Methods and compositions for predicting anticancer efficacy of compounds targeting the apoptosis pathway

Abstract

Provided are biomarkers for predicting the efficacy of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor in treating a cancer patient. Also provided are compositions, e.g., kits, for assessing gene levels of the biomarkers, and methods of using such gene levels to predict the response of a cancer patient to an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor. Such information can be used to determine the prognosis and treatment selection of a cancer patient.

Description

TECHNICAL FIELD

[0001] The present application relates generally to cancer treatment. BACKGROUND

[0002] Evasion of apoptosis is a hallmark of human cancer and a common cause of treatment resistance (Hanahan D, et al., Cell (2000) 100:57-70; Delbridge AR, et al., Cold Spring Harb Perspect Biol (2012) 4). Thus, targeting key players of apoptosis in human cancer is an attractive new strategy for developing a novel class of anticancer therapies.

[0003] Clinical responses to anticancer therapies are often limited to a fraction of patients. To maximize the efficiency of anticancer therapies, personalized chemotherapy based on molecular biomarkers has been proposed. However, the identification of predictive biomarkers that can predict the response to anticancer therapies remains a challenge. Thus, there is a continuous need to develop biomarkers for predicting the anticancer efficacy of compounds targeting the apoptosis pathway. SUMMARY

[0004] In one aspect, the disclosure provides a method for treating cancer in a subject in need thereof. In one embodiment, the method comprises: measuring the level of at least one biomarker comprising Noxa in a test sample derived from the subject; comparing the level of the at least one biomarker to a corresponding reference level of the at least one biomarker to determine a difference from the reference level; and administering to the subject an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor when the difference reaches a predetermined threshold.

[0005] In another aspect, the disclosure provides a method for identifying a subject having cancer who is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor. In one embodiment, the method comprises: measuring the level of at least one biomarker comprising Noxa in a test sample derived from the subject; and comparing the level of the at least one biomarker to a corresponding reference level of the at least one biomarker to determine a difference from the reference level; and identifying the subject who is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor when the difference reaches a predetermined threshold.

[0006] In one embodiment, the method for identifying a subject having cancer who is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor further comprises administering to the subject identified as likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor.

[0007] In another aspect, the disclosure provides a method for monitoring efficacy of treatment in a subject having cancer who has been treated with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor for a treatment period. In one embodiment, the method comprises: obtaining a test sample from the subject after the treatment period; measuring in the test sample the level of at least one biomarker comprising Noxa to obtain a post-treatment level of the at least one biomarker; comparing the post-treatment level to a baseline level of the at least one biomarker in a sample derived from the subject prior to the treatment period to determine a post-treatment change in the level of the at least one biomarker; and continuing administration of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-xL inhibitor or the Bcl-2 inhibitor to the subject, increasing the dose of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-xL inhibitor or the Bcl-2 inhibitor to the subject, or administering to the subject an effective amount of a second anti-cancer therapeutic agent in combination with the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-xL inhibitor or the Bcl-2 inhibitor, or discontinuing administration of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-xL inhibitor or the Bcl-2 inhibitor to the subject when the post-treatment change reaches a predetermined threshold, or when the post-treatment change does not reach a predetermined threshold.

[0008] In certain embodiments, an increase or maintenance of Noxa level in the post-treatment sample indicates a likelihood of sustained responsiveness to treatment with the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-xL inhibitor or the Bcl-2 inhibitor. In certain embodiments, a decrease in Noxa level in the sample indicates a likelihood of decreased responsiveness to treatment with the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-xL inhibitor or the Bcl-2 inhibitor.

[0009] In some embodiments, the second anti -therapeutic agent can be selected from the group consisting of an antineoplastic agent, an anti-angiogenic agent, a chemotherapeutic agent, and a peptide-based cancer therapeutic. In yet another embodiment, the antineoplastic agent is selected from the group consisting of antibiotic-type agents, alkylating agents, anti-metabolites, hormonal agents, immunological agents, interferon-type agents, kinase inhibitors, other agents, and combinations thereof.

[0010] In some embodiments, an immune checkpoint molecule is administered as a second anti-cancer therapeutic in combination with an MDM2 inhibitor. In some embodiments, a chemotherapeutic agent is administered as a second anti-cancer therapeutic in combination with a Bcl-2 / Bcl-xL inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor.

[0011] In some embodiments, the second anti-cancer therapeutic can be administered simultaneously, separately or sequentially with the MDM2 inhibitor or the Bcl-2 / Bcl-xL inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor.

[0012] In certain embodiments, the cancer is a solid tumor or a hematological cancer. In certain embodiments, the cancer is selected from adrenocortical carcinoma, anal cancer, astrocytic tumor, childhood cerebellar or cerebral cancer, basal cell carcinoma, bile duct cancer, bladder cancer (e.g., urothelial bladder cancer), bone tumor, brain cancer, cerebellar astrocytic tumor, cerebral astrocytic tumor / malignant glioma, ependymal tumor, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, Burkitt lymphoma, cervical cancer, colon cancer, emphysema, endometrial cancer, esophageal cancer, Ewing sarcoma, retinoblastoma, gastric / stomach cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (endocrine pancreatic cancer), Kaposi sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, gastrointestinal cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, Ewing tumor family, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, cholangiocarcinoma, vaginal cancer, and small cell carcinoma (e.g., small cell lung cancer (SCLC), extrapulmonary small cell carcinoma (EPSCC), prostate small cell carcinoma, or bladder small cell carcinoma), melanoma, cutaneous squamous cell carcinoma, glioblastoma, hysterocarcinoma, osteosarcoma, uterine cancer, colorectal cancer, cervical cancer, sarcoma, chromophobe carcinoma, renal cell carcinoma (RCC), clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low-grade glioma (LGG), mesothelioma, PCPG, or thymoma. In certain embodiments, the cancer is selected from chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), T-cell prolymphocytic leukemia, multiple myeloma (MM), Waldenstrom macroglobulinemia (WM), acute lymphoblastic leukemia (ALL), and lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma).

[0013] In one aspect, the disclosure provides a method for treating cancer in a subject in need thereof. In one embodiment, the method comprises: measuring the level of at least one biomarker comprising ASCL1 in a test sample derived from the subject; comparing the level of the at least one biomarker to a corresponding reference level of the at least one biomarker to determine a difference from the reference level; and administering to the subject an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor when the difference reaches a predetermined threshold.

[0014] In another aspect, the disclosure provides a method for identifying a subject having cancer who is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor. In one embodiment, the method comprises: measuring the level of at least one biomarker comprising ASCL1 in a test sample derived from the subject; and comparing the level of the at least one biomarker to a corresponding reference level of the at least one biomarker to determine a difference from the reference level; and identifying the subject as likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor when the difference reaches a predetermined threshold.

[0015] In one embodiment, the method for identifying a subject having cancer who is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor further comprises administering to the subject identified as likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor.

[0016] In certain embodiments, the cancer is a neuroendocrine cancer. In certain embodiments, the cancer is selected from the group consisting of lung large cell neuroendocrine carcinoma (LCNEC), thyroid tumor, midgut carcinoma, gallbladder carcinoma, ovarian carcinoma, cervical carcinoma, pheochromocytoma, Merkel cell carcinoma, gastric carcinoma, esophageal carcinoma, pancreatic carcinoma, gastrointestinal carcinoma, breast carcinoma, liver carcinoma, head and neck carcinoma, cholangiocarcinoma, or small cell carcinoma (e.g., small cell lung cancer, extrapulmonary small cell carcinoma (EPSCC), prostatic small cell carcinoma, or urothelial small cell carcinoma).

[0017] In one aspect, the disclosure provides a method for treating cancer in a subject in need thereof. In one embodiment, the method comprises: measuring in a test sample derived from the subject the level of at least one biomarker comprising both Noxa and ASCL1; comparing the level of the at least one biomarker to a corresponding reference level of the at least one biomarker to determine a difference from the reference level; and administering to the subject an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor when the difference reaches a predetermined threshold.

[0018] In another aspect, the disclosure provides a method for identifying a subject having cancer who is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor. In one embodiment, the method comprises: measuring in a test sample derived from the subject the level of at least one biomarker comprising both Noxa and ASCL1; and comparing the level of the at least one biomarker to a corresponding reference level of the at least one biomarker to determine a difference from the reference level; and identifying the subject as likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor when the difference reaches a predetermined threshold.

[0019] In one embodiment, the method for identifying a subject having cancer who is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor further comprises administering to the subject identified as likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor.

[0020] In another aspect, the disclosure provides methods for monitoring treatment efficacy in a subject having cancer who has been treated with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor over a treatment period. In one embodiment, the method comprises: obtaining a test sample from the subject after the treatment period; measuring in the test sample the level of at least one biomarker comprising both Noxa and ASCL1 to obtain a post-treatment level of the at least one biomarker; comparing the post-treatment level to a baseline level of the at least one biomarker in a sample derived from the subject prior to the treatment period to determine a post-treatment change in the level of the at least one biomarker; and when the post-treatment change meets a predetermined threshold, or when the post-treatment change does not meet a predetermined threshold, continuing administration of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-xL inhibitor or the Bcl-2 inhibitor to the subject, increasing the dose of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-xL inhibitor or the Bcl-2 inhibitor to the subject, administering to the subject an effective amount of a second anti-cancer therapeutic agent in combination with the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-xL inhibitor or the Bcl-2 inhibitor, or discontinuing administration of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-xL inhibitor or the Bcl-2 inhibitor to the subject.

[0021] In certain embodiments, the cancer is a neuroendocrine cancer.

[0022] In certain embodiments, the at least one biomarker comprises Noxa and ASCL1, and the cancer is a neuroendocrine cancer. In certain embodiments, the neuroendocrine cancer is a lung large cell neuroendocrine carcinoma (LCNEC), a thyroid tumor, a midgut cancer, a gallbladder cancer, an ovarian cancer, a cervical cancer, a pheochromocytoma, a Merkel cell carcinoma, a gastric cancer, an esophageal cancer, a pancreatic cancer, a gastrointestinal cancer, a breast cancer, a liver cancer, a head and neck cancer, a cholangiocarcinoma, or a small cell carcinoma (e.g., a small cell lung cancer, an extrapulmonary small cell carcinoma (EPSCC), a prostate small cell carcinoma, or a bladder small cell carcinoma). In certain embodiments, the neuroendocrine cancer is a small cell carcinoma. In certain embodiments, the at least one biomarker comprises Noxa and ASCL1, and the cancer is a small cell lung cancer.

[0023] In any aspect described herein, the level of the at least one biomarker is measured by mRNA level, protein level, or DNA level. In certain embodiments, the level of the at least one biomarker is measured by an amplification assay, a hybridization assay, a sequencing assay, or an immunoassay.

[0024] In certain embodiments, the amplification assay is a polymerase chain reaction (PCR)-based method.

[0025] In certain embodiments, the predetermined threshold is set by a statistical method.

[0026] In certain embodiments, the predetermined threshold for Noxa is reached when the level of Noxa in the test sample is at least 15% (e.g., at least 25%, at least 35%, at least 45%, at least 50%) higher than the corresponding reference level of Noxa.

[0027] In certain embodiments, the reference level of Noxa represents the average level of Noxa in a general population of subjects having cancer. In certain embodiments, the reference level of Noxa is measured in a control sample. In certain embodiments, the control sample is a sample having a level of Noxa that represents the average level of Noxa in the general cancer population.

[0028] In certain embodiments, the predetermined threshold for ASCL1 is reached when the level of ASCL1 in the test sample is at least 15% (e.g., at least 25%, at least 50%, at least 100%, at least 150%) higher than the corresponding reference level of ASCL1.

[0029] In certain embodiments, the reference level of ASCL1 represents the average level of ASCL1 in a general population of subjects having cancer.

[0030] In certain embodiments, the reference level of ASCL1 is measured in a control sample. In certain embodiments, the control sample is a sample having a level of ASCL1 that represents the average level of ASCL1 in the general cancer population.

[0031] In certain embodiments, the sample is a bodily fluid sample or a tissue sample.

[0032] In certain embodiments, the at least one biomarker further comprises one or more additional biomarkers selected from the group consisting of Bcl-xL, Bcl-2, Mcl-1, a protein complex comprising a Bcl-xL protein, a protein complex comprising Bcl-2, or any combination thereof.

[0033] In certain embodiments, the step of comparing is performed with an algorithm.

[0034] In certain embodiments, the algorithm comprises a classification algorithm.

[0035] In certain embodiments, the difference comprises a difference between a test score for the level in the test sample and a reference score for the reference level, and wherein the test score and the reference score are calculated by the algorithm.

[0036] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitors described herein are compounds having the structure of Formula (I), (II), or (III):

[0037] (I)

[0038] (II)

[0039] (III)

[0040] or a pharmaceutically acceptable salt thereof;

[0041] wherein A ring is

[0042] substituted or unsubstituted X is selected from the group consisting of alkylene, alkenylene, cycloalkylene, cycloalkenylene, and heterocycloalkylene;

[0043] Y is selected from the group consisting of (CH2) n -N(R a )2and

[0044] Q is selected from the group consisting of O, O(CH2) 1-3 , NR c , NR c (C 1-3 alkylene), OC(=0)(C 1-3 alkylene), C(=0)0, C(=0)0(C 1-3 alkylene), NHC(=0)(C 1-3 alkylene), C(=0)NH, and C(=0)NH(C 1-3 alkylene);

[0045] Z is O or NR c ;

[0046] R1and R2are independently selected from the group consisting of H, CN, NO2, halo, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl, OR', SR', NR'R", COR', CO2R', OCOR', CONR'R", CONR'SO2R", NR'COR", NR'CONR"R"', NR'C=SNR"R"', NR'SO2R", SO2R', and SO2NR'R";

[0047] R3is selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl, OR', NR'R", OCOR', CO2R', COR', CONR'R", CONR'SO2R", NR'COR", NR'CONR"R"', NR'C=SNR"R"', NR'SO2R", SO2R', and SO2NR'R"; 1-3 alkylene heterocycloalkyl, or heterocycloalkyl;

[0048] R', R", and R'" are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, C 1-3 alkylene heterocycloalkyl, or heterocycloalkyl;

[0049] R' and R", or R" and R'" can together with the atoms to which they are bonded form a 3- to 7-membered ring;

[0050] R4is hydrogen, halo, C 1-3 alkyl, CF3, or CN;

[0051] R5is hydrogen, halo, C 1-3 alkyl, substituted C 1-3 alkyl, hydroxyalkyl, alkoxy, or substituted alkoxy;

[0052] R6is selected from the group consisting of H, CN, NO2, halo, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl, OR', SR', NR'R", CO2R', OCOR', CONR'R", CONR'SO2R", NR'COR", NR'CONR"R"', NR'C=SNR"R"', NR'SO2R", SO2R', and SO2NR'R";

[0053] substituted or unsubstituted R7is selected from the group consisting of hydrogen, alkyl, alkenyl, (CH2) 0-3 cycloalkyl, (CH2) 0-3 cycloalkenyl, (CH2) 0-3 heterocycloalkyl, (CH2) 0-3 aryl, and (CH2) 0-3 heteroaryl;

[0054] R8is selected from the group consisting of hydrogen, halo, NO2, CN, CF3SO2, and CF3;

[0055] R a is selected from the group consisting of hydrogen, alkyl, heteroalkyl, alkenyl, hydroxyalkyl, alkoxy, substituted alkoxy, cycloalkyl, cycloalkenyl, and heterocycloalkyl;

[0056] R b is hydrogen or alkyl;

[0057] R c is selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxyalkyl, alkoxy, and substituted alkoxy; and

[0058] n, r, and s are independently 1, 2, 3, 4, 5, or 6.

[0059] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitors described herein are compounds having the structure of Formula (IV):

[0060] (IV)

[0061]

[0062] or a pharmaceutically acceptable salt thereof;

[0063] R 21 is SO2R2';

[0064] R 22 is alkyl, preferably C1-C4 alkyl, more preferably methyl, propyl, or isopropyl;

[0065] R 23 is alkyl, preferably C1-C4 alkyl, more preferably methyl, propyl, or isopropyl;

[0066] R 24 is halogen, preferably fluorine, chlorine;

[0067] R 25 is halogen, preferably fluorine, chlorine;

[0068] R 26 is selected from H, halogen, alkyl, preferably fluorine, chlorine, C1-C4 alkyl, more preferably methyl, propyl, isopropyl;

[0069] R 21b is H or alkyl, preferably C1-C4 alkyl, more preferably methyl, propyl, or isopropyl;

[0070] n2, r2, and s2 are independently 1, 2, 3, 4, 5, or 6, more preferably r2 and s2 are both 2, and n2 is 3, 4, or 5, more preferably n2, r2, and s2 are all 2; and

[0071] R2' is alkyl, preferably C1-C4 alkyl, more preferably methyl, propyl, or isopropyl.

[0072] In certain embodiments, the Bcl-2 inhibitors described herein are compounds having the structure of Formula (V):

[0073] (V)

[0074]

[0075] or a pharmaceutically acceptable salt thereof; A3is selected from the group consisting of:

[0076]

[0077] and

[0078] E3is a carbon atom, and is a double bond; or

[0079] E3is -C(H)-, and is a single bond; or

[0080] E3is a nitrogen atom, and is a single bond;

[0081] X 31 , X 32 , and X 33 are each independently selected from the group consisting of -CR 38 = and -N=;

[0082] R 31a and R 31b , together with the carbon atom to which they are attached, form a 3-, 4-, or 5-membered optionally substituted cycloalkyl; or

[0083] R 31a and R 31b , together with the carbon atom to which they are attached, form a 4- or 5-membered optionally substituted heterocycle;

[0084] R 32 is selected from the group consisting of -NO2, -SO2CH3, and -SO2CF3;

[0085] R 32a is selected from the group consisting of hydrogen and halogen;

[0086] R 33 is selected from the group consisting of hydrogen, -CN, -C≡CH, and -N(R 34a )(R 34b );

[0087] R 34a is selected from the group consisting of optionally substituted C 1-6 alkyl, optionally substituted C 3-6 cycloalkyl, heterocycle, heteroalkyl, (cycloalkyl)alkyl, and (heterocycle)alkyl;

[0088] R 34b is selected from the group consisting of hydrogen and C1-4 alkyl;

[0089] R 35 is selected from the group consisting of optionally substituted C 1-6 alkyl, heterocycle, heteroalkyl, (cycloalkyl)alkyl, and (heterocycle)alkyl;

[0090] R 36a , R 36c , R 36e , R 36f , and R 36g are each independently selected from the group consisting of hydrogen, optionally substituted C 1-6 alkyl, optionally substituted C 3-6 cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, heterocycle, heteroalkyl, (cycloalkyl)alkyl, and (heterocycle)alkyl;

[0091] R 36b , and R 36d are each independently selected from the group consisting of hydrogen, C 1-4 alkyl, and halo;

[0092] R 37 is selected from the group consisting of optionally substituted C 1-6 alkyl, heterocycle, heteroalkyl, (cycloalkyl)alkyl, and (heterocycle)alkyl; and

[0093] R 38 is selected from the group consisting of hydrogen and halo.

[0094] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitor or Bcl-xL inhibitor or Bcl-2 inhibitor is a compound selected from Table 1-A, Table 1-B, and Table 1-C, or a pharmaceutically acceptable salt thereof.

[0095] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitor is (R)-2-(1-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1- isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5- fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2- (trifluoromethylsulfanyl)phenylamino)-4-(phenylthio)butyl)piperidine-4- carbonyloxy)ethylphosphonic acid (also referred to herein as “Compound A15”), or a pharmaceutically acceptable salt thereof:

[0096]

[0097] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitor is (R)-1-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1- isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2- (trifluoromethylsulfonyl)phenylamino)-4-(phenylthio)butyl)piperidine-4-carboxylic acid (also referred to herein as “Compound B4”), or a pharmaceutically acceptable salt thereof:

[0098]

[0099] In certain embodiments, the Bcl-2 inhibitor is selected from the group consisting of compounds having the following structures

[0100]

[0101] or a pharmaceutically acceptable salt thereof.

[0102] In certain embodiments, the MDM2 inhibitor comprises a chemical structure having the following Formula (VI):

[0103]

[0104] or a pharmaceutically acceptable salt thereof, wherein

[0105] is selected from the group consisting of:

[0106]

[0107] B is C 4-7 carbocycle;

[0108] R 61 is H, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, OR 6a , or NR 6a R 6b ;

[0109] n3 is 0, 1, or 2;

[0110] R 62 , R 63 , R 64 , R 65 , R 67 , R 68 , R 69 and R 70 are independently selected from the group consisting of H, F, Cl, CH3, and CF3;

[0111] R 66 is

[0112] R 6a is hydrogen or substituted or unsubstituted C 1-4 alkyl;

[0113] R 6b is hydrogen or substituted or unsubstituted C 1-4 alkyl;

[0114] R 6c and R 6d are substituents on a carbon atom of ring B, wherein

[0115] R 6c is H, C 1-3 alkyl, C 1-3 alkylene-OR 6a , OR 6a , or halo;

[0116] R 6d is H, C 1-3 alkyl, C 1-3 alkylene-OR 6a , OR 6a , or halo; or

[0117] R 6c and R 6d together with the carbon to which they are attached form a 4- to 6- membered spiro substituent, optionally comprising an oxygen atom; and

[0118] R 6e is -C(=O)OR 6a , -C(=O)NR 6a R 6b , or -C(=O)NHSO2CH3.

[0119] In certain embodiments, is

[0120] B is and

[0121] R 6c and R 6d are F and F, H and H, OH and CH3, OH and H, CH3and CH3, CH3and OH, H and OH, CH2CH3and CH2CH3, or CH2OH and CH2OH.

[0122] In certain embodiments, is H, CH3, or CH2CH3.

[0123] In certain embodiments, R 62is H; R 63 is halo; R 64 and R 65 is H.

[0124] In certain embodiments, R 67 is fluoro; R 68 , R 69 and R 70 each is H; and R 6e is -C(=O)OH, -C(=O)NH2, or -C(=O)NHSO2CH3.

[0125] In certain embodiments, the MDM2 inhibitor is a compound selected from the group consisting of:

[0126]

[0127] or a pharmaceutically acceptable salt.

[0128] In certain embodiments, the MDM2 inhibitor is

[0129]

[0130] or a pharmaceutically acceptable salt thereof.

[0131] In certain embodiments, the MDM2 inhibitor is Compound C, or a pharmaceutically acceptable salt.

[0132] In yet another aspect, the disclosure provides kits for use in the methods described herein. In one embodiment, the kit comprises one or more reagents for measuring the level of at least one biomarker comprising Noxa. In one embodiment, the kit comprises one or more reagents for measuring the level of at least one biomarker comprising ASCL1. In one embodiment, the kit comprises one or more reagents for measuring the level of at least one biomarker comprising both Noxa and ASCL1.

[0133] In certain embodiments, the reagent for measuring Noxa level comprises a primer or a probe that can hybridize to a polynucleotide of Noxa, or an antibody that can specifically bind to a protein of Noxa. In certain embodiments, the reagent for measuring ASCL1 level comprises a primer or a probe that can hybridize to a polynucleotide of ASCL1, or an antibody that can specifically bind to a protein of ASCL1. In certain embodiments, the one or more reagents comprise a first primer or a first probe that can hybridize to a polynucleotide of Noxa, or a first antibody that can specifically bind to a protein of Noxa, and a second primer or a second probe that can hybridize to a polynucleotide of ASCL1, or a second antibody that can specifically bind to a protein of ASCL1.

[0134] In certain embodiments, the one or more reagents are detectably labeled.

[0135] In another aspect, the disclosure provides use of one or more reagents for measuring the level of at least one biomarker comprising Noxa, ASCL1, or both, in the manufacture of a diagnostic kit for performing the methods described herein. In one embodiment, the at least one biomarker comprises Noxa. In one embodiment, the at least one biomarker comprises ASCL1. In one embodiment, the at least one biomarker comprises both Noxa and ASCL1. BRIEF DESCRIPTION OF DRAWINGS

[0136] The following drawings form part of the specification and are included to further illustrate certain aspects of the present disclosure. The present disclosure can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0137] Figure 1A and 1B Illustrates that Noxa expression levels correlate with tumor regression in a gastric cancer PDX (patient-derived xenograft) model treated with Bcl-2 / Bcl-xL dual inhibitor Compound A15.

[0138] Figure 2A and 2B Illustrates that Noxa expression levels correlate with tumor regression in an esophageal cancer PDX (patient-derived xenograft) model treated with Bcl-2 / Bcl-xL dual inhibitor Compound A15.

[0139] Figures 3A to 3E Illustrates additional biomarkers in a gastric cancer PDX model, including PUMA Figure 3A ), BIM Figure 3B ), Bcl-xL Figure 3C ), and Mcl-1 Figure 3D), Bcl-2 Figure 3E ) and tumor regression.

[0140] Figure 4A and 4B illustrates the comparison of compound B4 and reference compound ABT-737 in reducing the level of Bcl-xL:BIM or Bcl2:BIM protein complex in Toledo cell line Figure 4A ) and in RS4;11 cell line Figure 4B ) and in RS4;11 cell line

[0141] Figure 5A , 5B illustrates the anti-tumor activity of compound A15 in ASCL-1-high and Noxa-high small cell lung cancer (SCLC) PDXs Figure 5A ), the correlation between the anti-tumor activity of compound A15 and Noxa expression in all SCLC PDX models Figure 5B ), and the correlation between the anti-tumor activity of compound A15 and Noxa expression in ASCL-1-high SCLC PDXs Figure 5C ) and in RS4;11 cell line

[0142] Figure 6 Exemplary sequences of biomarkers as provided herein are shown. DETAILED DESCRIPTION

[0143] Before the present disclosure is described in detail, it is to be understood that this disclosure is not limited to the particular embodiments described and as such can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present disclosure will be limited only by the appended claims.

[0144] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0145] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for the purpose of describing and disclosing materials and / or methods that are considered to be part of the state of the art, and are not an admission that the publication is prior art to the present disclosure and is expressly incorporated by reference. To the extent that any meaning or definition of a term in this document conflicts with the meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall control.

[0146] As will be clear to those of ordinary skill in the art upon reading the present disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0147] Definitions

[0148] The following definitions are provided to assist the reader. Unless otherwise defined, all technical terms, symbols, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art in the field of chemistry and medicine. In some instances, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and inclusion of such definitions herein should not necessarily be construed to mean that the terms are in any way

[0149] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0150] As used herein, the term "biomarker" refers to a biomolecule that is a measurable indicator of some biological state or condition. The term "biomarker" as used herein is intended to encompass, for example, a polynucleotide of interest, a polypeptide encoded by a polynucleotide of interest. Examples of biomarkers provided herein can be a gene (e.g., genomic DNA, cDNA), or a gene product (e.g., mRNA transcribed from a gene, or a protein encoded by a gene). Examples of particular biomarkers provided herein include, for example, Noxa and ASCL1.

[0151] The term "level" with respect to a biomarker refers to the amount or quantity of a biomarker of interest present in a sample. Such amount or quantity can be expressed in absolute terms (i.e., total quantity of biomarker in a sample), or in relative terms (i.e., concentration or percentage of biomarker in a sample). The level of a biomarker can be measured at the DNA level (e.g., as represented by the amount or quantity or copy number of a gene in a chromosomal region), at the RNA level (e.g., as represented by the amount or quantity of mRNA), or at the protein level (e.g., as represented by the amount or quantity of protein or the amount or quantity of a protein complex).

[0152] Notably, in this disclosure, terms such as "comprises", "comprised", "comprising", "contains", "containing" and the like are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0153] The terms "assay", "measurement" and "detection" are used interchangeably and refer to the determination of quantitative and semi-quantitative.

[0154] The term "hybridization" refers to the binding, duplexing, or pairing between at least partially complementary strands of nucleic acids. A nucleic acid strand can hybridize specifically to a target nucleic acid strand when there is sufficient complementarity to avoid non-specific binding to non-target nucleic acid sequences. Numerous guides to nucleic acid hybridization can be found in, for example, Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," (1993) Elsevier, New York).

[0155] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length (deoxyribonucleotides or ribonucleotides, or their analogs). Polynucleotides can have any three-dimensional structure and can perform any function for which polynucleotides can be used. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, short or long single- stranded RNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule can be linear or circular.

[0156] The term "complementarity" refers to the ability of a nucleic acid sequence to base pair with another nucleic acid sequence, either through traditional Watson-Crick types or other non-traditional types. Complementarity can be partial or complete. Partial complementarity occurs when one or more nucleic acid bases do not match according to the base pairing rules. Percent complementarity indicates the percentage of nucleic acid bases in a nucleic acid molecule that can form base pairs (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 bases pair is 50%, 60%, 70%, 80%, 90%, and 100% complementary).

[0157] Generally, a "protein" is a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). A protein can include moieties other than amino acids (e.g., can be a glycoprotein) and / or can be additionally processed or modified. One of ordinary skill in the art will appreciate that a "protein" can be the complete polypeptide chain produced by a cell (with or without a signal sequence), or can be a functional portion thereof. One of ordinary skill in the art will further appreciate that a protein can sometimes comprise more than one polypeptide chain, for example, linked by one or more disulfide bonds or associated by other means.

[0158] As used herein, "likelihood" and "likely" with respect to a subject's response to a treatment is a measure of the probability of occurrence of a therapeutic response in that subject. It can be used interchangeably with "probability." Likelihood refers to a probability that is greater than speculation but less than certainty. Thus, a therapeutic response is likely if a reasonable person using common sense, training, or experience concludes that, given the current circumstances, a therapeutic response is possible. In one embodiment, the terms "likelihood" and "likely" indicate the percentage chance of the probability of occurrence of a therapeutic response. In some embodiments, a subject having a cancer is identified as "likely to respond" means a subject having a cancer that has a greater than 30% chance, a greater than 40% chance, a greater than 50% chance, a greater than 60% chance, a greater than 70% chance, a greater than 80% chance, a greater than 90% chance of responding to treatment with a Bcl-2 / Bcl-xL inhibitor and a Bcl-2 inhibitor or a Bcl-xL inhibitor.

[0159] The terms "responsive" or "responsiveness" as used in the context of a subject's therapeutic response to a cancer therapy are used interchangeably and refer to a beneficial response of the subject to the therapy, as opposed to an adverse response (i.e., an adverse event). In a subject, a beneficial response can be expressed in terms of a number of clinical parameters including detectable disappearance of tumor (complete response), reduction in tumor size and / or number of cancer cells (partial response), stasis of tumor growth (stable disease), decreased rate of tumor growth (which lengthens overall survival), enhanced anti-tumor immune response that can lead to tumor regression or rejection; some degree of alleviation of one or more symptoms associated with the tumor; increased length of survival following treatment; and / or decreased mortality at a given time point following treatment. Continued increase in tumor size and / or number of cancer cells (without any decrease in growth rate that benefits overall survival), and / or tumor metastasis indicates a lack of beneficial response to the therapy and thus decreased responsiveness.

[0160] As described herein, the term "effective amount" refers to the amount of one or more active ingredients that, when administered by the methods of the present application, is sufficient to effect delivery of the one or more active ingredients to an individual in need thereof for the treatment of a disorder or disease for a therapeutic purpose. In the case of a cancer or other proliferative disorder, the effective amount of the agent can reduce (i.e., delay to some extent and preferably stop) unwanted cell proliferation; reduce the number of cancer cells; reduce tumor size; inhibit (i.e., delay to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., delay to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; reduce Bcl-2 / Bcl-xL signaling in target cells; and / or relieve to some extent one or more symptoms.

[0161] As used herein, "cancer" is a generic term used to describe a wide range of cellular malignancies characterized by uncontrolled growth, lack of differentiation, and potential or ability to invade local tissues and metastasize. These neoplastic malignancies affect every tissue and organ in the body with varying degrees of prevalence. Cancer involves the presence of cells having the typical characteristics of cancerous cells (e.g., uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain distinctive morphological features). Cancer cells often form tumors, but such cells can exist singly, or can circulate in the bloodstream as isolated cells (e.g., leukemia cells). The terms cancer and tumor are used interchangeably herein. The term includes all known cancers and neoplastic disorders (whether described as malignant, benign, hematologic, or solid), and all stages and grades of cancer (including pre-metastatic and metastatic cancers).

[0162] As used herein, the term "solid tumor" refers to any cancer that does not include cysts or fluid-filled areas. Solid tumors generally do not include leukemia (i.e., blood cancer). Solid tumors can be benign or malignant. As used herein, types of solid tumors include, but are not limited to, adrenocortical carcinoma, anal cancer, astrocytic tumor, childhood cerebellar or cerebral cancer, basal cell carcinoma, biliary tract cancer, bladder cancer (e.g., urothelial bladder cancer), bone tumor, brain cancer, cerebellar astrocytic tumor, cerebral astrocytic tumor / malignant glioma, ependymal tumor, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, Burkitt's lymphoma, cervical cancer, colon cancer, emphysema, endometrial cancer, esophageal cancer, Ewing's sarcoma, retinoblastoma, gastric / stomach cancer, glioma, head and neck cancer, heart cancer, Hodgkin's lymphoma, islet cell carcinoma (endocrine pancreatic cancer), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), neuroblastoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, gastrointestinal cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, Ewing's tumor family, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, cholangiocarcinoma, vaginal cancer, and small cell carcinoma (e.g., small cell lung cancer (SCLC), extrapulmonary small cell carcinoma (EPSCC), prostate small cell carcinoma, or bladder small cell carcinoma), melanoma, cutaneous squamous cell carcinoma, glioblastoma, uterine tumor, osteosarcoma, uterine cancer, colorectal cancer, cervical cancer, sarcoma, chromophobe carcinoma, renal cell carcinoma (RCC), clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low grade glioma (LGG), mesothelioma, PCPG, or thymoma.

[0163] As used herein, a "neuroendocrine cancer" is a cancer that arises from the neuroendocrine system, a diffuse system of interaction between the nervous system and endocrine glands hormones; or a cancer of non-endocrine cells that acquire some properties of neuroendocrine cells through oncogenic processes such as selective tumor gene expression of peptides essential for survival (STEPS) (see, North (2000) Exper. Physiol. 85S:27S-40S). Most well-described adult neuroendocrine tumors are unique and arise from known primary sites, including carcinoid, pheochromocytoma, and Merkel cell carcinoma. Carcinoid includes stomach cancer, pancreatic cancer, colon cancer, liver cancer, lung cancer, ovarian cancer, breast cancer, testicular cancer, and cervical cancer. Pheochromocytoma is an adrenal medulla cancer that usually causes the adrenal glands to produce too much catecholamine. Merkel cell carcinoma is a cancer that forms in the skin or just beneath the skin, but is sometimes also thought to arise from underlying soft tissue. They are also known as neuroendocrine cancers of the skin.

[0164] Examples of neuroendocrine cancers include, but are not limited to, small cell carcinoma, breast cancer, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma of the lung (LCNEC), thyroid tumor, gastric cancer, pancreatic cancer, midgut cancer, liver cancer, gallbladder cancer, ovarian cancer, cervical cancer, esophageal cancer, gastrointestinal cancer, head and neck cancer, cholangiocarcinoma, pheochromocytoma, and Merkel cell carcinoma.

[0165] As used herein, “small cell carcinoma” refers to a highly malignant type of cancer characterized by short doubling times, high growth fraction, and early development of metastases. Small cell carcinoma is most commonly found in the lung and occasionally in other body sites, such as the uterine cervix, prostate, and gastrointestinal tract. Exemplary small cell carcinomas include small cell lung cancer, extrapulmonary small cell carcinoma (EPSCC), prostate small cell carcinoma, bladder small cell carcinoma.

[0166] The term “hematological cancer” refers to any cancer that begins in the blood-forming tissue, such as the bone marrow, or in the cells of the immune system. In certain embodiments, the hematological cancer is chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), T-cell prolymphocytic leukemia, multiple myeloma (MM), Waldenstrom’s macroglobulinemia (WM), acute lymphoblastic leukemia (ALL), or lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma).

[0167] As used herein, the term “sample” refers to a biological sample derived from a subject and comprises one or more biomarkers of interest. Examples of samples include, but are not limited to, body fluids, such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal lavage fluid, pleural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, and the like; and tissues, such as a biopsy tissue (e.g., a biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tissue, lung tissue, liver tissue, prostate tissue, brain tissue, neural tissue, meningeal tissue, kidney tissue, endometrial tissue, cervical tissue, lymph node tissue, muscle tissue, or skin tissue), paraffin-embedded tissue. In certain embodiments, the sample can be a biological sample comprising cancer cells (including circulating cancer cells) or cells from tissue surrounding or adjacent to a tumor. In some embodiments, the biological sample is a fresh or archived sample obtained from tumor tissue or from tissue surrounding or adjacent to a tumor, for example, by tumor biopsy or fine needle aspiration. In some embodiments, the sample can be any biological fluid containing cancer cells or suspected of containing cancer cells (e.g., in peripheral blood mononuclear cells (PBMCs)). Collecting the sample from a subject is typically performed after a hospital or clinic visit, for example, during a biopsy, according to standard protocols.

[0168] As used herein, the term "test sample" refers to a sample derived from a subject in need of cancer treatment and is representative of the cancer condition of the subject. For example, the test sample can comprise cancer cells.

[0169] As used herein, the term "control sample" refers to a sample that is otherwise comparable to the test sample except that it expresses the biomarker of interest at a reference level. Examples of control samples include, but are not limited to, a reference cancer cell or tissue sample, or a healthy, non-cancerous tissue sample, or a diploid, non-transformed, non-cancerous, genomically stable healthy human cell line, as long as they express the biomarker of interest at a reference level.

[0170] As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). In many embodiments, the subject is a human. A subject can be a patient, referring to a person presented to a medical provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject can have or be predisposed to a disease or disorder, but can or can not display symptoms of the disease or disorder.

[0171] The term "treating" or "treatment" of a cancer, as used herein, unless otherwise indicated, means partially or completely reversing, alleviating, inhibiting or preventing the progression of a tumor growth, tumor metastasis, or other progression of cells or neoplastic cells that cause cancer in a subject.

[0172] As used herein, the term "prognose" or "prognosing" refers to the prediction or forecast of the future course or outcome of a disease or condition.

[0173] "Co-administration" or "combination therapy" as used herein is understood to mean administration of two or more active agents either using separate formulations or a single pharmaceutical formulation, or consecutively in any order such that both (or all) active agents are present in the body at the same time with the intention to improve the activity of the second agent, e.g., to sensitize target cells (e.g., cancer cells) to the activity of the second agent. Co-administration does not require that the agents be administered at the same frequency, at the same time, or by the same route of administration.

[0174] As used herein, the term "alkyl" refers to straight-chain and branched saturated C 1-10Hydrocarbon groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl.

[0175] The term C m-n denotes an alkyl group having "m" to "n" carbon atoms.

[0176] The term "alkylene" refers to an alkyl group having substituents. For example, an alkyl (e.g., methyl) or alkylene (e.g., -CH2-) group can be substituted with one or more (and typically one to three) independently selected halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or amino groups.

[0177] The term "alkenyl" is defined identically to "alkyl" except that it contains a carbon-carbon double bond. The term "alkenylene" is defined identically to "alkylene" except that it contains a carbon-carbon double bond. The terms "alkynyl" and "alkynylene" are defined identically to "alkyl" and "alkylene" except that the group contains a carbon-carbon triple bond.

[0178] As used herein, the term "halo" or "halogen" is defined as fluorine, chlorine, bromine, or iodine.

[0179] The term "hydroxy" is defined as -OH.

[0180] The term "alkoxy" is defined as -O-alkyl.

[0181] The term "amino" is defined as -NH2, and the term "alkylamino" is a substituent group in which the nitrogen is bonded to at least one alkyl group. Examples include NH-alkyl, N(alkyl)2, or a nitrogen bonded to one alkyl group and one substituent group (e.g., benzyl, phenethyl, etc.).

[0182] The term "carbamoyl" refers to H2NC(O)-, alkyl-NHC(O)-, (alkyl)2NC(O)-, aryl- NHC(O)-, alkyl(aryl)-NC(O)-, heteroaryl-NHC(O)-, alkyl(heteroaryl)-NC(O)-, aralkyl- NHC(O)-, alkyl(aralkyl)-NC(O)-, etc.

[0183] The term "carboxy" is defined as -C(=O)OH or a salt thereof.

[0184] The term "nitro" is defined as -NO2.

[0185] The term "cyano" is defined as -CN.

[0186] The term "trifluoromethyl" is defined as -CF3.

[0187] The term "trifluoromethoxy" is defined as -OCF3.

[0188] As used herein, the term "aryl" refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthracenyl, phenanthrenyl, pyrenyl, biphenyl, and terphenyl. Aryl also refers to bicyclic and tricyclic carbocyclic rings in which one ring is aromatic and the other rings are saturated, partially unsaturated, or aromatic, for example, indanyl, indenyl, indan, or tetrahydronaphthyl (tetralinyl). Unless otherwise indicated, aryl groups can be unsubstituted or substituted with one or more, and particularly one to four, groups independently selected from, for example, halo, alkyl, alkenyl, -OCF3, -NO2, -CN, -NC, -OH, alkoxy, amino, alkylamino, -CO2H, -CO2alkyl, -OCOalkyl, aryl, and heteroaryl.

[0189] As used herein, the term "heterocycle" refers to both heteroaryl and heterocycloalkyl ring systems.

[0190] As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in the aromatic ring. Each ring of the heteroaryl group can contain one or two O atoms, one or two S atoms, and / or one to four N atoms, provided that the total number of heteroatoms in each ring is four or less, and each ring contains at least one carbon atom. In certain embodiments, the heteroaryl group has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms. Examples of monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl, and triazolyl. Examples of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzothiophenyl, benzotriazolyl, benzoxazolyl, furopyridinyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridinyl, pyrrolopyridinyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidinyl, and thienopyridinyl. Unless otherwise indicated, a heteroaryl group can be unsubstituted or substituted by one or more, and particularly one to four, substituents selected from, for example, halo, alkyl, alkenyl, -OCF3, -NO2, -CN, -NC, -OH, alkoxy, amino, alkylamino, -CO2H, -CO2alkyl, -OCOalkyl, aryl, and heteroaryl.

[0191] As used herein, the term "cycloalkyl" denotes a monocyclic or bicyclic, saturated or partially unsaturated ring system containing three to eight carbon atoms, including, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl optionally substituted by one or more (and typically one to three) of independently selected halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or amino groups.

[0192] As used herein, the term "heterocycloalkyl" denotes a monocyclic or bicyclic, saturated or partially unsaturated ring system containing a total of 4 to 12 atoms, where one to five of the atoms are independently selected from nitrogen, oxygen, and sulfur, and the remaining atoms are carbon. Non-limiting examples of heterocycloalkyl groups are azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, dihydropyrrolyl, morpholinyl, thiomorpholinyl, dihydropyridinyl, oxepanyl, dioxepanyl, thioxepanyl, dinitroazepanyl, each optionally substituted with independently selected halo, C 1-6 alkyl, C1-6 alkoxy, cyano, amino, carbamoyl, nitro, carboxy, C 2-7 alkenyl, C 2-7 one or more (and typically one to three) of the substituents in alkyl, alkenyl, alkynyl, and the like.

[0193] Biomarkers for predicting efficacy of MDM2 inhibitors or Bcl-2 / Bcl-xL dual inhibitors or Bcl-2 inhibitors or Bcl-xL inhibitors iii. Detection reagents for biomarkers

[0194] The methods and compositions described herein are based, in part, on the discovery that the levels of biomarkers are predictive of the likelihood that a subject having cancer will respond to treatment with a compound targeting the apoptosis pathway, including an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor. The biomarkers are also useful for predicting the efficacy of treatment with a compound targeting the apoptosis pathway, including an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor, in a subject having cancer.

[0195] i. The Apoptosis Pathway and Compounds Targeting the Pathway

[0196] The apoptosis pathway includes multiple players in a complex network that combine to regulate the fate of a cell. Bcl-2 (B-cell lymphoma protein 2) family proteins are key regulators of apoptosis in the mitochondrial-mediated (also known as "intrinsic") pathway. Their activities are associated with the onset of lymphoid cancers and several solid tumor cancers, and are believed to be a key mediator of chemotherapy resistance in many cancers. Bcl-2 family proteins are characterized by structural homology domains BH1, BH2, BH3, and BH4, and can be further classified into three subfamilies according to how many homology domains each protein contains or according to their biological activity (i.e., whether the protein has anti-apoptotic (pro-survival) or pro-apoptotic (pro-death) function).

[0197] A first subset of Bcl-2 proteins includes proteins having all four homology domains (i.e., BH1, BH2, BH3, and BH4). Their general role is anti-apoptotic, i.e., to protect cells from initiating the cell death process. Proteins such as Bcl-2, Bcl-w, Bcl-xL, Mcl-1, and Bfl-1 / A1 are members of this first subset.

[0198] Proteins belonging to a second subset of Bcl-2 proteins contain three homology domains BH1, BH2, and BH3, and have a pro-apoptotic role. Two major representative proteins of this second subset are Bax and Bak.

[0199] A third subgroup of Bcl-2 proteins consists of proteins that contain only a BH3 domain, and members of this subgroup are commonly referred to as “BH3-only proteins.” Their biological effect on cells is pro-apoptotic. Bim, Bid, Bad, Bik, Noxa, Hrk, Bmf, and Puma are examples of this third subgroup of proteins.

[0200] The precise mechanism by which Bcl-2 family proteins regulate cell death is not fully understood. In one hypothesis for how Bcl-2 family proteins regulate cell death, these BH3-only proteins are further divided into “activators” (e.g., Bim and Bid), or “sensitizers” (e.g., Bad, Bik, Noxa, Hrk, Bmf, and Puma) based on their regulatory function.

[0201] Activator BH3-only proteins bind and directly activate pro-apoptotic proteins. These activators can also bind and inhibit anti-apoptotic Bcl-2 family proteins. This binding sequesters these activator proteins and prevents them from exerting their apoptotic activity.

[0202] Displacement of these activators by sensitizer peptides results in Bax / Bak-mediated apoptosis. Sensitizer BH3-only proteins only bind to anti-apoptotic Bcl-2 family proteins and block their anti-apoptotic function. Each sensitizer protein can have different specificity characteristics. For example, Noxa binds Mcl-1 with high affinity, BAD binds to Bcl-xL and Bcl-2 but only weakly to Mcl-1, while PUMA binds well to all three targets. These interactions can have a variety of outcomes, including homeostatic balance, cell death, sensitization to apoptosis, and blockade of apoptosis.

[0203] The balance between anti-apoptotic (pro-survival) and pro-apoptotic (pro-death) proteins determines the fate of cell survival or death. Overexpression of pro-survival proteins (e.g., Bcl-2 and Bcl-xL) is associated with tumorigenesis and is a common cause of resistance to anti-cancer therapies (Vaux DL et al., Nature (1988) 335:440-42; Delbridge AR et al., Cell Death Differ (2015) 22:1071-80). Thus, agents designed to target anti-apoptotic Bcl-2 family proteins (e.g., small molecule BH3 mimetics) can provide new strategies for the treatment of cancer patients. However, clinically useful inhibitors of anti-apoptotic Bcl-2 family proteins or BH3 mimetics, including those undergoing clinical evaluation, have shown limited efficacy in hematological cancers as well as solid tumors, which can be due to the complexity of signaling pathways and tumor microenvironments.

[0204] Bcl-2 / Bcl-xL dual inhibitors or Bcl-2 inhibitors or Bcl-xL inhibitors have been previously described as anti-cancer therapeutics (see, e.g., PCT Application WO 2014113413 Al, PCT / CN 2019 / 098576, PCT / CN 2019 / 086673, PCT US 2014011571, the entire contents of each of which are incorporated herein by reference), and evaluated in humans as monotherapy or in combination with standard-of-care chemotherapy agents used to treat diseases and disorders where inhibition of Bcl-2 family protein activity provides a benefit.

[0205] Because of the plethora of Bcl-2 family proteins involved in the apoptotic pathway, and the natural levels of these proteins can vary by different cell types, there are few biomarkers that are generally applicable to predict the anti-cancer efficacy of a particular Bcl-2 inhibitor, Bcl-xL inhibitor, or Bcl-2 / Bcl-xL dual inhibitor.

[0206] Upstream of the apoptotic pathway regulated by Bcl-2 family proteins, the tumor suppressor p53 integrates cell signals that lead to apoptosis. p53 promotes apoptosis apparently through both transcription-dependent and -independent mechanisms that act synergistically. For example, activation of p53 induces expression of specific apoptotic target genes, which shifts the balance of the Bcl-2 family toward pro-apoptotic members. Activation of p53 also allows for its rapid translocation to mitochondria, facilitating the release of pro-apoptotic members from their sequestration (McBride A. et al., Frontiers in Oncology, (2019) 9:192, Haupt S. et al., Journal of Cell Science, (2003) 116:4077). In addition to regulating apoptosis, activation of p53 also initiates cell cycle arrest or cellular senescence (Green DR, Nature, 2009; 458(7242): 1127). Emerging evidence further suggests that p53 dysfunction promotes inflammation and supports tumor immune evasion, thus p53 dysfunction can serve as an immunological driver of tumorigenesis (Guo G, Cancer Research, 2017; 77(9):2292). Therefore, manipulating p53 activity constitutes an attractive target for cancer therapy.

[0207] MDM2 (mouse double minute 2) is transcriptionally activated by p53, and MDM2 in turn inhibits p53 activity through at least three mechanisms (Wu et al., Genes Dev. 7:1126 (1993)). First, the MDM2 protein directly binds to the p53 transactivation domain, thereby inhibiting p53-mediated transactivation. Second, the MDM2 protein contains a nuclear export signal sequence, and upon binding to p53, this nuclear export signal sequence induces nuclear export of p53, thereby preventing p53 from binding to targeted DNA. Third, the MDM2 protein is an E3 ubiquitin ligase, and upon binding to p53, is able to promote p53 degradation. MDM2 and p53 are part of an autoregulatory feedback loop (Wu et al., Genes Dev. 7:1126 (1993)). As used herein, “MDM2” is intended to encompass the MDM2 gene, as well as MDM2 gene products (e.g., mRNA, protein). Exemplary sequences for human MDM2 are available under NCBI Accession Nos. ABT17086, ABT17084.1, ABT17085.1, or ABT17083.1.

[0208] MDM2 inhibitors interfere with the binding of the MDM2 oncoprotein to the tumor suppressor p53 protein and act as pharmacological p53 activators. MDM2 inhibitors have been previously described as anticancer therapeutics (see, e.g., U.S. Patent No. 9,745,314, incorporated by reference in its entirety herein), and are being evaluated in humans as monotherapy or in combination with standard-of-care chemotherapy agents used to treat diseases and disorders where inhibition of MDM2 and MDM2-related protein activity provides a benefit.

[0209] The MDM2 inhibitors disclosed in the present invention inhibit the interaction between p53 or p53-related proteins and MDM2 or MDM2-related proteins. By inhibiting the negative effects of MDM2 or MDM2-related proteins on p53 or p53-related proteins, the MDM2 inhibitors of the present invention sensitize cells to inducers of apoptosis and / or cell cycle arrest. In one embodiment, the MDM2 inhibitors of the present invention induce apoptosis and / or cell cycle arrest.

[0210] ii. Biomarkers

[0211] It has been identified herein that biomarkers are able to predict the likelihood of response or therapeutic efficacy of compounds that target the apoptosis pathway. The biomarkers provided herein include Noxa and ASCL1.

[0212] As used herein, the term “Noxa” refers to the Noxa gene and Noxa gene products (e.g., mRNA of the Noxa gene and a protein encoded by the Noxa gene). The Noxa gene (also known as the phorbol-12-myristate-13-acetate-induced protein 1 (PMA-induced protein 1, or PMAIP1) gene, the immediate early response protein APR gene, the adult T-cell leukemia-derived PMA-responsive gene, or the APR gene) encodes a protein that promotes activation of caspases and apoptosis at least in part by promoting mitochondrial membrane changes and pro-apoptotic protein efflux from mitochondria. The human Noxa gene has a Gene ID of 5366 in the NCBI database. The mRNA transcript of the human Noxa gene has a NCBI Reference Sequence of NM_021127.2. The protein encoded by the human Noxa gene has a NCBI Reference Sequence of NP_066950.1. Exemplary sequences for Noxa are provided herein as SEQ ID NO: 8 (DNA sequence) and SEQ ID NO: 7 (protein sequence).

[0213] As used herein, the term “ASCL1” refers to the ASCL1 gene and ASCL1 gene products (e.g., mRNA of the ASCL1 gene and a protein encoded by the ASCL1 gene). The ASCL1 gene (also known as the Achaete-Scute homolog 1 gene, the Achaete-Scute family BHLH transcription factor 1 gene, the Class A basic-helix-loop-helix protein 46 gene, the HASH1 gene, the ASH1 gene, the MASH1 gene) encodes a transcription factor that plays a key role in neuronal differentiation. Direct transcriptional targets of ASCL1 include Bcl2. The human ASCL1 gene has a Gene ID of 429 in the NCBI database. The mRNA transcript of the human ASCL1 gene has a NCBI Reference Sequence of NM_004316.4. The protein encoded by the human ASCL1 gene has a NCBI Reference Sequence of NP_004307.2. Exemplary sequences for ASCL1 are provided herein as SEQ ID NO: 22 (DNA sequence) and SEQ ID NO: 21 (protein sequence).

[0214] ASCL1 is critical for neuronal differentiation, for example, normal development of neuroendocrine cells. In addition, ASCL1 expression is associated with growth and survival of neuroendocrine lung cancers (Augustyn, A. et al. PNAS, 111 : 14788-14793 (2014)). A recent study on small cell lung cancer (SCLC) proposed that SCLC is divided into four subtypes by differential expression of four key transcriptional regulators (ASCL1, Neurogenic Differentiation Factor 1 (NeuroD1), yes-associated protein 1 (YAP1), and POU Class 2 Homeobox (POU2F3)), where expression of the four transcriptional regulators is essentially mutually exclusive, and SCLC expressing ASCL1 is the largest proportion of SCLC (i.e., about 70%) (Rudin, CM, et al., 19: 289-297, (2019)).

[0215] The inventors of the present disclosure have surprisingly found that the level of at least one biomarker comprising Noxa, or ASCL1, or both, is associated with the therapeutic efficacy of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor.

[0216] In certain embodiments, the biomarker comprises a Noxa gene comprising a genetic sequence of SEQ ID NO: 8, or an mRNA encoded therefrom. In certain embodiments, the biomarker comprises a Noxa protein comprising an amino acid sequence of SEQ ID NO: 7.

[0217] In certain embodiments, the biomarker comprises an ASCL1 gene comprising a genetic sequence of SEQ ID NO: 22, or an mRNA encoded therefrom. In certain embodiments, the biomarker comprises an ASCL1 protein comprising an amino acid sequence of SEQ ID NO: 21.

[0218] Accordingly, based on the measured level of at least one biomarker comprising Noxa, ASCL1, or both, the present disclosure provides assay reagents for measuring the level of at least one biomarker comprising Noxa, ASCL1, or both; and methods for identifying a subject having a cancer who is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor; methods for treating a cancer in a subject in need thereof; and methods for monitoring therapeutic efficacy in a subject who has a cancer and has been treated with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor for a treatment period.

[0219] In some embodiments, the at least one biomarker further comprises one or more additional biomarkers selected from the group consisting of Bcl-xL, Bcl-2, PUMA, Mcl-1, a protein complex comprising Bcl-xL, a protein complex comprising Bcl-2, and any combination thereof.

[0220] Exemplary sequences for Bcl-xL are provided herein as SEQ ID NO: 4 (DNA sequence) and SEQ ID NO: 3 (protein sequence). Exemplary sequences for Bcl-2 are provided herein as SEQ ID NO: 2 and 14 (DNA sequences) and SEQ ID NO: 1 and 13 (protein sequences). Exemplary sequences for PUMA are provided herein as SEQ ID NO: 10 (DNA sequence) and SEQ ID NO: 9 (protein sequence). Exemplary sequences for Mcl-1 are provided herein as SEQ ID NO: 12, 18, and 20 (DNA sequences) and SEQ ID NO: 11, 17, and 19 (protein sequences).

[0221] In some embodiments, the protein complex comprises a Bcl-xL protein complexed with a BH3-only protein or with a protein comprising a BH3 domain. In some embodiments, the protein complex further comprises a Bcl-2 protein complexed with a BH3-only protein or with a protein comprising a BH3 domain.

[0222] In certain embodiments, the BH3-only protein is selected from the group consisting of BIM, BID, BAD, BIK, HRK, BMF, and PUMA. In certain embodiments, the BH3-only protein can be selected from BIM and PUMA.

[0223] Exemplary sequences for BIM are provided herein as SEQ ID NO: 6 and 16 (DNA sequences) and SEQ ID NO: 5 and 15 (protein sequences).

[0224] In some embodiments, the protein complex comprises a complex selected from the group consisting of Bcl-xL:BIM, Bcl-xL:PUMA, Bcl-2:BIM, Bcl-2:PUMA, and any combination thereof.

[0225] Methods for patient identification, treatment guidance and prognosis

[0226] In an aspect, the disclosure provides a detection reagent for detecting or measuring the level of the at least one biomarker comprising Noxa, ASCL1, or both. The measurement can be at the RNA level, the DNA level, and / or the protein level. Suitable reagents for detecting a target RNA, a target DNA, or a target protein can be used.

[0227] In certain embodiments, the detection reagent comprises one or more primers or probes that can hybridize to a polynucleotide of Noxa, and / or one or more primers or probes that can hybridize to a polynucleotide of ASCL1. As used herein, the term "primer" refers to an oligonucleotide that, due to the sequence complementarity of at least a portion of the primer's sequence within the sequence of a target polynucleotide sequence, can hybridize specifically to the target polynucleotide sequence. The primer can be at least 8 nucleotides in length, typically from 8 to 70 nucleotides, often from 18 to 26 nucleotides. To hybridize properly to the target sequence, the primer can have at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence complementarity to the hybridizing portion of the target polynucleotide sequence. Oligonucleotides useful as primers can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. (1981) 22: 1859-1862, using an automated synthesizer, as described in Needham-VanDevanter et al., Nucleic Acids Res. (1984) 12: 6159-6168.

[0228] Primers can be used in nucleic acid amplification reactions, in which the primers are extended to produce new strands of polynucleotides. Primers can be readily designed by the skilled artisan using common general knowledge in the art, such that they can anneal specifically to the nucleotide sequence of a target nucleotide sequence of at least one biomarker provided herein. Typically, the 3' nucleotide of the primer is designed to be complementary to the target sequence at the corresponding nucleotide position to provide optimal primer extension by a polymerase.

[0229] As used herein, the term "probe" refers to an oligonucleotide or an analog thereof that, due to the sequence complementarity of at least a portion of the probe's sequence within the sequence of a target polynucleotide sequence, can specifically hybridize to a target polynucleotide sequence. Exemplary probes can be, for example, DNA probes, RNA probes, or protein nucleic acid (PNA) probes. The length of a probe can be at least 8 nucleotides, typically 8 to 70 nucleotides, often 18 to 26 nucleotides. To hybridize properly to a target sequence, a probe can have at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence complementarity to the hybridizing portion of the target polynucleotide sequence. And probes can also be chemically synthesized according to the solid phase phosphoramidite trister method as described above. Methods for preparing DNA and RNA probes, and conditions for hybridizing them to target nucleotide sequences, are described in Molecular Cloning: A Laboratory Manual, J. Sambrook et al. eds., 2nd ed. Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11.

[0230] In certain embodiments, a primer or probe provided herein comprises a polynucleotide sequence that can hybridize to a portion within the sequence of SEQ ID NO: 8 or 22. In certain embodiments, a primer or probe provided herein comprises a polynucleotide sequence that can hybridize to a portion within the sequence of SEQ ID NO: 2, 4, 6, 10, 12, 14, 16, 18, or 20. In certain embodiments, a primer or probe provided herein comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementary to a portion within the sequence of SEQ ID NO: 8 or 22. In certain embodiments, a primer or probe provided herein comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementary to a portion within the sequence of SEQ ID NO: 2, 4, 6, 10, 12, 14, 16, 18, or 20.

[0231] In certain embodiments, a detection reagent comprises one or more antibodies that can specifically bind to a protein of Noxa, and / or one or more antibodies that can specifically bind to a protein of ASCL1.

[0232] As used herein, the term "antibody" refers to an immunoglobulin or antigen binding fragment thereof that can specifically bind to a target protein antigen. Antibodies can be identified and prepared by selecting antibodies from a recombinant antibody library in a bacteriophage or similar vector, as well as by preparing polyclonal and monoclonal antibodies by immunizing an animal (e.g., a rabbit or a mouse) (see, e.g., Huse et al., Science (1989) 246: 1275-1281; Ward et al., Nature (1989) 341 : 544-546).

[0233] In certain embodiments, the antibodies provided herein comprise an antigen binding region that is capable of specifically binding to an epitope within a protein or polypeptide having the sequence of SEQ ID NO: 7 or 21. In certain embodiments, the antibodies provided herein comprise an antigen binding region that is capable of specifically binding to an epitope within a protein or polypeptide having the sequence of SEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17, or 19.

[0234] In certain embodiments, the primers, probes, and antibodies provided herein are detectably labeled. Examples of detectable labels suitable for labeling primers, probes, and antibodies include, for example, a chromophore, a radioisotope, a fluorophore, a chemiluminescent moiety, a particle (visible or fluorescent), a nucleic acid, a ligand, or a catalyst (e.g., an enzyme).

[0235] Examples of radioisotopes include, but are not limited to, 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C, 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 P.

[0236] Examples of fluorophores include, but are not limited to, acridine, 7-amino-4- methylcoumarin-3-acetic acid (AMCA), BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Edans, Eosin, Erythrosin, Fluorescein, 6-FAM, TET, JOC, HEX, Oregon Green, Rhodamine, Rhodol Green, Tamra, Rox, and Texas Red TM (Molecular Probes, Inc., Eugene, Oreg.).

[0237] Examples of enzymes include, but are not limited to, alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta-galactosidase, and ribonuclease.

[0238] Examples of ligands include, but are not limited to, biotin, avidin, an antibody, or an antigen.

[0239] It should be understood that a detectable label need not necessarily produce a detectable signal, for example, in some embodiments it can react with a detectable partner or with one or more additional compounds to produce a detectable signal. For example, a detectable label can be a ligand that is a member of a specific binding pair of ligands that can be used as a label (e.g., a second labeled antibody). As another example, an enzyme can be used as a detectable label due to its catalytic activity of a chromogenic, fluorogenic, or luminescent substrate that results in the production of a detectable signal.

[0240] In certain embodiments, the detectably labeled primers, probes, or antibodies provided herein can further comprise a quencher substance. A quencher substance refers to a substance that, when present in close enough proximity to a fluorescent substance, can quench the fluorescence emitted by the fluorescent substance due to, for example, fluorescence resonance energy transfer (FRET).

[0241] Examples of quencher substances include, but are not limited to, Tamra, Dabcyl, or Black Hole Quencher (BHQ, Biosearch Technologies), DDQ (Eurogentec), Iowa Black FQ (Integrated DNA Technologies), QSY-7 (Molecular Probes), and Eclipse Quencher (Epoch Biosciences).

[0242] Primers and probes can be labeled with high specificity activity by nick-translation methods or by random primer methods. Useful probe labeling techniques are described in the literature (Fan, Y-S, Molecular cytogenetics: protocols and applications, Humana Press, Totowa, N.J. xiv, 411 (2002)).

[0243] In certain embodiments, the one or more agents comprise a primer or probe that can hybridize to a polynucleotide of Noxa, or an antibody that can specifically bind to a protein of Noxa. In certain embodiments, an antibody provided herein comprises an antigen binding region that is capable of specifically binding to an epitope within a protein or polypeptide having the sequence of SEQ ID NO: 7.

[0244] In certain embodiments, the one or more agents comprise a primer or probe that can hybridize to a polynucleotide of ASCL1, or an antibody that can specifically bind to a protein of ASCL1. In certain embodiments, an antibody provided herein comprises an antigen binding region that is capable of specifically binding to an epitope within a protein or polypeptide having the sequence of SEQ ID NO: 21.

[0245] In certain embodiments, the one or more agents comprise a first primer or first probe that can hybridize to a polynucleotide of Noxa, or a first antibody that can specifically bind to a protein of Noxa, and a second primer or second probe that can hybridize to a polynucleotide of Noxa, or a second antibody that can specifically bind to a protein of Noxa. In certain embodiments, a first antibody provided herein comprises an antigen binding region that is capable of specifically binding to an epitope within a protein or polypeptide having the sequence of SEQ ID NO: 7, and a second antibody provided herein comprises an antigen binding region that is capable of specifically binding to an epitope within a protein or polypeptide having the sequence of SEQ ID NO: 21.

[0246] It can be appreciated that in certain embodiments, antibodies are modified or labeled for appropriate use in a variety of detection assays. In certain embodiments, an antibody is detectably labeled. In certain embodiments, an antibody can comprise a capture moiety, or can be immobilized.

[0247] Examples of capture moieties can include, for example, binding partners or solid substrates, such as porous and non-porous materials, latex particles, magnetic particles, microparticles, strips, beads, membranes, microtiter wells, and plastic tubes. The choice of solid phase material and the method of detectably labeling the antigen or antibody reagent is determined according to the desired performance characteristics of the assay format. In certain embodiments, the antibodies can be immobilized on a solid substrate. Immobilization can be by covalent linkage or non-covalent attachment (e.g., coating).

[0248] i. Sample preparation

[0249] In another aspect, the disclosure provides methods for identifying a subject having cancer who is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor. In certain embodiments, the method comprises measuring in a test sample derived from the subject the level of at least one biomarker comprising Noxa or ASCL1 or both; comparing the level of the at least one biomarker to a corresponding reference level of the at least one biomarker to determine a difference from the reference level; and determining that the subject is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor when the difference reaches a predetermined threshold.

[0250] In another aspect, the disclosure provides methods for treating cancer in a subject in need thereof. In certain embodiments, the method comprises measuring in a sample derived from the subject the level of at least one biomarker comprising Noxa or ASCL1 or both; comparing the level of the at least one biomarker to a corresponding reference level of the at least one biomarker to determine a difference from the reference level; and administering to the subject an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor when the difference reaches a predetermined threshold.

[0251] In another aspect, the disclosure provides methods for monitoring therapeutic efficacy or prognosis in a subject having cancer who has been treated with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor during a treatment period. In certain embodiments, the method comprises: obtaining a test sample from the subject after the treatment period; measuring the level of at least one biomarker comprising Noxa in the test sample to obtain a post-treatment level of the at least one biomarker; comparing the post-treatment level to a baseline level of the at least one biomarker in a test sample derived from the subject prior to the treatment period to determine a post-treatment change in the level of the at least one biomarker. In certain embodiments, when the post-treatment change meets a predetermined threshold, the method further comprises continuing administration of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor to the subject. In certain embodiments, when the post-treatment change does not meet the predetermined threshold, increasing the dose of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor to the subject, administering to the subject an effective amount of a second anti-cancer therapeutic agent in combination with the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor, or discontinuing administration of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor to the subject.

[0252] ii. Methods of measuring the level of biomarkers

[0253] Any biological sample suitable for use in practicing the methods provided herein can be derived from a subject. In certain embodiments, the sample can be further processed by any suitable method for performing the measurement of the level of the at least one biomarker.

[0254] In certain embodiments, the method further comprises isolating or extracting cancer cells (e.g., circulating tumor cells) from a biological fluid sample (e.g., a peripheral blood sample) or a tissue sample derived from the subject. Cancer cells can be isolated by immunomagnetic separation techniques, such as the Isolex® technology available from Immunicon (Huntingdon Valley, Pa.).

[0255] In certain embodiments, a tissue sample can be processed for in situ hybridization. For example, paraffin-embedding can be performed prior to fixation of the tissue sample on a glass microscope slide, followed by deparaffinization with a solvent (typically xylene).

[0256] In certain embodiments, the method further comprises isolating nucleic acids from the sample if the RNA or DNA level of the biomarker is to be measured. A variety of extraction methods are suitable for isolating DNA or RNA from cells or tissues, such as phenol and chloroform extraction, and a variety of other methods are described in, e.g., Ausubel et al., Current Protocols of Molecular Biology (1997) John Wiley & Sons, and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rded. (2001).

[0257] RNA can also be isolated using commercially available kits, including, e.g., NucliSens extraction kit (Biomerieux, Marcyl'Etoile, France), QIAamp RNA Blood Mini Kit (Qiagen), MagNA Pure 96 TM Mini Blood Kit, Agencourt Genfind TM 、 Mini Column (Qiagen), RNA Mini Kit (Thermo Fisher Scientific), and Eppendorf Phase Lock Gels TM A skilled artisan can readily extract or isolate RNA or DNA following the manufacturer's protocol.

[0258] a) Amplification assays

[0259] The methods of the disclosure comprise measuring the level of at least one biomarker described herein in a sample derived from a subject having or suspected of having cancer.

[0260] The biomarkers Noxa and / or ACSL1 (and optionally additional biomarkers, including Bcl-xL, Bcl-2, PUMA, Mcl-1, a protein complex comprising Bcl-xL, a protein complex comprising Bcl-2) provided herein are intended to encompass different forms including mRNA, protein, and DNA (e.g., genomic DNA). Thus, the level of the at least one biomarker can be measured by RNA level (e.g., mRNA level), protein level, or DNA level. The mRNA level and / or protein level can also be referred to as the expression level of the at least one biomarker. In certain embodiments, the protein complex is measured at the protein level.

[0261] The RNA (e.g., mRNA) or DNA level of the at least one biomarker comprising Noxa, or ASCL1, or both can be measured by any suitable nucleic acid assay known in the art (e.g., nucleic acid amplification assays, nucleic acid hybridization assays, nucleic acid sequencing assays), as well as other methods (e.g., high performance liquid chromatography (HPLC) fragment analysis, capillary electrophoresis, etc.). Protein levels of a biomarker can be measured by any method known in the art, for example, but not limited to, immunoassays. These methods are well known in the art and are described in detail below as illustrative examples.

[0262] b) Hybridization assays

[0263] Nucleic acid amplification assays involve replicating a target nucleic acid (e.g., DNA or RNA), thereby increasing the copy number of the amplified nucleic acid sequence. Amplification can be exponential or linear. Exemplary nucleic acid amplification methods include, but are not limited to, amplification using: polymerase chain reaction (“PCR” (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide To Methods And Applications (Innis et al. eds., 1990)), reverse-transcriptase polymerase chain reaction (RT-PCR), real-time quantitative PCR (qRT-PCR), quantitative PCR (e.g., ), nested PCR, ligase chain reaction (see Abravaya, K. et al., Nucleic Acids Research, 23: 675-682, (1995)), branched DNA signal amplification (see, Urdea, M.S. et al., AIDS, 7 (Suppl. 2): S11-S14, (1993)), amplifiable RNA reporter molecules, Q-beta replication (see, Lizardi et al., Biotechnology (1988) 6: 1197), transcription-based amplification (see, Kwoh et al., Proc. Natl. Acad. Sci. USA (1989) 86: 1173-1177), rolling circle DNA amplification, strand displacement activation, cycling probe technology, self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA (1990) 87: 1874-1878), rolling circle replication (U.S. Patent No. 5,854,033), nucleic acid sequence-based amplification (NASBA), and serial analysis of gene expression (SAGE).

[0264] In some embodiments, to measure the mRNA level of a biomarker, the target RNA of the biomarker is reverse transcribed into cDNA prior to amplification. A variety of reverse transcriptases can be used, including but not limited to MMLV RT, RNase H mutants of MMLV RT such as Superscript and Superscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, and thermostable reverse transcriptases from Thermus thermophilus. For example, one method that can be used to convert RNA to cDNA is a protocol adapted from the Superscript II Pre- Amplification System (Life Technologies, GIBCO BRL, Gaithersburg, Md.; Cat. No. 18089-011), as described by Rashtchian, A., PCR Methods Applic. 4:S83-S91, (1994).

[0265] In certain embodiments, the level of at least one biomarker provided herein is quantified after a nucleic acid amplification assay. For example, the amplified products can be separated on an agarose gel and stained with ethidium bromide and then detected and quantified using standard gel electrophoresis methods. Alternatively, the amplified products can be labeled en masse with a suitable detectable label (e.g., a radioactive or fluorescent nucleotide) and then visualized using X-ray film or visualized under the appropriate excitation spectrum.

[0266] In certain embodiments, the expression level of RNA (e.g., mRNA) or copy number changes of DNA of a biomarker is quantified during a nucleic acid amplification assay, also referred to as real-time amplification or quantitative amplification. Methods of quantitative amplification are disclosed in, for example, U.S. Patent Nos. 6,180,349, 6,033,854, and 5,972,602, and in, for example, Gibson et al., Genome Research (1996) 6:995-1001; DeGraves et al., Biotechniques (2003) 34(1): 106-10, 112-5; Deiman B et al., Mol Biotechnol. (2002) 20(2): 163-79. Quantification is typically based on the monitoring of a detectable signal that is representative of the copies of the template in the amplification (e.g., PCR) reaction cycle. The detectable signal can be generated by intercalating agents (e.g., SYBR GREEN TM and SYBR GOLD TM) or a labeled primer or labeled probe used in the amplification process to generate a detectable signal.

[0267] In certain embodiments, the labeled primer or labeled probe comprises a detectable label comprising a fluorophore. In certain embodiments, the labeled primer or labeled probe can further comprise a quencher substance. The presence of a fluorophore and a quencher substance in one primer or probe (“dual-labeled”) can help to provide a self-quenching probe such as a TaqMan (U.S. Patent Nos. 5,210,015 and 5,538,848) or a molecular beacon probe (U.S. Patent Nos. 5,118,801 and 5,312,728), or other branchless or linear beacon probes (Livak et al., 1995, PCR Method Appl. 4:357-362; Tyagi et al., 1996, Nature Biotechnology 14:303-308; Nazarenko et al., 1997, Nucl. Acids Res. 25:2516-2521; U.S. Patent Nos. 5,866,336 and 6,117,635). In the intact primer or probe, the quencher substance and the fluorophore are in close proximity, such that when the fluorophore is excited by radiation, the fluorophore transfers energy to the quencher substance in the same probe by fluorescence resonance energy transfer (FRET), thereby not emitting a signal.

[0268] In quantitative amplification assays (e.g., real-time PCR), the level of a detected biomarker can be quantified using methods known in the art. For example, during amplification, the fluorescent signal can be monitored and calculated during each PCR cycle. A threshold cycle or Ct value can be further calculated. The Ct value is the cycle at which fluorescence crosses a predetermined value. The Ct can be related to the initial amount of nucleic acid or starting cell number using a standard curve. The standard curve is constructed to correlate the difference between the Ct value and the log level of the measured biomarker.

[0269] As a quality control measure, the level of an internal control biomarker can be measured. The skilled person will appreciate that an internal control biomarker can be inherently present in the sample and its level can be used to normalize the measured level of at least one biomarker comprising Noxa, ASCL1, or both, to offset any differences in sample absolute numbers.

[0270] c) Sequencing methods

[0271] Nucleic acid hybridization assays use probes to hybridize to target nucleic acids, thereby allowing detection of the target nucleic acid. Non-limiting examples of hybridization assays include Northern blotting, Southern blotting, in situ hybridization, microarray analysis, and multiplex hybridization-based assays.

[0272] In certain embodiments, the probes used in the hybridization assay are detectably labeled. In certain embodiments, the nucleic acid-based probes used in the hybridization assay are unlabeled. Such unlabeled probes can be immobilized on a solid support (such as a microarray) and can be hybridized to detectably labeled target nucleic acid molecules.

[0273] In certain embodiments, the hybridization assay can be performed by isolating nucleic acids (e.g., RNA or DNA), separating the nucleic acids (e.g., by gel electrophoresis), and then transferring the separated nucleic acids to a suitable membrane filter (e.g., a nitrocellulose filter), where the probes are hybridized to the target nucleic acids and allowed to be detected. See, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd ed., Cold Spring Harbor Laboratory Press, 1989, Chapter 7. Hybridization of the probes and target nucleic acids can be detected or measured by methods known in the art. For example, autoradiographic detection of hybridization can be performed by exposing the hybridized filter to photographic film. Densitometric scanning of the photographic film exposed by the hybridized filter provides an accurate measurement of the level of target nucleic acid. Computer imaging systems can also be used to quantify the level of biomarker.

[0274] In some embodiments, the hybridization assay can be performed on a microarray. Microarrays provide a method for simultaneously measuring the levels of a large number of target nucleic acid molecules. The target nucleic acids can be RNA, DNA, cDNA reverse transcribed from mRNA, or chromosomal DNA. The target nucleic acids can be allowed to hybridize to a microarray comprising a substrate having a plurality of immobilized nucleic acid probes arranged at a density of up to several million probes per square centimeter of substrate surface. The RNA or DNA in the sample hybridizes to complementary probes on the array, which is then detected by laser scanning. The hybridization intensity of each probe on the array is determined and converted to a quantitative value representing the relative level of RNA or DNA. See, U.S. Patent Nos. 6,040,138, 5,800,992, and 6,020,135, 6,033,860, and 6,344,316.

[0275] Techniques for synthesizing these arrays using mechanical synthesis methods are described, for example, in U.S. Patent No. 5,384,261. Although planar array surfaces are often employed, the arrays can be fabricated on virtually any shaped surface or even multiple surfaces. Arrays can be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers (e.g., optical fibers), glass, or any other suitable substrate, see U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193, and 5,800,992. Arrays can be packaged in such a way as to allow for diagnosis or other manipulation of the entire package. Useful microarrays are also commercially available, for example, microarrays from Affymetrix, Nano String Technologies, QuantiGene 2.0 multiplex assays from Panomics.

[0276] In certain embodiments, the hybridization assay can be an in situ hybridization assay. In situ hybridization assays can be used to detect the presence of copy number changes (e.g., increases or amplifications) at the locus of a biomarker of interest (e.g., Noxa or ASCL1 or both). Probes useful for in situ hybridization assays can be locus-specific probes that hybridize to a specific locus on a chromosome to detect the presence or absence of a particular locus of interest (e.g., Noxa or ASCL1 or both). Other types of probes can also be useful, such as chromosome enumeration probes (e.g., that can hybridize to a repetitive sequence region in a chromosome of interest to indicate the presence or absence of the entire chromosome) and chromosome arm probes (e.g., that can hybridize to a region of a chromosome and indicate the presence or absence of an arm of a particular chromosome). Methods using unique sequence probes for in situ hybridization are described in U.S. Patent No. 5,447,841, which is incorporated herein by reference. Probes can be viewed using a fluorescence microscope and appropriate filters for each fluorophore, or by using a dual or triple bandpass filter set to observe multiple fluorophores. See, e.g., U.S. Patent No. 5,776,688 to Bittner et al., which is incorporated herein by reference. Any suitable microscopic imaging method, including automated digital imaging systems, can be used to visualize the hybridized probes. Alternatively, techniques such as flow cytometry can be used to examine the hybridization pattern of the probes.

[0277] d) Immunoassays

[0278] Sequencing methods useful in measuring the level of a biomarker of interest involve sequencing of a target nucleic acid and counting of the sequenced target nucleic acid. Examples of sequencing methods include, but are not limited to, RNA sequencing, pyrosequencing, and high-throughput sequencing.

[0279] High-throughput sequencing involves sequencing-by-synthesis, sequencing-by-ligation, and ultra-deep sequencing (e.g., described in Marguiles et al., Nature 437(7057): 376-80 (2005)). Sequencing-by-synthesis involves synthesizing the complementary strand of a target nucleic acid by incorporating labeled nucleotides or nucleotide analogs in a polymerase amplification. Upon or immediately after successful incorporation of a labeled nucleotide, the signal of the label is measured and the identity of the nucleotide is recorded. The detectable label on the incorporated nucleotide is removed prior to incorporation, and the detection and identification steps are repeated. Examples of sequencing-by-synthesis methods are known in the art and described in, e.g., U.S. Patent No. 7,056,676, U.S. Patent No. 8,802,368, and U.S. Patent No. 7,169,560, the contents of which are incorporated herein by reference. Sequencing-by-synthesis is performed on a solid surface (or microarray or chip) using a turn-around PCR and anchor primers. Target nucleic acid fragments can be attached to the solid surface by hybridization to anchor primers and bridge amplification is performed. This technology is used, e.g., on the Ion Torrent® sequencing platform.

[0280] Pyrosequencing involves hybridizing a target nucleic acid region to a primer and extending the new strand in the presence of a polymerase by sequentially incorporating deoxynucleotide triphosphates corresponding to bases A, C, G, and T (U). Each incorporation of a base is accompanied by the release of a pyrophosphate, which is converted to ATP by a sulfurylase enzyme, driving the synthesis of an oxidized luciferin and the release of visible light. Since the release of pyrophosphate is equimolar to the number of bases incorporated, the light emitted is directly proportional to the number of nucleotides added in any step. The process is repeated until the entire sequence is determined.

[0281] ​In certain embodiments, the level of a biomarker described herein is measured by whole transcriptome sequencing or RNA sequencing (e.g., RNA-Seq). Methods of RNA sequencing have been described (see Wang Z, Gerstein M, and Snyder M, Nature Review Genetics (2009) 10:57-63; Maher CA, et al., Nature (2009) 458:97-101; Kukurba K and Montgomery SB, Cold Spring Harbor Protocols (2015) 2015(11):951-969). Briefly, mRNA extracted from a sample is reverse transcribed into cDNA, which is then sheared into fragments. Fragments within an appropriate length range are selected and ligated to sequencing adapters, then amplified, sequenced, and reads are mapped to a reference genome.

[0282] The level of the at least one protein comprising Noxa or ASCL1 or both can be measured by any suitable protein assay known in the art, e.g., immunoassay, mass spectrometry, 2-D gel electrophoresis, protein array, etc. The level of protein complex comprising Bcl-xL and / or protein complex comprising Bcl-2 can be measured by any suitable assay known in the art for measuring protein-protein interactions, see generally, Protein-Protein Interactions: A Molecular Cloning Manual, 2nd Edition, Golemis and Adams, eds., Cold Spring Harbor Laboratory Press (2005)). In certain embodiments, the assay of protein-protein interactions is based on an immunoassay or proximity assay. Suitable methods are generally, e.g., Meso Scale Discovery (MSD) advanced enzyme-linked immunosorbent assay (MSD-ELISA), standard complex ELISA, proximity ligation assay (PLA), co-immunoprecipitation, immunoblot assay, or cross-linking assay, etc.

[0283] iii. Comparison with corresponding reference levels

[0284] Immunoassays typically involve the use of an antibody that specifically binds to a target polypeptide or protein (e.g., Noxa or ASCL1) to detect or measure the presence or level of the target polypeptide or protein. Such antibodies can be obtained using methods known in the art (see, e.g., Huse et al., Science (1989) 246: 1275-1281; Ward et al., Nature (1989) 341 : 544-546), or can be obtained from commercial sources. Examples of immunoassays include, but are not limited to, Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), sandwich assays, competitive assays, immunofluorescent staining and imaging, immunohistochemistry (IHC), and fluorescence-activated cell sorting (FACS). For a review of immunology and immunological procedures, see Basic and Clinical Immunology (Stites and Terr eds., 7th Ed. 1991). In addition, immunoassays can be performed in any of a variety of formats, which are extensively reviewed in Enzyme Immunoassay (Maggio ed., 1980) and Harlow and Lane (supra). For a review of general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, vol. 37 (Asai ed. 1993); Basic and Clinical Immunology (Stites and Terr eds., 7th Ed. 1991).

[0285] Immunohistochemistry (IHC) demonstrates in situ cellular or tissue composition by detecting specific antibody / aptamer-antigen interactions, where the antibody / aptamer has been tagged with a detectable label. The detectable label can be a fluorescent dye, a colloidal metal, a hapten, a radioactive marker, or more commonly, an enzyme. Experimental samples include formalin-fixed, paraffin-embedded (FFPE) samples. Ideally, maximum signal strength with minimal background or non-specific staining is required to give the best antigen display. IHC protocols are well known in the art; see, e.g., Immunocytochemical Methods and Protocols (2nd Edition) edited by Lorette C. Javois from Methods in Molecular Medicine, vol. 115, Humana Press, 1999 (ISBN 0-89603-570-0).

[0286] In certain embodiments, the antibody is detectably labeled, or is unlabeled but can react with a second molecule that is detectably labeled (e.g., a detectably labeled secondary antibody). Other detection systems are also available, such as time-resolved fluorescence, internal reflection fluorescence, amplification (e.g., polymerase chain reaction), and Raman spectroscopy.

[0287] In certain embodiments, the antibody can be immobilized on a solid substrate. Immobilization can be by covalent linkage or non-covalent attachment (e.g., coating). Examples of solid substrates include porous and non-porous materials, latex particles, magnetic particles, microparticles, strips, beads, membranes, microtiter wells, and plastic tubes. The choice of solid phase material and method of detectably labeling the antigen or antibody reagent is determined according to the desired performance characteristics of the assay format.

[0288] The level of a target polypeptide or protein can be determined, for example, by normalizing to an internal control value or a standard curve.

[0289] The level of each biomarker described herein can be normalized to a standard level of a standard marker. The standard level of the standard marker can be predetermined, contemporaneously determined, or determined after the sample is obtained from the subject. The standard marker can be run in the same assay, or can be a known standard marker from a previous assay. In the case where the level of a biomarker is determined by a sequencing assay (e.g., RNA sequencing), the level of the biomarker can be normalized to the total number of reads sequenced.

[0290] Any assay and method provided herein for measuring the level of a biomarker can be adapted or optimized for use in automated and semi-automated systems or point-of-care assay systems. Examples of automated and semi-automated systems are described in, e.g., Hu et al., Lab On a Chip 2017, 17(13):2225; Song et al., Anal Chem 2019, 91(1); 388; and Rusling et al., Analyst 2010, 135(10): 2496.

[0291] Methods for patient identification and treatment guidance

[0292] The level of the biomarker (e.g., optionally normalized) can then be compared to a corresponding reference level of the corresponding biomarker to determine a difference from the reference level. As used herein, the term “reference level” of the at least one biomarker (e.g., Noxa, or ASCL1, or both) refers to a level of the biomarker that is representative of a control level in normal subjects, or that is representative of an average level of the biomarker in a general population of subjects having cancer. In certain embodiments, the reference level of the at least one biomarker (e.g., Noxa, or ASCL1, or both) is representative of an average level of the biomarker in a general population of subjects having cancer.

[0293] In certain embodiments, the reference level can be a typical level, a measured level, or a range of levels of the corresponding biomarker level typically observed in one or more healthy cell or tissue samples or in one or more control (e.g., cancer) cell or tissue samples. In certain embodiments, the reference level can be an average level of the corresponding biomarker in a population of healthy subjects or in a general population of subjects having cancer (e.g., in a general population of cancer patients). As used herein, a “general population of subjects having cancer” or “general population of cancer patients” refers to a population of cancer subjects or patients having different kinds of cancer. For example, a general population of cancer patients can be a group of at least three (four, five, six, seven, eight, nine, ten, or more) types of cancer patients, some of whom have a first type of cancer, some of whom have a second type of cancer, some of whom have a third type of cancer, and so on. For example, a general population of cancer patients can be a population having various cancers or multiple cancer types. In certain embodiments, the reference level can also be an empiric level deemed to be representative of a general population of subjects having cancer. In certain embodiments, the reference level of a biomarker described herein is obtained using the same or comparable measurement method or assay as used in measuring the levels of the biomarkers provided herein.

[0294] In certain embodiments, the reference level can be predetermined. For example, the reference level can be calculated or extrapolated based on measurements of biomarker levels in a collection of normal tissues or samples. For another example, the reference level can be based on a statistic of biomarker levels typically observed in comparable samples from a normal population.

[0295] In certain embodiments, the reference level can be tested in parallel with the level of the biomarker in the test sample. In certain embodiments, the reference level is measured in a control sample. In certain embodiments, the control sample is a normal tissue sample derived from the same subject or from a healthy subject. In certain embodiments, the control sample is derived from a control cancer patient. In certain embodiments, the control sample is a comparable sample from one or more healthy subjects or from one or more cancer patients.

[0296] The level of the at least one biomarker in the test sample can exhibit a difference from the reference level in terms of being elevated or decreased. Depending on the particular biomarker, an increase can be sought in one biomarker, but a decrease in another biomarker, in order to predict the likelihood of responsiveness to an MDM2 inhibitor or Bcl-2 / Bcl-xL inhibitor dual inhibitor or Bcl-2 inhibitor or Bcl-xL inhibitor according to the methods provided herein. As used herein, the term "elevated" means that the level of a biomarker as measured in a test sample is higher than the corresponding reference level for that biomarker. Similarly, as used herein, "decreased" means that the level of a biomarker as measured in a sample is lower than the corresponding reference level for that biomarker. As used herein, the term "maintained" means no significant change.

[0297] In certain embodiments, an elevated level of Noxa is associated with the likelihood of responsiveness to an MDM2 inhibitor or Bcl-2 / Bcl-xL dual inhibitor or Bcl-2 inhibitor or Bcl-xL inhibitor provided herein. In certain embodiments, an elevated level of ASCL1 is associated with the likelihood of responsiveness to an MDM2 inhibitor or Bcl-2 / Bcl-xL dual inhibitor or Bcl-2 inhibitor or Bcl-xL inhibitor provided herein. In certain embodiments, an elevated level of Noxa and an elevated level of ASCL1 are associated with the likelihood of responsiveness to an MDM2 inhibitor or Bcl-2 / Bcl-xL dual inhibitor or Bcl-2 inhibitor or Bcl-xL inhibitor provided herein.

[0298] In certain embodiments, the level of the at least one biomarker (e.g., Bcl-xL, Bcl-2, Mcl-1, and / or a protein complex comprising a Bcl-xL protein and / or a protein complex comprising Bcl-2) can be further considered, e.g., to increase the sensitivity and specificity of the diagnosis. For example, an increase in the level of Bcl-xL, an increase in the level of Bcl-2, a decrease in the level of Mcl-1, and / or an increase in the level of a protein complex comprising a Bcl-xL protein or a Bcl-2 protein is also associated with the likelihood of responsiveness to a Bcl-2 / Bcl-xL dual inhibitor provided herein. In certain embodiments, an increase in the level of Noxa, and / or an increase in the level of ASCL1, in conjunction with an increase in the level of Bcl-xL, an increase in the level of Bcl-2, a decrease in the level of Mcl-1, or an increase in the level of a protein complex comprising a Bcl-xL protein or a Bcl-2 protein, is associated with the likelihood of responsiveness to an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor provided herein.

[0299] In certain embodiments, the difference from the reference level is further compared to a predetermined threshold. In certain embodiments, the predetermined threshold can be set by statistical methods such that if the difference from the reference level reaches the predetermined threshold, the difference can be considered statistically significant. Useful statistical analysis methods are described in L. D. Fisher and G. van Belle, Biostatistics: A Methodology for the Health Sciences (Wiley-Interscience, New York, 1993). Statistical significance can be determined based on a confidence ("p") value, which can be calculated using an unpaired two-tailed t-test. Statistical significance can be indicated using, e.g., a p value of less than or equal to 0.1, 0.05, 0.025, or 0.01. Confidence intervals and p values can be determined by methods well known in the art. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983.

[0300] In certain embodiments, the predetermined threshold for Noxa levels is an increase of at least 15%, at least 25%, at least 35%, at least 45%, or at least 50%. As used herein, a "percent increase" or "percent higher" refers to a percentage of an increment. For example, a 100% increase represents a 100% increase from a reference level, equivalent to a total of 200% of the reference level. In other words, when the Noxa levels measured in a test sample are at least 15%, at least 25%, at least 35%, at least 45%, or at least 50% higher than the corresponding reference levels for Noxa, such measured levels of Noxa in the test sample are considered to be significantly different from the reference levels.

[0301] In certain embodiments, the predetermined threshold for ASCL1 levels is an increase of at least 15%, at least 25%, at least 35%, at least 45%, or at least 50%, or at least 100%. In other words, when the ASCL1 levels measured in a test sample are at least 50%, at least 75%, at least 100%, at least 125%, or at least 150% higher than the corresponding reference levels for ASCL1, such measured levels of ASCL1 in the test sample are considered to be significantly different from the reference levels.

[0302] Bcl-2 / Bcl-xL dual inhibitors or Bcl-2 inhibitors or Bcl-xL inhibitors

[0303] In another aspect, the present disclosure provides methods for identifying a subject having cancer who is likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor. In one embodiment, the method comprises: measuring the levels of at least one biomarker comprising Noxa, ASCL1, or Noxa and ASCL1 in a test sample derived from the subject; and comparing the levels of the at least one biomarker to the corresponding reference levels for the at least one biomarker to determine a difference from the reference levels; and identifying the subject as likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor when the difference reaches a predetermined threshold.

[0304] In certain embodiments, if the difference does not reach the predetermined threshold, the subject is identified as being unlikely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor. These identified subjects can be recommended for additional testing to confirm the conclusion, or alternatively can be recommended not to be treated with the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor provided herein.

[0305] In certain embodiments, the methods provided herein further include administering to a subject identified as likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor.

[0306] In certain embodiments, the methods provided herein further include administering to a subject identified as likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor an effective amount of a second anti-cancer therapeutic agent in combination with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor.

[0307] In another aspect, the disclosure provides methods for treating cancer in a subject in need thereof. In certain embodiments, the method comprises: measuring the level of at least one biomarker comprising Noxa or ASCL1 or both in a test sample derived from the subject; comparing the level of the at least one biomarker to a corresponding reference level of the at least one biomarker to determine a difference from the reference level; and administering to the subject an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor when the difference reaches a predetermined threshold.

[0308] In another aspect, the disclosure provides methods for monitoring therapeutic efficacy in a subject having cancer who has been treated with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor during a treatment period. In certain embodiments, the method comprises: obtaining a test sample from the subject after the treatment period; measuring in the test sample the level of at least one biomarker comprising Noxa or ASCL1 or both to obtain a post-treatment level of the at least one biomarker; comparing the post-treatment level to a baseline level of the at least one biomarker in a test sample derived from the subject prior to the treatment period to determine a post-treatment change in the level of the at least one biomarker. If the post-treatment difference still meets a predetermined threshold, the subject is identified as still responsive to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor. Alternatively, if the post-treatment difference does not meet a predetermined threshold, the subject is identified as having reduced responsiveness or no longer responsive to the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor provided herein.

[0309] In certain embodiments, the method further comprises continuing administration of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor to the subject when the difference meets the predetermined threshold. In certain embodiments, when the difference does not meet the predetermined threshold, the method further comprises: administering to the subject an effective amount of a second anti-cancer therapeutic agent in combination with the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor, or discontinuing administration of the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor to the subject.

[0310] In certain embodiments, the at least one biomarker further comprises Bcl-xL, Bcl-2, PUMA, Mcl-1, a protein complex comprising Bcl-xL, or any combination thereof. A decrease in the post-treatment level of Noxa (and optionally Bcl-2, Bcl-xL) in the sample indicates a likelihood of reduced responsiveness to treatment with the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor. In certain embodiments, an increase in the post-treatment level of Mcl1 in the sample indicates a likelihood of reduced responsiveness to treatment with the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor.

[0311] MDM2 inhibitors

[0312] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitors described herein are compounds of structural formula (I), (II), or (III):

[0313] (I)

[0314] (II)

[0315] (III)

[0316] or a pharmaceutically acceptable salt of formula (I), (II), or (III);

[0317] wherein ring A is

[0318] substituted or unsubstituted X is selected from the group consisting of alkylene, alkenylene, cycloalkylene, cycloalkenylene, and heterocycloalkylene;

[0319] Y is selected from the group consisting of (CH2) n -N(R a )2and

[0320] Q is selected from the group consisting of O, O(CH2) 1-3 , NR c , NR c (C 1-3 alkylene), OC(=0)(C1-3alkylene), C(=0)0, C(=0)0(C 1-3 alkylene), NHC(=0)(C 1-3 alkylene), C(=0)NH, and C(=0)NH(C 1-3 alkylene);

[0321] Z is O or NR c ;

[0322] R1and R2are independently selected from the group consisting of H, CN, NO2, halo, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl, OR', SR', NR'R", COR', CO2R', OCOR', CONR'R", CONR'SO2R", NR'COR", NR'CONR"R"', NR'C=SNR"R"', NR'SO2R", SO2R', and SO2NR'R";

[0323] R3is selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl, OR', NR'R", OCOR', CO2R', COR', CONR'R", CONR'SO2R", C1-3alkyleneCH(OH)CH2OH, SO2R', and SO2NR'R";

[0324] R', R" and R'" are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, C 1-3 alkyleneheterocycloalkyl, or heterocycloalkyl;

[0325] R' and R", or R" and R'" can form, together with the atoms to which they are bonded, a 3- to 7-membered ring;

[0326] R4is hydrogen, halo, C 1-3 alkyl, CF3, or CN;

[0327] R5is hydrogen, halo, C 1-3 alkyl, substituted C 1-3 alkyl, hydroxyalkyl, alkoxy, or substituted alkoxy;

[0328] R6is selected from the group consisting of H, CN, NO2, halo, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl, OR', SR', NR'R", CO2R', OCOR', CONR'R", CONR'SO2R", NR'COR", NR'CONR"R'", NR'C=SNR"R'", NR'SO2R", SO2R', and SO2NR'R";

[0329] substituted or unsubstituted R7is selected from the group consisting of hydrogen, alkyl, alkenyl, (CH2) 0-3 cycloalkyl, (CH2) 0-3 cycloalkenyl, (CH2) 0-3 heterocycloalkyl, (CH2) 0-3 aryl, and (CH2) 0-3 heteroaryl;

[0330] R8is selected from the group consisting of hydrogen, halo, NO2, CN, CF3SO2, and CF3;

[0331] R a is selected from the group consisting of hydrogen, alkyl, heteroalkyl, alkenyl, hydroxyalkyl, alkoxy, substituted alkoxy, cycloalkyl, cycloalkenyl, and heterocycloalkyl;

[0332] R b is hydrogen or alkyl;

[0333] R c is selected from the group consisting of hydrogen, alkyl, substituted alkyl, hydroxyalkyl, alkoxy, and substituted alkoxy; and

[0334] n, r, and s are independently 1, 2, 3, 4, 5, or 6.

[0335] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitors described herein are compounds having the structure of Formula (IV):

[0336] (IV)

[0337]

[0338] or a pharmaceutically acceptable salt of (IV);

[0339] R 21 is SO2R2';

[0340] R 22 is alkyl, preferably C1-C4 alkyl, more preferably methyl, propyl, or isopropyl;

[0341] R 23 is alkyl, preferably C1-C4 alkyl, more preferably methyl, propyl, or isopropyl;

[0342] R 24 is halogen, preferably fluorine, chlorine;

[0343] R 25 is halogen, preferably fluorine, chlorine;

[0344] R 26 is selected from H, halogen, alkyl, preferably fluorine, chlorine, C1-C4 alkyl, more preferably methyl, propyl, isopropyl;

[0345] R 21b is H or alkyl, preferably C1-C4 alkyl, more preferably methyl, propyl, or isopropyl;

[0346] n2, r2, and s2 are independently 1, 2, 3, 4, 5, or 6, more preferably r2 and s2 are both 2, and n2 is 3, 4, or 5, more preferably n2, r2, and s2 are all 2; and

[0347] R2' is alkyl, preferably C1-C4 alkyl, more preferably methyl, propyl, or isopropyl.

[0348] In certain embodiments, the Bcl-2 inhibitors described herein are compounds having the structure of Formula (V):

[0349]

[0350] or a pharmaceutically acceptable salt of (V);

[0351] A3is selected from the group consisting of:

[0352]

[0353] and

[0354] E3is a carbon atom, and is a double bond; or

[0355] E3is -C(H)-, and is a single bond; or

[0356] E3is a nitrogen atom, and is a single bond;

[0357] X 31 , X 32 , and X 33 are each independently selected from the group consisting of -CR 38 = and -N=;

[0358] R 31a and R 31b together with the carbon atom to which they are attached form a 3-, 4-, or 5-membered optionally substituted cycloalkyl; or

[0359] R 31a and R 31b together with the carbon atom to which they are attached form a 4- or 5-membered optionally substituted heterocycle;

[0360] R 32 is selected from the group consisting of -NO2, -SO2CH3, and -SO2CF3;

[0361] R 32a is selected from the group consisting of hydrogen and halogen;

[0362] R 33 is selected from the group consisting of hydrogen, -CN, -C≡CH, and -N(R 34a )(R 34b );

[0363] R 34a is selected from the group consisting of optionally substituted C 1-6 alkyl, optionally substituted C 3-6 cycloalkyl, heterocycle, heteroalkyl, (cycloalkyl)alkyl, and (heterocycle)alkyl;

[0364] R 34b is selected from the group consisting of hydrogen and C1-4 alkyl;

[0365] R 35 selected from the group consisting of optionally substituted C 1-6 alkyl, heterocycle, heteroalkyl, (cycloalkyl)alkyl, and (heterocycle)alkyl;

[0366] R 36a , R 36c , R 36e , R 36f and R 36g each independently is selected from the group consisting of hydrogen, optionally substituted C 1-6 alkyl, optionally substituted C 3-6 cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, heterocycle, heteroalkyl, (cycloalkyl)alkyl, and (heterocycle)alkyl;

[0367] R 36b and R 36d each independently is selected from the group consisting of hydrogen, C 1-4 alkyl, and halogen;

[0368] R 37 selected from the group consisting of optionally substituted C 1-6 alkyl, heterocycle, heteroalkyl, (cycloalkyl)alkyl, and (heterocycle)alkyl; and

[0369] R 38 selected from the group consisting of hydrogen and halogen.

[0370] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitors having structural Formula (I-III) described herein are a compound selected from Table 1-A, or a pharmaceutically acceptable salt of the compound.

[0371] Table 1-A

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitors described herein having structural Formula (IV) are selected from the compounds of Table 1-B.

[0379] Table 1-B

[0380]

[0381]

[0382]

[0383]

[0384] In certain embodiments, the Bcl-2 inhibitors described herein having structural Formula (V) are selected from the compounds of Table 1-C or a pharmaceutically acceptable salt of such compound.

[0385] Table 1C

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitor is (R)-2-(l-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-l-isopropyl-5-methyl-4-(methylsulfonyl)-lH-pyrrol-3-yl)-5- fluorophenyl)piperazin-l-yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4- (phenylthio)butyl)piperidine-4-carbonyloxy)ethylphosphonic acid ("Compound A15") having the following structure:

[0406] or a pharmaceutically acceptable salt thereof.

[0407] In certain embodiments, the Bcl-2 / Bcl-xL dual inhibitor is (R)-l-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-l-isopropyl-5-methyl-4-(methylsulfonyl)-lH-pyrrol-3-yl)-5- fluorophenyl)piperazin-l-yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4- (phenylthio)butyl)piperidine-4-carboxylic acid ("Compound B4") having the following structure:

[0408] or a pharmaceutically acceptable salt thereof.

[0409] Compound A15 is a small molecule compound that binds to Bcl-2, Bcl-xL and Bcl-w proteins with very high affinity, with IC 50 values of 1.6 nM, 4.4 nM and 9.3 nM, respectively. Compound A15 has weak affinity for Mcl-1 protein. Compound A15 shows potent in vitro cell growth inhibition activity with nanomolar potency in a subset of cancer cell lines. Mechanistically, Compound A15 potently induces cleavage of caspase-3 and PARP, which are biochemical markers of apoptosis in human cancers in cancer cell lines and xenograft tumor tissues. Compound B4 is an active metabolite of Compound A15.

[0410] In some embodiments, the Bcl-2 inhibitor is selected from the group consisting of compounds having the following structure

[0411]

[0412] or a pharmaceutically acceptable salt thereof.

[0413] A number of Bcl-xL inhibitors are known in the art, for example those described in Urbaniak A, et al., Oncol Lett. 2019 Nov; 18(5): 5097-5106; Zhan Y, et al., Cell Biosci. 2019 Jul 23; 9: 60.; Hikita H, et al., Hepatology. 2010; 52: 1310-21.; Lee B, et al., Biochem Biophys Res Commun. 2019 Jun 25; 514(2): 518-523.; Henz K, et al., Biol Chem. 2019 Jan 28; 400(2): 181-185.; Wang Q, et al., Leuk Lymphoma. 2019 Sep; 60(9): 2170-2180.; Rello-Varona S, et al., Sci Rep. 2019 Mar 7; 9(1): 3816.; Levesley J, et al., Neuro Oncol. 2018 Jan 22; 20(2): 203-214; and Lucantoni F, et al., Oncotarget. 2018 May 25; 9(40): 26046-26063.

[0414] In certain embodiments, the Bcl-xL inhibitor described herein is selected from ABT-263, ABT-737, A-1331852, A-1155463, and WEHI-539.

[0415] Kits

[0416] In certain embodiments, the MDM2 inhibitor comprises a chemical structure having the following formula (VI):

[0417]

[0418] or a pharmaceutically acceptable salt thereof, wherein

[0419] is selected from the group consisting of:

[0420]

[0421] B is C 4-7 carbocyclic;

[0422] R 61H, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, OR 6a , or NR 6a R 6b ;

[0423] n3 is 0, 1, or 2;

[0424] R 62 , R 63 , R 64 , R 65 , R 67 , R 68 , R 69 and R 70 are independently selected from the group consisting of H, F, CI, CH3, and CF3;

[0425] R 66 is

[0426] R 6a is hydrogen or substituted or unsubstituted C 1-4 alkyl;

[0427] R 6b is hydrogen or substituted or unsubstituted C 1-4 alkyl;

[0428] R 6c and R 6d are substituents on one carbon atom of ring B, wherein

[0429] R 6c is H, C 1-3 alkyl, C 1-3 alkylene-OR 6a , OR 6a , or halo;

[0430] R 6d is H, C 1-3 alkyl, C 1-3 alkylene-OR 6a , OR 6a , or halo; or

[0431] R 6c and R 6d , together with the carbon to which they are attached, form a 4- to 6- membered spiro substituent, optionally comprising an oxygen atom; and

[0432] R 6e is -C(=O)OR 6a , -C(=O)NR 6a R 6bor -C(=O)NHSO2CH3.

[0433] In certain embodiments, is

[0434] B is and

[0435] R 6c and R 6d is F and F, H and H, OH and CH3, OH and H, CH3and CH3, CH3and OH, H and OH, CH2CH3and CH2CH3, or CH2OH and CH2OH.

[0436] In certain embodiments, is H, CH3, or CH2CH3.

[0437] In certain embodiments, R 62 is H; R 63 is halo; R 64 and R 65 is H.

[0438] In certain embodiments, R 67 is fluoro; R 68 , R 69 and R 70 each is H; and R 6e is -C(=O)OH, -C(=O)NH2, or -C(=O)NHSO2CH3.

[0439] In certain embodiments, the MDM2 inhibitor is a compound selected from the group consisting of:

[0440]

[0441] or a pharmaceutically acceptable salt.

[0442] In one embodiment, the MDM2 inhibitor is

[0443]

[0444] or a pharmaceutically acceptable salt thereof.

[0445] In one embodiment, the MDM2 inhibitor is

[0446]

[0447] or a pharmaceutically acceptable salt thereof.

[0448] Further MDM2 inhibitors and synthesis of MDM2 inhibitors that can be used in the present application are further disclosed in U.S. Patent No. 9,745,314, which is incorporated herein by reference.

[0449] Second Anti-Cancer Therapeutic

[0450] In some embodiments, an effective amount of a second anti-cancer therapeutic is administered to a subject in combination with an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor, the subject having been identified as likely to respond to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor by a method as provided herein.

[0451] In some embodiments, an effective amount of a second anti-cancer therapeutic is administered to a subject in combination with an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor, the subject having been identified as having a reduced likelihood of responding to treatment with an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-xL inhibitor or a Bcl-2 inhibitor by a method as provided herein.

[0452] In some embodiments, an effective amount of a second anti-cancer therapeutic is administered to a subject in combination with an effective amount of an MDM2 inhibitor or a Bcl-2 / Bcl-xL dual inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor, the subject having been determined to have a post-treatment change in at least one biomarker comprising Noxa, ASCL1, or both, that does not meet a predetermined threshold as described herein.

[0453] In some embodiments, the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor is administered at a dose that is different from (e.g., lower than) a standard dose under the standard of care for treating the particular cancer. In certain embodiments, the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor is administered at a dose that is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than a standard dose under the standard of care for treating the particular cancer. In certain embodiments, the MDM2 inhibitor or the Bcl-2 / Bcl-xL dual inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor is administered at a dose that is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of a standard dose under the standard of care for treating the particular cancer.

[0454] In a particular embodiment, the second anti-cancer therapeutic is an agent that can be used to treat cancer. For example, the second anti-cancer therapeutic can be selected from the group consisting of an anti-neoplastic agent, an anti-angiogenic agent, a chemotherapeutic agent, and a peptide-based cancer therapeutic. In yet another embodiment, the anti-neoplastic agent is selected from the group consisting of antibiotic-type agents, alkylating agents, anti-metabolite agents, hormonal agents, immunological agents, interferon-type agents, kinase inhibitors, other agents, and combinations thereof. Notably, the second anti-cancer therapeutic can be a traditional small organic chemical molecule, or can be a macromolecule (e.g., a protein, an antibody, a peptibody, DNA, RNA) or a fragment of such a macromolecule.

[0455] In some embodiments, the additional pharmaceutically active agent is a modulator of an immune checkpoint molecule.

[0456] As used herein, an "immune checkpoint" or "immune checkpoint molecule" is a molecule that is a regulatory signal in the immune system. An immune checkpoint molecule can be a co-stimulatory checkpoint molecule (i.e., upregulates a signal), or an inhibitory checkpoint molecule (i.e., downregulates a signal). As used herein, a "co-stimulatory checkpoint molecule" is a molecule that upregulates a signal or has co-stimulatory properties in the immune system. As used herein, an "inhibitory checkpoint molecule" is a molecule that downregulates a signal or has co-inhibitory properties in the immune system.

[0457] As used herein, a "modulator of an immune checkpoint molecule" is an agent that is capable of altering the activity of an immune checkpoint in a subject. In certain embodiments, a modulator of an immune checkpoint molecule alters the function of one or more immune checkpoint molecules including PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, CD160, 2B4, TGFp, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, ICAM-1, NKG2C, SLAMF7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, and CD83. A modulator of an immune checkpoint can be an activator (e.g., an agonist) or an inhibitor (e.g., an antagonist) of the immune checkpoint. In some embodiments, a modulator of an immune checkpoint molecule is an immune checkpoint binding protein (e.g., an antibody, an antibody Fab fragment, a diabody, an antibody drug conjugate, an scFv, a fusion protein, a diabody, or a tetrabody). In some embodiments, a modulator of an immune checkpoint molecule is a monoclonal antibody or an antigen binding fragment thereof. In other embodiments, a modulator of an immune checkpoint molecule is a small molecule. In a specific embodiment, a modulator of an immune checkpoint molecule is an anti-PD1 antibody. In a specific embodiment, a modulator of an immune checkpoint molecule is an anti-PD-L1 antibody. In a specific embodiment, a modulator of an immune checkpoint molecule is an anti-CTLA-4 antibody.

[0458] In some embodiments, a modulator of an immune checkpoint molecule restores anti-tumor T cell activity or blocks T cell inhibitory cell activity. In some embodiments, a modulator of an immune checkpoint molecule is an activator of a costimulatory checkpoint molecule, and the activator of a costimulatory checkpoint molecule alters a costimulatory signal required for full T cell activation.

[0459] In some embodiments, the modulator of an immune checkpoint molecule is pembrolizumab, ipilimumab, nivolumab, atezolizumab, avelumab, durvalumab, AGEN-1884, BMS-986016, CS-1002, LAG525, MBG453, MEDI-570, OREG-103 / BY40, lirilumab, tremelimumab, nivolumab, AMP-224, AMP-514, BGB-A317, cemiplimab, JS001, PDR-001, CS-1001, PF-06801591, IBI-308, pidilizumab, SHR-1210, or TSR-042, JS003, LY3300054, MDX-1105, SHR-1316, KN035, or CK-301.

[0460] In some embodiments, the second anti-cancer therapeutic is a chemotherapeutic drug, which is a biological (macromolecule) or chemical (small molecule) compound that can be used to treat cancer. Types of chemotherapeutic drugs include, but are not limited to, histone deacetylase inhibitors (HDACI), alkylating agents, antimetabolites, alkaloids, cytotoxic / anti-cancer antibiotics, topoisomerase inhibitors, tubulin inhibitors, proteins, antibodies, kinase inhibitors, and the like. Chemotherapeutic drugs include compounds used for targeted therapy and non-targeted compounds of traditional chemotherapy.

[0461] Non-limiting examples of chemotherapeutic drugs include: erlotinib, afatinib, docetaxel, doxorubicin, 5-FU (5-fluorouracil), paribulin, gemcitabine, cisplatin, carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab, dimethylsulfoxide, temozolomide, tamoxifen, doxorubicin, rapamycin, lapatinib, hydroxycamptothecin, trimetinib. Further examples of chemotherapeutic drugs include: oxaliplatin, bortezomib, sunitinib, letrozole, imatinib, PI3K inhibitors, fulvestrant, folinic acid, lonafarnib, sorafenib, gefitinib, crizotinib, irinotecan, topotecan, valrubicin, vemurafenib, telbivinib, capecitabine, vandetanib, chlorambucil, panitumumab, cetuximab, rituximab, tositumomab, temsirolimus, everolimus, pazopanib, carmustine, thiotepa, cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, benzodopa, camptothecin, meturedopa, uredopa, methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and temozolomide; a bulatacin, bulatacinone; a bryostatin; callystatin, CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues), a cryptophycin (particularly cryptophycin 1 and cryptophycin 8); a dolastatin, duocamycin (including the synthetic analogues, KW-2189 and CB1-TM1); an eleutherobin; a pancgoidin, a spongistatin; an nitrogen mustards such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; an nitrosurea such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; an antibiotic such as the enediyne antibiotics (e.g., calicheamicin, calicheamicin gammall, calicheamicin omegall, dynemicin, including dynemicin A;Bisphosphonates, such as clophosphonates, esperamycin, and neo-carcinogen chromophores and related chromopyrin diacetylene antibiotic chromophores), aclarubicin, actinomycin, all-trans retinoic acid, aminopicrine, diazoserine, bleomycin, actinomycin C, carabicin, erythromycin, carcinogens, cymoxanil, chromopycin, actinomycin D, daunorubicin, deoxyfluorouridine, detoxin, 6-diazo-5-oxo-L-leucine, morpholino-doxorubicin, cyano-morpholino-doxorubicin, 2-Pyrrolidone-Doxorubicin, Cyclodoxacin, Epirubicin, Isorubicin, Idarubicin, Ephedrine, Mitomycin, Mycophenolic Acid, Nogamycin, Oligomycin, Pelosimycin, Porphyromycin, Purobycin, Triamcinolone Acetonide, Rodoxine, Streptomycin, Streptozotocin, Tuberculin, Ubenimex, Netostatin, Zolarubicin; Antimetabolites, such as Methotrexate; Folic acid analogs, such as Dimethylfolate, Methotrexate, Pteroxate, Trimethyltrixazone; Purine analogs, such as Fludarabine, 6-Mercaptopurine, Methotrexate, Thiomilide, thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, azathioprine, bleomycin, 6-nitrouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, fluorouridine; androgens, calotestosterone, drotaldone propionate, cyclothionol, meandrolone, testrolide; antiadrenergic agents, such as aminoglutethimide, mitotane, trilosterone; folic acid supplements, such as folinic acid; acetoglucuronolactone; aldehydephosphoramide glycosides; aminolevulinic acid; enuracil, acridine, amustine (bestr... abucil), bismuth subtilis, idatraxa, defofamine, colchicine, diazinon, efornithine, erythritol, epothilone, etogluconate; gallium nitrate; hydroxyurea; lentinan, lonidainine, maytansine alkaloids, maytansine, anthraquinone, mitoxantrone, mopiperol, nitraerine, pentostatin, methamidophos, pirarubicin, loxoantrone, podophyllic acid; 2-ethylhydrazine; procarbazine; Polyglucosan complex (JHS Natural Products, Eugene, OR), razoxane, rhizoxin, sizofiran, spiromustine, tenuazonic acid, triaziquone; 2,2',2"-trichloro-triethylamine; trichothecenes (particularly T-2 toxin, verracurin A, orysastatin A, andanguidin); uramycin, vindesine, dacarbazine, mannomustine; dibromomannitol; dibromo- sulfophthalein; pipobroman, gacytosine, arabinoside ("Ara-C"); cyclophosphamide; thio- TEPA; thioguanine; 6-mercaptopurine; methotrexate; vinblastine; etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone; pemetrexed; teniposide, edatrexate, daunomycin; aminopterin; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; DMFO, retinoids such as retinoic acid; and pharmaceutically acceptable salts or derivatives thereof.

[0462] The chemotherapeutic drug used herein is preferably selected from the group consisting of panobinostat, dactinomycin, tretinoin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytosine arabinoside, daunorubicin, docetaxel, 5-fluorouracil, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, and hydroxycamptothecine.

[0463] In some embodiments, an immune checkpoint molecule is administered as a second anti-cancer therapeutic in combination with an MDM2 inhibitor. In some embodiments, a chemotherapeutic agent is administered as a second anti-cancer therapeutic in combination with a Bcl-2 / Bcl-xL inhibitor or a Bcl-2 inhibitor or a Bcl-xL inhibitor. The second anti-cancer therapeutic can be administered simultaneously, separately, or sequentially with the MDM2 inhibitor or the Bcl-2 / Bcl-xL inhibitor or the Bcl-2 inhibitor or the Bcl-xL inhibitor.

[0464] Materials and methods

[0465] In another aspect, the disclosure further provides one or more reagents useful in any of the assays as described herein for measuring the level of the at least one biomarker provided herein. The reagents can be primers, probes, and antibodies.

[0466] In some embodiments, the disclosure provides oligonucleotide probes attached to a solid support (e.g., an array slide or chip), e.g., as described in Bowtell and Sambrook, eds., DNA Microarrays: A Molecular Cloning Manual (2003) Cold Spring Harbor Laboratory Press. The construction of such devices is well known in the art, e.g., as described in the following U.S. patents and patent publications: U.S. Patent No. 5,837,832; PCT Application WO 95 / 11995; U.S. Patent No. 5,807,522; U.S. Patent Nos. 7,157,229, 7,083,975, 6,444,175, 6,375,903, 6,315,958, 6,295,153, and 5,143,854, 2007 / 0037274, 2007 / 0140906, 2004 / 0126757, 2004 / 0110212, 2004 / 0110211, 2003 / 0143550, 2003 / 0003032, and 2002 / 0041420. Nucleic acid arrays are also reviewed in Biotechnol Annu Rev (2002) 8:85-101; Sosnowski et al. Psychiatr Genet (2002) 12(4): 181-92; Heller, Annu Rev Biomed Eng (2002) 4:129-53; Kolchinsky et al., Hum. Mutat (2002) 19(4):343-60; and McGail et al., Adv Biochem Eng Biotechnol (2002) 77:21-42.

[0467] In another aspect, the disclosure contemplates kits for use in the above-described methods. Typically, a kit comprises reagents useful in any of the methods provided herein in a carrier or compartmentalized container. The carrier can be a container or support in the form of, e.g., a bag, a box, a tube, a rack, and optionally compartmentalized.

[0468] The kit contains one or more of the primers, probes, and / or antibodies or microarrays provided herein. The primers, probes, and / or antibodies may or may not be detectably labeled. In some embodiments, the kit may further contain other reagents for performing the methods described herein. In such applications, the kit may contain any or all of the following: suitable buffers, reagents for isolating nucleic acids, reagents for amplifying nucleic acids (e.g., polymerase, dNTP mixture), reagents for hybridizing nucleic acids, reagents for sequencing nucleic acids, reagents for quantifying nucleic acids (e.g., intercalating agents, detection probes), reagents for isolating proteins, and reagents for detecting proteins (e.g., antibodies).

[0469] In some embodiments, the kit may further include a standard negative control and / or a standard positive control.

[0470] Additionally, the kit may include instruction materials containing guidance (i.e., protocols) for implementing the methods provided herein. While instruction materials typically include written or printed materials, they are not limited to this.

[0471] In some embodiments, the kit may further comprise a computer program product stored on a computer-readable medium. When executed by a computer, the computer program product performs the following steps: comparing the level of the at least one biomarker with a corresponding reference level for the at least one biomarker to determine the difference from the reference level. Any medium capable of storing and transmitting such computer-executable instructions to an end user is considered in this description. Such media include, but are not limited to, electronic storage media (e.g., disks, magnetic tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. Such media may include the URL of an Internet website providing such guidance material.

[0472] Computer programs can also be encoded and transmitted using carrier signals, which are adapted for transmission over wired, optical, and / or wireless networks conforming to various protocols including the Internet. Therefore, such program-encoded data signals can be used to create computer-readable media according to embodiments of the invention. Computer-readable media with encoded program code can be packaged with compatible devices or provided separately from other devices (e.g., downloaded via the Internet). Any such computer-readable medium can reside on or within a single computer product (e.g., a hard disk drive, CD, or an entire computer system) and can exist on or within different computer products within a system or network.

[0473] The following examples are provided to better illustrate the claimed invention and are not to be construed as limiting the scope of the application. All specific compositions, materials and methods described herein (all or in part) fall within the scope of the application. These specific compositions, materials and methods are not intended to limit the scope of the application, but merely to illustrate particular embodiments falling within the scope of the application. Those skilled in the art can devise equally effective compositions, materials and methods without the use of the inventive ability and without departing from the scope of the application. It is understood that many variations can be made in the procedures described herein while still remaining within the scope of the application. It is the intention of the inventors that such variations are included within the scope of the application.

[0474] Example

[0475] Example 1

[0476] This example shows the correlation between Noxa expression and compound efficacy of Compound A15 in gastric cancer PDX models and esophageal cancer PDX models.

[0477] Results

[0478] Twelve gastric cancer PDX models and four esophageal cancer PDX models were selected for efficacy studies. Mice carrying patient-derived xenografts (PDX) were randomly assigned to 2 different study groups according to their tumor volume. The mean tumor volume at randomization was 100-200 mm3. 3 On Day 1, randomization was performed based on a "matched distribution" randomization method (Study Director™ software). Mice were administered vehicle or Compound A15 for 21 days (intravenous, twice a week).

[0479] Tumor volume was measured in two dimensions using calipers twice a week and volume was expressed in mm3using the following formula: Tumor volume (mm3) = 0.5 a x b2(where a and b are the long and short diameters of the tumor, respectively). Relative tumor volume (RTV) was calculated using the following formula: RTV = Vt / V1, where V1and Vtare the average tumor volume on the first day of treatment (Day 1) and the average tumor volume at a certain time point. The percent tumor growth inhibition (%T / C) was calculated as the average RTV of treated tumors (T) divided by the average RTV of control tumors (C) x 100%. The percent T / C value is an indicator of anti-tumor effect: a T / C value < 42% is considered by the NCI to be of significant anti-tumor activity. A T / C value < 10% is considered to indicate highly significant anti-tumor activity, and if toxicity and certain other requirements are met, this value is the level used by the NCI to justify clinical trials (referred to as DN-2 level activity). Greater than 20% body weight loss (group mean) or greater than 20% drug- induced mortality is considered to indicate a severely toxic dose. All data were analyzed in SPSS (Statistical Product and Service Solutions) version 18.0 (IBM Corporation, Armonk, New York, USA). Prism version 6 (GraphPad Software, Inc., San Diego, California) was used for image display. 2 (Decision Correlation) shows the percent change in y explained by all x variables ranging from 0 to 1. 1 indicates the strongest possible agreement, i.e., perfect correlation.

[0480] Figure 1A

[0481] In efficacy trials in gastric cancer PDX models, 12 PDX models (Bcl-xL hi , Bcl2 hi , Mcl1 低-正常 ) were employed and treated with 100 mg / kg of Compound A15 (intravenous, twice a week) for 21 days (total of 7 doses). The T / C values for these models varied between 17-117 Figure 2A and 1B (T / C value, average tumor volume in the treatment group compared to the vehicle control group). RNA sequencing has been performed for these models and the RNA sequencing data is expressed as Log2(FPKM), where FPKM refers to fragments per kilobase of transcript per million mapped reads. Based on the RNA sequencing data, the expression level of Noxa in these models ranged from 1.0354 to 7.5223 (expressed as Log2(FPKM)). The T / C values and the corresponding RNA expression were analyzed by linear regression and the R square was calculated as 0.8661, which indicates that 86.61% of the variation in T / C can be explained by the RNA expression of Noxa (p<0.0001).

[0482] From a set of independent experiments, although an esophageal cancer PDX model (n=4) was employed, a similar correlation between T / C and Noxa expression was observed Figure 3E and 2B ). R 2 The value was recorded as 0.7732 (where P<0.05).

[0483] The correlation of T / C with RNA expression of Bcl-2, Bcl-xL, Mcl-1, BIM and PUMA were also tested respectively, and the R square is shown in Figure 3: 0.2574 (Bcl-2, Figure 3C ); 0.0207 (Bcl-xL, Figure 3D ); 0.1737 (Mcl-1, Figure 3B ); 0.3382 (BIM, Figure 3A ) and 0.0517 (PUMA, Figure 4A ).

[0484] In summary, the RNA expression level of Noxa was highly correlated with the extent of tumor regression in gastric and esophageal cancer PDX models treated with the Bcl-2 / Bcl-xL dual inhibitor Compound A15. This correlation was much higher than any other tested biomarker: BIM, Mcl-2, Bcl-2, PUMA and Bcl-xL.

[0485] Example 2

[0486] This study shows that Compound B4 has a greater dependence on Bcl-xL than Bcl-2 in terms of functioning as a Bcl-2 / Bcl-xL dual inhibitor.

[0487] ABT-737 (IUPAC name: 4-{4-[(4'-chloro-2-biphenyl)methyl]-1-piperazinyl}-N-[(4-{[(2R)-4- (dimethylamino)-1-(phenylsulfanyl)-2-butyl]amino}-3-nitrophenyl)sulfonyl]benzamide) is one of the earliest BH3 mimetics developed by Abbott Laboratories (now Abbvie). It acts as a dual BCl-2 / Bcl-xL inhibitor and promotes tumor cell apoptosis. This study compares the binding of Bcl-2 and BCl-xL between Compound B4 or ABT-737.

[0488] The diffuse large B-cell lymphoma line Toledo and the acute lymphoblastic leukemia line RS4;11 were chosen for this study. Cells were treated with compound B4 or ABT-737 at the indicated concentrations (0.025 uM, 0.04 uM and 0.06 uM, respectively) for 24 hours and harvested for complex analysis. Both Bcl-2:BIM and Bcl-xL:BIM complexes were analyzed using the MSD high advanced ELISA method.

[0489] The results show that, in the Toledo ( Figure 4B ) and RS4;11 ( Figure 5A ) cell lines, compound B4 is more efficient in disrupting Bcl-xL:BIM than Bcl-2:BIM. In particular, compound B4 more strongly disrupts the Bcl-xL:BIM complex than ABT-737.

[0490] These cell line results are consistent with a related gastric / oesophageal cancer PDX trial using compound A15 (data not shown), which confirms that both compound A15 (in PDX) and compound B4 (in cell lines) target Bcl-xL more than Bcl-2 complex and are different from the reference compound ABT-737.

[0491] Example 3.

[0492] This study shows the anti-tumor activity of compound A15 in ASCL-1-high and Noxa-high SCLC PDX models.

[0493] SCLC is a heterogeneous disease with a very high mutational rate, but lacking identified driver oncogenes for molecularly targeted therapies. It is currently treated as a single disease entity in the clinic. Recent studies have improved our understanding of SCLC and characterized different subtypes based on the mutually exclusive expression of ASCL1, NEUROD1, POU2F3 or YAP1 (Rudin et al., 2019). ASCL1-expressing SCLC is the most common subtype, accounting for 70% of cases.

[0494] Noxa expression levels were obtained from 58 commercially available PDX models of SCLC (PDX models are available from Crownbio). Noxa expression levels of these 58 PDX models have been determined by RNA sequencing and are plotted in Figure 5A , where each dot represents one PDX model. Based on the RNA sequencing data, the expression levels of Noxa in these models range from below 2 to above 6 (expressed by Log2(FPKM)). In Figure 5A , the average levels of PMAIP1 (the encoded protein Noxa) are shown as straight lines with error bars on the graph.

[0495] The antitumor activity of compound A15 was tested in 13 of the 58 PDX models. Compound A15 monotherapy was administered intravenously at 50 mg / kg twice weekly, and a tumor inhibition rate of 60% was set as the cutoff value for tumor regression.

[0496] The results showed that compound A15 achieved tumor regression in 3 out of the 13 models tested in total. Figure 5A (represented as a hollow circle), but this was not achieved in the other 10 test models. Surprisingly, all three responsive models had above-average Noxa expression (e.g., Figure 5A In the mean, log2 FPKM was higher than 6, and ASCL1 was also expressed. In contrast, although NEUROD1, POU2F3, or YAP1 were expressed respectively, the other 10 non-responsive models ( Figure 5B (Represented as semi-solid circles) indicates models that do not show ASCL1 expression, or show ASCL1 expression but have below-average Noxa expression. The 45 models for which the antitumor activity of compound A15 was not tested in this study are represented as gray dots.

[0497] The results showed that SCLC with high Noxa expression in the ASCL1 subtype was highly sensitive to monotherapy with compound A15.

[0498] Further statistical analysis using linear regression showed that, across all tested models (n=13), the coefficient of determination (R²) between the antitumor activity of compound A15 and Noxa expression was 0.138. Figure 5C When ASCL-1 expression is taken into account (test model 5 / 13), the coefficient of determination (R²) between the antitumor activity of compound A15 and Noxa expression can increase to 0.5067. ​ Our results indicate that using ASCL1 and Noxa as co-biomarkers will identify SCLC patients most likely to respond to treatment with compound A15.

[0499] Example 4

[0500] The aim of this study was to evaluate the antitumor activity and Noxa level of the MDM2 inhibitor compound C in a gastric cancer PDX model.

[0501] The experimental methods and procedures are similar to those described in Example 1. The mediator or compound C was administered to the mice.

[0502] In this efficacy trial, a gastric cancer PDX model was used and treated with 100 mg / kg Compound C orally for 14 days (total of 7 doses) or with 10-50 mg / kg Compound C orally QOD. Expression levels of Noxa were determined by RNA sequencing. High expression levels of Noxa relative to reference levels were expected to correlate with the degree of tumor regression in Compound C treated gastric PDX models.

[0503] While the present disclosure has been particularly shown and described with reference to particular embodiments, which include preferred embodiments, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the disclosure as disclosed herein. SEQUENCE LISTING <110> Suzhou Yasheng Pharmaceutical Co., Ltd. Yasheng Pharmaceutical Group (Hong Kong) Limited <120> Methods and compositions for predicting anticancer efficacy of compounds targeting the apoptosis pathway <130> 1252-004-02 PCT <160> 22 <170> SIPO SequenceListing 1.0 <210> 1 <211> 239 <212> PRT <213> Homo sapiens <400> 1 Met Ala His Ala Gly Arg Thr Gly Tyr Asp Asn Arg Glu Ile Val Met 1 5 10 15 Lys Tyr Ile His Tyr Lys Leu Ser Gin Arg Gly Tyr Glu Trp Asp Ala 20 25 30 Gly Asp Val Gly Ala Ala Pro Pro Gly Ala Ala Pro Ala Pro Gly Ile 35 40 45 Phe Ser Ser Gin Pro Gly His Thr Pro His Pro Ala Ala Ser Arg Asp 50 55 60 Pro Val Ala Arg Thr Ser Pro Leu Gin Thr Pro Ala Ala Pro Gly Ala 65 70 75 80 Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His Leu Thr 85 90 95 Leu Arg Gin Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Arg Asp Phe 100 105 110 Ala Glu Met Ser Ser Gin Leu His Leu Thr Pro Phe Thr Ala Arg Gly 115 120 125 Arg Phe Ala Thr Val Val Glu Glu Leu Phe Arg Asp Gly Val Asn Trp 130 135 140 Gly Arg Ile Val Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu 145 150 155 160 Ser Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile Ala Leu Trp 165 170 175 Met Thr Glu Tyr Leu Asn Arg His Leu His Thr Trp Ile Gin Asp Asn 180 185 190 Gly Gly Trp Asp Ala Phe Val Glu Leu Tyr Gly Pro Ser Met Arg Pro 195 200 205 Leu Phe Asp Phe Ser Trp Leu Ser Leu Lys Thr Leu Leu Ser Leu Ala 210 215 220 Leu Val Gly Ala Cys lie Thr Leu Gly Ala Tyr Leu Gly His Lys 225 230 235 <210> 2 <211> 720 <212> DNA <213> Homo sapiens() <400> 2 atggcgcacg ctgggagaac agggtacgat aaccgggaga tagtgatgaa gtacatccat 60 tataagctgt cgcagagggg ctacgagtgg gatgcgggag atgtgggcgc cgcgcccccg 120 ggggccgccc ccgcaccggg catcttctcc tcccagcccg ggcacacgcc ccatccagcc 180 gcatcccggg acccggtcgc caggacctcg ccgctgcaga ccccggctgc ccccggcgcc 240 gccgcggggc ctgcgctcag cccggtgcca cctgtggtcc acctgaccct ccgccaggcc 300 ggcgacgact tctcccgccg ctaccgccgc gacttcgccg agatgtccag ccagctgcac 360 ctgacgccct tcaccgcgcg gggacgcttt gccacggtgg tggaggagct cttcagggac 420 ggggtgaact gggggaggat tgtggccttc tttgagttcg gtggggtcat gtgtgtggag 480 agcgtcaacc gggagatgtc gcccctggtg gacaacatcg ccctgtggat gactgagtac 540 ctgaaccggc acctgcacac ctggatccag gataacggag gctgggatgc ctttgtggaa 600 ctgtacggcc ccagcatgcg gcctctgttt gatttctcct ggctgtctct gaagactctg 660 ctcagtttgg ccctggtggg agcttgcatc accctgggtg cctatctggg ccacaagtga 720 <210> 3 <211> 233 <212> PRT <213> Homo sapiens () <400> 3 Met Ser Gin Ser Asn Arg Glu Leu Val Val Asp Phe Leu Ser Tyr Lys 1 5 10 15 Leu Ser Gin Lys Gly Tyr Ser Trp Ser Gin Phe Ser Asp Val Glu Glu 20 25 30 Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu Met Glu Thr Pro 35 40 45 Ser Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala Asp Ser Pro Ala 50 55 60 Val Asn Gly Ala Thr Gly His Ser Ser Ser Leu Asp Ala Arg Glu Val 65 70 75 80 Ile Pro Met Ala Ala Val Lys Gin Ala Leu Arg Glu Ala Gly Asp Glu 85 90 95 Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu Thr Ser Gin Leu 100 105 110 His lie Thr Pro Gly Thr Ala Tyr Gin Ser Phe Glu Gin Val Val Asn 115 120 125 Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg lie Val Ala Phe Phe 130 135 140 Ser Phe Gly Gly Ala Leu Cys Val Glu Ser Val Asp Lys Glu Met Gin 145 150 155 160 Val Leu Val Ser Arg lie Ala Ala Trp Met Ala Thr Tyr Leu Asn Asp 165 170 175 His Leu Glu Pro Trp lie Gin Glu Asn Gly Gly Trp Asp Thr Phe Val 180 185 190 Glu Leu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg Lys Gly Gin Glu 195 200 205 Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val Ala Gly Val Val 210 215 220 Leu Leu Gly Ser Leu Phe Ser Arg Lys 225 230 <210> 4 <211> 702 <212> DNA <213> Homo sapiens <400> 4 atgtctcaga gcaaccggga gctggtggtt gactttctct cctacaagct ttcccagaaa 60 ggatacagct ggagtcagtt tagtgatgtg gaagagaaca ggactgaggc cccagaaggg 120 actgaatcgg agatggagac ccccagtgcc atcaatggca acccatcctg gcacctggca 180 gacagccccg cggtgaatgg agccactggc cacagcagca gtttggatgc ccgggaggtg 240 atccccatgg cagcagtaaa gcaagcgctg agggaggcag gcgacgagtt tgaactgcgg 300 taccggcggg cattcagtga cctgacatcc cagctccaca tcaccccagg gacagcatat 360 cagagctttg aacaggtagt gaatgaactc ttccgggatg gggtaaactg gggtcgcatt 420 gtggcctttt tctccttcgg cggggcactg tgcgtggaaa gcgtagacaa ggagatgcag 480 gtattggtga gtcggatcgc agcttggatg gccacttacc tgaatgacca cctagagcct 540 tggatccagg agaacggcgg ctgggatact tttgtggaac tctatgggaa caatgcagca 600 gccgagagcc gaaagggcca ggaacgcttc aaccgctggt tcctgacggg catgactgtg 660 gccggcgtgg ttctgctggg ctcactcttc agtcggaaat ga 702 <210> 5 <211> 198 <212> PRT <213> H. sapiens <400> 5 Met Ala Lys Gin Pro Ser Asp Val Ser Ser Glu Cys Asp Arg Glu Gly 1 5 10 15 Arg Gin Leu Gin Pro Ala Glu Arg Pro Pro Gin Leu Arg Pro Gly Ala 20 25 30 Pro Thr Ser Leu Gin Thr Glu Pro Gin Gly Asn Pro Glu Gly Asn His 35 40 45 Gly Gly Glu Gly Asp Ser Cys Pro His Gly Ser Pro Gin Gly Pro Leu 50 55 60 Ala Pro Pro Ala Ser Pro Gly Pro Phe Ala Thr Arg Ser Pro Leu Phe 65 70 75 80 Ile Phe Met Arg Arg Ser Ser Leu Leu Ser Arg Ser Ser Ser Gly Tyr 85 90 95 Phe Ser Phe Asp Thr Asp Arg Ser Pro Ala Pro Met Ser Cys Asp Lys 100 105 110 Ser Thr Gin Thr Pro Ser Pro Pro Cys Gin Ala Phe Asn His Tyr Leu 115 120 125 Ser Ala Met Ala Ser Met Arg Gin Ala Glu Pro Ala Asp Met Arg Pro 130 135 140 Glu lie Trp lie Ala Gin Glu Leu Arg Arg lie Gly Asp Glu Phe Asn 145 150 155 160 Ala Tyr Tyr Ala Arg Arg Val Phe Leu Asn Asn Tyr Gin Ala Ala Glu 165 170 175 Asp His Pro Arg Met Val lie Leu Arg Leu Leu Arg Tyr lie Val Arg 180 185 190 Leu Val Trp Arg Met His 195 <210> 6 <211> 597 <212> DNA <213> Homo sapiens() <400> 6 atggcaaagc aaccttctga tgtaagttct gagtgtgacc gagaaggtag acaattgcag 60 cctgcggaga ggcctcccca gctcagacct ggggccccta cctccctaca gacagagcca 120 caaggtaatc ctgaaggcaa tcacggaggt gaaggggaca gctgccccca cggcagccct 180 cagggcccgc tggccccacc tgccagccct ggcccttttg ctaccagatc cccgcttttc 240 atctttatga gaagatcctc cctgctgtct cgatcctcca gtgggtattt ctcttttgac 300 acagacagga gcccagcacc catgagttgt gacaaatcaa cacaaacccc aagtcctcct 360 TGGCTGGAGA AGAAGAAGAA GAAGAAGAAG AAGAAGAAGA AGAAGAAGAA 60 GATATGCGCC CAGAGATATG GATCGCCCAA GAGTTGCGGC GTATTGGAGA CGAGTTT AAC 480 GCTTACTATG CAAGGAGGGT ATTTTTGAAT AATTACCAAG CAGCCGAAGA CCACCCACGA 540 ATGGTTATCT TACGACTGTT ACGTTACATT GTCCGCCTGG TGTGGAGAAT GCATTGA 597 <210> 7 <211> 54 <212> PRT <213> Homo sapiens <400> 7 Met Pro Gly Lys Lys Ala Arg Lys Asn Ala Gln Pro Ser Pro Ala Arg 1 5 10 15 Ala Pro Ala Glu Leu Glu Val Glu Cys Ala Thr Gln Leu Arg Arg Phe 20 25 30 Gly Asp Lys Leu Asn Phe Arg Gln Lys Leu Leu Asn Leu Ile Ser Lys 35 40 45 Leu Phe Cys Ser Gly Thr 50 <210> 8 <211> 165 <212> DNA <213> Homo sapiens <400> 8 ATGCCTGGGA AGAAGGCACG CAAGAACGCT CAACCCTGCC CCACCGCCCT CCAGCAGAG 60 ctggaagtcg agtgtgctac tcaactcagg agatttggag acaaactgaa cttccggcag 120 aaacttctga atctgatatc caaactcttc tgctcaggaa cctga 165 <210> 9 <211> 261 <212> PRT <213> Homo sapiens() <400> 9 Met Lys Phe Gly Met Gly Ser Ala Gln Ala Cys Pro Cys Gln Val Pro 1 5 10 15 Arg Ala Ala Ser Thr Thr Trp Val Pro Cys Gln Ile Cys Gly Pro Arg 20 25 30 [[ID=Z2]]Glu Arg His Gly Pro Arg Thr Pro Gly Gly Gln Leu Pro Gly Ala Arg 35 40 45 Arg Gly Pro Gly Pro Arg Arg Pro Ala Pro Leu Pro Ala Arg Pro Pro 50 55 60 Gly Ala Leu Gly Ser Val Leu Arg Pro Leu Arg Ala Arg Pro Gly Cys 65 70 75 80 Arg Pro Arg Arg Pro His Pro Ala Ala Arg Cys Leu Pro Leu Arg Pro 85 90 95 His Arg Pro Thr Arg Arg His Arg Arg Pro Gly Gly Phe Pro Leu Ala 100 105 110 Trp Gly Ser Pro Gin Pro Ala Pro Arg Pro Ala Pro Gly Arg Ser Ser 115 120 125 Ala Leu Ala Leu Ala Gly Gly Ala Ala Pro Gly Val Ala Arg Ala Gin 130 135 140 Arg Pro Gly Gly Ser Gly Gly Arg Ser His Pro Gly Gly Pro Gly Ser 145 150 155 160 Pro Arg Gly Gly Gly Thr Val Gly Pro Gly Asp Arg Gly Pro Ala Ala 165 170 175 Ala Asp Gly Gly Arg Pro Gin Arg Thr Val Arg Ala Ala Glu Thr Arg 180 185 190 Gly Ala Ala Ala Ala Pro Pro Leu Thr Leu Glu Gly Pro Val Gin Ser 195 200 205 His His Gly Thr Pro Ala Leu Thr Gin Gly Pro Gin Ser Pro Arg Asp 210 215 220 Gly Ala Gin Leu Gly Ala Cys Thr Arg Pro Val Asp Val Arg Asp Ser 225 230 235 240 Gly Gly Arg Pro Leu Pro Pro Pro Asp Thr Leu Ala Ser Ala Gly Asp 245 250 255 Phe Leu Cys Thr Met 260 <210> 10 <211> 786 <212> DNA <213> Homo sapiens <400> 10 atgaaatttg gcatggggtc tgcccaggca tgtccatgcc aggtgcccag ggctgcttcc 60 acgacgtggg tcccctgcca gatttgtggc cccagggagc gccatggccc gcgcacgcca 120 ggagggcagc tccccggagc ccgtagaggg cctggcccgc gacggcccgc gccccttccc 180 gctcggccgc ctggtgccct cggcagtgtc ctgcggcctc tgcgagcccg gcctggctgc 240 cgcccccgcc gcccccaccc tgctgcccgc tgcctacctc tgcgccccca ccgccccacc 300 cgccgtcacc gccgccctgg ggggttcccg ctggcctggg ggtccccgca gccggccccg 360 aggcccgcgc ccggacggtc ctcagccctc gctctcgctg gcggagcagc acctggagtc 420 gcccgtgccc agcgccccgg gggctctggc gggcggtccc acccaggcgg ccccgggagt 480 ccgcggggag gaggaacagt gggcccggga gatcggggcc cagctgcggc ggatggcgga 540 cgacctcaac gcacagtacg agcggcggag acaagaggag cagcagcggc accgcccctc 600 accctggagg gtcctgtaca atctcatcat gggactcctg cccttaccca ggggccacag 660 agcccccgag atggagccca attaggtgcc tgcacccgcc cggtggacgt cagggactcg 720 gggggcaggc ccctcccacc tcctgacacc ctggccagcg cgggggactt tctctgcacc 780 atgtag 786 <210> 11 <211> 197 <212> PRT <213> Homo sapiens <400> 11 Met Phe Gly Leu Lys Arg Asn Ala Val Ile Gly Leu Asn Leu Tyr Cys 1 5 10 15 Gly Gly Ala Gly Leu Gly Ala Gly Ser Gly Gly Ala Thr Arg Pro Gly 20 25 30 Gly Arg Leu Leu Ala Thr Gly Ala Lys Asp Thr Lys Pro Met Gly Arg 35 40 45 Ser Gly Ala Thr Ser Arg Lys Ala Leu Glu Thr Leu Arg Arg Val Gly 50 55 60 Asp Gly Val Gln Arg Asn His Glu Thr Ala Phe Gln Gly Met Leu Arg 65 70 75 80 Lys Leu Asp Ile Lys Asn Glu Asp Asp Val Lys Ser Leu Ser Arg Val 85 90 95 Met Ile His Val Phe Ser Asp Gly Val Thr Asn Trp Gly Arg Ile Val 100 105 110 Thr Leu Ile Ser Phe Gly Ala Phe Val Ala Lys His Leu Lys Thr Ile 115 120 125 Asn Gln Glu Ser Cys Ile Glu Pro Leu Ala Glu Ser Ile Thr Asp Val 130 135 140 Leu Val Arg Thr Lys Arg Asp Trp Leu Val Lys Gln Arg Gly Trp Asp 145 150 155 160 Gly Phe Val Glu Phe Phe His Val Glu Asp Leu Glu Gly Gly Ile Arg 165 170 175 Asn Val Leu Leu Ala Phe Ala Gly Val Ala Gly Val Gly Ala Gly Leu 180 185 190 Ala Tyr Leu Ile Arg 195 <210> 12 <211> 594 <212> DNA <213> Homo sapiens() <400> 12 atgtttggcc tcaaaagaaa cgcggtaatc ggactcaacc tctactgtgg gggggccggc 60 ttgggggccg gcagcggcgg cgccacccgc ccgggagggc gacttttggc caccggcgcc 120 aaggacacaa agccaatggg caggtctggg gccaccagca ggaaggcgct ggagacctta 180 cgacgggttg gggatggcgt gcagcgcaac cacgagacgg ccttccaagg catgcttcgg 240 aaactggaca tcaaaaacga agacgatgtg aaatcgttgt ctcgagtgat gatccatgtt 300 ttcagcgacg gcgtaacaaa ctggggcagg attgtgactc tcatttcttt tggtgccttt 360 gtggctaaac acttgaagac cataaaccaa gaaagctgca tcgaaccatt agcagaaagt 420 atcacagacg ttctcgtaag gacaaaacgg gactggctag ttaaacaaag aggctgggat 480 gggtttgtgg agttcttcca tgtagaggac ctagaaggtg gcatcaggaa tgtgctgctg 540 gcttttgcag gtgttgctgg agtaggagct ggtttggcat atctaataag atag 594 <210> 13 <211> 205 <212> PRT <213> Homo sapiens() <400> 13 Met Ala His Ala Gly Arg Thr Gly Tyr Asp Asn Arg Glu Ile Val Met 1 5 10 15 Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg Gly Tyr Glu Trp Asp Ala 20 25 30 Gly Asp Val Gly Ala Ala Pro Pro Gly Ala Ala Pro Ala Pro Gly Ile 35 40 45 Phe Ser Ser Gln Pro Gly His Thr Pro His Pro Ala Ala Ser Arg Asp 50 55 60 Pro Val Ala Arg Thr Ser Pro Leu Gin Thr Pro Ala Ala Pro Gly Ala 65 70 75 80 Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His Leu Thr 85 90 95 Leu Arg Gin Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Arg Asp Phe 100 105 110 Ala Glu Met Ser Ser Gin Leu His Leu Thr Pro Phe Thr Ala Arg Gly 115 120 125 Arg Phe Ala Thr Val Val Glu Glu Leu Phe Arg Asp Gly Val Asn Trp 130 135 140 Gly Arg Ile Val Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu 145 150 155 160 Ser Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile Ala Leu Trp 165 170 175 Met Thr Glu Tyr Leu Asn Arg His Leu His Thr Trp Ile Gin Asp Asn 180 185 190 Gly Gly Trp Val Gly Ala Leu Gly Asp Val Ser Leu Gly 195 200 205 <210> 14 <211> 618 <212> DNA <213> Homo sapiens <400> 14 atggcgcacg ctgggagaac agggtacgat aaccgggaga tagtgatgaa gtacatccat 60 tataagctgt cgcagagggg ctacgagtgg gatgcgggag atgtgggcgc cgcgcccccg 120 ggggccgccc ccgcaccggg catcttctcc tcccagcccg ggcacacgcc ccatccagcc 180 gcatcccggg acccggtcgc caggacctcg ccgctgcaga ccccggctgc ccccggcgcc 240 gccgcggggc ctgcgctcag cccggtgcca cctgtggtcc acctgaccct ccgccaggcc 300 ggcgacgact tctcccgccg ctaccgccgc gacttcgccg agatgtccag ccagctgcac 360 ctgacgccct tcaccgcgcg gggacgcttt gccacggtgg tggaggagct cttcagggac 420 ggggtgaact gggggaggat tgtggccttc tttgagttcg gtggggtcat gtgtgtggag 480 agcgtcaacc gggagatgtc gcccctggtg gacaacatcg ccctgtggat gactgagtac 540 ctgaaccggc acctgcacac ctggatccag gataacggag gctgggtagg tgcacttggt 600 gatgtgagtc tgggctga 618 <210> 15 <211> 138 <212> PRT <213> Homo sapiens <400> 15 Met Ala Lys Gin Pro Ser Asp Val Ser Ser Glu Cys Asp Arg Glu Gly 1 5 10 15 Arg Gin Leu Gin Pro Ala Glu Arg Pro Pro Gin Leu Arg Pro Gly Ala 20 25 30 Pro Thr Ser Leu Gin Thr Glu Pro Gin Asp Arg Ser Pro Ala Pro Met 35 40 45 Ser Cys Asp Lys Ser Thr Gin Thr Pro Ser Pro Pro Cys Gin Ala Phe 50 55 60 Asn His Tyr Leu Ser Ala Met Ala Ser Met Arg Gin Ala Glu Pro Ala 65 70 75 80 Asp Met Arg Pro Glu He Trp He Ala Gin Glu Leu Arg Arg He Gly 85 90 95 Asp Glu Phe Asn Ala Tyr Tyr Ala Arg Arg Val Phe Leu Asn Asn Tyr 100 105 110 Gln Ala Ala Glu Asp His Pro Arg Met Val He Leu Arg Leu Leu Arg 115 120 125 Tyr He Val Arg Leu Val Trp Arg Met His 130 135 <210> 16 <211> 417 <212> DNA <213> Homo sapiens <400> 16 atggcaaagc aaccttctga tgtaagttct gagtgtgacc gagaaggtag acaattgcag 60 cctgcggaga ggcctcccca gctcagacct ggggccccta cctccctaca gacagagcca 120 caagacagga gcccagcacc catgagttgt gacaaatcaa cacaaacccc aagtcctcct 180 tgccaggcct tcaaccacta tctcagtgca atggcttcca tgaggcaggc tgaacctgca 240 gatatgcgcc cagagatatg gatcgcccaa gagttgcggc gtattggaga cgagtttaac 300 gcttactatg caaggagggt atttttgaat aattaccaag cagccgaaga ccacccacga 360 atggttatct tacgactgtt acgttacatt gtccgcctgg tgtggagaat gcattga 417 <210> 17 <211> 350 <212> PRT <213> Homo sapiens <400> 17 Met Phe Gly Leu Lys Arg Asn Ala Val Ile Gly Leu Asn Leu Tyr Cys 1 5 10 15 Gly Gly Ala Gly Leu Gly Ala Gly Ser Gly Gly Ala Thr Arg Pro Gly 20 25 30 Gly Arg Leu Leu Ala Thr Glu Lys Glu Ala Ser Ala Arg Arg Glu Ile 35 40 45 Gly Gly Gly Glu Ala Gly Ala Val lie Gly Gly Ser Ala Gly Ala Ser 50 55 60 Pro Pro Ser Thr Leu Thr Pro Asp Ser Arg Arg Val Ala Arg Pro Pro 65 70 75 80 Pro lie Gly Ala Glu Val Pro Asp Val Thr Ala Thr Pro Ala Arg Leu 85 90 95 Leu Phe Phe Ala Pro Thr Arg Arg Ala Ala Pro Leu Glu Glu Met Glu 100 105 110 Ala Pro Ala Ala Asp Ala lie Met Ser Pro Glu Glu Glu Leu Asp Gly 115 120 125 Tyr Glu Pro Glu Pro Leu Gly Lys Arg Pro Ala Val Leu Pro Leu Leu 130 135 140 Glu Leu Val Gly Glu Ser Gly Asn Asn Thr Ser Thr Asp Gly Ser Leu 145 150 155 160 Pro Ser Thr Pro Pro Pro Ala Glu Glu Glu Glu Asp Glu Leu Tyr Arg 165 170 175 Gln Ser Leu Glu lie lie Ser Arg Tyr Leu Arg Glu Gin Ala Thr Gly 180 185 190 Ala Lys Asp Thr Lys Pro Met Gly Arg Ser Gly Ala Thr Ser Arg Lys 195 200 205 Ala Leu Glu Thr Leu Arg Arg Val Gly Asp Gly Val Gin Arg Asn His 210 215 220 Glu Thr Ala Phe Gin Gly Met Leu Arg Lys Leu Asp He Lys Asn Glu 225 230 235 240 Asp Asp Val Lys Ser Leu Ser Arg Val Met He His Val Phe Ser Asp 245 250 255 Gly Val Thr Asn Trp Gly Arg He Val Thr Leu He Ser Phe Gly Ala 260 265 270 Phe Val Ala Lys His Leu Lys Thr He Asn Gin Glu Ser Cys He Glu 275 280 285 Pro Leu Ala Glu Ser He Thr Asp Val Leu Val Arg Thr Lys Arg Asp 290 295 300 Trp Leu Val Lys Gin Arg Gly Trp Asp Gly Phe Val Glu Phe Phe His 305 310 315 320 Val Glu Asp Leu Glu Gly Gly He Arg Asn Val Leu Leu Ala Phe Ala 325 330 335 Gly Val Ala Gly Val Gly Ala Gly Leu Ala Tyr Leu He Arg 340 345 350 <210> 18 <211> 1053 <212> DNA <213> Homo sapiens <400> 18​​​​​​​​​ atgtttggcc tcaaaagaaa cgcggtaatc ggactcaacc tctactgtgg gggggccggc 60 ttgggggccg gcagcggcgg cgccacccgc ccgggagggc gacttttggc tacggagaag 120 gaggcctcgg cccggcgaga gataggggga ggggaggccg gcgcggtgat tggcggaagc 180 gccggcgcaa gccccccgtc caccctcacg ccagactccc ggagggtcgc gcggccgccg 240 cccattggcg ccgaggtccc cgacgtcacc gcgacccccg cgaggctgct tttcttcgcg 300 cccacccgcc gcgcggcgcc gcttgaggag atggaagccc cggccgctga cgccatcatg 360 tcgcccgaag aggagctgga cgggtacgag ccggagcctc tcgggaagcg gccggctgtc 420 ctgccgctgc tggagttggt cggggaatct ggtaataaca ccagtacgga cgggtcacta 480 ccctcgacgc cgccgccagc agaggaggag gaggacgagt tgtaccggca gtcgctggag 540 attatctctc ggtaccttcg ggagcaggcc accggcgcca aggacacaaa gccaatgggc 600 aggtctgggg ccaccagcag gaaggcgctg gagaccttac gacgggttgg ggatggcgtg 660 cagcgcaacc acgagacggc cttccaaggc atgcttcgga aactggacat caaaaacgaa 720 gacgatgtga aatcgttgtc tcgagtgatg atccatgttt tcagcgacgg cgtaacaaac 780 tggggcagga ttgtgactct catttctttt ggtgcctttg tggctaaaca cttgaagacc 840 ataaaccaag aaagctgcat cgaaccatta gcagaaagta tcacagacgt tctcgtaagg 900 acaaaacggg actggctagt taaacaaaga ggctgggatg ggtttgtgga gttcttccat 960 gtagaggacc tagaaggtgg catcaggaat gtgctgctgg cttttgcagg tgttgctgga 1020 gtaggagctg gtttggcata tctaataaga tag 1053 <210> 19 <211> 271<00018,64><212> PRT <213> Homo sapiens() <400> 19 Met Phe Gly Leu Lys Arg Asn Ala Val Ile Gly Leu Asn Leu Tyr Cys 1 5 10 15 Gly Gly Ala Gly Leu Gly Ala Gly Ser Gly Gly Ala Thr Arg Pro Gly 20 25 30 Gly Arg Leu Leu Ala Thr Glu Lys Glu Ala Ser Ala Arg Arg Glu Ile 35 40 45 Gly Gly Gly Glu Ala Gly Ala Val Ile Gly Gly Ser Ala Gly Ala Ser 50 55 60 Pro Pro Ser Thr Leu Thr Pro Asp Ser Arg Arg Val Ala Arg Pro Pro 65 70 75 80 Pro Ile Gly Ala Glu Val Pro Asp Val Thr Ala Thr Pro Ala Arg Leu 85 90 95 Leu Phe Phe Ala Pro Thr Arg Arg Ala Ala Pro Leu Glu Glu Met Glu 100 105 110 Ala Pro Ala Ala Asp Ala Ile Met Ser Pro Glu Glu Glu Leu Asp Gly 115 120 125 Tyr Glu Pro Glu Pro Leu Gly Lys Arg Pro Ala Val Leu Pro Leu Leu 130 135 140 Glu Leu Val Gly Glu Ser Gly Asn Asn Thr Ser Thr Asp Gly Ser Leu 145 150 155 160 Pro Ser Thr Pro Pro Pro Ala Glu Glu Glu Glu Asp Glu Leu Tyr Arg 165 170 175 Gln Ser Leu Glu Ile Ile Ser Arg Tyr Leu Arg Glu Gln Ala Thr Gly 180 185 190 Ala Lys Asp Thr Lys Pro Met Gly Arg Ser Gly Ala Thr Ser Arg Lys 195 200 205 Ala Leu Glu Thr Leu Arg Arg Val Gly Asp Gly Val Gln Arg Asn His 210 215 220 Glu Thr Ala Phe Gin Gly Trp Val Cys Gly Val Leu Pro Cys Arg Gly 225 230 235 240 Pro Arg Arg Trp His Gin Glu Cys Ala Ala Gly Phe Cys Arg Cys Cys 245 250 255 Trp Ser Arg Ser Trp Phe Gly lie Ser Asn Lys lie Ala Leu Leu 260 265 270 <210> 20 <211> 816 <212> DNA <213> Homo sapiens <400> 20 atgtttggcc tcaaaagaaa cgcggtaatc ggactcaacc tctactgtgg gggggccggc 60 ttgggggccg gcagcggcgg cgccacccgc ccgggagggc gacttttggc tacggagaag 120 gaggcctcgg cccggcgaga gataggggga ggggaggccg gcgcggtgat tggcggaagc 180 gccggcgcaa gccccccgtc caccctcacg ccagactccc ggagggtcgc gcggccgccg 240 cccattggcg ccgaggtccc cgacgtcacc gcgacccccg cgaggctgct tttcttcgcg 300 cccacccgcc gcgcggcgcc gcttgaggag atggaagccc cggccgctga cgccatcatg 360 tcgcccgaag aggagctgga cgggtacgag ccggagcctc tcgggaagcg gccggctgtc 420 ctgccgctgc tggagttggt cggggaatct ggtaataaca ccagtacgga cgggtcacta 480 ccctcgacgc cgccgccagc agaggaggag gaggacgagt tgtaccggca gtcgctggag 540 attatctctc ggtaccttcg ggagcaggcc accggcgcca aggacacaaa gccaatgggc 600 aggtctgggg ccaccagcag gaaggcgctg gagaccttac gacgggttgg ggatggcgtg 660 cagcgcaacc acgagacggc cttccaagga tgggtttgtg gagttcttcc atgtagagga 720 cctagaaggt ggcatcagga atgtgctgct ggcttttgca ggtgttgctg gagtaggagc 780 tggtttggca tatctaataa gatagcctta ctgtaa 816 <210> 21 <211> 236 <212> PRT <213> Homo sapiens () <400> 21 Met Glu Ser Ser Ala Lys Met Glu Ser Gly Gly Ala Gly Gln Gln Pro 1 5 10 15 Gln Pro Gln Pro Gln Gln Pro Phe Leu Pro Pro Ala Ala Cys Phe Phe 20 25 30 Ala Thr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gln 35 40 45 Ser Ala Gin Gin Gin Gin Gin Gin Gin Gin Gin Ala Pro 50 55 60 Gln Leu Arg Pro Ala Ala Asp Gly Gin Pro Ser Gly Gly Gly His Lys 65 70 75 80 Ser Ala Pro Lys Gin Val Lys Arg Gin Arg Ser Ser Ser Pro Gin Leu 85 90 95 Met Arg Cys Lys Arg Arg Leu Asn Phe Ser Gly Phe Gly Tyr Ser Leu 100 105 110 Pro Gin Gin Gin Pro Ala Ala Val Ala Arg Arg Asn Gin Arg Gin Arg 115 120 125 Asn Arg Val Lys Leu Val Asn Leu Gly Phe Ala Thr Leu Arg Gin His 130 135 140 Val Pro Asn Gly Ala Ala Asn Lys Lys Met Ser Lys Val Glu Thr Leu 145 150 155 160 Arg Ser Ala Val Gin Tyr He Arg Ala Leu Gin Gin Leu Leu Asp Gin 165 170 175 His Asp Ala Val Ser Ala Ala Phe Gin Ala Gly Val Leu Ser Pro Thr 180 185 190 He Ser Pro Asn Tyr Ser Asn Asp Leu Asn Ser Met Ala Gly Ser Pro 195 200 205 Val Ser Ser Tyr Ser Ser Asp Glu Gly Ser Tyr Asp Pro Leu Ser Pro 210 215 220 Glu Glu Gln Glu Leu Leu Asp Phe Thr Asn Trp Phe 225 230 235 <210> 22 <211> 711 <212> DNA <213> Homo sapiens() <400> 22 atggaaagct ctgccaagat ggagagcggc ggcgccggcc agcagcccca gccgcagccc 60 cagcagccct tcctgccgcc cgcagcctgt ttctttgcca cggccgcagc cgcggcggcc 120 gcagccgccg cagcggcagc gcagagcgcg cagcagcagc agcagcagca gcagcagcag 180 cagcaggcgc cgcagctgag accggcggcc gacggccagc cctcaggggg cggtcacaag 240 tcagcgccca agcaagtcaa gcgacagcgc tcgtcttcgc ccgaactgat gcgctgcaaa 300 cgccggctca acttcagcgg ctttggctac agcctgccgc agcagcagcc ggccgccgtg 360 gcgcgccgca acgagcgcga gcgcaaccgc gtcaagttgg tcaacctggg ctttgccacc 420 cttcgggagc acgtccccaa cggcgcggcc aacaagaaga tgagtaaggt ggagacactg 480 CGCTCGGGCGGTCGAGTACATCCGCgcgctgcagcagctgctggacgagcatgacgcggtg 540 AGCGCCGCCTTCCAGGCAGGCgtcctgtcGCCCACC ATCTCCCCA ACTACTCCAACGAC 600 TTGAAC TCCATGGCCGGCTCGCCG GTCTCATCCTACTCGTcGGACGAGGGCTCTTACGAC 660 CCGCTCAGCCCCGAGGAGCAGGAGCTTCTCGACTTCACCAACTGgtTCTGA 711

Claims

1. Use of a biomarker comprising Noxa or a biomarker comprising both Noxa and ASCL1 in the manufacture of a kit for use in a method of identifying whether a subject having a cancer will respond to treatment with a Bcl-2 / Bcl-xL dual inhibitor, the method comprising: a) measuring the level of at least one biomarker comprising Noxa and / or ASCL1 in a test sample derived from the subject; b) comparing the level of the at least one biomarker to a corresponding reference level of the at least one biomarker to determine a difference from the reference level; and c) identifying the subject as likely to respond to treatment with a Bcl-2 / Bcl-xL dual inhibitor when the difference reaches a predetermined threshold; the Bcl-2 / Bcl-xL dual inhibitor is (R)-2-(l-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-l- isopropyl-5-methyl-4-(methylsulfonyl)-lH-pyrrol-3-yl)-5-fluorophenyl)piperazin-l- yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfanyl)phenylamino)-4-(phenylthio)butyl)piperidine- 4-carbonyloxy)ethylphosphonic acid, or a pharmaceutically acceptable salt thereof: ； the cancer is selected from gastric cancer, esophageal cancer, or small cell lung cancer.

2. The use of claim 1, wherein the method further comprises: d) administering to the subject identified as likely to respond to treatment with a Bcl-2 / Bcl-xL dual inhibitor an effective amount of a Bcl-2 / Bcl-xL dual inhibitor.

3. Use of a biomarker comprising Noxa or a biomarker comprising both Noxa and ASCL1 in the manufacture of a kit for use in a method of monitoring efficacy of treatment in a subject having a cancer and who has been treated with a Bcl-2 / Bcl-xL dual inhibitor during a treatment period, the method comprising: a) obtaining a test sample from the subject after the treatment period; b) measuring the level of at least one biomarker comprising Noxa and / or ASCL1 in the test sample to obtain a post-treatment level of the at least one biomarker; c) comparing the post-treatment level to a baseline level of the at least one biomarker in a test sample derived from the subject prior to the treatment period to determine a post-treatment change in the level of the at least one biomarker; and d) continuing administration of the Bcl-2 / Bcl-xL dual inhibitor to the subject when the post-treatment change reaches a predetermined threshold, or increasing the dose of the Bcl-2 / Bcl-xL dual inhibitor to the subject, administering to the subject an effective amount of a second anti-cancer therapeutic agent in combination with the Bcl-2 / Bcl-xL dual inhibitor, or discontinuing administration of the Bcl-2 / Bcl-xL dual inhibitor to the subject when the post-treatment change does not reach the predetermined threshold. The Bcl-2 / Bcl-xL dual inhibitor is (R)-2-(1-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1- isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1- yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfanyl)phenylamino)-4-(phenylthio)butyl)piperidine- 4-carbonyloxy)ethylphosphonic acid, having the following structure: ； The cancer is selected from gastric cancer, esophageal cancer, or small cell lung cancer.

4. The use of any one of claims 1-3, wherein the level of the at least one biomarker is measured by mRNA level, protein level, or DNA level.

5. The use of claim 4, wherein the level of the at least one biomarker is measured by an amplification assay, a hybridization assay, a sequencing assay, or an immunoassay.

6. The use of claim 5, wherein the amplification assay is a polymerase chain reaction (PCR)- based method.

7. The use of any one of claims 1-3, wherein the predetermined threshold is set by a statistical method, or determined using a classification algorithm.

8. The use of claim 7, wherein the predetermined threshold of Noxa is reached when the level of Noxa in the test sample is at least 15% higher than the corresponding reference level of Noxa.

9. The use of claim 7, wherein the predetermined threshold of Noxa is reached when the level of Noxa in the test sample is at least 25% higher than the corresponding reference level of Noxa.

10. The use of claim 7, wherein the predetermined threshold of Noxa is reached when the level of Noxa in the test sample is at least 35% higher than the corresponding reference level of Noxa.

11. The use of claim 7, wherein the predetermined threshold of Noxa is reached when the level of Noxa in the test sample is at least 45% higher than the corresponding reference level of Noxa.

12. The use of claim 7, wherein the predetermined threshold of Noxa is reached when the level of Noxa in the test sample is at least 50% higher than the corresponding reference level of Noxa.

13. The use of any one of claims 8-12, wherein the reference level of Noxa represents the average level of Noxa in a general population of subjects having cancer.

14. The use of claim 13, wherein the reference level of Noxa is measured in a control sample.

15. The use of claim 7, wherein the predetermined threshold of ASCL1 is reached when the level of ASCL1 in the test sample is at least 15% higher than the corresponding reference level of ASCL1.

16. The use of claim 7, wherein the predetermined threshold of ASCL1 is reached when the level of ASCL1 in the test sample is at least 25% higher than the corresponding reference level of ASCL1.

17. The use of claim 7, wherein the predetermined threshold of ASCL1 is reached when the level of ASCL1 in the test sample is at least 50% higher than the corresponding reference level of ASCL1.

18. The use of claim 7, wherein the predetermined threshold of ASCLl is achieved when the level of ASCLl in the test sample is at least 100% higher than the corresponding reference level of ASCLl.

19. The use of claim 7, wherein the predetermined threshold of ASCLl is achieved when the level of ASCLl in the test sample is at least 150% higher than the corresponding reference level of ASCLl.

20. The use of any one of claims 15-19, wherein the reference level of ASCLl represents the average level of ASCLl in a general population of subjects having cancer.

21. The use of claim 20, wherein the reference level of ASCLl is measured in a control sample.

22. The use of claim 14 or 21, wherein the control sample is a sample having a level of ASCLl that represents the average level of ASCLl in a general cancer population; or a sample having a level of Noxa that represents the average level of Noxa in a general cancer population.

23. The use of any one of claims 1-3, wherein the sample is a bodily fluid sample or a tissue sample.

24. The use of claim 3, wherein an increase or maintenance of the level of Noxa in the sample after a treatment period indicates a likelihood of a sustained responsiveness to treatment with a Bcl-2 / Bcl-xL dual inhibitor.

25. The use of claim 3, wherein a decrease in the level of Noxa in the sample indicates a likelihood of a decreased responsiveness to treatment with a Bcl-2 / Bcl-xL dual inhibitor.

26. The use of any one of claims 1-3, wherein the kit comprises one or more reagents for measuring the level of at least one biomarker comprising Noxa or both Noxa and ASCLl.

27. The use of claim 26, wherein the one or more reagents for measuring the level of Noxa or both Noxa and ASCLl comprise a primer or a probe that can hybridize to a polynucleotide of Noxa or both Noxa and ASCLl, or an antibody that can specifically bind to a protein of Noxa and / or ASCLl.

28. The use of claim 27, wherein the one or more reagents comprise a first primer or a first probe that can hybridize to a polynucleotide of Noxa, or a first antibody that can specifically bind to a protein of Noxa, and a second primer or a second probe that can hybridize to a polynucleotide of ASCLl, or a second antibody that can specifically bind to a protein of ASCLl.

29. The use of claim 26, wherein the one or more reagents are detectably labeled.

30. Use of one or more reagents for measuring the level of at least one biomarker comprising Noxa or both Noxa and ASCLl in the manufacture of a kit for the use of any one of claims 1-25.

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