat GIST patients whose disease has progressed after TKI therapy.

Claims

1. A method for treating a subject with a disease, disorder, or condition associated with succinate dehydrogenase (SDH) deficiency, the method comprising administering to the subject a therapeutically effective amount of a multitarget tyrosine kinase inhibitor (TKI) thereby treating the subject with the disease, disorder, or condition associated with SDH deficiency.

2. A method for identifying subjects suffering from diseases, disorders, or conditions related to SDH deficiency and for selecting treatment for the identified subjects, the method comprising: a) Determine whether SDH defects are present in the biological samples from the subject; b) Based on the SDH deficiency identified in step a), the subject is identified as having the disease, disorder, or condition associated with the SDH deficiency, and c) It is recommended that the subjects receive treatment with a multi-target TKI.

3. The method according to claim 1 or 2, wherein the multi-target TKI comprises olabrutinib.

4. The method according to any one of claims 1-3, wherein the disease, disorder or condition associated with SDH deficiency is cancer (e.g., SDH-deficient cancer).

5. The method of claim 4, wherein the cancer is selected from: gastrointestinal cancer (e.g., gastrointestinal stromal tumor (GIST)), bile duct cancer, lung cancer, melanoma, breast cancer, colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), bladder cancer, pancreatic cancer, kidney cancer, esophageal cancer, head and neck cancer, thyroid cancer, squamous cell carcinoma of the skin, glioblastoma, neuroblastoma, urethral and bladder cancer, endometrial cancer, melanoma, osteosarcoma, lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma), leukemia. Diseases (e.g., T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia (ALL)), multiple myeloma, uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophageal cancer, sarcoma, chromophobe carcinoma, renal cell carcinoma, clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low-grade glioma (LGG), mesothelioma, pheochromocytoma, paraganglioma, thymoma, and adenoid cystic carcinoma (ACC).

6. The method of claim 4 or 5, wherein the cancer is an SDH-deficient GIST.

7. The method according to claim 4 or 5, wherein the SDH-deficient cancer is selected from SDH-deficient ALL, renal cell carcinoma, colon cancer, pheochromocytoma, thyroid adenoma (e.g., papillary thyroid adenoma, multifocal papillary thyroid adenoma, differentiated thyroid carcinoma, papillary (follicular variant) thyroid adenoma, follicular thyroid adenoma), pancreatic neuroendocrine tumor / pancreatic paraganglioma, breast cancer (e.g., triple-negative breast cancer), neuroblastoma, follicular lymphoma, B-cell lymphoma, Hodgkin lymphoma, complex paraganglioma / neuroblastoma, rectal adenocarcinoma, Burkitt lymphoma, thyroid paraganglioma, adrenocortical carcinoma, adrenal adenoma-like tumor, neuroblastoma, melanoma, synovial sarcoma, squamous cell carcinoma of the lung, embryonal rhabdomyosarcoma, lung adenocarcinoma, prostate adenocarcinoma, endometrial cancer, poorly differentiated gastric cancer, bladder urothelial carcinoma, colon adenocarcinoma, sigmoid colon adenocarcinoma, testicular seminoma, and pituitary adenoma.

8. The method according to any one of claims 1 and 3-7, wherein the subject has been identified as having an SDH deficiency.

9. The method according to any one of claims 1 and 3-8, wherein the SDH deficiency is determined in a biological sample from the subject.

10. The method according to any one of claims 1-3 and 9, wherein the biological sample is a tissue (e.g., tumor tissue), a fluid (e.g., whole blood, plasma, PBMC), or any biological sample that can be used to detect germline / somatic mutations in the SDH gene.

11. The method according to any one of claims 1-10, wherein the SDH defect is determined by immunohistochemistry, Western blotting, amplification-based assay, hybridization-based assay, or sequencing.

12. The method according to any one of claims 1-11, wherein the SDH deficiency is indicated by the presence of one or more loss-of-function mutations in SDHA, SDHB, SDHC and SDHD or any combination thereof, or by SDHC promoter methylation, resulting in reduced SDH enzyme activity, instability of the SDH protein complex, or reduced SDH expression.

13. The method of claim 12, wherein the one or more loss-of-function mutations comprise mutations selected from missense substitutions, deletions, insertions, translocations, and premature stop codons, or any combination thereof.

14. The method of claim 12 or 13, wherein the one or more loss-of-function mutations in SDHB comprise one or more mutations selected from p.A15T, p.W218Ter, and SDHB KO exon 2+3 (heterozygote).

15. The method of claim 12 or 13, wherein the one or more loss-of-function mutations in the SDHB comprise one or more mutations selected from c.17_42dup, c.71_72insA, c.136C>T, c.137G>A, c.274T>A, c.301delT, c.380T>G, c.423+1G>A, c.423+20T>A, c.566G>T, c.600G>T, c.643-1G>C, c.725G>A, and c.778_779insG, or any combination thereof.

16. The method of claim 12 or 13, wherein the one or more loss-of-function mutations in the SDHA comprise one or more mutations selected from p.G184R and p.P279S.

17. The method of claim 12 or 13, wherein the one or more loss-of-function mutations in the SDHA comprise: c.91C>T, c.113A>T, c.160C>T, c.511C>T, c.533C>T, c.457–3_457–1delCAG, c.562C>T, c.628C>T, c.666del, c.688delG, c.767C>T, c.800C>T, c.818C>T, c.923C>T, c.985C>T, c.1043–1055del, c.1046_1047delTG, c.1054C>T, c.1151C>G, c.121 5_1216insCC, c.1255G>A, c.1334C>T, c.1352G>A, c.1360G>A, c.1361C>A, c.1367C>T, c.1468delG, c.1513delA, c.1532T>C, c.1534C>T, c.1753C>T, c.1765C>T, c.1766G>A, c.1690G>A, c.1794G>C, c.1795–1G>T, c.1798C>T, and c.1799G>A, or any combination thereof.

18. The method of claim 12 or 13, wherein the one or more loss-of-function mutations in the SDHC comprise one or more mutations selected from the group consisting of c.1A>G, c.6delT, c.65G>A, c.160C>A, c.221A>G, c.224G>A, c.238C>T, c.301delT, c.380A>G, c.397C>T, and c.405+1G>A or any combination thereof.

19. The method of claim 12 or 13, wherein the one or more loss-of-function mutations in SDHD comprise one or more mutations selected from c.34G>A, c.352delG, c.416T>C, and c.455G>A, or any combination thereof.

20. The method according to any one of claims 1-19, wherein the subject (e.g., the biological sample of the subject) has been further identified as having wild-type KIT, wild-type PDGFR, or a combination thereof.

21. The method according to any one of claims 1-20, wherein the subject (e.g., the biological sample of the subject) has been identified as having one or more mutations comprising the NF1 / BRAF / RAS pathway gene, PIK3CA and a fusion affecting NTRK3 or any combination thereof.

22. The method according to any one of claims 1-21, wherein the subject (e.g., the biological sample of the subject) has been identified as overexpressing VEGFR or FGF ligands.

23. The method according to any one of claims 1-22, wherein the subject has been identified as insensitive to at least one existing TKI.

24. The method of claim 23, wherein the subject did not achieve complete remission (CR), partial remission (PR), or stable disease (SD) within 6 months of receiving treatment with the at least one existing TKI; or achieved initial remission or stable disease after ≥6 months of receiving treatment with the at least one existing TKI, but subsequently experienced disease progression.

25. The method according to any one of claims 1-24, wherein the subject has been determined to be insensitive to at least one existing TKI selected from: imatinib, riprinib, avatinib, cristatinib, masitinib, ponatinib, dasatinib, nilotinib, sunitinib, regorafenib, sorafenib, anlotinib, surufatinib, pazopanib, rogatinib, infelgratinib, erdatinib, pemitinib, fexotinib, and famitinib, or any combination thereof.

26. The method according to any one of claims 1-25, wherein the subject has been determined to be insensitive to at least one existing TKI selected from the group consisting of ripretinib, dasatinib, sunitinib, regorafenib, anlotinib, and famitinib, or any combination thereof.

27. The method according to any one of claims 1-26, wherein the multi-target TKI is capable of inhibiting at least two tyrosine kinases selected from: KIT, PDGFR, SRC, VEGFR (e.g., any one of VEGFR1, VEGFR2 and VEGFR3 or any combination thereof) and FGFR (e.g., any one of FGFR1, FGFR2, FGFR3 and FGFR4 or any combination thereof), or any combination thereof.

28. The method according to any one of claims 1-27, wherein the multi-target TKI is capable of inhibiting at least two tyrosine kinases selected from the group consisting of KIT, PDGFR, SRC, VEGFR2 and FGFR1, or any combination thereof.

29. The method according to any one of claims 1-28, wherein the multi-target TKI is capable of inhibiting the proliferation of SDH-deficient tumor cells.

30. The method according to any one of claims 1-29, wherein the multi-target TKI has an IC50 effect in inhibiting the proliferation of SDH-deficient tumor cells. 50 Not exceeding 10 μM (e.g., not exceeding 5 μM, 1 μM, 500 nM, 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM or 10 nM), as determined by CCK8 assay or CTG assay.

31. The method according to claim 29 or 30, wherein the SDH-deficient tumor cells are Jurkat clones E6-1, OS-RC-2, RKO, and PC12#5F7, or any combination thereof.

32. The method according to any one of claims 1-31, wherein the multi-target TKI comprises a heterocyclic acetylenoid compound having the following formula (I): Z is independently selected from CH or N; L 1 Independently selected from NH, -N=, CH; L 2 Independently selected from -CONH- or -NHCO-; R1 is independently selected from: 1) H； 2) C1-C6 alkyl groups; 3) C3-C6 cycloalkyl groups; 4) C1-C5 alkyl groups substituted with one or two hydroxyl groups; 5) Phenyl; 6) It can pass through L at the Z atomic site. 1 Fused A rings to form a ring containing 1-3 N atoms and L 1 Fused five-membered heterocyclic groups, such as ， in, X, Y, and Z are independently selected from N and CH; ring D is an aromatic heterocycle containing 1-3 N atoms; R2 is independently selected 1) H； 2) Halogens; 3) C1-C5 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C5 alkyl groups; R3 is selected independently 1) H； 2) Halogens; 3) C1-C4 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C4 alkyl groups; When R5 is H, R4 is independently selected. 1) H； 2) (CH2) n NR6R7; 3) (CH2) n -It 1 ; Alternatively, when R4 is H, R5 is independently selected. 1) H； 2) The 2 ; n is independently selected from 0 or 1; Het 1 Defined as a non-aromatic heterocycle containing 1-3 N atoms; Het 2 Defined as an aromatic five-membered heterocycle containing heteroatoms such as N, O and / or S; alkyl, cycloalkyl or NR6R7 may be incorporated into any C or N position of substituted non-aromatic heterocycles and aromatic heterocycles; R6 or R7 is selected independently from: 1) H； 2) C1-C3 alkyl groups; 3) F-containing C1-C3 alkyl groups; 4) C3-C6 cycloalkyl groups; Alternatively, R6 and R7 can be further formed into five-, six-, seven-, or eight-membered ring structures through C, O, N, and S atoms; Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

33. The method according to any one of claims 1-32, wherein the multi-target TKI is a compound having the following formula (II): ,(II) Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

34. The method according to any one of claims 1-31, wherein the multi-target TKI is a compound selected from the group consisting of olabatinib, ripretinib, avatinib, cristatinib, masitinib, ponatinib, dasatinib, nilotinib, sunitinib, regorafenib, sorafenib, anlotinib, surufatinib, infuratinib, erdatinib, pemitinib, rogatinib, and lincitinib, or a pharmaceutically acceptable salt or stereoisomer or prodrug of any of the foregoing.

35. The method according to any one of claims 1-34, wherein the multi-target TKI is administered at a dose of about 10 mg to 200 mg, 10 mg to 150 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 20 mg to 50 mg.

36. The method according to any one of claims 1-35, wherein the multi-target TKI is administered at a dose of about 10 mg, 20 mg, 30 mg, 40 mg or 50 mg.

37. The method according to any one of claims 1-36, wherein the multi-target TKI is administered once daily, once every other day, once weekly, once every other week, once monthly, or once every other month.

38. The method according to any one of claims 1-37, wherein the treatment comprises one or more 28-day treatment cycles.

39. The method according to any one of claims 1-38, wherein the treatment comprises at least 1, 2, 3, 6, 14, 16 or 36 treatment cycles.

40. The method according to any one of claims 1-39, wherein the multi-target TKI is administered via an oral or non-enteric route (e.g., intravenous, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, or intraocular injection).

41. The method according to any one of claims 1-40, wherein the multi-target TKI is administered orally once every other day during a 28-day treatment cycle.

42. The method according to any one of claims 1-41, wherein the method further comprises further administering a therapeutically effective amount of one or more other therapies.

43. The method according to any one of claims 1-41, wherein the multitarget TKI is administered in combination with one or more other therapies for treating the disease, disorder, or condition.

44. The method of claim 42 or 43, wherein the one or more additional therapies include radiotherapy, chemotherapy, targeted cancer therapy, therapy using immune checkpoint molecule modulators, or therapy using BCL-2 inhibitors.

45. The method according to any one of claims 1-44, wherein after treatment with the compound having formula (II), the subject experiences partial remission or disease stabilization.

46. ​​The method according to any one of claims 1-45, wherein after a 28-day treatment cycle with the compound having formula (II), the objective response rate (complete response and partial response) is about or greater than 20%, and / or the clinical benefit rate (complete response, partial response and stable disease) is about or greater than 90%.

47. A method of treating a subject's cancer, the method comprising administering, to the subject, a) A therapeutically effective dose of a multi-target tyrosine kinase inhibitor (TKI); and b) A therapeutically effective dose of Bcl-2 inhibitors.

48. The method of claim 47, wherein the cancer is selected from: gastrointestinal cancer (e.g., gastrointestinal stromal tumor (GIST)), bile duct cancer, lung cancer, melanoma, breast cancer, colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), bladder cancer, pancreatic cancer, kidney cancer, esophageal cancer, head and neck cancer, thyroid cancer, squamous cell carcinoma of the skin, glioblastoma, neuroblastoma, urethral and bladder cancer, endometrial cancer, melanoma, osteosarcoma, lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma), leukemia, etc. Blood disorders (e.g., T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia (ALL)), multiple myeloma, uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophageal cancer, sarcoma, chromophobe carcinoma, renal cell carcinoma, clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low-grade glioma (LGG), mesothelioma, pheochromocytoma, paraganglioma, thymoma, and adenoid cystic carcinoma (ACC).

49. The method of claim 47 or 48, wherein the cancer is GIST.

50. The method according to any one of claims 47-49, wherein the subject has been identified as comprising KIT PDGFR (For example, PDGFRA and PDGFRB ), SDH (For example, SDHA, SDHB, SDHC and SDHD ), NF1, BRAF, EGFR, HRAS NRAS, KRAS, ZNF217, MEN1, PIK3CA, ARID1A, ARID1B, FGFR1, FGFR2, FGFR3, FGFR4, SDHAF2, FGFR1-HOOK3, FGFR1-TACC1, NTRK1 (For example, NTRK1 Fusion NTRK2 (For example, NTRK2 Fusion NTRK3 (For example, NTRK3 Fusion KIT-PDGFRA and PRKAR1B-BRAF Or one or more mutations in any combination of them.

51. The method according to any one of claims 47-50, wherein the subject has been identified as insensitive to at least one existing TKI.

52. The method according to any one of claims 47-51, wherein the subject has not achieved complete remission (CR), partial remission (PR), or stable disease (SD) within 6 months of receiving treatment with the at least one existing TKI; or has achieved initial remission or stable disease after receiving treatment with the at least one existing TKI for ≥6 months, but subsequently experienced disease progression.

53. The method according to any one of claims 47-52, wherein the subject has been determined to be insensitive to at least one existing TKI selected from: imatinib, riprinib, avatinib, cristatinib, masatitinib, ponatinib, dasatinib, nilotinib, sunitinib, regorafenib, sorafenib, anlotinib, surufatinib, pazopanib, rogatinib, infelgratinib, erdatinib, pemitinib, fexotinib, and famitinib, or any combination thereof.

54. The method according to any one of claims 47-53, wherein the subject has been determined to be insensitive to at least one existing TKI selected from the group consisting of ripretinib, dasatinib, sunitinib, regorafenib, anlotinib, and famitinib, or any combination thereof.

55. The method according to any one of claims 47-54, wherein the multi-target TKI is capable of inhibiting at least two tyrosine kinases selected from: KIT, PDGFR, SRC, VEGFR (e.g., any one of VEGFR1, VEGFR2 and VEGFR3 or any combination thereof) and FGFR (e.g., any one of FGFR1, FGFR2, FGFR3 and FGFR4 or any combination thereof), or any combination thereof.

56. The method according to any one of claims 47-55, wherein the multi-target TKI is capable of inhibiting at least two tyrosine kinases selected from the group consisting of KIT, PDGFR, SRC, VEGFR2 and FGFR1, or any combination thereof.

57. The method according to any one of claims 47-56, wherein the multi-target TKI comprises a heterocyclic acetylenoid compound having the following formula (I): Z is independently selected from CH or N; L 1 Independently selected from NH, -N=, CH; L 2 Independently selected from -CONH- or -NHCO-; R1 is independently selected from: 1) H； 2) C1-C6 alkyl groups; 3) C3-C6 cycloalkyl groups; 4) C1-C5 alkyl groups substituted with one or two hydroxyl groups; 5) Phenyl; 6) It can pass through L at the Z atomic site. 1 Fused A rings to form a ring containing 1-3 N atoms and L 1 Fused five-membered heterocyclic groups, such as ， in, X, Y, and Z are independently selected from N and CH; ring D is an aromatic heterocycle containing 1-3 N atoms; R2 is independently selected 1) H； 2) Halogens; 3) C1-C5 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C5 alkyl groups; R3 is selected independently 1) H； 2) Halogens; 3) C1-C4 alkyl groups; 4) C3-C6 cycloalkyl groups; 5) F-containing C1-C4 alkyl groups; When R5 is H, R4 is independently selected. 1) H； 2) (CH2) n NR6R7; 3) (CH2) n -It 1 ; Alternatively, when R4 is H, R5 is independently selected. 1) H； 2) The 2 ; n is independently selected from 0 or 1; Het 1 Defined as a non-aromatic heterocycle containing 1-3 N atoms; Het 2 Defined as an aromatic five-membered heterocycle containing heteroatoms such as N, O and / or S; alkyl, cycloalkyl or NR6R7 may be incorporated into any C or N position of substituted non-aromatic heterocycles and aromatic heterocycles; R6 or R7 is selected independently from: 1) H； 2) C1-C3 alkyl groups; 3) F-containing C1-C3 alkyl groups; 4) C3-C6 cycloalkyl groups; Alternatively, R6 and R7 can be further formed into five-, six-, seven-, or eight-membered ring structures through C, O, N, and S atoms; Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

58. The method according to any one of claims 47-57, wherein the multi-target TKI is a compound having the following formula (II): ,(II) Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

59. The method according to any one of claims 47-56, wherein the multi-target TKI is a compound selected from the group consisting of olabatinib, riprinib, avatinib, cristatinib, masatitinib, ponatinib, dasatinib, nilotinib, sunitinib, regorafenib, sorafenib, anlotinib, surufatinib, infuratinib, erdatinib, pemitinib, rogatinib, and lincitinib, or a pharmaceutically acceptable salt or stereoisomer or prodrug of any of the foregoing.

60. The method according to any one of claims 47-59, wherein the multi-target TKI is administered at a dose of about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg or 200 mg / kg.

61. The method according to any one of claims 47-60, wherein the multi-target TKI is administered once daily, once every other day, once weekly, once every other week, once monthly, or once every other month.

62. The method according to any one of claims 47-61, wherein the multi-target TKI is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 4 months.

63. The method according to any one of claims 47-62, wherein the multi-target TKI is administered via an oral or enteral route (e.g., intravenous, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, or intraocular injection).

64. The method according to any one of claims 47-63, wherein the multi-target TKI is administered once every other day at a dose of about 15 mg / kg for 3 weeks.

65. The method according to any one of claims 47-64, wherein the Bcl-2 inhibitor is a compound having the following formula (III). (III), Or its pharmaceutically acceptable salt or stereoisomer or prodrug, wherein: A3 is selected from ; E3 is nitrogen and It is a single bond; X 31 X 32 and X 33 Each independently selected from -CR 38 = and -N=; R 31a and R 31b Together with the carbon atoms they are attached to, they form 3-, 4-, or 5-membered cycloalkyl groups; R 32 Selected from -NO2, -SO2CH3 and -SO2CF3; R 32a Selected from hydrogen and halogens; R 33 Selected from -N(R) 34a (R) 34b ); R 34a Selected from C (optional substitution) 1-6 Alkyl, optionally substituted C 3-6 Cycloalkyl, heterocyclic, heteroalkyl, (cycloalkyl)alkyl, and (heterocyclic)alkyl; R 34b Selected from hydrogen and C 1-4 alkyl; R 38 Selected from hydrogen and halogens.

66. The method according to any one of claims 47-65, wherein the Bcl-2 inhibitor is selected from: ， Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

67. The method according to any one of claims 47-66, wherein the Bcl-2 inhibitor is a compound having the following formula (IV). ,(IV) Or its pharmaceutically acceptable salt or stereoisomer or prodrug.

68. The method according to any one of claims 47-67, wherein the Bcl-2 inhibitor is administered at a dose of about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg or 200 mg / kg.

69. The method according to any one of claims 47-68, wherein the Bcl-2 inhibitor is administered twice daily, once daily, every other day, once weekly, every other week, once monthly, or every other month.

70. The method according to any one of claims 47-69, wherein the Bcl-2 inhibitor is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 4 months.

71. The method according to any one of claims 47-70, wherein the Bcl-2 inhibitor is administered via an oral or non-enteric route (e.g., intravenous, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, or intraocular injection).

72. The method according to any one of claims 47-71, wherein the Bcl-2 inhibitor is administered once daily at a dose of about 50 mg / kg for 3 weeks.

73. The method according to any one of claims 47-72, wherein the multi-target TKI and the Bcl-2 inhibitor are administered simultaneously.

74. The method according to any one of claims 47-72, wherein the multi-target TKI and the Bcl-2 inhibitor are administered sequentially.

75. The method according to any one of claims 47-74, wherein the multitarget TKI is administered every other day at a dose of about 15 mg / kg for 3 weeks, and the Bcl-2 inhibitor is administered once daily at a dose of about 50 mg / kg for 3 weeks.

76. The method according to any one of claims 47-75, wherein the combination of the multi-target TKI and the Bcl-2 inhibitor treats the subject's cancer in an additive or synergistic manner.

77. The method according to any one of claims 47-76, wherein after treatment with a compound having formula (II) in combination with a compound having formula (IV), the subject has a tumor growth inhibition (TGI) rate of at least 75%.

78. The method according to any one of claims 47-75, wherein the combination of the multi-target TKI and the Bcl-2 inhibitor can inhibit the proliferation of imatinib-resistant GIST cells.

79. The method according to any one of claims 47-75, wherein the combination of the multi-target TKI and the Bcl-2 inhibitor can inhibit the proliferation of imatinib-resistant GIST cells in an additive or synergistic manner.

80. The method according to claim 78 or 79, wherein the imatinib-resistant GIST cells are GIST430, GIST48, and GIST48B, or any combination thereof.

81. A method for treating a subject with a disease, disorder, or condition associated with upregulation of hypoxia-inducible factors (HIF-1α, HIF-2α), vascular endothelial growth factor-A (VEGFA), fibroblast growth factor receptor-1 (FGFR1), phosphorylated FGFR1, insulin-like growth factor-1 receptor (IGF-1R), phosphorylated IGF-1R, STAT3, phosphorylated STAT3, and / or phosphorylated Src, comprising administering to the subject a therapeutically effective amount of a multitarget tyrosine kinase inhibitor (TKI) thereby treating the subject with the disease, disorder, or condition related to lipid metabolism.

82. The method of claim 81, wherein the disease, disorder, or symptom may be cancer.

83. The method of claim 82, wherein the cancer is selected from: gastrointestinal cancer (e.g., gastrointestinal stromal tumor (GIST)), cholangiocarcinoma, lung cancer, melanoma, breast cancer, colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), bladder cancer, pancreatic cancer, kidney cancer, esophageal cancer, head and neck cancer, thyroid cancer, squamous cell carcinoma of the skin, glioblastoma, neuroblastoma, urethral and bladder cancer, endometrial cancer, melanoma, osteosarcoma, lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma), leukemia, etc. Blood disorders (e.g., T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia (ALL)), multiple myeloma, uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophageal cancer, sarcoma, chromophobe carcinoma, renal cell carcinoma, clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low-grade glioma (LGG), mesothelioma, pheochromocytoma, paraganglioma, thymoma, and adenoid cystic carcinoma (ACC).

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