WO2022136916A1

WO2022136916A1 - Wee1 inhibitors and methods for treating cancer

Info

Publication number: WO2022136916A1
Application number: PCT/IB2021/000891
Authority: WO
Inventors: Philippe PULTAR; Dimitrios VOLIOTIS; Anthony William Tolcher; Anthony Ya-Ning SUN; Fernando Donate; Petrus RUDOLF DE JONG; Ahmed Abdi SAMATAR; Kevin Duane BUNKER; Peter Qinhua HUANG
Original assignee: Recurium Ip Holdings, Llc
Priority date: 2020-12-22
Filing date: 2021-12-17
Publication date: 2022-06-30
Also published as: TW202242144A

Abstract

Disclosed herein is a method of determining subject sensitivity to a WEE1 inhibitor, comprising obtaining or having obtained a biological sample from the subject, and performing or having performed at least one assay on the biological sample to determine if the subject has an altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex. Also disclosed are methods of treating cancer with WEE1 inhibitors, and modifying the effective concentration based upon the sequence variation and function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex.

Description

WEE1 INHIBITORS AND METHODS FOR TREATING CANCER

INCORPORATION BY REFERENCE TO PRIORITY APPLICATION

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/129,452, filed December 22, 2020, which is hereby incorporated here by reference in its entirety.

BACKGROUND

Field

[0002] The present application relates generally to compounds that are WEE1 inhibitors and methods of using them to treat conditions characterized by excessive cellular proliferation, such as cancer. It also relates to methods of identifying mutations in subjects with cancer and the subsequent treatment of those subjects with a WEE1 inhibitor.

Description

[0003] DNA is constantly damaged from the environment. Light, chemicals, stress, and cellular replication lead to single- or double- stranded breakage along DNA’s backbone. Typically, organisms defend against DNA damage by repair proteins that either re-connect, or re-synthesize damaged DNA. The correct functioning of these proteins are essential for life. The incorrect replacement of nucleotides into DNA can cause mutations (and other genetic alterations including but not limited to insertions, deletions, and frameshifts), genetic disease, and loss of protein function. The altogether loss of DNA repair can cause cell death, tumor progression, and cancer.

[0004] Cell cycle checkpoints are important for proper DNA repair, ensuring that cells do not progress with cellular replication until their genomic integrity is restored. WEE1 is a nuclear kinase involved in the G2-M cell-cycle checkpoint arrest for DNA repair before mitotic entry. Normal cells repair damaged DNA during G1 arrest. Cancer cells often have a deficient Gl-S checkpoint and depend on a functional G2-M checkpoint for DNA repair. WEE1 is overexpressed in various cancer types.

SUMMARY [0005] Various embodiments provide a method of determining subject sensitivity to a WEE1 inhibitor, comprising: obtaining or having obtained a biological sample from the subject; and performing or having performed at least one assay on the biological sample to determine if the subject has an altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex.

[0006] Another embodiment provides a method of treating a cancer, comprising: obtaining or having obtained a biological sample from the subject; performing or having performed at least one assay on the biological sample to determine if the subject has altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and administering an effective amount of a cancer treatment to the subject based upon results of the assay.

[0007] Another embodiment provides a method of treating a cancer, comprising: identifying a subject having (a) the cancer and (b) endogenous or altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and administering an effective amount of a WEE1 inhibitor to the subject.

[0008] Another embodiment provides a method of treating cancer in a subject, comprising: determining whether the subject is sensitized to treatment with a WEE1 inhibitor, said determining comprising: obtaining or having obtained a biological sample from the subject; and performing or having performed at least one assay on the biological sample to determine if the subject has altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and selecting a treatment protocol for the subject on the basis of the determination of whether the subject is sensitized to treatment with the WEE1 inhibitor.

[0009] These and other embodiments are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS [0010] FIG. 1 depicts the quantitative analysis of CEA concentration (ng/mL) as a marker of tumor progression over time in a human subject with stage IV colorectal cancer (Patient 1) before and after administration of a WEE1 inhibitor.

[0011] FIG. 2 depicts a radiographical image of a cancerous mass in the liver of Patient 1. The outline in the baseline panel (left) indicates the size of the mass prior to administration of a WEE1 inhibitor, and the outline in the follow-up panel (right) indicates the size of the mass after administration.

[0012] FIG. 3 depicts a radiographical image of a cancerous mass in the lymph node of Patient 1. The outline in the baseline panel (left) indicates the size of the mass prior to administration of a WEE1 inhibitor, and the outline in the follow-up panel (right) indicates the size of the mass after administration.

[0013] FIG. 4 depicts the quantitative analysis of CA-125 concentration (units/mL) as a marker of tumor progression over time in a human subject with stage IV ovarian cancer (Patient 2) before and after administration of a WEE1 inhibitor.

[0014] FIG. 5 depicts a radiographical image of a cancerous pleural mass in Patient 2. The outline in the baseline panel (left) indicates the size of the mass prior to administration of a WEE1 inhibitor, and the outline in the follow-up panel (right) indicates the cancerous pleural mass is no longer detected after administration.

[0015] FIG. 6 depicts a radiographical image of a cancerous mass at the porta hepatis node in Patient 2. The outline in the baseline panel (left) indicates the size of the mass prior to administration of a WEE1 inhibitor, and the outline in the follow-up panel (right) indicates the size of the mass after administration.

[0016] FIG. 7 depicts a radiographical image of a cancerous mass in the peritoneal lesion of Patient 2. The outline in the baseline panel (left) indicates the size of the mass prior to administration of a WEE1 inhibitor, and the outline in the follow-up panel (right) indicates the size of the mass after administration.

[0017] FIG. 8 depicts Western blot analysis of an ovarian cancer cell line UWB 1.289 with and without treatment of siRNA mediated knockdown of NBN.

[0018] FIG. 9 depicts the percent survival of UWB 1.289 cells with and without treatment of siRNA mediated knockdown of NBN over increasing WEE1 inhibitor concentrations. Percent survival is calculated using cell titer glow analysis. [0019] FIG. 10 depicts the percent cell density of HCC1428 (breast cancer cell line), HS-578T (breast cancer cell line), OVCAR3 (ovarian cancer cell line) and UWB 1.289 (ovarian cancer cell line), with varying levels of altered DNA repair function of NBN in each cell line, over increasing WEE1 inhibitor concentrations. Cell density is calculated using cell titer glow analysis.

[0020] FIG. 11 depicts the adjusted cell density of OVCAR3 cells upon concurrent treatment with 0 pM or 30 pM Mirin over increasing WEE1 inhibitor concentrations. Cell density is calculated using cell titer glow analysis.

[0021] FIG. 12 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of WO2014167347.

[0022] FIG. 13 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of W02015019037.

[0023] FIG. 14 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of WO2015092431.

[0024] FIG. 15A describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of W02018011569.

[0025] FIG. 15B describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 2 of W02018011569.

[0026] FIG. 16 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of W02018011570 .

[0027] FIG. 17 describes a chemical structure representing a WEE1 inhibitor disclosed in claim 1 of WO2018162932.

[0028] FIG. 18 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of WO2019138227.

[0029] FIG. 19A describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of W02018090939.

[0030] FIG. 19B describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of US20190308984 (US National Phase of W02018090939).

[0031] FIG. 20A describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of W02019011228. [0032] FIG. 20B describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of US20200131192 (US National Phase of W02019011228).

[0033] FIG. 21 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of W02020210380.

[0034] FIG. 22 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of W02020210381.

[0035] FIG. 23 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of W02020210383.

[0036] FIG. 24 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of W02020210375.

[0037] FIG. 25 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of W02020210377.

[0038] FIG. 26 describes a generic chemical structure representing WEE1 inhibitors disclosed in claim 1 of WO2019173082.

[0039] FIG. 27 depicts Western blot analysis of an ovarian cancer cell line SKOV3 with and without treatment of sgRNA mediated knockdown of NBN.

[0040] FIG. 28 depicts the percent cell density of SKOV3 (ovarian cancer cell line) with varying levels of altered DNA repair function of NBN over increasing WEE1 inhibitor concentrations. Cell density is calculated using cell titer glow analysis.

[0041] FIG. 29 depicts Western blot analysis of an ovarian cancer cell line SKOV3 with and without treatment of sgRNA mediated knockdown of NBN.

[0042] FIG. 30 depicts the percent cell density of SKOV3 (ovarian cancer cell line) with varying levels of altered DNA repair function of NBN over increasing WEE1 inhibitor concentrations. Cell density is calculated using cell titer glow analysis.

[0043] FIG. 31 depicts Western blot analysis of an ovarian cancer cell line SKOV3 with sgRNA and siRNA-mediated knockout and/or knockdown, respectively.

[0044] FIG. 32 depicts the percent cell density of SKOV3 (ovarian cancer cell line) with varying levels of altered DNA repair function of NBN over increasing WEE1 inhibitor concentrations. Cell density is calculated using cell titer glow analysis. [0045] FIG. 33 depicts tumor volume of B ALB/c nude mice in a colorectal cancer LoVo xenograft model over number of days after treatment with varying WEE1 inhibitor concentrations.

[0046] FIG. 34 depicts tumor volume of BALB/c nude mice in an ovarian cancer TOV-21G xenograft model over number of days after treatment with varying WEE1 inhibitor concentrations.

[0047] FIG. 35 depicts tumor volume of BALB/c nude mice in an acute lymphoblastic leukemia MOLT-4 xenograft model over number of days after treatment with WEE1 inhibitor.

[0048] FIG. 36 depicts tumor volume of BALB/c nude mice in a breast cancer MCF-7 xenograft model over number of days after treatment with WEE1 inhibitor.

DETAILED DESCRIPTION

[0049] WEE1 is a tyrosine kinase that is a critical component of the ATR- mediated G2 cell cycle checkpoint control that prevents entry into mitosis in response to cellular DNA damage. ATR phosphorylates and activates CHK1, which in turn activates WEE1, leading to the selective phosphorylation of cyclin-dependent kinase 1 (CDK1) at Tyrl5, thereby stabilizing the CDKl-cyclin B complex and halting cell-cycle progression. This process confers a survival advantage by allowing tumor cells time to repair damaged DNA prior to entering mitosis. Inhibition of WEE1 abrogates the G2 checkpoint, promoting cancer cells with DNA damage to enter into premature mitosis and undergo cell death via mitotic catastrophe. Therefore, WEE1 inhibition has the potential to sensitize tumors to DNA-damaging agents, such as cisplatin, and to induce tumor cell death.

[0050] The MRN protein complex, consisting of MRE11, RAD50, and NBN (also called NBS1), binds to BRCA1, p53, and H2AX and recognizes, signals, and assists in repair of double- stranded DNA breaks (DSBs) via homologous recombination (HR) repair, among other mechanisms. Disruption of any component of the MRN complex therefore impacts the ability of a cell to restore DNA from damage. Pathogenic NBN, MRE11 and RAD50 mutations are known to be associated with homologous recombination repair (HRR) pathway deficiency (HRD). Deletion of NBN in mice is embryonically lethal. Mutations to NBN have been associated with an increased risk of development of various cancers, including breast cancer, ovarian cancer, colorectal cancer, and gastric cancer. Overall, pathogenic germline NBN mutations are associated with increased risk of various malignancies, though mutations are relatively rare (-0.2-1%).

Definitions

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0052] As used herein, the term “about” has its usual meaning as understood by those skilled in the art and thus indicates that a value includes the inherent variation of error for the method being employed to determine a value, or the variation that exists among multiple determinations.

[0053] As used herein, the terms "modify" or "alter", or any forms thereof, mean to modify, alter, replace, delete, substitute, remove, vary, or transform.

[0054] As used herein, the terms “function” and “functional” have their usual meaning as understood by those skilled in the art and thus refer to a biological, enzymatic, or therapeutic function.

[0055] As used herein, the term “endogenous” has its usual meaning as understood by those skilled in the art and thus refers to the native, or wild type property of a gene, protein, or cell. In some embodiments, the endogenous gene is the wild type sequence of said gene. In some embodiment, the endogenous protein is the wild type sequence of said protein. In some embodiments, the endogenous protein function is the wild type function and activity level of said protein. In some embodiments, the endogenous cell is the wild type cell.

[0056] As used herein, the term “zn vivo” has its usual meaning as understood by those skilled in the art and thus refers to the performance of a method inside living organisms, usually mammals, including humans or mice, or living cells which make up these living organisms, as opposed to a tissue extract or dead organism. [0057] As used herein, the term “zzz vitro” has its usual meaning as understood by those skilled in the art and thus refers to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.

[0058] The term “gene” as used herein has its usual meaning as understood by those skilled in the art and thus generally refers to a portion of a nucleic acid that encodes a protein or functional RNA; however, the term may optionally encompass regulatory sequences. It will be appreciated by those of ordinary skill in the art that the term “gene” may include gene regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences. It will further be appreciated that definitions of gene include references to nucleic acids that do not encode proteins but rather encode functional RNA molecules such as tRNAs and miRNAs. In some cases, the gene includes regulatory sequences involved in transcription, or message production or composition. In other embodiments, the gene comprises transcribed sequences that encode for a protein, polypeptide or peptide. In keeping with the terminology described herein, an “isolated gene” may comprise transcribed nucleic acid(s), regulatory sequences, coding sequences, or the like, isolated substantially away from other such sequences, such as other naturally occurring genes, regulatory sequences, polypeptide or peptide encoding sequences, etc. In this respect, the term “gene” is used for simplicity to refer to a nucleic acid comprising a nucleotide sequence that is transcribed, and the complement thereof. As will be understood by those in the art, this functional term “gene” includes both genomic sequences, RNA or cDNA sequences, or smaller engineered nucleic acid segments, including nucleic acid segments of a non-transcribed part of a gene, including but not limited to the non-transcribed promoter or enhancer regions of a gene. Smaller engineered gene nucleic acid segments may express or may be adapted to express using nucleic acid manipulation technology, proteins, polypeptides, domains, peptides, fusion proteins, mutants and/or such like.

[0059] The terms “nucleic acid” or “nucleic acid molecule” as used herein have their usual meaning as understood by those skilled in the art and thus refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.

[0060] As used herein, the term “codon” has its usual meaning as understood by those skilled in the art and refers to a sequence of three nucleotides, either RNA or DNA, that correspond to a particular amino acid or termination signal. Such codons can include, as nonlimiting examples, the 61 natural occurring codons, 3 stop codons, start codon, and synthetic codons corresponding to a non-standard amino acid.

[0061] The terms “peptide”, “polypeptide”, and “protein” used herein have their usual meaning as understood by those skilled in the art and thus refer to macromolecules comprised of amino acids linked by peptide bonds. The term refers to both short chains (i.e. peptides, oligopeptides and oligomers) and to longer chains. The numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. The term “downstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the C-terminus of a previous sequence. The term “upstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the N-terminus of a subsequent sequence. Proteins may contain amino acids other than the 20 gene encoded amino acids. Proteins include those modified by natural processes (e.g. processing and other post-translational modifications) and by chemical modification techniques. The same type of modification may be present in the same or varying degree at several sites in a given protein and a protein may contain many modifications. Modifications may occur in the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Examples of modifications include acetylation; acylation; ADP-ribosylation; amidation; covalent attachment of flavin, a heme moiety, a nucleotide or nucleotide derivative, a lipid or lipid derivative, a carbohydrate, or phospho tidy lino sitol; cross-linking; cyclization; disulfide bond formation; demethylation, formation of covalent cross-links; glycosylation; hydroxylation; iodination; methylation; myristoylation; oxidation; proteoytic processing; phosphorylation; S-nitrosation; racemization; lipid attachment; sulfation, gamma- carboxylation of glutamic acid residues; or hydroxylation.

[0062] The term “genotype” has its usual meaning as understood by those skilled in the art and refers to a sum of the alleles of the gene contained in an individual or a sample. In some embodiments, the genotype of a cell line, tissue, tumor, or cancer is different than the genotype of the original organisms from which those cells, tissue, tumor, or cancer originate.

[0063] The term “mutation” has its usual meaning as understood by those skilled in the art and refers to an alteration of genetic sequence. In some embodiments, cells have multiple mutations. In some embodiments, mutations are in coding regions of the genome. Mutations can range in size from a single nucleotide, to a large segment of the chromosome that includes multiple genes. In some embodiments, at least one mutation is silent, having no significant impact on gene expression or function. In some embodiments, at least one mutation has an impact on gene expression or function, such as gene amplification or enhanced copy number. In some embodiments, at least one mutation is silent, having no significant impact on protein expression or function. In some embodiments, at least one mutation has a small impact on protein expression or function. In some embodiments, at least one mutation has a moderate impact on protein expression or function. In some embodiments, at least one mutation has a large impact on protein expression or function. In some embodiments, at least one mutation prevents protein expression or function. Nonlimiting examples of mutations include insertions, deletions, truncations, substitutions, duplications, translocations, and inversions. In some embodiments, mutations are “somatic,” or occurring in body cells and are not inheritable. 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, mutations are “germline,” or occurring in germ cells and are inheritable.

[0064] As disclosed herein, mutations can be monitored through a variety of sequencing, expression, or functional assays. Non-limiting examples include DNA sequencing, RNA sequencing, DNA hybridization, protein sequencing, targeted genomic sequencing, whole exome sequencing, whole genome sequencing, Sanger sequencing, PCR, qPCR, RT-PCR, RT-qPCR, Next Generation Sequencing, protein truncation test, DNA microarrays, heteroduplex analysis, denaturing gradient gel electrophoresis, nucleotide sequencing, single strand conformational polymorphism, restriction enzyme digestion assay, fluorescence in situ hybridization (FISH), comparative genomic hybridization, restriction fragment length polymorphism, amplification refractory mutation system PCR, nested PCR, multiplex ligation-dependent probe amplification, single strand conformational polymorphism, and oligonucleotide ligation assay.

[0065] The term “cancer” is used herein in its usual biological sense and understood by those skilled in the art. Thus, it can include the cancer of any cell type, such as but not limited to 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, endometrial cancer, esophagus cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, Hodgkin lymphoma, hematological tumor, Kaposi sarcoma, kidney cancer, laryngeal 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, stomach cancer, small intestine cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous carcinoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor, solid tumor, or liquid tumor.

[0066] As used herein, the term ’’tumor” has its usual meaning as understood by those skilled in the art and refers to an abnormal growth of cells or tissue. In some embodiments, the tumor is benign. In some embodiments, the tumor is malignant. A tumor becomes a cancer when it metastasizes, or spreads to other areas of the body. The term “solid tumor” as used herein has its usual 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 cancer 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, renal cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcomas, neuroblastomas 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. [0067] The term “cell” as used herein has its usual meaning as understood by those skilled in the art and can refer to any cell type. In some embodiments, said cells are mammalian cells. In some embodiments, said cells are human cells.

[0068] The terms “individual”, “subject”, or “patient” as used herein have their usual meaning as understood by those skilled in the art and thus includes a human or a nonhuman mammal. The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea or pigs. In some embodiments, the subject can be human. In some embodiments, the subject can be a child and/or an infant. In other embodiments, the subject can be an adult.

[0069] The term “cancer treatment” as used herein has its usual 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 medicant that is used to treat, inhibit, or prevent cancer. Non-limiting examples of common classes of anti-cancer agents usable with 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, anti-androgens, antiestrogens, anti-hypercalcaemia 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, imidazotetrazinones, 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, alitretinon, altretamine, aminopterin, aminolevulinic acid, amsacrine, asparaginase, atrasentan, bexarotene, carboquone, demecolcine, efaproxiral, elsamitrucin, etoglucid, hydroxycarbamide, leucovorin, lonidamine, lucanthone, masoprocol, methyl aminolevulinate, mitoguazone, mitotane, oblimersen, omacetaxine, pegaspargase, porfimer sodium, prednimustine, sitimagene ceradenovec, talaporfin, temoporfin, trabectedin, or verteporfin.

[0070] The term “pharmaceutically acceptable salt” refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate 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 inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), a sulfuric acid, a nitric acid and a phosphoric acid (such as 2,3- dihydroxypropyl dihydrogen phosphate). Pharmaceutical salts can also be obtained by reacting a compound with an organic acid such as aliphatic or aromatic carboxylic or sulfonic acids, for example formic, acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, trifluoroacetic, benzoic, salicylic, 2- oxopentanedioic or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium, a potassium or a lithium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of a carbonate, a salt of a bicarbonate, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine and salts with amino acids such as arginine and lysine. ForWEEl inhibitor compounds, those skilled in the art understand that when a salt is formed by protonation of a nitrogen-based group (for example, NH2), the nitrogen-based group can be associated with a positive charge (for example, NH2 can become NH3⁺) and the positive charge can be balanced by a negatively charged counterion (such as Cl ).

[0071] It is understood that, in any compound described herein having one or more chiral centers, if an absolute stereochemistry is not expressly indicated, then each center may independently be of R-configuration or S -configuration or a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, enantiomeric ally enriched, racemic mixture, diastereomerically pure, diastereomerically enriched or a stereoisomeric mixture. In addition, it is understood that, in any compound described herein having one or more double bond(s) generating geometrical isomers that can be defined as E or Z, each double bond may independently be E or Z a mixture thereof. Likewise, it is understood that, in any compound described, all tautomeric forms are also intended to be included.

[0072] It is to be understood that where compounds disclosed herein have unfilled valencies, then the valencies are to be filled with hydrogens or isotopes thereof, e.g., hydrogen- 1 (protium) and hydrogen-2 (deuterium).

[0073] It is understood that the compounds described herein can be labeled isotopically. Substitution with isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements. Each chemical element as represented in a compound structure may 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 of the compound that a hydrogen atom may be present, the hydrogen atom can be any isotope of hydrogen, including but not limited to hydrogen- 1 (protium) and hydrogen-2 (deuterium). Thus, reference herein to a compound encompasses all potential isotopic forms unless the context clearly dictates otherwise.

[0074] It is understood that the methods and combinations described herein include crystalline forms (also known as polymorphs, which include the different crystal 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 or the like. In other embodiments, the compounds described herein exist in unsolvated form. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol or the like. Hydrates are formed when the solvent is water or alcoholates are formed when the solvent is alcohol. In addition, the compounds provided herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the compounds and methods provided herein.

[0075] Where a range of values is provided, it is understood that the upper and lower limit, and each intervening value between the upper and lower limit of the range is encompassed within the embodiments.

[0076] Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation,’ ‘including but not limited to,’ or the like; the term ‘comprising’ as used herein is synonymous with ‘including,’ ‘containing,’ or ‘characterized by,’ and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term ‘having’ should be interpreted as ‘having at least;’ the term ‘includes’ should be interpreted as ‘includes but is not limited to;’ the term ‘example’ is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; and use of terms like ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words of similar meaning should not be understood as implying that certain features are critical, essential, or even important to the structure or function, but instead as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment. In addition, the term “comprising” is to be interpreted synonymously with the phrases "having at least" or "including at least". When used in the context of a compound, composition or device, the term "comprising" means that the compound, composition or device includes at least the recited features or components, but may also include additional features or components.

[0077] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The indefinite article “a” or “an” does 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 used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

WEE1 Inhibitor Compounds

[0078] Various embodiments disclosed herein relate to a use or method of using a WEE1 inhibitor. The term ”WEE1 inhibitor” refers to a small molecule, compound, drug, protein, or mixture thereof that reduces the function of the WEE1 nuclear kinase. In various embodiments, the WEE1 inhibitor is a WEE1 inhibitor compound, e.g., having a formula as described herein. In some embodiments, a WEE1 inhibitor inhibits the expression of WEE1. In some embodiments, a WEE1 inhibitor inhibits the function of at least one protein upstream of WEE1 activity. In some embodiments, a WEE1 inhibitor inhibits the function of at least one protein downstream of WEE1 activity. In some embodiments, a WEE1 inhibitor directly inhibits WEE1. In some embodiments, a WEE1 inhibitor binds allosterically to WEE1. In some embodiments, a WEE1 inhibitor binds to the active site of WEE1. In some embodiments, a WEE1 inhibitor is an uncompetitive inhibitor. In some embodiments, a WEE1 inhibitor is a noncompetitive inhibitor. In some embodiments, a WEE1 inhibitor is a competitive inhibitor.

[0079] A wide variety of WEE1 inhibitors are known to those skilled in the art and may be utilized in the methods described herein. For example, in various embodiments the WEE1 inhibitor is described in any one or more of the following publications: W02020210383, W02020210375, W02020210377, W02020210380, W02020210381, WO 2019173082, W02019011228, WO2019138227, WO2018162932, W02018011570, W02018011569, W02018090939, WO2015092431, W02015019037, WO2014167347,

WO2007126122, WO2011034743, US20070254892, US2008133866, US20160060258,

US20190308984, US20200131192, WO2019085933, WO2020221358, EP3712150, WO2018133829, WO2019085933, W02020083404, WO2019037678, WO2018171633,

WO2019096322, WO2019165204, WO2012161812, W02013012681, W02013013031,

WO2013059485, WO2013126656, US20120220572, US20130018045, KR2016035878, KR2020016567, WO2018056621, WO2017075629, WO2019169065, WO2019134539, W02020028814, W02020069105, WO2020192581, CN111718348, and WO9634867. Each of the foregoing publications is expressly incorporated herein by reference in its entirety, and particularly for the purposes of describing WEE1 inhibitors and methods of making them.

[0080] In various embodiments, the WEE1 inhibitor is described in WO 2019173082. For example, in some embodiments, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 26. In an embodiment, the WEE1 inhibitor is a compound ZN-c3 of the formula:

or a pharmaceutically acceptable salt thereof.

[0081] In an embodiment, the WEE1 inhibitor is described in WO2019138227. For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 18, or a pharmaceutically acceptable salt thereof.

[0082] In an embodiment, the WEE1 inhibitor is described in WO2018162932. For example, in an embodiment, the WEE1 inhibitor is a compound represented by the structure described in FIG. 17:

pharmaceutically acceptable salt or N-oxide thereof.

[0083] In an embodiment, the WEE1 inhibitor is described in W02018011570. For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 16, or a pharmaceutically acceptable salt thereof.

[0084] In an embodiment, the WEE1 inhibitor is described in WO2018011569. For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 15 A, or a pharmaceutically acceptable salt thereof. In another embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 15B, or a pharmaceutically acceptable salt thereof. [0085] In an embodiment, the WEE1 inhibitor is described in WO2015092431. For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 14, or a pharmaceutically acceptable salt thereof.

[0086] In an embodiment, the WEE1 inhibitor is described in WO2014167347.

For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 12, or a pharmaceutically acceptable salt thereof.

[0087] In an embodiment, the WEE1 inhibitor is described in W02015019037.

For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 13, or a pharmaceutically acceptable salt thereof.

[0088] In an embodiment, the WEE1 inhibitor is described in W02020210375.

For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 24, or a pharmaceutically acceptable salt thereof.

[0089] In an embodiment, the WEE1 inhibitor is described in W02020210377.

For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 25, or a pharmaceutically acceptable salt thereof

[0090] In an embodiment, the WEE1 inhibitor is described in W02020210380.

For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 21, or a pharmaceutically acceptable salt thereof.

[0091] In an embodiment, the WEE1 inhibitor is described in W02020210381.

For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 22, or a pharmaceutically acceptable salt thereof. In various embodiments, the WEE1 inhibitor is a compound represented by a formula selected from

, or a pharmaceutically acceptable salt thereof of any of the foregoing.

[0092] In an embodiment, the WEE1 inhibitor is described in W02020210383.

For example, in an embodiment, the WEE1 inhibitor is a compound represented by the generic structure described in FIG. 23, or a pharmaceutically acceptable salt thereof. In various embodiments, the WEE1 inhibitor is a compound represented by a formula selected

pharmaceutically acceptable salt thereof of any of the foregoing.

[0093] In an embodiment, the WEE1 inhibitor is described in W02019011228 and/or US20200131192 (US National Phase of W02019011228). For example, in an embodiment, the WEE1 inhibitor is a compound represented by one or both of the generic structures described in FIGs. 20A and 20B, or a pharmaceutically acceptable salt thereof. For example, in an embodiment, the WEE1 inhibitor is a compound of the formula

, or a pharmaceutically acceptable salt thereof. [0094] In an embodiment, the WEE1 inhibitor is described in WO2018090939 and/or US20190308984 (US National Phase of W02018090939). For example, in an embodiment, the WEE1 inhibitor is a compound represented by one or both of the generic structures described in FIGs. 19A and 19B, or a pharmaceutically acceptable salt thereof. In various embodiments, the WEE1 inhibitor is a compound represented by a formula selected

a pharmaceutically acceptable salt thereof of any of the foregoing.

[0095] In an embodiment, the WEE1 inhibitor is a compound known to those skilled in the art as AZD1775, of the formula:

(AZD1775) or a pharmaceutically acceptable salt thereof.

[0096] In an embodiment, the WEE1 inhibitor is a compound known to those skilled in the art as SC0191, of the formula:

(SC0191) or a pharmaceutically acceptable salt thereof. [0097] In an embodiment, the WEE1 inhibitor is a compound known to those skilled in the art as PD0166285, of the formula:

(PD0166285) or a pharmaceutically acceptable salt thereof.

[0098] In an embodiment, the WEE1 inhibitor is a compound known to those skilled in the art as NUV-569. In an embodiment, the WEE1 inhibitor is a compound known to those skilled in the art as IMP7068. In an embodiment, the WEE1 inhibitor is a compound known to those skilled in the art as Debio 0123.

Pharmaceutical Compositions

[0099] Some embodiments described herein relate to a pharmaceutical composition, that can include an effective amount of one or more compounds described herein (for example, a WEE1 inhibitor as described elsewhere herein, or a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable carrier, diluent, excipient or combination thereof.

[0100] The term “pharmaceutical composition” refers to a mixture of one or more compounds and/or salts disclosed herein with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. Pharmaceutical compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0101] The term “physiologically acceptable” refers to a carrier, diluent or excipient that does not abrogate the biological activity and properties of the compound nor cause appreciable damage or injury to an animal to which delivery of the composition is intended.

[0102] As used herein, a “carrier” refers to a compound that facilitates the incorporation of a compound into cells or tissues. For example, without limitation, dimethyl sulfoxide (DMSO) is a commonly utilized carrier that facilitates the uptake of many organic compounds into cells or tissues of a subject.

[0103] As used herein, a “diluent” refers to an ingredient in a pharmaceutical composition that lacks appreciable pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and/or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the pH and isotonicity of human blood.

[0104] As used herein, an “excipient” refers to an essentially inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition. For example, stabilizers such as anti-oxidants and metal-chelating agents are excipients. In an embodiment, the pharmaceutical composition comprises an anti-oxidant and/or a metalchelating agent. A “diluent” is a type of excipient.

[0105] The pharmaceutical compositions described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or carriers, diluents, excipients or combinations thereof. Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art.

[0106] The pharmaceutical compositions disclosed herein may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes. Additionally, the active ingredients are contained in an amount effective to achieve its intended purpose. Many of the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharmaceutically compatible counterions.

[0107] Multiple techniques of administering a WEE1 inhibitor compound, salt and/or composition exist in the art including, but not limited to, oral, rectal, pulmonary, topical, aerosol, injection, infusion and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal and intraocular injections. In some embodiments, a WEE1 inhibitor compound, or a pharmaceutically acceptable salt thereof, can be administered orally.

[0108] One may also administer the WEE1 inhibitor compound, salt and/or composition in a local rather than systemic manner, for example, via injection or implantation of the compound directly into the affected area, often in a depot or sustained release formulation. Furthermore, one may administer the compound in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the organ. For example, intranasal or pulmonary delivery to target a respiratory disease or condition may be desirable.

[0109] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions that can include a compound and/or salt described herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Uses and Methods

[0110] Various embodiments provide a method of determining subject sensitivity to a WEE1 inhibitor, comprising: obtaining or having obtained a biological sample from the subject; and performing or having performed at least one assay on the biological sample to determine if the subject has an altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex. [0111] Another embodiment provides a method of treating a cancer, comprising: obtaining or having obtained a biological sample from the subject; performing or having performed at least one assay on the biological sample to determine if the subject has altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and administering an effective amount of a cancer treatment to the subject based upon results of the assay.

[0112] Another embodiment provides a method of treating a cancer, comprising: identifying a subject having (a) the cancer and (b) endogenous or altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and administering an effective amount of a WEE1 inhibitor to the subject.

[0113] Another embodiment provides a method of treating cancer in a subject, comprising: determining whether the subject is sensitized to treatment with a WEE1 inhibitor, said determining comprising: obtaining or having obtained a biological sample from the subject; and performing or having performed at least one assay on the biological sample to determine if the subject has altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and selecting a treatment protocol for the subject on the basis of the determination of whether the subject is sensitized to treatment with the WEE1 inhibitor.

[0114] Various embodiments and implementation details of the above methods are described in greater detail elsewhere herein. All such embodiments and implementations will be understood to apply to all of the above methods unless the context clearly indicates otherwise.

[0115] As used herein, “DNA repair” refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. Consequently, the DNA repair process is constantly active as it responds to damage in the DNA structure. Disruption of DNA repair results in chromosomal instability and aneuploidy which frequently results in abnormal cell division, mutation accumulation, mitotic catastrophe (i.e., cell death), or cell cycle checkpoint failure resulting in apoptosis.

[0116] Loss of function of DNA repair pathways or cell cycle checkpoints can result in double-strand breaks (DSBs), in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to cell death or genome rearrangements. Two major mechanisms exist to repair DSBs: non-homologous end joining (NHEJ) and recombinational repair (also known as template-assisted repair or homologous recombination repair).

[0117] Many different assays can be used to assess DNA breaks and repair. Nonlimiting examples of assays include RAD51 foci assay, yH2AX (gamma-H2AX) detection or assay, UV-induced CPD by paper chromatography, radioactive labeling, repair replication, unscheduled DNA synthesis assay, acridine orange staining, halo assay with propidium iodide, HPLC-MS, GC-MS, EM, recombinant DNA technology, Sanger sequencing, comet assay, gel electrophoresis, radioimmunoassay, ELISA, PCR, qPCR, LMPCR, ICPCR, TUNEL assay, microarray, immunohistochemical assay, immunological assay, immunoprecipitation, next generation sequencing, RADAR-seq, SMRT sequencing, FISH assay, Comet- FISH assay, DBD-FISH assay, protein XRCC1, protein Ku, CometChip assay, immunoslot blot, excision repair assay, incision assay, repair patch assay, fluorescent-dye staining, affinity-based binding assay, enzyme-mediated fluorescent labeling, host cell reactivation assay, SPLseq, XR-seq, tXR-seq, Excision-seq, Ribose-seq, hydrolytic end sequencing, NMP-seq, Pu-seq, HydEn-seq, alkaline single-cell gel electrophoresis, neutral single-cell gel electrophoresis, and FCM- Annexin V labeling.

[0118] As used herein, a “cell cycle checkpoint” is a series of tests or evaluations performed by the cell that pause the cell cycle and gives the cell time to repair the damage before continuing to divide. DNA damage checkpoints occur at the Gl/S and G2/M boundaries. An intra-S checkpoint also exists. Checkpoint activation is controlled by two master kinases, ATM and ATR. ATM responds to DNA double-strand breaks and disruptions in chromatin structure, whereas ATR primarily responds to stalled replication forks. These kinases phosphorylate downstream targets in a signal transduction cascade, eventually leading to cell cycle arrest. A class of checkpoint mediator proteins are also required for transmitting the checkpoint activation signal to downstream proteins to promote or inhibit progress through the cell cycle.

[0119] A “functional assay” is a method to detect the activity of a gene, protein, or cell in response to a stimulus or insult. The specific functional assay performed depends on the specific mutation or mutations incorporated into the genome of the cell. Functional assays include, but are not limited to, kinase assays, transcription assays using, for example, reporter constructs, proliferation assays, apoptosis assays, migration/chemotaxis assays, nutrient sensitivity assay, agent (e.g., drug, chemotherapeutic agent, mutagen) or radiation sensitivity assays, nucleic acid-binding assay or protein-binding assay, all of which are within the ability of those of skill in the art.

[0120] Various embodiments provide a method of determining subject sensitivity to a WEE1 inhibitor, comprising: obtaining or having obtained a biological sample from the subject; and performing or having performed at least one assay on the biological sample to determine if the subject has an altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex.

[0121] In an embodiment, the subject has a genotype for the altered DNA repair function. In an embodiment, the genotype for the altered DNA repair function comprises a functional mutation to RAD50, NBN (NBS1), MRE11, a protein that associates with the MRN protein complex, or any combination thereof. In an embodiment, the functional mutation is a deletion, insertion, truncation, point mutation, frameshift, or other genetic alteration.

[0122] In an embodiment, the altered DNA repair function is a loss of DNA repair function. In an embodiment, Nibrin (NBN, or NBS1) has a loss of DNA repair function. In an embodiment, the altered DNA repair function is a reduction of DNA repair function. In an embodiment, NBN has a reduction of DNA repair function. In an embodiment, the altered DNA repair function is a gain of DNA repair function. In an embodiment, NBN has a gain of DNA repair function.

[0123] In an embodiment, the altered DNA repair function is due to altered expression of at least one gene associated with the MRN protein complex. In an embodiment, a gene associated with NBN has enhanced expression. In an embodiment, a gene associated with NBN has reduced expression. In an embodiment, the genotype for the altered DNA repair function is a somatic genotype. In an embodiment, the genotype for the altered DNA repair function is a germline genotype.

[0124] Another embodiment provides a method of treating a cancer, comprising: obtaining or having obtained a biological sample from the subject; performing or having performed at least one assay on the biological sample to determine if the subject has altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and administering an effective amount of a cancer treatment to the subject based upon results of the assay.

[0125] In an embodiment of a method of treating cancer as described herein, if the subject has a reduced or endogenous DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBS1 (MRN) protein complex, then administering the cancer treatment comprises administering an effective amount of a WEE1 inhibitor to the subject; or if the subject has a gain in DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBS1 (MRN) protein complex, then administering the cancer treatment does not include administering a WEE1 inhibitor to the subject.

[0126] In an embodiment of a method of treating cancer as described herein, the cancer comprises a tumor. In an embodiment, the subject is mammalian. In an embodiment, the subject is human. In an embodiment, the subject has a reduced DNA repair function and the effective amount of the WEE1 inhibitor administered to the subject comprises a lower dosage as compared to a normal dosage given in treatment. In an embodiment, the subject has endogenous DNA repair function and the effective amount of WEE1 inhibitor administered to the subject is a normal dosage given in treatment.

[0127] Another embodiment provides a method of treating a cancer, comprising: identifying a subject having (a) the cancer and (b) endogenous or altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and administering an effective amount of a WEE1 inhibitor to the subject.

[0128] In an embodiment of a method of treating cancer as described herein, the altered DNA repair function is a loss of function. In an embodiment, the altered DNA repair function is a gain of function. In an embodiment, the altered DNA repair function is a reduction of function. In an embodiment, the subject has endogenous DNA repair function.

[0129] Another embodiment provides a method of treating cancer in a subject, comprising: determining whether the subject is sensitized to treatment with a WEE1 inhibitor, said determining comprising: obtaining or having obtained a biological sample from the subject; and performing or having performed at least one assay on the biological sample to determine if the subject has altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and selecting a treatment protocol for the subject on the basis of the determination of whether the subject is sensitized to treatment with the WEE1 inhibitor.

[0130] In an embodiment of a method of treating cancer as described herein, the method further comprises treating the subject in accordance with the treatment protocol. In an embodiment, the altered DNA repair function is a loss of function. In an embodiment, the altered DNA repair function is a reduction of function. In an embodiment, the altered DNA repair function is a gain of function. In an embodiment, the subject has an endogenous DNA repair function. In an embodiment, the altered DNA repair function is enhanced expression of at least one gene associated with the MRN protein complex. In an embodiment, the altered DNA repair function is reduced expression of at least one gene associated with the MRN protein complex.

[0131] In an embodiment of a method of treating cancer as described herein, if the subject has a genotype for endogenous or reduced expression of at least one gene associated with NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex, then the treatment protocol comprises administering an effective amount of a WEE1 inhibitor to the subject; or if the subject has a genotype for enhanced expression of at least one gene associated with NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex, then the treatment protocol comprises administering an effective amount of a cancer treatment other than a WEE1 inhibitor to the subject.

[0132] In an embodiment of a method of treating cancer as described herein, if the subject has a genotype for endogenous or reduced expression of at least one gene associated with NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex, then the treatment protocol comprises administering the WEE1 inhibitor to the subject at a first effective dosage; or if the subject has a genotype for enhanced expression of at least one gene associated with NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex, then the treatment protocol comprises administering the WEE1 inhibitor to the subject at second effective dosage that is higher than the first effective dosage.

[0133] In an embodiment of a method of treating cancer as described herein, the genotype for altered DNA repair function, reduced DNA repair function, loss of DNA repair function, gain of DNA repair function, enhanced gene expression, or reduced gene expression comprises an NBN mutation In an embodiment, the genotype for altered DNA repair function, reduced DNA repair function, loss of DNA repair function, gain of DNA repair function, enhanced gene expression, or reduced gene expression comprises an MRE11 mutation. In an embodiment, the genotype for altered DNA repair function, reduced DNA repair function, loss of DNA repair function, gain of DNA repair function, reduced gene expression, or enhanced gene expression comprises a RAD50 mutation.

[0134] In an embodiment of a method of treating cancer as described herein, the method further comprises determining that the treatment protocol at the first effective dosage poses a lower toxicity risk to the subject than the treatment protocol at the second effective dosage.

[0135] In any of the various embodiment of the methods described herein, the WEE1 inhibitor can be selected from among the various WEE1 inhibitor compounds described in the section above under the heading “WEE1 Inhibitor Compounds”. [0136] Some embodiments described herein relate to a method for ameliorating and/or treating a cancer described herein that can include administering an effective amount of a WEE1 inhibitor compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes an effective amount of a WEE1 inhibitor compound described herein or a pharmaceutically acceptable salt thereof, to a subject having a cancer described herein. Other embodiments described herein relate to the use of an effective amount of a WEE1 inhibitor compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes an effective amount of a WEE1 inhibitor compound described herein or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for ameliorating and/or treating a cancer described herein. Still other embodiments described herein relate to an effective amount of a WEE1 inhibitor compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes an effective amount of a WEE1 inhibitor compound described herein or a pharmaceutically acceptable salt thereof, for ameliorating and/or treating a cancer described herein.

[0137] Some embodiments described herein relate to a method for inhibiting replication of a malignant growth or a tumor that can include contacting the growth or the tumor with an effective amount of a WEE1 inhibitor compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes an effective amount of a WEE1 inhibitor compound described herein or a pharmaceutically acceptable salt thereof, wherein the malignant growth or tumor is due to a cancer described herein. Other embodiments described herein relate to the use of an effective amount of a WEE1 inhibitor compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes an effective amount of a WEE1 inhibitor compound described herein or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting replication of a malignant growth or a tumor, wherein the malignant growth or tumor is due to a cancer described herein. Still other embodiments described herein relate to an effective amount of a WEE1 inhibitor compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes an effective amount of a WEE1 inhibitor compound described herein or a pharmaceutically acceptable salt thereof, for inhibiting replication of a malignant growth or a tumor, wherein the malignant growth or tumor is due to a cancer described herein.

[0138] Some embodiments described herein relate to a method for ameliorating or treating a cancer described herein that can include contacting a malignant growth or a tumor with an effective amount of a WEE1 inhibitor compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes an effective amount of a WEE1 inhibitor compound described herein or a pharmaceutically acceptable salt thereof, to a subject having a cancer described herein. Other embodiments described herein relate to the use of an effective amount of a WEE1 inhibitor compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes an effective amount of a WEE1 inhibitor compound described herein or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for ameliorating or treating a cancer that can include contacting a malignant growth or a tumor, wherein the malignant growth or tumor is due to a cancer described herein. Still other embodiments described herein relate to an effective amount of a WEE1 inhibitor compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition that includes an effective amount of a WEE1 inhibitor compound described herein or a pharmaceutically acceptable salt thereof, for ameliorating or treating a cancer that can include contacting a malignant growth or a tumor, wherein the malignant growth or tumor is due to a cancer described herein.

[0139] Some embodiments described herein relate to a method for inhibiting the activity of WEE1 (for example, inhibiting the activity of WEE1 in NBN mutated cells, inhibiting the activity of WEE1 in NBN wild type cells, inhibiting the activity of WEE1 in MRE11 mutated cells, inhibiting the activity of WEE1 in MRE11 wildtype cells, inhibiting the activity of WEE1 in RAD50 mutated cells, inhibiting the activity of WEE1 in RAD50 wild type cells, and/or decreasing the overexpression of WEE1 in cells) that can include providing an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) to a cancer cell from a cancer described herein. Other embodiments described herein relate to the use of an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament for inhibiting the activity of WEE1 (for example, inhibiting the activity of WEE1 in NBN mutated cells, inhibiting the activity of WEE1 in NBN wild type cells, inhibiting the activity of WEE1 in MRE11 mutated cells, inhibiting the activity of WEE1 in MRE11 wildtype cells, inhibiting the activity of WEE1 in RAD50 mutated cells, inhibiting the activity of WEE1 in RAD50 wild type cells, and/or decreasing the overexpression of WEE1 in cells). Still other embodiments described herein relate to an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) for inhibiting the activity of WEE1 (for example, inhibiting the activity of WEE1 inhibiting the activity of WEE1 in NBN mutated cells, inhibiting the activity of WEE1 in NBN wild type cells, inhibiting the activity of WEE1 in MRE11 mutated cells, inhibiting the activity of WEE1 in MRE11 wildtype cells, inhibiting the activity of WEE 1 in RAD50 mutated cells, inhibiting the activity of WEE 1 in RAD50 wild type cells, and/or decreasing the overexpression of WEE1 in cells). Some embodiments described herein relate to a method for inhibiting the activity of WEE1 (for example, inhibiting the activity of WEE1 in NBN mutated cells, inhibiting the activity of WEE1 in NBN wild type cells, inhibiting the activity of WEE1 in MRE11 mutated cells, inhibiting the activity of WEE 1 in MRE11 wildtype cells, inhibiting the activity of WEE 1 in RAD50 mutated cells, inhibiting the activity of WEE1 in RAD50 wild type cells, and/or decreasing the overexpression of WEE1 in cells) that can include providing an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) to a cancer cell from a cancer described herein. Other embodiments described herein relate to a method for inhibiting the activity of WEE1 (for example, inhibiting the activity of WEE1 in NBN mutated cells, inhibiting the activity of WEE1 in NBN wild type cells, inhibiting the activity of WEE1 in MRE11 mutated cells, inhibiting the activity of WEE 1 in MRE11 wildtype cells, inhibiting the activity of WEE 1 in RAD50 mutated cells, inhibiting the activity of WEE1 in RAD50 wild type cells, and/or decreasing the overexpression of WEE1 in cells) that can include contacting a cancer cell from a cancer described herein with an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, a WEE1, or a pharmaceutically acceptable salt thereof), and thereby inhibiting the activity of WEE 1.

[0140] Some embodiments described herein relate to a method for ameliorating or treating a cancer described herein that can include inhibiting the activity of WEE1 (for example inhibiting the activity of WEE1 in NBN mutated cells, inhibiting the activity of WEE1 in NBN wild type cells, inhibiting the activity of WEE1 in MRE11 mutated cells, inhibiting the activity of WEE 1 in MRE11 wildtype cells, inhibiting the activity of WEE 1 in RAD50 mutated cells, inhibiting the activity of WEE1 in RAD50 wild type cells, and/or decreasing the overexpression of WEE1 in cells) using an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof). Other embodiments described herein relate to the use of an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament for ameliorating or treating a cancer described herein by inhibiting the activity of WEE1 (for example inhibiting the activity of WEE1 in NBN mutated cells, inhibiting the activity of WEE1 in NBN wild type cells, inhibiting the activity of WEE1 in MRE11 mutated cells, inhibiting the activity of WEE1 in MRE11 wildtype cells, inhibiting the activity of WEE1 in RAD50 mutated cells, inhibiting the activity of WEE1 in RAD50 wild type cells, and/or decreasing the overexpression of WEE1 in cells). Still other embodiments described herein relate to an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof) for ameliorating or treating a cancer described herein by inhibiting the activity of WEE1 (for example inhibiting the activity of WEE1 in NBN mutated cells, inhibiting the activity of WEE1 in NBN wild type cells, inhibiting the activity of WEE 1 in MRE11 mutated cells, inhibiting the activity of WEE 1 in MRE11 wildtype cells, inhibiting the activity of WEE1 in RAD50 mutated cells, inhibiting the activity of WEE1 in RAD50 wild type cells, and/or decreasing the overexpression of WEE1 in cells). Some embodiments described herein relate to a method for ameliorating or treating a cancer described herein that can include contacting a cancer cell with an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof), wherein the compound inhibits the activity of WEE1 (for example inhibiting the activity of WEE1 in NBN mutated cells, inhibiting the activity of WEE1 in NBN wild type cells, inhibiting the activity of WEE1 in MRE11 mutated cells, inhibiting the activity of WEE1 in MRE11 wildtype cells, inhibiting the activity of WEE1 in RAD50 mutated cells, inhibiting the activity of WEE1 in RAD50 wild type cells, and/or decreasing the overexpression of WEE1 in cells).

[0141] Some embodiments disclosed herein relate to a method for inhibiting the activity of WEE1 that can include providing an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) to a subject having a cancer described herein or a cancer cell from a cancer described herein. Other embodiments disclosed herein relate to the use of an effective amount of a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament for inhibiting the activity of WEE1. Still other embodiments disclosed herein relate to a compound described herein (for example, a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition that includes an effective amount of a compound described herein (for example, ZN-c3, or a pharmaceutically acceptable salt thereof) for inhibiting the activity of WEE 1.

[0142] As used herein, the terms “treat,” “treating,” “treatment,” “therapeutic,” and “therapy” do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of the disease or condition, to any extent can be considered treatment and/or therapy. Furthermore, treatment may include acts that may worsen the subject’s overall feeling of well-being or appearance.

[0143] The terms “administration” or “administering” as used herein have their usual meaning as understood by those skilled in the art and refer to providing or giving a subject an agent, such as the composition disclosed herein, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as intracranial, subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal, intranasal, vaginal, intraocular, or inhalation routes.

[0144] The terms “therapeutically effective amount” and “effective amount” are used to indicate an amount of an active compound, or pharmaceutical agent, that elicits the biological or medicinal response indicated. For example, a therapeutically effective amount of compound, salt or composition can be the amount needed to prevent, alleviate or ameliorate symptoms of the disease or condition, or prolong the survival of the subject being treated. This response may occur in a tissue, system, animal or human and includes alleviation of the signs or symptoms of the disease or condition being treated. Determination of an effective amount is well within the capability of those skilled in the art, in view of the disclosure provided herein. The therapeutically effective amount of the compounds disclosed herein required as a dose will depend on the route of administration, the type of animal, including human, being treated and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.

[0145] For example, an effective amount of a compound, or radiation, is the amount that results in: (a) the reduction, alleviation or disappearance of one or more symptoms caused by the cancer, (b) the reduction of tumor size, (c) the elimination of the tumor, and/or (d) long-term disease stabilization (growth arrest) of the tumor. In the treatment of lung cancer (such as non- small cell lung cancer) a therapeutically effective amount is that amount that alleviates or eliminates cough, shortness of breath and/or pain. As another example, an effective amount, or a therapeutically effective amount of an WEE1 inhibitor is the amount which results in the reduction in WEE1 activity and/or phosphorylation (such as phosphorylation of CDC2, also known as CDK1). The reduction in WEE1 activity is known to those skilled in the art and can be determined by the analysis of WEE1 intrinsic kinase activity and downstream substrate phosphorylation.

[0146] The amount of the WEE1 inhibitor compound, or a pharmaceutically acceptable salt thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature and/or symptoms of the disease or condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free base. As will be understood by those of skill in the art, in certain situations it may be necessary to administer the compounds disclosed herein in amounts that exceed, or even far exceed, the dosage ranges described herein in order to effectively and aggressively treat particularly aggressive diseases or conditions.

[0147] In general, however, a suitable dose will often be in the range of from about 0.05 mg/kg to about 10 mg/kg. For example, a suitable dose may be in the range from about 0.10 mg/kg to about 7.5 mg/kg of body weight per day, such as about 0.15 mg/kg to about 5.0 mg/kg of body weight of the recipient per day, about 0.2 mg/kg to 4.0 mg/kg of body weight of the recipient per day, or any amount in between. The compound may be administered in unit dosage form; for example, containing 1 to 500 mg, 10 to 100 mg, 5 to 50 mg or any amount in between, of active ingredient per unit dosage form.

[0148] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0149] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, the mammalian species treated, the particular compounds employed and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials, in vivo studies and in vitro studies. For example, useful dosages of a WEE1 inhibitor compound, or pharmaceutically acceptable salts thereof, can be determined by comparing their in vitro activity, and in vivo activity in animal models. Such comparison can be done by comparison against an established drug, such as cisplatin and/or gemcitabine)

[0150] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vivo and/or in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0151] It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the disease or condition to be treated and to the route of administration. The severity of the disease or condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0152] WEE1 inhibitor compounds, salts and compositions disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular compound, or of a subset of the compounds, sharing certain chemical moieties, may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular compounds in an animal model, such as mice, rats, rabbits, dogs or monkeys, may be determined using known methods. The efficacy of a particular compound may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, route of administration and/or regime.

EXAMPLES

Example 1 - Clinical trials demonstrate superior ability of WEE 1 inhibitors to treat cancer in subjects with NBN mutations

[0153] The effectiveness of WEE1 inhibitors in human subjects having NBN mutations was assessed through clinical trials. Two human subjects were examined: Patient 1, with stage IV colorectal cancer, and Patient 2, with stage IV ovarian cancer. Part of the genomes of both subjects were sequenced. Patient 1 was found to have a germline mutation for NBN (c.l23delC, truncated exon 2; pathogenic mutation) The NBN c.l23delC variant in Patient 1 is predicted to result in reduced DNA repair function of NBN. Patient 2 was found to have a somatic mutation for NBN (truncated intron 8), which is predicted to be associated with reduced DNA repair function of NBN.

[0154] Both patients were assessed for response to WEE1 inhibitors over time. Tumor progression was monitored using tumor markers; CEA was used for Patient 1, and CA-125 was used for Patient 2. Prior to treatment, Patient 1 showed elevated CEA levels (> 300 units/mL), significantly above the reference range (FIG. 1). However, within two weeks of the start of treatment with ZN-c3 (a WEE1 inhibitor), CEA levels rescinded to the normal range, suggestive of substantial tumor regression. From subsequent imaging analysis of the tumor, Patient 1 was estimated to have a 42% tumor size reduction (partial response) to treatment with the WEE1 inhibitor (FIG. 2-3), which further increased up to 51% tumor size reduction (partial response) in the course of treatment thereafter. Similarly, tumor progression in Patient 2 rescinded dramatically approximately 1 month following the start of treatment with ZN-c3, reflected by the significant decrease in CA-125 levels (FIG. 4). From subsequent imaging analysis of the tumor, Patient 2 had a 56% tumor size reduction (partial response) to treatment with a WEE1 inhibitor (FIG. 5-7) , which further increased up to 71% tumor size reduction (partial response) in the course of treatment thereafter. These results illustrate the enhanced effectiveness of WEE1 inhibition in human subjects having reduced NBN function under a clinical setting.

Example 2 - Cell culture analysis indicates that NBN function correlates inversely to WEE1 inhibitor effectiveness in halting cellular proliferation

[0155] The ovarian cell culture line UWB 1.289 was treated with siRNA mediated knockdown of the NBN gene, as confirmed by Western blot analysis (FIG. 8). These cells were then treated with increasing concentrations of ZN-c3 over 72 hours and monitored for survival (FIG. 9). Compared to wild type, cells lacking NBN function were 1.5-fold more sensitized to WEE1 inhibition (TABLE 1). These results are consistent with the clinical trial data of Example 1 indicating that reduced NBN function enhances the effectiveness of WEE1 inhibitors.

TABLE 1

TABLE 1 summarizes the statistical analysis of the experiment depicted in FIG. 9.

[0156] The effectiveness of a WEE1 inhibitor was evaluated across cell lines having differing levels of NBN expression. HCC1428 (breast cancer) and HS-578T (breast cancer) are cell lines in which NBN is amplified due to copy number amplifications. In contrast, NBN expression is not amplified in ovarian cell cancer lines OVCAR3 and UWB 1.289. Cells from all four lines were treated with ZN-c3 (FIG. 10). The expression level of NBN correlated inversely with WEE1 inhibition effectiveness; cells with amplified NBN expression were less sensitive to treatment with the WEE1 inhibitor than cells without amplified NBN expression, as evidenced by higher IC50 values and/or lower maximal cell density inhibition (TABLE 2). This data is consistent with the clinical trial data of Example 1 and the above cell culture data.

TABLE 2

TABLE 2 summarizes the statistical analysis of the experiment depicted in FIG. 10.

[0157] The effectiveness of WEE 1 inhibition in combination with the compound Mirin was also evaluated. Mirin acts as a specific MRE11-RAD50-NBN (MRN) protein complex inhibitor, by blocking MRE 11 -associated nuclease activity. The ovarian cancer cell line OVCAR3 was treated with increasing concentrations of ZN-c3 for 96 hours, with or without 30 pM Mirin (FIG. 11). The data shown in TABLE 3 indicates that cells co-treated with 30 pM Mirin were approximately 3-fold more sensitive to ZN-c3 than cells treated with ZN-c3 alone. OVCAR3 cells have native MRN-complex function, which would be inhibited through the consequent targeting of complex member MRE 11 through Mirin treatment. As Mirin treatment enhanced the effectiveness of the WEE1 inhibitor, MRN complex function, and through it NBN function, is important for inhibitor resistance. This data is consistent with the clinical trial data of Example 1 and the cell culture data discussed above.

TABLE 3

TABLE 3 summarizes the statistical analysis of the experiment depicted in FIG. 11

[0158] The ovarian cell culture line SKOV3 was treated with sgRNA mediated knockdown of the NBN gene, as confirmed by Western blot analysis (FIG. 27). These cells were then treated with increasing concentrations of ZN-c3 over 72 hours in 2D growth assays with or without 300 pM hydroxyurea (HU), a replication stress inducing compound, and monitored for survival (FIG. 28). Compared to wild type, cells lacking NBN function were 1.8-fold more sensitized to WEE1 inhibition in the presence of hydroxyurea (TABLE 4). These results are consistent with the clinical trial data of Example 1 indicating that reduced NBN function enhances the effectiveness of WEE1 inhibitors.

TABLE 4

TABLE 4 summarizes the statistical analysis of the experiment depicted in FIG. 28. sgNT = non targeting; sgl NBN = NBN knockout; HU = hydroxyurea.

[0159] The ovarian cell culture line SKOV3 was treated with sgRNA mediated knockdown of the NBN gene, as confirmed by Western blot analysis (FIG. 29). These cells were then cultured in 3D spheroid growth assays and treated with increasing concentrations of ZN-c3 over 11 days and monitored for survival (FIG. 30). Compared to wild type, cells lacking NBN function were 1.9-fold more sensitized to WEE1 inhibition (TABLE 5). These results are consistent with the clinical trial data of Example 1 indicating that reduced NBN function enhances the effectiveness of WEE 1 inhibitors.

TABLE 5

TABLE 5 summarizes the statistical analysis of the experiment depicted in FIG. 30. sgNT = non targeting; sgl NBN = NBN knockout.

[0160] The ovarian cell culture line SKOV3 was treated with sgRNA and siRNA- mediated knockout and/or knockdown, respectively, of the NBN gene, as confirmed by Western blot analysis (FIG. 31). These cells were then cultured in 2D growth assays and treated with increasing concentrations of ZN-c3 over 72 hours and monitored for survival (FIG. 32). Compared to wild type, cells lacking NBN function (mediated by sgRNA mediated knockout and siRNA-mediated knockdown) were 2.0-fold more sensitized to WEE1 inhibition (TABLE 6). These results are consistent with the clinical trial data of Example 1 indicating that reduced NBN function enhances the effectiveness of WEE1 inhibitors.

TABLE 6

TABLE 6 summarizes the statistical analysis of the experiment depicted in FIG. 32. sgNT = non-targeting; sgl NBN = NBN knockout; siCTRL = non-targeting (siRNA); siNBN = NBN knockdown.

[0161] The anti-tumor effects of ZN-c3 were tested in xenograft models established from cell lines with genetic variants of MRN genes that have been described to lead to loss-of-function of the corresponding proteins. Four different models were tested, including colorectal cancer LoVo cell line (relevant genetic variant: NBN L490fs), ovarian cancer TOV-21G cell line (relevant genetic variant: NBN R466Gfs*18), acute lymphoblastic leukemia MOLT-4 cell line (relevant genetic variants: RAD50 R656*, Q882*, R385C) and breast cancer MCF-7 cell line (relevant genetic variants: NBN P325H, R43*, N321Y).

[0162] The antitumor activity of ZN-c3 was assessed using the colorectal cancer LoVo xenograft model with BALB/c nude mice. Each mouse was inoculated on the right flank subcutaneously with 5 x 10⁶/100 pL LoVo tumor cells for the tumor development, when the mean tumor size reached 207 mm³, animals were randomized into 4 groups (10 animals/group) and the treatments were initiated according to TABLE 7 below.

TABLE 7

TABLE 7 summarizes the treatment groups of the experiment depicted in FIG. 33. [0163] Study endpoints included daily body weight, clinical observations and tumor volume. Figure 33 showed that ZN-c3 as a single agent produced robust inhibition of tumor growth increasing with the dose level (40 mg/kg/day, 60 mg/kg/day, and 80 mg/kg/day) with tumor growth inhibition (TGI) of 21.4%, 32.1% and 70.3%, respectively. There were no adverse clinical observations in any dose group and there was no significant impact of treatments on mean body weights. These results are consistent with the clinical trial data of Example 1 indicating that reduced MRN function associated with a gene mutation is associated with a robust response of cancer cells to WEE1 inhibitors.

TABLE 8

TABLE 8 summarizes the results of the experiment depicted in FIG. 33. ^a TGI = Tumor growth inhibition, calculated as TGI = (l - (Td - To) / (Cd - Co)) x 100%, Td and Cd were the mean tumor volumes of the treated and control animals, and To and Co were the mean tumor volumes of the treated and control animals at the start of the experiment; ^b calculated vs. Vehicle Control by LSD Test.

[0164] The antitumor activity of ZN-c3 was assessed using the ovarian cancer TOV-21G xenograft model with BALB/c nude mice. Each mouse was inoculated at the right flank with TOV-21G tumor cells (5xl0⁶ cells /mouse) in 0.2 mL mixture of base media with 50% BD Matrigel for tumor development. When the tumor sizes reached 100-150 mm³, animals were randomized into 4 groups (8 animals/group) and the treatments were initiated according to TABLE 9 below.

TABLE 9

TABLE 9 summarizes the treatment groups of the experiment depicted in FIG. 34.

[0165] Study endpoints included daily body weight, clinical observations and tumor volume. Figure 34 showed that ZN-c3 as a single agent produced robust inhibition of tumor growth increasing with the dose level (40 mg/kg/day, 60 mg/kg/day, and 80 mg/kg/day) with TGIs of 65.6%, 94.4% and 104.2%, respectively. There were no adverse clinical observations in any dose group and there was no significant impact on mean body weights of treatment compared to vehicle controls. These results are consistent with the clinical trial data of Example 1 indicating that reduced MRN function associated with a gene mutation is associated with a robust response of cancer cells to WEE1 inhibitors.

TABLE 10

TABLE 10 summarizes the results of the experiment depicted in FIG. 34. ^a TGI = Tumor growth inhibition, calculated as TGI (%) = (1 - (TVlreatment/Dn - TVTreatment/Do)/ (TVcontrol/Dii— TVcontroi/oo)) x 100%; ^b' calculated by Two-way ANOVA followed by Bonferroni post-test to compare means by row vs. Vehicle group.

[0166] The antitumor activity of ZN-c3 was assessed using the acute lymphoblastic leukemia MOLT-4 xenograft model with BALB/c nude mice. Each mouse was inoculated on the right flank with the single cell suspension of 95% viable tumor cells (5 x 10⁶) in 100 pL RPMI 1640 with Matrigel mixture (1:1 ratio) without serum for the tumor development. When the tumor sizes reached a mean tumor volume of approximately 163 mm³, animals were randomized into 2 groups (8 animals/group) and the treatments were initiated according to TABLE 11 below.

TABLE 11

TABLE 11 summarizes the treatment groups of the experiment depicted in FIG. 35.

[0167] Study endpoints included daily body weight, clinical observations and tumor volume. Figure 35 showed that ZN-c3 as a single agent produced robust inhibition of tumor growth at 60 mg/kg/day with TGI of 70.2%. There were no adverse clinical observations and there was no significant impact on mean body weights. These results are consistent with the clinical trial data of Example 1 indicating that reduced MRN function associated with a gene mutation is associated with a robust response of cancer cells to WEE1 inhibitors.

TABLE 12

TABLE 12 summarizes the results of the experiment depicted in FIG. 35. ^a TGI = Tumor growth inhibition, calculated as TGI = (1 - (Td - To) / (Cd - Co)) x 100%; ^b calculated vs. Vehicle Control by Mann- Whitney U Test.

[0168] The antitumor activity of ZN-c3 was assessed using the breast cancer MCF-7 xenograft model with BALB/c nude mice. Each mouse was inoculated subcutaneously on the right flank with the single cell suspension of 95% viable tumor cells (1.5 x 10⁷) in 200 pL DMEM Matrigel mixture (1:1 ratio) without serum for the tumor development. When the tumor sizes reached a mean tumor volume of approximately 202 mm³, animals were randomized into 2 groups (10 animals/group) and the treatments were initiated according to TABLE 13 below. In addition, estradiol benzoate injection was delivered by s.c. (40 ug/20 uL, twice weekly).

TABLE 13

TABLE 13 summarizes the treatment groups of the experiment depicted in FIG. 36.

[0169] Study endpoints included daily body weight, clinical observations and tumor volume. Figure 36 showed that ZN-c3 as a single agent produced robust inhibition of tumor growth with TGI of 116.0%. There were no adverse clinical observations and there was no significant impact on mean body weights. These results are consistent with the clinical trial data of Example 1 indicating that reduced MRN function associated with a gene mutation is associated with a robust response of cancer cells to WEE1 inhibitors.

TABLE 14

TABLE 14 summarizes the results of the experiment depicted in FIG. 36. ^a TGI = Tumor growth inhibition, calculated as TGI = (1 - (Td - To) / (Cd - Co)) x 100%; ^b calculated vs. Vehicle Control by Mann- Whitney U Test.

[0170] Furthermore, although the foregoing has been described in some detail by way of illustrations and examples for purposes of clarity and understanding, it will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present disclosure. Therefore, it should 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 also cover all modification and alternatives coming with the true scope and spirit of the disclosure.

Claims

WHAT IS CLAIMED IS:

1. A method of determining subject sensitivity to a WEE1 inhibitor, comprising: obtaining or having obtained a biological sample from the subject; and performing or having performed at least one assay on the biological sample to determine if the subject has an altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex.

2. The method of claim 1, wherein the subject has a genotype for the altered DNA repair function.

3. The method of any of the claims 1 to 2, wherein the genotype for the altered DNA repair function comprises a functional mutation to RAD50, NBN (NBS1), MRE11, a protein that associates with the MRN protein complex, or any combination thereof.

4. The method of claim 3, wherein the functional mutation is a deletion, insertion, truncation, point mutation, or other genetic alteration.

5. The method of any one of claims 1 to 4, wherein the altered DNA repair function is a loss of DNA repair function.

6. The method of claim 5, wherein Nibrin (NBN, or NBS1) has a loss of DNA repair function.

7. The method of any one of claims 1 to 4, wherein the altered DNA repair function is a reduction of function.

8. The method of claim 7, wherein NBN has a reduction of DNA repair function.

9. The method of any one of claims 1 to 4, wherein the altered DNA repair function is a gain of DNA repair function.

10. The method of claim 9, wherein NBN has a gain of DNA repair function.

11. The method of any one of claims 1 to 4, wherein the altered DNA repair function is due to altered expression of at least one gene associated with the MRN protein complex.

12. The method of claim 11, wherein a gene associated with NBN has enhanced expression.

13. The method of claim 11, wherein a gene associated with NBN has reduced expression.

47

14. The method of any one of claims 2 to 13, wherein the genotype for the altered

DNA repair function is a somatic genotype.

15. The method of any one of claims 2 to 13, wherein the genotype for the altered DNA repair function is a germline genotype.

16. A method of treating a cancer, comprising: obtaining or having obtained a biological sample from the subject; performing or having performed at least one assay on the biological sample to determine if the subject has altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and administering an effective amount of a cancer treatment to the subject based upon results of the assay.

17. The method of claim 16, wherein: if the subject has a reduced or endogenous DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBS1 (MRN) protein complex, then administering the cancer treatment comprises administering an effective amount of a WEE1 inhibitor to the subject; or if the subject has a gain in DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBS1 (MRN) protein complex, then administering the cancer treatment does not include administering a WEE1 inhibitor to the subject.

18. The method of claim 16 or 17, wherein the cancer is a tumor.

19. The method of any one of claims 16 to 18, wherein the subject is mammalian.

20. The method of claim 19, wherein the subject is human.

21. The method of any one of claims 17 to 20, wherein the subject has a reduced

DNA repair function and the effective amount of the WEE1 inhibitor administered to the subject comprises a lower dosage as compared to a normal dosage given in treatment.

22. The method of any one of claims 17 to 20, wherein the subject has endogenous DNA repair function and the effective amount of WEE1 inhibitor administered to the subject is a normal dosage given in treatment.

23. A method of treating a cancer, comprising:

48 identifying a subject having (a) the cancer and (b) endogenous or altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and administering an effective amount of a WEE1 inhibitor to the subject.

24. The method of claim 23, wherein the altered DNA repair function is a loss of function.

25. The method of claim 23, wherein the altered DNA repair function is a gain of function.

26. The method of claim 23, wherein the altered DNA repair function is a reduction of function.

27. The method of claim 23, wherein the subject has endogenous DNA repair function.

28. A method of treating cancer in a subject, comprising: determining whether the subject is sensitized to treatment with a WEE1 inhibitor, said determining comprising: obtaining or having obtained a biological sample from the subject; and performing or having performed at least one assay on the biological sample to determine if the subject has altered DNA repair function of NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex; and selecting a treatment protocol for the subject on the basis of the determination of whether the subject is sensitized to treatment with the WEE1 inhibitor.

29. The method of claim 28, further comprising treating the subject in accordance with the treatment protocol.

30. The method of any one of claims 28 to 29, wherein the altered DNA repair function is a loss of function.

31. The method of any one of claims 28 to 29, wherein the altered DNA repair function is a reduction of function.

32. The method of any one of claims 28 to 29, wherein the altered DNA repair function is a gain of function.

33. The method of any one of claims 28 to 29, wherein the subject has an endogenous DNA repair function.

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34. The method of any one of claims 28 to 29, wherein the altered DNA repair function is enhanced expression of at least one gene associated with the MRN protein complex.

35. The method of any one of claims 28 to 29, wherein the altered DNA repair function is reduced expression of at least one gene associated with the MRN protein complex.

36. The method of any one of claims 28 to 35, wherein: if the subject has a genotype for endogenous or reduced expression of at least one gene associated with NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex, then the treatment protocol comprises administering an effective amount of a WEE1 inhibitor to the subject; or if the subject has a genotype for enhanced expression of at least one gene associated with NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex, then the treatment protocol comprises administering an effective amount of a cancer treatment other than a WEE1 inhibitor to the subject.

37. The method of any of claims 28 to 35, wherein: if the subject has a genotype for endogenous or reduced expression of at least one gene associated with NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex, then the treatment protocol comprises administering the WEE1 inhibitor to the subject at a first effective dosage; or if the subject has a genotype for enhanced expression of at least one gene associated with NBN, MRE11, RAD50 or MRE11-RAD50-NBN (MRN) protein complex, then the treatment protocol comprises administering the WEE1 inhibitor to the subject at second effective dosage that is higher than the first effective dosage.

38. The method of any one of claims 2 to 37, wherein the genotype for altered DNA repair function, reduced DNA repair function, loss of DNA repair function, gain of DNA repair function, enhanced gene expression, or reduced gene expression comprises an NBN mutation.

39. The method of any one of claims 2 to 38, wherein the genotype for altered DNA repair function, reduced DNA repair function, loss of DNA repair function, gain of

50 DNA repair function, enhanced gene expression, or reduced gene expression comprises an MRE11 mutation.

40. The method of any one of claims 2 to 39, wherein the genotype for altered DNA repair function, reduced DNA repair function, loss of DNA repair function, gain of DNA repair function, reduced gene expression, or enhanced gene expression comprises a RAD50 mutation.

41. The method of any one of claims 37 to 40, further comprising determining that the treatment protocol at the first effective dosage poses a lower toxicity risk to the subject than the treatment protocol at the second effective dosage.

42. The method of any one of claim 1 to 41, wherein the WEE1 inhibitor is described in any one or more of the following publications: W02020210383, W02020210375, W02020210377, W02020210380, W02020210381, WO 2019173082, W02019011228, WO2019138227, WO2018162932, W02018011570, W02018011569, W02018090939, WO2015092431, W02015019037, WO2014167347, WO2007126122, WO2011034743, US20070254892, US2008133866, US20160060258, US20190308984, US20200131192, WO2019085933, WO2020221358, EP3712150, WO2018133829, WO2019085933,

W02020083404, WO2019037678, WO2018171633, WO2019096322, WO2019165204,

WO2012161812, W02013012681, W02013013031, WO2013059485, WO2013126656,

US20120220572, US20130018045, KR2016035878, KR2020016567, WO2018056621,

WO2017075629, WO2019169065, WO2019134539, W02020028814, W02020069105,

WO2020192581, CN111718348, and WO9634867.

43. The method of any one of claims 1 to 42, wherein the WEE1 inhibitor is AZD1775, SC0191, PD0166285, NUV-569, IMP7068, Debio 0123, or any combination thereof.

44. The method of any one of claims 1 to 42, wherein the WEE1 inhibitor is a compound ZN-c3 of the formula:

or a pharmaceutically acceptable salt thereof.

45. The method of any one of claims 1 to 43, wherein the WEE1 inhibitor is a compound of the formula:

or a pharmaceutically acceptable salt or N-oxide thereof.

46. The method of any one of claims 1 to 43, wherein the WEE1 inhibitor is a

pharmaceutically acceptable salt thereof of any of the foregoing.

47. The method of any one of claims 1 to 43, wherein the WEE1 inhibitor is a compound having a formula selected from

a pharmaceutically acceptable salt thereof of any of the foregoing.

48. The method of any one of claims 1 to 43, wherein the WEE1 inhibitor is a

compound of the formula , or a pharmaceutically acceptable salt thereof.

49. The method of any one of claims 1 to 43, wherein the WEE1 inhibitor is a compound having a formula selected from:

or a pharmaceutically acceptable salt thereof of any of the foregoing.

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