Use of cyclin E1 status as a predictive biomarker for cancer treatment with WEE1 inhibitors

Abstract

The present disclosure provides, inter alia, methods of treating cancer comprising administering an effective dose of azenosertib to a subject selected to have a cyclin E1 status above a predetermined threshold.

Description

[Technical Field]

[0001] Cross-reference to related art All applications for which a foreign or domestic priority claim is identified in the Application Data Sheet filed with this application are expressly incorporated herein by reference pursuant to 37 CFR § 1.57 and Rules 4.18 and 20.6, including U.S. Provisional Patent Application Nos. 63 / 382,817, filed November 8, 2022; 63 / 485,764, filed February 17, 2023; 63 / 459,520, filed April 14, 2023; 63 / 504,166, filed May 24, 2023; 63 / 506,023, filed June 2, 2023; and 63 / 588,235, filed October 5, 2023, each of which is incorporated by reference in its entirety, including any drawings. [Background technology]

[0002] DNA damage is typically resolved by repair proteins, which either reconnect or resynthesize the damaged DNA. However, incorrect nucleotide substitutions into DNA can lead to mutations and other genetic alterations, inherited diseases, and loss of protein function. Improper DNA repair can lead to cell death, tumor progression, and cancer. Cell cycle checkpoints are important for proper DNA repair and ensure that cells do not progress with cell replication until genomic integrity is restored. Cyclin E1 (encoded by the CCNE1 gene) is involved in cell cycle regulation by binding to cyclin-dependent kinases (CDKs), including CDK2, thereby promoting cell cycle progression. WEE1 is a nuclear kinase that is involved in the G2-M cell cycle checkpoint arrest for DNA repair before the onset of mitosis and is overexpressed in various cancers. Summary of the Invention

[0003] The present disclosure is based, in part, on the discovery that certain cyclin E1 conditions, e.g., increased cyclin E1 protein expression levels, sensitize subjects with diseases with DNA damage repair deficiencies (or defects or alterations) (e.g., cancer) to treatment with the WEE1 inhibitor azenosertib (also identified as ZN-c3) as monotherapy or in combination with at least one second chemotherapeutic agent or a pharmaceutically acceptable salt thereof, and that cyclin E1 biomarker levels (e.g., overexpression of cyclin E1 protein with or without CCNE1 gene amplification) can be used to select subjects for treatment with azenosertib. Subjects selected based on a predetermined threshold of the cyclin E1 biomarker experienced significantly improved response (e.g., tumor growth inhibition and increased progression-free survival (PFS)) when treated with azenosertib or a pharmaceutically acceptable salt thereof as monotherapy or in combination with at least one second chemotherapeutic agent or a pharmaceutically acceptable salt thereof.

[0004] Combination therapy with azenosertib, or a pharmaceutically acceptable salt thereof, and a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, can further provide synergistic effects and improve subject outcomes. Thus, the present disclosure provides, inter alia, a method for treating cancer with azenosertib (including a pharmaceutically acceptable salt), a WEE1 inhibitor, alone or in combination with a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, using a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. In some embodiments, the cyclin E1 status or cyclin E1 biomarker The levels are used as predictive biomarkers.

[0005] In a first aspect, the present disclosure provides a method for treating cancer, comprising administering an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, to a subject selected for having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 2+ greater than 30%. In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 125.

[0006] In a second aspect, the present disclosure provides a method of treating cancer, comprising administering an effective dose of azenosertib or a pharmaceutically acceptable salt thereof and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof to a subject selected for having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. In some embodiments, the predetermined threshold is the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 10%. In some embodiments, the predetermined threshold is the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 30%. In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 50. In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 125.

[0007] In a fourth aspect, the present disclosure provides a method for treating ovarian cancer, comprising administering an effective dose of azenosertib or a pharmaceutically acceptable salt thereof to a subject selected for having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. In some embodiments, the predetermined threshold is the proportion of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 30%. In some embodiments, the predetermined threshold is a cyclin E1 IHC H score greater than 125.

[0008] In a fifth aspect, the present disclosure provides a method of treating ovarian cancer, the method comprising administering an effective dose of azenosertib or a pharmaceutically acceptable salt thereof one or more times during a treatment cycle to a subject selected for having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold, and administering a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof one or more times during the treatment cycle. In some embodiments, the predetermined threshold is the percentage of viable tumor cells having a cyclin E1 IHC staining intensity of 2+ greater than 10%. In some embodiments, the predetermined threshold is the percentage of viable tumor cells having a cyclin E1 IHC staining intensity of 2+ greater than 30%. In some embodiments, the predetermined threshold is a cyclin E1 IHC H score greater than 50. In some embodiments, the predetermined threshold is a cyclin E1 IHC H score greater than 125.

[0009] Various embodiments of the first, second, third, fourth, and fifth aspects of the present disclosure described above are summarized in the following paragraphs and also described in the detailed description.

[0010] In some embodiments, the predetermined cyclin E1 status is cyclin E1 positive or cyclin E1-high. In some embodiments, the predetermined cyclin E1 status is cyclin E1 positive (low) or cyclin E1 positive (high). In one embodiment, the predetermined cyclin E1 status is a cyclin E1 protein expression level above a predetermined cutoff.

[0011] In some embodiments, a predetermined cyclin E1 status or cyclin E1 biomarker The level is measured by cyclin E1 protein expression level.In some embodiments, the cyclin E1 protein expression level is determined by CCNE1 mRNA or transcript level.In some embodiments, the cyclin E1 protein expression level is determined by protein level.

[0012] In some embodiments, the predetermined cyclin E1 status or cyclin E1 biomarker level is an immunochemistry (IHC) status.

[0013] In one embodiment, the predetermined cutoff or predetermined threshold is measured by the percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 2+.

[0014] In one embodiment, the predetermined cutoff or predetermined threshold is greater than 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, or 62% of cyclin E1 2+. Percentage of viable tumor cells with IHC staining intensity.

[0015] In one embodiment, the predetermined cutoff or threshold is the percentage of viable tumor cells having a cyclin E1 IHC staining intensity of 2+ greater than 10%. In one embodiment, the predetermined cutoff or threshold is the percentage of viable tumor cells having a cyclin E1 IHC staining intensity of 2+ greater than 30%.

[0016] In some embodiments, the cyclin E1 expression level is measured by a cyclin E1 IHC H score.

[0017] In some embodiments, the predetermined cutoff or predetermined threshold for the Cyclin E1 IHC H-score is greater than 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160.

[0018] In some embodiments, the predetermined cutoff or predetermined threshold for the Cyclin E1 IHC H score is greater than 50.

[0019] In some embodiments, the predetermined cutoff or predetermined threshold for the Cyclin E1 IHC H score is greater than 125.

[0020] In some embodiments, a given cyclin 1 status or cyclin E1 biomarker level is independent of the CCNE1 gene amplification status of the subject. In alternative embodiments, a given cyclin 1 status or cyclin E1 biomarker level is associated with the CCNE1 gene amplification status of the subject.

[0021] In some embodiments, CCNE1 gene amplification status is measured by CCNE1 gene copy number.

[0022] In some embodiments, the CCNE1 gene amplification status is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34. The CCNE1 gene copy number is 4.

[0023] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.

[0024] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.

[0025] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 3. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 4. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 5. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 6. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 7. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 8. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 9. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 10. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 11. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 12. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 14.

[0026] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 7.

[0027] In some embodiments, subjects are selected without determining the levels and status of other oncogenes.

[0028] In some embodiments, the other oncogene is selected from BRCA1, BRCA2, TP53, PKMYT1, and PPP2R1A.

[0029] In some embodiments, subjects are selected without determining the levels of BRCA1 and / or BRCA2.

[0030] In some embodiments, the subject is selected without determining the level of TP53.

[0031] In some embodiments, the cancer is a cyclin E1-driven cancer.

[0032] In some embodiments, the cancer is glioblastoma (GBM), astrocytoma, meningioma, craniopharyngioma, medulloblastoma, other brain cancer, head and neck cancer, leukemia, AML (acute myeloid leukemia), CLL (chronic lymphocytic leukemia), ALL (acute lymphocytic leukemia), myelodysplastic syndrome (MDS), skin cancer, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, uterine cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hematologic malignancies, head cancer, hematologic malignancies, Kaposi's sarcoma, kidney cancer, pharynx and hypopharynx cancer, liver cancer, lung cancer, non-small cell lung cancer (NSCLC). SCLC), small cell lymphoma, lymphoma, mesothelioma, melanoma, multiple myeloma, neuroblastoma, nasopharyngeal cancer, cervical cancer, ovarian cancer, osteosarcoma, sarcoma, gastrointestinal stromal tumor (GIST), pancreatic cancer, pituitary cancer, prostate cancer, kidney cancer, retinoblastoma, salivary gland cancer, skin cancer, stomach cancer, small intestine cancer, sarcoma, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous adenocarcinoma (USC), uterine CS, vaginal cancer, vulvar cancer, Waldenström's macroglobulinemia, Wilms' tumor, solid tumors, or liquid tumors, HGSOC, invasive breast cancer, triple-negative breast cancer (TN) BC), esophagogastric cancer, gastric cancer, esophageal cancer, pRCC, ccRCC, chromophobe RCC, head and neck cancer, adenoid cystic carcinoma (ACC), diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), cholangiocarcinoma, acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), ALL (acute lymphocytic leukemia), myelodysplastic syndrome (MDS), thymoma, BRAF-mutated metastatic colorectal cancer, uveal melanoma, high-grade serous ovarian, fallopian tube, or primary peritoneal cancer, BRAF Selected from V600E-mutated colorectal cancer, platinum-sensitive ovarian cancer, poly(ADP-ribose) polymerase inhibitor (PARPi)-resistant ovarian cancer, platinum-resistant ovarian cancer, platinum-refractory ovarian cancer, advanced pancreatic ductal adenocarcinoma, pancreatic ductal adenocarcinoma, neuroendocrine tumor, neuroendocrine prostate cancer, pancreatic neuroendocrine tumor, small cell lung cancer (SCLC), germ cell cancer, and stromal cancer.

[0033] In some embodiments, the cancer is a cancer of an organ selected from adrenal gland, ampulla of Vater, biliary tract, bladder / urinary tract, bone, intestine, breast, cervix, CNS / brain, esophagus / stomach, eye, head and neck, kidney, liver, lung, lymphatic system, bone marrow, ovaries / fallopian tubes, pancreas, penis, peripheral nervous system, peritoneum, pleura, prostate, skin, soft tissue, testicle, thymus, thyroid, uterus, vulva / vagina, adenocarcinoma in situ, extragonadal germ cell tumor (EGCT), mixed adenocarcinoma, high-grade ovarian neuroendocrine carcinoma, high-grade serous fallopian tube carcinoma (HGSFT), ovarian choriocarcinoma, and ovarian cancer NOS (OCNOS).

[0034] In some embodiments, the cancer is a solid tumor or a hematological malignancy.

[0035] In some embodiments, the cancer is a solid tumor.

[0036] In some embodiments, the solid tumor is selected from uterine cancer, endometrial cancer, gallbladder cancer, ovarian cancer (e.g., HGSOC), endometrial cancer, melanoma, colorectal cancer, bladder cancer, breast cancer (e.g., invasive triple-negative breast cancer (TNBC)), prostate cancer, lung cancer (e.g., NSCLC, SCLC), esophagogastric cancer, gastric cancer, esophageal cancer, renal cancer (e.g., pRCC, ccRCC, chromophobe RCC), head and neck cancer, osteosarcoma cancer, pancreatic cancer, brain cancer, uterine CS, uterine cancer, adenoid cystic carcinoma (ACC), mesothelioma, cervical cancer, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), liver cancer, glioblastoma (GBM), testicular cancer, low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), cholangiocarcinoma, thyroid cancer, thymoma, and uveal melanoma.

[0037] In some embodiments, the cancer is acute myeloid leukemia (AML).

[0038] In some embodiments, the tumor is selected from the group consisting of SCLC, neuroendocrine tumor, neuroendocrine prostate cancer, and pancreatic neuroendocrine tumor.

[0039] In some embodiments, the solid tumor is ovarian cancer.

[0040] In some embodiments, the ovarian cancer is epithelial ovarian cancer, germ cell cancer, or stromal cancer.

[0041] In some embodiments, the ovarian cancer is epithelial ovarian cancer.

[0042] In some embodiments, the ovarian cancer is high-grade serous ovarian cancer (HGSOC). In some embodiments, the ovarian cancer is platinum-resistant ovarian cancer (PROC). In some embodiments, the ovarian cancer is PARP inhibitor-resistant. In some embodiments, the ovarian cancer is CCNE1 gene-amplified ovarian cancer. In some embodiments, the ovarian cancer is a cyclin E1-overexpressing cancer. In some embodiments, the ovarian cancer is a cyclin E1-overexpressing / non-CCNE1 gene-amplified cancer.

[0043] In some embodiments, the cancer is histologically and / or cytologically confirmed, or the cancer is pathologically confirmed.

[0044] In some embodiments, the cancer is recurrent or persistent, in some embodiments, the cancer is metastatic, in some embodiments, the cancer is unresectable.

[0045] In some embodiments, the subject has received no more than one, at least one, one, two, three, four, one or two, one to two, one to three, or one to four prior lines of therapy, prior lines of therapy in the advanced or metastatic setting, prior lines of chemotherapy, prior lines of platinum-based chemotherapy, prior regimens, or prior treatment regimens.

[0046] In some embodiments, the cancer is platinum-resistant, platinum-sensitive, or platinum-refractory.

[0047] In some embodiments, the cancer is PARP inhibitor resistant.

[0048] In one embodiment, the method of treatment comprises administering an effective dose of azenosertib or a pharmaceutically acceptable salt thereof in combination with a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof. In an alternative embodiment, the method of treatment comprises administering an effective dose of azenosertib or a pharmaceutically acceptable salt thereof in combination with a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof.

[0049] In some embodiments, the second chemotherapeutic agent is selected from carboplatin, cisplatin, paclitaxel, docetaxel, pegylated liposomal doxorubicin (PLD), doxorubicin, gemcitabine, cytarabine, fludarabine, fluorouracil (5-FU), irinotecan, topotecan, temozolomide, triapine, 5-azacytidine, capecitabine, AraC-FdUMP

[10] (CF-10), cladribine, decitabine, hydroxyurea, oxaliplatin, niraparib, encorafenib, and cetuximab, or a pharmaceutically acceptable salt of any of the foregoing.

[0050] In some embodiments, the second chemotherapeutic agent is selected from azacitidine, bendamustine, bortezomib, carfilzomib, ixazomib, busulfan, carboplatin, cytarabine, cyclophosphamide, cladribine, cisplatin, capecitabine, decitabine, dexamethasone, etoposide, fludarabine, gemcitabine, daunorubicin, doxorubicin, ifosfamide, methotrexate, and vincristine, or a pharmaceutically acceptable salt of any of the foregoing.

[0051] In some embodiments, the second chemotherapeutic agent is carboplatin, paclitaxel, gemcitabine, or pegylated liposomal doxorubicin (PLD), or a pharmaceutically acceptable salt of any of the foregoing. In some embodiments, the second chemotherapeutic agent is carboplatin or a pharmaceutically acceptable salt thereof. In some embodiments, the second chemotherapeutic agent is paclitaxel. In some embodiments, the second chemotherapeutic agent is clitaxel or a pharmaceutically acceptable salt thereof. In some embodiments, the second chemotherapeutic agent is gemcitabine or a pharmaceutically acceptable salt thereof. In some embodiments, the second chemotherapeutic agent is pegylated liposomal doxorubicin (PLD) or a pharmaceutically acceptable salt thereof.

[0052] In some embodiments, the methods include selecting subjects with a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold.

[0053] In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, and the second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, are administered simultaneously.

[0054] In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, and the second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, are administered sequentially.

[0055] In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, and / or the second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, are administered intermittently.

[0056] In some embodiments, the treatment method comprises selecting a subject with a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold.

[0057] In some embodiments, the treatment method involves cyclin E1 status or cyclin E1 biomarker levels being first determined prior to the selection step.

[0058] In some embodiments, the treatment method results in an overall response rate (ORR) of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or greater of subjects. In some embodiments, the overall response rate (ORR) is measured by complete response (CR), partial response (PR), 50% CA-125 response, or a combination thereof.

[0059] In some embodiments, the treatment method results in a subject having a median progression-free survival (mPFS) of 5, 6, 7, 8, 9, 10, 11, 12 months or greater.

[0060] In a sixth aspect, the present disclosure provides a method of treating ovarian cancer, the method comprising administering to a subject selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, during a treatment cycle, and administering a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, one or more times during the treatment cycle. All of the various embodiments described above for the first, second, third, fourth, and fifth aspects of the present disclosure expressly apply to this sixth aspect.

[0061] In some embodiments, the treatment cycle is 21 or 28 days.

[0062] In some embodiments, the treatment cycle is repeated.

[0063] Other features, objects, and advantages will be apparent in the following detailed description. It should be understood, however, that the detailed description, while illustrating embodiments, is given by way of example only and not by way of limitation. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the detailed description. The drawings are for purposes of illustration only and are not limiting. [Brief explanation of the drawings]

[0064] [Figure 1A]Figures 1A-1G show that overexpression of cyclin E1 protein is associated with increased sensitivity to azenosertib in ovarian cancer cell lines. Figure 1A shows exemplary results demonstrating that azenosertib sensitivity correlates with cyclin E1 protein expression in cancer cell lines OV90, Kuramochi, TYK-nu, and OVCAR3, assessed by CellTiter Glo after 96 hours of culture. Figure 1B shows that OV90 cells transduced with a lentiviral vector expressing the CCNE1 gene (low endogenous expression of cyclin E1) exhibited increased sensitivity to azenosertib. Figure 1C shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, and OV90 control (empty vector), as well as lentivirus-induced cyclin E1 protein overexpression. Figure 1D shows growth rate inhibition and IC50 for empty vector and cyclin E1. Figure 1E shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, JOM1, ES2, TYK-nu, CAVO3, OAW28, OVCAR4, OVCAR3, and OV90 control (empty vector). Figure 1F shows a graph of the H-score of cyclin E1 protein expression levels as a function of cyclin E1 levels. Figure 1G shows a graph of the growth rate (GRmax) values ​​as a function of cyclin E1 levels. [Figure 1B]Figures 1A-1G show that overexpression of cyclin E1 protein is associated with increased sensitivity to azenosertib in ovarian cancer cell lines. Figure 1A shows exemplary results demonstrating that azenosertib sensitivity correlates with cyclin E1 protein expression in cancer cell lines OV90, Kuramochi, TYK-nu, and OVCAR3, assessed by CellTiter Glo after 96 hours of culture. Figure 1B shows that OV90 cells transduced with a lentiviral vector expressing the CCNE1 gene (low endogenous expression of cyclin E1) exhibited increased sensitivity to azenosertib. Figure 1C shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, and OV90 control (empty vector), as well as lentivirus-induced cyclin E1 protein overexpression. Figure 1D shows growth rate inhibition and IC50 for empty vector and cyclin E1. Figure 1E shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, JOM1, ES2, TYK-nu, CAVO3, OAW28, OVCAR4, OVCAR3, and OV90 control (empty vector). Figure 1F shows a graph of the H-score of cyclin E1 protein expression levels as a function of cyclin E1 levels. Figure 1G shows a graph of the growth rate (GRmax) values ​​as a function of cyclin E1 levels. [Figure 1C]Figures 1A-1G show that overexpression of cyclin E1 protein is associated with increased sensitivity to azenosertib in ovarian cancer cell lines. Figure 1A shows exemplary results demonstrating that azenosertib sensitivity correlates with cyclin E1 protein expression in cancer cell lines OV90, Kuramochi, TYK-nu, and OVCAR3, assessed by CellTiter Glo after 96 hours of culture. Figure 1B shows that OV90 cells transduced with a lentiviral vector expressing the CCNE1 gene (low endogenous expression of cyclin E1) exhibited increased sensitivity to azenosertib. Figure 1C shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, and OV90 control (empty vector), as well as lentivirus-induced cyclin E1 protein overexpression. Figure 1D shows growth rate inhibition and IC50 for empty vector and cyclin E1. Figure 1E shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, JOM1, ES2, TYK-nu, CAVO3, OAW28, OVCAR4, OVCAR3, and OV90 control (empty vector). Figure 1F shows a graph of the H-score of cyclin E1 protein expression levels as a function of cyclin E1 levels. Figure 1G shows a graph of the growth rate (GRmax) values ​​as a function of cyclin E1 levels. [Figure 1D]Figures 1A-1G show that overexpression of cyclin E1 protein is associated with increased sensitivity to azenosertib in ovarian cancer cell lines. Figure 1A shows exemplary results demonstrating that azenosertib sensitivity correlates with cyclin E1 protein expression in cancer cell lines OV90, Kuramochi, TYK-nu, and OVCAR3, assessed by CellTiter Glo after 96 hours of culture. Figure 1B shows that OV90 cells transduced with a lentiviral vector expressing the CCNE1 gene (low endogenous expression of cyclin E1) exhibited increased sensitivity to azenosertib. Figure 1C shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, and OV90 control (empty vector), as well as lentivirus-induced cyclin E1 protein overexpression. Figure 1D shows growth rate inhibition and IC50 for empty vector and cyclin E1. Figure 1E shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, JOM1, ES2, TYK-nu, CAVO3, OAW28, OVCAR4, OVCAR3, and OV90 control (empty vector). Figure 1F shows a graph of the H-score of cyclin E1 protein expression levels as a function of cyclin E1 levels. Figure 1G shows a graph of the growth rate (GRmax) values ​​as a function of cyclin E1 levels. [Figure 1E]Figures 1A-1G show that overexpression of cyclin E1 protein is associated with increased sensitivity to azenosertib in ovarian cancer cell lines. Figure 1A shows exemplary results demonstrating that azenosertib sensitivity correlates with cyclin E1 protein expression in cancer cell lines OV90, Kuramochi, TYK-nu, and OVCAR3, assessed by CellTiter Glo after 96 hours of culture. Figure 1B shows that OV90 cells transduced with a lentiviral vector expressing the CCNE1 gene (low endogenous expression of cyclin E1) exhibited increased sensitivity to azenosertib. Figure 1C shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, and OV90 control (empty vector), as well as lentivirus-induced cyclin E1 protein overexpression. Figure 1D shows growth rate inhibition and IC50 for empty vector and cyclin E1. Figure 1E shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, JOM1, ES2, TYK-nu, CAVO3, OAW28, OVCAR4, OVCAR3, and OV90 control (empty vector). Figure 1F shows a graph of the H-score of cyclin E1 protein expression levels as a function of cyclin E1 levels. Figure 1G shows a graph of the growth rate (GRmax) values ​​as a function of cyclin E1 levels. [Figure 1F]Figures 1A-1G show that overexpression of cyclin E1 protein is associated with increased sensitivity to azenosertib in ovarian cancer cell lines. Figure 1A shows exemplary results demonstrating that azenosertib sensitivity correlates with cyclin E1 protein expression in cancer cell lines OV90, Kuramochi, TYK-nu, and OVCAR3, assessed by CellTiter Glo after 96 hours of culture. Figure 1B shows that OV90 cells transduced with a lentiviral vector expressing the CCNE1 gene (low endogenous expression of cyclin E1) exhibited increased sensitivity to azenosertib. Figure 1C shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, and OV90 control (empty vector), as well as lentivirus-induced cyclin E1 protein overexpression. Figure 1D shows growth rate inhibition and IC50 for empty vector and cyclin E1. Figure 1E shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, JOM1, ES2, TYK-nu, CAVO3, OAW28, OVCAR4, OVCAR3, and OV90 control (empty vector). Figure 1F shows a graph of the H-score of cyclin E1 protein expression levels as a function of cyclin E1 levels. Figure 1G shows a graph of the growth rate (GRmax) values ​​as a function of cyclin E1 levels. [Figure 1G]Figures 1A-1G show that overexpression of cyclin E1 protein is associated with increased sensitivity to azenosertib in ovarian cancer cell lines. Figure 1A shows exemplary results demonstrating that azenosertib sensitivity correlates with cyclin E1 protein expression in cancer cell lines OV90, Kuramochi, TYK-nu, and OVCAR3, assessed by CellTiter Glo after 96 hours of culture. Figure 1B shows that OV90 cells transduced with a lentiviral vector expressing the CCNE1 gene (low endogenous expression of cyclin E1) exhibited increased sensitivity to azenosertib. Figure 1C shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, and OV90 control (empty vector), as well as lentivirus-induced cyclin E1 protein overexpression. Figure 1D shows growth rate inhibition and IC50 for empty vector and cyclin E1. Figure 1E shows cyclin E1 protein expression by Western blot in the cell lines KURAMOCHI, COV362, JOM1, ES2, TYK-nu, CAVO3, OAW28, OVCAR4, OVCAR3, and OV90 control (empty vector). Figure 1F shows a graph of the H-score of cyclin E1 protein expression levels as a function of cyclin E1 levels. Figure 1G shows a graph of the growth rate (GRmax) values ​​as a function of cyclin E1 levels. [Figure 2] FIG. 2 shows exemplary growth rate inhibition (GR) of OV90, Kuramochi, OVCAR8, TYK-nu, Cov362, OVCAR3, and Caov3 cells in the presence of azenosertib. [Figure 3A] FIG. 3A shows that downregulation of CDK2 was assessed by Western blot after treatment with siRNA. [Figure 3B] FIG. 3B shows a graph of percent viability with increasing concentrations of azenosertib. [Figure 3C] FIG. 3C shows a graph of growth rate inhibition with increasing concentrations of azenosertib. [Figure 4A]Figures 4A-4D show changes in replicative stress markers in SKOV3 cyclin E1-low cells and OVCAR3 cyclin E1-high cells after treatment with azenosertib (80 mg / kg). Figure 4A shows baseline cyclin E1 protein expression in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells examined by immunohistochemistry (IHC). Figure 4B shows a graph showing the decrease in CDK1Y15 levels before and after treatment with azenosertib. Figure 4C shows γH2AX (a replicative stress marker) levels in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells. Figure 4D shows γH2AX Western blots in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells after treatment with azenosertib. [Figure 4B] Figures 4A-4D show changes in replicative stress markers in SKOV3 cyclin E1-low cells and OVCAR3 cyclin E1-high cells after treatment with azenosertib (80 mg / kg). Figure 4A shows baseline cyclin E1 protein expression in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells examined by immunohistochemistry (IHC). Figure 4B shows a graph showing the decrease in CDK1Y15 levels before and after treatment with azenosertib. Figure 4C shows γH2AX (a replicative stress marker) levels in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells. Figure 4D shows γH2AX Western blots in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells after treatment with azenosertib. [Figure 4C]Figures 4A-4D show changes in replicative stress markers in SKOV3 cyclin E1-low cells and OVCAR3 cyclin E1-high cells after treatment with azenosertib (80 mg / kg). Figure 4A shows baseline cyclin E1 protein expression in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells examined by immunohistochemistry (IHC). Figure 4B shows a graph showing the decrease in CDK1Y15 levels before and after treatment with azenosertib. Figure 4C shows γH2AX (a replicative stress marker) levels in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells. Figure 4D shows γH2AX Western blots in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells after treatment with azenosertib. [Figure 4D] Figures 4A-4D show changes in replicative stress markers in SKOV3 cyclin E1-low cells and OVCAR3 cyclin E1-high cells after treatment with azenosertib (80 mg / kg). Figure 4A shows baseline cyclin E1 protein expression in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells examined by immunohistochemistry (IHC). Figure 4B shows a graph showing the decrease in CDK1Y15 levels before and after treatment with azenosertib. Figure 4C shows γH2AX (a replicative stress marker) levels in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells. Figure 4D shows γH2AX Western blots in SKOV3 cyclin E1-low and OVCAR3 cyclin E1-high cells after treatment with azenosertib. [Figure 5A] Figures 5A-5D show exemplary reductions in tumor volume (Figures 5A and 5C) and changes in body weight (Figures 5B and 5D) in SKOV3 (non-CCNE1 amplified, CN=2) CDX mice treated with azenosertib compared to vehicle controls. [Figure 5B] Figures 5A-5D show exemplary reductions in tumor volume (Figures 5A and 5C) and changes in body weight (Figures 5B and 5D) in SKOV3 (non-CCNE1 amplified, CN=2) CDX mice treated with azenosertib compared to vehicle controls. [Figure 5C] Figures 5A-5D show exemplary reductions in tumor volume (Figures 5A and 5C) and changes in body weight (Figures 5B and 5D) in SKOV3 (non-CCNE1 amplified, CN=2) CDX mice treated with azenosertib compared to vehicle controls. [Figure 5D] Figures 5A-5D show exemplary reductions in tumor volume (Figures 5A and 5C) and changes in body weight (Figures 5B and 5D) in SKOV3 (non-CCNE1 amplified, CN=2) CDX mice treated with azenosertib compared to vehicle controls. [Figure 6A] Figures 6A and 6B show exemplary reductions in tumor volume (Figure 6A) and changes in body weight (Figure 6B) in OVCAR8 (non-CCNE1-amplified) tumor-bearing mice treated with azenosertib compared to vehicle controls. [Figure 6B] Figures 6A and 6B show exemplary reductions in tumor volume (Figure 6A) and changes in body weight (Figure 6B) in OVCAR8 (non-CCNE1-amplified) tumor-bearing mice treated with azenosertib compared to vehicle controls. [Figure 7A] Figures 7A and 7B show exemplary reductions in tumor volume (Figure 7A) and changes in body weight (Figure 7B) in HCC1806 (CCNE1 amplified, CN=7) CDX mice treated with azenosertib compared to vehicle controls. [Figure 7B] Figures 7A and 7B show exemplary reductions in tumor volume (Figure 7A) and changes in body weight (Figure 7B) in HCC1806 (CCNE1 amplified, CN=7) CDX mice treated with azenosertib compared to vehicle controls. [Figure 8A] Figures 8A-8D show exemplary reductions in tumor volume (Figure 8A and Figure 8C) and changes in body weight (Figure 8B and Figure 8D) in OVCAR3 (CCNE1 amplified, CN=14) CDX mice treated with azenosertib compared to vehicle controls. [Figure 8B] Figures 8A-8D show exemplary reductions in tumor volume (Figure 8A and Figure 8C) and changes in body weight (Figure 8B and Figure 8D) in OVCAR3 (CCNE1 amplified, CN=14) CDX mice treated with azenosertib compared to vehicle controls. [Figure 8C] Figures 8A-8D show exemplary reductions in tumor volume (Figure 8A and Figure 8C) and changes in body weight (Figure 8B and Figure 8D) in OVCAR3 (CCNE1 amplified, CN=14) CDX mice treated with azenosertib compared to vehicle controls. [Figure 8D] Figures 8A-8D show exemplary reductions in tumor volume (Figure 8A and Figure 8C) and changes in body weight (Figure 8B and Figure 8D) in OVCAR3 (CCNE1 amplified, CN=14) CDX mice treated with azenosertib compared to vehicle controls. [Figure 9] FIG. 9 shows an exemplary synergy analysis of azenosertib in combination with gemcitabine in OV90, OVCAR8, and OVCAR3 cells. [Figure 10A] Figures 10A and 10B show exemplary reductions in tumor volume (Figure 10A) and changes in body weight (Figure 10B) in A2780 (non-CCNE1-amplified, CN=2) tumor-bearing mice treated with azenosertib alone or in combination with paclitaxel compared to vehicle controls. [Figure 10B] Figures 10A and 10B show exemplary reductions in tumor volume (Figure 10A) and changes in body weight (Figure 10B) in A2780 (non-CCNE1-amplified, CN=2) tumor-bearing mice treated with azenosertib alone or in combination with paclitaxel compared to vehicle controls. [Figure 11A] Figures 11A and 11B show exemplary reductions in tumor volume (Figure 11A) and changes in body weight (Figure 11B) in OVCAR3 (CCNE1-amplified, CN=14) tumor-bearing mice treated with azenosertib alone or in combination with paclitaxel compared to vehicle controls. [Figure 11B]Figures 11A and 11B show exemplary reductions in tumor volume (Figure 11A) and changes in body weight (Figure 11B) in OVCAR3 (CCNE1-amplified, CN=14) tumor-bearing mice treated with azenosertib alone or in combination with paclitaxel compared to vehicle controls. [Figure 11C-1] FIG. 11C shows a heat map illustrating the synergistic effect of chemotherapy (paclitaxel) and azenosertib treatment in cyclin E1 high cells (OVCAR3) compared to cyclin E1 low cells (OV90 and TYL-nu). [Figure 11C-2] FIG. 11C shows a heat map illustrating the synergistic effect of chemotherapy (paclitaxel) and azenosertib treatment in cyclin E1 high cells (OVCAR3) compared to cyclin E1 low cells (OV90 and TYL-nu). [Figure 11C-3] FIG. 11C shows a heat map illustrating the synergistic effect of chemotherapy (paclitaxel) and azenosertib treatment in cyclin E1 high cells (OVCAR3) compared to cyclin E1 low cells (OV90 and TYL-nu). [Figure 12A] 12A-12C show exemplary cyclin E1 IHC H-score correlations with response in human subjects treated with azenosertib in combination with carboplatin, paclitaxel, PLD, or gemcitabine. [Figure 12B] 12A-12C show exemplary cyclin E1 IHC H-score correlations with response in human subjects treated with azenosertib in combination with carboplatin, paclitaxel, PLD, or gemcitabine. [Figure 12C] 12A-12C show exemplary cyclin E1 IHC H-score correlations with response in human subjects treated with azenosertib in combination with carboplatin, paclitaxel, PLD, or gemcitabine. [Figure 13A]Figures 13A-13E show exemplary cyclin E1 IHC H score correlations with tumor response (Figures 13A and 13C), progression-free survival (Figure 13B), and CA125 response (Figure 13D) in human subjects. Subjects were grouped by low cyclin E1 IHC H score (<70), intermediate cyclin E1 IHC H score (70-130), and high cyclin E1 IHC H score (>130) (Figure 13E). [Figure 13B] Figures 13A-13E show exemplary cyclin E1 IHC H score correlations with tumor response (Figures 13A and 13C), progression-free survival (Figure 13B), and CA125 response (Figure 13D) in human subjects. Subjects were grouped by low cyclin E1 IHC H score (<70), intermediate cyclin E1 IHC H score (70-130), and high cyclin E1 IHC H score (>130) (Figure 13E). [Figure 13C] Figures 13A-13E show exemplary cyclin E1 IHC H score correlations with tumor response (Figures 13A and 13C), progression-free survival (Figure 13B), and CA125 response (Figure 13D) in human subjects. Subjects were grouped by low cyclin E1 IHC H score (<70), intermediate cyclin E1 IHC H score (70-130), and high cyclin E1 IHC H score (>130) (Figure 13E). [Figure 13D] Figures 13A-13E show exemplary cyclin E1 IHC H score correlations with tumor response (Figures 13A and 13C), progression-free survival (Figure 13B), and CA125 response (Figure 13D) in human subjects. Subjects were grouped by low cyclin E1 IHC H score (<70), intermediate cyclin E1 IHC H score (70-130), and high cyclin E1 IHC H score (>130) (Figure 13E). [Figure 13E]Figures 13A-13E show exemplary cyclin E1 IHC H score correlations with tumor response (Figures 13A and 13C), progression-free survival (Figure 13B), and CA125 response (Figure 13D) in human subjects. Subjects were grouped by low cyclin E1 IHC H score (<70), intermediate cyclin E1 IHC H score (70-130), and high cyclin E1 IHC H score (>130) (Figure 13E). [Figure 14A] 14A-14C show CCNE1 gene amplification status and cyclin E1 IHC H scores of subjects receiving azenosertib. [Figure 14B] 14A-14C show CCNE1 gene amplification status and cyclin E1 IHC H scores of subjects receiving azenosertib. [Figure 14C] 14A-14C show CCNE1 gene amplification status and cyclin E1 IHC H scores of subjects receiving azenosertib. [Figure 14D] FIG. 14D is an exemplary scatter plot showing that cyclin E1 IHC H scores were highly correlated with CCNE1 transcript levels. [Figure 14E] FIG. 14E is an exemplary image of tumor cells designated in a cyclin E1-positive state, while FIG. 14F is an exemplary image of tumor cells designated in a cyclin E1-negative state. [Figure 14F] FIG. 14E is an exemplary image of tumor cells designated in a cyclin E1-positive state, while FIG. 14F is an exemplary image of tumor cells designated in a cyclin E1-negative state. [Figure 14G] Figures 14G and 14H show exemplary cyclin E1 IHC H-score correlations with response in human subjects treated with azenosertib in combination with carboplatin, gemcitabine, paclitaxel, or PLD, and show that subjects with either a clinical partial or complete response (PR or CR, respectively) were associated with cyclin E1 IHC protein expression levels and an H-score of >50. [Figure 14H]Figures 14G and 14H show exemplary cyclin E1 IHC H-score correlations with response in human subjects treated with azenosertib in combination with carboplatin, gemcitabine, paclitaxel, or PLD, and show that subjects with either a clinical partial or complete response (PR or CR, respectively) were associated with cyclin E1 IHC protein expression levels and an H-score of >50. [Figure 14I] Figures 14I and 14J show exemplary percentages of viable tumor cells with a 2+ cyclin E1 IHC staining intensity correlation with response in human subjects treated with azenosertib in combination with carboplatin, gemcitabine, paclitaxel, or PLD, and also show that subjects with either a clinical partial or complete response (PR or CR, respectively) were associated with cyclin E1 IHC protein expression levels and percentages of viable tumor cells >10%. [Figure 14J] Figures 14I and 14J show exemplary percentages of viable tumor cells with a 2+ cyclin E1 IHC staining intensity correlation with response in human subjects treated with azenosertib in combination with carboplatin, gemcitabine, paclitaxel, or PLD, and also show that subjects with either a clinical partial or complete response (PR or CR, respectively) were associated with cyclin E1 IHC protein expression levels and percentages of viable tumor cells >10%. [Figure 14K] FIG. 14K is a graph mapping the percentage of viable tumor cells with a staining intensity of 2+ to the H-score cutoff for cyclin E1 immunohistochemistry (IHC). [Figure 15A]Figure 15A shows systemic therapy and response prior to azenosertib study enrollment. Subjects were classified as "platinum-sensitive" or "platinum-resistant" at the time of sample collection. For each subject (y-axis), the timeline (x-axis) is centered at the time of sample collection. Each treatment line (data segment) and event (data point) is shown. Treatment classification + response and event type are coded by color (platinum = platinum) and shape, respectively. CR: complete response, PR: partial response, SD: stable disease, PD: progressive disease, NE: not evaluable, and NA: not applicable. [Figure 15B-1] Figure 15B shows the relative distribution of cyclin E1 protein expression status (i.e., cyclin E1 negative, cyclin E1 low positive, cyclin E1 high positive) (y-axis) across major subsets (x-axis) for the clinical variables: platinum response, previous platinum exposure, HRR status, and subject age at harvest (panel). The data show that platinum exposure, response, or HRR mutation status did not significantly affect cyclin E1 protein expression, suggesting that cyclin E1 positivity is frequent in high-grade serous ovarian cancer and is independent of previous platinum treatment. [Figure 15B-2] Figure 15B shows the relative distribution of cyclin E1 protein expression status (i.e., cyclin E1 negative, cyclin E1 low positive, cyclin E1 high positive) (y-axis) across major subsets (x-axis) for the clinical variables: platinum response, previous platinum exposure, HRR status, and subject age at harvest (panel). The data show that platinum exposure, response, or HRR mutation status did not significantly affect cyclin E1 protein expression, suggesting that cyclin E1 positivity is frequent in high-grade serous ovarian cancer and is independent of previous platinum treatment. [Figure 16A] 16A and 16B are exemplary pathology scans of a subject with CCNE1-amplified platinum-resistant ovarian cancer at screening (FIG. 16A) and after treatment with azenosertib (FIG. 16B). [Figure 16B]16A and 16B are exemplary pathology scans of a subject with CCNE1-amplified platinum-resistant ovarian cancer at screening (FIG. 16A) and after treatment with azenosertib (FIG. 16B). DETAILED DESCRIPTION OF THE INVENTION

[0065] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, applications, published applications, and other non-patent publications referenced herein are incorporated by reference in their entirety unless otherwise stated. In the event that there are multiple definitions for terms herein, those in this section prevail unless otherwise stated.

[0066] As used herein, the term "about" has its ordinary meaning as understood by one of ordinary skill in the art, and thus indicates that a value includes the inherent variation of error for the method being used to determine the value or the variation that exists among multiple determinations.

[0067] As used herein, the term "modify" or "alter" or any form thereof means to modify, change, replace, delete, substitute, remove, alter, or convert.

[0068] As used herein, the terms "function" and "functional" have their ordinary meaning as understood by those of skill in the art and thus refer to biological, enzymatic, or therapeutic function.

[0069] As used herein, the term "endogenous" has its ordinary meaning as understood by those of skill in the art, and thus refers to the native or wild-type characteristics of a gene, protein, or cell. In some embodiments, an endogenous gene is the wild-type sequence of said gene. In some embodiments, an endogenous protein is the wild-type sequence of said protein. In some embodiments, an endogenous protein function is the wild-type function and activity level of said protein. In some embodiments, an endogenous cell is a wild-type cell.

[0070] The term "mutation" has its ordinary meaning as understood by those skilled in the art and refers to a change in a gene sequence. In some embodiments, a cell has multiple mutations. In some embodiments, the mutations are in coding regions of the genome. Mutations can range in size from a single nucleotide to large segments of a chromosome containing multiple genes. In some embodiments, at least one mutation is silent and does not significantly affect gene expression or function. In some embodiments, at least one mutation affects gene expression or function, such as gene amplification, overexpression, or increased copy number. In some embodiments, at least one mutation is silent and does not significantly affect protein expression or function. In some embodiments, at least one mutation has a small effect on protein expression or function. In some embodiments, at least one mutation has a moderate effect on protein expression or function. In some embodiments, at least one mutation has a large effect on protein expression or function. In some embodiments, at least one mutation disrupts protein expression or function. Non-limiting examples of mutations include insertions, deletions, truncations, substitutions, duplications, translocations, and inversions. In some embodiments, the mutations are "somatic," meaning they occur in somatic cells and are not heritable. In some embodiments, a subset of somatic cells in an organism have at least one mutation that other somatic cells do not have. In some embodiments, the mutation occurs in the "germline," or reproductive cells, and is heritable.

[0071] As disclosed herein, mutations can be monitored through various sequencing, expression, or functional assays.Non-limiting examples include DNA sequencing, RNA sequencing, DNA hybridization, protein sequencing, targeted genome sequencing, whole exome sequencing, whole genome sequencing, ATAC sequencing, Sanger sequencing, PCR, qPCR, RT-PCR, RT-qPCR, next-generation sequencing, protein truncation test, DNA microarray, heteroduplex analysis, denaturing gradient gel electrophoresis, nucleotide sequencing, single-strand conformation polymorphism, restriction enzyme digestion assay, fluorescence in situ hybridization (FISH), comparative genomic hybridization, restriction fragment length polymorphism, amplification-refractory mutation PCR, nested PCR, multiplex ligation-dependent probe amplification, single-strand conformation polymorphism, and oligonucleotide ligation assay. Mutations can also be monitored via various antibody-based methods using biological samples, including, but not limited to, Western blotting, fluorescence-activated cell sorting, immunofluorescence, immunohistochemistry, immunocytochemistry, immunoprecipitation, enzyme-linked immunosorbent assay, radioimmunoassay, and electrochemiluminescence assay.

[0072] The term "cancer" is used herein in its ordinary biological sense and is understood by those skilled in the art. As such, it can include cancer of any cell type, including, but not limited to: However, there are also cases of glioblastoma, astrocytoma, meningioma, craniopharyngioma, medulloblastoma, and other brain cancers. Leukemia, skin cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colorectal cancer, uterine cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, Hodgkin's lymphoma, hematologic tumors, hematologic malignancies, Kaposi's sarcoma, kidney cancer, pharyngeal and hypopharyngeal cancer, liver cancer, lung cancer, lymphoma, mesothelioma, melanoma, and multiple myeloma. Includes neuroblastoma, nasopharyngeal cancer, ovarian cancer, osteosarcoma, pancreatic cancer, pituitary cancer, retinoblastoma, salivary gland cancer, gastric cancer, small intestine cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous adenocarcinoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, solid tumors, and / or liquid tumors.

[0073] As used herein, the term "tumor" has its ordinary meaning as understood by those skilled in the art and refers to an abnormal growth of cells or tissue. In some embodiments, tumors are benign. In some embodiments, tumors are malignant. A tumor becomes cancerous when it metastasizes or spreads to other areas of the body. As used herein, the term "solid tumor" has its ordinary meaning as understood by those skilled in the art and refers to an abnormal mass of tissue that does not contain liquid areas or cysts. Non-limiting examples of solid tumors include sarcomas, carcinomas, or lymphomas. Many cancerous tissues can form solid tumors, such as, but not limited to, breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, and / or ovarian cancer. The terms "cancer" and "tumor" may generally be used interchangeably unless the context clearly indicates that a more specific meaning is intended.

[0074] As used herein, the term "cell" has its ordinary meaning as understood by one of ordinary skill in the art and can refer to any cell type. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0075] As used herein, the terms "individual," "subject," or "patient" have their ordinary meanings as understood by those skilled in the art, and thus include human or non-human mammals. The term "mammal" is used in its ordinary biological sense. Thus, it specifically includes, but is not limited to, monkeys (chimpanzees, apes, monkeys) and primates, including humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or pigs. In some embodiments, the subject may be a human. In some embodiments, the subject may be a child and / or infant. In other embodiments, the subject may be an adult.

[0076] As used herein, the term "cancer therapy" has its ordinary meaning as understood by those skilled in the art and refers to a therapeutic modality (such as surgery and / or radiation) or an anti-cancer agent such as a small molecule, compound, protein, or other drug used to treat, inhibit, or prevent cancer. Non-limiting examples of common classes of anti-cancer agents that can be used in any one or more of the alternatives described herein include alkylating agents, anti-EGFR antibodies, anti-Her-2 antibodies, antimetabolites, vinca alkaloids, platinum-based agents, anthracyclines, topoisomerase inhibitors, taxanes, antibiotics, immunomodulators, immune cell antibodies, interferons, interleukins, HSP90 inhibitors, antiandrogens, antiestrogens, antihypercalcemic agents, apoptosis inducers, Aurora kinase inhibitors, Bruton's tyrosine kinase inhibitors, calcineurin inhibitors, CaM kinase II inhibitors, CD45 tyrosine phosphatase inhibitors, CDC25 phosphatase inhibitors, CHK kinase inhibitors, cyclooxygenase inhibitors, bRAF kinase inhibitors, cRAF kinase inhibitors, Ras inhibitors, cyclin-dependent kinase inhibitors, cysteine ​​protease inhibitors, DNA intercalators, DNA strand breakers, E3 ligase inhibitors, EGF pathway inhibitors , farnesyltransferase inhibitors, Flk-1 kinase inhibitors, glycogen synthase kinase-3 (GSK3) inhibitors, histone deacetylase (HDAC) inhibitors, I-kappa B-alpha kinase inhibitors, imidazotetrazinone, insulin tyrosine kinase inhibitors, c-Jun-N-terminal kinase (JNK) inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, MDM2 inhibitors, MEK inhibitors, ERK inhibitors, MMP inhibitors, mTor inhibitors, NGFR tyrosine kinase inhibitors, p38 MAP kinase inhibitors, p56 tyrosine kinase inhibitors, PDGF pathway inhibitors, phosphatidylinositol 3-kinase inhibitors, phosphatase inhibitors, protein phosphatase inhibitors, PKC inhibitors, PKC delta kinase inhibitors, polyamine synthesis inhibitors, PTP1B inhibitors, protein tyrosine kinase inhibitors, SRC family tyrosine kinase inhibitors, Syk tyrosine kinase inhibitors, Janus (JAK-2 and / or JAK-3) tyrosine kinase inhibitors, retinoids, RNA polymerase II elongation inhibitors, serine / threonine kinase inhibitors, sterol biosynthesis inhibitors, VEGF pathway inhibitors, chemotherapeutic agents, aryletinone, altretamine, aminopterin, aminolevulinic acid, amsacrine asparaginase, atrasentan, bexarotene, carboquone, demecolcine, efaproxiral, elsamitrucin, etoglucide, hydroxycarbamide, leucovorin, lonidamine, lucantone, masoprocol, methyl aminolevulinate, mitoguazone, mitotane, oblimersen, omacetaxine, pegaspargase, porfimer sodium, prednimustine, citimagine seradenovec, talaporfin, temoporfin, trabectedin, or verteporfin.Examples of chemotherapeutic agents useful in treating cancer include carboplatin, cisplatin, paclitaxel, docetaxel, pegylated liposomal doxorubicin, doxorubicin, gemcitabine, cytarabine, fludarabine, fluorouracil (5-FU), irinotecan, topotecan, temozolomide, triapine, 5-azacytidine, capecitabine, AraC-FdUMP

[10] (CF-10), cladribine, decitabine, hydroxyurea, and / or oxaliplatin, or a pharmaceutically acceptable salt of any of the foregoing. Other examples of chemotherapeutic agents useful in treating cancer include azacitidine, bendamustine, bortezomib, carfilzomib, ixazomib, busulfan, carboplatin, cytarabine, cyclophosphamide, cladribine, cisplatin, capecitabine, decitabine, dexamethasone, etoposide, fludarabine, gemcitabine, daunorubicin, doxorubicin, ifosfamide, methotrexate, and / or vincristine, or a pharmaceutically acceptable salt of any of the foregoing.

[0077] The term "pharmaceutically acceptable salt" refers to a salt of a compound that does not cause significant irritation to the organism to which it is administered and does not interfere with the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting a compound with an inorganic acid such as a hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid (e.g., 2,3-dihydroxypropyl dihydrogen phosphate). Pharmaceutical salts can also be obtained by reacting a compound with an organic acid such as formic acid, acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, benzoic acid, salicylic acid, 2-oxopentanedioic acid, or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form salts such as ammonium salts, alkali metal salts (such as sodium, potassium, or lithium salts), alkaline earth metal salts (such as calcium or magnesium salts), carbonate salts, bicarbonate salts, salts of organic bases (such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamines, cyclohexylamine, triethanolamine, ethylenediamine, and the like), and salts with amino acids (such as arginine and lysine).

[0078] When the compounds disclosed herein have unfilled valences, the valences are not hydrogen. or isotopes thereof, such as hydrogen-1 (protium) and hydrogen-2 (deuterium).

[0079] It is understood that the compounds described herein can be isotopically labeled. Substitution with isotopes such as deuterium can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Each chemical element represented in a compound structure can include any isotope of said element. For example, in a compound structure, a hydrogen atom may be explicitly disclosed or understood to be present in the compound. At any position in the compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including, but not limited to, hydrogen-1 (protium) and hydrogen-2 (deuterium). Therefore, reference to a compound herein encompasses all possible isotopic forms, unless the context clearly indicates otherwise.

[0080] It is understood that the compounds described herein include crystalline forms (also known as polymorphs, which include different crystalline packing arrangements of the same elemental composition of a compound), amorphous phases, salts, solvates, and hydrates. In some embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. In other embodiments, the compounds described herein exist in unsolvated forms. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and can be formed during the crystallization process using pharmaceutically acceptable solvents such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Furthermore, the compounds provided herein can exist in unsolvated and solvated forms. Generally, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods provided herein.

[0081] When a range of values ​​is provided, it is understood that the upper and lower limits, and each intervening value between the upper and lower limits of the range, are encompassed within an embodiment.

[0082] Terms and phrases used in this application, and variations thereof, should be construed as open-ended rather than limiting, unless expressly stated otherwise, particularly in the appended claims. As an example of the foregoing, the term "including" should be read to mean "including, without limitation," "including but not limited to," or similar expressions. As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," is open-ended, and does not exclude additional, unrecited elements or method steps. The term "having" should be interpreted as "having at least." The term "including" should be interpreted as "including but not limited to." The term "example" is used to provide illustrative examples of the items under discussion, not an exhaustive or limiting list thereof. Additionally, the use of terms such as "preferably," "preferred," "desired," "desirable," or words of similar import should not be understood to imply that any particular feature is critical, essential, or even crucial to structure or function, but are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment. Additionally, the term "comprising" should be interpreted synonymously with the phrases "having at least" or "including at least." When used in the context of a compound, composition, or device, In this case, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may include additional features or components.

[0083] With respect to the use of virtually any plural and / or singular term herein, those skilled in the art can translate from the plural to the singular and / or from the singular to the plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity. The indefinite articles "a" or "an" do not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be advantageously used. Any reference signs in the claims should not be construed as limiting the scope.

[0084] As used herein, the term "equivalent dose" refers to the above-mentioned effective amount of a compound, for example, azenosertib in other salt forms.

[0085] The term "rest" or "rest day" refers to a period of time during which azenosertib is not administered, or a day without administration, a day off treatment, or a rest day. For example, a rest refers to the period following a dosing cycle or the intervening period during a dosing week during which azenosertib administration is suspended.

[0086] The term "platinum resistance" or "platinum refractory" when referring to cancer refers to a cancer that initially responds to treatment with a platinum-containing drug, but then returns within a certain period of time. For example, ovarian cancer that recurs within six months of treatment is considered platinum resistant. In one embodiment, a cancer is platinum refractory if it progresses in any line within 90 days of the last administration of a platinum-based regimen.

[0087] The term "cyclin E1" refers to a protein involved in cell cycle regulation by binding to cyclin-dependent kinases, including CDK2. The cyclin E1 protein is encoded by the CCNE1 gene, an oncogene in many cancers. The term "cyclin E1 status" refers to the expression level of the cyclin E1 protein, which can be classified as, for example, cyclin E1-negative, cyclin E1-low, cyclin E1-positive, cyclin E1-positive (low), cyclin E1-positive (high), or cyclin E1-high. Overexpression of cyclin E1 refers to overexpression of the protein, not overexpression or amplification of the CCNE1 gene. Depending on the subject's stratification, in one embodiment, cyclin E1 overexpression is indicated by cyclin E1-positive, cyclin E1-positive (low), cyclin E1-positive (high), and cyclin E1-high cyclin E1 status, or by cyclin E1-high cyclin E1 status, or by cyclin E1-positive cyclin E1 status. Similarly, in one embodiment, lack of expression or overexpression of cyclin E1 is indicated by cyclin E1-negative or cyclin E1-low cyclin E1 status. In clinical settings, cyclin E1 protein expression levels can be determined, for example, by immunohistochemistry (IHC) or Western blot, whereby a sample (e.g., a target tumor tissue, target tumor cells) is contacted with an anti-cyclin E1 antibody, stained, and subjected to histological evaluation by a board-certified pathologist, who rates the intensity of nuclear staining of viable tumor cells in the sample as 0 (negative), 1+ (negative, weak, or low), 2+ (positive, slightly positive, neutral, weak to moderate, or moderate), or 3+ (positive, strongly positive, or high). In some embodiments, the percentage of viable tumor cells with a staining intensity of 0, 1+, 2+, or 3+ is obtained. In some embodiments, the percentage is used to obtain a histological score or H score. As used herein, the terms "H score," "IHC H score," and "cyclin E1 IHC H score," as well as their full and / or plural forms, are used interchangeably.

[0088] Various aspects are described in detail in the following sections. The use of sections is not intended to limit the disclosure. Each section may apply to any aspect of the disclosure. In this application, the use of "or" means "and / or" unless stated otherwise.

[0089] Detailed Description The present disclosure provides, inter alia, methods for treating cancer in subjects selected for a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. In particular, the present disclosure provides methods for treating conditions characterized by excessive cell proliferation, such as cancer, by administering to a subject the WEE1 inhibitor azenosertib. In some embodiments, the present disclosure provides methods for treating conditions characterized by excessive cell proliferation, such as cancer, by administering to a subject azenosertib (WEE1 inhibitor), or a pharmaceutically acceptable salt thereof, as monotherapy or in combination with one or more chemotherapeutic agents at an effective dose. The present disclosure also relates to treating cancer in a subject by identifying the subject as having a cyclin E1 biomarker level above a predetermined threshold without determining the levels of other cancer biomarkers in the subject.

[0090] The present disclosure further provides a method of treating ovarian cancer, comprising administering an effective dose of azenosertib or a pharmaceutically acceptable salt thereof to a subject selected to have a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold.The present disclosure further provides a method of treating ovarian cancer, comprising administering an effective dose of azenosertib or a pharmaceutically acceptable salt thereof to a subject selected to have a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold during a treatment cycle, and administering a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof one or more times during the treatment cycle.

[0091] [ka]

[0092] The compound azenosertib and its pharmaceutically acceptable salts are WEE1 inhibitors. The chemical structure of the compound azenosertib is shown above. The compound azenosertib and its pharmaceutically acceptable salts can be prepared by various methods. See, for example, WO 2019 / 173082. WO 2019 / 173082 and WO 2021 / 231653 describe the compound azenosertib and methods of using it to treat cancer.

[0093] Cyclin E1 (encoded by the CCNE1 gene) is involved in cell cycle regulation by binding to cyclin-dependent kinases (CDKs), including CDK2, thereby promoting cell cycle progression. Cyclin E1 and cyclin E2 are encoded by the CCNE1 gene at 19q12 and the CCNE2 gene at 8q22.1, respectively. Cyclin E1 plays an important role in cell proliferation and carcinogenesis, and cyclin E2 is considered to be largely functionally redundant with cyclin E1. Cyclin E Cyclin E accumulates at the G1-S phase boundary and is degraded as cells progress through S phase. Cyclin E has multiple functions in cell cycle progression, both CDK2-dependent and CDK2-independent. The cyclin E / CDK2 complex controls the G1 / S phase transition and S phase progression by phosphorylating numerous proteins, regulates the apoptotic response to DNA damage via FOXO1 phosphorylation, and plays a role in epigenetic regulation via EZH2 phosphorylation.

[0094] WEE1 is a tyrosine kinase that is a key component of the ATR-mediated G2 cell cycle checkpoint control, which prevents entry into mitosis in response to cellular DNA damage. WEE1 activation leads to selective phosphorylation of CDK2, thereby regulating the CDK2-cyclin A / E complex, which controls G1 / S phase progression. Inhibition of WEE1 can result in excessive replicative activity, thereby leading to replicative collapse. WEE1 inhibition can sensitize tumors to induce tumor cell death.

[0095] Target history and selection The CCNE1 gene is overexpressed and / or amplified in various cancers. Changes in CCNE1 levels (e.g., gene amplification and / or gene overexpression) deregulate cell cycle progression and make cells more vulnerable to WEE1 inhibition. As CCNE1 levels increase, sensitivity to WEE1 inhibitors increases, leading to improved efficacy in cancer treatment using WEE1 inhibitors (e.g., azenosertib).

[0096] Thus, the methods described herein use CCNE1 gene amplification status, a predetermined cyclin E1 status, and / or a cyclin E1 biomarker level to select subjects for cancer treatment with azenosertib or a pharmaceutically acceptable salt thereof. In some embodiments, the methods described herein include selecting subjects with a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold.

[0097] In some embodiments, the method further comprises first determining the cyclin E1 biomarker level prior to the selection step.

[0098] In some embodiments, the subject has received one or more prior lines of therapy, in some embodiments, the subject has received two prior lines of therapy, in some embodiments, the subject has received three prior lines of therapy, in some embodiments, the subject has received more than three prior lines of therapy.

[0099] In some embodiments, the subject has a cancer that is relapsed or refractory. In some embodiments, the cancer is platinum-resistant. In some embodiments, the cancer is platinum-refractory. In some embodiments, the cancer is PARP inhibitor-resistant.

[0100] Predetermined cyclin E1 status and predetermined cutoff cyclin E1 biomarker level and predetermined threshold The cyclin E1 status and its predetermined cutoff and the predetermined threshold of the cyclin E1 biomarker can be determined by various methods. In some embodiments, the predetermined threshold is an absolute value or a standard. In some embodiments, the predetermined threshold is obtained from a literature source. In some embodiments, the predetermined threshold is obtained from the subject's own past cyclin E1 status and cyclin E1 biomarker levels. In some embodiments, the predetermined threshold is obtained from the cyclin E1 status and cyclin E1 biomarker levels in subjects without cancer.

[0101] In some embodiments, the predetermined threshold is expressed by comparison with a reference or control. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test cyclin E1 biomarker level in the subject. In embodiments, the reference or control is a past reference or control. In some embodiments, the reference or control may be based on the subject's cyclin E1 level before treatment with azenosertib or a pharmaceutically acceptable salt thereof.

[0102] Cyclin E1 overexpression In some embodiments, the predetermined cyclin E1 status, or the cyclin E1 biomarker predetermined threshold, is measured by cyclin E1 protein expression levels.

[0103] In some embodiments, the cyclin E1 protein expression level is determined by detecting the amount of CCNE1 mRNA (transcript) or cyclin E1 protein. In some embodiments, the cyclin E1 protein overexpression level is determined by the mRNA level or transcript level. In some embodiments, the cyclin E1 protein expression level is determined by the protein level. In some embodiments, the cyclin E1 protein expression level is a cyclin E1 protein expression level above a predetermined cutoff. In one embodiment, the predetermined cutoff or predetermined threshold I is measured by the proportion of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 2+. In some embodiments, the cyclin E1 protein expression level is determined by cyclin E1 IHC It is measured by the H score.

[0104] CCNE1 mRNA levels or cyclin E1 protein expression levels can be measured by any method known in the art, including, but not limited to, reporter gene, Northern blot, Western blot, fluorescent in situ hybridization (FISH), reverse transcription PCR, or RNA-Seq-based assays.

[0105] In some embodiments, cyclin E1 protein expression is determined using RNA detection of CCNE1 mRNA or transcript. In some embodiments, cyclin E1 protein expression is determined using RNA sequencing.

[0106] In some embodiments, cyclin E1 protein expression is measured by a quantitative readout. In some embodiments, cyclin E1 protein expression is measured by a qualitative readout.

[0107] In some embodiments, cyclin E1 protein expression is measured by signal intensity. In some embodiments, the signal intensity is determined using Western blot. In some embodiments, the relative signal intensity is quantified.

[0108] IHC percentage of viable tumor cells with staining intensity of 0, 1+, 2+, or 3+, and H score In some embodiments, the cyclin E1 protein expression level is measured by the percentage of viable tumor cells having a specific cyclin E1 immunohistochemistry (IHC) staining intensity, i.e., an IHC staining intensity of 0, 1+, 2+, or 3+. In some embodiments, the cyclin E1 protein expression level is measured by a cyclin E1 IHC H score. In some embodiments, the cyclin E1 protein expression level is measured by the percentage of viable tumor cells having a specific cyclin E1 IHC staining intensity combined with a cyclin E1 IHC H score. In some embodiments, the H score is calculated using the method described in Diar Aziz, et. al., Gynecologic Oncology 151, 327-336 (2018). Briefly, the H score is The core is a semi-quantitative measurement derived by immunohistochemical staining of the tumor cells of interest. The tumor cells of interest were stained using a Vetana Bench Mark ULTRA TM Using an automated staining platform and the Optiview™ Detection Kit, staining with cyclin E1 antibodies was performed. Expression of each protein was assessed by trained, qualified observers, with pathologist-confirmed classification of equivocal cases, using a staining intensity of 0, 1+, 2+, or 3+. For cyclin E1, the percentage of tumor cells with a cyclin E1 IHC staining intensity (SI) of 0, 1+, 2+, or 3+ was obtained and used to determine cyclin E1 protein expression levels. Cyclin E1 was assessed based on nuclear staining, while URI1 expression was assessed based on cytoplasmic staining.

[0109] A subject's H-score is determined by adding 3 times the percentage of strongly stained cells (SI = 3+) + 2 times the percentage of moderately stained cells (SI = 2+) + 1 times the percentage of weekly stained cells (SI = 1+). Thus, a subject's H-score can range from 0 to 300. The formula for calculating the histological score (H-score) is <score = a*f1 + b*f2 + c*f3>, where f1 is the percentage of cells with a staining intensity of i, and i = 1 (1+), 2 (2+), or 3 (3+). In some embodiments, a = 1, b = 2, and c = 3. In some embodiments, a = 0, b = 1, and c = 1, and the score is the percentage of cells with a staining intensity of 2+. In some embodiments, a = 0, b = 0, and c = 1, and the score is the percentage of cells with a staining intensity of 3+.

[0110] In some embodiments, the predetermined cutoff of cyclin E1 status and the predetermined threshold of cyclin E1 biomarker level are measured by the percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 2+. In some embodiments, the predetermined cutoff or predetermined threshold is greater than 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, or 62% of cyclin E1 2+. The percentage of viable tumor cells having IHC staining intensity of 2+. In some embodiments, the predetermined cutoff or predetermined threshold is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% or more of viable tumor cells having Cyclin E1 IHC staining intensity of 2+.

[0111] In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 10%. In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 8-12%, or greater than 8-11%, or greater than 8-10%, or greater than 8-9%, or greater than 9-12%, or greater than 9-11%, or greater than 9-10%, or greater than 10-12%, or greater than 10-11%, or greater than 11-12%.

[0112] In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 15%. In some embodiments, the predetermined cutoff or threshold is greater than 13-17%, or greater than 13-16%, or greater than 13-15%, or greater than 13-14%, or greater than 14-16%, or greater than 14-15%, or greater than 15-17%, or greater than 15- The percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 16% or greater than 16-17%.

[0113] In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 20%. In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 18-22%, or greater than 18-21%, or greater than 18-20%, or greater than 18-19%, or greater than 19-22%, or greater than 19-21%, or greater than 19-20%, or greater than 20-22%, or greater than 20-21%, or greater than 21-22%.

[0114] In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 30%. In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 28-32%, or greater than 28-31%, or greater than 28-30%, or greater than 28-29%, or greater than 29-32%, or greater than 29-31%, or greater than 29-30%, or greater than 30-32%, or greater than 30-31%, or greater than 31-32%.

[0115] In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 35%. In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 33-37%, or greater than 33-36%, or greater than 33-35%, or greater than 33-34%, or greater than 34-36%, or greater than 34-35%, or greater than 35-37%, or greater than 35-36%, or greater than 36-37%.

[0116] In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 40%. In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 38-42%, or greater than 38-41%, or greater than 38-40%, or greater than 38-39%, or greater than 39-42%, or greater than 39-41%, or greater than 39-40%, or greater than 40-42%, or greater than 40-41%, or greater than 41-42%.

[0117] In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 45%. In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 43-47%, or greater than 43-46%, or greater than 43-45%, or greater than 43-44%, or greater than 44-46%, or greater than 44-46%, or greater than 44-35%, or greater than 45-47%, or greater than 45-46%, or greater than 46-47%.

[0118] In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 50%. In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 48-52%, or greater than 48-51%, or greater than 48-50%, or greater than 48-49%, or greater than 49-52%, or greater than 49-51%, or greater than 49-50%, or greater than 50-52%, or greater than 50-51%, or greater than 51-52%.

[0119] In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 55%. In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 53-57%, or greater than 53-56%, or greater than 53-55%, or greater than 53-54%, or greater than 54-56%, or greater than 54-56%, or greater than 54-55%, or greater than 55-57%, or greater than 55-56%, or greater than 56-57%.

[0120] In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 60%. In some embodiments, the predetermined cutoff or threshold is the percentage of viable tumor cells with a 2+ cyclin E1 IHC staining intensity greater than 58-62%, or greater than 58-61%, or greater than 58-60%, or greater than 58-69%, or greater than 59-62%, or greater than 59-61%, or greater than 59-60%, or greater than 60-62%, or greater than 60-61%, or greater than 61-62%.

[0121] In some embodiments, the predetermined cutoff of cyclin E1 status and the predetermined threshold of cyclin E1 biomarker level are measured by the percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 3+.

[0122] In some embodiments, the predetermined cutoff or predetermined threshold is a cyclin E1 IHC H score of greater than 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300. The IHC H-score thresholds are >40, 50, 60, 65, 70, 75, 80, 90, 95, 130, or >180.

[0123] In some embodiments, the predetermined cutoff or predetermined threshold is a Cyclin E1 IHC H-score of greater than 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160.

[0124] In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 40. In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 50. In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 60. In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 65. In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 70. In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 75. In some embodiments, the cyclin E1 IHC H score threshold is greater than 80. In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 90. In some embodiments, the predetermined cutoff or threshold is a cyclin E1 IHC H score greater than 95. In some embodiments, the cyclin E1 IHC H score threshold is greater than 125. In some embodiments, the predetermined cutoff or predetermined threshold is a Cyclin E1 IHC score greater than 130. In some embodiments, the predetermined cutoff or predetermined threshold is a cyclin E1 IHC H score of greater than 135. In some embodiments, the cyclin E1 IHC H score is greater than 135. The threshold for IHC H score is >150.

[0125] In some embodiments, the predetermined cutoff or predetermined threshold is a cyclin E1 IHC H score greater than 25. In some embodiments, the predetermined cutoff or predetermined threshold is a cyclin E1 IHC H score greater than 70. In some embodiments, the predetermined cutoff or predetermined threshold is a cyclin E1 IHC H score greater than 130.

[0126] In some embodiments, the proportion of viable tumor cells having a pre-determined cutoff or pre-determined threshold of Cyclin E1 IHC staining intensity of 2+, and / or the pre-determined cutoff or pre-determined threshold of Cyclin E1 IHC H-score, whether as monotherapy or in combination with one or more second chemotherapeutic agents or pharmaceutically acceptable salts thereof, is a predictive biomarker for treating a subject with a WEE1 inhibitor, such as azenosertib (including pharmaceutically acceptable salts thereof), for example, to predict a subject's sensitivity, responsiveness to treatment (including predicting a subject's tumor response, overall response rate (ORR) and / or median progression-free survival (mPFS)).

[0127] Of course, in both the cyclin E1 IHC H score and the percentage of viable tumor cells with a cyclin E1 IHC staining intensity scoring system of 2+, comparison to a score threshold or cutoff X is generally expressed using exact inequality to determine a positive status (e.g., cyclin E1 positive) versus a negative status (e.g., cyclin E1 negative). Because scores are typically rounded to the nearest integer, calling a positive result using a score strictly greater than X is equivalent to calling a positive result using a score equal to or greater than X+1.

[0128] CCNE1 gene amplification CCNE1 gene amplification is a differential increase in the CCNE1 portion of the genome compared to the entire genome. In some embodiments, "gene amplification" or "increased CCNE1 gene amplification level" refers to any increase in gene copies relative to endogenous copies.

[0129] CCNE1 gene amplification level can be determined by methods known in the art.In some embodiments, CCNE1 gene amplification level is determined using in situ hybridization (ISH) assay.In some embodiments, CCNE1 gene amplification level is determined using fluorescence in situ hybridization (FISH) amplification.In some embodiments, CCNE1 gene amplification level is determined using quantitative polymerase chain reaction.In some embodiments, CCNE1 gene amplification level is determined using next-generation sequencing.

[0130] Number of copies In some embodiments, the CCNE1 gene amplification level or CCNE1 gene amplification status of a subject is measured by CCNE1 gene copy number. In some embodiments, CCNE1 gene copy number is determined using the ISH assay described in Aziz et al. (supra). Briefly, a pre-diluted, ready-to-use 19q12 DNP ISH probe covering the coding sequences of CCNE1 and URI1 is used to measure CCNE1 gene amplification, along with an ISNR DIG ISH probe, which serves as a surrogate standard for diploid copy number located at 19p13.2 in an ISH assay of a subject's tumor cells optimized on the Vetana ULTRA™ platform. Copy number is measured in at least 50 malignant cells and a minimum of 19p13.2. In normal cells and malignant cells with background staining, the presence of interpretable black (19q12) and red (INSR) signals can be determined, and the average number of interpretable black signals per cell is determined. In some embodiments, CCNE1 gene copy number is determined using whole genome sequencing or whole exome sequencing.

[0131] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 7. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 8. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 14.

[0132] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 3. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 4. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 5. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 6. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 7. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 8. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 9. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 10. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 11. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 12. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 13. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 14.

[0133] In some embodiments, a CCNE1 copy number of 2 (CN=2) is non-amplified. In some embodiments, a CCNE1 copy number between 2 and 5 (CN=2-5) is copy number increased. In some embodiments, a CCNE1 copy number greater than 5 (CN>5) is amplified.

[0134] In some embodiments, CCNE1 gene amplification status based on the CCNE1 gene copy number cutoff is a predictive biomarker. In some embodiments, the CCNE1 gene copy number cutoff is a predictive biomarker for treating cancer with azenosertib or a pharmaceutically acceptable salt thereof. In some embodiments, the CCNE1 gene copy number cutoff is selected based on progression-free survival. In some embodiments, the CCNE1 gene copy number cutoff is selected based on tumor response. In some embodiments, the CCNE1 gene copy number cutoff is selected based on clinical benefit rate (CBR). In some embodiments, the CCNE1 gene copy number cutoff is selected based on disease control rate (DCR). In some embodiments, the CCNE1 gene copy number cutoff is selected based on overall survival (OS).

[0135] Additional biomarkers In some embodiments, the subject is selected based on a predetermined status of cyclin E1 or cyclin E1 biomarker level and the level of one or more additional biomarkers. In some embodiments, the subject is previously identified as having one or more additional biomarkers. In some embodiments, additional biomarkers are included in the selection criteria. In some embodiments, additional biomarkers are not included in the selection criteria.

[0136] In some embodiments, subjects are selected without determining the levels of other cancer biomarkers. In some embodiments, subjects are selected based solely on Cyclin E1 biomarker levels. In some embodiments, subjects are selected without determining the levels of BRCA1 and / or BRCA2. In some embodiments, subjects are selected without determining the levels of TP53. In some embodiments, subjects are selected without determining the levels of CA125.

[0137] In other embodiments, the subject is selected without determining the level of other cancer biomarkers. In some embodiments, the subject is selected to have a predetermined level of a cancer biomarker other than cyclin E1. In some embodiments, the subject is selected to have a BCRA1 and / or BRCA2 biomarker level below a predetermined threshold. In some embodiments, the subject is selected to have a TP53 biomarker level below a predetermined cutoff or threshold. In some embodiments, the subject is selected to have a CA125 biomarker level below a predetermined cutoff threshold. In some embodiments, the subject is selected to have a BCRA1 and / or BRCA2 biomarker level above a predetermined cutoff or threshold. In some embodiments, the subject is selected to have a TP53 biomarker level above a predetermined cutoff or threshold. In some embodiments, the subject is selected to have a CA125 biomarker level above a predetermined cutoff or threshold.

[0138] Treatment method The present disclosure provides a method of treating cancer using a compound known as azenosertib, or a pharmaceutically acceptable salt thereof, in which a subject is selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. Azenosertib is a WEE1 inhibitor of the following formula:

[0139] [ka]

[0140] WO 2019 / 173082 and WO 2021 / 231653 describe the compound azenosertib, both of which are incorporated herein by reference in their entireties. Azenosertib is also known as ZN-c3, and these terms are used interchangeably.

[0141] In embodiments, the methods described herein result in a therapeutic effect (e.g., a desired pharmacological and / or physiological effect). A therapeutic effect can include partially or completely curing a disease, alleviating one or more adverse symptoms resulting from a disease, and / or slowing disease progression. To this end, the methods of the present invention comprise administering a therapeutically effective amount of a therapeutic agent (e.g., azenosertib, or a pharmaceutically acceptable salt thereof, and / or a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof). A therapeutically effective amount is a therapeutically effective amount that results in a desired therapeutic outcome (e.g., tumor growth inhibition, progression-free survival, complete response, partial ... A therapeutically effective amount can be an amount effective at dosages and for periods of time necessary to achieve a therapeutically effective effect (e.g., a partial response). A therapeutically effective amount can vary depending on factors such as the condition, age, sex, and weight of the individual, and the ability of the binding agent to elicit a desired response in the individual.

[0142] Administration route In some embodiments, an effective dose of azenosertib or a pharmaceutically acceptable salt thereof is administered orally, intravenously, or subcutaneously. In some embodiments, an effective dose of azenosertib or a pharmaceutically acceptable salt thereof is administered orally. Alternative suitable techniques for administering an effective dose of azenosertib or a pharmaceutically acceptable salt thereof known to those skilled in the art may also be used, including, but not limited to, oral, rectal, topical pulmonary, aerosol, injection, infusion, and parenteral delivery, including, but not limited to, intramuscular, subcutaneous, intravenous, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, and intraocular injection. In other embodiments, azenosertib or a pharmaceutically acceptable salt thereof and / or a chemotherapeutic agent may be administered orally.

[0143] In some embodiments, an effective dose of azenosertib or a pharmaceutically acceptable salt thereof is administered orally, intravenously, subcutaneously, intrathecally, intramuscularly, intracavitary, intrapleurally, intralesionally, or intraarterially. In some embodiments, an effective dose of azenosertib or a pharmaceutically acceptable salt thereof is administered orally, intravenously, or subcutaneously. In some embodiments, an effective dose of azenosertib or a pharmaceutically acceptable salt thereof is administered intrathecally, intramuscularly, intracavitary, intrapleurally, intralesionally, or intraarterially.

[0144] In some embodiments, an effective dose of azenosertib or a pharmaceutically acceptable salt thereof is administered orally.

[0145] Azenosertib dose and schedule In some embodiments, the methods described herein include intermittent administration, i.e., including consecutive administration days followed by rest days, in one or more administration cycles, including intervening rest weeks. In some embodiments, the methods described herein include consecutive administration. In some embodiments, the methods described herein include combination therapy and include consecutive administration of one of the agents. In some embodiments, the methods described herein include combination therapy including consecutive administration of one of the agents and intermittent administration of azenosertib or a pharmaceutically acceptable salt thereof.

[0146] In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered based on the subject's body weight. In some embodiments, an effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 2 mg / kg to 20 mg / kg. In some embodiments, an effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 2 mg / kg to 18 mg / kg, 2 mg / kg to 16 mg / kg, 2 mg / kg to 14 mg / kg, 2 mg / kg to 12 mg / kg, 2 mg / kg to 10 mg / kg, 2 mg / kg to 8 mg / kg, 2 mg / kg to 6 mg / kg, 3 mg / kg to 4 mg / kg, 3 mg / kg to 5 mg / kg, or 4 mg / kg to 6 mg / kg. In some embodiments, the effective dose is at least 2 mg / kg, at least 3 mg / kg, at least 4 mg / kg, at least 5 mg / kg, at least 6 mg / kg, at least 7 mg / kg, at least 8 mg / kg, at least 9 mg / kg, at least 10 mg / kg, at least 11 mg / kg, at least 12 mg / kg, at least 13 mg / kg, at least 14 mg / kg, at least 15 mg / kg, at least 16 mg / kg, at least 17 mg / kg, at least 18 mg / kg, or at least 19 mg / kg.

[0147] In some embodiments, azenosertib may also be in the form of an equivalent dose (e.g., another salt form of the compound). In some embodiments, the effective dose is a fixed dose. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is administered once daily, twice daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is in the range of 00 to 800 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is in the range of 200 to 600 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is in the range of 300 to 600 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is in the range of 400 to 600 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is in the range of 400 to 800 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is in the range of 50-350 mg, 50-290 mg, 100-290 mg, 100-250 mg, 150-250 mg, or 180-220 mg once daily, or equivalents thereof. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is in the range of 50-400 mg, 100-400 mg, 150-400 mg, 200-400 mg, 200-375 mg, 200-350 mg, 200-300 mg, 200-400 mg, or 400-600 mg once daily, or equivalents thereof.

[0148] In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 450 mg, 500 mg, 550 mg, or 600 mg, or its equivalent, once daily.

[0149] In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 200 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 300 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 350 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 400 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 450 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 500 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 600 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 700 mg or its equivalent once daily. In some embodiments, the effective dose of azenosertib or a pharmaceutically acceptable salt thereof is 800 mg or its equivalent once daily.

[0150] In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 550 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, or equivalents thereof or more. In some embodiments, the present disclosure provides for the administration of high doses of azenosertib or a pharmaceutically acceptable salt thereof, e.g., the dose is 375 mg or more.

[0151] Treatment Cycle The method of the present disclosure includes administering azenosertib or a pharmaceutically acceptable salt thereof, and / or a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof according to a suitable administration schedule. For example, azenosertib or a pharmaceutically acceptable salt thereof, and / or a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof, as described herein, may be administered once or multiple times daily (e.g., once, twice, or three times daily), followed by a period of no administration for a specific number of days. This treatment cycle (including days of administration and days of no administration) is then repeated. ) may be repeated.

[0152] In some embodiments, the treatment cycle is 3 to 28 days in duration. In some embodiments, the treatment cycle is 5, 7, 10, or 14 days. In some embodiments, the treatment cycle is 21 or 28 days. In some embodiments, the treatment cycle is repeated.

[0153] In some aspects, provided herein is a method for treating cancer, comprising administering to a subject in need thereof a daily dose of 350 mg or more, or its equivalent, of azenosertib or a pharmaceutically acceptable salt thereof, according to an intermittent administration cycle, wherein the intermittent administration cycle comprises one or more administration weeks, each administration week comprising at least three consecutive administration days and at least one day without administration. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is 400 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is 450 mg.

[0154] In some embodiments, the disclosure provides for high doses of azenosertib, or a pharmaceutically acceptable salt thereof, e.g., about 350 mg to about 800 mg once daily, or about 175 mg to about 400 mg twice daily in an intermittent dosing regimen, e.g., 5 days of administration ("on" days) followed by 2 days of rest ("off" days) (i.e., 5 / 2), 4 days of administration followed by 3 days of rest (i.e., 4 / 3), or 3 days of administration followed by 4 days of rest (i.e., 3 / 4), or 6 days of administration followed by 1 day of rest (i.e., 6 / 1). Alternatively, an intermittent dosing regimen of azenosertib or a pharmaceutically acceptable salt thereof may also be expressed as about 350 mg to about 800 mg once daily, or about 175 mg to about 400 mg twice daily, with an intermittent frequency, e.g., 5 on / 2 off, 4 on / 2 off, or 3 on / 4 off, among others.

[0155] In some embodiments, one or more weeks of administration are separated by at least one week of rest. In some embodiments, the intermittent administration regimen described herein (e.g., 7 / 0, 6 / 1, 5 / 2, 4 / 3, or 3 / 4) is implemented with 2 weeks followed by a one-week rest, or 1 week followed by a one-week rest, thereby achieving high efficacy while increasing safety and tolerability in the treatment of cancer. In some embodiments, the intermittent administration regimen described herein (e.g., 7 / 0, 5 / 2, 6 / 1, 4 / 3, or 3 / 4) is implemented with 3 weeks followed by a one-week rest, or 1 week followed by a one-week rest, thereby achieving high efficacy while increasing safety and tolerability in the treatment of cancer. In some embodiments, the intermittent dosing regimens described herein (e.g., 7 / 0, 6 / 1, 5 / 2, 4 / 3, or 3 / 4) are implemented for periods of more than 3 weeks followed by a week of rest, or a week followed by a week of rest, thereby achieving high efficacy while increasing safety and tolerability in the treatment of cancer.

[0156] In some aspects, provided herein is a method of treating cancer comprising administering to a subject in need thereof a daily dose of 100 mg or more of azenosertib or a pharmaceutically acceptable salt thereof according to an intermittent dosing cycle, wherein the intermittent dosing cycle comprises one or more dosing weeks, each dosing week comprising at least three consecutive dosing days and at least one dosing-free day, followed by at least one week of rest. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, or equivalents thereof or more. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered once daily at a dose of about 200 mg in an intermittent dosing regimen. In some embodiments, azenosertib In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 225 mg once daily in an intermittent dosing regimen. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 250 mg once daily in an intermittent dosing regimen. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 275 mg once daily in an intermittent dosing regimen. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of more than about 300 mg once daily in an intermittent dosing regimen. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg once daily in an intermittent dosing regimen. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 350 mg once daily in an intermittent dosing regimen.

[0157] In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 550 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, or equivalents thereof or more. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is 375 mg or more. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 400 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 425 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 450 mg. In some embodiments, the daily dose of azenosertib is about 475 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 500 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 550 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 600 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 625 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 650 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 675 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 700 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 725 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 750 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 775 mg. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is about 800 mg or its equivalent.

[0158] In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is administered once daily.

[0159] In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is divided into two doses per day.

[0160] In some embodiments, each dosing week includes at least 4, 5, or 6 consecutive dosing days.

[0161] In some embodiments, each dosing week includes 5 consecutive dosing days and 2 dosing-free days.

[0162] In some embodiments, each dosing week includes 4 consecutive dosing days and 3 dosing-free days.

[0163] In some embodiments, each dosing week includes 3 consecutive dosing days and 4 dosing-free days.

[0164] In some embodiments, each dosing week includes 7 consecutive dosing days and 7 dosing-free days.

[0165] In some embodiments, each intermittent administration cycle comprises about 7 to about 10 consecutive administration days. In some embodiments, each intermittent administration cycle comprises about 8 consecutive administration days. In some embodiments, each intermittent administration cycle comprises about 9 consecutive administration days. In some embodiments, each intermittent administration cycle comprises about 10 consecutive administration days.

[0166] In some embodiments, the intermittent dosing cycle comprises 21 consecutive dosing days and 7 dosing-free days.

[0167] In some embodiments, the intermittent dosing cycle comprises two consecutive dosing weeks.

[0168] In some aspects, provided herein are methods of treating cancer, comprising administering to a subject in need thereof a daily dose of 350 mg or more of azenosertib, or a pharmaceutically acceptable salt thereof, according to an intermittent dosing cycle, wherein the intermittent dosing cycle includes at least two consecutive days of dosing and at least one day of no dosing.

[0169] In some embodiments, the intermittent administration cycle comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive administration days. In some embodiments, the intermittent administration cycle comprises more than 14 consecutive administration days. In some embodiments, the intermittent administration cycle comprises 21 consecutive administration days. In some embodiments, the intermittent administration cycle comprises 28 consecutive administration days. In some embodiments, the intermittent administration cycle comprises 32 consecutive administration days. In some embodiments, the intermittent administration cycle comprises 42 consecutive administration days.

[0170] In some embodiments, an intermittent administration cycle includes at least 1, 2, 3, 4, 5, 6, or 7 days without administration. In some embodiments, an intermittent administration cycle includes 1 day without administration. In some embodiments, an intermittent administration cycle includes about 2-7 days without administration. In some embodiments, an intermittent administration cycle includes 2 days without administration. In some embodiments, an intermittent administration cycle includes 3 days without administration. In some embodiments, an intermittent administration cycle includes 4 days without administration. In some embodiments, an intermittent administration cycle includes 5 days without administration. In some embodiments, an intermittent administration cycle includes 6 days without administration. In some embodiments, an intermittent administration cycle includes 7 days of administration.

[0171] In some embodiments, an intermittent administration cycle comprises about 2-7 consecutive administration days ("on" days) followed by about 1-7 rest periods ("off" days).

[0172] In some embodiments, the intermittent dosing cycle includes 5 consecutive dosing days and 2 dosing-free days.

[0173] In some embodiments, the intermittent dosing cycle includes 4 consecutive dosing days and 3 dosing-free days.

[0174] In some embodiments, the intermittent dosing cycle includes 3 consecutive days of dosing and 4 days without dosing.

[0175] In some embodiments, the intermittent dosing cycle includes six consecutive dosing days and one dosing-free day.

[0176] In some embodiments, an intermittent dosing cycle includes 7 consecutive dosing days and 7 days without dosing. nothing.

[0177] In some embodiments, the intermittent dosing cycle comprises 14 consecutive dosing days and 7 dosing-free days.

[0178] In some embodiments, the intermittent dosing cycle comprises 21 consecutive dosing days and 7 dosing-free days.

[0179] In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 550 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, or equivalents thereof or more. In some embodiments, the present disclosure provides for the administration of high-dose azenosertib or a pharmaceutically acceptable salt thereof, for example, the dose is 375 mg or more. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 400 mg once daily in an intermittent dosing regimen. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 450 mg once daily in an intermittent dosing regimen. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered in an intermittent dosing regimen at a dose of about 500 mg once daily. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered in an intermittent dosing regimen at a dose of about 550 mg once daily. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered in an intermittent dosing regimen at a dose of about 600 mg once daily. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered in an intermittent dosing regimen at a dose of more than about 600 mg once daily. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered in an intermittent dosing regimen at a dose of about 650 mg once daily. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 700 mg once daily in an intermittent dosing regimen. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 750 mg once daily in an intermittent dosing regimen. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 775 mg once daily in an intermittent dosing regimen. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered at a dose of about 800 mg once daily in an intermittent dosing regimen.

[0180] In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is administered once daily.

[0181] In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is divided equally twice daily.

[0182] In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is divided equally into three doses per day. In some embodiments, the daily dose of azenosertib or a pharmaceutically acceptable salt thereof is divided equally into four doses per day.

[0183] In some embodiments, the twice-daily dose of azenosertib or a pharmaceutically acceptable salt thereof is 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, or equivalents thereof or more.

[0184] In some aspects, provided herein is a method of administering to a subject in need thereof a daily dose of 400 mg or more of azenosertib, or a pharmaceutically acceptable salt thereof, according to an intermittent dosing cycle, wherein the intermittent dosing cycle includes 5 consecutive days of dosing and 2 consecutive days of no dosing. A method of treating cancer is provided, comprising administering,

[0185] In some aspects, provided herein are methods of treating cancer, comprising administering to a subject in need thereof a daily dose of 450 mg or more of azenosertib, or a pharmaceutically acceptable salt thereof, according to an intermittent dosing cycle, wherein the intermittent dosing cycle includes 5 consecutive days of dosing and 2 consecutive days of no dosing.

[0186] In some aspects, provided herein are methods of treating cancer, comprising administering to a subject in need thereof a daily dose of 400 mg or more of azenosertib, or a pharmaceutically acceptable salt thereof, according to an intermittent dosing cycle, wherein the intermittent dosing cycle comprises four consecutive dosing days and three dosing days with no dosing.

[0187] In some aspects, provided herein are methods of treating cancer, comprising administering to a subject in need thereof a daily dose of 450 mg or more of azenosertib, or a pharmaceutically acceptable salt thereof, according to an intermittent dosing cycle, wherein the intermittent dosing cycle comprises four consecutive dosing days and three dosing days with no dosing.

[0188] In some embodiments, the intermittent administration cycle is repeated.

[0189] In some embodiments, the method further comprises administering a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof during the intermittent administration cycle. Without wishing to be bound by any particular theory, administering azenosertib or a pharmaceutically acceptable salt thereof in combination with a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof may enable a subject resistant to treatment with the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof alone to respond, or may prevent or reduce drug-induced toxicity, and / or improve the efficacy of treatment compared to monotherapy. Combination therapy using intermittent administration cycles may provide additional benefits to administration, for example, by requiring a lower effective dose of the second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, and / or azenosertib or a pharmaceutically acceptable salt thereof.

[0190] In some embodiments, azenosertib, or a pharmaceutically acceptable salt thereof, is administered in combination with one or more second chemotherapeutic agents (including pharmaceutically acceptable salts thereof) in intermittent dosing cycles.

[0191] Type of cancer The methods of the present disclosure can be used to treat cancer.

[0192] In some embodiments, the cancer is glioblastoma (GBM), astrocytoma, meningioma, craniopharyngioma, medulloblastoma, other brain cancer, head and neck cancer, leukemia, AML (acute myeloid leukemia), CLL (chronic lymphocytic leukemia), ALL (acute lymphocytic leukemia), myelodysplastic syndrome (MDS), skin cancer, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, uterine cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hematologic malignancies, head cancer, hematologic malignancies, Kaposi's sarcoma, kidney cancer, pharynx and hypopharyngeal cancer, liver cancer, lung cancer, non-small cell lung cancer (NSCLC), small cell lymphoma, mesothelioma, melanoma, multiple myeloma. Neuroblastoma, nasopharyngeal cancer, cervical cancer, ovarian cancer, osteosarcoma, sarcoma, gastrointestinal stromal tumor (GIST), pancreatic cancer, pituitary cancer, prostate cancer, kidney cancer, retinoblastoma, salivary gland cancer, skin cancer, stomach cancer, small intestine cancer, sarcoma, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous adenocarcinoma (USC), uterine CS, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, solid tumors, or liquid tumors, HGSOC, invasive breast cancer, triploidy Trans-negative breast cancer (TNBC), esophagogastric cancer, gastric cancer, esophageal cancer, pRCC, ccRCC, chromophobe RCC, head and neck cancer, adenoid cystic carcinoma (ACC), diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), cholangiocarcinoma, acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), ALL (acute lymphocytic leukemia), myelodysplastic syndrome (MDS), thymoma, BRAF-mutated metastatic colorectal cancer, uveal melanoma, high-grade serous ovarian, fallopian tube, or primary peritoneal cancer, BRAF V600E-mutated colorectal cancer, platinum-sensitive ovarian cancer, poly(ADP-ribose) polymerase inhibitor (PARPi)-resistant ovarian cancer, platinum-resistant ovarian cancer, platinum-refractory ovarian cancer, advanced pancreatic ductal adenocarcinoma, pancreatic ductal adenocarcinoma, neuroendocrine tumors, neuroendocrine prostate cancer, pancreatic neuroendocrine tumors, small cell lung cancer (SCLC), germ cell cancer, and stromal cancer.

[0193] In some embodiments, the subject has cancer, hi some embodiments, the cancer is breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, or ovarian cancer.

[0194] In some embodiments, the cancer is glioblastoma, astrocytoma, meningioma, craniopharyngioma, medulloblastoma, and other brain cancers, leukemia, skin cancer, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colorectal cancer, uterine cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, Hodgkin's lymphoma, blood tumors, hematological malignancies, Kaposi's sarcoma, kidney cancer, pharyngeal and hypopharyngeal cancer, liver cancer, lung cancer, lymphoma, mesothelioma, melanoma, multiple myeloma. Neuroblastoma, nasopharyngeal cancer, ovarian cancer, osteosarcoma, pancreatic cancer, pituitary cancer, retinoblastoma, salivary gland cancer, gastric cancer, small intestine cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous adenocarcinoma (USC), vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, solid tumor, or liquid tumor.

[0195] In some embodiments, the cancer is a solid tumor or a hematological malignancy. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from endometrial cancer, gallbladder cancer, ovarian cancer, HGSOC, endometrial cancer, melanoma, colorectal cancer, bladder cancer, breast cancer, invasive breast cancer, triple-negative breast cancer (TNBC), prostate cancer, lung cancer, NSCLC, SCLC, esophagogastric cancer, gastric cancer, esophageal cancer, renal cancer, pRCC, ccRCC, chromophobe RCC, head and neck cancer, osteosarcoma cancer, pancreatic cancer, brain cancer, uterine CS, uterine cancer, adenoid cystic carcinoma (ACC), mesothelioma, cervical cancer, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), liver cancer, glioblastoma (GBM), testicular cancer, low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), cholangiocarcinoma, thyroid cancer, thymoma, and uveal melanoma.

[0196] In some embodiments, the solid tumor is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer, germ cell cancer, or stromal cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, the epithelial ovarian cancer is high-grade serous ovarian cancer (HGSOC).

[0197] In some embodiments, cancer may be a "homologous recombination repair deficiency," which refers to a reduction or impairment of the homologous recombination process. In some embodiments, the cancer is associated with increased levels of cyclin E1 or a cyclin E1 biomarker (e.g., CCNE1 gene amplified cancer, cyclin E1 overexpressing / non-CCNE1 gene amplified cancer, cyclin E1 driven cancer).

[0198] In some embodiments, the cancer has a homologous recombination repair deficiency (HRD) positive status. In some embodiments, the cancer is an HRD positive cancer selected from ovarian cancer (including recurrent ovarian cancer), breast cancer (such as triple-negative breast cancer and / or metastatic breast cancer), prostate cancer (e.g., metastatic castration-resistant prostate cancer), fallopian tube cancer, and primary peritoneal cancer.

[0199] In some embodiments, the cancer is associated with an organ selected from adrenal gland, ampulla of Vater, biliary tract, bladder / urinary tract, bone, intestine, breast, cervix, CNS / brain, esophagus / stomach, eye, head and neck, kidney, liver, lung, lymphatic system, bone marrow, ovary / fallopian tube, pancreas, penis, peripheral nervous system, peritoneum, pleura, prostate, skin, soft tissue, testicle, thymus, thyroid, uterus, vulva / vagina, adenocarcinoma in situ, extragonadal germ cell tumor (EGCT), mixed adenocarcinoma, high-grade ovarian neuroendocrine carcinoma, high-grade serous fallopian tube carcinoma (HGSFT), ovarian choriocarcinoma, and ovarian cancer NOS (OCNOS).

[0200] In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is uterine cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is prostate cancer.

[0201] In some embodiments, the cancer is a primary cancer originating from a relevant organ, hi some embodiments, the cancer is a primary peritoneal cancer.

[0202] In some embodiments, the cancer has metastasized to related organs.

[0203] In some embodiments, the cancer is a solid tumor or a hematological malignancy.

[0204] In some embodiments, the cancer is a solid tumor.

[0205] In some embodiments, the solid tumor is selected from endometrial cancer, gallbladder cancer, ovarian cancer (e.g., HGSOC), endometrial cancer, melanoma, colorectal cancer, bladder cancer, breast cancer (e.g., invasive triple-negative breast cancer (TNBC)), prostate cancer, lung cancer (e.g., NSCLC, SCLC), esophagogastric cancer, gastric cancer, esophageal cancer, renal cancer (e.g., pRCC, ccRCC, chromophobe RCC), head and neck cancer, osteosarcoma cancer, pancreatic cancer, brain cancer, uterine CS, uterine cancer, adenoid cystic carcinoma (ACC), mesothelioma, cervical cancer, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), liver cancer, glioblastoma (GBM), testicular cancer, low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), cholangiocarcinoma, thyroid cancer, thymoma, and uveal melanoma.

[0206] In some embodiments, the cancer is acute myeloid leukemia (AML).

[0207] In some embodiments, the tumor is a neuroendocrine tumor, neuroendocrine prostate cancer, and pancreatic neuroendocrine tumor.

[0208] In some embodiments, the solid tumor is ovarian cancer.

[0209] In some embodiments, the ovarian cancer is epithelial ovarian cancer, germ cell cancer, or stromal cancer.

[0210] In some embodiments, the ovarian cancer is epithelial ovarian cancer.

[0211] In some embodiments, the ovarian cancer is high-grade serous ovarian cancer (HGSOC). In some embodiments, the ovarian cancer is platinum-resistant ovarian cancer (PROC). In embodiments, the ovarian cancer is a cyclin E-amplified ovarian cancer. In some embodiments, the ovarian cancer is a cyclin E1-overexpressing cancer. In some embodiments, the ovarian cancer is a cyclin E1-overexpressing / non-amplified cancer.

[0212] In some embodiments, the cancer is platinum-resistant. In some embodiments, the cancer is resistant to one or more chemotherapy regimens. In some embodiments, the cancer is refractory to one or more chemotherapy regimens.

[0213] In some embodiments, the cancer is uterine serous adenocarcinoma (USC).

[0214] In some embodiments, the cancer is osteosarcoma.

[0215] In some embodiments, the solid tumor is uterine serous adenocarcinoma, ovarian cancer, peritoneal cancer, fallopian tube cancer, osteosarcoma, pancreatic cancer, or BRAF-mutated metastatic colorectal cancer.

[0216] In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), chronic myelomonocytic leukemia (CMML), cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, or multiple myeloma (MM).

[0217] In some embodiments, the cancer is a platinum-refractory or platinum-resistant cancer. In some embodiments, the cancer is a platinum-resistant cancer. In some embodiments, the cancer is platinum-resistant. In some embodiments, the cancer is resistant to one or more chemotherapy regimens. In some embodiments, the cancer is refractory to one or more chemotherapy regimens.

[0218] Combination therapy The present disclosure provides methods of using azenosertib, or a pharmaceutically acceptable salt thereof, in combination with one or more additional agents (e.g., combination therapy with a chemotherapeutic agent). In one aspect, the present disclosure provides a method of treating cancer, comprising administering to a subject selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, and a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof.

[0219] Combination therapy refers to a clinical intervention in which a subject is exposed to two or more treatment regimens (e.g., azenosertib or a pharmaceutically acceptable salt thereof, and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof) simultaneously. In some embodiments, the two or more chemotherapy regimens may be administered simultaneously. In some embodiments, the two or more chemotherapy regimens may be administered sequentially (e.g., a first regimen is administered before any dose of a second regimen is administered). In some embodiments, the two or more chemotherapy regimens are administered in overlapping administration regimens.

[0220] In some embodiments, combination therapy does not necessarily require that the individual agents be administered together (or necessarily simultaneously) in a single composition. In some embodiments, two or more treatment regimens of the combination therapy (e.g., azenosertib or a pharmaceutically acceptable salt thereof, and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof) are administered to a subject separately, e.g., in separate compositions, via separate routes of administration (e.g., one agent administered orally and another agent administered intravenously), and / or at different times. In some embodiments, the two or more chemotherapeutic agents are administered in a combination composition or as a combination compound (e.g., a single chemical complex). or as part of a covalently linked entity), may be administered together via the same route of administration and / or simultaneously.

[0221] In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof and the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered simultaneously. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof and the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered sequentially. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered before the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof. In other embodiments, azenosertib or a pharmaceutically acceptable salt thereof is administered after the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof. In some embodiments, azenosertib or a pharmaceutically acceptable salt thereof and the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered intermittently.

[0222] In some embodiments, the second chemotherapeutic agent is selected from bendamustine, bortezomib, carfilzomib, ixazomib, busulfan, carboplatin, cisplatin, cyclophosphamide, cladribine, paclitaxel, docetaxel, pegylated liposomal doxorubicin (PLD), dexamethasone, doxorubicin, gemcitabine, cytarabine, fludarabine, fluorouracil (5-FU), irinotecan, topotecan temozolomide, triapine, azacitidine, 5-azacytidine, capecitabine, AraC-FdUMP

[10] (CF-10), cladribine, etoposide, decitabine, daunorubicin, doxorubicin, ifosfamide, methotrexate, vincristine, hydroxyurea, oxaliplatin, or a pharmaceutically acceptable salt of any of the foregoing.

[0223] In some embodiments, the cancer treatment is selected from the group consisting of alkylating agents, anti-EGFR antibodies, anti-Her-2 antibodies, antimetabolites, vinca alkaloids, platinum-based agents, anthracyclines, topoisomerase inhibitors, taxanes, antibiotics, immunomodulators, immune cell antibodies, interferons, interleukins, HSP90 inhibitors, antiandrogens, antiestrogens, antihypercalcemic agents, apoptosis inducers, aurora kinase inhibitors, Bruton's tyrosine kinase inhibitors, calcineurin inhibitors, CaM kinase II inhibitors, CD45 tyrosine phosphatase inhibitors, CDC25 phosphatase inhibitors, CHK kinase inhibitors, cyclooxygenase inhibitors, bRAF kinase inhibitors, cRAF kinase inhibitors, Ras inhibitors, cyclin-dependent kinase inhibitors, cysteine ​​protease inhibitors, DNA intercalators, DNA strand breakers, E3 ligase inhibitors, EGF pathway inhibitors, farnesyltransferase inhibitors, Flk-1 kinase inhibitors, glycogen synthase kinase-3 (GSK3) inhibitors, histone deacetylase (HDAC) inhibitors, I-kappa B-alpha kinase inhibitors, imidazotetrazinone, insulin tyrosine kinase inhibitors, c-Jun N-terminal kinase (JNK) inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, MDM2 inhibitors, MEK inhibitors, ERK inhibitors, MMP inhibitors, mTor inhibitors, NGFR tyrosine kinase inhibitors, p38 MAP kinase inhibitors, p56 tyrosine kinase inhibitors, PDGF pathway inhibitors, phosphatidylinositol 3-kinase inhibitors, phosphatase inhibitors, protein phosphatase inhibitors, PKC inhibitors, PKC delta kinase inhibitors, polyamine synthesis inhibitors, PTP1B inhibitors, protein tyrosine kinase inhibitors, SRC family tyrosine kinase inhibitors, Syk tyrosine kinase inhibitors, Janus (JAK-2 and / or JAK-3) tyrosine kinase inhibitors, retinoids, RNA polymerase II elongation inhibitors, serine / threonine kinase inhibitors, sterol biosynthesis inhibitors, VEGF pathway inhibitors, chemotherapeutic agents, aryletinone, altretamine, aminopterin, aminolevulinic acid, amsacrineAsparaginase, atrasentan, bexarotene, carboquone, demecolcine, efaproxiral, elsamitrucin, etoglucide, hydroxycarbamide, leucovorin, lonidamine, lucantone, masoprocol, methyl aminolevulinate, mitoguazone, mitotane, oblimersen The agent is selected from the group consisting of omacetaxine, pegaspargase, porfimer sodium, prednimustine, citimagine seradenovec, talaporfin, temoporfin, trabectedin, and verteporfin.

[0224] In some embodiments, the chemotherapeutic agent is carboplatin, cisplatin, paclitaxel, docetaxel, pegylated liposomal doxorubicin, doxorubicin, gemcitabine, cytarabine, fludarabine, fluorouracil (5-FU), irinotecan, topotecan, temozolomide, triapine, 5-azacytidine, capecitabine, AraC-FdUMP

[10] (CF-10), cladribine, decitabine, hydroxyurea, and oxaliplatin, or a pharmaceutically acceptable salt of any of the foregoing. In other embodiments, the chemotherapeutic agent is azacitidine, bendamustine, bortezomib, carfilzomib, ixazomib, busulfan, carboplatin, cytarabine, cyclophosphamide, cladribine, cisplatin, capecitabine, decitabine, dexamethasone, etoposide, fludarabine, gemcitabine, daunorubicin, doxorubicin, ifosfamide, methotrexate, and vincristine, or a pharmaceutically acceptable salt of any of the foregoing.

[0225] In some embodiments, the second chemotherapeutic agent is carboplatin, paclitaxel, gemcitabine, or pegylated liposomal doxorubicin (PLD), or a pharmaceutically acceptable salt of any of the foregoing.

[0226] In some embodiments, the second chemotherapeutic agent is encorafenib or a pharmaceutically acceptable salt thereof. In some embodiments, the second chemotherapeutic agent is cetuximab or a pharmaceutically acceptable salt thereof. In some embodiments, the second chemotherapeutic agent consists of a combination of encorafenib and cetuximab, or a pharmaceutically acceptable salt of either of the foregoing.

[0227] Administration of a second chemotherapy agent In some embodiments, the second chemotherapeutic agent is carboplatin, paclitaxel, gemcitabine, or pegylated liposomal doxorubicin (PLD), or a pharmaceutically acceptable salt of any of the foregoing.

[0228] In some embodiments, the second chemotherapeutic agent is carboplatin or a pharmaceutically acceptable salt thereof, wherein the carboplatin is administered intravenously at a dose ranging from 1 to 10 mg / mL*min over 15 minutes during a treatment cycle. In some embodiments, the second chemotherapeutic agent is carboplatin or a pharmaceutically acceptable salt thereof, wherein the carboplatin or a pharmaceutically acceptable salt thereof is administered intravenously at a dose ranging from 3 to 6 mg / mL*min over 15 minutes during a treatment cycle. In some embodiments, the second chemotherapeutic agent is carboplatin or a pharmaceutically acceptable salt thereof, and the carboplatin or a pharmaceutically acceptable salt thereof is administered intravenously over 15 minutes or more once during a treatment cycle at a dose ranging from 1-10 mg / mL*min, 2-10 mg / mL*min, 3-10 mg / mL*min, 4-10 mg / mL*min, 5-10 mg / mL*min, 6-10 mg / mL*min, 7-10 mg / mL*min, 8-10 mg / mL*min, 9-10 mg / mL*min, 2-8 mg / mL*min, 2-7 mg / mL*min, 3-7 mg / mL*min, 4-7 mg / mL*min, 5-7 mg / mL*min, 4-6 mg / mL*min, 2-6 mg / mL*min, 3-8 mg / mL*min, 9-10 mg / mL*min.

[0229] In some embodiments, the second chemotherapeutic agent is PLD or a pharmaceutically acceptable salt thereof, and the PLD or a pharmaceutically acceptable salt thereof is administered at a dose of 10-100 mg / m over 60 minutes once during a treatment cycle. 2 In some embodiments, the second chemotherapeutic agent is PLD or a pharmaceutically acceptable salt thereof, and the second chemotherapeutic agent is administered intravenously at a dose ranging from 0.1 to 1.0 mg / kg. Clinically acceptable salts are administered at a dose of 5 to 50 mg / m over 60 minutes once during a treatment cycle. 2 In some embodiments, the second chemotherapeutic agent is PLD or a pharmaceutically acceptable salt thereof, wherein the PLD or a pharmaceutically acceptable salt thereof is administered intravenously at a dose ranging from 10 to 40 mg / m over 60 minutes once during the treatment cycle. 2 It is administered intravenously in doses ranging from 100mg to 100mg.

[0230] In some embodiments, the second chemotherapeutic agent is PLD or a pharmaceutically acceptable salt thereof, and the PLD or a pharmaceutically acceptable salt thereof is administered at a dose of 10-100 mg / m over 60 minutes once during a treatment cycle. 2 , 10-90 mg / m 2 , 10-80 mg / m 2 , 10-70 mg / m 2 , 10-60 mg / m 2 , 10-50 mg / m 2 , 10-40 mg / m 2 , 10-30 mg / m 2 , 10-20 mg / m 2 , 20-90 mg / m 2 , 30-90 mg / m 2 , 40-90 mg / m 2 , 50-90 mg / m 2 , 60-90 mg / m 2 , 70-90 mg / m 2 , 20-80 mg / m 2 , 20-70 mg / m 2 , 20-60 mg / m 2 , 20-50 mg / m 2 , 20-40 mg / m 2 , or 30-40 mg / m 2 It is administered intravenously in doses ranging from 100mg to 100mg.

[0231] In some embodiments, the second chemotherapeutic agent is paclitaxel or a pharmaceutically acceptable salt thereof, and the paclitaxel or a pharmaceutically acceptable salt thereof is administered at a dose of 10-120 mg / m over 60 minutes (+10 minutes) three times during the treatment cycle. 2 In some embodiments, the second chemotherapeutic agent is paclitaxel or a pharmaceutically acceptable salt thereof, and the paclitaxel or a pharmaceutically acceptable salt thereof is administered intravenously at a dose ranging from 10 to 100 mg / m for up to 3 hours, three times during the treatment cycle. 2 , 20-100 mg / m 2 , 30-100mg / m 2 , 40-100mg / m 2 , 50-100mg / m 2 , 60-100mg / m 2 , 70-100mg / m 2 , 80-100 mg / m 2 , 90-100mg / m 2 , 10-90 mg / m 2 , 10-80 mg / m 2 , 10-70 mg / m 2 , 10-60 mg / m 2 , 10-50 mg / m 2 , 10-40 mg / m 2 , 10-30 mg / m 2 , 30-70 mg / m 2 , 40-70 mg / m 2 , 50-70 mg / m 2 , 60-70 mg / m 2 , 30-90 mg / m 2 , 30-80 mg / m 2 It is administered intravenously in doses ranging from 100mg to 100mg.

[0232] In some embodiments, the second chemotherapeutic agent is paclitaxel or a pharmaceutically acceptable salt thereof, and the paclitaxel or a pharmaceutically acceptable salt thereof is administered at a dose of 40-100 mg / m for up to 3 hours, up to 3 times during a treatment cycle. 2 It is administered intravenously in doses ranging from 100mg to 100mg.

[0233] In some embodiments, the second chemotherapeutic agent is gemcitabine or a pharmaceutically acceptable salt thereof, and the gemcitabine or a pharmaceutically acceptable salt thereof is administered at a dose of 500 to 1500 mg / m for more than 15 minutes once during a treatment cycle. 2 It is administered intravenously in doses ranging from 100mg to 100mg.

[0234] In some embodiments, the second chemotherapeutic agent is gemcitabine or a pharmaceutically acceptable salt thereof, and the gemcitabine or a pharmaceutically acceptable salt thereof is administered at a dose of 100-1000 mg / m over 15 minutes or more up to three times during a treatment cycle. 2 , 100-1000mg / m 2 , 100-900 mg / m 2 , 100-800 mg / m 2 , 100-700 mg / m 2 , 100-600 mg / m 2 , 100-500 mg / m 2 , 100-400 mg / m 2 , 100-300 mg / m 2 , 100-200 mg / m 2 , 200-1000mg / m 2 , 300-1000mg / m 2 , 400-1000mg / m 2 , 500-1000mg / m 2 , 600-1000mg / m 2 , 700-1000mg / m 2 , 800-1000mg / m 2 , 200-800 mg / m 2 , 200-700 mg / m 2 , 200-600 mg / m 2 , 200-500mg / m 2 , 300-900 mg / m 2 , 300-800 mg / m 2 , 400-700 mg / m 2 , 500-700mg / m 2 , 500-800mg / m 2 , 600-900 mg / m 2 It is administered intravenously in doses ranging from 100mg to 100mg.

[0235] In some embodiments, the second chemotherapeutic agent is gemcitabine or a pharmaceutically acceptable salt thereof, and the gemcitabine or a pharmaceutically acceptable salt thereof is administered at a dose of 100-1000 mg / m over 15 minutes or more up to three times during a treatment cycle. 2 It is administered intravenously in doses ranging from 100mg to 100mg.

[0236] Responsiveness In some embodiments, the treatment methods described herein result in a response rate of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or greater. In some embodiments, the response rate is measured by complete response (CR), partial response (PR), CA-125 50% response, or a combination thereof. In some embodiments, the response is determined based on progression-free survival. In some embodiments, the response is determined based on tumor response. In some embodiments, the response is determined based on clinical benefit rate (CBR). In some embodiments, the response is determined based on disease control rate (DCR). In some embodiments, the response is determined based on overall survival (OS).

[0237] Progression-free survival (PFS) refers to the period that the subject with disease (for example, cancer) lives without significant deterioration of the condition.Progression-free survival can be evaluated as the period that tumor growth does not progress and / or the period that the subject's disease state is not determined to be progressive disease.In embodiments, the progression-free survival of the subject with cancer is evaluated by evaluating tumor size, tumor number and / or metastasis.

[0238] In some embodiments, the treatment results in a progression-free survival (PFS) of 5, 6, 7, 8, 9, 10, 11, 12 months or more. In some embodiments, the treatment results in a progression-free survival (PFS) of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more. In some embodiments, the treatment results in a median progression-free survival (mPFS) of 1 year, 1.5 years, 2 years, 2.5 years or more.

[0239] As used herein, the term "progression" of tumor growth or "progressive disease" (PD), as used herein in relation to a cancer condition, refers to an increase in the sum of the diameters of the target tumor. Progression for the purposes of determining progression-free survival is defined as 1) tumor assessment by CT / MRI clearly demonstrating progressive disease according to RECIST 1.1 criteria, or 2) additional diagnostic testing (e.g., histology / cytology, ultrasound techniques, endoscopy, positron emission tomography) identifies a new tumor or an existing tumor progresses beyond the threshold of the Gynecologic Cancer PD may also be determined when at least one of the following criteria is met: definite disease progression and / or CA-125 eligibility by the Global Cancer Institute of Cancer Intergroup (GCIG) criteria (see Rustin et al., Int J Gynecol Cancer 2011;21:419-423, incorporated herein in its entirety); or 3) definite clinical signs and symptoms of PD unrelated to non-malignant or iatrogenic causes ([i] refractory cancer-related pain, [ii] worsening malignant bowel obstruction / functional disability, or [iii] definite symptomatic worsening of ascites or pleural effusion) and / or CA-125 progression by GCIG criteria.

[0240] As used herein, the term "partial response" or "PR" refers to a reduction in tumor progression in a subject, as indicated by a reduction in the sum of diameters of the target tumor relative to the baseline sum of diameters. In some embodiments, PR refers to a reduction of at least 30% in the sum of diameters relative to the baseline sum of diameters. Exemplary methods for assessing partial response are specified by the RECIST guidelines. EA Eisenhauer, et al. al., “New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1.),” Eur. J. of Cancer, 45: See 228-247 (2009).

[0241] As used herein, "stable" or "stable disease" (SD) tumor growth refers to neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD. In embodiments, stable refers to a change (increase or decrease) of less than 30%, 25%, 20%, 15%, 10%, or 5% in the sum of the diameters of the target tumor relative to the baseline sum of the diameters. Exemplary methods for evaluating stable tumor growth or stable disease are specified by the RECIST guidelines. See EA Eisenhauer, et al., "New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1)," Eur. J. of Cancer, 45: 228-247 (2009).

[0242] As used herein, the term "complete response" or "CR" refers to the disappearance of all or substantially all target lesions. In several embodiments, CR refers to a reduction of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% in the sum of the diameters of the target tumor (i.e., tumor loss) based on the sum of the diameters at baseline. In embodiments, CR indicates that less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the sum of the diameters of the lesions remains after treatment. Exemplary methods for assessing complete response are specified by the RECIST guidelines. See EA Eisenhauer, et al., "New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1)," Eur. J. of Cancer, 45: 228-247 (2009). [Example]

[0243] Additional embodiments are disclosed in more detail in the following examples, which are not intended to limit the scope of the claims in any way.

[0244] Example 1. Cancer cell lines with cyclin E1 overexpression are sensitive to azenosertib Overexpression of cyclin E1 (encoded by the CCNE1 gene) protein is associated with increased sensitivity to the WEE1 inhibitor azenosertib in ovarian cancer cell lines. As shown in Figure 1A, azenosertib sensitivity correlates with cyclin E1 protein expression in OV90, Kuramochi, TYK-nu, and OVCAR3 cells. After 96 hours of culture, cells were assessed for protein expression and azenosertib sensitivity by CellTiter Glo.

[0245] To determine whether cyclin E1 expression levels were responsible for the increased sensitivity to azenosertib, OV90, Kuramochi, and COV362 cells (which express low levels of cyclin E1 endogenously) were transduced with a lentiviral vector carrying the CCNE1 gene or with an empty vector control. Stable OV90, Kuramochi, and COV362 cell lines were established by puromycin selection. Cyclin E1 overexpression in the stable OV90 cell line compared with the empty vector control cells was confirmed by Western blotting (Figure 1C). As shown in Figure 1B, overexpression increased sensitivity to azenosertib. Azenosertib expression of cyclin E1 was also observed. The sensitivity of HGSOC cell lines to azenosertib by overexpression of cyclin E1 was assessed by plotting the growth rate (GR) inhibition in Figure 1D. It was observed that cyclin E1 overexpression sensitized HGSOC cell lines to azenosertib by further reducing the growth rate and cell viability compared to the control empty vector cell line (Figure 1D). 50 Value and IC 50 Values ​​were determined by CellTiter-Glo (CTG) assay and GR calculator.

[0246] Similar assays were performed on other cell lines, such as JOM1, ES2, TYK-nu, CAOV3, OAW28, OVCAR4, and OVCAR3. OVCAR3 (CCNE1 amplified, CN=12), OAW28, and OVCAR4 cells express high levels of cyclin E1, as determined by Western blot (WB) and immunohistochemistry (IHC). Cyclin E1 expression was normalized to vinculin. Figure 1E shows the overexpression status of cyclin E1 protein by Western blot. These values ​​are plotted using the H-score as a function of cyclin E1 levels (Figure 1F). Figure 1G shows a graph of GR values ​​as a function of cyclin E1 levels.

[0247] HGSOC cells were evaluated for growth rate inhibition (GR) in the presence of azenosertib (compared to untreated controls). GR values ​​are independent of cell division rate and were determined as described by Hafner et al. (Curr. Protoc. Chem. Biol. 9, 96-116 (2017)). HGSOC cells with high cyclin E1 protein expression were most sensitive to azenosertib. Low or absent cyclin E1-expressing cells indicate decreased sensitivity or resistance to azenosertib (Figure 2). Inhibitory effects were observed in OV90 and Kuramochi cells, cytotoxic effects in ES2 and TYK-nu cells, and cytotoxic effects in azenosertib-treated COV362, JHOMI, OAW28, OVCAR3, CAOV3, and OVCAR4 cells. Ovarian cancer cell lines overexpressing cyclin E1 protein (cyclin E1-high) were more sensitive to azenosertib than ovarian cancer cell lines with low levels of cyclin E1 protein (cyclin E1-low), inducing significant cytotoxic effects (GR max < -0.5). In cyclin E1-low ovarian cancer cell lines, lentiviral overexpression of cyclin E1 increased sensitivity to azenosertib.

[0248] Taken together, these data suggest that high cyclin E1 protein expression is associated with sensitivity to azenosertib and that artificial overexpression of cyclin E1 in cell lines with low endogenous cyclin E1 expression sensitizes cells to azenosertib.

[0249] Example 2. CDK2-dependent sensitivity of cyclin E1-high HGSOC to azenosertib The effect of CDK2 on the sensitization of cyclin E1-high HGSOC to azenosertib was assessed by knocking down CDK2 protein expression using two different siRNAs. CDK2 downregulation was assessed by Western blot at 3 and 6 days after transfection (Figure 3A). Three days after CDK2 siRNA transfection, OVCAR4 cells were treated with azenosertib for 3 days. Viability and proliferation rate values ​​were determined using the CellTiter-Glo assay and proliferation rate calculator. Figures 3B and 3C show the decrease in viability and proliferation rate after treatment with azenosertib following siRNA treatment. The sensitivity of cyclin E1-high HGSOC to azenosertib was observed to be CDK2-dependent.

[0250] These data suggest that overexpression of cyclin E1 protein acts as a biomarker of replication stress. We provide fundamental details on the mechanistic basis of cyclin E1 sensitization to Wee1 inhibition, including that it leads to the accumulation of cyclin E1 and that azenosertib sensitivity is mediated by CDK2 activity.

[0251] Example 3. Effect of azenosertib in a cyclin E1-high tumor mouse model with increased replication stress This example demonstrates that the greater antitumor efficacy of azenosertib in a cyclin E1-high tumor model is associated with increased replication stress.

[0252] Corresponding tumor-bearing NOD / SCID mice (SKOV3-cyclin E1-low and OVCAR3-cyclin E1-high) or BALB / c nude mice (HCC1806) were orally administered daily for the times indicated in Figures 5A and 8A. Treatment was well tolerated, and changes in tumor volume were plotted against administration time. Baseline cyclin E1 protein expression in each model was examined by IHC and is shown in Figure 4A. Target engagement of azenosertib (reduction of pCDK1) and γH2AX was examined by IHC in tumors 12 hours after azenosertib treatment in SKOV3-cyclin E1-low and OVCAR3-cyclin E1-high cells. The Y axis represents the sum of H scores assessed by three independent pathologists (Figures 4B and 4C). Replication stress markers pCHK1 and γH2AX were determined by OVCAR3 Western blot in tumor samples with or without azenosertib treatment over a 12-hour period ( Fig. 4D ).

[0253] Example 4. Mouse models bearing CCNE1-amplified cell line-derived xenografts (CDX) show increased sensitivity to azenosertib This example shows that treatment with azenosertib results in a reduction in tumor volume and an increase in CCNE1 gene amplification, which correlates with increased sensitivity in the CDX model.

[0254] Mice were treated with 95% viable tumor cells (1 × 10) in a McCoy's 5a and Matrigel mixture (CCNE 1:1 ratio) in 100 μL without serum for tumor development. 7 ) were inoculated into the right flank with SKOV3 cells (non-CCNE1 amplified, wild-type CCNE1 CN=2). Animals were randomized and treated daily with vehicle (20% HP-β-CD) or 80 mg / kg azenosertib for 21 days, resulting in a mean tumor volume of 215 mm. 3Tumor growth inhibition (TGI) was calculated using the following formula: TGI = (1 - (Td - T0) / (Cd - C0)) x 100%. Td and Cd are the mean tumor volumes of treated and control animals, and T0 and C0 are the mean tumor volumes of treated and control animals at the start of the experiment. Mice treated with azenosertib showed reduced tumor growth compared to vehicle-treated control animals (Figures 5A and 5B). Additional doses and schedules are included as summarized in Table 1 below and shown in Figures 5C and 5D. Daily treatment with 80 mg / kg azenosertib resulted in a TGI of 51.5% at day 28. (80mpk、28日目) Similarly, OVCAR8 (non-CCNE1 amplified) CDX models were treated with 80 mg / kg azenosertib daily for 39 days, resulting in a mean tumor growth inhibition of 52.8%. (80mpk、28日目) The mean tumor growth inhibition was 1.0% (Figures 6A and 6B). These results demonstrate that azenosertib moderately inhibits tumor growth in a non-CCNE1-amplified ovarian model.

[0255] [Table 1]

[0256] 1×10 6 HCC1806 human triple-negative breast cancer cells (CCNE1 amplified, CN=7) were inoculated into the right flank of 10 female BALB / c nude mice, 6–8 weeks old. Animals were randomized and tumors were grown until a mean tumor volume of 155 mm was achieved. 3 Treatment was initiated when tumor volume reached 100 μg / mL. Animals were treated daily with vehicle (20% HP-β-CD) or 80 mg / kg azenosertib, and tumor volume and body weight were measured twice weekly. Compared to vehicle-treated mice, azenosertib-treated mice showed a significant reduction (63.5%) in tumor growth after 28 days (Figures 7A and 7B).

[0257] Six- to eight-week-old female NOD / SCID mice were inoculated with OVCAR-3 cells (CCNE1 amplified, CN=14) into the right flank. Animals were randomized and tumors were grown to a mean tumor volume of 111 mm. 3Once tumor growth reached 100% (TGI), mice were treated daily with 80 mg / kg azenosertib vehicle (20% HP-β-CD) or 80 mg / kg azenosertib. Tumor volume and body weight were measured twice weekly. Azenosertib-treated mice showed a significant reduction in tumor growth by 88% after 28 days compared to vehicle controls (TGI). (80mpk、28日目) ) (Figures 8A and 8B). Decreased tumor growth was also observed in animals treated with 40 mg / kg azenosertib daily, 60 mg / kg azenosertib daily, and 80 mg / kg azenosertib daily (Figures 8C and 8D).

[0258] Taken together, these results indicate that CCNE1 gene amplification (e.g., high copy number) leads to increased efficacy of azenosertib treatment (e.g., improved tumor growth inhibition).

[0259] Example 5. CCNE1 gene amplification increases sensitivity to azenosertib in combination with a second chemotherapeutic agent Ovarian cancer cell lines were evaluated for sensitivity to azenosertib in combination with gemcitabine. As shown in Figure 9, 37% of the combination conditions tested were responsive to OVCAR3 (CCNE1 amplified, CN=14) compared to 16% and 21% in OV90 (non-CCNE1 amplified, CN=2) and OVCAR8 (non-CCNE1 amplified, CN=2), respectively. It was synergistic in this regard.

[0260] To evaluate the effect of azenosertib in combination with paclitaxel, mice were inoculated with A2780 (non-CCNE1-amplified, CN=2) (Figures 10A and 10B) or OVCAR3 (CCNE1-amplified, CN=14) (Figures 11A and 11B). When the mean tumor volume reached the designated size, animals were randomized and treated as summarized in Table 2. Mice treated with azenosertib and paclitaxel showed reduced tumor growth compared with vehicle-treated control animals. Tumor regression was observed in animals bearing CCNE1-amplified tumors, as shown in Figures 11A and 11B. Cyclin E1-high OVCAR3 model (46% reduction in initial tumor volume / 104% TGI) and cyclin E1-low A2780 model (85% TGI) (Table 2). These results indicate that CCNE1-amplified tumors are more sensitive to combination therapy (e.g., azenosertib and paclitaxel combination) compared to non-CCNE1-amplified tumors.

[0261] [Table 2]

[0262] Cyclin E1-high OVCAR3 cells demonstrated greater synergy in all chemotherapy and azenosertib combinations than cyclin E1-low OV90 and TYK-nu cells (Loewe synergy scores >10 indicate synergy, <-10 indicates antagonism) (Figure 11C). Drug combination effects were assessed by measuring cell viability and using four calculation methods (ZIP, Bliss, Loewe, and HSA) according to the SynergyFinder guidelines. The Loewe score was consistent with other methods for calculating synergy. For visualization purposes, scores were capped at 30. Each panel represents an overview of 3–6 replicates. Chemotherapy concentrations were rank-transformed, and the ranges were cell line and chemotherapy specific: oxaliplatin: 0–10 μM (OVCAR3, OV90), 0–3.3 μM (TYK-nu); paclitaxel: 0–0.005 μM (OVCAR3), 0–0.02 μM (OV90, TYK-nu); gemcitabine: 0~1μM (OVCAR3), 0~0.02μM (OV90), 0~0.01μM (TYK-nu).

[0263] Example 6. Clinical Trial to Treat Cancer in Subjects with CCNE1 Gene Amplification The efficacy of azenosertib in combination with a second chemotherapy agent was evaluated in human subjects with CCNE1 gene amplification through a clinical trial. Selected subjects had high-grade serous ovarian cancer (HGSOC), platinum-resistant or refractory disease, had received one or two prior chemotherapy regimens, and measurable disease. Azenosertib was administered in combination with PLD, carboplatin, paclitaxel, and gemcitabine.

[0264] Cyclin E1 expression levels were determined using immunohistochemistry (IHC) and summarized as an H score for each tumor sample from 63 subjects enrolled in the clinical study. Of the 63 subjects included in the IHC study, a total of 58 were evaluated for best overall response (BOR), including partial response (PR) (N = 17), stable disease (SD) (N = 34), progressive disease (PD) (N = 7), and non-evaluable disease (NE) (N = 5). These included subjects from all four arms of the study: carboplatin (N = 18), paclitaxel (N = 11), PLD (N = 28), and gemcitabine (N = 6). Each IHC microscopic image was reviewed by a board-certified pathologist and used to assess cyclin E1 expression levels. Epithelial cells from the tumor area were classified by cyclin E1 staining intensity (SI): absent (SI = 0), low (SI = 1+), moderate (SI = 2+), and high (SI = 3+). (Figure 12A) Summary H-scores were calculated from the proportions of cells in each class weighted by SI as follows: H score = f1+2*f2+3*f3 where fi is the fraction of cells with SI=i (i=1+, 2+, or 3+).

[0265] Best overall response was assessed using the (PR) criterion versus the (PD+SD+NE+uPR+) criterion. Subjects with a cyclin E1 H score greater than 130 were more likely to respond (ORR = 47% vs. 16%, p = 0.01 Fisher's exact test), and cyclin E1 H scores were higher in PR subjects compared with PD subjects (Figures 12B and 12C). Subjects were assessed based on the change in tumor size from baseline. As shown in Figure 13A, subjects with intermediate (70-130) and high (>130) cyclin E1 H scores demonstrated superior tumor responses compared with subjects with low (<70) cyclin E1 H scores. Subjects with the highest expression (H score >130) had greater tumor responses (-34% vs. -12%, p = 0.001 Wilcoxon test). Low-expressing subjects (H<70, N=10) had significantly shorter progression-free survival (PFS) (3.25 vs. 10.35 months, p=0.0027 log-rank test) (Figure 13B). The proportion of overall responders (Figure 13C) or CA125 responders (Figure 13D) decreased with increasing H-score. Groups were defined based on optimal association with tumor response (high group) and PFS (low group), using an H-score of 130 as the threshold associated with tumor response and an H-score of 70 associated with PFS (Figure 13E).

[0266] Example 7. Clinical trial to treat cancer in subjects with CCNE1 gene amplification. This example describes a clinical trial conducted to evaluate the safety and efficacy of treatment with azenosertib in combination with a second exemplary chemotherapeutic agent in subjects with metastatic high-grade epithelial ovarian, peritoneal, or fallopian tube cancer (EOC) after two or fewer lines of chemotherapy, including, in some embodiments, platinum chemotherapy.

[0267] Azenosertib was administered continuously or intermittently once daily in a 21-day or 28-day cycle with a second chemotherapeutic agent. In some embodiments, the second chemotherapeutic agent , pegylated liposomal doxorubicin, carboplatin, paclitaxel, and gemcitabine. This study was designed to evaluate safety and establish the maximum tolerated dose of each combination in addition to clinical activity. In one embodiment, azenosertib (azenosertib) was tested in combination with paclitaxel. Azenosertib was administered orally once daily for 28-day treatment cycles at two doses, beginning with 200 mg QD 5 days on / 2 days off (5 / 2), followed by an intermittent dosing regimen of 300 mg QD 5 / 2. Paclitaxel was administered at 80 mg / m on days D1, D8, and D15 of each 28-day cycle. 2 was administered intravenously over 60 minutes (±10 minutes).

[0268] In one embodiment, azenosertib (azenosertib) was tested in combination with carboplatin. Azenosertib was administered orally once daily for a 28-day treatment cycle, starting with two doses of 300 mg QD 5 / 2 followed by two doses of 200 mg QD 5 / 2 in an intermittent dosing regimen for four doses. Carboplatin was administered intravenously at 5 mg / mL*min over 15 minutes on day 1 of each 21-day cycle (±3 days).

[0269] In one embodiment, azenosertib (azenosertib) was tested in combination with gemcitabine. Azenosertib was administered orally once daily for a 28-day treatment cycle, starting with three doses of 200 mg QD 5 / 2, followed by four doses of 200 mg QD 5 / 2 in an intermittent dosing regimen. Gemcitabine was administered at 1000 mg / m over 30 minutes on days 1 and 8 of each 21-day cycle. 2 and 600 mg / m 2 It was administered intravenously in two doses.

[0270] In one embodiment, azenosertib (azenosertib) was tested in combination with pegylated liposomal doxorubicin (PLD). Azenosertib was administered orally once daily for 28-day treatment cycles at three doses, starting with 200 mg QD followed by 400 mg QD 5 / 2 in an intermittent dosing regimen. PLD was administered at one dose of 40 mg / m over 60 minutes every 4 weeks on day 1 of each 28-day cycle. 2 was administered intravenously.

[0271] The endpoints were to determine the recommended phase II dose (RP2D), safety, and preliminary clinical activity. From these clinical trials, the RP2D was determined to be (a) azenosertib 300 mg QD 5 / 2 in combination with paclitaxel 80 mg / m2 on D1, D8, and D15 (28-day cycle), (b) azenosertib 200 mg QD 5 / 2 in combination with carboplatin AUC 5 mg / mL* min on D1 (21-day cycle), (c) PLD 40 mg / m 2 The most effective treatment regimen was determined to be azenosertib 400 mg QD 5 / 2 (28-day cycle) in combination with gemcitabine D1. Azenosertib in combination with gemcitabine has sustained activity, and dose cohorts are ongoing to determine the maximum tolerated dose (MTD).

[0272] The overall response rate and median progression-free survival of the study are shown in Table 3 below. Overall response rate (ORR) refers to the proportion of subjects in the study whose tumor is significantly reduced or destroyed upon treatment. Median progression-free survival (mPFS) refers to the length of time from either the date of diagnosis or the start of treatment that half of the subjects diagnosed with the disease or tumor are still alive. This provides an indication of the success of the treatment.

[0273] [Table 3]

[0274] Of the 103 subjects enrolled, 26.6% had a partial response, and the median progression-free survival was 9.03 months (95% CI: 5.52-11.01). Results showed that azenosertib and paclitaxel had the highest overall response rate (ORR) (9 / 18 (50%)), followed by carboplatin (9 / 27 (33.3%)).

[0275] The overall response rate of azenosertib when administered in combination with pegylated liposomal doxorubicin or gemcitabine was 14.3% (5 / 35 and 2 / 14, respectively).

[0276] Among 80 subjects evaluated for cyclin E1 expression data by immunohistochemistry (IHC), higher cyclin E1 levels (using a threshold of H-score >50) correlated with a higher overall response rate (ORR = 31.3% vs. 7.7%) and longer progression-free survival (PFS = 10.35 months vs. 3.25 months, HR = 0.3). For example, a hazard ratio (HR) of 0.5 means that half of subjects in the treatment arm will experience an adverse event at any time point compared with placebo. The most frequent grade ≥3 treatment-emergent adverse events (TEAEs) observed (%) were neutropenia (44.4%), thrombocytopenia (30.3%), anemia (12.1%), leukopenia (11.1%), fatigue (10.1%), diarrhea (6.1%), nausea (5.1%), and vomiting (5.1%).

[0277] At separate time points, 115 subjects were enrolled in the study, 94 of whom were evaluable for efficacy, with a median progression-free survival (mPFS) of 9.0 months (95% CI: 5.8-13.7 months). Azenosertib in combination with paclitaxel demonstrated the highest confirmed ORR of 50% (mPFS 7.4 months), followed by gemcitabine at 38.5% (mPFS 10.4 months), carboplatin at 35.7% (mPFS 8.3 months), and PLD at 19.4% (mPFS 6.3 months). A total of 82 response-evaluable subjects had cyclin E1 expression data available by IHC. Cyclin E1-positive status (H-score >50) correlated with higher ORR and longer PFS (ORR = 40.0% vs. 8.3%, PFS = 9.86 vs. 3.25 months, HR = 0.37, P = 0.0078). Frequent grade ≥3 related TEAEs (%) in the intermittent azenosertib treatment group were thrombocytopenia (12.2%), neutropenia (11.3%), anemia (7.0%), fatigue (4.3%), nausea (1.7%), vomiting (1.7%), and diarrhea (0.9%).

[0278] Control samples were also evaluated for CCNE1 gene amplification. See Figures 14A-14C. An H score of >50 includes all CCNE1-amplified tumors. The H score was calculated by multiplying the percentage of cells (0-100%) by the intensity of cyclin E1 expression (0, 1, 2+, 3+).

[0279] Subjects were classified as cyclin E1-positive (H > 50) or cyclin E1-high (H > 135, the lowest score observed in samples with CCNE1 gene amplification). 90% (151 / 167) were cyclin E1-positive, 59% (99 / 167) were cyclin E1-high, 9% (13 / 141) of evaluable samples were CCNE1-amplified, and 85% (71 / 84) of cyclin E1-high evaluable samples were CCNE1-unamplified. Cyclin E1-positive expression is highly prevalent, even in subjects without CCNE1 gene amplification. CCNE1 transcript levels were assessed in subject samples and were highly correlated with cyclin E1 H scores (rho = 0.5, p < 0.001) (Figure 14D).

[0280] Cyclin E1 status, including CCNE1 gene amplification, predicts benefit from azenosertib in addition to chemotherapy, suggesting that azenosertib restores chemotherapy sensitivity in heavily pretreated platinum-resistant ovarian cancer.

[0281] These results demonstrated that administration of azenosertib in combination with chemotherapy was well tolerated, had clinical activity, and produced durable responses in subjects with platinum-resistant or refractory metastatic high-grade epithelial ovarian, peritoneal, or fallopian tube cancer. Subjects with cyclin E1-overexpressing tumors (cyclin E1-positive), a subgroup known to experience suboptimal benefit from chemotherapy, demonstrated significantly improved ORR and PFS compared with subjects with tumors with low cyclin E1 expression (cyclin E1-negative). Figure 14E shows an exemplary image of tumor cells designated by cyclin E1-positive status, while Figure 14F shows an exemplary image of tumor cells designated by cyclin E1-negative status.

[0282] Azenosertib in combination with chemotherapy demonstrated potent antitumor activity in heavily pretreated populations, with ORRs of 50% when combined with paclitaxel, 35.7% when combined with carboplatin, and 38.5% when combined with gemcitabine. Azenosertib confers higher objective response rates than previous chemotherapy alone or chemotherapy combined with other WEE1 inhibitors. The response to azenosertib combination therapy was increased regardless of the type of chemotherapy used. Subjects with cyclin E1-positive tumors (e.g., H-score >50) benefited from the chemotherapy combination arm, demonstrating synergy between azenosertib and chemotherapy in this subject population. As shown in Figures 14G-14J, subjects with an IHC H-score >50 or viable tumor cells with a cyclin E1 IHC staining intensity of 2+ in >10% of the tumor cells were resistant to chemotherapy. A proportion achieved a partial response after combination therapy with azenosertib. Figure 14K is a graph mapping the percentage of viable tumor cells with a staining intensity of 2+ to the H-score cutoff for cyclin E1 immunohistochemistry (IHC), showing that an H-score of >50 correlates with >10% of viable tumor cells with a staining intensity of 2+, and an H-score of >125 correlates with >30% of viable tumor cells with a staining intensity of 2+.

[0283] Azenosertib was well tolerated in combination with multiple chemotherapy regimens and showed promising clinical activity, including durable objective responses, in patients with platinum-resistant ovarian cancer. The addition of azenosertib increased the objective response rate (ORR) and median progression-free survival (mPFS) compared with those previously observed with chemotherapy alone or in combination with azenosertib. Particularly encouraging are the improvements in ORR and mPFS observed in patients with cyclin E1-positive tumors, a subgroup recognized as poor prognosis and refractory to chemotherapy. Furthermore, the tolerability and durable efficacy of azenosertib in combination with paclitaxel or carboplatin compare favorably with previous data from either paclitaxel-carboplatin or PLD-carboplatin chemotherapy doublets.

[0284] Example 8. Cyclin E1 positive status is independent of prior platinum therapy in high-grade serous ovarian cancer HGSOC tumor samples from 167 subjects were obtained from the ongoing azenosertib clinical trial (N=111) (NCT04516447), which is evaluating azenosertib in combination with chemotherapy in subjects with platinum-resistant ovarian, peritoneal, or fallopian tube cancer. Cyclin E1 protein expression levels were measured via immunohistochemistry using an anti-cyclin E1 mouse monoclonal antibody (Abcam Cyclin E1 / 2460). The H score was defined as the percentage (pc) or weighted sum of percentages stained with increasing intensity (1*pc1 + 2*pc2 + 3*pc3). Subjects were classified as cyclin E1-positive (H>50, the predictive threshold previously reported in this study) or cyclin E1-high (H>135, the lowest score observed in samples with CCNE1 gene amplification).

[0285] CCNE1 gene copy number (N = 141) and homologous recombination repair (HRR) gene mutation status (N = 86) were obtained from tissue-based genomic profiling. CCNE1 gene amplification status (amplified, unamplified, or nonamplified) was obtained from clinical assays or using at least six copies to call gene amplification (research assay). Transcript abundance (N = 49) was obtained using the Central Caris MI Profile assay and measured as transcripts per million reads (TPM). Clinical and pathological variables, including treatment modality and outcome, were primarily obtained from clinical data in this study and supplemented, when available, by data related to the procured specimen (e.g., age, collection method). There was no statistically significant association between the histological variables examined (anatomical location, collection site, collection method, tumor cellularity, received format, or tissue age in the IHC assay) and the cyclin E1 IHC H-score (data not shown). The distribution of cyclin E1 expression measured by IHC was independent of preanalytical variables.

[0286] A classification of platinum response was established from analysis of prior systemic therapy time course and response (FIG. 15A). Subjects whose sample collection met one of the following criteria were classified as platinum-sensitive at the time of collection (PS-aCT, N=63 / 107): ● Samples taken before platinum exposure • Specimens collected after the first platinum exposure but before subsequent platinum treatment in the subject, with a platinum-free interval (PFI) of at least 6 months after the last dose. ● In subjects with PFI at least 6 months after the last dose, Samples are taken during Chinna treatment In subjects with PFI at least 6 months after the last dose, specimens will be collected at interventional surgery between neoadjuvant and adjuvant platinum treatment.

[0287] All other evaluable cases were classified as platinum-resistant / refractory (PR-aCT, N=44 / 107) at the time of collection. As shown in Figure 15B, platinum exposure, response, or HRR mutation status did not significantly affect the cyclin E1 expression-based classification of subjects.

[0288] Example 9. Treatment of cancer in subjects selected to have cyclin E1 biomarker levels above a predetermined threshold As described above, CCNE1 gene amplification and / or cyclin E1 expression serve as markers for enriching a subject population for treatment with azenosertib. These data demonstrate that azenosertib induces cancer cell death in cyclin E1-high tumor cells in vitro and substantially inhibits the growth of cyclin E1-high subject-derived in vivo tumor models. Furthermore, these data support the use of CCNE1 gene copy number and / or cyclin E1 protein expression as predictive markers for significantly improving subject outcomes by enabling optimal subject selection for azenosertib treatment.

[0289] This example demonstrates that an effective dose of azenosertib (e.g., 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 450 mg, or 600 mg) is administered alone or in combination with a second chemotherapeutic agent to treat subjects selected for a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. The subjects are selected based on whether the target tumor tissue (1) has CCNE1 gene amplification (e.g., a copy number of 5 or more) or (2) has cyclin E1 overexpression (e.g., an mRNA or IHC H score of greater than 50, or a proportion of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 10%). Additional inclusion criteria may include subjects with specific cancer types (e.g., high-grade serous ovarian cancer (HGSOC)), platinum resistance or refractory, or 1-3 prior lines of therapy (e.g., bevacizumab).

[0290] Subjects are also selected based on having high-grade serous ovarian cancer, ECOG PS 0-1, platinum resistance (excluding platinum refractory), 1-3 prior lines of chemotherapy, measurable disease by RECIST v 1.1, cyclin E1 positivity (e.g., IHC+), and / or CCNE1 amplification.

[0291] This Phase III trial compares azenosertib in combination with either carboplatin or paclitaxel to conventional doublet chemotherapy in platinum-sensitive ovarian cancer. The Phase III trial focused on cyclin E1-positive ovarian cancer and was supported by promising Phase Ib clinical data. Subjects with cyclin E1-positive tumors have been shown to be refractory to chemotherapy alone and generally have a poor prognosis.

[0292] Subjects will receive 400 mg QD weekly for 5 days followed by 2 days without azenosertib (5:2). Subjects will be evaluated for ORR after treatment and divided into the following treatment groups:

[0293] Cohort 1 (N=30) subjects were confirmed Cyclin E1 positive (e.g., IHC+) and / or CCNE1 amplified

[0294] Cohort 2A (N=60) subjects had confirmed CCNE1 amplification.

[0295] Cohort 2B (N=80) subjects have non-CCNE1 amplified tumors and are cyclin E1 positive (IHC+).

[0296] Cohort 2C (N=40) subjects have non-CCNE1 amplified tumors and are cyclin E1-low (eg, IHC low or IHC-negative (IHC-)).

[0297] Additional subject groups will receive azenosertib and chemotherapy in recurrent platinum-sensitive ovarian cancer that is cyclin E1-positive. Eligibility criteria include confirmed high-grade serous ovarian cancer, ECOG performance status 0-1, ≥1L prior platinum-based chemotherapy line, platinum sensitivity (platinum-free interval ≥6 months), prior bevacizumab and PARPi if eligible and according to local standard of care, and cyclin E1 positivity (either CCNE1 amplification and / or cyclin E1 IHC+).

[0298] Subjects receiving combination therapy will be stratified based on stratification factors including prior lines of therapy (1 vs. 2-3), prior PARPi (yes vs. no), and CCNE1 amplification (yes vs. no). Subjects will be randomized into two groups. Group 1 will receive azenosertib plus chemotherapy (paclitaxel or carboplatin) for six cycles, followed by azenosertib maintenance at 400 mg QD 5:2. Group 2 will receive azenosertib plus carboplatin doublet (paclitaxel or pegylated liposomal doxorubicin) for six cycles without a subsequent azenosertib maintenance period. Exemplary dosing for the combination therapy groups is shown in Table 4 below.

[0299] [Table 4]

[0300] Subjects will be evaluated for primary and secondary endpoints, including progression-free survival and overall survival determined by blinded independent central review.

[0301] Example 10. Azenosertib demonstrates cancer responses in heavily pretreated subjects Azenosertib demonstrated efficacy in an exemplary human subject with CCNE1-amplified platinum-resistant ovarian cancer. The subject was selected to have CCNE1-amplified status (confirmed by the Foundation assay) and was administered an intermittent dosing schedule of at least 400 mg of azenosertib once daily for 11 months, with 5 consecutive days on treatment followed by 2 days off. The subject was a 73-year-old woman who had previously received 10 lines of therapy: (1) avelumab (SD), (2) doxorubicin liposomal (PD), (3) topotecan / bevacizumab (PD), (4) cyclophosphamide / bevacizumab (unk), (5) cyclophosphamide / bevacizumab (unk), (6) cyclophosphamide / bevacizumab (unk), (7) cyclophosphamide / bevacizumab (unk), (8) cyclophosphamide / bevacizumab (unk), (9) cyclophosphamide / bevacizumab (unk), (10) cyclophosphamide / bevacizumab (unk), (11) cyclophosphamide / bevacizumab (unk), (12) cyclophosphamide / bevacizumab (unk), (13) cyclophosphamide / bevacizumab (unk), (14) cyclophosphamide / bevacizumab (unk), (15) cyclophosphamide / bevacizumab (unk), (16) cyclophosphamide / bevacizumab (unk), (17) cyclophosphamide / bevacizumab (unk), (18) cyclophosphamide / bevacizumab (unk), (19 ...20) cyclophosphamide / bevacizuma 5)XMT1536(NaPi2b ADC)(PR),(6)APG115(MDM2 (inh) / pembrolizumab (SD), (7) ABBV-155 (CD275 ADC) (PD), (8) NC318 (Siglec-15 mAb) (SD), (9) SM08502 (CLK inhibitor) (PD), (10) NBMBMX (HDAC8 inh) (SD). After treatment with azenosertib, the subject demonstrated a sustained cPR of -71% and a visible reduction in target lesions (Figures 16A and 16B). This exemplary subject demonstrates further support for treating platinum-resistant cancer using azenosertib and CCNE1 gene amplification status as a biomarker.

[0302] Equivalence and Scope Furthermore, although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be understood by those skilled in the art that numerous and various modifications can be made without departing from the spirit of the present disclosure. It should therefore be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, but rather to cover all modifications and alternatives that come within the true scope and spirit of the present disclosure. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. 1. A method of treating cancer, comprising: A method comprising administering to a subject selected as having a predetermined cyclin E1 status an effective dose of azenosertib or a pharmaceutically acceptable salt thereof.

2. 1. A method of treating cancer, comprising: A method comprising administering to a subject selected as having a cyclin E1 biomarker level above a predetermined threshold an effective dose of azenosertib or a pharmaceutically acceptable salt thereof.

3. 1. A method of treating cancer, comprising: A method comprising administering to a subject selected as having a cyclin E1 biomarker level above a predetermined threshold an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, and a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof.

4. 2. The method of claim 1, wherein the predetermined cyclin E1 status is cyclin E1 positive, cyclin E1 positive (low), cyclin E1 positive (high), or cyclin E1-high.

5. The method of claim 4, wherein the predetermined cyclin E1 status is cyclin E1 positive.

6. The method of claim 4, wherein the predetermined cyclin E1 state is cyclin E1-high.

7. The method of any one of claims 1 to 6, wherein the cyclin E1 status or the cyclin E1 biomarker level is measured by the cyclin E1 protein expression level.

8. The method of claim 7, wherein the cyclin E1 protein expression level is determined by CCNE1 mRNA or transcript levels.

9. The method of claim 7, wherein the cyclin E1 protein expression level is determined by protein level.

10. The method of any one of claims 1 and 5 to 9, wherein the predetermined cyclin E1 status is a cyclin E1 protein expression level above a predetermined cutoff.

11. The method of any one of claims 1 and 5 to 10, wherein the predetermined cyclin E1 status is an immunohistochemistry (IHC) status.

12. 13. The method of any one of claims 2, 4, and 8-12, wherein the predetermined cutoff or predetermined threshold is measured by the percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 2+.

13. the predetermined cutoff or the predetermined threshold is 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 28%, 29%, 30%, 31%, 32%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 13. The method of claim 12, wherein the cyclin E1 IHC staining intensity is greater than 40%, 41%, 42%, 43%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, or 62%.

14. 14. The method of claim 13, wherein the predetermined cutoff or predetermined threshold is the percentage of viable tumor cells with a Cyclin E1 IHC staining intensity of IHC 2+ greater than 10%.

15. 14. The method of claim 13, wherein the predetermined cutoff or predetermined threshold is the percentage of viable tumor cells with a Cyclin E1 IHC staining intensity of IHC 2+ greater than 30%.

16. The method of any one of claims 2, 4, and 10-15, wherein the predetermined cutoff or predetermined threshold is measured by a cyclin E1 immunohistochemistry (IHC) H score.

17. 17. The method of claim 16, wherein the predetermined cutoff or predetermined threshold is a Cyclin E1 IHC H score of greater than 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160.

18. The predetermined cutoff or the predetermined threshold is greater than 50. The method of claim 17, wherein the H score is an H score.

19. 18. The method of claim 17, wherein the predetermined cutoff or predetermined threshold is a Cyclin E1 IHC H score of greater than 125.

20. The method of any one of claims 1 to 19, wherein the predetermined cyclin 1 status or the cyclin E1 biomarker level is independent of CCNE1 gene amplification in the subject.

21. The method of any one of claims 1 to 19, wherein the predetermined cyclin 1 status or cyclin E1 biomarker level is associated with CCNE1 gene amplification status in the subject.

22. 22. The method of claim 20 or claim 21, wherein the CCNE1 gene amplification or the CCNE1 gene amplification status is measured by CCNE1 gene copy number.

23. 23. The method of claim 22, wherein the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.

24. 24. The method of claim 23, wherein the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 7.

25. The method of any one of claims 1 to 24, wherein the subject is selected without determining the levels and status of other cancer genes.

26. 26. The method of claim 25, wherein the other cancer gene is selected from BRCA1, BRCA2, TP53, PKMYT1, and PPP2R1A.

27. 27. The method of any one of claims 1 to 26, wherein the cancer is a solid tumor or a hematological malignancy.

28. The method of any one of claims 1 to 27, wherein the cancer is a cyclin E1-driven cancer.

29. The cancer is glioblastoma (GBM), astrocytoma, meningioma, craniopharyngioma, medulloblastoma, other brain cancer, head and neck cancer, leukemia, AML (acute myeloid leukemia), CLL (chronic lymphocytic leukemia), ALL (acute lymphocytic leukemia), myelodysplastic syndrome (MDS), skin cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, uterine cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, Intestinal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hematologic tumors, head cancer, hematologic malignancies, Kaposi's sarcoma, kidney cancer, pharyngeal and hypopharyngeal cancer, liver cancer, lung cancer, non-small cell lung cancer (NSCLC), small cell, lymphoma, mesothelioma, melanoma, multiple myeloma, neuroblastoma, nasopharyngeal cancer, cervical cancer, ovarian cancer, osteosarcoma, sarcoma, gastrointestinal stromal tumor (GIST), pancreatic cancer, pituitary cancer, prostate cancer, renal cancer, retinoblastoma, salivary gland cancer, skin cancer, stomach cancer, small intestine Cancer, spleen cancer, sarcoma, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous adenocarcinoma (USC), uterine CS, vaginal cancer, vulvar cancer, Waldenström's macroglobulinemia, Wilms' tumor, solid tumor, or liquid tumor, HGSOC, invasive breast cancer, triple-negative breast cancer (TNBC), esophagogastric cancer, gastric cancer, esophageal cancer, pRCC, ccRCC, chromophobe RCC, head and neck cancer, adenoid cystic carcinoma (ACC), diffuse large intestine DLBCL, non-Hodgkin's lymphoma (NHL), low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), cholangiocarcinoma, acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), ALL (acute lymphocytic leukemia), myelodysplastic syndrome (MDS), thymoma, BRAF-mutated metastatic colorectal cancer, uveal melanoma, high-grade serous ovarian cancer, fallopian tube cancer, or primary peritoneal cancer, BRAF 29. The method of any one of claims 1 to 28, wherein the cancer is selected from V600E mutant colorectal cancer, platinum-sensitive ovarian cancer, poly(ADP-ribose) polymerase inhibitor (PARPi)-resistant ovarian cancer, platinum-resistant ovarian cancer, platinum-refractory ovarian cancer, advanced pancreatic ductal adenocarcinoma, pancreatic ductal adenocarcinoma, neuroendocrine tumors, neuroendocrine prostate cancer, pancreatic neuroendocrine tumors, small cell lung cancer (SCLC), germ cell cancer, and stromal cancer.

30. 30. The method of any one of claims 1 to 29, wherein the cancer is histologically or cytologically confirmed, or the cancer is pathologically confirmed.

31. The method of any one of claims 1 to 30, wherein the cancer is recurrent or persistent.

32. The method of any one of claims 1 to 31, wherein the cancer is metastatic.

33. The method of any one of claims 1 to 32, wherein the cancer is unresectable.

34. 34. The method of any one of claims 1-33, wherein the subject has received at least 1, 1, 2, 3, 4, 1 or 2, 1-2, 1-3, or 1-4 prior lines of therapy, a prior line of therapy in the advanced or metastatic setting, a previous line of chemotherapy, a previous line of platinum-based chemotherapy, a previous regimen, or a previous treatment regimen, but not more than 1 line of therapy.

35. 35. The method of any one of claims 1 to 34, wherein the cancer is platinum-resistant, platinum-sensitive, or platinum-refractory.

36. The method of any one of claims 1 to 35, wherein the cancer is resistant to a PARP inhibitor.

37. 37. The method of any one of claims 1, 2, and 4-36, comprising administering an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, in combination with a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof.

38. 37. The method of any one of claims 1 and 4-36, comprising administering an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof, in combination with a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof.

39. The second chemotherapeutic agent is selected from the group consisting of bendamustine, bortezomib, carfilzomib, ixazomib, busulfan, carboplatin, cisplatin, cyclophosphamide, cladribine, paclitaxel, docetaxel, pegylated liposomal doxorubicin (PLD), dexamethasone, doxorubicin, gemcitabine, cytarabine, fludarabine, fluorouracil (5-FU), irinotecan, topotecan, temozolomide, and triapine. , azacitidine, 5-azacytidine, capecitabine, AraC-FdUMP[10] (CF-10), cladribine, etoposide, decitabine, daunorubicin, doxorubicin, ifosfamide, methotrexate, vincristine, oxaliplatin hydroxyurea, niraparib, encorafenib, cetuximab, or a pharmaceutically acceptable salt of any of the foregoing.

40. 40. The method of claim 39, wherein the second chemotherapeutic agent is carboplatin, paclitaxel, gemcitabine, or pegylated liposomal doxorubicin (PLD), or a pharmaceutically acceptable salt of any of the foregoing.

41. The method of any one of claims 3 to 36 and 38 to 40, wherein azenosertib, or a pharmaceutically acceptable salt thereof, and the second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, are administered simultaneously.

42. The method of any one of claims 3 to 36 and 38 to 40, wherein azenosertib, or a pharmaceutically acceptable salt thereof, and the second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, are administered sequentially.

43. The method of any one of claims 3 to 36 and 38 to 42, wherein azenosertib or a pharmaceutically acceptable salt thereof, and / or the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof, is administered intermittently.

44. 44. The method of any one of claims 1 to 43, wherein the method comprises selecting said subjects having said predetermined cyclin E1 status or said cyclin E1 biomarker level above said predetermined threshold.

45. 45. The method of claim 44, wherein the method further comprises first determining the cyclin E1 status or the cyclin E1 biomarker level prior to the selecting step.

46. The method may be performed at 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 46. ​​The method of any one of claims 1 to 45, wherein the method results in an overall response rate (ORR) of 50% or greater in subjects.

47. 47. The method of claim 46, wherein the overall response rate is measured by complete response (CR), partial response (PR), CA-125 50% response, or a combination thereof.

48. 49. The method of claim 47 or claim 48, wherein the method results in a median progression-free survival (mPFS) of 5, 6, 7, 8, 9, 10, 11, 12 months or more in the subject.

49. 1. A method of treating ovarian cancer, comprising: administering, during a treatment cycle, to a subject selected to have a predetermined cyclin E1 status, an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof; and optionally administering a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof one or more times during said treatment cycle.

50. 1. A method of treating ovarian cancer, comprising: administering, during a treatment cycle, to subjects selected to have a cyclin E1 biomarker level above a predetermined threshold, an effective dose of azenosertib, or a pharmaceutically acceptable salt thereof; administering a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof one or more times during said treatment cycle.

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