Methods for treating cancers with biallelic loss-of-function or gene overexpression mutations

Abstract

A method for treating cancer with both allele ATM loss-of-function mutations is disclosed. A method for inducing cell death in cancer cells with both allele ATM loss-of-function mutations is also disclosed. The method typically comprises administering an effective amount of an ATR inhibitor to a subject in need thereof or contacting cancer cells with both allele ATM loss-of-function mutations with an ATR inhibitor. A method for treating cancer with CCNE1 amplification is also disclosed.

Description

[Technical field]

[0001] The present invention relates to methods of treating cancers with biallelic ATM loss of function using ATR inhibitors, and methods of identifying whether the mutation is biallelic or monoallelic, and whether it is germline or somatic. [Background technology]

[0002] ATR is essential for dividing cells and is therefore recognized as an important cancer target. ATR-deficient mice are embryonic lethal, whereas conditional ATR knockout adult mice are viable, affecting rapidly proliferating tissues and stem cell populations. Mouse embryonic stem cells lacking ATR divide only 1–2 times and then die. Interestingly, mice carrying a hypomorphic ATR mutation that reduced ATR expression to 10% of normal levels showed reduced H-rasG12D-induced tumor growth with minimal effects on proliferating normal cells, e.g., bone marrow or intestinal epithelial cells.

[0003] There is a need for new anti-cancer therapies, particularly those targeted at patient populations that are highly susceptible to such treatments. Summary of the Invention

[0004] Generally, the invention provides methods of treating cancer (e.g., those with biallelic ATM loss-of-function mutations or amplified CCNE1), inducing cell death in cancer cells (e.g., those with biallelic ATM loss-of-function mutations or amplified CCNE1), or identifying that a subject has biallelic loss-of-function of a target gene (e.g., ATM) or an amplified gene (e.g., CCNE1).

[0005] In one aspect, the present invention provides a method of treating a cancer harboring biallelic ATM loss-of-function mutations in a subject, comprising administering to a subject in need thereof an effective amount of an ATR inhibitor.

[0006] In another aspect, the present invention provides a method of treating cancer in a subject, comprising administering to a subject in need thereof an effective amount of an ATR inhibitor, wherein the cancer has been previously identified as having biallelic ATM loss-of-function mutations.

[0007] In yet another aspect, the invention provides a method of inducing cell death in a cancer cell harboring biallelic ATM loss-of-function mutations, comprising contacting the cell with an ATR inhibitor.

[0008] In some embodiments, the cell is in a subject. In some embodiments, the method further comprises identifying the cancer as having a biallelic ATM loss-of-function mutation prior to the administering or contacting step.

[0009] In some embodiments, the identifying step comprises: determining an integer total copy number of a locus segment and / or two integer allele-specific copy numbers of a locus segment in a cancer cell or ATM gene region of a cancer cell from a subject from read counts (e.g., from next generation sequencing) of a plurality of SNVs, including homozygous single nucleotide variants (SNVs) and heterozygous SNVs, obtained from sequencing a sample comprising the cancer cell and a reference read count; Here, cancer is at least one of the integer total copy number and the integer allele-specific copy number is zero (provided that the remaining ATM allele, if present, contains an inactivating mutation); or A biallelic ATM loss-of-function mutation is identified as occurring when none of the integer allele-specific copy numbers are zero, an ATM allele is present, and each of the ATM alleles independently has an inactivating mutation.

[0010] In some embodiments, the determining step comprises: determining total copy number log ratios, allele copy number log odds ratios, and target coverage values ​​for the SNVs from the read counts and the reference read counts; Segmenting the total copy number log ratio and the allele copy number log odds ratio; estimating sample purity and sample ploidy of the cancer cells from the total copy number log ratio and the target coverage value; and generating an integer total copy number and two integer allele-specific copy numbers of the segment containing multiple SNVs within the ATM gene region of the cancer cell from the target coverage value, sample purity, sample ploidy, total copy number log ratio, and allele copy number log odds ratio.

[0011] In some embodiments, the method further comprises adjusting the misregistration rate. In some embodiments, the plurality of SNVs includes SNVs with uniform coverage (e.g., each of the SNVs with uniform coverage has an average coverage of at least 200x reads across the panel of normal samples). In some embodiments, each of the SNVs with uniform coverage has an average coverage of at least 300x reads across the panel of normal samples. In some embodiments, the plurality of SNVs includes a high frequency SNV, the high frequency SNV having an allele frequency in humans of 33%-66%. In some embodiments, the plurality of SNVs includes an SNV adjacent to the high frequency SNV (e.g., located within 300 contiguous nucleobases downstream from the high frequency SNV). In some embodiments, the plurality of SNVs includes a plurality of SNVs, each of the plurality of SNVs having a 5'-flanking sequence of at least 20 contiguous nucleobases with a GC content of 25-75%, the 5'-flanking sequence being unique and free of other SNVs. In some embodiments, the plurality of SNVs comprises at least 20 heterozygous SNVs. In some embodiments, the reference read count is derived from a panel of normal samples. In some embodiments, the plurality of SNVs comprises scaffold SNVs (e.g., scaffold SNVs can be useful to constrain the solution space of integer total copy numbers and integer allele-specific copy numbers). In some embodiments, the ATM gene region comprises ATM and up to 10 kilobases of each flanking region. In some embodiments, the ATM gene region comprises ATM and up to 5 kilobases of each flanking region. In some embodiments, the ATM gene region comprises ATM and up to 2 kilobases of each flanking region. In some embodiments, the ATM gene region is an ATM exome region. In some embodiments, the ATM gene region is an ATM transcriptome region. In some embodiments, the ATM gene region is an ATM genomic region. In some embodiments, the biallelic ATM loss-of-function mutation comprises at least one somatic ATM loss-of-function mutation.In some embodiments, the biallelic ATM loss-of-function mutations comprise at least one germline ATM loss-of-function mutation, hi some embodiments, the biallelic ATM loss-of-function mutations comprise one somatic ATM loss-of-function mutation and one germline ATM loss-of-function mutation.

[0012] In some embodiments, the cancer is listed in FIG. 15A. In some embodiments, the cancer is listed in FIG. 15B. In some embodiments, the cancer is lung adenocarcinoma. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is invasive breast cancer. In some embodiments, the cancer is LumB positive invasive breast cancer. In some embodiments, the cancer is Her2 positive invasive breast cancer. In some embodiments, the cancer is basal-like invasive breast cancer. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is bladder urothelial carcinoma. In some embodiments, the cancer is rectal adenocarcinoma. In some embodiments, the cancer is gastric adenocarcinoma. In some embodiments, the cancer is cutaneous melanoma. In some embodiments, the cancer is colon adenocarcinoma. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is glioblastoma multiforme. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is uterine endometrial cancer. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is uterine endometrial cancer. In some embodiments, the cancer is lung squamous cell carcinoma. In some embodiments, the cancer is sarcoma. In some embodiments, the cancer is ovarian serous cystadenocarcinoma. In some embodiments, the ATR inhibitor is a compound of formula (I):

[0013] [ka]

[0014] or a pharma- ceutically acceptable salt thereof (In the formula,

[0015] [ka]

[0016] is a double bond, and each Y is independently N or CR 4 or

[0017] [ka]

[0018] is a single bond, and each Y is independently NR Y , carbonyl, or C(R Y ) 2 where each R Y are independently H or optionally substituted C 1-6 is alkyl, R 1 is optionally replaced by C 1-6 alkyl or H, R 2 is optionally replaced by C 2-9 Heterocyclyl, optionally substituted C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 2-9 Heterocyclyl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, optionally substituted C 1-9 Heteroaryl C 1-6 Alkyl, halogen, -N(R 5 ) 2 , -OR 5 , -CON(R 6 ) 2 , -SO 2 N(R 6 ) 2 , -SO 2 R 5A , or -QR5B and R 3 is optionally replaced by C 1-9 Heteroaryl or optionally substituted C 1-9 Heteroaryl C 1-6 is alkyl, Each R 4 are independently hydrogen, halogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 alkenyl, or optionally substituted C 2-6 is alkynyl, Each R 5 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, or -SO 2 R 5A or both R 5 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Each R 5A independently represents an optionally substituted C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 6-10 is aryl, R 5B is hydroxyl, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, -N(R 5 ) 2 , -CON(R 6 ) 2 , -SO 2 N(R 6 ) 2 , -SO 2 R 5Aor optionally substituted alkoxy; Each R 6 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkoxyalkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 1-9 Heteroaryl or both R 6 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Q is optionally substituted C 2-9 Heterocyclylene, optionally substituted C 3-8 Cycloalkylene, optionally substituted C 1-9 Heteroarylene or optionally substituted C 6-10 is an arylene, X is hydrogen or a halogen.

[0019] In some embodiments, the ATR inhibitor is a compound of formula (II):

[0020] [ka]

[0021] or a pharma- ceutically acceptable salt thereof (In the formula, Each Y is independently N or CR 4 and R 1 is optionally replaced by C 1-6 alkyl or H, R 2 is optionally replaced by C 2-9 Heterocyclyl, optionally substituted C 1-6Alkyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 2-9 Heterocyclyl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, optionally substituted C 1-9 Heteroaryl C 1-6 Alkyl, halogen, -N(R 5 ) 2 , -OR 5 , -CON(R 6 ) 2 , -SO 2 N(R 6 ) 2 , -SO 2 R 5A , or -QR 5B and R 3 is optionally replaced by C 1-9 Heteroaryl or optionally substituted C 1-9 Heteroaryl C 1-6 is alkyl, Each R 4 are independently hydrogen, halogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 alkenyl, or optionally substituted C 2-6 is alkynyl, Each R 5 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, or -SO 2 R 5A or both R 5 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Each R 5Aindependently represents an optionally substituted C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 6-10 is aryl, R 5B is hydroxyl, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, -N(R 5 ) 2 , -CON(R 6 ) 2 , -SO 2 N(R 6 ) 2 , -SO 2 R 5A or optionally substituted alkoxy; Each R 6 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkoxyalkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 1-9 Heteroaryl or both R 6 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Q is optionally substituted C 2-9 Heterocyclylene, optionally substituted C 3-8 Cycloalkylene, optionally substituted C 1-9 Heteroarylene or optionally substituted C 6-10 is an arylene, X is hydrogen or a halogen.

[0022] In some embodiments, the ATR inhibitor is selected from the group consisting of compounds 43, 57, 62, 87, 93, 94, 95, 99, 100, 106, 107, 108, 109, 111, 112, 113, 114, 115, 116, 118, 119, 120, 121, 122, 123, 135, 147, 148 in Table 1, and pharma- ceutically acceptable salts thereof. In some embodiments, the ATR inhibitor is compound 43 in Table 1, or a pharma- ceutically acceptable salt thereof. In some embodiments, the ATR inhibitor is compound 121 in Table 1, or a pharma- ceutically acceptable salt thereof. In some embodiments, the ATR inhibitor is compound 122 in Table 1, or a pharma- ceutically acceptable salt thereof.

[0023] In some embodiments, the ATR inhibitor is

[0024] [ka]

[0025] or a pharma- ceutically acceptable salt thereof. In yet another aspect, the present invention provides a method for identifying a cell from a subject that has a biallelic mutation in a target gene, comprising: determining an integer total copy number of a locus segment and / or two integer allele-specific copy numbers of a locus segment within a target gene region in cells from the subject from read counts of a plurality of SNVs (e.g., uniform coverage SNVs) including homozygous single nucleotide variants (SNVs) and heterozygous SNVs (e.g., homozygous and heterozygous uniform coverage SNVs) obtained from sequencing a sample comprising cells, the target gene region comprising a mutation, and the reference read counts are derived from a reference population of normal subjects; Here, the cells are at least one of the integer total copy number and the integer allele-specific copy number is zero (provided that the remaining target gene allele, if present, contains a mutation); or The method provides a method for identifying a target gene as having a biallelic mutation if none of the integer allele-specific copy numbers are zero, and if the target gene alleles are present and each of the target gene alleles independently has a mutation.

[0026] In some embodiments, the determining step comprises: determining a total copy number log ratio, an allele copy number log odds ratio, and a target coverage value for the SNV from the read counts (e.g., read counts of alternative alleles) and the reference read counts (e.g., read counts of reference alleles); Segmenting the total copy number log ratio and the allele copy number log odds ratio; estimating the sample purity and sample ploidy of the cells from the total copy number log ratio and the target coverage value; and generating an integer total copy number of the segment containing the plurality of SNVs and two integer allele-specific copy numbers of the segment from the target coverage value, the sample purity, the sample ploidy, the total copy number log ratio, and the allele copy number log odds ratio in the target gene region of the cell.

[0027] In some embodiments, the method further comprises adjusting the misregistration rate. In yet another aspect, the invention provides a method for identifying a cell from a subject that has amplification in a target gene, comprising: determining a total copy number and a sample ploidy of a locus segment within a target genomic region of cells from a subject from read counts of a plurality of uniform coverage single nucleotide variants (SNVs), including homozygous and heterozygous uniform coverage SNVs, and reference read counts obtained from sequencing a sample comprising cells, wherein the target genomic region comprises a mutation, and the reference read counts are derived from a panel of normal samples; Here, the cells are If the total copy number is at least twice the sample ploidy, or The method provides that the target gene is identified as having amplification if the total copy number is at least 2 greater than the sample ploidy.

[0028] In some embodiments, the determining step comprises: determining a total copy number log ratio of the SNVs and a target coverage value from the read counts and the reference read counts; Segmenting the total copy number log ratio; Estimating the sample purity and sample ploidy of the cells from the total copy number log ratio and the target coverage value; and generating a total copy number of the locus segment within the target gene region from the target coverage value, the sample purity, the sample ploidy, and the total copy number log ratio.

[0029] In some embodiments, the total copy number is a normalized total copy number. In some embodiments, the cell is identified as having an amplification in the target gene if the total copy number is at least 2 times the sample ploidy. In some embodiments, the cell is identified as having an amplification in the target gene if the total copy number is at least 3 times the sample ploidy. In some embodiments, the total copy number is a normalized total copy number. In some embodiments, the cell is identified as having an amplification in the target gene if the total copy number is at least 2 times greater than the sample ploidy. In some embodiments, the cell is identified as having an amplification in the target gene if the total copy number is at least 4 times greater than the sample ploidy.

[0030] In some embodiments, the method further includes adjusting the misregistration rate. In some embodiments, the target gene is CCNE1. In yet another aspect, the present invention provides a method of treating cancer in a subject, comprising: identifying the cancer as a CCNE1 amplified cancer according to the methods described herein; and administering to a subject in need thereof a therapeutically effective amount of a membrane-bound tyrosine- and threonine-specific cdc2-inhibitory kinase (Myt1) inhibitor.

[0031] In some embodiments, the Myt1 inhibitor is a compound of formula (III):

[0032] [ka]

[0033] or a pharma- ceutically acceptable salt thereof (In the formula, Each of X, Y, and Z is independently N or CR 2 and R 1 and each R 2 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 3-8 Cycloalkenyl, optionally substituted C 2-9 Heterocyclyl, optionally substituted C 2-9 Heterocyclyl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, optionally substituted C 1-9 Heteroaryl C 1-6 Alkyl, halogen, cyano, -N(R 7 ) 2 , -OR 7 , -C(O)N(R 8 ) 2 , -SO 2 N(R 8 ) 2 , -SO 2 R 7A , or -QR 7B or R1 is R 1 One R that is vicinal to 2 In combination with, and optionally substituted, C 3-6 Forming an alkylene R 3 and R 4 each independently represents an optionally substituted C 1-6 is alkyl or halogen; R 5 is H or -N(R 7 ) 2 and R 6 is -C(O)NH(R 8 ), -C(O)R 7A , or -SO 2 R 7A and Each R 7 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 6-10 Aryl, optionally substituted C 2-9 Heterocyclyl, optionally substituted C 1-9 Heteroaryl, optionally substituted C 1-9 Heteroaryl C 1-6 Alkyl, or -SO 2 R 7A or two R 7 The group, together with the atoms to which both are attached, may be an optionally substituted C 2-9 forming a heterocyclyl, Each R 7A independently represents an optionally substituted C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 6-10 is aryl, Each R 7B independently represents hydroxyl, optionally substituted C1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 2-9 Heterocyclyl, optionally substituted C 1-9 Heteroaryl, -N(R 7 ) 2 , -C(O)N(R 8 ) 2 , -SO 2 N(R 8 ) 2 , -SO 2 R 7A or optionally substituted alkoxy; Each R 8 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkoxyalkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 1-9 Heteroaryl or two R 8 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Q is optionally substituted C 1-6 Alkylene, optionally substituted C 2-6 Alkenylene, optionally substituted C 2-6 Alkynylene, optionally substituted C 3-8 Cycloalkylene, optionally substituted C 3-8 Cycloalkenylene, optionally substituted C 6-10 Arylene, optionally substituted C 2-9 Heterocyclylene, or optionally substituted C 1-9 Heteroarylene.

[0034] In some embodiments, the compound is an atropisomer of formula (IIIA):

[0035] [ka]

[0036] or a pharma- ceutically acceptable salt thereof. In some embodiments, X is CR 2 It is. In some embodiments, the compound has formula (IV):

[0037] [ka]

[0038] or a pharma- ceutically acceptable salt thereof. In some embodiments, the compound is an atropisomer of formula (IVA):

[0039] [ka]

[0040] or a pharma- ceutically acceptable salt thereof. In some embodiments, the compound has formula (V):

[0041] [ka]

[0042] or a pharma- ceutically acceptable salt thereof (In the formula, R 2A is hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 3-8Cycloalkenyl, optionally substituted C 2-9 Heterocyclyl, optionally substituted C 2-9 Heterocyclyl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, optionally substituted C 1-9 Heteroaryl C 1-6 Alkyl, halogen, -N(R 7 ) 2 , -OR 7 , -C(O)N(R 8 ) 2 , -SO 2 N(R 8 ) 2 , -SO 2 R 7A , or -QR 7B (where:

[0043] In some embodiments, the compound is an atropisomer of formula (VA):

[0044] [ka]

[0045] or a pharma- ceutically acceptable salt thereof. In some embodiments, the Myt1 inhibitor is any one of compounds 1-328 in Table 2, and pharma- ceutically acceptable salts thereof. In some embodiments, the Myt1 inhibitor is compound 181 in Table 2, or a pharma- ceutically acceptable salt thereof. In some embodiments, the Myt1 inhibitor is compound 182 in Table 2, or a pharma- ceutically acceptable salt thereof.

[0046] In some embodiments, the method further comprises administering to the subject an effective amount of a WEE1 inhibitor, a FEN1 inhibitor, a TOP1 inhibitor, a RRM1 inhibitor, a RRM2 inhibitor, an AURKB inhibitor, a TOP2A inhibitor, an ATR inhibitor, a TTK inhibitor, a SOD1 inhibitor, a SOD2 inhibitor, a BUB1 inhibitor, a CDC7 inhibitor, a SAE1 inhibitor, a PLK1 inhibitor, a UBA2 inhibitor, a DUT inhibitor, a HDAC3 inhibitor, a CHEK1 inhibitor, an AURKA inhibitor, a MEN1 inhibitor, a DOT1L inhibitor, a CREBBP inhibitor, an EZH2 inhibitor, a PLK4 inhibitor, a HASPIN inhibitor, a METTL3 inhibitor, a nucleoside analog, a platinum-based DNA damaging agent, or a combination thereof.

[0047] In some embodiments, the method further comprises adjusting the misalignment rate. In some embodiments, the plurality of SNVs comprises SNVs with uniform coverage. In some embodiments, each of the SNVs with uniform coverage has an average coverage of at least 200x reads across the panel of normal samples. In some embodiments, the plurality of SNVs comprises high frequency SNVs, the high frequency SNVs having an allele frequency of 33%-66% in humans. In some embodiments, the plurality of SNVs comprises SNVs adjacent to the high frequency SNV.

[0048] In some embodiments, the plurality of SNVs comprises a plurality of SNVs, each of the plurality of SNVs having a 5'-flanking sequence of at least 20 contiguous nucleobases with 25-75% GC content, wherein the 5'-flanking sequence is unique and does not include other SNVs. In some embodiments, the plurality of SNVs comprises at least 20 heterozygous SNVs. In some embodiments, the reference read count is derived from a panel of normal samples.

[0049] In some embodiments, the target gene region includes the target gene and up to 10 kilobases of each of the flanking regions. In some embodiments, the target gene region includes the target gene and up to 5 kilobases of each of the flanking regions. In some embodiments, the target gene region includes the target gene and up to 2 kilobases of each of the flanking regions.

[0050] In some embodiments, the target gene region is a target gene exome region. In some embodiments, the target gene region is a target gene transcriptome region. In some embodiments, the target gene region is a target gene genomic region.

[0051] In a further aspect, the invention provides a method for identifying a target mutation in a cell from a subject as being germline or somatic, comprising the steps of: determining an observed allele fraction of a locus segment within a target genomic region in cells from the subject from a plurality of single nucleotide variants (SNVs) with uniform coverage, including homozygous and heterozygous uniform coverage SNVs, obtained from sequencing a sample comprising the cells, the target genomic region comprising a target mutation; determining expected allele fractions of germline and somatic targeted mutations; comparing the observed allele fraction to an expected allele fraction to identify the most likely germline and somatic mutations; and identifying the most likely of the germline and somatic mutations as the germline or somatic targeted mutation.

[0052] In a further aspect, the invention provides a method for identifying a target mutation in a cell from a subject as being germline or somatic, comprising identifying the target mutation in a matched normal sample from the subject, Where: If the target mutation present in the cells from the subject is identified in a matched normal sample, the target mutation is germline; Provided is a method, wherein the target mutation is somatic, if the target mutation present in cells from the subject is not identified in a matched normal sample.

[0053] In some embodiments of any of the aspects, the comparing step is performed using a Bayesian model comparison. In some embodiments of any of the aspects, each of the uniform coverage SNVs has an average coverage of at least 200x reads across the panel of normal samples. In some embodiments of any of the aspects, the plurality of SNVs includes SNVs with an allele frequency in humans of 33%-66%. In some embodiments, the plurality of SNVs includes SNVs adjacent to a high frequency SNV (e.g., located within 300 contiguous nucleobases downstream from the high frequency SNV). In some embodiments, the plurality of SNVs includes a plurality of SNVs, each of the plurality of SNVs having a 5'-flanking sequence of at least 20 contiguous nucleobases with a GC content of 25-75%, the 5'-flanking sequence being unique and free of other SNVs. In some embodiments of any of the aspects, the plurality of SNVs includes at least 20 heterozygous SNVs. In some embodiments of any of the aspects, the plurality of SNVs includes scaffold SNVs (e.g., scaffold SNVs can be useful to constrain the solution space of integer total copy numbers and integer allele-specific copy numbers). In some embodiments of any of the aspects, the target genetic region includes the target gene and up to 10 kilobases of each of the flanking regions. In some embodiments of any of the aspects, the target genetic region includes the target gene and up to 5 kilobases of each of the flanking regions. In some embodiments of any of the aspects, the target genetic region includes the target gene and up to 2 kilobases of each of the flanking regions. In some embodiments of any of the aspects, the target genetic region is a target exome region. In some embodiments of any of the aspects, the target genetic region is a target transcriptome region. In some embodiments of any of the aspects, the target genetic region is a target genomic region. In some embodiments of any of the aspects, the cell from the subject is a cancer cell from the subject. In some embodiments of any of the aspects, the mutation is a germline mutation.

[0054] definition The term "acyl," as used herein, refers to the group -C(=O)-R, where R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heterocyclyl. Acyl can be optionally substituted as described herein for each R group.

[0055] The term "alkanoyl" as used herein represents a hydrogen or alkyl group attached to the parent molecular group through a carbonyl group, and is exemplified by formyl (i.e., a carboxaldehyde group), acetyl, propionyl, butyryl, and iso-butyryl. Unsubstituted alkanoyl groups contain 1 to 7 carbons. Alkanoyl groups may be unsubstituted or substituted as described herein for alkyl groups (e.g., optionally substituted C1-7 alkanoyl). The ending "-oyl" can be attached to other groups defined herein, such as aryl, cycloalkyl, and heterocyclyl, to define "aryloyl", "cycloalkanoyl", and "(heterocyclyl)oyl". These groups represent a carbonyl group substituted with aryl, cycloalkyl, or heterocyclyl, respectively. Each of "aryloyl", "cycloalkanoyl", and "(heterocyclyl)oyl" can be optionally substituted as defined for "aryl", "cycloalkyl", or "heterocyclyl", respectively.

[0056] The term "alkenyl" as used herein refers to an acyclic monovalent straight or branched chain hydrocarbon group containing one, two, or three carbon-carbon double bonds. Non-limiting examples of alkenyl groups include ethenyl, prop-1-enyl, prop-2-enyl, 1-methylethenyl, but-1-enyl, but-2-enyl, but-3-enyl, 1-methylprop-1-enyl, 2-methylprop-1-enyl, and 1-methylprop-2-enyl. Alkenyl groups can be optionally substituted as defined herein for alkyl.

[0057] The term "alkoxy" as used herein refers to a chemical substituent of formula -OR, where R is C, unless otherwise specified. 1-6 is an alkyl group. In some embodiments, the alkyl group may be further substituted as defined herein. The term "alkoxy" may be combined with other terms defined herein, such as aryl, cycloalkyl, or heterocyclyl, to define "arylalkoxy", "cycloalkylalkoxy" and "(heterocyclyl)alkoxy" groups. These groups represent alkoxy substituted with aryl, cycloalkyl, or heterocyclyl, respectively. Each of "arylalkoxy", "cycloalkylalkoxy" and "(heterocyclyl)alkoxy" may be optionally substituted as defined herein for the individual moieties.

[0058] The term "alkoxyalkyl" as used herein refers to a chemical substituent of formula -LOR, where L is C 1-6 alkylene and R is C 1-6 Optionally substituted alkoxyalkyl is optionally substituted alkoxyalkyl as described herein for alkyl.

[0059] The term "alkyl" as used herein refers to an acyclic, straight or branched chain, saturated hydrocarbon group, which, if unsubstituted, has 1 to 12 carbons, unless otherwise specified. In certain preferred embodiments, unsubstituted alkyls have 1 to 6 carbons. Examples of alkyl groups include methyl; ethyl; n- and iso-propyl; n-, sec-, iso- and tert-butyl; neopentyl, and the like, and, where valence permits, includes amino, aryl, aryloxy, azido, cycloalkyl, cycloalkoxy, cycloalkenyl, cycloalkynyl, halo, heterocyclyl, (heterocyclyl)oxy, heteroaryl, hydroxy, nitro, thiol, silyl, cyano, alkylsulfonyl, alkylsulfinyl, alkylsulfenyl, =O, =S, -SO 2 R, where R is amino or cycloalkyl, =NR', where R' is H, alkyl, aryl, or heterocyclyl, may be optionally substituted with one, two, three, or, in the case of an alkyl group of two or more carbons, four or more substituents independently selected from the group consisting of: each of the substituents may itself be unsubstituted or, if valence permits, substituted with an unsubstituted substituent(s) as defined herein for the respective group.

[0060] The term "alkylene" as used herein refers to a divalent alkyl group. Optionally substituted alkylene is optionally substituted as described herein for alkyl.

[0061] The term “alkylamino” as used herein refers to a group of the formula —N(R N1 ) 2 or -NHR N1 In the formula, R N1is alkyl as defined herein. The alkyl portion of the alkylamino may be optionally substituted as defined for alkyl. Each of the optional substituents on a substituted alkylamino may itself be unsubstituted or, if valence allows, substituted with unsubstituted substituent(s) as defined herein for the respective group.

[0062] The term "alkylsulfenyl" as used herein refers to a group of the formula -S-(alkyl). Alkylsulfenyl can be optionally substituted as defined for alkyl.

[0063] The term "alkylsulfinyl" as used herein represents a group of the formula -S(O)-(alkyl). Alkylsulfinyl can be optionally substituted as defined for alkyl.

[0064] The term "alkylsulfonyl" as used herein represents a group of formula -S(O)-(alkyl). Alkylsulfonyl can be optionally substituted as defined for alkyl.

[0065] The term "alkynyl," as used herein, refers to a monovalent straight or branched chain hydrocarbon group of 2 to 6 carbon atoms containing at least one carbon-carbon triple bond, and is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groups can be unsubstituted or substituted as defined for alkyl (e.g., optionally substituted alkynyl).

[0066] The term "allele fraction" as used herein refers to a normalized measure of the allele intensity ratio of variant alleles, with an allele fraction of 1 or 0 indicating the complete absence of one of the two alleles. For ploidy of 2, an allele fraction of 0.5 indicates that both alleles are present equally. For ploidy of 3, an allele fraction of 0.33 or 0.66 indicates that one allele is present in one copy and the other in two copies. For ploidy of 4, an allele fraction of 0.25 or 0.75 indicates that one allele is present in one copy and the other in three copies, and an allele fraction of 0.5 indicates that both alleles are present equally. Allele fraction can be measured as B allele frequency.

[0067] The term "allele copy number log odds ratio" as used herein refers to the ratio of parental copy numbers in cancer cells (E[logOR]=[p1·Φ+(1-Φ)] / [p2·Φ+(1-Φ)]), where E[logOR] is the expected value of logOR, p1 is the parental copy number of the variant allele, p2 is the parental copy number of the allele from the other parent, and Φ is the cell fraction that is a function of tumor purity and clonal frequency (in case of subclonal mutations).

[0068] The term “amino” as used herein refers to —N(R N1 ) 2 where if amino is unsubstituted, then both R N1 is H, or if amino is substituted, each R N1 are independently H, -OH, and -NO 2 , -N(R N2 ) 2 , -SO 2 OR N2 , -SO 2 R N2 , -SOR N2 , -COOR N2 , an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, aryloxy, cycloalkyl, cycloalkenyl, heteroalkyl, or heterocyclyl, provided that at least one RN1 is not H, but R N2 is independently H, alkyl, or aryl. Each of the substituents may itself be unsubstituted or substituted with the unsubstituted substituent(s) defined herein for each group. In some embodiments, amino is an unsubstituted amino (i.e., -NH 2 ), or a substituted amino (e.g., NHR N1 ) in which R N1 are independently -OH, SO 2 OR N2 , -SO 2 R N2 , -SOR N2 , -COOR N2 , optionally substituted alkyl, or optionally substituted aryl, and each R N2 may be an optionally substituted alkyl or an optionally substituted aryl. In some embodiments, the substituted amino may be an alkylamino, where the alkyl group is optionally substituted as described herein for alkyl. In some embodiments, the amino group is -NHR N1 where R N1 is optionally substituted alkyl.

[0069] The term "aryl" as used herein refers to a monocyclic, bicyclic, or polycyclic carbocyclic ring system having one or two aromatic rings. An aryl group may contain from 6 to 10 carbon atoms. All atoms in an unsubstituted carbocyclic aryl group are carbon atoms. Non-limiting examples of carbocyclic aryl groups include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, and the like. An aryl group may be unsubstituted or substituted with one, two, three, four, or five substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylsulfinyl, alkylsulfenyl, alkylsulfonyl, amino, aryl, aryloxy, azido, cycloalkyl, cycloalkoxy, cycloalkenyl, cycloalkynyl, halo, heteroalkyl, heterocyclyl, (heterocyclyl)oxy, hydroxy, nitro, thiol, silyl, and cyano. Each of the substituents can itself be unsubstituted or substituted with unsubstituted substituent(s) as defined herein for the respective group.

[0070] The term "arylalkyl" as used herein refers to an alkyl group substituted with an aryl group. The aryl and alkyl portions may be optionally substituted as with the individual groups described herein.

[0071] The term "arylene" as used herein refers to a divalent aryl group. An optionally substituted arylene is an optionally substituted arylene as described herein for aryl.

[0072] The term "aryloxy" as used herein represents a chemical substituent of formula -OR, where R is an aryl group, unless otherwise specified. In an optionally substituted aryloxy, the aryl group is optionally substituted as described herein for aryl.

[0073] The term "ATM," as used herein, refers to ATM serine / threonine kinase. The term "ATR inhibitor," as used herein, refers to an agent that, upon contact with the enzyme ATR kinase, whether in vitro, in cell culture, or in an animal, reduces the IC of ATR kinase measured. 50 is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM).

[0074] The term "ATR kinase" as used herein refers to ataxia-telangiectasia- and RAD-3-related protein kinase. The term "azido" as used herein means -N 3 Represents a group.

[0075] The term "biallelic loss-of-function mutation" as used herein refers to a mutation in a cell that results in the loss of an active form of a target gene in a subject's cell (e.g., a cancer cell). For example, "biallelic ATM loss-of-function mutation" refers to a mutation in a cell that results in the loss of an active form of the ATM gene in a subject's cell (e.g., a cancer cell).

[0076] The term "carbocyclic" as used herein refers to an optionally substituted C3-16 monocyclic, bicyclic, or tricyclic structure, the rings of which may be aromatic or non-aromatic and are formed by carbon atoms. Carbocyclic structures include cycloalkyl, cycloalkenyl, cycloalkynyl, and certain aryl groups.

[0077] The term "carbonyl" as used herein refers to a -C(O)- group. The term "cyano" as used herein refers to a -CN group. The term "cycloalkenyl," as used herein, unless otherwise specified, refers to a non-aromatic carbocyclic group having at least one double bond and 3 to 10 carbons in the ring (e.g., C 3-10 Cycloalkenyl refers to cycloalkyl, cycloprop-1-enyl, cycloprop-2-enyl, cyclobut-1-enyl, cyclobut-1-enyl, cyclobut-2-enyl, cyclopent-1-enyl, cyclopent-2-enyl, cyclopent-3-enyl, norbornen-1-yl, norbornen-2-yl, norbornen-5-yl, and norbornen-7-yl. Cycloalkenyl groups can be unsubstituted or substituted as described for cycloalkyl (e.g., optionally substituted cycloalkenyl). Non-limiting examples of cycloalkenyl include cycloprop-1-enyl, cycloprop-2-enyl, cyclobut-1-enyl, cyclobut-1-enyl, cyclobut-2-enyl, cyclopent-1-enyl, cyclopent-2-enyl, cyclopent-3-enyl, norbornen-1-yl, norbornen-2-yl, norbornen-5-yl, and norbornen-7-yl. Cycloalkenyl groups can be unsubstituted or substituted as described for cycloalkyl (e.g., optionally substituted cycloalkenyl).

[0078] The term "cycloalkenylalkyl," as used herein, refers to an alkyl group substituted with a cycloalkenyl group, each of which is as defined herein. The cycloalkenyl and alkyl portions can be substituted as individual groups defined herein.

[0079] The term "cycloalkoxy" as used herein refers to a chemical substituent of formula -OR, where R is a cycloalkyl group, unless otherwise specified. In some embodiments, the cycloalkyl group can be further substituted as defined herein.

[0080] The term "cycloalkyl," as used herein, unless otherwise specified, refers to a cyclic alkyl group having 3 to 10 carbons (e.g., C 3-C10Cycloalkyl groups may be monocyclic or bicyclic. Bicyclic cycloalkyl groups may be of the type bicyclo[pq0]alkyl, where each of p and q is independently 1, 2, 3, 4, 5, 6, or 7, with the proviso that the sum of p and q is 2, 3, 4, 5, 6, 7, or 8. Alternatively, bicyclic cycloalkyl groups may include bridged cycloalkyl structures, such as bicyclo[pqr]alkyl, where r is 1, 2, or 3, and each of p and q is independently 1, 2, 3, 4, 5, or 6, with the proviso that the sum of p, q, and r is 3, 4, 5, 6, 7, or 8. A cycloalkyl group can be a spirocyclic group, such as spiro[pq]alkyl, where each of p and q is independently 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 4, 5, 6, 7, 8, or 9. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2.2.1.]heptyl, 2-bicyclo[2.2.1.]heptyl, 5-bicyclo[2.2.1.]heptyl, 7-bicyclo[2.2.1.]heptyl, and decalinyl. Cycloalkyl groups can be unsubstituted or can be selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylsulfinyl, alkylsulfenyl, alkylsulfonyl, amino, aryl, aryloxy, azido, cycloalkyl, cycloalkoxy, cycloalkenyl, cycloalkynyl, halo, heteroalkyl, heterocyclyl, (heterocyclyl)oxy, heteroaryl, hydroxy, nitro, thiol, silyl, cyano, =O, =S, -SO 2 R (wherein R is amino or cycloalkyl), =NR' (wherein R' is H, alkyl, aryl, or heterocyclyl), or -CON(R A ) 2 (In the formula, each R A are independently H or alkyl, or both R Atogether with the atom to which they are attached form a heterocyclyl) (e.g., an optionally substituted cycloalkyl). Each of the substituents may itself be unsubstituted or substituted with an unsubstituted substituent(s) as defined herein for the respective group.

[0081] The term "cycloalkylalkyl," as used herein, refers to an alkyl group substituted with a cycloalkyl group, each as defined herein. The cycloalkyl and alkyl portions can be optionally substituted as the individual groups described herein.

[0082] The term "cycloalkylene" as used herein refers to a divalent cycloalkyl group. Optionally substituted cycloalkylene is cycloalkylene optionally substituted as described herein for cycloalkyl.

[0083] The term "cycloalkynyl," as used herein, unless otherwise specified, refers to a monovalent carbocyclic group having one or two carbon-carbon triple bonds and having 8 to 12 carbons. Cycloalkynyls can include one transannular bond or bridge. Non-limiting examples of cycloalkynyls include cyclooctynyl, cyclononynyl, cyclodecynyl, and cyclodecadinyl. Cycloalkynyl groups can be unsubstituted or substituted as defined for cycloalkyl (e.g., optionally substituted cycloalkynyl).

[0084] "Disease" or "Condition" refers to a condition or medical state of a patient or subject that can be treated with the compounds or methods provided herein. The term "halo," as used herein, refers to a halogen selected from bromine, chlorine, iodine, and fluorine.

[0085] The term "heteroalkyl" as used herein refers to an alkyl, alkenyl, or alkynyl group interrupted once by one or two heteroatoms, interrupted twice by one or two heteroatoms, each time independently, interrupted three times by one or two heteroatoms, each time independently, or interrupted four times by one or two heteroatoms, each time independently. Each heteroatom is independently O, N, or S. In some embodiments, the heteroatom is O or N. No heteroalkyl group contains two consecutive oxygen or sulfur atoms. Heteroalkyl groups may be unsubstituted or substituted (e.g., optionally substituted heteroalkyl). When a heteroalkyl is substituted and a substituent is attached to a heteroatom, the substituent is selected according to the nature and valence of the heteroatom. Thus, substituents attached to a heteroatom may be O, -N(R ), if valence permits, or any other group. N2 ) 2 , -SO 2 OR N3 , -SO 2 R N2 , -SOR N3 , -COOR N3 , an N-protecting group, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, or cyano, N2 is independently H, alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, or heterocyclyl, and each R N3 is independently alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, or heterocyclyl. Each of these substituents may itself be unsubstituted or substituted with the unsubstituted substituent(s) defined herein for the respective group. When a heteroalkyl is substituted and a substituent is attached to a carbon, the substituent is selected from those described for alkyl, except that the substituent on the carbon atom attached to the heteroatom is not Cl, Br, or I. It is understood that the carbon atom is at the terminal end of the heteroalkyl group.

[0086] The term "heteroarylalkyl," as used herein, refers to an alkyl group substituted with a heteroaryl group, each of which is as defined herein. The heteroaryl and alkyl portions can be optionally substituted as with the individual groups described herein.

[0087] The term "heteroarylene" as used herein refers to a divalent heteroaryl. Optionally substituted heteroarylene is optionally substituted as described herein for heteroaryl.

[0088] The term "heteroaryloxy" as used herein refers to the structure -OR, where R is a heteroaryl. The heteroaryloxy can be optionally substituted as defined for heterocyclyl.

[0089] The term "heterocyclyl" as used herein refers to a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having fused, bridged, and / or spiro 3-, 4-, 5-, 6-, 7-, or 8-membered rings, and unless otherwise specified, containing 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, a "heterocyclyl" is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having fused or bridged 5-, 6-, 7-, or 8-membered rings, and unless otherwise specified, containing 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. A heterocyclyl can be aromatic or non-aromatic. Non-aromatic 5-membered heterocyclyls have zero or one double bond, non-aromatic 6- and 7-membered heterocyclyl groups have zero to two double bonds, and non-aromatic 8-membered heterocyclyl groups have zero to two double bonds and / or zero or one carbon-carbon triple bond. Heterocyclyl groups contain 1 to 16 carbon atoms, unless otherwise specified. Certain heterocyclyl groups can contain up to 9 carbon atoms. Non-aromatic heterocyclyl groups include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, thiazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, etc. When a heterocyclic ring system has at least one aromatic resonance structure or at least one aromatic tautomer, such structure is an aromatic heterocyclyl (i.e., heteroaryl).Non-limiting examples of heteroaryl groups include benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, isoindazolyl, isoquinolinyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl, quinazolinyl, quinolinyl, thiadiazolyl (e.g., 1,3,4-thiadiazole), thiazolyl, thienyl, triazolyl, tetrazolyl, etc. The term "heterocyclyl" also refers to heterocyclic compounds having bridged polycyclic structures in which one or more carbon and / or heteroatoms bridge two non-adjacent members of a monocyclic ring, such as, for example, quinuclidine, tropane, or diaza-bicyclo[2.2.2]octane. The term "heterocyclyl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocycles are fused to one, two, or three carbon rings, such as an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentene ring, or another monocyclic heterocycle. Examples of fused heterocyclyls include 1,2,3,5,8,8a-hexahydroindolizine, 2,3-dihydrobenzofuran, 2,3-dihydroindole, and 2,3-dihydrobenzothiophene. Heterocyclyl groups can be unsubstituted or substituted with one, two, three, four, five, or six substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylsulfinyl, alkylsulfenyl, alkylsulfonyl, amino, aryl, aryloxy, azido, cycloalkyl, cycloalkoxy, cycloalkenyl, cycloalkynyl, halo, heteroalkyl, heterocyclyl, (heterocyclyl)oxy, hydroxy, nitro, thiol, silyl, cyano, =0, =S, =NR', where R' is H, alkyl, aryl, or heterocyclyl. Each of the substituents can itself be unsubstituted or substituted with the unsubstituted substituent(s) defined herein for the respective group.

[0090] The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with a heterocyclyl group, each of which is as defined herein. The heterocyclyl and alkyl portions can be optionally substituted as the individual groups described herein.

[0091] The term "heterocyclylene" as used herein refers to a divalent heterocyclyl. Optionally substituted heterocyclylene is optionally substituted as described herein for heterocyclyl.

[0092] The term "(heterocyclyl)oxy" as used herein represents a chemical substituent of formula -OR, where R is a heterocyclyl group, unless otherwise specified. (Heterocyclyl)oxy can be optionally substituted in the manner described for heterocyclyl.

[0093] The terms "hydroxyl" and "hydroxy", as used interchangeably herein, refer to the -OH group. The term "isotopically enriched" as used herein refers to a pharmaceutical active agent in which one isotope at a given position in a molecule has an isotopic content that is at least 100 times greater than the natural abundance of the isotope. For example, a composition that is isotopically enriched for deuterium includes an active agent that has at least one hydrogen atom position with an abundance of deuterium that is at least 100 times greater than the natural abundance of deuterium. Preferably, the isotopic enrichment of deuterium is at least 100 times greater than the natural abundance of deuterium. More preferably, the isotopic enrichment of deuterium is at least 4000 times greater than the natural abundance of deuterium (e.g., at least 4750 times greater, e.g., up to 5000 times greater).

[0094] The term "Myt1," as used herein, refers to the membrane-bound tyrosine- and threonine-specific cdc2-inhibitory kinase (Myt1) (gene name PKMYT1). The term "Myt1 inhibitor," as used herein, refers to an agent that, upon contact with the enzyme Myt1, either in vitro, in cell culture, or in an animal, reduces the IC of Myt1 as measured. 50 is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 3 nM or less) and can be as low as 100 pM or 10 pM. 50 More preferably, the IC of Myt1 is 1 nM to 1 μM (e.g., 1 nM to 750 nM, 1 nM to 500 nM, or 1 nM to 250 nM). 50 is less than 20 nm (for example, 1 nM to 20 nM).

[0095] The term "next generation sequencing (NGS)" as used herein refers to a sequencing method that allows for massively parallel sequencing of clonally amplified molecules and single nucleic acid molecules.Non-limiting examples of NGS include sequencing by synthesis using reversible dye terminators and sequencing by ligation.

[0096] The term "nitro" as used herein means -NO 2 Represents a group. The term "oxo," as used herein, represents a divalent oxygen atom (eg, the structure of oxo can be depicted as ═O).

[0097] The term "Ph," as used herein, represents phenyl. The term "pharmaceutical composition" as used herein refers to a composition that contains a compound described herein, is formulated with a pharma- ceutical acceptable excipient, and is manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., tablets, capsules, caplets, gelcaps, or syrups), for topical administration (e.g., as creams, gels, lotions, or ointments), for intravenous administration (e.g., as a sterile solution in a solvent system suitable for intravenous use without particulate insoluble matter), or any other formulation described herein.

[0098] The term "pharmaceutical acceptable excipient" or "pharmaceutical acceptable carrier" is used interchangeably herein and refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound) that has non-toxic and non-inflammatory properties in patients. Excipients can include, for example, anti-adherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (coloring agents), emollients, emulsifiers, bulking agents (diluents), film-forming agents or coating agents, flavors, fragrances, glidants (glidants), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, or hydrating water. Examples of excipients include butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0099] The term "pharmaceutically acceptable salts" as used herein refers to salts that are suitable for use in contact with human and animal tissues, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic reactions, etc., and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or can be prepared separately by reacting a free base group with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogensulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Examples of the salts include phosphate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate.Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0100] The term "protecting group" as used herein refers to a group intended to protect a hydroxy, amino, or carbonyl from participating in one or more undesired reactions during chemical synthesis. The term "O-protecting group" as used herein refers to a group intended to protect a hydroxy or carbonyl group from participating in one or more undesired reactions during chemical synthesis. The term "N-protecting group" as used herein refers to a group intended to protect a nitrogen-containing (e.g., amino, amido, heterocyclic NH, or hydrazine) group from participating in one or more undesired reactions during chemical synthesis. Commonly used O-protecting groups and N-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis," 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. O- and N-protecting groups include alkanoyl, aryloyl, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl, tri-isopropylsilyloxymethyl, 4,4′-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4-isopropylphenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl.

[0101] O-protecting groups for protecting carbonyl-containing groups include, but are not limited to, acetals, acylals, 1,3-dithianes, 1,3-dioxanes, 1,3-dioxolanes, and 1,3-dithiolanes.

[0102] Other O-protecting groups include substituted alkyl, aryl, and aryl-alkyl ethers (e.g., trityl, methylthiomethyl, methoxymethyl, benzyloxymethyl, siloxymethyl, 2,2,2-trichloroethoxymethyl, tetrahydropyranyl, tetrahydrofuranyl, ethoxyethyl, 1-[2-(trimethylsilyl)ethoxy]ethyl, 2-trimethylsilylethyl, t-butyl ether, p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl), silyl, silyl ethers (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, triphenylsilyl, and diphenylmethylsilyl), carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, vinyl, allyl, nitrophenyl, benzyl, methoxybenzyl, 3,4-dimethoxybenzyl, and nitrobenzyl).

[0103] Other N-protecting groups include chiral auxiliaries such as protected or unprotected D, L or D,L-amino acids, such as alanine, leucine, phenylalanine, and the like; sulfonyl-containing groups, such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate-forming groups, such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5 dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4 methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5 trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl Examples of the aryl groups include, but are not limited to, aryl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like; aryl-alkyl groups such as benzyl, p-methoxybenzyl, 2,4-dimethoxybenzyl, triphenylmethyl, benzyloxymethyl, and the like; silylalkyl acetal groups such as [2-(trimethylsilyl)ethoxy]methyl, and silyl groups such as trimethylsilyl, and the like. Useful N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, dimethoxybenzyl, [2-(trimethylsilyl)ethoxy]methyl (SEM), tetrahydropyranyl (THP), t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0104] The term "purity," as used herein with respect to methods involving identification of loss of function, refers to the proportion of target cells (e.g., cancer cells) to total cells in a sample. The term "sarcoma" generally refers to a tumor composed of a substance like embryonic connective tissue, and generally consists of closely packed cells embedded in a fibrous or homogeneous substance. Non-limiting examples of sarcomas that may be treated with the compounds or methods provided herein include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy sarcoma, liposarcoma (adipose tissue), and sarcoma (sarcoma). sarcoma, liposarcoma), alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chlorosarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multifocal pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cell, immunoblastic sarcoma of T cell, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulum cell sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0105] The term "scaffold SNVs" as used herein refers to frequent, well-covered single-nucleotide variants that lie outside of the target gene region and are spaced throughout the chromosome that bears the target gene region.

[0106] The term "subject" as used herein refers to a human or non-human animal (e.g., a mammal) suffering from or at risk of a disease or condition as determined by a qualified professional (e.g., a physician or clinical nurse) with or without clinical test(s) known in the art of a sample(s) from the subject. Preferably, the subject is a human. Non-limiting examples of diseases and conditions include diseases with symptoms of cellular hyperproliferation, such as cancer.

[0107] The term "target coverage," as used herein, refers to the average number of reads that align to the chromosomal locations of a target region. The term "tautomer" refers to structural isomers that are readily interconverted, often by rearrangement of a proton. Tautomers are distinct chemical species that can be distinguished by different spectroscopic characteristics, but generally cannot be individually isolated. Non-limiting examples of tautomers include ketone-enol, enamine-imine, amide-imidic acid, nitroso-oxime, ketene-ynol, and amino acid-ammonium carboxylate.

[0108] The term "therapeutically effective amount" as used herein means an amount of an ATR inhibitor sufficient to treat cancer. The term "total copy number log ratio" as used herein refers to the signal ratio of cancer cells to control cells. For a given region, deviation of the total copy number log ratio from the mean of 0 indicates that the signal intensity is higher (if greater than 0) or lower (if less than 0) than expected for two chromosome copies. Total copy number log ratio, also known as LogR, can be estimated using Illumina's GenomeStudio™ software.

[0109] "Treatment" and "treating" as used herein refer to the medical management of a subject with the intent to improve, ameliorate, stabilize, prevent or cure a disease or condition. The terms include active treatment (treatment directed at improving the disease or condition), causal treatment (treatment directed at the cause of the associated disease or condition), palliative treatment (treatment designed to relieve the symptoms of the disease or condition), preventive treatment (treatment directed at minimizing or partially or completely suppressing the occurrence of the associated disease or condition), and supportive treatment (treatment employed to complement another treatment). The disease or condition may be cancer. [Brief description of the drawings]

[0110] [Figure 1]1 shows the structure of germline ATM loss-of-function mutations. A and C show monoallelic germline ATM loss-of-function mutations, and B shows biallelic germline ATM loss-of-function mutations. [Figure 2A] 1 is a chart showing copy number calls across all chromosomes determined by whole genome sequencing (WGS) for a 46 year old female subject with papillary serous carcinoma. [Figure 2B] FIG. 1 is a chart showing copy number calls across all chromosomes determined using single nucleotide variant (SNV) panel version 1 for a 46 year old female subject with papillary serous carcinoma. [Figure 3A] 1 is a chart showing copy number calls across all chromosomes determined by WGS for a 62-year-old female subject with pancreatic ductal adenocarcinoma. [Figure 3B] 1 is a chart showing copy number calls across all chromosomes determined using SNV panel version 1 for a 62-year-old female subject with pancreatic ductal adenocarcinoma. [Figure 4A] 1 is a chart showing copy number calls across all chromosomes determined by WGS for a 72 year old female subject with metastatic breast cancer (ER-, PR-, Her2-). [Figure 4B] 1 is a chart showing copy number calls across all chromosomes determined using SNV panel version 1 for a 72 year old female subject with metastatic breast cancer (ER-, PR-, Her2-). [Figure 5A] 1 is a chart showing single nucleotide variant (SNV) coverage of SNV panel version 1 on chromosome 1. [Figure 5B] 1 is a chart showing SNV coverage of SNV panel version 2 on chromosome 1. [Figure 6] FIG. 1 is a set of charts showing sequencing coverage depth (top graph), b allele fraction (middle graph), and copy number profile (bottom graph) for cancer biopsies from subjects with biallelic germline ATM loss-of-function mutations. [Figure 7A]1 is a chart showing copy number calls across all chromosomes determined by WGS for a 55 year old subject with lung adenocarcinoma. [Figure 7B] 1 is a chart showing copy number calls across all chromosomes determined using SNV panel version 2 for a 55 year old subject with lung adenocarcinoma. [Figure 8A] 1 is a chart showing copy number calls across all chromosomes determined by WGS for a 72 year old subject with breast cancer lum B. [Figure 8B] 1 is a chart showing copy number calls across all chromosomes determined using SNV panel version 2 for a 72 year old subject with breast cancer lum B. [Figure 9A] 1 is a chart showing copy number calls across all chromosomes determined by WGS for a 41 year old subject with bladder cancer. [Figure 9B] 1 is a chart showing copy number calls across all chromosomes determined using SNV panel version 2 for a 41 year old subject with bladder cancer. [Figure 10A] 1 is a chart showing copy number calls across all chromosomes determined by WGS for an 80 year old subject with bladder cancer. [Figure 10B] 1 is a chart showing copy number calls across all chromosomes determined using SNV panel version 2 for an 80 year old subject with bladder cancer. [Figure 11A] 1 is a chart showing copy number calls across all chromosomes determined by WGS for a 66 year old subject with luminal B breast cancer. [Figure 11B] 1 is a chart showing copy number calls across all chromosomes determined using SNV panel version 2 for a 66 year old subject with luminal B breast cancer. [Figure 12A] 1 is a chart showing copy number calls across all chromosomes determined by WGS for a 62 year old subject with prostate cancer. [Figure 12B]1 is a chart showing copy number calls across all chromosomes determined using SNV panel version 2 for a 62 year old subject with prostate cancer. [Figure 13A] 1 is a chart showing copy number calls across all chromosomes determined by WGS for a 69 year old subject with uterine cancer. [Figure 13B] 1 is a chart showing copy number calls across all chromosomes determined using SNV panel version 2 for a 69 year old subject with uterine cancer. [Figure 14A] 1 is a chart showing copy number calls across all chromosomes determined by WGS for a 46 year old subject with triple negative breast cancer. [Figure 14B] 1 is a chart showing copy number calls across all chromosomes determined using SNV panel version 2 for a 46 year old subject with triple negative breast cancer. [Figure 15A] 1 is a bar graph showing the frequency of biallelic ATM mutations in various cancers. [Figure 15B] 1 is a bar graph showing the frequency of biallelic germline ATM mutations in various cancers. [Figure 16] 1 is a chart showing the observed sequencing coverage (x reads) for a series of downstream positions downstream from the primer binding site. The chart shows that better quality samples yield higher sequencing coverage across all positions. A panel of normal samples included good and moderate quality samples. [Figure 17A] 1 is a chart showing the total copy number log ratio of a sample containing cells with amplified CCNE1. Using the methods described herein, the sample was identified as having 31 total copies of CCNE1 and 30 copies of CCNE1 after normalization to diploidy. [Figure 17B]1 is a chart showing the total copy number log ratio of a sample containing cells with amplified CCNE1. Using the methods described herein, the sample was identified as having 31 total copies of CCNE1 and 17 copies of CCNE1 after normalization to diploidy. [Figure 17C] 1 is a chart showing the total copy number log ratio of a sample containing cells with amplified CCNE1. Using the methods described herein, the sample was identified as having 9 total copies of CCNE1 and 5 copies of CCNE1 after normalization to diploidy. [Figure 18A] 1 is a chart showing total copy number determined using SNV panel version 2. The total copy number of CCNE1 was determined to be 13, and after normalization to diploidy, was 9. [Figure 18B] 1 is a chart showing the total copy number determined using WGS. The total copy number of CCNE1 was determined to be 26, and after normalization to diploidy, it was 19. [Figure 19A] 1 is a chart showing total copy number determined using SNV panel version 2. The total copy number of CCNE1 was determined to be 8, and after normalization to diploidy, it was 8. [Figure 19B] 1 is a chart showing the total copy number determined using WGS. The total copy number of CCNE1 was determined to be 40, and after normalization to diploidy, it was 19. [Figure 20A] 1 is a chart showing total copy number determined using SNV panel version 2. The total copy number of CCNE1 was determined to be 10, and after normalization to diploidy, it was 6. [Figure 20B] 1 is a chart showing the total copy number determined using WGS. The total copy number of CCNE1 was determined to be 19, and after normalization to diploidy, it was 11. [Figure 21] Total copy numbers determined using SNV panel version 2 and WGS Ascat for various genes (N=24 samples, 605 genes) including CCNE1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0111] Generally, the present invention relates to methods of treating cancers that have biallelic ATM loss-of-function mutations, methods of inducing cell death in cancer cells that have biallelic ATM loss-of-function mutations, or methods of identifying target gene mutations (e.g., ATM).

[0112] The method of treating cancer or inducing cell death described herein typically utilizes ATR inhibitor.Cancer can be, for example, lung adenocarcinoma, adrenocortical carcinoma, invasive breast cancer (e.g., invasive breast cancer: LumB; invasive breast cancer: Her2; or invasive breast cancer: basal cell), pancreatic adenocarcinoma, bladder urothelial carcinoma, rectal adenocarcinoma, gastric adenocarcinoma, skin melanoma, colon adenocarcinoma, prostate adenocarcinoma, glioblastoma multiforme, esophageal carcinoma, uterine endometrial carcinoma, hepatocellular carcinoma, uterine endometrial carcinoma, lung squamous cell carcinoma, sarcoma, or ovarian serous cystadenocarcinoma.

[0113] Advantageously, the methods of the present invention use ATR inhibitors to treat cancers that have been found to be particularly responsive to ATR inhibition, these cancers harboring biallelic ATM loss-of-function mutations.

[0114] The method of the present invention also addresses the challenge of distinguishing between biallelic and uniallelic loss-of-function mutations, as well as between germline and somatic mutations. Advantageously, the method of the present invention explicitly considers sample purity, and is therefore substantially unaffected by contaminated samples. A further advantage of the method of the present invention is that it can utilize existing data from a panel of normal samples (normal non-cancerous tissues from a reference population) and does not require normal tissue samples from the subject.

[0115] Typically, subjects have a monoallelic germline (e.g., ATM) loss-of-function mutation and subsequently acquire a somatic loss-of-function mutation in the same gene (e.g., ATM). Thus, these subjects have biallelic (e.g., ATM) loss-of-function mutations.

[0116] Identification of biallelic loss of function A subject or its cancer cells may be identified as having biallelic loss of function of a gene, for example, using whole genome sequencing (WGS) or whole exome sequencing (WES). The method of the present invention addresses the need to identify biallelic loss-of-function mutations. Three exemplary structures of loss-of-function mutations are illustrated in Figures 1A-1C. Figures 1A and 1C show monoallelic loss-of-function mutations, and Figure 1B shows biallelic loss-of-function mutations. Typical next generation sequencing techniques used for cancer testing cannot distinguish between these structures. Immunohistochemistry (IHC) cannot distinguish between the biallelic mutation in Figure 1B and the monoallelic mutation in Figure 1C, resulting in apparent ATM protein loss. As described herein, a method involving identification of biallelic ATM loss-of-function mutations distinguishes between the biallelic mutation in Figure 1B and the monoallelic mutation in Figures 1A and 1C.

[0117] Advantageously, the methods presented herein identify that a subject or their cancer cells have biallelic loss of function of a gene with greater cost efficiency and targeted gene coverage than WGS and WES techniques.

[0118] Typically, the method of the present invention may include determining the total integer copy number of a locus segment and / or two integer allele-specific copy numbers of a locus segment in a target gene (e.g., ATM) region of a cancer cell or cancer cell from a subject from a read count and a reference read count of a plurality of SNVs, including homozygous single nucleotide variants (SNVs) and heterozygous SNVs, obtained from sequencing a sample containing cancer cells, wherein the cancer is identified as having a biallelic (e.g., ATM) loss-of-function mutation when at least one of the total integer copy number and the allele-specific copy number of the integer is 0. When the total integer copy number is 0, the detected mutation is a homozygous deletion. Thus, a homozygous deletion indicates a biallelic loss-of-function mutation of a target gene (e.g., ATM). When the total integer copy number is greater than 0 and the allele-specific copy number of the integer is 0 (e.g., in a locus where an ATM inactivating mutation is found), the detected mutation is a heterozygous loss. Thus, if the remaining alleles of the target gene (e.g., ATM) contain inactivating mutations, an integer allele-specific copy number of 0 indicates that the subject has both alleles of the target gene (e.g., ATM). For example, the determining step may include: determining a total copy number log ratio, an allele copy number log odds ratio, and a target coverage of heterozygous SNVs from the read counts and reference read counts of a plurality of SNVs, including homozygous SNVs and heterozygous SNVs, obtained from sequencing a sample containing cancer cells; segmenting the total copy number log ratio and the allele copy number log odds ratio; estimating the sample purity and sample ploidy of the cancer cells from the total copy number log ratio and the target coverage value; and generating an integer total copy number and two integer allele-specific copy numbers of the segment containing a plurality of heterozygous single nucleotide variants (SNVs) in the target gene region (e.g., ATM gene region) of the cancer cells from the target coverage value, sample purity, sample ploidy, total copy number log ratio, and allele copy number log odds ratio. Typically, cells derived from a subject are provided as a biopsy.Read counts can be obtained using next generation sequencing of cells in the sample.

[0119] Alternatively, the method of the present invention can utilize B allele frequency analysis to identify biallelic (e.g., ATM) loss of function. For example, the method can include determining a plurality of allele fractions of SNVs in a cancer cell or a target gene region (e.g., ATM gene region) from a subject, and segmenting the plurality of allele fractions to generate a plurality of constant allele fraction segments, where the cancer is identified as having a biallelic loss of function mutation (e.g., biallelic ATM loss of function mutation) if the target gene region (e.g., ATM gene region) includes a segmental locus lacking an SNV with an allele fraction of 0.05 to 0.95.

[0120] Among the methods described herein, the methods that utilize integer allele-specific copy numbers and integer total copy numbers are more advantageous than other methods because these methods are robust and can be used to process low purity samples.Furthermore, the methods described herein that utilize integer allele-specific copy numbers and integer total copy numbers can utilize existing data from a panel of normal samples from a reference population, and do not require normal tissue samples from the subject.Therefore, such methods allow for the determination of both allele loss-of-function mutations based on a single sample (e.g., biopsy) from the subject.

[0121] Target SNV The target SNVs used in the methods of the present invention can be selected from those known in the art according to several selection criteria specified below. SNVs can be searched, for example, at gnomad.broadinstitute.org.

[0122] The target SNV preferably has uniform coverage across samples. The target SNV has uniform coverage across samples if its average coverage across a panel of normal samples is at least 50x reads (e.g., at least 100x reads, at least 200x reads, at least 300x reads, at least 400x reads, or at least 500x reads). The panel of targeted SNVs may have an average coverage of at least 50x (e.g., at least 100x, at least 200x, at least 300x, at least 400x, at least 500x, at least 600x, at least 700x, at least 800x, at least 900x, or at least 1000x (e.g., 100x to 2500x, 200x to 2500x, 300x to 2500x, 400x to 2500x, 500x to 2500x, 600x to 2500x, 700x to 2500x, 800x to 2500x, 900x to 2500x, or 1000x to 2500x)) across a panel of normal samples. The panel of normal samples is derived from normal tissues of a reference population that is assumed to be chromosomally normal. A panel of normal samples has an SNV allele fraction of 0–0.1 for homozygous variants, 0.4–0.6 for heterozygous variants, and 0.9–1 for absent variants. Typically, a panel of normal samples is compiled from samples of the same tissue type as the subject samples.

[0123] The target SNV may be a high frequency SNV, for example, a high frequency SNV may have an allele frequency in humans greater than 33% (e.g., 33%-66%). Here, the assessment of the allele frequency in humans may be based on an SNV source, for example, Gnomad. The inbreeding coefficient of the reference population may be 0-0.2. Furthermore, the target SNV may be a close SNV, which is an SNV with uniform coverage located within the 3'-flanking sequence relative to the high frequency SNV, and the 3'-flanking sequence comprises a total of 300 contiguous nucleic acid bases.

[0124] The target SNV may have a 5'-flanking sequence of at least 20 contiguous nucleobases (e.g., 20-50 contiguous nucleobases, e.g., 50 contiguous nucleobases) that contains 25-75% GC content. Typically, the 5'-flanking sequence is unique (i.e., the sequence of 20 contiguous nucleobases is not found anywhere else in the target genome) and does not contain other SNVs.

[0125] The target SNV may be a clean SNV, which has a variant allele fraction (VAF) value within the ranges of 0-0.1, 0.4-0.6, and 0.9-1 in at least 95% of samples of a reference population.

[0126] Typically, the target SNV can be detected using a primer-based detection technique (e.g., next-generation sequencing technique). For multiple target SNVs, multiple primers can be designed using techniques and methods known in the art. When selecting target SNVs from a sequenced sample containing cancer cells from a subject, the target SNVs can be selected that are located within the 3'-flanking sequence relative to the binding sites of the multiple primers used. The 3'-flanking sequence is typically a sequence that contains 300 or less (e.g., 200 or less) consecutive nucleic acid bases in the 3' direction relative to the binding site of the primer used. The number of consecutive nucleic acid bases selected for the 3'-flanking sequence can be influenced by the level of DNA damage and the length of the DNA fragments of each patient sample. For example, when the average coverage is 100x or more (e.g., 200x or more), a 3'-flanking sequence with 200 or less consecutive nucleic acid bases can be used. For example, for samples with more than 17% of the input DNA fragments longer than 130 bp, a 3'-flanking sequence of 300 bp will be used, otherwise a 3'-flanking sequence of 200 bp will be used. As a general matter, the length of the 3'-flanking sequence can be adjusted taking into account the sequencing technique utilized for sample analysis and sample quality, with lower quality samples (i.e. samples with a high degree of DNA fragmentation) typically requiring the use of shorter 3'-flanking sequences and / or higher average coverage levels.

[0127] Advantageously, the method described herein does not require a normal tissue sample of the subject to determine whether the mutation is monoallelic or biallelic.Instead, the method described herein can utilize a reference population sample.For example, the reads from a panel of normal samples can be used instead of the normal reads in the BAM file.

[0128] Total copy number log ratio Total copy number log ratios (LogR) can be generated from the total read counts of the cancer vs. reference for all targeted SNVs with at least a minimum depth of coverage in the reference. LogR provides information on the total copy number ratio. Sequence read count information can be first parsed from the paired cancer and reference files. A normalization constant is calculated for each cancer / reference pair to correct for the total library size. Subsampling within 150-250 bp intervals may be applied to reduce hyper-segmentation of genomic regions with high density of SNVs. Specifically, the expected value of LogR can be expressed as:

[0129] E[logR]=log{(p1 * +p2 * ) / 2}+w(·)+λ In the formula, p1 * = p1 Φ + (1-Φ) and p2 * =p1·Φ+(1-Φ) is the parental copy number in the tumor sample resulting from a mixture of normal (1,1) and abnormal (p1,p2) copy number genotypes with a mixture ratio Φ. Φ is the cell fraction associated with the abnormal genotype and is a function of tumor purity and clonal frequency (in case of subclonal mutations). The term w(·) denotes lineage bias. GC content can be explicitly taken into account, using loess regression of logR against GC in 1 kb windows along the genome to estimate the effect of GC on read counts and subtracting it from logR. In addition, since LogR quantifies relative copy number, the constant λ is included in the conversion to absolute copy number.

[0130] For LogR generation, sequence read count information may be parsed from paired cancer and control BAM files. A normalization constant may be calculated for each cancer / control pair to correct for total library size. Subsampling within 150-250 bp intervals may be applied to reduce hypersegmentation of genomic regions with high density of SNVs.

[0131] Log odds ratio of allele copy numbers The log odds ratio (logOR) of the allelic copy number of the variant allele count and the reference allele count in cancer, which is an unbiased estimator of the allelic ratio: E[logOR] = [p1·Φ + (1 - Φ)] / [p2·Φ + (1 - Φ)] (where E[logOR] is the expected value of logOR, p1 is the parental copy number of the variant allele, p2 is the parental copy number of the allele from the other parent, and Φ is the cell fraction that is a function of tumor purity and clonal frequency (in the case of subclonal mutations)). In the absence of phase data, log 2 The squared logOR can be used to infer ([p1·Φ + (1 - Φ)] / [p2·Φ + (1 - Φ)]).

[0132] Joint segmentation Segmentation analysis can be used to identify genomic regions with a constant copy number using a change point detection method. Conventional methods (e.g., BIC-seq, exome CNV) typically perform one-dimensional segmentation using only logR or apply one-dimensional segmentation separately to logR and B allele frequency (BAF).

[0133] Preferably, the bivariate Hotelling T 2 Based on statistics, the circular binary segmentation (CBS) algorithm is used for joint segmentation of logR and logOR.

[0134]

Number

[0135] where T 1ij is the Mann-Whitney statistic that compares the observed logR set shown as {X 1k : i < k ≦ j} and its complement {X 1k : 1 < k ≦ i or j < k ≦ n}, and T 2ij is the Mann-Whitney statistic that compares the observed logR set shown as {X 2k : i < k ≦ j} and its complement {X 2k:1 < k ≦ i or j < k ≦ n}, which is the Mann-Whitney statistic for comparing the observed logOR sets shown as. In the above, c is a scale factor inversely proportional to the heterozygosity rate.

[0136] Here, when the maximum statistic is greater than a predetermined critical value, the change is stated, and the change point is estimated as i, j that maximizes the statistic. This approach repeatedly searches for change points between pairs of possible breakpoints and their complements to identify genomic regions with a certain allele-specific copy number. For each segment, the logR data is summarized using the median of the logR values

[0137]

Number

[0138] and the logOR data takes the form of Σ{x 2 2 -s 2 ) / s 2} / Σ{1 / s 2}(where s 2 is the estimated variance of logOR).

[0139]

Number

[0140] is summarized by. logR is defined for all SNVs (loci of both homozygosity and heterozygosity), while logOR is defined only for heterozygous loci (het-loci or het-SNVs). Thus, in the integrated statistics, there may be an imbalance between the two. To address this problem, a weighting inversely proportional to the heterozygosity rate is introduced to increase the contribution of het-SNVs in subsequent segmentation analysis. Specifically, the scale factor c is introduced into the T 2 statistic. This is empirically

[0141]

number

[0142] where γ is the proportion of het-SNVs in the cancer cell sample. Weighting the contribution of the logOR of het-SNVs increases the ability to detect allelic imbalance in regions with low frequency of het-SNVs.

[0143] Alternatively, segmentation can be performed using, for example, a moving average method. Alternatively, the LogR and LogOR data can be divided into predefined short segments (based on the SNV loci).

[0144] After segmentation, the segments are clustered into groups of the same underlying genotype, which reduces the number of potential copy number and cell fraction states required for subsequent modeling.

[0145] Position deviation determination The LogR estimate is proportional to the absolute total copy number up to a position constant λ. For a diploid genome, logR=0 (library size normalized by logR) is the position of the 2-copy state. However, due to aneuploidy, the position may shift in tumors. Therefore, it is necessary to determine the 2-copy state in the tumor genome and quantify the position constant λ.

[0146] Copy number status can be indicated using total copy number and integer minor copy number (e.g., 1-0 indicates monosomy with total copy number 1 and minor copy number 0). Estimates of λ should correspond to the logR level at which the segment is in 2-1 (normal diploid) or 2-0 (LOH with no copy number change) state. In order to estimate λ, normal diploid segments need to be allelically balanced. Thus, candidate values ​​of λ (λ c ), is close to zero

[0147]

number

[0148] of segment clusters with values

[0149]

number

[0150] is obtained from On the other hand, homozygous deletions (0-0) and balanced gains (4-2, 6-3, etc.) also have balanced alleles,

[0151]

number

[0152] Large homozygous deletions of multiple genes do not contribute to cell survival,

[0153]

number

[0154] Non-focal segments with small λ can be excluded as homozygous deletions. Additionally, for simplicity, higher order balanced gain states (6-3, 8-4, etc.) across large portions of the genome are not considered. Samples with segments of allelic balance in only a small portion of the target region are flagged and their λ estimates can be manually reviewed.

[0155] In samples with many allelic balanced segments, some

[0156]

number

[0157] There exist values ​​from which λc can be selected. Integer number of copies The integer allele-specific copy numbers (major and minor) and associated cell fractions for each segment cluster are estimated using a combination of parametric and nonparametric methods by modeling the expected values ​​of logR and logOR given the total copy number (t) and each parental copy number (p1, p2) as a function of the cf parameter Φ, allowing both clonal and subclonal events to be modeled.

[0158] First, the total copy number of segment cluster i is

[0159]

number

[0160] The moment estimates of

[0161]

number

[0162] (In the formula,

[0163]

number

[0164] where denotes the logR median of segment cluster i, corrected for sequence bias and tumor ploidy (λ normalized). Once the total counts are obtained, the allele-specific copy numbers m and p and the cell fraction Φ are calculated using the logOR summary measure

[0165]

number

[0166] But log 2 μ equal to ({p1·Φ+(1-Φ)} / {p2·Φ+(1-Φ)}) 2This is calculated by taking advantage of the fact that it is a moment estimator of To further refine the initial estimate, we use a Gaussian non-centred χ 2 Specifically, the logR of SNV locus j in segment cluster i is expressed as X 1ij (corrected for alignment bias and misalignment) and is assumed to follow a normal distribution: X 1ij ~N(ν ig , τ i 2 ) In the formula, ν ig is the expectation of logR given the underlying copy number state g, and is of the form ν ig =log 2 (2(1-Φ k )+t g Φ k ) / 2 In the formula, t g =p1 g +p2 g denotes the total copy number (the sum of the copy numbers of the two parents) given the underlying copy number state g, and Φ k denotes the cell fraction of clonal cluster k, and τ i 2 are the independent variance parameters. In practice, we assume equal variances and τ i 2 = τ 2 It is reasonable to set it as ∀i.

[0167] Furthermore, the logOR of SNV locus j in segment cluster i is X 2ij (X 2ij / σ ij ) 2 is assumed to follow a non-central chi-squared distribution: (X 2ij / σ ij ) 2 ~χ 2 (δ ijg ) In the formula, σ ij 2is the variance parameter of logOR, and δ ijg =μ ig 2 / σ ij 2 is the non-centrality parameter: μ ig 2 =log 2 ((m g Φ k +(1-Φ k )) / (p g Φ k +(1-Φ k ))) X 1ij and X 2ij Assuming that x, y, and y are independent random variables given the underlying copy number state g, the likelihood of the combined data can be written as:

[0168]

number

[0169] where P(g) is the prior probability of the potential copy number state g. The expectation-maximization (EM) algorithm can be applied to improve the likelihood of the combined data. This can be viewed as an estimation problem with the latent copy number states as missing data. The E-step of the EM procedure uses Bayes' theorem to calculate the posterior probability that copy number state g is assigned to segment cluster i given the parameter estimates at the tth iteration.

[0170]

number

[0171] In the M step, first, the parameters of the normal distribution and the non-central chi-square distribution are updated.

[0172]

number

[0173] s in the ceremony 2 is the sample variance estimate of logOR. After obtaining an estimate of ν, the cell fraction parameter Φ is given by k (t+1) Update.

[0174]

number

[0175] g in the formula * is the most likely genotype (highest posterior probability) given the data and current parameter estimates at the tth iteration. The E and M steps are repeated until convergence.

[0176] Cell fraction Φ k A clonal structure is imposed on the cell fraction parameter Φ 1 This is done in a sequential approach where the algorithm starts with a single clonal cluster (k=1) with k = 0. The method may then involve identifying segment clusters where the segment cluster-specific estimate is non-trivially (at least 0.05) lower than the clonally constrained estimate that does not lead to the best fit at k=1. These segment clusters with discordant cell fraction estimates are then analyzed to determine the lower cell fraction Φ 2 We form candidate subclonal clusters of events in and fit a model with optimized joint likelihood with k = 2. This procedure is repeated until no further discrepancies in cell fraction estimates are found or until a maximum k (e.g., k = 5) is reached, as desired and depending on intratumor heterogeneity. The output is

[0177]

number

[0178] is an estimate of the cell fraction of clonal events, which is also the tumor purity by definition,

[0179]

number

[0180] are any subclonal clusters identified in the sample. Distinguishing between germline and somatic mutations The methods described herein can be used to identify target mutations as germline or somatic.

[0181] Using the methods described herein, identifying that the target mutation is a germline or somatic mutation can be achieved with or without the use of a matched normal sample from the subject (in addition to the sample containing cancer cells from the subject, e.g., biopsy).A matched normal sample from the same subject is a sample containing normal (non-cancerous) cells from the subject, e.g., a blood sample.

[0182] When a sample containing cancer cells from a subject (e.g., a biopsy from a subject) and a matched normal sample from the subject (e.g., a blood sample from a subject) are available, the methods described herein may include identifying a mutation in the matched normal sample from the subject. If the target mutation present in the cancer cells from the subject is identified in the matched normal sample, the target mutation is germline. If the target mutation present in the cancer cells from the subject is not identified in the matched normal sample, the target mutation is somatic.

[0183] For example, if a matched normal sample from the subject is not available, the methods described herein include: determining an observed allele fraction of a target mutation from read counts of a plurality of uniform coverage single nucleotide variants (SNVs), including homozygous and heterozygous uniform coverage SNVs, obtained from sequencing a sample comprising the cells; Determining the expected allele fraction (E[a]) of germline and somatic mutations, the allele fraction of germline mutations is E[a]=[(1-Φ) 1+Φ m cn ] / [(1-Φ) 2+Φ t tum ], and m cn is p1 or p2, and t tum is the total copy number of the locus in the tumor, and the allele fraction of the somatic mutation is m cn ≦min(p1,p2) for all enumerated E[a]=[Φ m cn ] / [(1-Φ) 2+Φ t tum ], wherein m cn is the variant allele copy number of the genomic region containing the target mutation, Φ is the cell fraction (used as a measure of sample purity), p1 is the parent copy number of the variant allele, and p2 is the parent copy number of the allele from the other parent; comparing the observed allele fraction to an expected allele fraction to identify the most likely germline and somatic mutations; and identifying the most likely of the germline and somatic mutations as the germline or somatic targeted mutation.

[0184] Mutant allele copy number (m cn ) is 1~t tum is an integer, and t tum is the total copy number of the allele of interest in the cancer cells from the subject. The comparing step can be performed using a Bayesian model comparison (Bayes factor).

[0185] This approach assumes that normal cells are diploid and that samples from subjects are not pure (Φ<1). As Φ approaches 1, germline and somatic mutations become indistinguishable in this approach in the absence of a matched normal sample from the subject.

[0186] Alternatively, for example, if a matched normal sample from the subject is not available and the sample containing cancer cells from the subject is not pure (Φ<0.9, or 90%), the methods described herein can be used to identify the target mutation as somatic if the observed allele fraction is outside the range of the expected allele fraction (without purity adjustment). For example, if the total copy number is 2, SNVs are expected to occur within the range of less than 10% (no homozygous SNVs), 40-60% (e.g., 45-55%) (heterozygous SNVs), and more than 90% (with homozygous SNVs) of the allele fraction (without purity adjustment), and therefore, if the observed allele fraction is outside the range of the expected allele fraction, the target mutation is somatic. If the observed allele fraction is within the range of the expected allele fraction, this identification approach cannot characterize the target mutation.

[0187] Identification of overexpression of target genes A subject or cancer cells thereof may be identified as having an amplification in a target gene if the total copy number is at least twice the sample ploidy, or if the total copy number is at least 2 units greater than the sample ploidy, using the methods described for identifying biallelic loss-of-function mutations, except that the cells are identified as having an amplification in the target gene.

[0188] Here, the total copy number is defined as t=p1+p2, or t=[2 (logR-λ+1) -2+2Φ] / Φ where p1*=p1·Φ+(1-Φ) and p2*=p1·Φ+(1-Φ) are the parental copy numbers in the tumor sample resulting from a mixture of normal (1,1) and abnormal (p1,p2) copy number genotypes with a mixture ratio Φ. The total copy number defined is therefore the unnormalized copy number. Φ is the cell fraction associated with the abnormal genotype, which is a function of the tumor purity and clonal frequency (in case of subclonal mutations). The term w(·) denotes lineage bias. GC content can be explicitly taken into account, using loess regression of logR against GC in 1 kb windows along the genome to estimate the effect of GC on read counts and subtracting it from logR. In addition, since LogR quantifies relative copy number, the constant λ is included in the conversion of absolute copy number.

[0189] For LogR generation, sequence read count information may be parsed from paired cancer and control BAM files. A normalization constant may be calculated for each cancer / control pair to correct for total library size. Subsampling within 150-250 bp intervals may be applied to reduce hypersegmentation of genomic regions with high density of SNVs.

[0190] Accordingly, the present invention provides a method for identifying cells derived from a subject that have amplification in a target gene, comprising the steps of: determining a total copy number and a sample ploidy of a locus segment within a target genomic region of cells from a subject from read counts of a plurality of uniform coverage single nucleotide variants (SNVs), including homozygous and heterozygous uniform coverage SNVs, and reference read counts obtained from sequencing a sample comprising cells, wherein the target genomic region comprises a mutation, and the reference read counts are derived from a panel of normal samples; Here, methods are provided in which a cell is identified as having amplification in a target gene if the total copy number is at least twice (e.g., at least 2x) the sample ploidy, or if the total copy number is at least 2 (e.g., at least 3) greater than the sample ploidy.

[0191] In some embodiments, the determining step comprises: determining a total copy number log ratio of the SNVs and a target coverage value from the read counts and the reference read counts; Segmenting the total copy number log ratio; Estimating the sample purity and sample ploidy of the cells from the total copy number log ratio and the target coverage value; and generating a total copy number of the locus segment within the target gene region from the target coverage value, the sample purity, the sample ploidy, and the total copy number log ratio.

[0192] Total copy number can be normalized for ploidy as follows: n =2·t / p2(where t n is the normalized total copy number, t is the non-normalized copy number, and p2 is as defined above).

[0193] Identification of biallelic ATM loss-of-function mutations Thus, the methods described herein may include identifying cancers that have biallelic ATM loss-of-function mutations using the techniques described above.

[0194] Identification of CCNE1 overexpression Thus, the methods described herein may include a step of identifying cancers in which CCNE1 is amplified using the techniques described above.

[0195] Detection of SNVs Detection techniques for evaluating nucleic acid for the presence of SNV include procedures well known in the field of molecular genetics. Many, but not all, of these methods involve nucleic acid amplification. Sufficient guidelines for carrying out amplification are provided in the art. Exemplary references include manuals such as PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Current Protocols in Molecular Biology, Ausubel, 1994-1999 (including supplements up to April 2004); Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001). A general method for detecting single base variants is disclosed in Single Nucleotide Variants: Methods and Protocols, Pui-Yan Kwok, ed., 2003, Humana Press. SNV detection methods often employ labeled oligonucleotides. Oligonucleotides can be labeled by incorporating a label that can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful labels include fluorescent dyes, radioactive labels such as 32P, electron-dense reagents, enzymes such as peroxidase or alkaline phosphatase, biotin, or haptens and proteins that can be used against antisera or monoclonal antibodies. Labeling techniques are well known in the art (see, for example, Current Protocols in Molecular Biolog, supra; Sambrook & Russell, supra).

[0196] The methods typically employ a PCR step, although other amplification protocols may be used. Suitable amplification methods include the ligase chain reaction (see, e.g., Wu & Wallace, Genomics 4:560-569, 1988); strand displacement assays (see, e.g., Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992; U.S. Pat. No. 5,455,166); as well as several transcription-based amplification systems, such as those described in U.S. Pat. Nos. 5,437,990; 5,409,818; and 5,399,491; the transcription amplification system (TAS) (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173-1177, 1989); and self-sustained sequence replication (3SR) (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990; WO92 / 08800). Alternatively, methods for amplifying the probe to detectable levels, such as Qβ-replicase amplification, can be used (Kramer & Lizardi, Nature 339:401-402, 1989; Lomeli et al., Clin. Chem. 35:1826-1831, 1989). A review of known amplification methods is provided, for example, by Abramson and Myers in Curr. Op Biotechnol. 4:41-47, 1993.

[0197] Detection of individual genotypes, haplotypes, SNVs, microsatellites, or other variants can be performed using oligonucleotide primers and / or probes. Oligonucleotides can be prepared by any suitable method, usually chemical synthesis. Oligonucleotides can be synthesized using commercially available reagents and equipment. Alternatively, they can be purchased commercially. Methods for synthesizing oligonucleotides are well known in the art (see, for example, Narang et al., Meth. Enzymol. 68:90-99, 1979; Brown et al., Meth. Enzymol. 68:109-151, 1979; Beaucage et al., Tetrahedron Lett. 22:1859-1862, 1981; and the solid-phase method of U.S. Pat. No. 4,458,066). In addition, modifications of the above-mentioned synthesis methods can be used to affect enzyme behavior on synthesized oligonucleotides in a desired way. For example, incorporation of modified phosphodiester linkages (e.g., phosphorothioates, methylphosphonates, phosphoamidates, or boranophosphates) or linkages other than phosphorous derivatives into oligonucleotides can be used to prevent cleavage at selected sites. In addition, the use of 2'-amino modified sugars tends to favor displacement over digestion of the oligonucleotide when hybridized to a nucleic acid that is also a template for the synthesis of a new nucleic acid strand.

[0198] The genotype of an individual can be determined using many detection methods well known in the art. Most assays require one of several general protocols: hybridization using allele-specific oligonucleotides, primer extension, allele-specific ligation, sequencing, or electrophoretic separation techniques, such as single-stranded conformation variants (SSCP) and heteroduplex analysis. Exemplary assays include 5'-nuclease assays, template-directed dye terminator incorporation, molecular beacon allele-specific oligonucleotide assays, single-base extension assays, and SNV scoring by real-time pyrophosphate sequences. Analysis of the amplified sequences can be performed using a variety of techniques, such as microchips, fluorescence polarization assays, and MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) mass spectrometry. Two methods that can be used as well are the assay based on invasive cleavage by Flap nucleases and the methodology employing padlock probes.

[0199] The determination of the presence or absence of a particular allele is generally carried out by analyzing a nucleic acid sample obtained from an individual to be analyzed.In many cases, the nucleic acid sample comprises genomic DNA.The genomic DNA is typically obtained from a blood sample, but may also be obtained from other cells or tissues.

[0200] It is also possible to analyze RNA samples for the presence of polymorphic alleles.For example, mRNA can be used to determine the genotype of an individual at one or more polymorphic sites.In this case, a nucleic acid sample is obtained from a cell in which the target nucleic acid is expressed, for example, an adipocyte.Such analysis can be carried out by first reverse transcribing the target RNA, for example, using viral reverse transcriptase, and then amplifying the obtained cDNA, or by combining the use of high-temperature reverse transcription polymerase chain reaction (RT-PCR) as described in U.S. Patent Nos. 5,310,652; 5,322,770; 5,561,058; 5,641,864; and 5,693,517.

[0201] A brief description of methodologies commonly used in the analysis of nucleic acid samples to detect SNVs is provided below. However, any method known in the art can be used in the present invention to detect the presence of a single base substitution.

[0202] Allele-specific hybridization Allele-specific hybridization, also commonly referred to as allele-specific oligonucleotide hybridization (ASO) (e.g., Stoneking et al., Am. J. Hum. Genet. 48:70-382, 1991; Saiki et al., Nature 324, 163-166, 1986; EP 235, 726; and WO 89 / 11548), relies on distinguishing between two DNA molecules that differ by only one base by hybridizing an oligonucleotide probe specific for one of the variants to an amplification product resulting from amplification of a nucleic acid sample. This method typically employs short, e.g., 15-20 base long, oligonucleotides. The probes are designed to differentially hybridize with one variant from another. Principles and guidelines for designing such probes are available in the art, e.g., in the references cited herein. Hybridization conditions must be sufficiently stringent so that there is a significant difference in hybridization intensity between the alleles, resulting in an essentially binary response, with the probe hybridizing to only one of the alleles. Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site is aligned with the central position of the probe (e.g., the 7th position for a 15-base oligonucleotide; the 8th or 9th position for a 16-base oligonucleotide), although this design is not required.

[0203] The amount and / or presence of an allele is determined by measuring the amount of allele-specific oligonucleotide hybridized to a sample. Typically, the oligonucleotide is labeled with a label, such as a fluorescent label. For example, the allele-specific oligonucleotide is applied to an immobilized oligonucleotide representing an SNV sequence. After stringent hybridization and washing conditions, the fluorescence intensity of each SNV oligonucleotide is measured.

[0204] In one embodiment, the nucleotides present at the polymorphic site are identified by hybridization under sequence-specific hybridization conditions with an oligonucleotide probe or primer that is exactly complementary to one of the polymorphic alleles in the region encompassing the polymorphic site. The hybridization sequence of the probe or primer and the sequence-specific hybridization conditions are selected such that a single mismatch at the polymorphic site sufficiently destabilizes the hybridization duplex such that a hybridization duplex is not substantially formed. Therefore, under sequence-specific hybridization conditions, a stable duplex is formed only between the probe or primer and the exactly complementary allele sequence. Thus, oligonucleotides of about 10 to about 35 nucleotides in length, usually about 15 to about 35 nucleotides in length, that are exactly complementary to the allele sequence in the region encompassing the polymorphic site, are within the scope of the present invention.

[0205] In an alternative embodiment, the nucleotide present at the polymorphic site is identified by hybridization under sufficiently stringent hybridization conditions using an oligonucleotide that is substantially complementary to one of the SNV alleles in the region encompassing the polymorphic site and is exactly complementary to the allele at the polymorphic site.Since the mismatch occurring at the non-polymorphic site is a mismatch with both allele sequences, the difference between the number of mismatches in the duplex formed with the target allele sequence and the number of mismatches in the duplex formed with the corresponding non-target allele sequence is the same as when an oligonucleotide that is exactly complementary to the target allele sequence is used.In this embodiment, the hybridization conditions are sufficiently relaxed to allow the formation of a stable duplex with the target sequence while maintaining sufficient stringency to exclude the formation of a stable duplex with non-target sequences.Under such sufficiently stringent hybridization conditions, a stable duplex is formed only between the probe or primer and the target allele. Thus, oligonucleotides from about 10 to about 35 nucleotides in length, typically from about 15 to about 35 nucleotides in length, that are substantially complementary to the allelic sequence in the region encompassing the polymorphic site and that are exactly complementary to the allelic sequence at the polymorphic site are within the scope of the present invention.

[0206] In assay formats where optimization of hybridization conditions is limited, the use of substantially complementary oligonucleotides rather than exactly complementary oligonucleotides may be desirable. For example, in a typical multi-target immobilized oligonucleotide assay format, a probe or primer for each target is immobilized on a single solid support. Hybridization is performed simultaneously by contacting the solid support with a solution containing the target DNA. Since all hybridizations are performed under identical conditions, hybridization conditions cannot be optimized separately for each probe or primer. If the assay format does not allow adjustment of hybridization conditions, the incorporation of mismatches into the probe or primer can be used to adjust duplex stability. The effect of the particular mismatch introduced on duplex stability is well known, and duplex stability can be routinely estimated or empirically determined as described above. Suitable hybridization conditions depend on the exact size and sequence of the probe or primer, and can be empirically selected using the guidelines provided herein and well known in the art. The use of oligonucleotide probes or primers to detect single base pair differences in sequence is described, for example, in Conner et al., Proc. Natl. Acad. Sci. USA 80:278-282, 1983, and U.S. Pat. Nos. 5,468,613 and 5,604,099, each of which is incorporated herein by reference.

[0207] The proportional change in stability between a perfectly matched hybridization duplex and a single base mismatched hybridization duplex depends on the length of the hybridized oligonucleotide. The duplex formed with the shorter probe sequence is proportionally more destabilized by the presence of mismatches. Oligonucleotides of about 15 to about 35 nucleotides in length are often used for sequence-specific detection. Furthermore, because the ends of the hybridized oligonucleotide undergo continuous random separation and reannealing by thermal energy, mismatches at either end do not destabilize the hybridization duplex as much as mismatches occurring internally. To identify a single base pair change in a target sequence, the probe sequence hybridized to the target sequence is selected such that the polymorphic site is present in the internal region of the probe.

[0208] The above criteria for selecting the probe sequence that hybridizes to specific alleles are applied to the hybridization region of the probe, i.e., the part of the probe that is involved in hybridization with the target sequence.The probe can be bound to additional nucleic acid sequences, such as poly-T tails, that are used for immobilizing the probe, without significantly changing the hybridization properties of the probe.Those skilled in the art will recognize that when used in the present method, the probe that is bound to additional nucleic acid sequences that are not complementary to the target sequence and therefore are not involved in hybridization is essentially equivalent to a non-bound probe.

[0209] Suitable assay formats for detecting hybrids formed between a probe and a target nucleic acid sequence in a sample are known in the art and include immobilized target (dot blot) and immobilized probe (reverse dot blot or line blot) assay formats. Dot blot and reverse dot blot assay formats are described in U.S. Patent Nos. 5,310,893; 5,451,512; 5,468,613; and 5,604,099, each of which is incorporated herein by reference.

[0210] In the dot blot format, amplified target DNA is immobilized on a solid support such as a nylon membrane, the membrane-target complex is incubated with a labeled probe under suitable hybridization conditions, unhybridized probe is removed by washing under suitable stringency conditions, and the membrane is monitored for the presence of bound probe.

[0211] In the reverse dot blot (or line blot) format, the probe is immobilized on a solid support such as a nylon membrane or a microtiter plate. Typically, the target DNA is labeled by incorporation of a labeled primer during amplification. One or both of the primers can be labeled. The membrane-probe complex is incubated with the labeled amplified target DNA under suitable hybridization conditions, unhybridized target DNA is removed by washing under suitable stringent conditions, and the membrane is monitored for the presence of bound target DNA. A reverse line blot detection assay is described in the Examples.

[0212] In many cases, an allele-specific probe specific for one variant mutation and an allele-specific probe for the other variant mutation are used together. In some embodiments, the probe is fixed to a solid support, and the target sequence in an individual is analyzed using both probes simultaneously. Examples of nucleic acid arrays are described in WO95 / 11995. The same array or different arrays can be used to analyze characterized variants. WO95 / 11995 also describes subarrays that are optimized for detecting precharacterized variant types. Such subarrays can be used to detect the presence of variants described herein.

[0213] Allele-specific primers Variants can also be detected using allele-specific amplification or primer extension methods. These reactions typically involve the use of primers designed to specifically target variants via a mismatch at the 3' end of the primer. The presence of a mismatch affects the ability of the polymerase to extend the primer if the polymerase does not have error-correcting activity. For example, to detect an allele sequence using allele-specific amplification or extension-based methods, a primer complementary to one allele of the variant is designed such that the 3' terminal nucleotide hybridizes at the polymorphic position. The presence of a particular allele can be determined by the ability of the primer to initiate extension. If the 3' end is mismatched, extension is inhibited.

[0214] In some embodiments, the primer is used together with a second primer in the amplification reaction. The second primer hybridizes at a site unrelated to the polymorphic position. Amplification proceeds from the two primers, resulting in a detectable product that indicates the presence of a specific allele type. Methods based on allele-specific amplification or extension are described, for example, in WO93 / 22456; U.S. Patent No. 5,137,806; U.S. Patent No. 5,595,890; U.S. Patent No. 5,639,611; and U.S. Patent No. 4,851,331.

[0215] Using allele-specific amplification-based genotyping, allele identification is simply a matter of detecting the presence or absence of an amplified target sequence. Methods for detecting an amplified target sequence are well known in the art. For example, the presence of a nucleic acid can often be detected using gel electrophoresis and probe hybridization assays as described.

[0216] In an alternative probeless method, amplified nucleic acid is detected by monitoring the increase in the total amount of double-stranded DNA in the reaction mixture, as described, for example, in U.S. Patent No. 5,994,056 and European Patent Publications Nos. 487,218 and 512,334. Detection of double-stranded target DNA relies on the increased tendency of various DNA binding dyes, such as SYBR Green, to bind to double-stranded DNA.

[0217] As will be understood by those skilled in the art, allele-specific amplification methods can be carried out in a reaction that employs multiple allele-specific primers that target specific alleles.The primers for such multiplex applications are generally labeled with distinguishable labels or selected so that the amplification products generated from alleles can be distinguished by size.Therefore, for example, one round of amplification can be used to identify both alleles in a single sample by gel analysis of the amplification products.

[0218] As with the allele-specific probes, the allele-specific oligonucleotide primers may be exactly complementary to one of the polymorphic alleles in the hybridization region, or may have some mismatches at positions other than the 3' end of the oligonucleotide, which mismatches are present at non-polymorphic sites in both allele sequences.

[0219] Detectable Probes 5'-Nuclease Assay Probe Genotyping can also be performed using "TaqMan®" or "5'-nuclease assays", for example, as described in U.S. Pat. Nos. 5,210,015; 5,487,972; and 5,804,375; and Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-72801988. In the TaqMan® assay, a labeled detection probe that hybridizes within the amplified region is added during the amplification reaction. The probe is modified so that it does not act as a primer for DNA synthesis. Amplification is performed using a DNA polymerase with 5'→3' exonuclease activity. During each synthesis step of amplification, any probe that hybridizes to the target nucleic acid downstream from the primer being extended is degraded by the 5'→3' exonuclease activity of the DNA polymerase. Thus, synthesis of a new target strand also results in degradation of the probe, and accumulation of degradation products is indicative of synthesis of the target sequence.

[0220] The hybridization probe may be an allele-specific probe that distinguishes between SNV alleles. Alternatively, the method may be performed using an allele-specific primer and a labeled probe that binds to the amplification product.

[0221] Any method suitable for detecting degradation products can be used in the 5'-nuclease assay. Often, the detection probe is labeled with two fluorescent dyes, one capable of quenching the fluorescence of the other dye. A dye is attached to the probe, usually one at the 5' end and the other at an internal position, so that quenching occurs when the probe is unhybridized and cleavage of the probe occurs between the two dyes due to the 5'→3' exonuclease activity of DNA polymerase. Amplification cleaves the probe between the dyes, removing the quenching and increasing the observable fluorescence from the originally quenched dye. The accumulation of degradation products is monitored by measuring the increase in reaction fluorescence. U.S. Pat. Nos. 5,491,063 and 5,571,673 describe alternative methods for detecting the degradation of the probe that occurs with amplification, both of which are incorporated herein by reference.

[0222] Secondary structure probes Probes detectable by changes in secondary structure are also suitable for detecting variants, including SNVs. Exemplary secondary structure or stem-loop structure probes include molecular beacons or Scorpion® primers / probes. Molecular beacon probes are single-stranded oligonucleic acid probes that can typically form a hairpin structure with a fluorophore and a quencher on either side of the oligonucleotide. Short complementary sequences at both ends of the probe allow the formation of an intramolecular stem, which allows the fluorophore and quencher to be in close proximity. The loop portion of the molecular beacon is complementary to the target nucleic acid of interest. When the probe binds to its target nucleic acid of interest, a hybrid is formed and the stem is forced apart. This causes a conformational change that separates the fluorophore and quencher from each other, resulting in a stronger fluorescent signal. However, molecular beacon probes are extremely sensitive to slight sequence changes in the probe target (Tyagi and Kramer, Nat. Biotechnol. Vol. 14, pages 303-308, 1996; Tyagi et al., Nat. Biotech, Vol. 16, pages 49-53, 1998; Piatek et al., Nat Biotechnol, 16:359-363 (1998); Marras et al., Genetic Analysis: Biomolecular Engineering, Vol 14, pages 151-156 (1999); Tapp I. et al, BioTechniques. Vol 28, pages 732-738, 2000). Scorpion® primer / probes contain a stem-loop structure probe covalently attached to the primer.

[0223] Electrophoresis Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on differences in sequence-dependent melting properties and electrophoretic migration of DNA in solution (see, e.g., Erlich, ed., PCR Technology: Principles and Applications for DNA Amplification, WH Freeman and Co, New York, 1992, Chapter 7).

[0224] Microsatellite variants can be distinguished using capillary electrophoresis. Capillary electrophoresis can easily identify the number of repeats of a particular microsatellite allele. The application of capillary electrophoresis to DNA variant analysis is well known in the art (see, for example, Szantai, et al, J Chromatogr A. 1079(1-2):41-49, 2005; Bjorheim and Ekstrom, Electrophoresis 26(13):2520-2530, 2005 and Mitchelson, Mol Biotechnol. 24(1):41 ​​68, 2003).

[0225] Single-strand conformation polymorphism analysis Alleles of a target sequence can be distinguished using single-strand conformation polymorphism analysis, which identifies base differences by changes in the electrophoretic mobility of single-stranded PCR products, for example, as described in Orita et al., Proc. Natl. Acad. Sci. USA 86(8), 2766-2770, 1989. Amplified PCR products can be generated as described above and heated or otherwise denatured to form single-stranded amplification products. Single-stranded nucleic acids can refold or form secondary structures that depend in part on the base sequence. Differences in the electrophoretic mobility of single-stranded amplification products can be related to differences in base sequence between alleles of the target.

[0226] DNA sequencing and single base extension SNVs can also be detected by direct sequencing, including methods based on dideoxy sequencing and others such as Maxam-Gilbert sequences (see, e.g., Sambrook and Russell, supra).

[0227] Other detection methods include Pyrosequencing™ of oligonucleotide-length products. Such methods often utilize amplification techniques such as PCR. For example, in pyrosequencing, a sequencing primer is hybridized to a PCR-amplified single-stranded DNA template and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase, and the substrates adenosine 5′ phosphosulfate (APS) and luciferin. The first of four deoxynucleotide triphosphates (dNTPs) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxynucleotide triphosphate into the DNA strand if it is complementary to a base in the template strand. Each incorporation event is accompanied by the release of pyrophosphate (PPi) in an amount equimolar to the amount of incorporated nucleotide. ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5′ phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin, producing visible light proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge-coupled device (CCD) camera and displayed as a peak on a Pyrogram™. Each light signal is proportional to the number of nucleotides incorporated. Apyrase, a nucleolytic enzyme, continuously degrades unincorporated dNTPs and excess ATP. Once degradation is complete, another dNTP is added.

[0228] Another similar method for characterizing SNVs does not require the use of full PCR, but typically uses only primer extension with a single fluorescently labeled dideoxyribonucleic acid molecule (ddNTP) complementary to the nucleotide to be interrogated. The nucleotide at the polymorphic site can be identified through detection of a primer that is extended by a single base and fluorescently labeled (e.g., Kobayashi et al, Mol. Cell. Probes, 9:175-182, 1995).

[0229] Furthermore, SNVs can be determined from the analysis (e.g., computational analysis) of data obtained from next-generation sequencing (NGS) experiments (Buermans and Dunnen. Biochimica et Biophysica Acta. 1842:1932-1941, 2014). Sequencing can be performed by various systems currently available, such as, but not limited to, sequencing systems by ILLUMINA®, Pacific Biosciences (PACBIO®), Oxford NANOPORE®, or Life Technologies (ION TORRENT®). Methods, reagents, and equipment for performing these different sequencing systems are available from the respective manufacturers. Alternatively or additionally, sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real-time PCR), or isothermal amplification. Such systems may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., a human) generated by the system from a sample provided by the subject. In some instances, such systems provide sequencing reads, which may include a string of nucleobases that corresponds to the sequence of the sequenced nucleic acid molecule.

[0230] SNVs can be identified from data generated by NGS experiments by comparing the occurrence of different nucleic acid base pairs at the same locus in multiple samples. Due to errors that occur in NGS sequencing, probabilistic models (e.g., Bayesian models) are often used to distinguish and correct for read errors and true SNVs. A wide variety of methods and algorithms have been developed to detect SNVs from NGS data (see, e.g., Nielsen et al. Nat. Rev. Genet. 12(6):443-451, 2011; Bansal, Bioinformatics. 26(12):i318-i324, 2010; Roth et al. Bioinformatics. 28(7):907-913, 2012; You et al. Bioinformatics. 28(5):643-650, 2012; Li et al., Genome Res. 19(6):1124-1132, 2009; Abecasis et al. Nature. 467(7319):1061-1073, 2010; Larson et al. Bioinformatics. 28(3):311-317, 2012). Resources for identifying SNVs found in the human genome include databases of sequenced genomes (e.g., gnomAD, Bravo, ClinVar, 1000 Genome Project, and TopMed) and databases of identified SNVs (e.g., dbSNP, HapMap, Biomart, SPSmart, and Genome Variation Server (GVS)).

[0231] ATR inhibitors When ATR inhibitors come into contact with the enzyme ATR kinase, whether in vitro, in cell culture, or in animals, they increase the measured IC 50 is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM).

[0232] Examples of ATR inhibitors are

[0233] [ka]

[0234] and pharma-ceutically acceptable salts thereof. Additional examples of ATR inhibitors are those described in International Application Nos. PCT / US2019 / 051539 and PCT / US2018 / 034729, U.S. Pat. Nos. 9,663,535, 9,549,932, 8,552,004, and 8,841,308, and U.S. Patent Application Publication No. 2019 / 0055240, each of which is incorporated herein by reference.

[0235] In one embodiment, the ATR inhibitor is a compound of formula (I):

[0236] [ka]

[0237] or a pharma- ceutically acceptable salt thereof Where:

[0238] [ka]

[0239] is a double bond, and each Y is independently N or CR 4 or

[0240] [ka]

[0241] is a single bond, and each Y is independently NR Y , carbonyl, or C(R Y ) 2 where each R Y are independently H or optionally substituted C 1-6 is alkyl, R 1 is optionally replaced by C 1-6 alkyl or H, R 2 is optionally replaced by C 2-9 Heterocyclyl, optionally substituted C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 2-9 Heterocyclyl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, optionally substituted C 1-9 Heteroaryl C 1-6 Alkyl, halogen, -N(R 5 ) 2 , -OR 5 , -CON(R 6 ) 2 , -SO 2 N(R 6 ) 2 , -SO 2 R 5A , or -QR 5B and R 3 is optionally replaced by C 1-9 Heteroaryl or optionally substituted C 1-9 Heteroaryl C 1-6 is alkyl, Each R 4 are independently hydrogen, halogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6alkenyl, or optionally substituted C 2-6 is alkynyl, Each R 5 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, or -SO 2 R 5A or both R 5 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Each R 5A independently represents an optionally substituted C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 6-10 is aryl, R 5B is hydroxyl, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, -N(R 5 ) 2 , -CON(R 6 ) 2 , -SO 2 N(R 6 ) 2 , -SO 2 R 5A or optionally substituted alkoxy; Each R 6 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkoxyalkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 3-8Cycloalkyl, or optionally substituted C 1-9 Heteroaryl or both R 6 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Q is optionally substituted C 2-9 Heterocyclylene, optionally substituted C 3-8 Cycloalkylene, optionally substituted C 1-9 Heteroarylene or optionally substituted C 6-10 is an arylene, X is hydrogen or a halogen.

[0242] ATR inhibitors include, for example, compounds of formula (II):

[0243] [ka]

[0244] or a pharma- ceutically acceptable salt thereof Where: Each Y is independently N or CR 4 and R 1 is optionally replaced by C 1-6 alkyl or H, R 2 is optionally replaced by C 2-9 Heterocyclyl, optionally substituted C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 2-9 Heterocyclyl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, optionally substituted C 1-9 Heteroaryl C 1-6 Alkyl, halogen, -N(R 5 ) 2 , -OR5 , -CON(R 6 ) 2 , -SO 2 N(R 6 ) 2 , -SO 2 R 5A , or -QR 5B and R 3 is optionally replaced by C 1-9 Heteroaryl or optionally substituted C 1-9 Heteroaryl C 1-6 is alkyl, Each R 4 are independently hydrogen, halogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 alkenyl, or optionally substituted C 2-6 is alkynyl, Each R 5 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, or -SO 2 R 5A or both R 5 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Each R 5A independently represents an optionally substituted C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 6-10 is aryl, R 5B is hydroxyl, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, -N(R 5) 2 , -CON(R 6 ) 2 , -SO 2 N(R 6 ) 2 , -SO 2 R 5A or optionally substituted alkoxy; Each R 6 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkoxyalkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 1-9 Heteroaryl or both R 6 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Q is optionally substituted C 2-9 Heterocyclylene, optionally substituted C 3-8 Cycloalkylene, optionally substituted C 1-9 Heteroarylene or optionally substituted C 6-10 is an arylene, X is hydrogen or a halogen.

[0245] In some embodiments, in a compound of Formula (II), (I), or (Ib), Each Y is independently N or CR 4 and R 1 is H or optionally substituted C 1-6 is alkyl, R 2 is optionally replaced by C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 2-9Heterocyclyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, optionally substituted C 1-9 Heteroaryl C 1-6 Alkyl, -N(R 5 ) 2 , -CON(R 6 ) 2 , -SO 2 N(R 6 ) 2 , or -SO 2 R 5A and R 3 is optionally replaced by C 1-9 is heteroaryl, Each R 4 are independently H or optionally substituted C 1-6 is alkyl, Each R 5 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, or -SO 2 R 5A where each R 5A independently represents an optionally substituted C 1-6 Alkyl or optionally substituted C 3-8 cycloalkyl or both R 5 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Each R 5A independently represents an optionally substituted C 1-6 Alkyl or optionally substituted C 3-8 is cycloalkyl, Each R 6 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, or optionally substituted C 1-9 Heteroaryl or both R 6 together with the atoms to which they are attached, optionally substituted C 2-9 Forming a heterocyclyl.

[0246] Methods for making compounds of formula (I) are described, for example, in International Application No. PCT / US2019 / 051539, which is incorporated herein by reference. ATR inhibitors include, for example, compounds of formula (Ia):

[0247] [ka]

[0248] or a pharma- ceutically acceptable salt thereof (wherein Y, R 1 , R 2 , R 3 , and R 4 is as described in formula (I). ATR inhibitors include, for example, compounds of formula (Ib):

[0249] [ka]

[0250] or a pharma- ceutically acceptable salt thereof (wherein Y, R 1 , R 2 , R 3 , and R 4 is as described in formula (I). ATR inhibitors include, for example, compounds of formula (IA):

[0251] [ka]

[0252] or a pharma- ceutically acceptable salt thereof, 1 , R 2 , R 3 , and R 4 is as described in formula (I). ATR inhibitors include, for example, compounds of formula (IA-a):

[0253] [ka]

[0254] or a pharma- ceutically acceptable salt thereof, 1 , R 2 , R 3 , and R 4 is as described in formula (I). ATR inhibitors include, for example, compounds of formula (IB):

[0255] [ka]

[0256] or a pharma- ceutically acceptable salt thereof, 1 , R 2 , R 3 , and R 4 is as described in formula (I). ATR inhibitors include, for example, compounds of formula (IB-a):

[0257] [ka]

[0258] or a pharma- ceutically acceptable salt thereof, 1 , R 2 , R 3 , and R 4 is as described in formula (I). ATR inhibitors include, for example, compounds of formula (IC):

[0259] [ka]

[0260] or a pharma- ceutically acceptable salt thereof, 1 , R 2 , R 3 , and R 4 is as described in formula (I). ATR inhibitors include, for example, compounds of formula (IC-a):

[0261] [ka]

[0262] or a pharma- ceutically acceptable salt thereof, 1 , R 2 , R 3 , and R 4 is as described in formula (I). ATR inhibitors include, for example, compounds of formula (ID):

[0263] [ka]

[0264] or a pharma- ceutically acceptable salt thereof, 1 , R 2 , R 3 , and R 4 is as described in formula (I). ATR inhibitors include, for example, compounds of formula (ID-a):

[0265] [ka]

[0266] or a pharma- ceutically acceptable salt thereof, 1 , R 2 , R3 , and R 4 is as described in formula (I). Preferably, R 1 is methyl.

[0267] In some embodiments, R 2 can be, for example, optionally substituted C 3-8 For example, R 2 is a group of formula (A):

[0268] [ka]

[0269] Where: n is 0, 1, 2, or 3; R 7 is hydrogen, alkylsulfonyl, cyano, -CON(R A ) 2 , -SON(R A ) 2 , optionally replaced by C 1-9 heteroaryl, hydroxy, or alkoxy, where each R A are independently H or alkyl, or both R A together with the atoms to which they are attached, C 2-9 forming a heterocyclyl).

[0270] In some embodiments, R 2 can be, for example, optionally substituted C 1-6 Alkyl (e.g., optionally substituted tertiary C 3-6 For example, R 2 is a group of formula (B):

[0271] [ka]

[0272] (In the formula, R7 is hydrogen, alkylsulfonyl, cyano, -CON(R A ) 2 , -SON(R A ) 2 , optionally replaced by C 1-9 heteroaryl, hydroxy, or alkoxy, where each R A are independently H or alkyl, or both R A , together with the atoms to which they are attached, C 2-9 forming a heterocyclyl).

[0273] In some embodiments, R 2 is, for example, an optionally substituted non-aromatic C 2-9 It may be a heterocyclyl. In some embodiments, R 2 For example,

[0274] [ka]

[0275] [ka]

[0276] [ka]

[0277] It could be. In some embodiments, R 3 is, for example, a monocyclic C ring containing at least one optionally substituted nitrogen atom (e.g., two nitrogen atoms). 1-9 For example, R 3 is a group of formula (C):

[0278] [ka]

[0279] wherein A is an optionally substituted monocyclic C 1-9 heteroaryl ring). In some embodiments, A is, for example, a group of formula (C1):

[0280] [ka]

[0281] (In the formula, R 8 is hydrogen, halogen, or optionally substituted C 1-6 It can be alkyl. In some embodiments, R 3 For example,

[0282] [ka]

[0283] It could be. In some embodiments, R 3 For example,

[0284] [ka]

[0285] It could be. In some embodiments, R 4 can be, for example, hydrogen. The ATR inhibitor can be, for example, a compound listed in Table 1, or a pharma- ceutically acceptable salt thereof.

[0286] [Table 1-1]

[0287] [Table 1-2]

[0288] [Table 1-3]

[0289] [Table 1-4]

[0290] [Table 1-5]

[0291] [Table 1-6]

[0292] [Table 1-7]

[0293] [Table 1-8]

[0294] [Table 1-9]

[0295] [Table 1-10]

[0296] [Table 1-11]

[0297] The ATR inhibitor may be isotopically enriched (eg, enriched with deuterium). The methods disclosed herein that utilize ATR inhibitors may further include the use of a PARP inhibitor (e.g., administering a PARP inhibitor to a subject).Non-limiting examples of PARP inhibitors include AZD5305, olaparib, rucaparib, veliparib (ABT-888), niraparib (ZL-2306), iniparib (BSI-201), talazoparib (BMN673), 2X-121, CEP-9722, KU-0059436 (AZD2281), PF-01367338, pharmaceutically acceptable salts thereof, and combinations thereof.

[0298] Myt1 inhibitors Myt1 inhibitors are known in the art, for example, in WO2021 / 195781 and WO2021 / 195782. Myt1 inhibitors for use in the methods of the present invention include, for example, compounds of formula (III):

[0299] [ka]

[0300] or a pharma- ceutically acceptable salt thereof Where: Each of X, Y, and Z is independently N or CR 2 and R 1 and each R 2 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 3-8 Cycloalkenyl, optionally substituted C 2-9 Heterocyclyl, optionally substituted C 2-9 Heterocyclyl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, optionally substituted C1-9 Heteroaryl C 1-6 Alkyl, halogen, cyano, -N(R 7 ) 2 , -OR 7 , -C(O)N(R 8 ) 2 , -SO 2 N(R 8 ) 2 , -SO 2 R 7A , or -QR 7B or R 1 is R 1 One R that is vicinal to 2 In combination with, and optionally substituted, C 3-6 Forming an alkylene R 3 and R 4 each independently represents an optionally substituted C 1-6 is alkyl or halogen; R 5 is H or -N(R 7 ) 2 and R 6 is -C(O)NH(R 8 ), -C(O)R 7A , or -SO 2 R 7A and Each R 7 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 6-10 Aryl, optionally substituted C 2-9 Heterocyclyl, optionally substituted C 1-9 Heteroaryl, optionally substituted C 1-9 Heteroaryl C 1-6 Alkyl, or -SO 2 R 7A or two R 7The group, together with the atoms to which both are attached, may be an optionally substituted C 2-9 forming a heterocyclyl, Each R 7A independently represents an optionally substituted C 1-6 Alkyl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 6-10 is aryl, Each R 7B independently represents hydroxyl, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 2-9 Heterocyclyl, optionally substituted C 1-9 Heteroaryl, -N(R 7 ) 2 , -C(O)N(R 8 ) 2 , -SO 2 N(R 8 ) 2 , -SO 2 R 7A or optionally substituted alkoxy; Each R 8 are independently hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkoxyalkyl, optionally substituted C 6-10 Aryl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 3-8 Cycloalkyl, or optionally substituted C 1-9 Heteroaryl or two R 8 together with the atoms to which they are attached, optionally substituted C 2-9 forming a heterocyclyl, Q is optionally substituted C 1-6 Alkylene, optionally substituted C 2-6 Alkenylene, optionally substituted C 2-6Alkynylene, optionally substituted C 3-8 Cycloalkylene, optionally substituted C 3-8 Cycloalkenylene, optionally substituted C 6-10 Arylene, optionally substituted C 2-9 Heterocyclylene, or optionally substituted C 1-9 Heteroarylene.

[0301] Preferably, the compound of formula (III) is an atropisomer of formula (IIIA):

[0302] [ka]

[0303] where all variables are as described herein. The compound used in the method of the present invention is, for example, a compound of formula (IV):

[0304] [ka]

[0305] where all variables are as described herein. Preferably, the compound of formula (IV) is an atropisomer of formula (IVA):

[0306] [ka]

[0307] where all variables are as described herein. The compound used in the method of the present invention is, for example, a compound of formula (V):

[0308] [ka]

[0309] (In the formula, R 2A is hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 2-6 Alkynyl, optionally substituted C 3-8 Cycloalkyl, optionally substituted C 3-8 Cycloalkenyl, optionally substituted C 2-9 Heterocyclyl, optionally substituted C 2-9 Heterocyclyl C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 1-9 Heteroaryl, optionally substituted C 1-9 Heteroaryl C 1-6 Alkyl, halogen, -N(R 7 ) 2 , -OR 7 , -C(O)N(R 8 ) 2 , -SO 2 N(R 8 ) 2 , -SO 2 R 7A , or -QR 7B (It can be.)

[0310] Preferably, the compound of formula (V) is an atropisomer of formula (VA):

[0311] [ka]

[0312] is concentrated relative to The compound used in the methods of the invention can be, for example, a compound listed in Table 2 below, or a pharma- ceutically acceptable salt thereof.

[0313] [Table 2-1]

[0314]

Table 2-2

[0315]

Table 2-3

[0316]

Table 2-4

[0317]

Table 2-5

[0318]

Table 2-6

[0319]

Table 2-7

[0320]

Table 2-8

[0321]

Table 2-9

[0322]

Table 2-10

[0323]

Table 2-11

[0324]

Table 2-12

[0325]

Table 2-13

[0326]

Table 2-14

[0327]

Table 2-15

[0328]

Table 2-16

[0329]

Table 2-17

[0330]

Table 2-18

[0331]

Table 2-19

[0332]

Table 2-20

[0333]

Table 2-21

[0334] [Table 2-22]

[0335] [Table 2-23]

[0336] [Table 2-24]

[0337] [Table 2-25]

[0338] [Table 2-26]

[0339] [Table 2-27]

[0340] [Table 2-28]

[0341] Myt1 inhibitors may be isotopically enriched (eg, enriched with deuterium). The methods disclosed herein may be used to treat diseases or conditions that depend on the activity of membrane-bound tyrosine and threonine-specific cdc2 inhibitory kinase (Myt1) (gene name PKMYT1), such as cancers with CCNE1 amplification. The methods disclosed herein may include administering to a subject in need thereof a therapeutically effective amount of a membrane-bound tyrosine and threonine-specific cdc2 inhibitory kinase (Myt1) inhibitor. Without wishing to be bound by theory, it is believed that CCNE1 amplification may result in overexpression of CCNE1 gene products, such as CCNE1 transcripts and / or CCNE1 protein.

[0342] The method disclosed herein may include administering a therapeutically effective amount of a second therapeutic agent.The second therapeutic agent may be, for example, a WEE1 inhibitor, a FEN1 inhibitor, a TOP1 inhibitor, a RRM1 inhibitor, a RRM2 inhibitor, an AURKB inhibitor, a TOP2A inhibitor, an ATR inhibitor, a TTK inhibitor, a SOD1 inhibitor, a SOD2 inhibitor, a BUB1 inhibitor, a CDC7 inhibitor, a SAE1 inhibitor, a PLK1 inhibitor, a UBA2 inhibitor, a DUT inhibitor, a HDAC3 inhibitor, a CHEK1 inhibitor, an AURKA inhibitor, a MEN1 inhibitor, a DOT1L inhibitor, a CREBBP inhibitor, an EZH2 inhibitor, a PLK4 inhibitor, a HASPIN inhibitor, a METTL3 inhibitor, a nucleoside analog, a platinum-based DNA damaging agent, or a combination thereof.

[0343] The disease or condition may have a symptom of cellular hyperproliferation. For example, the disease or condition may be cancer (e.g., CCNE1 amplified cancer). Cancers with a high incidence of CCNE1 overexpression include, for example, uterine cancer, ovarian cancer, bladder cancer, pancreatic cancer, mesothelioma, kidney cancer, bladder cancer, gastric cancer, ovarian cancer, breast cancer, gastric cancer, esophageal cancer, lung cancer, and endometrial cancer. Preferably, the cancer is uterine cancer, colon cancer, breast cancer, lung cancer, or esophageal cancer.

[0344] The compounds disclosed herein may be administered by a route selected from the group consisting of oral, sublingual, buccal, transdermal, intradermal, intramuscular, parenteral, intravenous, intraarterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal, intranasal, inhalation, intratumor, and topical administration.

[0345] In some embodiments, the Myt1 inhibitor is administered prior to the second agent (e.g., within 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, or 12 hours). In some embodiments, the Myt1 inhibitor is administered after the second agent (e.g., within 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, or 12 hours). In some embodiments, the Myt1 inhibitor is co-administered with the second agent. In some embodiments, the Myt1 inhibitor is administered intermittently (e.g., 1 day / week, 2 days / week, or 3 days / week). In some embodiments, the second agent is administered consecutively every day.

[0346] AURKA inhibitors AURKA inhibitors, when exposed to AURKA, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of AURKA such that the IC of AURKA is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of AURKA inhibitors are MK0547, barasertib (AZD1152), PHA739358, AT9283, AMG900, SNS-314, TAK-901, CYC116, GSK1070916, PF03814735, and pharma- ceutical acceptable salts thereof. Exemplary AURKA inhibitors are also disclosed in US6,977,259; US6,919,338; US7,105,669; US7,214,518; US7,235,559; US7,402,585; US7,709,479; US8,026,246; US8,138,338; US8,377,983; US9,567,329; US9,637,474; US20060178382; US20090029992; and US20190352297, the AURKA inhibitors disclosed therein are incorporated by reference in their entireties herein.

[0347] AURKB inhibitors AURKB inhibitors, when exposed to AURKB in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be one that reduces the activity of AURKB such that the IC of AURKB for a particular AURKB inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of AURKB inhibitors are MLN8237, MK0547, MLN8054, PHA739358, AT9283, AMG900, MK5108, SNS314, TAK901, CYC116, ENMD2076, and pharma- ceutically acceptable salts thereof. Exemplary AURKB inhibitors are also disclosed in US7,560,551; US7,977,477; US8,110,573; and US20110293745, the AURKB inhibitors disclosed therein are incorporated herein by reference in their entirety.

[0348] BUB1 inhibitors BUB1 inhibitors, when exposed to BUB1, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of BUB1 such that the IC of BUB1 is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (for example, 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of the BUB1 inhibitor are BAY-320, BAY-419, BAY1816032, and pharma- ceutically acceptable salts thereof. Exemplary BUB1 inhibitors are also disclosed in US9,265,763; US9,416,125; US9,745,285; US10,266,548; US10,428,044; US20150141372; US20160145267; US20160046604; US20160046610; US20170275269; US20170305882, the BUB1 inhibitors disclosed therein are incorporated by reference in their entireties.

[0349] CDC7 inhibitors CDC7 inhibitors, when exposed to CDC7 in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of CDC7 such that the IC of CDC7 for a particular CDC7 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (for example, 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of the CDC7 inhibitor are SRA141, TAK931, and pharma- ceutically acceptable salts thereof. Exemplary CDC7 inhibitors are also disclosed in US7,279,575; US8,314,121; US8,383,624; US8,658,662; US8,691,828; US9,156,824; US9,180,105; US9,974,795; US10,745,510; US20050043346; US20050256121; US20070293491; US20190336502; and US20200093828, the CDC7 inhibitors disclosed therein are incorporated by reference in their entireties herein.

[0350] CHEK1 inhibitors CHEK1 inhibitors, when exposed to CHEK1, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of CHEK1 such that the IC of CHEK1 for a particular CHEK1 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). An example of a CHEK1 inhibitor is SRA737 and its pharma- ceutically acceptable salts. Exemplary CHEK1 inhibitors are also disclosed in US 7,067,506; US 8,093,244; US 8,410,279; US 8,530,468; US 8,618,121; US ​​8,916,591; US ​​9,067,920; US 9,440,976; US 10,189,818; US 10,822,327; US 200901820 01; US20090233896; US20090258852; US20090270416; US20090275570; US20150368244; US20180369202; and US20200397796, the CHEK1 inhibitors disclosed therein are incorporated by reference in their entireties herein.

[0351] CREBBP inhibitors CREBBP inhibitors, when exposed to CREBBP, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of CREBBP such that the IC of CREBBP is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of CREBBP inhibitors are CPI4, CCS1477, E7386, NEO1132, NEO2734, PRI724, C82, BC001, C646, EML425, CBP30, and pharma- ceutical acceptable salts thereof. Exemplary CREBBP inhibitors are also disclosed in US9,763,922; US10,206,931; US10,696,655; US10,870,648; US20190270797; US20190298729; and US20190308978, the CREBBP inhibitors disclosed therein are incorporated by reference in their entireties.

[0352] DOT1L inhibitors DOT1L inhibitors, when exposed to DOT1L, either in vitro, in cell culture, or in animals, have been shown to reduce the measured IC 50 The compound may be a compound that reduces the activity of DOT1L such that the IC of DOT1L for a particular DOT1L inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). An example of a DOT1L inhibitor is pinometostat (EPZ5676) and its pharma- ceutically acceptable salts. Exemplary DOT1L inhibitors are also disclosed in US8,722,877; US9,458,165; US10,112,968; US20140100184; US20150342979; and US20170335402, the DOT1L inhibitors disclosed therein are incorporated herein by reference in their entirety.

[0353] DUT inhibitors A DUT inhibitor, when exposed to a DUT, whether in vitro, in cell culture, or in animals, increases the measured IC 50 The IC of a particular DUT inhibitor may be a compound that reduces the activity of the DUT such that the IC of the DUT is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (for example, 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). An example of a DUT inhibitor is TAS114 and pharma- ceutically acceptable salts thereof. Exemplary DUT inhibitors are also disclosed in US7,601,702; US8,530,490; US9,790,214; US9,809,571; US10,544,105; US10,562,860; US10,570,100; US10,577,321; US10,829,457; US10,858,344; US20110212467; US20190270756; US20190330158; US20190330210; and US20200039966, the DUT inhibitors disclosed therein are incorporated by reference in their entireties herein.

[0354] EZH2 inhibitors EZH2 inhibitors, when exposed to EZH2, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of EZH2 such that the IC of EZH2 for a particular EZH2 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of EZH2 inhibitors are EPZ-6438, GSK126, and pharma- ceutically acceptable salts thereof. Exemplary EZH2 inhibitors are also disclosed in US8,691,507; US9,394,283; US9,889,138; US10,166,238; US10,040,782; US10,457,640; US10,633,371; US10,647,700; US10,786,511; US20190328743; US20190345139; and US20210052595, the EZH2 inhibitors disclosed therein are incorporated by reference in their entireties.

[0355] HASPIN inhibitors HASPIN inhibitors, when exposed to HASPIN, either in vitro, in cell culture, or in animals, have been shown to reduce the IC 50 The compound may be a compound that reduces the activity of HASPIN such that the IC of HASPIN is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). An example of a HASPIN inhibitor is SEL120 and pharma- ceutically acceptable salts thereof. Exemplary HASPIN inhibitors are also disclosed in US20130102627 and US20130231360, the HASPIN inhibitors disclosed therein being incorporated by reference in their entirety herein.

[0356] HDAC3 inhibitors HDAC3 inhibitors, when exposed to HDAC3, either in vitro, in cell culture, or in animals, increase the measured IC 50The compound may be one that reduces the activity of HDAC3 such that the IC of HDAC3 for a particular HDAC3 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). An example of an HDAC3 inhibitor is RGFP966 and its pharma- ceutically acceptable salts. Exemplary HDAC3 inhibitors are also disclosed in US8,716,344; US9,096,549; US10,029,988; US10,059,723; and US20190216754, the HDAC3 inhibitors disclosed therein are incorporated herein by reference in their entirety.

[0357] FEN1 inhibitors FEN1 inhibitors, when exposed to FEN1, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of FEN1 such that the IC of FEN1 for a particular FEN1 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of FEN1 inhibitors are C8 (PMID: 32719125), SC13, FEN1-IN-3, and pharma- ceutically acceptable salts thereof. Exemplary FEN1 inhibitors are also disclosed in US20200237763 and US7,927,790, the FEN1 inhibitors disclosed therein are incorporated herein by reference in their entirety.

[0358] MEN1 inhibitors MEN1 inhibitors, when exposed to MEN1, either in vitro, in cell culture, or in animals, have been shown to reduce the IC 50 The compound may be one that reduces the activity of MEN1 such that the IC of MEN1 for a particular MEN1 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of MEN1 inhibitors are MI3454, SNDX5613, VTP50469, KO539, and pharma- ceutically acceptable salts thereof. Exemplary MEN1 inhibitors are also disclosed in US8,242,078' US9,212,180; US10,077,271; US10,526,341; US10,611,778; US10,745,409; US10,752,639; US10,781,218; US10,899,738; US20170119769; US20190010167; US20190211036; US20200022953; US20200216471; and US20200223853, the MEN1 inhibitors disclosed therein are incorporated by reference in their entireties.

[0359] METTL3 inhibitors METTL3 inhibitors, when exposed to METTL3, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of METTL3 such that the IC of METTL3 is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of METTL3 inhibitors are UZH1a, sTC-15, and pharma- ceutically acceptable salts thereof. Exemplary METTL3 inhibitors are also disclosed in US20160264934 and WO2020201773, and the METTL3 inhibitors disclosed therein are incorporated by reference in their entirety.

[0360] Nucleoside Analogues Nucleoside analogs can be compounds that can act as antimetabolites by inhibiting nucleotide production, either in cell culture or in animals, or by acting as chain terminators in DNA elongation by polymerase enzymes. For certain nucleoside analogs, biological activity occurs at 10 μM or less (e.g., 5 μM or less or 1 μM or less) and can be as low as 100 pM or 10 pM. Preferably, nucleoside analog activity occurs at 1 nM to 1 μM (e.g., 1 nM to 750 nM, 1 nM to 500 nM, or 1 nM to 250 nM). Examples of nucleoside analogs are cytarabine, gemcitabine, mercaptopurine, azacytidine, cladribine, decitabine, fluorouracil, floxuridine, fludarabine, or nelarabine.

[0361] PLK1 inhibitors PLK1 inhibitors, when exposed to PLK1, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of PLK1 such that the IC of PLK1 for a particular PLK1 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50is 0.1 nM to 1 μM (for example, 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of PLK1 inhibitors are BI2536, BI6727, TAK960, NMSP937, GSK461364, and pharma- ceutically acceptable salts thereof. Exemplary PLK1 inhibitors are also disclosed in US 7,504,513; US 7,517,873; US 7,851,495; US 7,977,336; US 8,101,628; US 8,129,387; US 8,278,299; US 9,175,038; US 9,175,357; US 20070185133; US 20080015192; US 20100278833; US 20150368209; US 20170283445; and US 20200247796, the PLK1 inhibitors disclosed therein are incorporated by reference in their entireties herein.

[0362] PLK4 inhibitors PLK4 inhibitors, when exposed to PLK4, either in vitro, in cell culture, or in animals, increase the measured IC 50 The compound may be a compound that reduces the activity of PLK4 such that the IC of PLK4 is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of PLK4 inhibitors are centrinone, CFI-400945, and pharma- ceutically acceptable salts thereof. Exemplary PLK4 inhibitors are also disclosed in US10,752,612; US20190070190; and US20200383990, the PLK4 inhibitors disclosed therein are incorporated herein by reference in their entirety.

[0363] RRM1 and RRM2 inhibitors RRM1 inhibitors, when exposed to RRM1, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of RRM1 such that the IC of RRM1 for a particular RRM1 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM).

[0364] RRM2 inhibitors, when exposed to RRM2, either in vitro, in cell culture, or in animals, have been shown to increase the measured IC 50 The compound may be a compound that reduces the activity of RRM2 such that the IC of RRM2 for a particular RRM2 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of RRM2 inhibitors are motexafine gadolinium, hydroxyurea, fludarabine, cladribine, tezacitabine, triapine, and pharma- ceutically acceptable salts thereof. Exemplary RRM2 inhibitors are also disclosed in US4,188,378; US4,357,324; and US2019 / 0161461, the RRM2 inhibitors disclosed therein being incorporated herein by reference in their entirety.

[0365] SAE1 inhibitors SAE1 inhibitors, when exposed to SAE1, either in vitro, in cell culture, or in animals, have a measured IC 50The compound may be a compound that reduces the activity of SAE1 such that the IC of SAE1 for a particular SAE1 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). An example of an SAE1 inhibitor is ML792 and its pharma- ceutically acceptable salts. Exemplary SAE1 inhibitors are also disclosed in US 7,951,810; US 8,008,307; US 8,207,177; US 9,683,003; and US 9,695,154, the SAE1 inhibitors disclosed therein are incorporated herein by reference in their entirety.

[0366] SOD1 inhibitors SOD1 inhibitors, when exposed to SOD1, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of SOD1 such that the IC of SOD1 for a particular SOD1 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (for example, 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of the SOD1 inhibitor are LCS1, ATN-224, pyrimethamine, and pharma- ceutically acceptable salts thereof.

[0367] SOD2 inhibitors SOD2 inhibitors, when exposed to SOD2, either in vitro, in cell culture, or in animals, reduce the measured IC 50The compound may be a compound that reduces the activity of SOD2 such that the IC of SOD2 for a particular SOD2 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (for example, 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of the SOD2 inhibitor are LCS1, ATN-224, pyrimethamine, and pharma- ceutically acceptable salts thereof.

[0368] TOP1 inhibitors TOP1 inhibitors, when exposed to TOP1, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of TOP1 such that the IC of TOP1 for a particular TOP1 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of TOP1 inhibitors are irinotecan, topotecan, camptothecin, lamellarin D, and pharmaceutically acceptable salts thereof. Exemplary TOP1 inhibitors are also disclosed in US 4,604,463; US 4,894,456; and US 5,004,758, the TOP1 inhibitors disclosed therein are incorporated herein by reference in their entirety.

[0369] TOP2 inhibitors TOP2 inhibitors, when exposed to TOP2, either in vitro, in cell culture, or in animals, reduce the measured IC 50The compound may be a compound that reduces the activity of TOP2 such that the IC of TOP2 is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of TOP2 inhibitors are etoposide, teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticine, and pharma- ceutically acceptable salts thereof. Exemplary TOP2 inhibitors are also disclosed in US3,590,028; US3,933,827; US3,989,598; US4,258,191; US4,464,529; and US4,965,348, the TOP2 inhibitors disclosed therein are incorporated herein by reference in their entirety.

[0370] TTK inhibitors TTK inhibitors, when exposed to TTK, either in vitro, in cell culture, or in animals, increase the measured IC 50 The compound may be a compound that reduces the activity of TTK such that the IC of TTK for a particular TTK inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50is 0.1 nM to 1 μM (for example, 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of TTK inhibitors are BAY1217389 and pharma- ceutically acceptable salts thereof. Exemplary TTK inhibitors are also described in US8,551,980; US8,729,082; US9,199,999; US9,212,184; US9,284,317; US9,340,528; US9,388,140; US9,388,177; US9,468,642; US9,512,126; US9,512,130; US9,555,022; US9,586,958; US9,663,510; US9,670,202; US2017 / 0217946; US2017 / 0305912; US2017 / 0334899; US2017 / 0342064; US9,676,766; Wengner et al. al., Mol. Cancer Ther., 15:583-592, 2016; Zaman et al., Mol. Cancer Ther., 16:2609-2617, 2017; Mason et al., Proc. Nat'l Acad. Sci. USA, 21:3127-3132, 2017; and Riggs et al., J. Med. Chem., 62:4401-4410, 2019, the TTK inhibitors disclosed therein are incorporated by reference in their entireties.

[0371] UBA2 inhibitors UBA2 inhibitors, when exposed to UBA2, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of UBA2 such that the IC of UBA2 for a particular UBA2 inhibitor is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). An example of a UBA2 inhibitor is TAK981 and its pharma- ceutically acceptable salts. Exemplary UBA2 inhibitors are also disclosed in US 9,045,483, the UBA2 inhibitors disclosed therein being incorporated herein by reference in their entirety.

[0372] WEE1 inhibitors WEE1 inhibitors, when exposed to WEE1, either in vitro, in cell culture, or in animals, reduce the measured IC 50 The compound may be a compound that reduces the activity of WEE1 such that the IC of WEE1 is 10 μM or less (e.g., 5 μM or less or 1 μM or less). 50 can be 100 nM or less (e.g., 10 nM or less, or 1 nM or less), and can be as low as 100 pM or 10 pM. 50 is 0.1 nM to 1 μM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM). Examples of WEE1 inhibitors are adavosertib (AZD1775), Debio-0123, ZN-c3, and pharma- ceutically acceptable salts thereof. Exemplary WEE1 inhibitors are also disclosed in US8,791,125; US9,850,247, WO2020210320; WO2019028008; WO2019173082; and WO2020210377, the WEE1 inhibitors disclosed therein are incorporated herein by reference in their entirety.

[0373] Platinum-based DNA-damaging agents Platinum-based DNA damaging agents are coordination compounds of Pt(II) or Pt(IV), typically known in the art as platinum drugs. Platinum-based DNA damaging agents contain at least two coordination sites at the platinum center, occupied by nitrogen-based spectator ligand(s). The nitrogen-based spectator ligands are monodentate or bidentate ligands, whose donor atoms are sp 3 or sp 2A hybridized nitrogen atom. Non-limiting examples of nitrogen-based spectator ligands are ammonia, 1,2-cyclohexanediamine, picoline, phenanthroline, or 1,6-hexanediamine. Non-limiting examples of platinum-based DNA damaging agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthroplatin, picoplatin, and satraplatin.

[0374] Isomers and compositions thereof The present invention includes the individual diastereomers, enantiomers, epimers, and atropisomers of the compounds disclosed herein, as well as mixtures of the diastereomers and / or enantiomers, including racemic mixtures, where possible. The specific stereochemistry disclosed herein is preferred, but other stereoisomers, including diastereomers, enantiomers, epimers, atropisomers, and mixtures thereof, may also be useful in treating disease. Inactive or less active diastereoisomers and enantiomers may also be useful, for example, in scientific studies of receptors and mechanisms of activation.

[0375] It is understood that a particular molecule can exist in more than one tautomeric form, and although only one tautomer may be shown in the examples, the invention includes all tautomers.

[0376] The invention also includes pharma- ceutically acceptable salts of the compounds, and pharmaceutical compositions comprising the compounds and a pharma- ceutically acceptable carrier. The compounds are particularly useful, for example, in certain types of cancer, and for slowing the progression of cancer once it has developed in a patient.

[0377] The compounds disclosed herein may be used in pharmaceutical compositions that include (a) the compound(s) or a pharma- ceutically acceptable salt thereof, and (b) a pharma- ceutically acceptable carrier. The compounds may be used in pharmaceutical compositions that include one or more other active pharmaceutical ingredients. The compounds may also be used in pharmaceutical compositions in which the compounds disclosed herein or a pharma- ceutically acceptable salt thereof is the only active ingredient.

[0378] Optical Isomers - Diastereomers - Geometric Isomers - Tautomers The compounds disclosed herein may, for example, contain one or more stereocenters and may exist as racemates, racemic mixtures, single enantiomers, individual diastereomers, and mixtures of diastereomers and / or enantiomers. The present invention includes all such isomeric forms of the compounds disclosed herein. All possible stereoisomers (e.g., enantiomers and / or diastereomers) as mixtures and pure or partially purified compounds are intended to be included within the scope of the present invention (i.e., all possible combinations of stereocenters as pure compounds or mixtures).

[0379] Some of the compounds described herein may contain bonds that are hindered in rotation, such that two distinct rotamers, or atropisomers, can be separated and found to have different biological activities that may be advantageous. It is intended that all possible atropisomers are included within the scope of the present invention.

[0380] Some of the compounds described herein may contain olefinic double bonds, and unless otherwise specified, are meant to include both E and Z geometric isomers. Some of the compounds described herein may exist as different points of attachment of hydrogen, called tautomers. One example is a ketone and its enol form, known as keto-enol tautomers. Individual tautomers and mixtures thereof are encompassed by the present invention.

[0381] Compounds disclosed herein that possess one or more asymmetric centers can be separated into diastereoisomers, enantiomers, and the like by methods well known in the art.

[0382] Alternatively, enantiomers and other compounds containing chiral centers may be synthesized by stereospecific synthesis using optically pure starting materials and / or reagents of known configuration.

[0383] Metabolites-Prodrugs The present invention includes therapeutically active metabolites, and the metabolites themselves are within the scope of the claims. The present invention also includes prodrugs, which are compounds that are converted to the claimed compounds when or after administration to a patient. The claimed chemical structures of the present application may in some cases themselves be prodrugs.

[0384] Isotopically enriched derivatives The present invention includes molecules that are isotopically enriched at one or more positions within the molecule, and therefore compounds enriched with deuterium are also within the scope of the claims.

[0385] Methods for preparing ATR inhibitors ATR inhibitors can be prepared using reactions and techniques known in the art.For example, certain ATR inhibitors can be prepared using techniques and methods disclosed in, for example, International Application Nos. PCT / US2019 / 051539 and PCT / US2018 / 034729, U.S. Patent Nos. 9,663,535, 9,549,932, 8,552,004 and 8,841,308, and U.S. Patent Application Publication No. 2019 / 0055240, each of which is incorporated herein by reference.

[0386] Pharmaceutical Compositions The compound used in the method described herein is preferably formulated into a pharmaceutical composition for administration to human subjects in a biologically compatible form suitable for in vivo administration.The pharmaceutical composition typically comprises the compound described herein and a pharmaceutically acceptable excipient.Certain pharmaceutical compositions may comprise one or more additional pharmacoactive agents described herein.

[0387] The compounds described herein may also be used in the form of free base, salts, zwitterions, solvates, or as prodrugs or pharmaceutical compositions thereof. All forms are within the scope of the present invention. The compounds, their salts, zwitterions, solvates, prodrugs, or pharmaceutical compositions may be administered to patients in various forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds used in the methods described herein may be administered, for example, by oral, parenteral, oral mucosal, sublingual, nasal, rectal, patch, pump, or transdermal administration, and pharmaceutical compositions may be formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, pulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[0388] For use in humans, the compounds of the present invention can be administered alone or in admixture with a pharmaceutical carrier selected with respect to the intended route of administration and standard pharmaceutical practice. Thus, pharmaceutical compositions for use according to the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries, that facilitate the processing of the compounds of the present invention into pharma- ceutical preparations that can be used.

[0389] The present invention also includes pharmaceutical compositions that may contain one or more pharma- ceutically acceptable carriers. In making pharmaceutical compositions of the present invention, active ingredients are typically mixed with or diluted by excipients, or enclosed within such carriers, for example in the form of capsules, sachets, paper, or other containers. When excipients function as diluents, they can be solid, semi-solid, or liquid materials (e.g., normal saline), and act as vehicles, carriers, or media for the active ingredients. Thus, the compositions can be in the form of tablets, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, and soft and hard gelatin capsules. As known in the art, the type of diluent can vary depending on the intended route of administration. The resulting compositions can include additional agents, for example, preservatives.

[0390] Excipient or carrier is selected based on the formulation and route of administration. Suitable pharmaceutical carriers and pharmaceutical requirements for use in pharmaceutical formulations are described in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippincott Williams & Wilkins (2005), well-known references in the field, and USP / NF (United States Pharmacopeia and the National Formulary). Examples of suitable excipients are lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulation may further include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl benzoate and propyl hydroxybenzoate; sweeteners; and flavoring agents. Other exemplary excipients are described in Handbook of Pharmaceutical Excipients, 6th Edition, Rowe et al., Eds., Pharmaceutical Press (2009).

[0391] These pharmaceutical compositions can be manufactured in a conventional manner, for example, by conventional mixing, dissolving, granulating, sugar-coating, levigating, emulsifying, encapsulating, encapsulating or lyophilizing processes. Methods for preparing formulations well known in the art can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippincott Williams & Wilkins (2005), and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York. Appropriate formulations depend on the route of administration selected. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulations. When preparing formulations, the active compound can be milled and then mixed with other ingredients to obtain a suitable particle size. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, eg, 40 mesh.

[0392] Dosage The dosage of the compound used in the methods described herein, or its pharma- ceutically acceptable salt or prodrug, or its pharmaceutical composition, may vary depending on many factors, such as, for example, the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of symptoms; the frequency of treatment, and the type of concomitant treatment (if any); and the clearance rate of the compound in the treated animal. Those skilled in the art can determine the appropriate dosage based on the above factors. The compound used in the methods described herein can be administered at a suitable dosage initially, which may be adjusted according to clinical response, if necessary. In general, the suitable daily dose of the compound of the present invention is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends on the above factors.

[0393] The compounds of the invention may be administered to a patient in a single dose or multiple doses. When multiple doses are administered, the doses may be separated from each other by, for example, 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, or 1 to 12 months. The compounds may be administered according to a schedule, or the compounds may be administered without a set schedule. The active compounds may be administered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per day, every 2nd, 3rd, 4th, 5th, or 6th day, 1, 2, 3, 4, 5, 6, or 7 times per week, 1, 2, 3, 4, 5, or 6 times per month, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per year. It is understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[0394] Although the attending physician will ultimately determine the appropriate amount and dosing regimen, an effective amount of the compound of the present invention can be, for example, 0.05 mg to 3000 mg of any of the compounds described herein in a total daily dose. Alternatively, the dose can be calculated using the patient's body weight. Such dose ranges include, for example, 0.05 to 1000 mg (e.g., 0.25 to 800 mg). In some embodiments, 0.05, 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg of the compound is administered.

[0395] formulation Compounds identified as capable of treating any of the conditions described herein using any of the methods described herein may be administered to a patient or animal in unit dosage form together with a pharma- ceutically acceptable diluent, carrier, or excipient. Chemical compounds used in such treatments may be produced and isolated by any standard technique known in the art of medicinal chemistry. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions for administering the identified compounds to a subject in need thereof. Administration may begin before the patient becomes symptomatic.

[0396] Exemplary routes of administration of the compounds used in the present invention (e.g., compounds of the present invention), or pharmaceutical compositions thereof, include oral, sublingual, oral mucosal, transdermal, intradermal, intramuscular, parenteral, intravenous, intraarterial, intracranial, subcutaneous, intraorbital, intravenous, intraspinal, intraperitoneal, intranasal, inhalation, and topical administration. Compounds are desirably administered with a pharmaceutically acceptable carrier. Pharmaceutical formulations of the compounds described herein that are formulated for the treatment of the disorders described herein are also part of the present invention. Oral administration is the preferred route of administration in the method of the present invention.

[0397] Oral Formulations Pharmaceutical compositions contemplated by the present invention include those formulated for oral administration ("oral dosage forms"). Oral dosage forms may be in the form of, for example, tablets, capsules, liquid solutions or suspensions, powders, or liquid or solid crystals, and contain the active ingredient(s) in admixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, glidants, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc). Other pharma- ceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0398] Formulations for oral administration may also be provided as chewable tablets, hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, e.g., peanut oil, liquid paraffin, or olive oil. Powders, granules, and pellets may be prepared in a conventional manner using the ingredients of the above-mentioned tablets and capsules, for example, using a mixer, fluid bed equipment, or spray drying equipment.

[0399] Controlled release compositions for oral use can be configured to release the active drug by controlling the dissolution and / or diffusion of the active drug substance. Any of a number of strategies can be implemented to obtain controlled release and a target plasma concentration versus time profile. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, for example, various types of controlled release compositions and coatings. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes. In certain embodiments, the composition includes a biodegradable, pH-sensitive, and / or temperature-sensitive polymer coating.

[0400] Controlled release by dissolution or diffusion can be achieved by suitable coating of the compound's tablet, capsule, pellet or granule formulation, or by incorporating the compound into a suitable matrix. Controlled release coatings can include one or more of the above coating materials and / or, for example, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethyl cellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxymethacrylate, methacrylate hydrogel, 1,3 butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. In controlled release matrix formulations, the matrix material may also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbons.

[0401] Liquid forms into which the compounds and compositions of the present invention can be incorporated for oral administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils, such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0402] Parenteral Formulations The compounds described herein for use in the methods of the present invention can be administered in pharma- ceutically acceptable parenteral (e.g., intravenous or intramuscular) formulations described herein. Pharmaceutical formulations can also be administered parenterally (intravenously, intramuscularly, subcutaneously, etc.) in dosage forms or formulations containing conventional non-toxic pharma- ceutically acceptable carriers and adjuvants. In particular, suitable formulations for parenteral administration include aqueous and nonaqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions that may contain suspending agents and thickening agents. For example, to prepare such compositions, the compounds of the present invention can be dissolved or suspended in parenterally acceptable liquid vehicles. Acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide, or suitable buffers, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution. The aqueous formulation may also contain one or more preservatives, such as methyl benzoate, ethyl benzoate, or n-propyl p-hydroxybenzoate. Additional information regarding parenteral formulations can be found, for example, in the United States Pharmacopeia-National Formulary (USP-NF), incorporated herein by reference.

[0403] Parenteral formulations can be any of the five general types of preparations identified by the USP-NF as suitable for parenteral administration. (1) "Drug injection": a liquid preparation that is a drug substance (e.g., a compound of the present invention) or a solution thereof; (2) "Drug for injection": a drug substance (e.g., a compound of the invention) that is a dry solid that is combined with a sterile vehicle suitable for parenteral administration as a drug injection; (3) "Drug Injectable Emulsion": a liquid preparation of a drug substance (e.g., a compound of the invention) dissolved or dispersed in a suitable emulsion medium; (4) "Drug Injectable Suspension": A liquid preparation of a drug substance (e.g., a compound of the invention) suspended in a suitable liquid medium; and (5) "Drug for injectable suspension": A drug substance (eg, a compound of the invention) that is a dry solid that is combined with a sterile vehicle suitable for parenteral administration as a drug for injectable suspension.

[0404] Exemplary formulations for parenteral administration include solutions of the compound prepared in water suitably mixed with a surfactant, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof, with or without alcohol, and in oils. These preparations may contain preservatives to prevent the growth of microorganisms under normal storage and use conditions. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippincott Williams & Wilkins (2005) and The United States Pharmacopeia: The National Formulary (USP 36 NF31) (published in 2013).

[0405] Formulations for parenteral administration may contain, for example, excipients, sterilized water or saline, polyalkylene glycols, such as polyethylene glycol, oils derived from plants, or hydrogenated naphthalene. Biocompatible biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compound. Other potentially useful parenteral delivery systems for the compound include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, such as lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions or gels for administration in the form of nasal drops.

[0406] Parenteral formulations can be formulated for immediate release or sustained / sustained release of the compound. Exemplary formulations for parenteral release of the compound include aqueous solutions, powders for reconstitution, co-solvent solutions, oil / water emulsions, suspensions, oily solutions, liposomes, microspheres, and polymer gels.

[0407] The following examples are intended to illustrate the invention without, however, limiting it in any way. EXAMPLES

[0408] Example 1. Design of SNV panel version 1 To quantify genetic alterations in the ATM gene, an SNV panel was designed based on Anchored Multiplex PCR (AMP™) technology. Gene-specific primers were designed to amplify genomic regions of interest using AMP chemistry.

[0409] Gene-specific primers were designed for up to 30 single nucleotide variants (SNVs) occurring in 20–80% of the global population (as defined in the gnomAD database v.2.1.1) for ATM targets.

[0410] Samples were assessed for ploidy and chromosome arm losses using gene-specific primers designed against 1000 SNVs randomly distributed across the genome. Example 2. Performance evaluation of SNV Panel version 1 for whole genome sequencing The SNV panel designed according to the process outlined in Example 1 can quantify ATM loss of heterozygosity and copy number deletion. Furthermore, the SNV panel design shows complete exon coverage and also provides genome-wide SNV quantification of purity and ploidy calls. The performance of the SNV panel was evaluated against whole genome sequencing (WGS) of three samples from subjects with various cancers. Specifically, the three subjects were a 46-year-old woman with papillary serous carcinoma, a 62-year-old woman with pancreatic ductal adenocarcinoma, and a 72-year-old woman with ER-, PR-, HER2- metastatic breast cancer.

[0411] To prepare each SNV panel sample, DNA from the sample was amplified using SNV panel primers using VariantPlex® cycling optimized for a large panel of over 3500 primer pairs: i.PCR1 / 2 cycle number 10 / 15 ii. Holding time: 15 minutes iii. Annealing / extension temperature 60℃ / 65℃ iv. PCR primer amount increase test Amplified DNA libraries were sequenced and normalized to 12 million reads per library.

[0412] Quantification and comparison of copy number calls across all chromosomes determined by WGS and SNV panels for three exemplary samples are shown in Figures 2A, 2B, 3A, 3B, 4A, and 4B. Specifically, the ability of the SNV panel to accurately identify LOH was evaluated using WGS as a basis for comparison. The SNV panel and WGS showed good concordance of LOH calls in the three exemplary samples, especially in tumors with simple karyotypes and high cancer cell fractions in the samples (see Tables 3-5).

[0413] [Table 3]

[0414] [Table 4]

[0415] [Table 5]

[0416] Example 3. Design of SNV panel version 2 We designed SNV panel version 2 with increased SNV coverage, which has a 5-fold higher density of heterozygous SNVs compared to SNV panel version 1 (see Figures 5A and 5B), and therefore can provide more accurate LOH identification when assessed against WGS.

[0417] For SNV panel version 2, SNVs were selected according to the following selection criteria based on population and sequencing characteristics: The population characteristics were as follows: (1) 33%< allele frequency < 66%, and (2) 0< inbreeding coefficient<0.2.

[0418] The 5'-flanking sequence (50 base pairs) was characterized as follows: (1) GC percentage between 25% and 75%; (2) Unique; and (3) does not contain other common SNVs.

[0419] Additional primers for four thousand (4000) single nucleotide variants (SNVs) were added to obtain further coverage. To improve compatibility with low-quality FFPE input (multiple clinical origins), primer pairs close to each target are preferred. To include SNVs useful for detecting loss of heterozygosity (LOH) structural variants without copy number changes, select SNVs that are commonly heterozygous across subpopulations according to the current gnomAD release (v3). To select SNVs likely to generate the highest quality and quality NGS reads using AMP chemistry, avoid SNVs in amplicons adjacent to repetitive or GC-rich regions of the genome and prioritize SNVs that are less susceptible to noise during PCR or sequencing (e.g., not adjacent to polynucleotide tracts). Finally, to select SNVs that take into account the spatial granularity of genome calls, select SNVs that are as evenly distributed as possible across the genome.

[0420] Genomic DNA (>50ng) was extracted from FFPE samples (n=43) of multiple solid tumor types. Next generation sequencing was performed on anchored multiplex PCR libraries constructed using probes incorporating unique molecular identifiers and spanning 26 genes and 5,000 genome-wide common germline SNVs. Unmatched non-tumor samples (n=24) were used to generate a reference baseline dataset. Copy number imbalance at heterozygous SNVs was assessed and tumor purity was quantified using the FACETS algorithm, optimized to account for differences in DNA fragmentation between samples. Allele fractions of each heterozygous SNV were used to estimate allelic imbalance across chromosomal regions. The reference dataset was derived from matched FFPE tumor samples by whole genome sequencing (WGS) and sequence data analysis using three complementary algorithms. Allele-specific copy number analysis and tumor purity estimation from SNV Panel version 2 and WGS data were compared.

[0421] Copy number was assessable for 605 genes in 24 matched tumor samples that passed quality control filters. Median sequencing depth across samples by SNV Panel version 2 and WGS was 1346x and 18.6x, respectively. LOH detection by SNV Panel version 2 was reproducible (100%) across 170 genes in seven samples analyzed in duplicate. A strong correlation was observed between WGS and SNV Panel version 2 sample purity estimates (Pearson's r=0.81, p<0.001). Compared with WGS calls, the sensitivity and specificity of LOH detection by SNV Panel version 2 was 95% and 90%, respectively, increasing to 97% and 91% in regions with concordant LOH across all three WGS algorithms and 99% and 97% in diploid regions without subclonal mutations.

[0422] Figure 6 shows an example of a copy number profile compiled using SNV panel version 2 for a cancer cell with biallelic ATM loss of function. Quantification and comparison of copy number calls across all chromosomes determined by WGS and SNV panel version 2 for eight exemplary samples are shown in Figures 7A-14B. Specifically, the ability of SNV panel version 2 to accurately identify LOH was evaluated using WGS as a basis for comparison. The SNV panel and WGS showed good concordance of LOH calls in eight exemplary samples (see Tables 6-13).

[0423] [Table 6]

[0424] [Table 7]

[0425] [Table 8]

[0426] [Table 9]

[0427] [Table 10]

[0428] [Table 11]

[0429] [Table 12]

[0430] [Table 13]

[0431] Example 4. Biallelic ATM loss-of-function mutations in certain cancers The frequency of biallelic ATM deletions in tumors was estimated based on genomic data available in The Cancer Genome Atlas (Nature Genetics, 45:1113-1120, 2013). This dataset allowed for molecular characterization of tumor samples from over 10,000 patients classified into 33 cancer indications. Data indicating the presence of somatic mutations, copy number variation, or loss of heterozygosity were downloaded from the Genomic Data Commons Data Portal (portal.gdc.cancer.gov). Identification of pathogenic germline mutations in TCGA samples was obtained from published literature (Nature Commun. 8:857, 2017). Samples with the simultaneous presence of loss of heterozygosity and either germline or somatic pathogenic mutations were classified as biallelic deletions. Summarizing the mutation data by tumor indication showed that ATM biallelic deletions in the TCGA cohort were more frequent in cancers such as bladder urothelial carcinoma, pancreatic adenocarcinoma, and lung adenocarcinoma. The frequency of biallelic ATM mutations in various cancers is shown in Figures 7A and 7B: Figure 7A is a chart showing the frequency of biallelic ATM mutations in various cancers; Figure 7B is a chart showing the frequency of biallelic germline ATM mutations in various cancers.

[0432] Example 5. CCNE1 gene overexpression detection using SNV panel version 2 and WGS The techniques described in Example 3 were adapted to detect overexpression of the genes, and the results are shown in Figures 17A-21.

[0433] Other embodiments Various modifications and variations of the invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unnecessarily limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

[0434] Other embodiments are found in the claims.

Claims

1. 1. An ATR inhibitor for use in a method of treating a cancer having biallelic ATM loss-of-function mutations in a subject, said method comprising administering to said subject in need thereof an effective amount of an ATR inhibitor.

2. The method further comprises, prior to the administering step, identifying the cancer as having a biallelic ATM loss-of-function mutation, the identifying step comprising: determining an integer total copy number of a locus segment within the cancer cell of the subject or the ATM gene region of the cancer cell and / or two integer allele-specific copy numbers of the locus segment from read counts of a plurality of SNVs, including homozygous single nucleotide variants (SNVs) and heterozygous SNVs, and a reference read count obtained from sequencing a sample comprising the cancer cell; The cancer is at least one of the total copy number of said integer and the allele-specific copy number of said integer is zero (provided that the remaining ATM allele, if present, contains an inactivating mutation); or The ATR inhibitor according to claim 1, wherein the ATR inhibitor is identified as having a biallelic ATM loss-of-function mutation when none of the integer allele-specific copy numbers are 0, an ATM allele is present, and each of the ATM alleles independently has an inactivating mutation.

3. The determining step further comprises: determining a total copy number log ratio, an allele copy number log odds ratio, and a target coverage value for the SNV from the read counts and the reference read counts; Segmenting the total copy number log ratio and the allele copy number log odds ratio; estimating a sample purity and a sample ploidy of the cancer cells from the total copy number log ratio and the target coverage value; and generating an integer total copy number and two integer allele-specific copy numbers of a segment containing a plurality of SNVs in the ATM gene region of the cancer cell from the target coverage value, the sample purity, the sample ploidy, the total copy number log ratio, and the allele copy number log odds ratio. The ATR inhibitor of claim 2.

4. The ATR inhibitor of claim 3 , wherein the method further comprises adjusting the misregistration rate.

5. The ATR inhibitor according to any one of claims 2 to 4, wherein the plurality of SNVs includes SNVs having uniform coverage.

6. 6. The ATR inhibitor of claim 5, wherein each of the uniform coverage SNVs has an average coverage of at least 200x reads across a panel of normal samples.

7. (a) the plurality of SNVs includes a high-frequency SNV, the high-frequency SNV having an allele frequency in humans of 33% to 66%; or (b) the plurality of SNVs comprises a plurality of SNVs each having a 5'-flanking sequence of at least 20 contiguous nucleobases having a GC content of 25-75%, wherein the 5'-flanking sequences are unique and do not contain other SNVs; or (c) the plurality of SNVs comprises at least 20 heterozygous SNVs; The ATR inhibitor according to any one of claims 2 to 4, which is any combination thereof.

8. The ATR inhibitor according to claim 7 , wherein the plurality of SNVs includes SNVs adjacent to the high frequency SNV.

9. The ATR inhibitor of any one of claims 2 to 4, wherein the reference read counts are derived from a panel of normal samples. (a) the ATM gene region comprises ATM and up to 10 kilobases of each of flanking regions; or (b) the ATM gene region comprises ATM and up to 5 kilobases of each of the flanking regions; or (c) The ATR inhibitor according to claim 2, wherein the ATM gene region comprises ATM and up to 2 kilobases each of the flanking regions. (a) the ATM gene region is an ATM exome region, (b) the ATM gene region is the ATM transcriptome region; or (c) The ATR inhibitor according to claim 2, wherein the ATM gene region is an ATM genomic region.

12. 2. The ATR inhibitor of claim 1, wherein the biallelic ATM loss-of-function mutations comprise at least one somatic ATM loss-of-function mutation.

13. The ATR inhibitor according to any one of claims 1 to 4, wherein the biallelic ATM loss-of-function mutations comprise at least one germline ATM loss-of-function mutation.

14. The ATR inhibitor according to any one of claims 1 to 4, wherein the cancer is lung adenocarcinoma, adrenocortical carcinoma, invasive breast cancer, LumB-positive invasive breast cancer, Her2-positive invasive breast cancer, basal-like invasive breast cancer, pancreatic adenocarcinoma, bladder urothelial carcinoma, rectal adenocarcinoma, gastric adenocarcinoma, skin melanoma, colon adenocarcinoma, prostate cancer, glioblastoma multiforme, esophageal cancer, uterine endometrial cancer, hepatocellular carcinoma, lung squamous cell carcinoma, sarcoma, or ovarian serous cystadenocarcinoma.

15. The ATR inhibitor is a compound of formula (I): 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof; During the ceremony, 【Chemistry 2】 is a double bond, and each Y is independently N or CR ４ or 【Chemistry 3】 is a single bond, and each Y is independently NR Ｙ , carbonyl, or C(R Ｙ ) ２ wherein each R Ｙ is independently H or optionally substituted C １－６ is alkyl, R １ is optionally substituted C １－６ alkyl or H; R ２ is optionally substituted C ２－９ Heterocyclyl, optionally substituted C １－６ Alkyl, optionally substituted C ３－８ Cycloalkyl, optionally substituted C ２－９ Heterocyclyl C １－６ Alkyl, optionally substituted C ６－１０ Aryl, optionally substituted C １－９ Heteroaryl, optionally substituted C １－９ Heteroaryl C １－６ Alkyl, halogen, -N(R ５ ) ２ , -OR ５ , -CON(R ６ ) ２ , -SO ２ N (R ６ ) ２ , -SO ２ R ５Ａ , or -Q-R ５Ｂ and R ３ is optionally substituted C １－９ Heteroaryl or optionally substituted C １－９ Heteroaryl C １－６ is alkyl, Each R ４ are independently hydrogen, halogen, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ alkenyl, or optionally substituted C ２－６ is alkynyl, Each R ５ are independently hydrogen, optionally substituted C １－６ Alkyl, optionally substituted C ６－１０ Aryl C １－６ Alkyl, optionally substituted C ６－１０ Aryl, optionally substituted C １－９ Heteroaryl, or -SO ２ R ５Ａ or both R ５ together with the atoms to which they are attached, optionally substituted C ２－９ forming a heterocyclyl, Each R ５Ａ is independently an optionally substituted C １－６ Alkyl, optionally substituted C ３－８ Cycloalkyl, or optionally substituted C ６－１０ is aryl, R ５Ｂ is hydroxyl, optionally substituted C １－６ Alkyl, optionally substituted C ６－１０ Aryl, optionally substituted C １－９ Heteroaryl, -N(R ５ ) ２ , -CON(R ６ ) ２ , -SO ２ N (R ６ ) ２ , -SO ２ R ５Ａ or optionally substituted alkoxy; Each R ６ are independently hydrogen, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ Alkoxyalkyl, optionally substituted C ６－１０ Aryl C １－６ Alkyl, optionally substituted C ６－１０ Aryl, optionally substituted C ３－８ Cycloalkyl, or optionally substituted C １－９ heteroaryl, or both R ６ together with the atoms to which they are attached, optionally substituted C ２－９ forming a heterocyclyl, Q is an optionally substituted C ２－９ Heterocyclylene, optionally substituted C ３－８ Cycloalkylene, optionally substituted C １－９ Heteroarylene, or optionally substituted C ６－１０ is an arylene, The ATR inhibitor according to any one of claims 1 to 4, wherein X is hydrogen or halogen.

16. The ATR inhibitor is a compound of formula (II): 【Chemistry 4】 or a pharma- ceutically acceptable salt thereof; During the ceremony, Each Y is independently N or CR ４ and R １ is optionally substituted C １－６ alkyl or H; R ２ is optionally substituted C ２－９ Heterocyclyl, optionally substituted C １－６ Alkyl, optionally substituted C ３－８ Cycloalkyl, optionally substituted C ２－９ Heterocyclyl C １－６ Alkyl, optionally substituted C ６－１０ Aryl, optionally substituted C １－９ Heteroaryl, optionally substituted C １－９ Heteroaryl C １－６ Alkyl, halogen, -N(R ５ ) ２ , -OR ５ , -CON(R ６ ) ２ , -SO ２ N (R ６ ) ２ , -SO ２ R ５Ａ , or -Q-R ５Ｂ and R ３ is optionally substituted C １－９ Heteroaryl or optionally substituted C １－９ Heteroaryl C １－６ is alkyl, Each R ４ are independently hydrogen, halogen, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ alkenyl, or optionally substituted C ２－６ is alkynyl, Each R ５ are independently hydrogen, optionally substituted C １－６ Alkyl, optionally substituted C ６－１０ Aryl C １－６ Alkyl, optionally substituted C ６－１０ Aryl, optionally substituted C １－９ Heteroaryl, or -SO ２ R ５Ａ or both R ５ together with the atoms to which they are attached, optionally substituted C ２－９ forming a heterocyclyl, Each R ５Ａ is independently an optionally substituted C １－６ Alkyl, optionally substituted C ３－８ Cycloalkyl, or optionally substituted C ６－１０ is aryl, R ５Ｂ is hydroxyl, optionally substituted C １－６ Alkyl, optionally substituted C ６－１０ Aryl, optionally substituted C １－９ Heteroaryl, -N(R ５ ) ２ , -CON(R ６ ) ２ , -SO ２ N (R ６ ) ２ , -SO ２ R ５Ａ or optionally substituted alkoxy; Each R ６ are independently hydrogen, optionally substituted C １－６ Alkyl, optionally substituted C ２－６ Alkoxyalkyl, optionally substituted C ６－１０ Aryl C １－６ Alkyl, optionally substituted C ６－１０ Aryl, optionally substituted C ３－８ Cycloalkyl, or optionally substituted C １－９ heteroaryl, or both R ６ together with the atoms to which they are attached, optionally substituted C ２－９ forming a heterocyclyl, Q is an optionally substituted C ２－９ Heterocyclylene, optionally substituted C ３－８ Cycloalkylene, optionally substituted C １－９ Heteroarylene, or optionally substituted C ６－１０ is an arylene, The ATR inhibitor according to claim 15, wherein X is hydrogen or halogen.

17. 16. The ATR inhibitor of claim 15, wherein the ATR inhibitor is selected from the group consisting of compounds 43, 57, 62, 87, 93, 94, 95, 99, 100, 106, 107, 108, 109, 111, 112, 113, 114, 115, 116, 118, 119, 120, 121, 122, 123, 135, 147, 148 in Table 1, and pharma- ceutically acceptable salts thereof.

18. 18. The ATR inhibitor of claim 17, wherein the ATR inhibitor is compound 121 of Table 1 or a pharma- ceutically acceptable salt thereof.

19. The ATR inhibitor is 【Chemistry 5】 The ATR inhibitor according to any one of claims 1 to 4, which is an ATR inhibitor or a pharma- ceutically acceptable salt thereof.

20. The ATR inhibitor of any one of claims 1 to 4, wherein the method further comprises administering to the subject a PARP inhibitor.

Images