Tricyclic AKR1C3 dependent KARS inhibitor administration method

Abstract

The present invention relates to methods of identifying a subject for treatment with a tricyclic AKR1C3 dependent KARS inhibitor of formula (I), or a pharmaceutically acceptable salt thereof, or treating a subject with the inhibitor, or a pharmaceutically acceptable salt thereof. The methods can include determining a level of at least one of the following biomarkers in a sample of the subject: AKR1C3, NFE2L2, KEAP1, or CUL3, wherein an elevated level of biomarkers identifies the subject as in need of treatment; or detecting a somatic mutation of at least one of the following genes in a sample of the subject: NFE2L2, KEAP1 or CUL3, wherein detecting the somatic mutation identifies the subject as in need of treatment.

Description

Technical Field

[0001] The present invention relates to methods for identifying a subject for treatment with a tricyclic aldo-keto reductase 1C3 (AKR1C3)-dependent lysyl tRNA synthase (KARS) inhibitor of formula (I) or a pharmaceutically acceptable salt thereof, or treating a subject with the inhibitor or a pharmaceutically acceptable salt thereof. In embodiments described herein, the methods may include determining the level of a biomarker (e.g., AKR1C3) in a sample from a subject, wherein an elevated level of the biomarker identifies the subject as requiring treatment; or detecting a somatic mutation in at least one of the following genes in a sample from a subject: NFE2-like Bzip transcription factor 2 (NFE2L2), Kelch-like ECH-associated protein 1 (KEAP1), or Cullin 3 (CUL3), wherein detection of a somatic mutation identifies the subject as requiring treatment. Background Art

[0002] The NFE2L2 / NFE2L2-KEAP1 pathway has a strong genetic basis in cancer. The Cancer Genome Atlas (TCGA) sequencing effort reported that this pathway is altered in 34% of lung squamous cell carcinomas (Hammerman et al., (2012) Nature 489:519-25). In addition, TCGA and other groups have reported significant mutations in this pathway in other solid tumor indications, including head and neck squamous cell carcinoma and hepatocellular carcinoma. Aberrant activation of the NFE2L2 pathway may occur due to gain-of-function genetic changes in NFE2L2 or loss-of-function genetic changes in KEAP1 or CUL3, which stabilize NFE2L2 and increase the expression of its target genes. Uncontrolled transcription of these target genes confers an advantage to cancer cells (such as malignancies) and protection against oxidative stress, chemotherapy, and radiation therapy (Jaramillo and Zhang (2013) Genes Dev. [Genes and Development] 27:2179-91). Increased NFE2L2 activity in tumors is associated with poor prognosis (Shibata et al., (2008) Proc Natl Acad Sci USA 105:13568–73). Currently there are no approved therapies that can selectively target cancers with genetic alterations in the NFE2L2 / KEAP1 pathway, and this represents an unmet medical need.

[0003] Aldo-keto reductase 1C3 (AKR1C3) is one of the many target genes of the transcription factor NFE2L2, and its expression is upregulated in NFE2L2 / KEAP1 mutant cancers (MacLeod et al. (2016) Br J Cancer 115:1530–9). AKR1C3 (also known as type 2 3α(17β)-hydroxysteroid dehydrogenase) is a NADP(H)-dependent ketosteroid reductase and a member of the aldo-keto reductase (AKR) superfamily that plays a role in steroid hormone metabolism and signaling and xenobiotic detoxification. Some known substrates of AKR1C3 are the endogenous substrates 5α-dihydrotestosterone, Δ4-androstene-3,17-dione and progesterone (Penning et al., (2000) Biochem. J. [Journal of Biological Chemistry] 351: 67-77), as well as the synthetic prodrugs coumberone (Halim et al., (2008) J. Am. Chem. Soc. [Journal of the American Chemical Society] 130: 14123–8), PR104 (Jamieson et al., (2014) Biochem Pharmacol. [Biochemical Pharmacology] 88: 36-45) and TH3424 / OBI3424 (International Publication No. WO 2016 / 145092). Tricyclic ketone compounds that are converted by AKR1C3 into lysine t-RNA synthetase (KARS) inhibitors in the presence of NADPH have also been identified.

[0004] AKR1C3-dependent KARS inhibitors provide an attractive strategy to selectively treat tumors that overexpress AKR1C3 compared to normal tissues, such as NFE2L2 / KEAP1 mutant cancers and other types of cancer reported to overexpress AKR1C3 (Guise et al., (2010) Cancer Res. 70:1573-84), such as breast cancer (Lewis et al., (2004) BMC Cancer 4:27) and prostate cancer (Fung et al., (2006) Endocr Relat Cancer 13:169–80).

[0005] Compounds of formula (I) and pharmaceutically acceptable salts thereof described in International Publication No. WO 2021 / 005586 have been identified as potent tricyclic AKR1C3-dependent KARS inhibitors. Currently, identifying subjects for treatment with tricyclic AKR1C3-dependent KARS inhibitors and determining appropriate doses for treating such patients remain a challenge. It is necessary to provide accurate and reliable methods to identify and treat such patients. Summary of the invention

[0006] AKR1C3-dependent KARS inhibitors provide treatment and therapy for patients with cancers with genetic alterations in the NFE2L2 / KEAP1 pathway. The invention described herein provides methods for identifying a subject for treatment with an AKR1C3-dependent KARS inhibitor compound of formula (I) or a pharmaceutically acceptable salt thereof. The invention described herein also provides methods for selecting a compound of formula (I) or a pharmaceutically acceptable salt thereof to treat a subject. The invention described herein also provides methods for treating a subject with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0007] In some embodiments, the methods described herein include determining the level of a biomarker (e.g., an AKR1C3 biomarker) in a subject sample, such as the level of an AKR1C3 protein or the level of an AKR1C3 mRNA. In some embodiments of the methods described herein, the subject sample is characterized as having elevated AKR1C3 levels, such as elevated AKR1C3 protein levels or elevated AKR1C3 mRNA levels. In some embodiments, elevated AKR1C3 levels identify the subject as a subject in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0008] In some embodiments, the methods described herein comprise detecting a somatic mutation in at least one of the following genes in a sample from a subject: NFE2L2, KEAP1, or CUL3. In some embodiments of the methods described herein, the sample from a subject is characterized by the presence of a somatic mutation in at least one of the following genes: NFE2L2, KEAP1, or CUL3. In some embodiments, the somatic mutation identifies the subject as being in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the methods described herein include administering to a subject a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes administering to a subject an amount (e.g., an effective amount, e.g., a therapeutically effective amount) of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0010] Various embodiments of the invention are described herein.

[0011] In one aspect of the invention, described herein is a method of identifying a subject for treatment with a compound of formula (I):

[0012]

[0013] in is a single bond or a double bond;

[0014] Z OH when it is a single bond; or When it is a double bond, it is O;

[0015] Each R 1 independently selected from the group consisting of: (C1-C6)alkyl, (C1-C6)alkoxy, (C0-C4)alkylN(R 8 )2 and halogenated;

[0016] R 2a and R 2b Each independently selected from the group consisting of: H, (C1-C6)alkyl and halo;

[0017] Each R 3 are independently selected from the group consisting of: H and halo;

[0018] R 4 is selected from the group consisting of: aryl; 5- to 6-membered heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; and 9- to 10-membered fused bicyclic heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; wherein any of the foregoing groups is optionally replaced by one or more R 6 replace;

[0019] R 5 Selected from the group consisting of: H, (C1-C6) alkyl, (C2-C6) alkenyl, (C0-C4) alkyl, OR 8 、(C1-C4)alkyl(C3-C 10 )cycloalkyl, halo(C1-C6)alkyl, (C2-C3)alkynyl, (C1-C4)alkylN(R 10 )2;

[0020] Each R 6 independently selected from the group consisting of halo, (C1-C6) alkyl, (C1-C6) alkoxy, halo (C1-C6) alkyl, OH, aryl, 3- to 6-membered heterocyclic, 5- to 6-membered heteroaryl, (C0-C4) alkyl, S(O) m (C1-C6) alkyl, halogenated (C1-C6) alkoxy, (C0-C4) alkyl S(O) m N(R 8 )2、(C0-C4)alkyl N(R 8 )2、(C0-C4)alkyl(CO)OR 7 、N(R 8 )S(O) m (C1-C6) alkyl, N(R 8 )S(O) m(C3-C6)cycloalkyl, OP(O)(OH)2, (C0-C3)alkyl (CO)NHR 11 、(C0-C3)alkylOR 7 and (C3-C 10 )cycloalkyl; wherein each R 6 When not halogenated, OH or OP(O)(OH)2, it is optionally substituted with one to three R 9 Substitution; or two adjacent R 6 Together with the atoms to which they are attached, they form a 5- to 7-membered heterocyclic ring or a (C5-C8)cycloalkyl group;

[0021] Each R 7 and R 8 are independently selected from the group consisting of: H or optionally with one to three R 9 Substituted (C1-C6) alkyl;

[0022] Each R 9 Independently selected from the group consisting of: halo, -OH, amino, (C 1- C4) alkylamino, di(C 1- C4)alkylamino, OP(O)(OH)2, (C1-C6)alkyl, (C1-C3)alkynyl, (C1-C6)alkoxy, halo(C1-C6)alkyl, (C0-C4)alkylS(O) m (C1-C6) alkyl, halo (C1-C6) alkoxy, 3- to 6-membered heterocyclic ring optionally substituted with oxo (=O), (C0-C4) alkyl S (O) m N(R 10 )2、(C0-C4)alkyl(CO)R 10 、(C0-C4)alkyl(CO)OR 10 、(C0-C4) alkyl NR 10 S(O) m (C1-C6)alkyl, (C0-C4)alkylOR 10 、(C0-C4)alkyl N(R 10 )2, (C0-C4) alkyl CN, (C0-C4) alkyl N (R 10 )2 and (C0-C4)alkyl (CO)N(R 10 )2;

[0023] Each R 10 independently selected from the group consisting of: H, (C1-C6) alkyl, or a 3- to 6-membered heterocycle, wherein the 3- to 6-membered heterocycle is optionally substituted with one or more of: (C1-C6) alkyl and oxo (=O);

[0024] Each R 11Selected from the group consisting of: H, optionally with one to four R 12 substituted 4 to 6 membered heterocyclic ring, optionally substituted by one to four R 12 substituted (C3-C6) cycloalkyl, optionally substituted (C0-C3) alkyl, (C3-C6) cycloalkyl (C1-C3) alkyl, optionally substituted by one to three R 12 substituted CH2-aryl, (C1-C6)alkyl, (C2-C6)alkenyl or (C2-C6)alkynyl, wherein each of the (C1-C6)alkyl, (C2-C6)alkenyl and (C2-C6)alkynyl is optionally substituted with one or more R 13 replace;

[0025] Each R 12 independently selected from the group consisting of OH, (C1-C3)alkoxy, NH2 or (C1-C3)alkyl optionally substituted with one or more OH;

[0026] Each R 13 Independently selected from the group consisting of: halo, OH, amino, (C 1- C4) alkylamino, di(C 1- C4) alkylamino, (C1-C3) alkoxy and C(O)-(C3-C8) cycloalkyl;

[0027] m is 0, 1 or 2; and

[0028] n is 0, 1, or 2,

[0029] or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining the level of AKR1C3 in a sample from a subject. In some embodiments, elevated levels of AKR1C3 identify the subject as a subject in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0030] In another aspect of the invention, described herein is a method of selecting a compound of formula (I) or a pharmaceutically acceptable salt thereof to treat a subject. In some embodiments, the method comprises determining the level of AKR1C3 in a sample from the subject. In some embodiments, elevated AKR1C3 levels identify the subject as a subject in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0031] In another aspect of the present invention, a method of treating a subject is described herein. In some embodiments, the method comprises: determining the level of AKR1C3 in a sample of the subject, wherein the elevated level of AKR1C3 identifies the subject as requiring treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof; and administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0032] In some embodiments, the methods of treating a subject described herein include administering to the subject an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. For example, described herein is a method of treating a subject, the method comprising administering to the subject an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein prior to said administration, the subject sample is characterized as having a certain AKR1C3 level.

[0033] Also described herein is a method of treating a subject with a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein a sample from the subject is characterized as having elevated levels of AKR1C3.

[0034] In some embodiments of the methods described herein, the compound of formula (I) is a specific compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is selected from a subset of the compound of formula (I) or a pharmaceutically acceptable salt thereof. For example, in some embodiments, the compound of formula (I) is 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3', 4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3', 4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) is selected from the group consisting of: 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof.

[0035] In some embodiments of the methods described herein, the subject sample is characterized as having elevated levels of AKR1C3. In some embodiments of the methods described herein, the method comprises determining the level of AKR1C3 in a subject sample.

[0036] In various embodiments described herein, a sample (e.g., a subject sample) is or comprises a cell, cell group, cell lysate, tissue, or fluid of a subject. In certain embodiments, a subject sample comprises a cell, cell group, cell lysate, tissue, or fluid of a subject, such as an mRNA or protein content of a cell, cell group, cell lysate, tissue, or fluid of a subject. In certain embodiments, a subject sample comprises a subject genome, transcriptome, or proteome. In certain embodiments, a subject sample comprises a subject tumor genome, transcriptome, or proteome.

[0037] Subject samples can be obtained by any suitable means (e.g., by biopsy or cell or tissue extraction). Similarly, control samples can be obtained from control subjects by any suitable means.

[0038] In some embodiments, the subject sample is or comprises a cell, wherein the cell is a cancer cell, such as a tumor cell, such as a lung cancer tumor cell, a non-small cell lung cancer tumor cell, a lung adenocarcinoma tumor cell, a lung squamous cell carcinoma cell, a bladder tumor cell, a cervical tumor cell, an esophageal tumor cell, a head and neck tumor cell, a kidney tumor cell or a liver tumor cell. In some embodiments, the subject sample is or comprises a cell, wherein the cell is a lung cell, a bladder cell, a stomach cell, a prostate cell, an esophageal cell, a gastrointestinal cell, a lymphocyte, a nervous system cell, an ovarian cell, a cervical cell, a vaginal cell, a pancreatic cell, a throat cell, an esophageal cell, a kidney cell, a small intestinal cell, a large intestinal cell, a blood cell, a red blood cell, a white blood cell, a platelet cell or a liver cell. In some embodiments, the subject sample is or comprises a cell, wherein the cell is a lung cell. In some embodiments, the subject sample is or comprises a cell, wherein the cell is a respiratory cell. For example, in some embodiments, the cell is or comprises a bronchial cell, a bronchiolar cell, an alveolar cell (e.g., an alveolar type I cell, an alveolar type II cell, or an alveolar macrophage), an epithelial basement membrane cell, an endothelial cell, an airway epithelial cell (e.g., a goblet cell, a ciliated cell, a Clara cell, a neuroendocrine cell, a basal cell, an intermediate cell, a serous cell, a brush cell, a tumor cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara-mucous cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a ductal cell), an interstitial connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a medullary cell, a fat tissue cell, or a neural cell), a vascular cell (e.g., an endothelial cell, a smooth muscle cell, a In some embodiments, the subject sample is a cell, a fibroblast, a myofibroblast, a pericyte, a lymphocyte, a hematopoietic cell, a lymphoid tissue cell (e.g., a lymphocyte, a plasma cell, a megakaryocyte, a macrophage, a Langerhans cell, a mast cell, an eosinophil, a neutrophil, or a basophil), a pleural cell (e.g., a mesothelial cell, a multipotent submesothelial fibroblast, or an adipocyte), a stem cell, a perivascular epithelial-like cell, a multipotent epithelial stem cell, a medullary cell, or an endothelial progenitor cell. In some embodiments, the subject sample is or comprises a fluid, wherein the fluid is selected from the group consisting of: blood, plasma, mucus, urine, and lymph. In some embodiments, the subject sample comprises a subject genome, transcriptome, or proteome, such as a subject tumor genome, transcriptome, or proteome of any of the aforementioned cells or fluids.

[0039] In some embodiments described herein, the subject is diagnosed with a disease or disorder (e.g., cancer), needs treatment for the disease or disorder, is being treated for the disease or disorder, is in remission of the disease or disorder, is at risk of developing the disease or disorder, or is prone to developing the disease or disorder. In some embodiments, the disease or disorder is selected from the group consisting of non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer (e.g., bladder urothelial carcinoma), cervical cancer (e.g., cervical squamous cell carcinoma), uterine cancer (e.g., endometrial cancer), esophageal cancer (e.g., esophageal squamous cell carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), kidney cancer (e.g., papillary renal cell carcinoma), breast cancer, colorectal cancer, melanoma, gastric cancer, castration-resistant prostate cancer (CRPC), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and liver cancer (e.g., hepatocellular carcinoma). In some embodiments, the subject is diagnosed with a disease or disorder selected from the group consisting of non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer (e.g., bladder urothelial carcinoma), cervical cancer (e.g., cervical squamous cell carcinoma), uterine cancer (e.g., endometrial cancer), esophageal cancer (e.g., esophageal squamous cell carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), kidney cancer (e.g., papillary renal cell carcinoma), breast cancer, colorectal cancer, melanoma, gastric cancer, castration-resistant prostate cancer (CRPC), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and liver cancer (e.g., hepatocellular carcinoma). In specific embodiments, the subject is diagnosed with a disease or disorder, such as cancer.

[0040] In some embodiments of the methods described herein, the subject's tumor genome comprises a somatic mutation associated with a disease or disorder (e.g., cancer). For example, in some embodiments, the subject's tumor genome comprises a somatic mutation in one or more of the NFE2L2, KEAP1, or CUL3 gene sequences. In some embodiments, the presence of somatic mutations in the NFE2L2, KEAP1, or CUL3 gene sequences in the subject's tumor genome may indicate that the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0041] In some embodiments, the AKR1C3 level detected or characterized in the subject sample is compared to a control AKR1C3 level. In some embodiments, the AKR1C3 level in the subject sample is elevated relative to the control AKR1C3 level. An elevated AKR1C3 level in the subject sample relative to the control AKR1C3 level may indicate that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0042] In some embodiments, the control level comprises the AKR1C3 level of a control sample or a control data set. For example, in some embodiments, the control level comprises the AKR1C3 level of a control sample or a control data set, wherein the biomarker is a biomarker characterized or determined in a subject sample (e.g., AKR1C3 protein or AKR1C3 mRNA).

[0043] In various embodiments described herein, the control sample is or comprises a non-cancer cell of a subject, a non-cancer cell group of a subject, a non-cancerous tissue of a subject, a non-cancerous fluid of a subject, a non-cancerous cell group of a control subject, a non-cancerous tissue of a control subject, or a non-cancerous fluid of a control subject. In some embodiments, the control sample is or comprises the contents of a cell, such as the protein or mRNA contents of the following items: a non-cancerous cell group of a subject, a non-cancerous tissue of a subject, a non-cancerous fluid of a control subject, a non-cancerous cell group of a control subject, a non-cancerous tissue of a control subject, or a non-cancerous fluid of a control subject. In some embodiments, the control sample comprises a non-cancerous cell genome, transcriptome, or proteome, such as a genome, transcriptome, or proteome of a non-cancerous cell of a subject or a genome, transcriptome, or proteome of a non-cancerous cell of a control subject.

[0044] In some embodiments, the control data set comprises biomarker (e.g., AKR1C3) level data from non-cancer cells of a subject, a non-cancer cell population of a subject, a non-cancer tissue of a subject, a non-cancer fluid of a subject, a non-cancer cell of a control subject, a non-cancer cell population of a control subject, a non-cancer tissue of a control subject, a non-cancer fluid of a control subject, or a combination thereof. In some embodiments, the control data set comprises biomarker (e.g., AKR1C3) level data obtained from the contents of a cell (e.g., the protein or mRNA content of the following items): non-cancer cells of a subject, a non-cancer cell population of a subject, a non-cancer tissue of a subject, a non-cancer fluid of a control subject, a non-cancer cell population of a control subject, a non-cancer tissue of a control subject, a non-cancer fluid of a control subject, or a combination thereof. In some embodiments, the control data set comprises biomarker (e.g., AKR1C3) level data from one or more non-cancer cell genomes, transcriptomes, or proteomes (e.g., genomes, transcriptomes, or proteomes of one or more non-cancer cells of a subject or genomes, transcriptomes, or proteomes of one or more non-cancer cells of a control subject).

[0045] In some embodiments, the control sample is or comprises a cell, wherein the cell is a lung cell, a bladder cell, a stomach cell, a prostate cell, an esophageal cell, a gastrointestinal cell, a lymphocyte, a nervous system cell, an ovarian cell, a cervical cell, a vaginal cell, a pancreatic cell, a throat cell, an esophageal cell, a kidney cell, a small intestinal cell, a large intestinal cell, a blood cell, a red blood cell, a white blood cell, a platelet cell, or a liver cell. In some embodiments, the control sample is or comprises a cell, wherein the cell is a lung cell. In some embodiments, the control sample is or comprises a cell, wherein the cell is a respiratory cell. For example, in some embodiments, the cell is or comprises a bronchial cell, a bronchiolar cell, an alveolar cell (e.g., an alveolar type I cell, an alveolar type II cell, or an alveolar macrophage), an epithelial basement membrane cell, an endothelial cell, an airway epithelial cell (e.g., a goblet cell, a ciliated cell, a Clara cell, a neuroendocrine cell, a basal cell, an intermediate cell, a serous cell, a brush cell, a tumor cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara-mucous cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a ductal cell), a mesenchymal connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a medullary cell, a fat tissue cell, or a neural cell), a vascular cell (e.g., an endothelial cell, a smooth muscle cell, a fibroblast, a myofibroblast, Pericytes, lymphocytes, hematopoietic cells, lymphoid tissue cells (e.g., lymphocytes, plasma cells, megakaryocytes, macrophages, Langerhans cells, mast cells, eosinophils, neutrophils or basophils), pleural cells (e.g., mesothelial cells, multipotent submesothelial fibroblasts or adipocytes), stem cells, perivascular epithelial-like cells, multipotent epithelial stem cells, medullary cells or endothelial progenitor cells. In some embodiments, the control sample is or comprises a fluid, wherein the fluid is selected from the group consisting of: blood, plasma, mucus, urine and lymph. In some embodiments, the control sample comprises a non-cancerous cell genome, transcriptome or proteome of any of the aforementioned cells or fluids (e.g., a genome, transcriptome or proteome of a non-cancerous cell of a subject or a genome, transcriptome or proteome of a non-cancerous cell of a control subject).

[0046] In some embodiments, the control data set comprises biomarker level data from cells, wherein the cells are lung cells, bladder cells, stomach cells, prostate cells, esophageal cells, gastrointestinal cells, lymphocytes, nervous system cells, ovarian cells, cervical cells, vaginal cells, pancreatic cells, pharyngeal cells, esophageal cells, kidney cells, small intestinal cells, large intestinal cells, blood cells, red blood cells, white blood cells, platelet cells or liver cells. In some embodiments, the control data set comprises biomarker level data from cells, wherein the cells are lung cells. In some embodiments, the control data set comprises biomarker (e.g., AKR1C3) level data from cells, wherein the cells are respiratory cells. For example, in some embodiments, the cell is or comprises a bronchial cell, a bronchiolar cell, an alveolar cell (e.g., an alveolar type I cell, an alveolar type II cell, or an alveolar macrophage), an epithelial basement membrane cell, an endothelial cell, an airway epithelial cell (e.g., a goblet cell, a ciliated cell, a Clara cell, a neuroendocrine cell, a basal cell, an intermediate cell, a serous cell, a brush cell, a tumor cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara-mucous cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a ductal cell), an interstitial connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a medullary cell, an adipose tissue cell, or a neural cell), a vascular cell (e.g., an endothelial cell, a smooth muscle cell, a fibroblast, a myofibroblast, a pericyte, a lymphocyte, a hematopoietic cell, a lymphoid tissue cell (e.g., a lymphocyte, a plasma cell, a megakaryocytic cell, In some embodiments, the control data set comprises biomarker level data from a fluid, wherein the fluid is selected from the group consisting of: blood, plasma, mucus, urine, and lymph. In some embodiments, the control data set comprises biomarker (e.g., AKR1C3) level data from one or more non-cancerous cell genomes, transcriptomes, or proteomes (e.g., genomes, transcriptomes, or proteomes of one or more non-cancerous cells of a subject or genomes, transcriptomes, or proteomes of one or more non-cancerous cells of a control subject), wherein the biomarker level data is from one or more of the aforementioned cell types and fluids.

[0047] In some embodiments, the subject sample and the control sample comprise cells, cell groups, cell lysates, tissues or fluids (or their protein or mRNA contents) of the same type. In some embodiments, the subject sample AKR1C3 level is determined or characterized in cells, cell groups, cell lysates, tissues or fluids (or from their protein or mRNA contents), and the control level of ARK1C3 is determined or characterized from a control data set, which comprises biomarker level data from cells, cell groups, cell lysates, tissues or fluids (or from their protein or mRNA contents) of the same or comparable type. For example, in some embodiments, the subject sample comprises lung cells, and the control sample comprises lung cells. For example, in some embodiments, the subject sample comprises lung cells, and the control data set comprises biomarker (e.g., AKR1C3) level data from lung cells. For example, in some embodiments, the subject sample comprises a genome, transcriptome or proteome of lung cells, and the control sample comprises a genome, transcriptome or proteome of lung cells. For example, in some embodiments, the subject sample comprises a genome, transcriptome, or proteome of a lung cell, and the control data set comprises biomarker (eg, AKR1C3) level data from the genome, transcriptome, or proteome of the lung cell.

[0048] In the methods described herein, the AKR1C3 biomarker data can be protein or nucleic acid biomarker data. For example, in some embodiments, the level of the biomarker is a protein level, a ribonucleic acid (e.g., messenger ribonucleic acid (mRNA)) level, or the level of another suitable substance of the biomarker.

[0049] In the embodiments described herein, an increase in AKR1C3 levels in a subject sample relative to a control AKR1C3 level (eg, AKR1C3 levels in a control sample or control dataset) indicates that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. For example, in some embodiments, if the level of AKR1C3 in a subject's sample is about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 1500 times, or about 2000 times the level of AKR1C3 in a control sample or control dataset, then the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0050] In some embodiments, if the level of AKR1C3 in a subject's sample is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1000 times, at least about 1500 times, or at least about 2000 times the level of AKR1C3 in a control sample or control dataset, then the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0051] In some embodiments, the level of AKR1C3 is determined or characterized by a specific assay. In some embodiments, determining the level of AKR1C3 includes performing an antigen detection assay. For example, in some embodiments, the antigen detection assay is selected from the group consisting of: a protein blot assay, an enzyme-linked immunosorbent assay (ELISA), an immunohistochemistry (IHC) assay, an immunocytochemistry assay, a flow cytometry assay, an immunoprecipitation assay, an immunoelectrophoresis assay, and an immunoelectron microscopy assay. In a specific embodiment, the antigen detection assay is an IHC assay.

[0052] In some embodiments, performing an antigen detection assay comprises probing the subject sample with an AKR1C3 antibody.The AKR1C3 antibody can be anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6 (Catalog No. ab49680; Abcam, Waltham, MA). Examples of AKR1C3 antibodies suitable for use with the methods described herein include anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6; mouse monoclonal antibody clone 871701 (Catalog No. MAB7678; R&D Systems, Inc., Minneapolis, MN); anti-AKR1C3 rabbit polyclonal antibody ab84327 (Catalog No. ab84327; Abcam, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody 11194-1-AP (Catalog No. 11194-1-AP; Thermo Fisher Scientific, Waltham, MA). Scientific, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody PA5-106891 (Catalog No. PA5-106891; Thermo Fisher Scientific, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody PA5-97446 (Catalog No. PA5-97446; Thermo Fisher Scientific, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody PA5-29779 (Catalog No. PA5-29779; Thermo Fisher Scientific, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody PA5-76071 (Catalog No. PA5-7607 1; Thermo Fisher Scientific, Waltham, MA); anti-AKR1C3 goat polyclonal antibody PA5-18339 (Catalog No. PA5-18339; Thermo Fisher Scientific, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody BS-11401R (Catalog No. BS-11401R; Thermo Fisher Scientific, Waltham, MA); and anti-AKR1C3 rabbit polyclonal antibody NBP1-33556 (Catalog No. NBP1-33556; Novus Biologicals, Centennial, CO). In some embodiments, the AKR1C3 antibody comprises a CDR sequence that shares at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with the CDR sequences of an anti-AKR1C3 antibody (e.g., an anti-AKR1C3 mouse monoclonal antibody, e.g., anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6).

[0053] The AKR1C3 antibody used in the methods described herein can be conjugated to an element that allows antibody detection using a detection assay. For example, in some embodiments, the AKR1C3 antibody is conjugated to horseradish peroxidase (HRP). In some embodiments, the AKR1C3 antibody is not conjugated to HRP or another element that allows antibody detection using a detection assay. In such embodiments, performing an antigen detection assay further includes probing the subject sample with a secondary antibody (e.g., a secondary antibody conjugated to HRP) or a probe that can bind to a primary antibody (e.g., streptavidin). The presence of HRP can be detected by reacting with 3,3'-diaminobenzidine (DAB). Therefore, in some embodiments (e.g., embodiments in which a primary or secondary antibody is conjugated to HRP), the antigen detection assay further includes applying 3,3'-diaminobenzidine (DAB) to the subject sample.

[0054] In some embodiments of the methods described herein, determining the AKR1C3 level (e.g., the AKR1C3 level of a subject sample or a control sample) may include generating a signal intensity score. Thus, in some embodiments including performing an IHC assay, determining the AKR1C3 level of a subject sample further includes generating an IHC signal intensity score for the subject sample. In some embodiments including performing an IHC assay, determining the AKR1C3 level of a control sample further includes generating an IHC signal intensity score for the control sample. An increase in the IHC signal intensity score of the subject sample or the IHC signal intensity score of the subject sample relative to the IHC signal intensity score of the control sample may indicate that the subject needs treatment. For example, in some embodiments, if the IHC signal intensity score of the subject sample is 0.5 or greater, 1.0 or greater, 1.5 or greater, 2 or greater, 2.5 or greater, 2.6 or greater, 2.7 or greater, 2.8 or greater, or 2.9 or greater, and the IHC signal intensity score may be in the range of 0-3, the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, if the IHC signal intensity score of the subject's sample is 50 or greater, 100 or greater, 150 or greater, 200 or greater, 250 or greater, 260 or greater, 270 or greater, 280 or greater, or 290 or greater, and the IHC signal intensity score can be in the range of 0-300, then the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments of the methods described herein, the subject sample is characterized as having elevated AKR1C3 levels by an antigen detection assay, such as a Western blot assay, an enzyme-linked immunosorbent assay (ELISA), an immunohistochemistry (IHC) assay, an immunocytochemistry assay, a flow cytometry assay, an immunoprecipitation assay, an immunoelectrophoresis assay, or an immunoelectron microscopy assay. In specific embodiments, the antigen detection assay is an IHC assay.

[0056] In other embodiments of the methods described herein, determining the AKR1C3 level (e.g., the level of AKR1C3 mRNA) in a sample (e.g., a subject sample or a control sample) includes performing a polymerase chain reaction (PCR). In such embodiments, PCR effectively determines the AKR1C3 level in the subject sample and / or the control sample. In some embodiments, determining the AKR1C3 level further includes performing PCR that effectively determines the control marker level in the subject sample and / or the control sample. The control marker can be, for example, beta actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In some embodiments, PCR is quantitative PCR (qPCR), reverse transcription PCR (RT-PCR) or reverse transcription qPCR (RT-qPCR).

[0057] In some embodiments of the methods described herein, the level of AKR1C3 is characterized in a sample (e.g., a subject sample and / or a control sample) by PCR. For example, in some embodiments, by PCR (e.g., a PCR that effectively determines the AKR1C3 level in the subject sample), the subject sample is characterized as having an elevated AKR1C3 level (e.g., an elevated AKR1C3 mRNA level). In some embodiments, for example, as determined by PCR, the subject sample is characterized as having an elevated AKR1C3 level relative to the AKR1C3 level of a control sample or a control data set. Therefore, in some embodiments, the level of AKR1C3 is determined in the control sample by PCR that effectively determines the AKR1C3 level in the control sample. In some embodiments, the level of a control marker is determined in the subject sample by PCR that effectively determines the control marker level in the subject sample. In some embodiments, the level of a control marker is determined in the control sample by PCR that effectively determines the control marker level in the control sample. The control marker can be, for example, beta actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In some embodiments, PCR is quantitative PCR (qPCR).

[0058] In another aspect of the invention, described herein is a method for identifying a subject in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein the method comprises detecting a somatic mutation in at least one of the following genes in a sample from the subject: NFE2L2, KEAP1, or CUL3. In some embodiments, somatic mutations are detected in NFE2L2. In some embodiments, somatic mutations are detected in KEAP1. In some embodiments, somatic mutations are detected in CUL3. In some embodiments, somatic mutations are detected in each of NFE2L2 and KEAP1. In some embodiments, somatic mutations are detected in each of NFE2L2 and CUL3. In some embodiments, somatic mutations are detected in each of KEAP1 and CUL3. In some embodiments, somatic mutations are detected in each of NFE2L2, KEAP1, and CUL3.

[0059] Also described herein is a method of selecting a compound of Formula (I) or a pharmaceutically acceptable salt thereof to treat a subject, the method comprising detecting a somatic mutation in at least one of the following genes in a sample from the subject: NFE2L2, KEAP1, or CUL3. In some embodiments, somatic mutations are detected in NFE2L2. In some embodiments, somatic mutations are detected in KEAP1. In some embodiments, somatic mutations are detected in CUL3. In some embodiments, somatic mutations are detected in each of NFE2L2 and KEAP1. In some embodiments, somatic mutations are detected in each of NFE2L2 and CUL3. In some embodiments, somatic mutations are detected in each of KEAP1 and CUL3. In some embodiments, somatic mutations are detected in each of NFE2L2, KEAP1, and CUL3.

[0060] Also described herein is a method of treating a subject, the method comprising: detecting a somatic mutation in at least one of the following genes in a sample from the subject: NFE2L2, KEAP1, or CUL3; and administering to the subject an effective amount (e.g., a therapeutically effective amount) of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, detecting a somatic mutation identifies the subject as needing treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, somatic mutations are detected in NFE2L2. In some embodiments, somatic mutations are detected in KEAP1. In some embodiments, somatic mutations are detected in CUL3. In some embodiments, somatic mutations are detected in each of NFE2L2 and KEAP1. In some embodiments, somatic mutations are detected in each of NFE2L2 and CUL3. In some embodiments, somatic mutations are detected in each of KEAP1 and CUL3. In some embodiments, somatic mutations are detected in each of NFE2L2, KEAP1, and CUL3.

[0061] Also described herein is a method of treating a subject, comprising administering to the subject an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein prior to said administering, a sample from the subject is characterized by the presence of a somatic mutation in at least one of the following genes: NFE2L2, KEAP1, or CUL3. In some embodiments, the somatic mutation is in NFE2L2. In some embodiments, the somatic mutation is in KEAP1. In some embodiments, the somatic mutation is in CUL3. In some embodiments, the somatic mutation is in each of NFE2L2 and KEAP1. In some embodiments, the somatic mutation is in each of NFE2L2 and CUL3. In some embodiments, the somatic mutation is in each of KEAP1 and CUL3. In some embodiments, the somatic mutation is in each of NFE2L2, KEAP1, and CUL3.

[0062] Also described herein is a method of treating a subject with a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein the subject sample is characterized by the presence of a somatic mutation in at least one of the following genes: NFE2L2, KEAP1, or CUL3. In some embodiments, the somatic mutation is in NFE2L2. In some embodiments, the somatic mutation is in KEAP1. In some embodiments, the somatic mutation is in CUL3. In some embodiments, the somatic mutation is in each of NFE2L2 and KEAP1. In some embodiments, the somatic mutation is in each of NFE2L2 and CUL3. In some embodiments, the somatic mutation is in each of KEAP1 and CUL3. In some embodiments, the somatic mutation is in each of NFE2L2, KEAP1, and CUL3.

[0063] In some embodiments of the methods described herein, the compound of formula (I) is a specific compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is selected from a subset of the compound of formula (I) or a pharmaceutically acceptable salt thereof. For example, in some embodiments, the compound of formula (I) is 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3', 4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3', 4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) is selected from the group consisting of: 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof.

[0064] In some embodiments of the methods described herein, the somatic mutation is a disease-associated nucleotide gene sequence associated with a KEAP1, CUL3, NFE2L2, or AKR1C3 gene sequence. Somatic mutations and disease-associated nucleotide gene sequences associated with a KEAP1, CUL3, NFE2L2, or AKR1C3 gene sequence include nucleotide sequences associated with cancer (e.g., lung cancer, e.g., NSCLC). In some embodiments, the somatic mutation is a disease-associated mutation that indicates that an individual carrying a somatic mutation has an increased likelihood of suffering from, developing into, or being prone to developing a specific disease. For example, an individual carrying a somatic mutation may have an increased likelihood of suffering from, developing into, or being prone to developing cancer. In some embodiments, a subject tumor genome comprising a somatic mutation indicates that the subject has an increased likelihood of suffering from, developing into, or being prone to developing cancer.

[0065] Somatic mutations and disease-associated nucleotide sequences associated with KEAP1 are described, for example, in Campbell et al., (2016) Nature Genetics, 48:607-16; Chen, R. (2020) “Cullin 3 and Its Role in Tumorigenesis” in Cullin-RING Ligases and Protein Neddylation, 187-210; Collisson et al., (2014) Nature, 511:543–550; Delgobo et al., (2021) Freed Radical Biology and Medicine, 177:58-71; Gong et al., (2020) Cell Communication and Signaling, 18, 98; Hammerman et al. (2012) Nature, 489: 519–525; Hayes and McMahon (2009) Trends in Biochemical Sciences, 34(4): 176–88; Jin et al. (2021) Cancer Medicine, 10(23): 8673–92; Kandoth et al. (2013) Nature, 502: 333–339; Konstantinopoulos et al. (2011) Cancer Res, 71: 5081–5089; Ohta et al. (2008) Cancer Res, 68: 1303–1309; Padmanabhan et al. (2006) Mol. Cell, 21:689–700; Romero et al., (2020) Nature Cancer, 1(6):589-602; Saleh et al., (2021) Journal of Thoracic Oncology, 17(1):76-88; Shibata et al., (2008) Gastroenterology, 135:1358–1368; Shibata et al., (2011) Neoplasia, 13:864–873; Singh et al., (2006) PLoS Med, 3:e420;Taguchi and Yamamoto (2017) Frontiers in Oncology, 7:85; Wang et al. (2020) “CRL3s: The BTB-CUL3-RING E3 Ubiquitin Ligases” in Cullin-RING Ligases and Protein Neddylation, 211–223; and Yoo et al. (2012) Histopathology, 60:943–952, which are incorporated herein by reference. ;

[0066] Somatic mutations and disease-associated nucleotide sequences associated with CUL3 are described in, for example, Campbell et al., (2016) Nature Genetics, 48:607-16; Chen, R. (2020) “Cullin 3 and Its Role in Tumorigenesis” in Cullin-RING Ligases and Protein Neddylation, 187-210; Collisson et al., (2014) Nature, 511:543–550; Delgobo et al., (2021) Freed Radical Biology and Medicine, 177:58-71; Hammerman et al., (2012) Nature, 489:519–25; Jin et al., (2021) Cancer Medicine, 10(23): 8673-92; Ooi et al., (2013) Cancer Res, 73: 2044–51; and Wang et al., (2020) “CRL3s: The BTB-CUL3-RING E3 Ubiquitin Ligases” in Cullin-RING Ligases and Protein Neddylation, 211–23, which are incorporated herein by reference.

[0067] Somatic mutations and disease-associated nucleotide sequences associated with NFE2L2 are described in, for example, Campbell et al., (2016) Nature Genetics, 48:607-16; Chen, R. (2020) "Cullin 3 and Its Role in Tumorigenesis" in Cullin-RING Ligases and Protein Neddylation, 187-210; Collisson et al., (2014) Nature, 511:543–50; Delgobo et al., (2021) Freed Radical Biology and Medicine, 177:58-71; Goldstein et al. (2016) Cell Rep, 16:2605-2617; Hammerman et al. (2012) Nature, 489:519-25; Jin et al. (2021) Cancer Medicine, 10(23):8673-92; Ooi et al. (2013) Cancer Res, 73:2044-51; Shibata et al. (2011) Neoplasia, 13:864-873; and Wang et al. (2020) “CRL3s: The BTB-CUL3-RING E3 Ubiquitin Ligases” in Cullin-RING Ligases and Protein Neddylation [“CRL3: BTB-CUL3-RING E3 ubiquitin ligase” in Cullin-RING ligases and protein ubiquitination], 211–23, which are incorporated herein by reference.

[0068] Somatic mutations may include mutations relative to the wild-type nucleotide sequence of a gene (e.g., NFE2L2, KEAP1, or CUL3). In some embodiments, somatic mutations include mutations selected from the group consisting of nonsense mutations, missense mutations, substitution mutations, frameshift mutations, point mutations, insertion mutations, deletion mutations (e.g., gene sequence deletions), amplification mutations (e.g., gene amplification), inversion mutations, and duplication mutations. For example, in some embodiments, somatic mutations include mutations in the wild-type nucleotide sequence of NFE2L2, KEAP1, or CUL3, wherein the mutations are nonsense mutations, missense mutations, substitution mutations, frameshift mutations, point mutations, insertion mutations, deletion mutations, inversion mutations, or duplication mutations. In some embodiments, somatic mutations include single nucleotide polymorphisms (SNPs), such as SNPs of NFE2L2, KEAP1, or CUL3 genes. In some embodiments, somatic mutations are mutations in the tumor cell genome (e.g., the subject's tumor cell genome).

[0069] In various embodiments described herein, a sample (e.g., a subject sample) is or comprises a subject's cell, cell mass, cell lysate, tissue, or fluid. In certain embodiments, a subject sample is or comprises a subject's cell, cell mass, cell lysate, tissue, or fluid nucleic acid, e.g., genomic deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) (e.g., messenger RNA (mRNA)). In certain embodiments, a subject sample is or comprises a cell genome or transcriptome, e.g., a tumor cell genome or transcriptome.

[0070] The subject sample can be obtained from the subject by any suitable means. Similarly, the control sample can be obtained from the control subject by any suitable means.

[0071] In some embodiments, the subject sample is or comprises a cell (or its nucleic acid material, e.g., genomic DNA or mRNA), wherein the cell is a cancer cell, e.g., a tumor cell, e.g., a lung cancer cell, a non-small cell lung cancer cell, a lung adenocarcinoma cell, a lung squamous cell carcinoma cell, a bladder tumor cell, a cervical tumor cell, an esophageal tumor cell, a head and neck tumor cell, a kidney tumor cell or a liver tumor cell. In some embodiments, the subject sample is or comprises a cell (or its nucleic acid material, e.g., genomic DNA or mRNA), wherein the cell is a lung cell, a bladder cell, a stomach cell, a prostate cell, an esophageal cell, a gastrointestinal cell, a lymphocyte, a nervous system cell, an ovarian cell, a cervical cell, a vaginal cell, a pancreatic cell, a throat cell, an esophageal cell, a kidney cell, a small intestinal cell, a large intestinal cell, a blood cell, an erythrocyte, a leukocyte, a platelet cell or a liver cell. In some embodiments, the subject sample is or comprises a cell (or its nucleic acid material, e.g., genomic DNA or mRNA), wherein the cell is a lung cell. In some embodiments, the subject sample is or comprises a cell (or its nucleic acid material, e.g., genomic DNA or mRNA), wherein the cell is a respiratory cell. For example, in some embodiments, the cell is or comprises one of the following cell types or nucleic acid material (e.g., genomic DNA or mRNA) thereof: a bronchial cell, a bronchiolar cell, an alveolar cell (e.g., an alveolar type I cell, an alveolar type II cell, or an alveolar macrophage), an epithelial basement membrane cell, an endothelial cell, an airway epithelial cell (e.g., a goblet cell, a ciliated cell, a Clara cell, a neuroendocrine cell, a basal cell, an intermediate cell, a serous cell, a brush cell, a tumor cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara-mucous cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a ductal cell), a mesenchymal connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a medullary cell, a fat tissue cell, or a neural cell), a vascular cell (e.g., an endothelial cell, a smooth muscle cell, a , fibroblasts, myofibroblasts, pericytes, lymphocytes, hematopoietic cells, lymphoid tissue cells (e.g., lymphocytes, plasma cells, megakaryocytes, macrophages, Langerhans cells, mast cells, eosinophils, neutrophils or basophils), pleural cells (e.g., mesothelial cells, multipotent submesothelial fibroblasts or adipocytes), stem cells, perivascular epithelial cells, multipotent epithelial stem cells, medullary cells or endothelial progenitor cells. In some embodiments, the subject sample is or comprises a fluid (or related genetic material), wherein the fluid is selected from the group consisting of: blood, plasma, mucus, urine and lymph. In some embodiments, the subject sample is or comprises the genome or transcriptome of any of the aforementioned cells or fluids. In some embodiments, the subject sample is or comprises the genome or transcriptome of a tumor cell, wherein the tumor cell is selected from the group consisting of the aforementioned cells and fluids.In some embodiments, the subject sample is or comprises the genome or transcriptome of a tumor, wherein the tumor is composed of one or more cells selected from the group consisting of the aforementioned cells. In some embodiments, the subject sample is or comprises the genome or transcriptome of a tumor, wherein the tumor is composed of one or more fluids selected from the group consisting of the aforementioned fluids.

[0072] In some embodiments described herein, the subject is diagnosed with a disease or disorder (e.g., cancer), needs treatment for the disease or disorder, is being treated for the disease or disorder, is in remission of the disease or disorder, is at risk of developing the disease or disorder, or is prone to developing the disease or disorder. In some embodiments, the subject is diagnosed with a disease or disorder selected from the group consisting of non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer (e.g., bladder urothelial carcinoma), cervical cancer (e.g., cervical squamous cell carcinoma), uterine cancer (e.g., endometrial cancer), esophageal cancer (e.g., esophageal squamous cell carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), kidney cancer (e.g., papillary renal cell carcinoma), breast cancer, colorectal cancer, melanoma, gastric cancer, castration-resistant prostate cancer (CRPC), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and liver cancer (e.g., hepatocellular carcinoma). In certain embodiments, the subject is diagnosed with a disease or disorder, such as cancer.

[0073] In some embodiments of the methods described herein, the subject tumor genome comprises one or more mutations associated with a disease or disorder (e.g., cancer). In some embodiments, the subject tumor genome comprises somatic mutations in one or more of the NFE2L2, KEAP1, or CUL3 gene sequences. For example, in some embodiments, the subject tumor genome comprises somatic mutations in the following: in NFE2L2; in KEAP1; in CUL3; in each of NFE2L2 and KEAP1; in each of NFE2L2 and CUL3; in each of KEAP1 and CUL3; or in each of NFE2L2, KEAP1, and CUL3.

[0074] In some embodiments of the methods described herein, a somatic mutation present or detected in a subject sample is not present in a control sample or control data set. For example, in some cases, the subject tumor genome comprises or is characterized by a somatic mutation of one or more of the NFE2L2, KEAP1, or CUL3 gene sequences, and the somatic mutation is not present in the control sample or control data set, or the control sample or control data set is characterized by the absence of the somatic mutation. In some cases, a somatic mutation of one or more of the NFE2L2, KEAP1, or CUL3 gene sequences is detected in the subject tumor genome, and the somatic mutation is not detected in the control sample or control data set.

[0075] In some embodiments, a subject sample characterized by the presence of a somatic mutation or a somatic mutation group can be compared to a control sample or control data set characterized by the absence of a somatic mutation or a somatic mutation group. Therefore, in some embodiments, a somatic mutation or a somatic mutation group detected or present in a subject sample does not exist in a control sample or a control data set. In some embodiments, a somatic mutation or a somatic mutation group is detected or present in a subject sample at a higher frequency relative to a control sample or a control data set. Detecting, detecting or having a somatic mutation or a somatic mutation group in a subject sample can indicate that the subject needs to be treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof. For example, in some embodiments, detecting, detecting or having a somatic mutation or a somatic mutation group in a subject sample and not having a somatic mutation or a somatic mutation group in a control sample or a control data set can indicate that the subject needs to be treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, detecting, detecting or having a somatic mutation or a somatic mutation group in a subject sample at a higher frequency relative to a control sample or a control data set can indicate that the subject needs to be treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0076] In various embodiments described herein, the control sample is or comprises a non-cancer cell of a subject, a non-cancer cell group of a subject, a non-cancerous tissue of a subject, a non-cancerous fluid of a subject, a non-cancerous cell group of a control subject, a non-cancerous tissue of a control subject, or a non-cancerous fluid of a control subject. In some embodiments, the control sample comprises a nucleic acid substance, such as a genomic DNA or mRNA of the following items: a non-cancerous cell group of a subject, a non-cancerous tissue of a subject, a non-cancerous fluid of a control subject, a non-cancerous cell group of a control subject, a non-cancerous tissue of a control subject, or a non-cancerous fluid of a control subject. For example, in some embodiments, the control sample comprises genomic sequence data from a non-cancerous cell group of a subject, a non-cancerous tissue of a subject, a non-cancerous fluid of a control subject, a non-cancerous cell group of a control subject, a non-cancerous tissue of a control subject, a non-cancerous fluid of a control subject, or a combination thereof. In some embodiments, the control sample comprises a non-cancerous cell genome or transcriptome, such as a genome or transcriptome of a non-cancerous cell of a subject or a genome or transcriptome of a non-cancerous cell of a control subject.

[0077] In some embodiments, the control data set comprises nucleic acid (e.g., genomic DNA or mRNA) data from a non-cancer cell of a subject, a non-cancer cell population of a subject, a non-cancerous tissue of a subject, a non-cancerous fluid of a subject, a non-cancerous cell of a control subject, a non-cancerous cell population of a control subject, a non-cancerous tissue of a control subject, a non-cancerous fluid of a control subject, or a combination thereof. For example, in some embodiments, the control data set comprises genomic sequence data from a non-cancerous cell of a subject, a non-cancerous cell population of a subject, a non-cancerous tissue of a subject, a non-cancerous fluid of a control subject, a non-cancerous cell population of a control subject, a non-cancerous tissue of a control subject, a non-cancerous fluid of a control subject, or a combination thereof. In some embodiments, the control data set comprises genomic or transcriptome data from one or more non-cancerous cell genomes or transcriptomes (e.g., genomes or transcriptomes of one or more non-cancerous cells of a subject or genomes or transcriptomes of one or more non-cancerous cells of a control subject).

[0078] In some embodiments, the control sample is or comprises a cell (or its genomic DNA or mRNA), wherein the cell is a lung cell, a bladder cell, a stomach cell, a prostate cell, an esophageal cell, a gastrointestinal cell, a lymphocyte, a nervous system cell, an ovarian cell, a cervical cell, a vaginal cell, a pancreatic cell, a throat cell, an esophageal cell, a kidney cell, a small intestinal cell, a large intestinal cell, a blood cell, a red blood cell, a white blood cell, a platelet cell or a liver cell. In some embodiments, the control sample is or comprises a cell (or its genomic DNA or mRNA), wherein the cell is a lung cell. In some embodiments, the control sample is or comprises a cell (or its genomic DNA or mRNA), wherein the cell is a respiratory cell. For example, in some embodiments, the cell is or comprises a bronchial cell, a bronchiolar cell, an alveolar cell (e.g., an alveolar type I cell, an alveolar type II cell, or an alveolar macrophage), an epithelial basement membrane cell, an endothelial cell, an airway epithelial cell (e.g., a goblet cell, a ciliated cell, a Clara cell, a neuroendocrine cell, a basal cell, an intermediate cell, a serous cell, a brush cell, a tumor cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara-mucous cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a ductal cell), a mesenchymal connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a medullary cell, a fat tissue cell, or a neural cell), a vascular cell (e.g., an endothelial cell, a smooth muscle cell, a fibroblast, a Myofibroblasts, pericytes, lymphocytes, hematopoietic cells, lymphoid tissue cells (e.g., lymphocytes, plasma cells, megakaryocytes, macrophages, Langerhans cells, mast cells, eosinophils, neutrophils or basophils), pleural cells (e.g., mesothelial cells, multipotent submesothelial fibroblasts or adipocytes), stem cells, perivascular epithelial-like cells, multipotent epithelial stem cells, medullary cells or endothelial progenitor cells. In some embodiments, the control sample is or comprises a fluid, wherein the fluid is selected from the group consisting of: blood, plasma, mucus, urine and lymph. In some embodiments, the control sample comprises a non-cancerous cell genome or transcriptome of any of the foregoing cells or fluids (e.g., a genome or transcriptome of a non-cancerous cell of a subject or a genome or transcriptome of a non-cancerous cell of a control subject).

[0079] In some embodiments, the control data set comprises genomic DNA or mRNA data from cells, wherein the cells are lung cells, bladder cells, stomach cells, prostate cells, esophageal cells, gastrointestinal cells, lymphocytes, nervous system cells, ovarian cells, cervical cells, vaginal cells, pancreatic cells, pharyngeal cells, esophageal cells, kidney cells, small intestinal cells, large intestinal cells, blood cells, red blood cells, white blood cells, platelet cells or liver cells. In some embodiments, the control data set comprises genomic DNA or mRNA data from cells, wherein the cells are lung cells. In some embodiments, the control data set comprises genomic DNA or mRNA data from cells, wherein the cells are respiratory cells. For example, in some embodiments, the cell is or comprises a bronchial cell, a bronchiolar cell, an alveolar cell (e.g., an alveolar type I cell, an alveolar type II cell, or an alveolar macrophage), an epithelial basement membrane cell, an endothelial cell, an airway epithelial cell (e.g., a goblet cell, a ciliated cell, a Clara cell, a neuroendocrine cell, a basal cell, an intermediate cell, a serous cell, a brush cell, a tumor cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara-mucous cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a ductal cell), an interstitial connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a medullary cell, an adipose tissue cell, or a neural cell), a vascular cell (e.g., an endothelial cell, a smooth muscle cell, a fibroblast, a myofibroblast, a pericyte, a lymphocyte, a hematopoietic cell, a lymphoid tissue cell (e.g., In some embodiments, the control data set comprises biomarker level data from a fluid, wherein the fluid is selected from the group consisting of: blood, plasma, mucus, urine, and lymph. In some embodiments, the control data set comprises genomic or transcriptome data from one or more non-cancerous cell genomes or transcriptomes (e.g., genomes or transcriptomes of one or more non-cancerous cells of a subject, or genomes or transcriptomes of one or more non-cancerous cells of a control subject), wherein the genomic or transcriptome data is from one or more of the aforementioned cell types and fluids.

[0080] In certain embodiments, subject sample and control sample comprise the same type of cell, cell group, cell lysate, tissue or fluid (or its protein, mRNA or genomic DNA content).In certain embodiments, subject sample genomic DNA or mRNA is characterized in cell, cell group, cell lysate, tissue or fluid, and the genomic DNA or mRNA of control data set is characterized in the same or quite type of cell, cell group, cell lysate, tissue or fluid.For example, in certain embodiments, subject sample comprises pneumonocyte, and control sample comprises pneumonocyte.For example, in certain embodiments, subject sample comprises pneumonocyte, and control data set comprises genomic DNA or mRNA data from pneumonocyte.For example, in certain embodiments, subject sample comprises genome or transcriptome of pneumonocyte, and control sample comprises genome or transcriptome of pneumonocyte.For example, in certain embodiments, subject sample comprises genome or transcriptome of pneumonocyte, and control data set comprises genome or transcriptome data from genome or transcriptome of pneumonocyte.

[0081] Detecting somatic mutations (e.g., detecting somatic mutations in a subject sample) can be performed using techniques known to those skilled in the art. For example, in some embodiments, detecting somatic mutations (e.g., detecting somatic mutations in at least one of NFE2L2, KEAP1, or CUL3) comprises, for example, sequencing genomic DNA of a subject sample by PCR (e.g., by quantitative PCR (qPCR) or digital PCR). In some embodiments, detecting somatic mutations (e.g., detecting somatic mutations in at least one of NFE2L2, KEAP1, or CUL3) comprises, for example, sequencing mRNA of a subject sample by RNA-Seq (e.g., mRNA-Seq), reverse transcription polymerase chain reaction (RT-PCR), reverse transcription quantitative PCR (RT-qPCR), or digital PCR. In some embodiments, genomic DNA of the NFE2L2, KEAP1, and / or CUL3 gene sequences of a subject sample is sequenced. In some embodiments, NFE2L2, KEAP1 and / or CUL3 mRNA (eg, mRNA transcribed from a NFE2L2, KEAP1 and / or CUL3 gene sequence) is sequenced in a sample from a subject.

[0082] In some embodiments of the methods described herein, detecting somatic mutations includes sequencing the genomic DNA of subject samples and / or control samples. In certain embodiments, genomic DNA sequencing methods are high-throughput or next-generation sequencing methods. In certain embodiments, sequencing or the method for sequencing genomic DNA is selected from the group consisting of: exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrophosphate sequencing, sequencing while synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing and Heliscope single molecule sequencing. In certain embodiments, genomic DNA sequencing methods are single molecule sequencing methods. In certain embodiments, sequencing or the method for sequencing genomic DNA is or includes performing PCR, quantitative PCR (qPCR) or Sanger sequencing. Therefore, in some embodiments, detecting somatic mutations includes sequencing the genomic DNA of the subject's sample, and the sequencing or sequencing method is selected from the group consisting of: exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrophosphate sequencing, sequencing by synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing, digital PCR, Heliscope single molecule sequencing, PCR, quantitative PCR (qPCR) and Sanger sequencing. In some embodiments, detecting somatic mutations includes performing in situ hybridization (ISH) (e.g., fluorescent in situ hybridization (FISH), multicolor FISH, in situ PCR and chromogenic in situ hybridization (CISH)).

[0083] Detection of somatic mutations in a subject sample (e.g., somatic mutations in NFE2L2, KEAP1 or CUL3 genomic DNA or mRNA sequences) can indicate that the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Therefore, in some embodiments, if sequencing of the genomic DNA or RNA (e.g., mRNA) of the subject sample detects or causes the detection of a somatic mutation in the subject sample (e.g., a somatic mutation in the NFE2L2, KEAP1 or CUL3 gene sequence), the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0084] In some embodiments, the methods described herein include sequencing a control sample, such as sequencing genomic DNA or mRNA of the control sample (e.g., sequencing genomic DNA or mRNA of at least one of NFE2L2, KEAP1, or CUL3 of the control sample). In some embodiments, the method includes sequencing genomic DNA of the NFE2L2, KEAP1, and / or CUL3 gene sequence of the control sample. In some embodiments, the method includes sequencing NFE2L2, KEAP1, and / or CUL3 mRNA of the control sample. In some embodiments, the methods described herein include sequencing mRNA of the control sample, such as by RNA-Seq (e.g., mRNA-Seq), reverse transcription polymerase chain reaction (RT-PCR), reverse transcription quantitative PCR (RT-qPCR), or digital PCR. In some embodiments, the method described herein comprises sequencing the genomic DNA of a control sample, and the sequencing or sequencing method is selected from the group consisting of: exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, PCR, quantitative PCR (qPCR), digital PCR and Sanger sequencing.

[0085] The methods described herein may include a step of comparing sequencing data (e.g., mRNA or genomic sequencing data) from a subject sample with a control sample or a control data set. The presence or detection of somatic mutations in the subject sample and the absence or non-detection of somatic mutations in the control sample or control data set may indicate that the subject needs to be treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof. Therefore, in some embodiments, the methods described herein include comparing the sequencing data of the subject sample (e.g., sequencing data of genomic DNA sequencing or sequencing data of mRNA sequencing) with the sequencing data of the control sample or control data set. Therefore, in some embodiments, if sequencing of the genomic DNA or RNA (e.g., mRNA) of the subject sample detects or causes the detection of somatic mutations in the subject sample (e.g., somatic mutations of NFE2L2, KEAP1 or CUL3 gene sequences), and sequencing of the genomic DNA or RNA (e.g., mRNA) of the control sample does not detect or causes the detection of somatic mutations in the control sample, then the subject needs to be treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0086] In some embodiments, the detection of somatic mutations in the subject sample at a higher frequency relative to the detection of somatic mutations in the control sample or control data set can indicate that the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Therefore, in some embodiments, if the genomic DNA or RNA (e.g., mRNA) of the subject sample and the control sample is sequenced to detect or cause the detection of somatic mutations in the subject sample (e.g., somatic mutations of NFE2L2, KEAP1 or CUL3 gene sequences) at a higher frequency relative to the control sample, then the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, if the genomic DNA or RNA (e.g., mRNA) of the subject sample is sequenced to detect or cause the detection of somatic mutations in the subject sample (e.g., somatic mutations of NFE2L2, KEAP1 or CUL3 gene sequences) at a higher frequency relative to the control data set or the frequency of somatic mutations detected in the control data set, then the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0087] In some embodiments, the subject sample is characterized by the presence of somatic mutations. In some embodiments, the subject sample is characterized by the presence of somatic mutations by sequencing the genomic DNA of the subject sample. The sample (e.g., a subject sample or a control sample) may be characterized by the presence of somatic mutations using techniques known to those skilled in the art. For example, in some embodiments, the subject sample is characterized by the presence of somatic mutations (e.g., somatic mutations of at least one of NFE2L2, KEAP1, or CUL3 gene sequences) by sequencing the genomic DNA of the subject sample. In some embodiments, the subject sample is characterized by the presence of somatic mutations (e.g., somatic mutations of at least one of NFE2L2, KEAP1, or CUL3 gene sequences) by sequencing the mRNA of the subject sample (e.g., by RNA-Seq (e.g., mRNA-Seq), reverse transcription polymerase chain reaction (RT-PCR), or reverse transcription quantitative PCR (RT-qPCR)). In some embodiments, the genomic DNA of the NFE2L2, KEAP1, and / or CUL3 gene sequences of the subject sample is sequenced. In some embodiments, the NFE2L2, KEAP1, and / or CUL3 mRNA of the subject sample is sequenced.

[0088] In some embodiments of the methods described herein, the subject sample is characterized by the presence of somatic mutations (e.g., somatic mutations of at least one of the NFE2L2, KEAP1, or CUL3 gene sequences) by sequencing the genomic DNA of the subject sample. In some embodiments of the methods described herein, the control sample is characterized by the absence of somatic mutations (e.g., somatic mutations of at least one of the NFE2L2, KEAP1, or CUL3 gene sequences) by sequencing the genomic DNA of the control sample. In some embodiments, the genomic DNA sequencing method is a high-throughput or next-generation sequencing method. In some embodiments, the sequencing method is selected from the group consisting of: exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrophosphate sequencing, sequencing by synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing, and Heliscope single molecule sequencing. In some embodiments, the genomic DNA sequencing method is a single molecule sequencing method. In some embodiments, sequencing or the method of sequencing genomic DNA is or comprises performing PCR, quantitative PCR (qPCR), digital PCR, or Sanger sequencing.

[0089] Thus, in some embodiments, the subject sample is characterized by the presence of somatic mutations (e.g., somatic mutations in at least one of the NFE2L2, KEAP1, or CUL3 gene sequences) by sequencing the genomic DNA of the subject sample, and the sequencing or sequencing method is selected from the group consisting of: exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, PCR, quantitative PCR (qPCR), digital PCR, and Sanger sequencing. In some embodiments, the subject sample is characterized by the presence of somatic mutations (e.g., somatic mutations in at least one of the NFE2L2, KEAP1, or CUL3 gene sequences) by sequencing the genomic DNA of the subject sample, and the sequencing or sequencing method is PCR.

[0090] A subject sample characterized by the presence of somatic mutations (e.g., the presence of somatic mutations in the NFE2L2, KEAP1, or CUL3 gene sequences) may indicate that the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Thus, in some embodiments, if the subject sample is characterized by the presence of somatic mutations (e.g., if sequencing of the genomic DNA or RNA (e.g., mRNA) of the subject sample characterizes the presence of somatic mutations in the subject sample (e.g., the presence of somatic mutations in the NFE2L2, KEAP1, or CUL3 gene sequences)), the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0091] In some embodiments of the methods described herein, the control sample is characterized by the absence of somatic mutations (e.g., somatic mutations of at least one of NFE2L2, KEAP1, or CUL3) by sequencing genomic DNA or RNA (e.g., mRNA) of the control sample. In some embodiments, the method for sequencing genomic DNA is a high-throughput or next-generation sequencing method. In some embodiments, the control sample is characterized by the absence of somatic mutations by sequencing mRNA of the control sample (e.g., by RNA-Seq (e.g., mRNA-Seq), reverse transcription polymerase chain reaction (RT-PCR), digital PCR, or reverse transcription quantitative PCR (RT-qPCR)). In some embodiments, the control sample is characterized in that there is no somatic mutation by sequencing the genomic DNA of the control sample, and the sequencing or sequencing method is selected from the group consisting of: exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrophosphate sequencing, sequencing by synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, PCR, quantitative PCR (qPCR), digital PCR and Sanger sequencing. In some embodiments, the sequencing or sequencing method is PCR.

[0092] In some embodiments, the subject sample is characterized by the presence of somatic mutations and / or the control sample or control data set is characterized by the absence of somatic mutations, indicating that the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Therefore, in some embodiments, if sequencing of the subject sample (e.g., sequencing of the genomic DNA or RNA (e.g., mRNA) of the subject sample) detects or causes the detection of somatic mutations (e.g., the presence of somatic mutations in the NFE2L2, KEAP1 or CUL3 gene sequences), and sequencing of the control sample (e.g., sequencing of the genomic DNA or RNA (e.g., mRNA) of the control sample) does not detect or causes the detection of somatic mutations, then the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0093] In some embodiments, the subject sample is characterized by the presence of somatic mutations at a higher frequency relative to a control sample or a control data set, indicating that the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Therefore, in some embodiments, if the subject sample is characterized by the presence of somatic mutations at a higher frequency relative to a control sample or a control data set, the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0094] The invention described herein includes, in another aspect, the use of AKR1C3 levels for selecting a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof. Accordingly, also described herein is the use of AKR1C3 levels for selecting a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein if a sample from the subject is characterized as having elevated AKR1C3 levels, the subject is treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0095] The invention described herein includes, in another aspect, the use of somatic mutations for selecting a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof. Thus, also described herein is the use of somatic mutations for selecting a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein if a sample of the subject is characterized by the presence of somatic mutations, and wherein a somatic mutation is detected in one of the following genes, the subject is treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof: NFE2L2, KEAP1, or CUL3.

[0096] In some embodiments of the uses described herein, the compound of formula (I) is selected from the group consisting of: 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof. In some embodiments of the uses described herein, the compound of formula (I) is 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide or a pharmaceutically acceptable salt thereof. In some embodiments of the uses described herein, the compound of formula (I) is N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide or a pharmaceutically acceptable salt thereof.

[0097] Also described herein is a kit suitable for performing the methods described herein or for the uses described herein. For example, described herein is a kit for: identifying a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof; selecting a compound of formula (I) or a pharmaceutically acceptable salt thereof to treat a subject; determining the level of AKR1C3 in a subject sample; or characterizing a subject sample as having an elevated level of AKR1C3. Also described herein is a kit for: detecting a somatic mutation in at least one of the following genes in a subject sample: NFE2L2, KEAP1, or CUL3; or characterizing the presence of a somatic mutation in at least one of the following genes in a subject sample: NFE2L2, KEAP1, or CUL3. The kits described herein may include components suitable for determining the level of AKR1C3 in a sample (e.g., a subject sample or a control sample). The kits described herein may include components suitable for detecting somatic mutations in one or more gene sequences (e.g., NFE2L2, KEAP1, or CUL3 gene sequences). The kits described herein may include components suitable for detecting somatic mutations of NFE2L2, KEAP1 or CUL3 gene sequences, wherein the method includes sequencing RNA (e.g., mRNA) transcribed from NFE2L2, KEAP1 or CUL3 gene sequences. In some embodiments, the kits described herein include components suitable for determining mRNA or protein levels of biomarkers. In some embodiments, the kits described herein include components suitable for performing antigen detection assays (e.g., IHC assays). In some embodiments, the kits described herein include components suitable for sequencing genomic DNA or RNA (e.g., mRNA).

[0098] Also described herein are assays suitable for: identifying a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof; selecting a compound of formula (I) or a pharmaceutically acceptable salt thereof to treat a subject; determining the level of AKR1C3 in a subject sample; or characterizing a subject sample as having elevated AKR1C3 levels. Also described herein are assays for: detecting a somatic mutation in at least one of the following genes in a subject sample: NFE2L2, KEAP1, or CUL3; or characterizing the presence of a somatic mutation in at least one of the following genes in a subject sample: NFE2L2, KEAP1, or CUL3. The assays described herein may include steps suitable for determining the level of AKR1C3 in a sample (e.g., a subject sample or a control sample). The assays described herein may include steps suitable for detecting somatic mutations in one or more gene sequences (e.g., NFE2L2, KEAP1, or CUL3 gene sequences). The assays described herein may include steps suitable for detecting somatic mutations of NFE2L2, KEAP1 or CUL3 gene sequences by sequencing RNA (e.g., mRNA) transcribed from NFE2L2, KEAP1 or CUL3 gene sequences. In some embodiments, the assays described herein include steps suitable for determining mRNA or protein levels of AKR1C3. In some embodiments, the assays described herein include steps suitable for performing antigen detection assays (e.g., IHC assays). In some embodiments, the assays described herein include steps suitable for sequencing genomic DNA or RNA (e.g., mRNA).

[0099] For a subject of about 50-70kg, the pharmaceutical composition comprising the compound of formula (I) or its pharmaceutically acceptable salt can be in the unit dosage of about 1-1000mg active ingredient or about 1-500mg or about 1-250mg or about 1-150mg or about 0.5-100mg or about 1-50mg active ingredient. The therapeutically effective dose of the compound of formula (I) or its pharmaceutical composition depends on the species, body weight, age and individual illness, obstacle or disease or its severity of the subject being treated. A physician, clinician or veterinarian with ordinary skills can easily determine the effective amount of each active ingredient necessary for preventing, treating or suppressing obstacle or disease progression.

[0100] The above dosage properties are advantageously demonstrated using in vitro and in vivo tests using mammals (e.g., mice, rats, dogs, monkeys) or their isolated organs, tissues and preparations. The compounds of formula (I) can be applied in vitro in the form of solutions (e.g., aqueous solutions), and in vivo, for example, as suspensions or in aqueous solutions, enterally, parenterally (advantageously, intravenously). In vitro doses can be in the range of about 10 -3 Molar concentration and 10 -9Depending on the route of administration, the in vivo therapeutically effective amount may range from about 0.1-500 mg / kg or from about 1-100 mg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 is a graph showing H-scores calculated for samples of various tumor types, including: non-small cell lung cancer ("NSCLC") (adenocarcinoma and squamous cell carcinoma subtypes), prostate cancer ("prostate"), and hepatocellular carcinoma ("HCC"). Also shown are H-scores for clinical trial biopsies of NSCLC, HCC, and head and neck cancer ("H&N") patient tumors carrying NFE2L2 / KEAP1 mutations. The percentage of samples and clinical trial biopsies assigned an H-score greater than 250 (out of 300) is shown.

[0102] Figure 2 is a schematic diagram of the clinical trial study design for a clinical trial to determine dosing of an AKR1C3-dependent KARS inhibitor.

[0103] Figure 3 is a schematic diagram showing the design for determining the safety and efficacy assessment of dosing of an AKR1C3-dependent KARS inhibitor during the screening / baseline period and during the treatment period of a clinical trial. DETAILED DESCRIPTION

[0104] Various embodiments of the invention are described herein. It should be appreciated that the features specified in each embodiment may be combined with other specified features to provide further embodiments of the invention.

[0105] The present disclosure is based, at least in part, on the identification of compounds that inhibit AKR1C3 and methods of using the same to treat AKR1C3-related diseases. Compounds (I) and (II) and their pharmaceutical compositions are disclosed herein:

[0106]

[0107] Compound of formula (I): 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide;

[0108]

[0109] The compound of formula (II), (R)-6'-fluoro-N-(4-fluorobenzyl)-4'-hydroxy-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, is activated in multiple assays and therapeutic models, acting as a selective AKR1C3 inhibitor.

[0110] In one aspect, the invention provides a method of identifying a subject for treatment with a compound of formula (I):

[0111]

[0112] in is a single bond or a double bond;

[0113] Z OH when it is a single bond; or When it is a double bond, it is O;

[0114] Each R 1 independently selected from the group consisting of: (C1-C6)alkyl, (C1-C6)alkoxy, (C0-C4)alkylN(R 8 )2 and halogenated;

[0115] R 2a and R 2b Each independently selected from the group consisting of: H, (C1-C6)alkyl and halo;

[0116] Each R 3 are independently selected from the group consisting of: H and halo;

[0117] R 4 is selected from the group consisting of: aryl; 5- to 6-membered heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; and 9- to 10-membered fused bicyclic heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; wherein any of the foregoing groups is optionally replaced by one or more R 6 replace;

[0118] R 5 Selected from the group consisting of: H, (C1-C6) alkyl, (C2-C6) alkenyl, (C0-C4) alkyl, OR 8 、(C1-C4)alkyl(C3-C 10 )cycloalkyl, halo(C1-C6)alkyl, (C2-C3)alkynyl, (C1-C4)alkylN(R 10 )2;

[0119] Each R 6 independently selected from the group consisting of halo, (C1-C6) alkyl, (C1-C6) alkoxy, halo (C1-C6) alkyl, OH, aryl, 3- to 6-membered heterocyclic, 5- to 6-membered heteroaryl, (C0-C4) alkyl, S(O) m (C1-C6) alkyl, halogenated (C1-C6) alkoxy, (C0-C4) alkyl S(O) m N(R 8)2、(C0-C4)alkyl N(R 8 )2、(C0-C4)alkyl(CO)OR 7 、N(R 8 )S(O) m (C1-C6) alkyl, N(R 8 )S(O) m (C3-C6)cycloalkyl, OP(O)(OH)2, (C0-C3)alkyl (CO)NHR 11 、(C0-C3)alkylOR 7 and (C3-C 10 )cycloalkyl; wherein each R 6 When not halogenated, OH or OP(O)(OH)2, it is optionally substituted with one to three R 9 Substitution; or two adjacent R 6 Together with the atoms to which they are attached, they form a 5- to 7-membered heterocyclic ring or a (C5-C8)cycloalkyl group;

[0120] Each R 7 and R 8 are independently selected from the group consisting of: H or optionally with one to three R 9 Substituted (C1-C6) alkyl;

[0121] Each R 9 Independently selected from the group consisting of: halo, -OH, amino, (C 1- C4) alkylamino, di(C 1- C4)alkylamino, OP(O)(OH)2, (C1-C6)alkyl, (C1-C3)alkynyl, (C1-C6)alkoxy, halo(C1-C6)alkyl, (C0-C4)alkylS(O) m (C1-C6) alkyl, halo (C1-C6) alkoxy, 3- to 6-membered heterocyclic ring optionally substituted by oxo (=O), (C0-C4) alkyl S (O) m N(R 10 )2、(C0-C4)alkyl(CO)R 10 、(C0-C4)alkyl(CO)OR 10 、(C0-C4) alkyl NR 10 S(O) m (C1-C6)alkyl, (C0-C4)alkylOR 10 、(C0-C4)alkyl N(R 10 )2, (C0-C4) alkyl CN, (C0-C4) alkyl N (R 10 )2 and (C0-C4)alkyl (CO)N(R 10 )2;

[0122] Each R10 independently selected from the group consisting of: H, (C1-C6) alkyl, or a 3- to 6-membered heterocycle, wherein the 3- to 6-membered heterocycle is optionally substituted with one or more of: (C1-C6) alkyl and oxo (=O);

[0123] Each R 11 Selected from the group consisting of: H, optionally with one to four R 12 substituted 4 to 6 membered heterocyclic ring, optionally substituted by one to four R 12 substituted (C3-C6) cycloalkyl, optionally substituted (C0-C3) alkyl, (C3-C6) cycloalkyl (C1-C3) alkyl, optionally substituted by one to three R 12 substituted CH2-aryl, (C1-C6)alkyl, (C2-C6)alkenyl or (C2-C6)alkynyl, wherein each of the (C1-C6)alkyl, (C2-C6)alkenyl and (C2-C6)alkynyl is optionally substituted with one or more R 13 replace;

[0124] Each R 12 independently selected from the group consisting of OH, (C1-C3)alkoxy, NH2 or (C1-C3)alkyl optionally substituted with one or more OH;

[0125] Each R 13 Independently selected from the group consisting of: halo, OH, amino, (C 1- C4) alkylamino, di(C 1- C4) alkylamino, (C1-C3) alkoxy and C(O)-(C3-C8) cycloalkyl;

[0126] m is 0, 1 or 2; and

[0127] n is 0, 1, or 2,

[0128] or a pharmaceutically acceptable salt thereof,

[0129] The method comprises determining the level of AKR1C3 in a sample from the subject, wherein elevated levels of AKR1C3 identify the subject as a subject in need of treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0130] Also described herein is a method of selecting a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating a subject, the method comprising determining the level of AKR1C3 in a sample from the subject, wherein an elevated level of AKR1C3 identifies the subject as a subject in need of treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0131] In another aspect, the present invention provides a method of treating a subject, the method comprising: determining the level of AKR1C3 in a sample from the subject, wherein elevated levels of AKR1C3 identify the subject as in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof; and administering to the subject an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0132] In another aspect, the present invention provides a method of treating a subject, the method comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein prior to said administering, a sample from the subject is characterized as having elevated AKR1C3 levels.

[0133] In another aspect, the invention provides a method of treating a subject with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein a sample from the subject is characterized as having elevated levels of AKR1C3.

[0134] In some embodiments, the compound of formula (I) is selected from the group consisting of: 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3', 4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3', 4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3', 4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide or a pharmaceutically acceptable salt thereof.

[0135] In some embodiments of the methods described herein, the AKR1C3 levels are levels of AKR1C3 protein. In some embodiments of the methods described herein, the AKR1C3 levels are levels of AKR1C3 nucleic acid species (eg, AKR1C3 mRNA).

[0136] In some embodiments, the subject sample includes a cell, a cell group, a cell lysate, a tissue or a fluid of the subject. In some embodiments of the methods described herein, the cell is a cancer cell. In some embodiments, the cancer cell is a tumor cell. In some embodiments, the tumor cell is selected from the group consisting of: lung cancer tumor cells, non-small cell lung cancer tumor cells, lung adenocarcinoma tumor cells, lung squamous cell carcinoma cells, bladder tumor cells, cervical tumor cells, esophageal tumor cells, head and neck tumor cells, kidney tumor cells and liver tumor cells. In some embodiments, the cell is a lung cell. In some embodiments, the fluid is selected from the group consisting of: blood, plasma and lymph. In some embodiments, the subject sample comprises a genome, transcriptome or proteome of a cell (e.g., a subject tumor cell). In some embodiments, the subject sample comprises a genome, transcriptome or proteome of any of the aforementioned cells or fluids.

[0137] In some embodiments of the methods described herein, the subject is diagnosed with a disease or disorder selected from the group consisting of non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer, cervical cancer, esophageal cancer, head and neck cancer, kidney cancer, and liver cancer.

[0138] In some embodiments of the methods described herein, the subject's tumor genome comprises a somatic mutation in one or more of the NFE2L2, KEAP1, or CUL3 gene sequences.

[0139] In some embodiments of the methods described herein, the AKR1C3 level is elevated relative to the control AKR1C3 level. In some embodiments, the control level includes the AKR1C3 level of a control sample or a control data set. In some embodiments, the control sample includes a sample selected from the group consisting of: non-cancer cells of the subject, non-cancer cell populations of the subject, non-cancerous tissues of the subject, non-cancerous fluids of the subject, non-cancer cells of the control subject, non-cancerous tissues of the control subject, and non-cancerous fluids of the control subject. In some embodiments, the control data set includes AKR1C3 level data from a source selected from the group consisting of: non-cancer cells of the subject, non-cancer cell populations of the subject, non-cancerous tissues of the subject, non-cancerous fluids of the subject, non-cancer cells of the control subject, non-cancerous tissues of the control subject, and combinations thereof. In some embodiments, the control sample includes the genome, transcriptome, or proteome of the non-cancer cells of the subject, non-cancer cell populations of the subject, non-cancerous tissues of the subject, non-cancerous fluids of the subject, non-cancer cells of the control subject, non-cancerous tissues of the control subject, or non-cancerous fluids of the control subject. In some embodiments, the control sample includes the genome, transcriptome, or proteome of any of the aforementioned cells or fluids. In some embodiments, the control data set comprises AKR1C3 level data from a source selected from the group consisting of: a genome, transcriptome, or proteome of a non-cancerous cell of a subject, a population of non-cancerous cell of a subject, a non-cancerous tissue of a subject, a non-cancerous fluid of a subject, a non-cancerous cell of a control subject, a population of non-cancerous cell of a control subject, a non-cancerous tissue of a control subject, a non-cancerous fluid of a control subject, and combinations thereof.

[0140] In some embodiments of the methods described herein, the AKR1C3 levels are AKR1C3 protein levels.

[0141] In some embodiments of the methods described herein, AKR1C3 levels are AKR1C3 RNA levels. In some embodiments, AKR1C3 RNA levels are AKR1C3 mRNA levels.

[0142] In some embodiments of the methods described herein, if the level of AKR1C3 in a sample from a subject is about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 1500 times, or about 2000 times the level of AKR1C3 in a control sample or control dataset, then the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0143] In some embodiments of the methods described herein, if the level of AKR1C3 in a sample from a subject is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1000 times, at least about 1500 times, or at least about 2000 times the level of AKR1C3 in a control sample or control dataset, then the subject is in need of treatment with a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0144] In some embodiments of the methods described herein, determining the level of AKR1C3 in a subject sample further comprises performing an antigen detection assay. In some embodiments, the antigen detection assay is selected from the group consisting of: a Western blot assay, an enzyme-linked immunosorbent assay (ELISA), an immunohistochemistry (IHC) assay, an immunocytochemistry assay, a flow cytometry assay, an immunoprecipitation assay, an immunoelectrophoresis assay, and an immunoelectron microscopy assay. In some embodiments, the antigen detection assay is an IHC assay.

[0145] In some embodiments, performing an antigen detection assay comprises probing a subject sample with an AKR1C3 antibody. In some embodiments, the AKR1C3 antibody is an anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6. In some embodiments, the AKR1C3 antibody comprises a CDR sequence that shares at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with the CDR sequence of the anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6. In some embodiments, the AKR1C3 antibody is conjugated to horseradish peroxidase (HRP).

[0146] In some embodiments, performing the antigen detection assay further comprises probing the subject sample with a secondary antibody. In some embodiments, the secondary antibody is conjugated to HRP.

[0147] In some embodiments, the antigen detection assay further comprises applying 3,3'-diaminobenzidine (DAB) to the subject sample.

[0148] In some embodiments of the methods described herein, determining the level of AKR1C3 in the subject sample further comprises generating an IHC signal intensity score for the subject sample. In some embodiments, if the IHC signal intensity score for the subject sample is 0.5 or greater, 1.0 or greater, 1.5 or greater, 2.0 or greater, 2.5 or greater, 2.6 or greater, 2.7 or greater, 2.8 or greater, or 2.9 or greater, and the IHC signal intensity score is in the range of 0-3.0, then the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, if the IHC signal intensity score for the subject sample is 50 or greater, 100 or greater, 150 or greater, 200 or greater, 250 or greater, 260 or greater, 270 or greater, 280 or greater, or 290 or greater, and the IHC signal intensity score is in the range of 0-300, then the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0149] In some embodiments of the methods described herein, the subject sample is characterized as having elevated AKR1C3 levels by an antigen detection assay. In some embodiments, the antigen detection assay is selected from the group consisting of: Western blot assay, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC) assay, immunocytochemistry assay, flow cytometry assay, immunoprecipitation assay, immunoelectrophoresis assay, and immunoelectron microscopy assay. In some embodiments, the antigen detection assay is an IHC assay.

[0150] In some embodiments, the antigen detection assay comprises probing a subject sample with an AKR1C3 antibody. In some embodiments, the AKR1C3 antibody is an anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6. In some embodiments, the AKR1C3 antibody comprises a CDR sequence that shares at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with the CDR sequence of the anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6. In some embodiments, the AKR1C3 antibody is conjugated to horseradish peroxidase (HRP).

[0151] In some embodiments, the antigen detection assay further comprises probing the subject sample with a secondary antibody. In some embodiments, the secondary antibody is conjugated to HRP.

[0152] In some embodiments, the antigen detection assay further comprises applying 3,3'-diaminobenzidine (DAB) to the subject sample.

[0153] In some embodiments, the antigen detection assay further comprises generating an IHC signal intensity score for the subject's sample. In some embodiments, if the IHC signal intensity score of the subject's tissue sample is 0.5 or greater, 1.0 or greater, 1.5 or greater, 2.0 or greater, 2.5 or greater, 2.6 or greater, 2.7 or greater, 2.8 or greater, or 2.9 or greater, and the IHC signal intensity score is in the range of 0-3.0, the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, if the IHC signal intensity score of the subject's tissue sample is 50 or greater, 100 or greater, 150 or greater, 200 or greater, 250 or greater, 260 or greater, 270 or greater, 280 or greater, or 290 or greater, and the IHC signal intensity score is in the range of 0-300, the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, if the IHC signal intensity score of the subject's tissue sample is greater than the IHC signal intensity score of the control sample or control data set, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, if the IHC signal intensity score of the subject's tissue sample is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% higher than the IHC signal intensity score of the control sample or control data set, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0154] In some embodiments of the methods described herein, determining the level of AKR1C3 in the subject sample comprises performing a polymerase chain reaction (PCR) effective to determine the level of AKR1C3 in the subject sample. In some embodiments, determining the level of AKR1C3 in the subject sample further comprises performing a PCR effective to determine the level of a control marker in the subject sample. In some embodiments, the control marker is selected from the group consisting of: beta actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0155] In some embodiments, determining the level of AKR1C3 in the subject sample further comprises performing PCR effective to determine the level of AKR1C3 in the control sample. In some embodiments, determining the level of AKR1C3 in the subject sample further comprises performing PCR effective to determine the level of a control marker in the control sample. In some embodiments, the control marker is beta actin or GAPDH.

[0156] As described above, in some embodiments of the methods described herein, determining the level of AKR1C3 in a subject sample comprises performing a polymerase chain reaction (PCR) effective to determine the level of AKR1C3 and / or a control marker in a subject sample or a control sample. In some embodiments, PCR is quantitative PCR (qPCR). In some embodiments, PCR is RT-PCR. In some embodiments, PCR is RT-qPCR. In some embodiments, PCR is digital PCR.

[0157] In some embodiments of the methods described herein, the subject sample is characterized as having an elevated AKR1C3 level by PCR that effectively determines the AKR1C3 level in the subject sample. In some embodiments, the level of AKR1C3 is determined in the control sample by PCR that effectively determines the AKR1C3 level in the control sample. In some embodiments, the level of the control marker is determined in the subject sample by PCR that effectively determines the control marker level in the subject sample. In some embodiments, the level of the control marker is determined in the control sample by PCR that effectively determines the control marker level in the control sample. In some embodiments, the control marker is selected from the group consisting of: beta actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0158] As described above, in some embodiments of the methods described herein, the level of AKR1C3 or the level of a control marker is determined in a subject sample or a control sample by performing a polymerase chain reaction (PCR) effective to determine the level of AKR1C3 or the level of a control marker in the subject sample or the control sample. In some embodiments, the PCR is qPCR. In some embodiments, the PCR is RT-PCR. In some embodiments, the PCR is RT-qPCR. In some embodiments, the PCR is digital PCR.

[0159] In another aspect, the present invention provides a method for identifying a subject in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, the method comprising detecting a somatic mutation in at least one of the following genes in a sample from the subject: NFE2L2, KEAP1 or CUL3.

[0160] In another aspect, the present invention provides a method for selecting a compound of formula (I) or a pharmaceutically acceptable salt thereof to treat a subject, the method comprising detecting a somatic mutation in at least one of the following genes in a sample from the subject: NFE2L2, KEAP1 or CUL3.

[0161] In another aspect, the present invention provides a method for treating a subject, the method comprising: detecting a somatic mutation in at least one of the following genes in a sample from the subject: NFE2L2, KEAP1 or CUL3, wherein the detection identifies the subject as needing treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof; and administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof to the subject.

[0162] In another aspect, the present invention provides a method of treating a subject, the method comprising administering to the subject an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein prior to said administration, a sample from the subject is characterized by the presence of a somatic mutation in at least one of the following genes: NFE2L2, KEAP1, or CUL3.

[0163] In another aspect, the invention provides a method of treating a subject with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the subject sample is characterized by the presence of a somatic mutation in at least one of the following genes: NFE2L2, KEAP1 or CUL3.

[0164] In some embodiments of the methods described herein, somatic mutations include mutations selected from the group consisting of nonsense mutations, missense mutations, substitution mutations, frameshift mutations, point mutations, insertion mutations, amplification mutations (e.g., gene amplification), deletion mutations (e.g., gene deletion), inversion mutations, and duplication mutations. In some embodiments, somatic mutations are mutations in the genome of tumor cells.

[0165] In some embodiments of the methods described herein, the somatic mutation comprises a single nucleotide polymorphism (SNP).

[0166] In some embodiments of the methods described herein, the subject sample includes a cell, a cell group, a cell lysate, a tissue or a fluid of the subject. In certain embodiments, the subject sample includes the genomic DNA of the cell, a cell group, a cell lysate, a tissue or a fluid of the subject. In certain embodiments, the cell is a cancer cell. In certain embodiments, the cancer cell is a tumor cell. In certain embodiments, the tumor cell is selected from the group consisting of: lung cancer tumor cells, non-small cell lung cancer tumor cells, lung adenocarcinoma tumor cells, lung squamous cell cancer cells, bladder tumor cells, cervical tumor cells, esophageal tumor cells, head and neck tumor cells, kidney tumor cells and liver tumor cells. In certain embodiments, the cell is a lung cell. In certain embodiments, the fluid is selected from the group consisting of: blood, plasma and lymph. In certain embodiments, the subject sample includes the genome or transcriptome of the cell (e.g., tumor cell) of the subject. In certain embodiments, the subject sample includes the genome or transcriptome of any one of the aforementioned cells or fluids of the subject.

[0167] In some embodiments of the methods described herein, somatic mutations are not present in the control sample or control data set. In some embodiments, the control sample includes a sample selected from the group consisting of: non-cancer cells of the subject, non-cancer cell groups of the subject, non-cancerous tissues of the subject, non-cancerous fluids of the subject, non-cancer cells of the control subject, non-cancerous tissues of the control subject, and non-cancerous fluids of the control subject. In some embodiments, the control sample includes genomic DNA of the following items: non-cancer cells of the subject, non-cancer cell groups of the subject, non-cancerous tissues of the subject, non-cancerous fluids of the subject, non-cancer cells of the control subject, non-cancerous tissues of the control subject, or non-cancerous fluids of the control subject. In some embodiments, the control data set includes genomic sequence data from a source selected from the group consisting of: non-cancer cells of the subject, non-cancerous cell groups of the subject, non-cancerous tissues of the subject, non-cancerous fluids of the subject, non-cancerous cells of the control subject, non-cancerous tissues of the control subject, and combinations thereof. In some embodiments, the control sample includes genome or transcriptome of non-cancer cells of the subject, non-cancerous cell groups of the subject, non-cancerous tissues of the subject, non-cancerous fluids of the subject, non-cancerous cells of the control subject, non-cancerous tissues of the control subject, or non-cancerous fluids of the control subject. In some embodiments, the control sample comprises the genome or transcriptome of any of the aforementioned cells or fluids. In some embodiments, the control data set comprises genome or transcriptome data from a source selected from the group consisting of: a non-cancer cell of a subject, a non-cancer cell population of a subject, a non-cancerous tissue of a subject, a non-cancerous fluid of a subject, a genome or transcriptome of a control subject, a non-cancerous cell population of a control subject, a non-cancerous tissue of a control subject, a non-cancerous fluid of a control subject, and a combination thereof.

[0168] In some embodiments of the methods described herein, the level of AKR1C3 (e.g., AKR1C3 mRNA or AKR1C3 protein) in a sample from a subject is about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 1500-fold, or about 2000-fold higher than the level of AKR1C3 (e.g., AKR1C3 mRNA or AKR1C3 protein) in a control sample or control dataset.

[0169] In some embodiments of the methods described herein, the level of AKR1C3 (eg, AKR1C3 mRNA or AKR1C3 protein) in a sample from a subject is a level of AKR1C3 (eg, AKR1C3 mRNA or AKR1C3 protein) in a control sample or control dataset. mRNA or AKR1C3 protein) levels by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, at least about 1500-fold, or at least about 2000-fold.

[0170] In some embodiments of the methods described herein, detecting a somatic mutation in at least one of the following genes in a subject sample comprises sequencing genomic DNA of the subject sample: NFE2L2, KEAP1, or CUL3. In some embodiments of the methods described herein, detecting a somatic mutation in at least one of the following genes in a subject sample comprises sequencing mRNA of the subject sample: NFE2L2, KEAP1, or CUL3. In some embodiments, sequencing is selected from the group consisting of: exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing, and Heliscope single molecule sequencing. In some embodiments, sequencing includes performing exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrophosphate sequencing, sequencing by synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing or Heliscope single molecule sequencing. In some embodiments, sequencing includes performing polymerase chain reaction (PCR). In some embodiments, PCR is selected from the group consisting of: qPCR, RT-PCR, RT-qPCR and digital PCR.

[0171] In some embodiments of the methods described herein, if sequencing detects a somatic mutation in a sample from a subject, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0172] In some embodiments of the methods described herein, the method further comprises sequencing the genomic DNA of the control sample. In some embodiments of the methods described herein, the method further comprises comparing the sequencing data of sequencing the genomic DNA of the subject sample with the sequencing data of the control sample or the control data set.

[0173] Alternatively, in some embodiments of the methods described herein, the method further comprises sequencing the mRNA of the control sample. In some embodiments of the methods described herein, the method further comprises comparing the sequencing data of sequencing the mRNA of the subject sample with the sequencing data of the control sample or the control data set.

[0174] In some embodiments of the method described herein, the subject sample is characterized in that there is somatic mutation by sequencing the genomic DNA of the subject sample. In some embodiments of the method described herein, the subject sample is characterized in that there is somatic mutation by sequencing the mRNA of the subject sample. In certain embodiments, sequencing is selected from the group consisting of: exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrophosphate sequencing, sequencing while synthesizing, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing and Heliscope single molecule sequencing. In some embodiments, sequencing includes performing exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrophosphate sequencing, sequencing by synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing or Heliscope single molecule sequencing. In some embodiments, sequencing includes performing polymerase chain reaction (PCR). In some embodiments, PCR is selected from the group consisting of: qPCR, RT-PCR, RT-qPCR and digital PCR.

[0175] In some embodiments of the methods described herein, if sequencing detects a somatic mutation in a sample from a subject, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0176] In some embodiments of the methods described herein, the control sample is characterized by the absence of somatic mutations by sequencing the genomic DNA of the control sample. In some embodiments, the methods described herein further include comparing the sequencing data of the genomic DNA of the subject sample to the sequencing data of the control sample or the control data set.

[0177] In some embodiments of the methods described herein, the control sample is characterized by the absence of somatic mutations by sequencing the mRNA of the control sample. In some embodiments, the methods described herein further include comparing the sequencing data of the mRNA of the subject sample to the sequencing data of the control sample or the control data set.

[0178] In another aspect, the present invention provides the use of AKR1C3 levels for selecting a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein if a sample from the subject is characterized as having elevated AKR1C3 levels, the subject is treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0179] In another aspect, the present invention provides the use of somatic mutations for selecting a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the subject is treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof if the subject's sample is characterized by the presence of somatic mutations and wherein the somatic mutation is detected in one of the following genes: NFE2L2, KEAP1 or CUL3.

[0180] In some embodiments of the uses described herein, the compound of formula (I) is selected from the group consisting of: 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof.

[0181] definition

[0182] For the purpose of interpreting this specification, the following definitions shall apply unless otherwise specified and, where appropriate, terms used in the singular shall also include the plural, and vice versa.

[0183] As used herein, the terms "a / an", "the", and similar terms used in the context of the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0184] As used herein, the term “ 1- "C6) alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms, and attached to the remainder of the molecule by a single bond. 1- C4) alkyl" should be interpreted accordingly. 1- Examples of C6) alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, and 1,1-dimethylethyl (tert-butyl).

[0185] As used herein, the term “ 2- "C6)alkenyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to six carbon atoms, attached to the remainder of the molecule by a single bond. 2- C4) alkenyl" should be interpreted accordingly. 2- Examples of C6) alkenyl groups include, but are not limited to, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-4-enyl, and pent-1,4-dienyl.

[0186] As used herein, the term “ 2- C6) alkynyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to six carbon atoms, and attached to the remainder of the molecule by a single bond. 2- C4) alkynyl" should be interpreted accordingly. 2- C6) Examples of alkynyl groups include, but are not limited to, ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl, and pent-1,4-diynyl.

[0187] As used herein, the term “ 1- "C6) alkoxy" refers to the formula -OR a A group in which R a is generally defined as above (C 1- C6) alkyl. 1-Examples of C6) alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentyloxy, and hexyloxy.

[0188] As used herein, the term “ 1- C6) alkoxy (C 1- "C6) alkyl" refers to the formula -R a -OR a A group in which each R a are independently as defined above (C 1- C6) alkyl. The oxygen atom may be bonded to any carbon atom in any alkyl group. 1- C6) alkoxy (C 1- Examples of C6) alkyl include, but are not limited to, methoxy-methyl, methoxy-ethyl, ethoxy-ethyl, 1-ethoxy-propyl and 2-methoxy-butyl.

[0189] As used herein, the term “ 1- "C4) alkylcarbonyl" refers to a group of the formula -C(=O)-R a A group in which R a is defined as above (C 1- C4) alkyl.

[0190] As used herein, the term “ 1- C6) alkylcarbonyl (C 1- "C6) alkyl" refers to the formula -R a -C(=O)-R a A group in which each R a are independently as defined above (C 1- C6) alkyl. The carbon atom of the carbonyl group may be bonded to any carbon atom in any alkyl group.

[0191] As used herein, the term “ 1- C6) alkoxycarbonyl" refers to the formula -C(=O)-OR a A group in which R a is defined as above (C 1- C6) alkyl.

[0192] As used herein, the term “ 1- C6) alkoxycarbonyl (C 1- "C6) alkyl" refers to the formula -R a -C(=O)-OR a A group in which each R a are independently as defined above (C 1- C6) alkyl.

[0193] As used herein, the term “ 1-C4) "alkoxycarbonylamino" refers to the formula -NH-C(=O)-OR a A group in which R a is defined as above (C 1- C4) alkyl.

[0194] As used herein, the term "hydroxyl (C 1- "C6) alkyl" means a (C 1- C6) alkyl, wherein C 1-6 One of the hydrogen atoms of the alkyl group is replaced by OH. 1- Examples of C6) alkyl include, but are not limited to, hydroxy-methyl, 2-hydroxy-ethyl, 2-hydroxy-propyl, 3-hydroxy-propyl and 5-hydroxy-pentyl.

[0195] As used herein, the term "amino (C 1- "C6) alkyl" means a (C 1- C6) alkyl radical, wherein (C 1- One of the hydrogen atoms of an amino (C6) alkyl group is replaced by a primary amino group. 1- Representative examples of C6) alkyl include, but are not limited to, amino-methyl, 2-amino-ethyl, 2-amino-propyl, 3-amino-propyl, 3-amino-pentyl, and 5-amino-pentyl.

[0196] As used herein, the term “ 1- "C4) alkylamino" refers to the formula -NH-R a The free radical, R a is defined as above (C 1- C4) alkyl radicals.

[0197] As used herein, the term “ 1- C4) alkylamino (C 1- "C6) alkyl" refers to the formula -R a1 -NH-R a2 The free radical, R a1 is defined as above (C 1- C6) alkyl radical, and R a2 is defined as above (C 1- C4) alkyl radicals. The nitrogen atom can be bonded to any carbon atom in any alkyl radical.

[0198] As used herein, the term " 1- "C4) alkylamino" refers to the formula -N(R a )-R a A free radical, where each R a are as defined above and may be the same or different (C 1-C4) alkyl radicals.

[0199] As used herein, the term " 1- C4) alkylamino (C 1- "C6) alkyl" refers to the formula -R a1 -N(R a2 )-R a2 The free radical, R a1 is defined as above (C 1- C6) alkyl radical, and each R a2 are as defined above and may be the same or different (C 1- C4) alkyl radicals. The nitrogen atom can be bonded to any carbon atom in any alkyl radical.

[0200] As used herein, the term "aminocarbonyl" refers to a free radical of the formula -C(=O)-NH2.

[0201] As used herein, the term "aminocarbonyl C 1-6 "Alkyl" refers to a group of the formula -R a -C(=O)-NH2 free radical, where R a is defined as above (C 1- C6) alkyl radicals.

[0202] As used herein, the term “ 1- "C4) alkylaminocarbonyl" refers to a group of the formula -C(=O)-NH-R a The free radical, R a is defined as above (C 1- C4) alkyl radicals.

[0203] As used herein, the term “ 1- C4) alkylaminocarbonyl 1-6 "Alkyl" refers to a group of the formula -R a1 -C(=O)-NH-R a2 The free radical, R a1 is defined as above (C 1- C6) alkyl radical, and R a2 is defined as above (C 1- C4) alkyl radicals.

[0204] As used herein, the term " 1- "C4) alkylaminocarbonyl" refers to a group of the formula -C(=O)-N(R a )-R a A free radical, where each R a are as defined above and may be the same or different (C 1- C4) alkyl radicals.

[0205] As used herein, the term " 1- C4) alkylaminocarbonyl 1-6 "Alkyl" refers to a group of the formula -R a1 -C(=O)-N(R a2 )-R a2 The free radical, R a1 is C as defined above 1-6 alkyl radical, and each R a2 are as defined above and may be the same or different (C 1- C4) alkyl radicals.

[0206] As used herein, the term “ 3- C8) Cycloalkyl (C 0- "C6) alkyl" means a group consisting of only carbon and hydrogen atoms, having three to eight carbon atoms and connected by a single bond or by a (C 1- C6) Alkyl radical A stable monocyclic saturated hydrocarbon radical attached to the rest of the molecule. 3- C8) Cycloalkyl (C 0- Examples of C6) alkyl include, but are not limited to, cyclopropyl, cyclopropyl-methyl, cyclobutyl, cyclobutyl-ethyl, cyclopentyl, cyclopentyl-propyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0207] The term "aryl" refers to a 6- to 10-membered aromatic carbocyclic moiety having a single ring system (eg, phenyl) or a fused ring system (eg, naphthalene). A typical aryl group is a phenyl group.

[0208] As used herein, the term "phenyl (C 0- "C6) alkyl" means a group consisting of (C 1- C6) alkyl radical attached to the benzene ring of the rest of the molecule. 0- Examples of C6) alkyl groups include, but are not limited to, phenyl and benzyl.

[0209] As used herein, the term "phenyl (C 0- C6) alkylamino (C 1- "C6) alkyl" refers to the formula -R a -NH-R b The free radical, R a is defined as above (C 1- C6) alkyl radical, and R b is a phenyl group as defined above (C 0- C6) alkyl radicals.

[0210] As used herein, the term "phenyl (C 0- C6)alkylamino 1-C4) alkyl) (C 1- "C6) alkyl" refers to the formula -R a1 -N(R a2 )-R b The free radical, R a1 is defined as above (C 1- C6) alkyl radical, R a2 is defined as above (C 1- C4) an alkyl radical, and R b is a phenyl group as defined above (C 0- C6) alkyl radicals.

[0211] As used herein, halo refers to bromo, chloro, fluoro or iodo.

[0212] As used herein, the term "halo (C 1- "C6) alkyl" refers to a (C6) alkyl group as defined above substituted by one or more halogen groups as defined above 1- C6) alkyl radical. Halogen (C 1- Examples of C6) alkyl include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,3-dibromopropan-2-yl, 3-bromo-2-fluoropropyl, and 1,4,4-trifluorobutan-2-yl.

[0213] The term "heterocyclic group" refers to a saturated or partially saturated (but non-aromatic) ring or ring system, including monocycles, fused rings, bridged rings and spirocycles with a specified number of ring atoms. For example, heterocyclic groups include but are not limited to 5 to 6-membered heterocyclic groups, 4 to 10-membered heterocyclic groups, 4 to 14-membered heterocyclic groups and 5 to 14-membered heterocyclic groups. Unless otherwise specified, heterocyclic groups contain 1 to 7, 1 to 5, 1 to 3 or 1 to 2 heteroatoms independently selected from the group consisting of the following as ring members: nitrogen, oxygen and sulfur, wherein N and S can also be optionally oxidized to various oxidation states. The heterocyclic group can be attached to a heteroatom or a carbon atom. Examples of such heterocyclic groups include, but are not limited to, azetidine, oxetane, piperidine, piperazine, pyrroline, pyrrolidine, imidazolidine, imidazoline, morpholine, tetrahydrofuran, tetrahydrothiophene, tetrahydrothiopyran, tetrahydropyran, 1,4-dioxane, 1,4-oxathiinane, hexahydropyrimidinyl, 3-azabicyclo[3.1.0]hexane, azepane, 3-azabicyclo[3.2.2]nonane, decahydroisoquinoline, 2-azaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3] heptane, 8-aza-bicyclo[3.2.1]octane, 3,8-diazabicyclo[3.2.1]octane, 3-oxa-8-aza-bicyclo[3.2.1]octane, 8-oxa-3-aza-bicyclo[3.2.1]octane, 2-oxa-5-aza-bicyclo[2.2.1]heptane, 2,5-diaza-bicyclo[2.2.1]heptane, 1,4-dioxa-8-aza-spiro[4.5]decane, 3-oxa-1,8-diazaspiro[4.5]decane, octahydropyrrolo[3,2-b]pyrrole, and the like.

[0214] The term "fused heterocyclic group" refers to a heterocyclic group as defined above fused to an aryl (e.g., phenyl) or heteroaryl ring as defined above. Examples of such fused heterocyclic groups include, but are not limited to, 1,2,3,4-tetrahydroisoquinoline, indoline, isoindoline, 1,2,3,4-tetrahydro-2,7-naphthyridine, 5,6,7,8-tetrahydro-1,7-naphthyridine, 1,2,3,4-tetrahydro-2,6-naphthyridine, 5,6,7,8-tetrahydro-1,6-naphthyridine, 2,3,4,5-tetrahydro-1H-benzo[d]azepine, 1,2,3,4-tetrahydro-1,4-epiaminonaphthalene, 2,3-dihydrobenzofuran, 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine, and the like. As used herein, the term "heterocyclic group (C 0- "C6) alkyl" means a group consisting of (C 1- A C6) alkyl radical is attached to a heterocycle as defined above to the remainder of the molecule.

[0215] The term "heteroaryl" refers to an aromatic moiety containing at least one heteroatom (e.g., oxygen, sulfur, nitrogen, or a combination thereof) within a 5- to 10-membered aromatic ring system (e.g., pyrrolyl, pyridyl, pyrazolyl, indolyl, indazolyl, thienyl, furanyl, benzofuranyl, oxazolyl, isoxazolyl, imidazolyl, triazolyl, tetrazolyl, triazinyl, pyrimidinyl, pyrazinyl, thiazolyl, purinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl, benzopyranyl, benzothienyl, benzimidazolyl, benzoxazolyl, 1H-benzo[d][1,2,3]triazolyl, etc.). The heteroaromatic moiety can consist of a single ring system or a fused ring system. A typical single heteroaryl ring is a 5- to 6-membered ring containing one to three heteroatoms independently selected from oxygen, sulfur and nitrogen, and a typical fused heteroaryl ring system is a 9- to 10-membered ring system containing one to four heteroatoms independently selected from oxygen, sulfur and nitrogen. A fused heteroaryl ring system may consist of two heteroaryl rings fused together or a heteroaryl fused to an aryl (e.g., phenyl). As used herein, the term “heteroaryl (C 0- "C6) alkyl" means a group consisting of (C 1- A C6) alkyl radical is attached to a heteroaryl ring as defined above of the rest of the molecule.

[0216] Unless otherwise specified, the term "compounds of the invention" refers to compounds of formula (I) and salts thereof as defined herein, as well as all stereoisomers (including diastereomers and enantiomers), rotamers, tautomers and isotopically labeled compounds (including deuterated substitutions) and inherently formed moieties. The term "compounds of the invention" refers to compounds as defined in any of the Examples mentioned below.

[0217] Compounds of formula (I)

[0218] Embodiments of the invention described herein relate, in part, to a compound of formula (I): or a pharmaceutically acceptable salt thereof:

[0219]

[0220] in:

[0221] is a single bond or a double bond;

[0222] Z When it is a single bond, it is OH; or When it is a double bond, it is O;

[0223] Each R 1 independently selected from the group consisting of: (C1-C6)alkyl, (C1-C6)alkoxy, (C0-C4)alkylN(R 8 )2 and halogenated;

[0224] R 2a and R 2b Each independently selected from the group consisting of: H, (C1-C6)alkyl and halo;

[0225] Each R 3 are independently selected from the group consisting of: H and halo;

[0226] R 4 is selected from the group consisting of: aryl; 5- to 6-membered heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; and 9- to 10-membered fused bicyclic heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; wherein any of the foregoing groups is optionally replaced by one or more R 6 replace;

[0227] R 5 Selected from the group consisting of: H, (C1-C6) alkyl, (C2-C6) alkenyl, (C0-C4) alkyl, OR 8 、(C1-C4)alkyl(C3-C 10 )cycloalkyl, halo(C1-C6)alkyl, (C2-C3)alkynyl, (C1-C4)alkylN(R 10 )2;

[0228] Each R 6 independently selected from the group consisting of halo, (C1-C6) alkyl, (C1-C6) alkoxy, halo (C1-C6) alkyl, OH, aryl, 3- to 6-membered heterocyclic, 5- to 6-membered heteroaryl, (C0-C4) alkyl, S(O) m (C1-C6) alkyl, halogenated (C1-C6) alkoxy, (C0-C4) alkyl S(O) m N(R 8 )2、(C0-C4)alkyl N(R 8 )2、(C0-C4)alkyl(CO)OR 7 、N(R 8 )S(O) m (C1-C6) alkyl, N(R 8 )S(O) m (C3-C6)cycloalkyl, OP(O)(OH)2, (C0-C3)alkyl (CO)NHR 11 、(C0-C3)alkylOR 7 and (C3-C 10 )cycloalkyl; wherein each R 6 When not halogenated, OH or OP(O)(OH)2, it is optionally substituted with one to three R 9 Substitution; or two adjacent R 6Together with the atoms to which they are attached, they form a 5- to 7-membered heterocyclic ring or a (C5-C8)cycloalkyl group;

[0229] Each R 7 and R 8 are independently selected from the group consisting of: H or optionally with one to three R 9 Substituted (C1-C6) alkyl;

[0230] Each R 9 Independently selected from the group consisting of: halo, -OH, amino, (C 1- C4) alkylamino, di(C 1- C4)alkylamino, OP(O)(OH)2, (C1-C6)alkyl, (C1-C3)alkynyl, (C1-C6)alkoxy, halo(C1-C6)alkyl, (C0-C4)alkylS(O) m (C1-C6) alkyl, halo (C1-C6) alkoxy, 3- to 6-membered heterocyclic ring optionally substituted by oxo (=O), (C0-C4) alkyl S (O) m N(R 10 )2、(C0-C4)alkyl(CO)R 10 、(C0-C4)alkyl(CO)OR 10 、(C0-C4) alkyl NR 10 S(O) m (C1-C6)alkyl, (C0-C4)alkylOR 10 、(C0-C4)alkyl N(R 10 )2, (C0-C4) alkyl CN, (C0-C4) alkyl N (R 10 )2 and (C0-C4)alkyl (CO)N(R 10 )2;

[0231] Each R 10 independently selected from the group consisting of: H, (C1-C6) alkyl, or a 3- to 6-membered heterocycle, wherein the 3- to 6-membered heterocycle is optionally substituted with one or more of: (C1-C6) alkyl and oxo (=O);

[0232] Each R 11 Selected from the group consisting of: H, optionally with one to four R 12 substituted 4 to 6 membered heterocyclic ring, optionally substituted by one to four R 12 substituted (C3-C6) cycloalkyl, optionally substituted (C0-C3) alkyl, (C3-C6) cycloalkyl (C1-C3) alkyl, optionally substituted by one to three R 12substituted CH2-aryl, (C1-C6)alkyl, (C2-C6)alkenyl or (C2-C6)alkynyl, wherein each of the (C1-C6)alkyl, (C2-C6)alkenyl and (C2-C6)alkynyl is optionally substituted with one or more R 13 replace;

[0233] Each R 12 independently selected from the group consisting of OH, (C1-C3)alkoxy, NH2 or (C1-C3)alkyl optionally substituted with one or more OH;

[0234] Each R 13 Independently selected from the group consisting of: halo, OH, amino, (C 1- C4) alkylamino, di(C 1- C4) alkylamino, (C1-C3) alkoxy and C(O)-(C3-C8) cycloalkyl;

[0235] m is 0, 1 or 2; and

[0236] n is 0, 1 or 2.

[0237] In some embodiments, the compound of formula (I) is selected from the group consisting of: 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3', 4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3', 4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3', 4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide or a pharmaceutically acceptable salt thereof. The compound of formula (I) is described in International Publication No. WO 2021 / 005586, which is incorporated herein by reference. In some embodiments, the compound of formula (I) is a compound of formula (I) described in International Publication No. WO 2021 / 005586 or a pharmaceutically acceptable salt thereof.

[0238] Pharmaceutical composition

[0239] The compounds of formula (I) or pharmaceutically acceptable salts thereof described herein may be a component of a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

[0240] As used herein, the term "pharmaceutical composition" refers to a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier in a form suitable for oral or parenteral administration. In some embodiments, the pharmaceutical compositions described herein are suitable for oral administration.

[0241] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that can be used to prepare or use a pharmaceutical composition, and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffers, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegrants, lubricants, wetting agents, sweeteners, flavorings, dyes, and combinations thereof, as known to those skilled in the art (see, e.g., Remington The Science and Practice of Pharmacy, 22nd ed., Pharmaceutical Press, 2013, pp. 1049-1070).

[0242] The term "therapeutically effective amount" of a compound of formula (I) refers to an amount of a compound of formula (I) that will elicit a biological or medical response in a subject (e.g., a reduction or inhibition of enzyme or protein activity) or improve symptoms, alleviate a condition, slow or delay disease progression, or prevent a disease. In one non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of formula (I) that, when administered to a subject, is effective to: (1) at least partially alleviate, inhibit, prevent and / or ameliorate a condition or disorder or disease that is (i) mediated by KARS, or (ii) sensitive to KARS inhibition, or (iii) characterized by KARS activity (normal or abnormal); or (2) reduce or inhibit a disease that is sensitive to KARS inhibition. The present invention also provides a method for treating or preventing a disease and / or disorder associated with high AKR1C3 expression or sensitivity to KARS inhibition, the method comprising administering to a subject in need thereof a therapeutically effective amount of an AKR1C3-dependent KARS inhibitor. In some embodiments, the therapeutically effective amount of a compound of formula (I) is effective to inhibit KARS activity when administered to a subject. In some embodiments, inhibition of KARS activity improves disease symptoms, alleviates disease symptoms, slows or delays disease progression, or prevents diseases that are sensitive to KARS inhibition. In some embodiments, a therapeutically effective amount of a compound of formula (I) effectively reduces the number of cancer cells in a subject; reduces the size of a primary tumor; inhibits or prevents cancer cells from infiltrating into surrounding organs; inhibits tumor metastasis; inhibits or prevents tumor growth; and / or alleviates one or more symptoms associated with a disease or disorder to a certain extent. In vivo efficacy can be measured, for example, by assessing survival duration, time to disease progression (TTP), time to recurrence, response rate (e.g., CR and PR), duration of response, and / or quality of life. In some embodiments, in vivo efficacy can be measured, for example, by assessing the enzyme activity or expression level (e.g., mRNA level or protein level) of a biomarker (e.g., the level of AKR1C3). In some embodiments, in vivo efficacy can be measured, for example, by assessing the level of KARS enzyme activity.

[0243] As used herein, the term "subject" refers to primates (e.g., humans (male or female)), monkeys, dogs, rabbits, guinea pigs, pigs, rats, and mice. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human. Unless otherwise indicated, as used herein, the term "subject" is interchangeable with the term "patient."

[0244] As used herein, the term "inhibit" refers to reducing or suppressing a given condition, symptom or disorder or disease, or significantly reducing the baseline activity of a biological activity or process.

[0245] As used herein, the term "treatment" of any disease or disorder refers to relieving or ameliorating the disease or disorder (i.e., slowing or stopping the development of the disease or at least one clinical symptom thereof); or relieving or improving at least one physical parameter or biomarker associated with the disease or disorder (e.g., reducing the level of a biomarker, such as AKR1C3 levels), including those that may not be discernible by the patient. "Treatment" may also refer to regulating a disease or disorder physically (e.g., stabilization of discernible symptoms) or physiologically (e.g., stabilization of a physical parameter) or both. More specifically, "treatment" means any action that improves or maintains the anatomical function affected by a particular disease or disorder and / or the quality of life of a subject suffering from the disease or disorder. As used herein, "treatment" may mean any manner in which one or more symptoms of a disease or disorder are improved or otherwise beneficially altered. As used herein, improvement of the symptoms of a disease or disorder refers to any alleviation that can be attributed to treatment by the methods of the present invention or associated with treatment by the methods of the present invention, whether permanent or temporary, lasting or transient.

[0246] As used herein, the term "prevention" of any disease or disorder refers to the preventive treatment of the disease or disorder; or, for example, delaying the onset or progression of a disease or disorder by preventive treatment. Prevention can include any action to prevent or slow the deterioration of a patient who suffers from a particular disease or disorder and is at risk of deterioration in function, quality of life, and / or another parameter associated with the particular disease or disorder.

[0247] As used herein, a subject is "in need of" a treatment if the subject would benefit biologically, medically, or in quality of life from such treatment.

[0248] All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradictory to the context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention, without limiting the scope of the present invention otherwise claimed.

[0249] The compounds of formula (I) may be in the form of one of the possible stereoisomers, rotamers, atropisomers, tautomers or mixtures thereof, for example as substantially pure geometric (cis or trans) stereoisomers, diastereomers, optical isomers (enantiomers), racemates or mixtures thereof.

[0250] Any resulting mixtures of stereoisomers can be separated on the basis of the physicochemical differences of the components, into the pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and / or fractional crystallization.

[0251] Any resulting racemate of the final product or intermediate can be resolved into optical antipodes by known methods, for example by separating its diastereomeric salts obtained with optically active acids or bases, and releasing optically active acidic or basic compounds to resolve. In particular, basic compounds can therefore be used to resolve the compounds of the present invention into their optical antipodes, for example by fractional crystallization of salts formed with optically active acids, such as tartaric acid, dibenzoyltartaric acid, diacetyltartaric acid, di-O,O'-p-toluoyltartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid. The racemic product can also be resolved by chiral chromatography, for example, using high performance liquid chromatography (HPLC) using a chiral adsorbent.

[0252] In some embodiments, the compound of formula (I) is a component of a pharmaceutical composition. For example, a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier is described herein. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein. The pharmaceutical composition can be formulated for a specific route of administration, such as oral administration, parenteral administration (e.g., by injection, infusion, transdermal or topical administration) and rectal administration. In some embodiments, the pharmaceutical composition described herein is formulated for oral administration. Topical administration may also involve inhalation or intranasal application. The pharmaceutical composition of formula (I) described herein may be prepared in solid form (including but not limited to capsules, tablets, pills, granules, powders or suppositories) or in liquid form (including but not limited to solutions, suspensions or emulsions). Tablets may be film-coated or enteric-coated according to methods known in the art. Typically, a pharmaceutical composition is a tablet or gelatin capsule comprising an active ingredient and one or more of the following:

[0253] a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine;

[0254] b) lubricants, such as silicon dioxide, talc, stearic acid, its magnesium or calcium salts and / or polyethylene glycol; for tablets, also

[0255] c) binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; if desired,

[0256] d) disintegrants, for example starch, agar, alginic acid or its sodium salt or effervescent mixtures; and

[0257] e) Adsorbents, colorants, flavorings and sweeteners.

[0258] In some embodiments, a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a subject (e.g., a patient). Administration can be carried out by any suitable means, including direct delivery to desired organs, cells or tissues, oral, inhaled, intranasal, intratracheal, oral, sublingual, intrathecal, intravenous, intramuscular, intraarticular, subcutaneous, intradermal, intraperitoneal, intraspinal, epidural, intradural, subdural, behind the eyeball, intraocular, intracorneal, conjunctival, intraocular, intravitreal, parenteral, intracranial, intracerebral, intraventricular, directly delivered to the lung and other parenteral administration routes. If desired, administration routes can be combined. In some embodiments, a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof is orally administered to a subject. Administration can be carried out by any suitable route (e.g., by injection, such as intravenous or subcutaneous injection, which depends in part on whether administration is short-lived or long-term). In some embodiments, administration is by oral route. Various dosing plans are contemplated herein, including but not limited to single administration or multiple administrations, push administration and pulse infusion at different time points.

[0259] A composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof may be prescribed or administered to a subject (e.g., a subject requiring treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof) at an appropriate dosage level. For example, a dose of a composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof may be prescribed or administered to a subject. In some embodiments, the dose comprises about 10 mg, about 25 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg or about 1500 mg of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the dose comprises at least about 10 mg, at least about 25 mg, at least about 50 mg, at least about 100 mg, at least about 150 mg, at least about 200 mg, at least about 250 mg, at least about 300 mg, at least about 350 mg, at least about 400 mg, at least about 450 mg, at least about 500 mg, at least about 550 mg, at least about 600 mg, at least about 650 mg, at least about 700 mg, at least about 750 mg, at least about 800 mg, at least about 850 mg, at least about 900 mg, at least about 950 mg, at least about 1000 mg, at least about 1100 mg, at least about 1200 mg, at least about 1300 mg, at least about 1400 mg, or at least about 1500 mg of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the dosage comprises about 10 mg to about 1500 mg, about 50 mg to about 1000 mg, about 50 mg to about 1500 mg, about 50 mg to about 250 mg, about 50 mg to about 500 mg, about 100 mg to about 1000 mg, about 500 mg to about 1000 mg, about 250 mg to about 500 mg, about 500 mg to about 750 mg, about 750 mg to about 1000 mg, about 100 mg to about 400 mg, about 200 mg to about 500 mg, about 300 mg to about 600 mg, about 400 mg to about 700 mg, about 500 mg to about 800 mg, about 600 mg to about 900 mg, or about 700 mg to about 1000 mg of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the dose is administered every 3 hours, every 6 hours, every 8 hours, every 12 hours, every 24 hours, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every week.

[0260] In some embodiments, a composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof is administered orally.

[0261] In some embodiments, the composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof is formulated for oral administration.

[0262] Biomarkers

[0263] Described herein are methods for identifying a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, and methods for treating a subject with a compound of formula (I) or a pharmaceutically acceptable salt thereof. The methods described herein may include a step of detecting or determining the level of a biomarker (e.g., AKR1C3 protein) or a nucleic acid (e.g., AKR1C3 mRNA transcript) in a subject or a subject sample. In some embodiments, an elevated AKR1C3 level identifies the subject as needing treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the methods described herein may include a step of detecting somatic mutations of one or more genes (e.g., NFE2L2, KEAP1, or CUL3) in a subject or a subject sample. In some embodiments, somatic mutations of one or more genes (e.g., somatic mutations of NFE2L2, KEAP1, or CUL3) are biomarkers. In some embodiments, detecting somatic mutations of one or more genes identifies the subject as needing treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0264] As used herein, the term "determine" includes any means of determination, including direct and indirect determination. For example, "determine" may include any means of determining the presence or level (e.g., the level of AKR1C3 protein or mRNA) of a biomarker (e.g., AKR1C3 biomarker) in a subject or subject sample. As used herein, the term "detection" includes any detection means, including direct and indirect detection. For example, "detection" may include any means of detecting the presence or level (e.g., the level of AKR1C3 protein or mRNA) of a biomarker (e.g., AKR1C3 biomarker) in a subject or subject sample. Methods for detecting or determining the presence or level of an AKR1C3 protein biomarker include, but are not limited to, antigen detection and quantitative assays (e.g., Western blot, quantitative Western blot, immunohistochemistry, immunocytochemistry, enzyme-linked absorbent assay (ELISA), immunoprecipitation, immunoelectrophoresis or dot blot, immunostaining of cells, fluids, tissues, or their extracts or lysates, and other immunodetection methods). Methods for detecting or determining the presence or level of AKR1C3 nucleic acid biomarkers (e.g., mRNA) include, but are not limited to, RNA detection and quantitative assays (e.g., RNA-Seq (e.g., mRNA-Seq), RT-PCR, digital PCR, and RT-qPCR). Methods for detecting or determining the presence of nucleic acid biomarkers (e.g., somatic mutations of NFE2L2, CUL3, or KEAP1 gene sequences) (e.g., DNA (e.g., genomic DNA)) include, but are not limited to, methods for sequencing DNA (e.g., genomic DNA) (e.g., exome sequencing, targeted genome sequencing, whole genome sequencing, SMRT sequencing, ion semiconductor sequencing, pyrophosphate sequencing, sequencing by synthesis, cPAS sequencing, cPAL sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, PCR, qPCR, digital PCR, and Sanger sequencing).

[0265] As used herein, the term "biomarker" refers to an indicator (e.g., predictive, diagnostic, and / or prognostic) that can be detected in a sample, such as a specific gene (including but not limited to changes in gene sequence relative to the wild-type sequence (e.g., somatic mutations) or changes in gene expression levels relative to a control sample or control data set (e.g., as determined by the mRNA transcript of the gene)) or a protein encoded by the gene (including but not limited to changes in protein expression levels relative to a control sample or control data set). A biomarker may include one or more somatic mutations of a gene (e.g., NFE2L2, CUL3, or KEAP1). A biomarker can be used as an indicator of a specific disease or disorder or a specific subtype of a disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and / or clinical features (e.g., responsiveness to treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof). In some embodiments, a biomarker is a collection of genes or proteins (e.g., single or multiple genes and protein expression levels) or the collective number of mutations / alterations (e.g., somatic mutations) in a collection of genes. Biomarkers include, but are not limited to, polynucleotides, polynucleotide changes (e.g., gene sequence mutations, such as somatic mutations), polypeptides, and proteins. In some embodiments described herein, the biomarker is AKR1C3 protein level. In some embodiments described herein, the biomarker is AKR1C3 nucleotide sequence expression level. In some embodiments described herein, the biomarker is AKR1C3 gene expression level. In some embodiments described herein, the biomarker is AKR1C3 mRNA transcript level. In some embodiments described herein, the biomarker is NFE2L2, CUL3, or KEAP1 gene sequence. In some embodiments described herein, the biomarker is a somatic mutation of NFE2L2, CUL3, or KEAP1 gene sequence.

[0266] The term "level" refers to the presence or amount of a biomarker in a sample (eg, a subject sample or a control sample) or a data set (eg, a control data set).

[0267] "Increased," "elevated," or "high" biomarker levels refer to an increase in the level of a biomarker (e.g., a protein or mRNA biomarker) in a sample (e.g., a subject sample) relative to a control sample (such as one or more individuals not suffering from a disease or disorder (e.g., cancer)) or a control data set (such as a data set consisting of biomarker levels from one or more individuals not suffering from a disease or disorder (e.g., cancer). In some embodiments, the increased biomarker level can be detected in a subject or subject sample.

[0268] "Decreased," "reduced," or "low" biomarker levels refer to a decrease in the level of a biomarker (e.g., a protein or mRNA biomarker) in a sample (e.g., a subject sample) relative to a control sample (such as one or more individuals not suffering from a disease or disorder (e.g., cancer)) or a control dataset (such as a dataset consisting of biomarker levels from one or more individuals not suffering from a disease or disorder (e.g., cancer). In some embodiments, increased biomarker levels can be detected in a subject or subject sample.

[0269] The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a biological sample. "Expression" generally refers to the process of converting information (e.g., genetic coding and / or epigenetic information) into structures that exist and operate in a cell. Therefore, as used herein, "expression" may refer to transcription into polynucleotides, translation into polypeptides, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides). Transcribed polynucleotides, fragments of translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered to be expressed, whether they originate from transcripts or degraded transcripts produced by alternative splicing, or from post-translational processing of polypeptides, such as by proteolysis. "Expressed genes" include those that are transcribed as mRNA into polynucleotides and then translated into polypeptides, as well as those that are transcribed into RNA but not translated into polypeptides (e.g., transport and ribosomal RNA).

[0270] As used herein, "amplification" generally refers to the process of producing multiple copies of a desired sequence. "Multiple copies" means at least two copies. "Copy" does not necessarily mean having perfect sequence complementarity or identity with the template sequence. For example, copies can include nucleotide analogs (such as deoxyinosine), intentional sequence changes (such as sequence changes introduced by primers containing sequences that can hybridize with the template but are not complementary), and / or sequence errors that occur during amplification.

[0271] As used herein, "polymerase chain reaction" or "PCR" technology generally refers to a procedure in which trace amounts of specific nucleic acid fragments, i.e., RNA and / or DNA, are amplified, as described, for example, in U.S. Pat. No. 4,683,195. Typically, sequence information from the ends or further away of the region of interest needs to be available so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the opposite strand of the template to be amplified. The 5' terminal nucleotides of the two primers can be consistent with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cell RNA, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. [Cold Spring Harbor Quantitative Biology Symposium] 51: 263 (1987) and Erlich, ed., PCR Technology [PCR Technology] (Stockton Press [Stockton Press], NY [New York], 1989). As used herein, PCR is considered to be one but not the only example of a nucleic acid polymerase reaction method for amplifying nucleic acids (e.g., nucleic acids in a sample (e.g., a control sample or a subject sample)), which includes using known nucleic acids (DNA or RNA) as primers and using a nucleic acid polymerase to amplify or generate a specific nucleic acid fragment or amplify or generate a specific nucleic acid fragment complementary to a specific nucleic acid. In some embodiments, PCR is used to detect somatic mutations (e.g., somatic mutations of KEAP1, NFE2L2, or CUL3) or to detect or determine the expression level of a gene of interest (e.g., KEAP1, NFE2L2, or CUL3).

[0272] The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acids obtained from a single source (eg, an individual sample from a subject) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction.

[0273] The term "quantitative PCR" (also called "qPCR" or "real-time PCR") refers to a PCR reaction used to monitor the amplification of a target nucleic acid species. qPCR typically relies on fluorescent dyes that intercalate into double-stranded DNA or fluorescently labeled sequence-specific DNA probes that can hybridize to the PCR products of interest in order to detect and quantify the amplification of the nucleic acid species of interest. qPCR can be quantitative or semi-quantitative.

[0274] "Reverse transcription PCR" or "RT-PCR" refers to a form of PCR that uses a reverse transcriptase to convert an RNA template (e.g., an mRNA transcript) into complementary DNA (cDNA). The cDNA produced by this reaction is then amplified by PCR.

[0275] "Reverse transcription quantitative PCR" or "RT-qPCR" refers to a quantitative or semi-quantitative form of qPCR. Generally speaking, RT-qPCR relies on the same methodology as qPCR, but employs a reverse transcriptase to generate cDNA from mRNA. RT-qPCR is used to monitor the amplification of a targeted mRNA species, thereby allowing for quantification of the mRNA in a sample.

[0276] "Digital PCR" refers to a PCR method for direct quantitative and clonally amplified nucleic acid chains (including DNA, cDNA or RNA). Digital PCR performs a single PCR reaction within a sample, similar to traditional PCR. However, in contrast to traditional PCR, the sample is divided into a large number of partitions (e.g., 10 4 Partitions), and PCR reactions were performed separately in each partition.

[0277] The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer). For example, "diagnosis" may refer to the identification of a particular type of cancer. "Diagnosis" may also refer to the classification of a particular subtype of cancer, for example, by histopathological criteria or by molecular features (e.g., a subtype characterized by the expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).

[0278] The term "sample" or "biological sample" as used herein refers to a composition obtained or derived from a subject and / or individual of interest, the composition containing, for example, cells, fluids and / or other molecular entities characterized and / or identified based on physical, biochemical, chemical and / or physiological characteristics. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor cells, tumor lysates and tissue culture media, tissue extracts (such as homogenized tissue), tumor tissue, cell extracts and combinations thereof. In some embodiments, the sample is a tissue sample, a blood sample or a cell sample. In some embodiments, the sample is or comprises a protein or genetic material. In some embodiments, the sample comprises a cell genome, a transcriptome or a proteome, such as a tumor cell genome, a transcriptome or a proteome.

[0279] The so-called "tissue sample" means a collection of similar cells obtained from a subject or individual's tissue. The source of the tissue or cell sample can be a solid tissue from a fresh, frozen and / or preserved organ, tissue sample, biopsy and / or aspirate; blood or any blood component, such as plasma; body fluid, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from any time during the pregnancy or development of the subject. The tissue sample can also be a primary or cultured cell or cell line. Optionally, the tissue or cell sample is obtained from a diseased tissue / organ. For example, a "tumor sample" is a tissue sample obtained from a tumor or other cancerous tissue. The tissue sample may contain a mixed population of cell types (e.g., tumor cells and non-tumor cells, cancer cells and non-cancer cells). The tissue sample may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. In some cases, the tissue sample or tumor tissue sample is not a blood sample or sample or blood component, such as plasma. In a preferred embodiment, the tissue sample or cell sample is a tumor sample.

[0280] As used herein, "subject sample" can be a sample from or derived from a subject (e.g., a subject in need of treatment or prevention), such as a biological sample. Subject sample can include any sample of cells and / or molecular entities (e.g., biomarkers) to be characterized that are expected or known to be obtained from a subject of interest. In certain embodiments, subject sample includes components that are not directly from a subject. For example, subject sample can be a mixture of material directly from a subject (e.g., a subject's tissue) or a material derived from a subject (e.g., a cell line derived from a subject's tissue) and other materials (e.g., buffer or cell culture fluid). Subject sample can be composed of one or more cells, cell groups, cell lysates, tissues or fluids of a subject (e.g., one or more cells, cell groups, cell lysates, tissues or fluids obtained from a subject or provided by a subject) of the subject. In some embodiments, the subject sample can include one or more cancer cells, such as one or more tumor cells (e.g., liquid tumor cells or solid tumor cells), whole blood, plasma, serum, blood-derived cells, platelets, lymph, urine, feces, mucus, sputum, sweat, saliva, semen, cerebrospinal fluid, bone marrow, amniotic fluid, one or more tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, vitreous fluid, synovial fluid, follicular fluid, milk, tears, tumor lysates, tissue culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof. In some embodiments, the subject sample is or includes a protein or genetic material from or derived from a subject. For example, in some embodiments, the subject sample is or includes a genome, transcriptome, or proteome from or derived from a subject, such as a genome, transcriptome, or proteome of a tumor cell of a subject.

[0281] As used herein, "tumor cell" refers to any tumor cell present in a tumor or a sample thereof. Tumor cells may be distinguished from other cells that may be present in a tumor sample, such as stromal cells and tumor-infiltrating immune cells, using methods known in the art and / or described herein. Tumor cells may be liquid tumor cells or solid tumor cells.

[0282] As used herein, "control sample" refers to a sample, tissue, cell, data set, standard or level for comparison purposes. In one embodiment, the control sample is obtained from a healthy and / or non-diseased portion of the body (e.g., tissue or cell) of the same subject or individual. For example, the control sample can be a healthy and / or non-diseased tissue or cell adjacent to a diseased tissue or cell (e.g., tissue or cell adjacent to a tumor). In another embodiment, the control sample is obtained from a healthy and / or non-diseased portion of the body (e.g., tissue or cell) of an individual that is not the same subject or individual. In some embodiments, the control sample is or comprises a genome, transcriptome or proteome of a control cell, tissue or fluid.

[0283] As used herein, "control data set" refers to a data set containing one or more samples, tissues, cells, standards or levels for comparison purposes (e.g., for comparison with a subject sample). In one embodiment, the control data set is composed of data obtained from the healthy and / or non-diseased part of the body (e.g., tissue or cell) of the same subject or individual. In some embodiments, the control data set is composed of data obtained from one or more healthy and / or non-disease individuals that are not subjects. In some embodiments, the control data set includes data on the level (e.g., protein or RNA level) of one or more biomarkers in one or more healthy control individuals. In some embodiments, the control data set includes data on the presence, absence or prevalence (e.g., the presence, absence or prevalence of one or more disease-related nucleotide sequences or somatic mutations) of one or more biomarkers in one or more healthy control individuals. In some embodiments, the control data set includes genome, transcriptome or proteome data.

[0284] The term "AKR1C3" as used herein refers to a protein encoded by the aldehyde-keto reductase family 1 member C3 gene sequence (also known as DD3, DDX, PGFS, HAKRB, HAKRe, HA1753, HSD17B5 or hluPGFS) (described in, e.g., NCBI Gene ID: 8644) and its orthologs, or a nucleotide sequence (e.g., mRNA sequence) or gene sequence encoding the protein.

[0285] The term "KEAP1" as used herein refers to a protein encoded by the A Kelch-like ECH-associated protein 1 gene sequence (also known as INFE2L2 or KLHL19) (described in, e.g., NCBI Gene ID: 9817) and its orthologs, or a nucleotide sequence (e.g., mRNA sequence) or gene sequence encoding the protein.

[0286] The term "CUL3" as used herein refers to a protein encoded by a Cullin 3 gene sequence (also known as CUL-3, PHA2E or NEDAUS) (described in, e.g., NCBI Gene ID: 8452) and its orthologs, or a nucleotide sequence (e.g., mRNA sequence) or gene sequence encoding the protein.

[0287] The term "NFE2L2" as used herein refers to a protein encoded by the NFE2-like Bzip transcription factor 2 gene sequence (also known as NRF2, HEBP1, Nrf-2 or IMDDHH) (described in, e.g., NCBI Gene ID: 4780) and its orthologs, or a nucleotide sequence (e.g., mRNA sequence) or gene sequence encoding the protein.

[0288] The term "KARS" as used herein refers to a protein encoded by the lysyl-TRNA synthetase 1 gene sequence (also known as KRS, KARS, KARS2, LEPID, CMTRIB, DEAPLE or DFNB89) (described in, e.g., NCBI Gene ID: 3735) and its orthologs, or a nucleotide sequence (e.g., mRNA sequence) or gene sequence encoding the protein.

[0289] Genetic mutations

[0290] Methods for identifying a subject in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof are described herein. In some embodiments, the methods described herein include a step of detecting somatic mutations in a subject sample, such as detecting somatic mutations in at least one of the following genes: NFE2L2, KEAP1, or CUL3A. Somatic mutations include genetic sequences that cause an individual carrying a somatic mutation to develop or be prone to developing a particular disease with an increased likelihood. For example, an individual carrying a somatic mutation may have an increased likelihood of developing or being prone to developing cancer. Somatic mutations can be associated with disease through genomic studies (e.g., genome-wide association studies (GWAS)).

[0291] Various types of genetic mutations are known in the art, and include, for example, point mutations, single nucleotide polymorphism (SNP) mutations, substitution mutations, missense mutations, nonsense mutations, frameshift mutations, nucleotide repeat expansion mutations, inversion mutations, insertion mutations, deletion mutations, copy number variation mutations, amplification mutations, gene duplication mutations, somatic mutations, germline mutations, homozygous mutations, heterozygous mutations, chromosome rearrangement mutations, splice site mutations, gain of function mutations, hypomorphic mutations, and de novo mutations. In certain embodiments, somatic mutations are point mutations, single nucleotide polymorphism (SNP) mutations, substitution mutations, missense mutations, nonsense mutations, frameshift mutations, nucleotide repeat expansion mutations, inversion mutations, insertion mutations, deletion mutations, copy number variation mutations, amplification mutations, gene duplication mutations, somatic mutations, homozygous mutations, heterozygous mutations, chromosome rearrangement mutations, splice site mutations, gain of function mutations, hypomorphic mutations, or de novo mutations of the gene sequence of at least one of the following genes: NFE2L2, KEAP1, or CUL3A. Somatic mutations can occur in any part of the gene sequence, including, for example, protein coding regions, gene enhancers, exons, introns, promoters, splice sites, 5'UTRs or 3'UTRs. In some embodiments, somatic mutations include amplification of the NFE2L2 gene sequence or a portion thereof. In some embodiments, somatic mutations include deletions of the KEAP1 or CUL3 gene sequence or a portion thereof.

[0292] Disease-associated nucleotide sequences of KEAP1 are described, for example, in Campbell et al., (2016) Nature Genetics, 48:607-16; Chen, R. (2020) "Cullin 3 and Its Role in Tumorigenesis" in Cullin-RING Ligases and Protein Neddylation, 187-210; Collisson et al., (2014) Nature, 511:543-550; Delgobo et al., (2021) Freed Radical Biology and Medicine, 177:58-71; Gong et al., (2020) Cell Communication and Signaling, 18, 98; Hammerman et al. (2012) Nature, 489: 519–525; Hayes and McMahon (2009) Trends in Biochemical Sciences, 34(4): 176–88; Jin et al. (2021) Cancer Medicine, 10(23): 8673–92; Kandoth et al. (2013) Nature, 502: 333–339; Konstantinopoulos et al. (2011) Cancer Res, 71: 5081–5089; Ohta et al. (2008) Cancer Res, 68:1303–1309; Padmanabhan et al. (2006) Mol Cell, 21:689–700; Romero et al. (2020) Nature Cancer, 1(6):589–602; Saleh et al. (2021) Journal of Thoracic Oncology, 17(1):76–88; Shibata et al. (2008) Gastroenterology, 135:1358–1368; Shibata et al. (2011) Neoplasia, 13:864–873; Singh et al. (2006) PLoS Med, 3:e420;Taguchi and Yamamoto (2017) Frontiers in Oncology, 7:85; Wang et al. (2020) “CRL3s: The BTB-CUL3-RING E3 Ubiquitin Ligases” in Cullin-RING Ligases and Protein Neddylation, 211–223; and Yoo et al. (2012) Histopathology, 60:943–952, which are incorporated herein by reference.

[0293] Disease-associated nucleotide sequences of CUL3 are described, for example, in Campbell et al., (2016) Nature Genetics, 48:607-16; Chen, R. (2020) “Cullin 3 and Its Role in Tumorigenesis” in Cullin-RING Ligases and Protein Neddylation, 187-210; Collisson et al., (2014) Nature, 511:543–550; Delgobo et al., (2021) Freed Radical Biology and Medicine, 177:58-71; Hammerman et al., (2012) Nature, 489:519–25; Jin et al., (2021) Cancer Medicine, 10(23): 8673-92; Ooi et al., (2013) Cancer Res, 73: 2044–51; and Wang et al., (2020) “CRL3s: The BTB-CUL3-RING E3 Ubiquitin Ligases” in Cullin-RING Ligases and Protein Neddylation, 211–23, which are incorporated herein by reference.

[0294] Disease-associated nucleotide sequences of NFE2L2 are described, for example, in Campbell et al., (2016) Nature Genetics, 48:607-16; Chen, R. (2020) “Cullin 3 and Its Role in Tumorigenesis” in Cullin-RING Ligases and Protein Neddylation, 187-210; Collisson et al., (2014) Nature, 511:543–50; Delgobo et al., (2021) Freed Radical Biology and Medicine, 177:58-71; Goldstein et al., (2016) Cell Biology, 197:57-61; Rep [Cell Reports], 16:2605–2617; Hammerman et al., (2012) Nature [Nature], 489:519–25; Jin et al., (2021) Cancer Medicine [Cancer Medicine], 10(23):8673-92; Ooi et al., (2013) Cancer Res [Cancer Research], 73:2044–51; Shibata et al., (2011) Neoplasia [Neoplasia], 13:864–873; and Wang et al., (2020) “CRL3s: The BTB-CUL3-RING E3 Ubiquitin Ligases” in Cullin-RING Ligases and Protein Neddylation [“CRL3: BTB-CUL3-RINGE3 ubiquitin ligase” in Cullin-RING ligases and protein ubiquitination], 211–23, which are incorporated herein by reference.

[0295] As used herein, "subject tumor genome" refers to a complete set of genetic information (including coding and non-coding portions of chromosomal DNA) included in the cells of the subject's tumor. The subject's tumor genome may include genetic mutations associated with a disease or disorder. For example, in some embodiments of the methods described herein, the subject's tumor genome comprises somatic mutations associated with a disease or disorder (e.g., cancer). For example, in some embodiments, the subject's tumor genome comprises somatic mutations of one or more of the NFE2L2, KEAP1, or CUL3 gene sequences. In some embodiments, the presence of somatic mutations in the subject's tumor genome may indicate that the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the subject's tumor genome may include genetic mutations that are unknown to be associated with a disease or disorder (e.g., cancer) but may indicate that the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0296] Disease and cancer

[0297] Methods of treating a subject with a compound of formula (I) or a pharmaceutically acceptable salt thereof or identifying a subject for treatment with the compound or a pharmaceutically acceptable salt thereof are described herein. In particular embodiments, the subject is diagnosed with, suffers from, or is predisposed to develop a particular disease or disorder. The diseases and disorders described herein may include conditions that would benefit from treatment, including but not limited to chronic and acute disorders or diseases, including those pathological conditions that predispose the subject to the disease or disorder in question. In particular embodiments, the subject is diagnosed with, suffers from, or is predisposed to develop cancer.

[0298] The terms "cancer" and "carcinoma" refer to or describe a physiological condition that is usually characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. By "early stage cancer" or "early stage tumor" is meant a cancer that is not invasive or metastatic or is classified as a stage I or II cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.Examples of cancer also include, but are not limited to, lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer (e.g., renal urothelial carcinoma or RCC), bladder cancer (e.g., bladder urothelial (transitional cell) carcinoma (e.g., locally advanced or metastatic urothelial carcinoma, including 1L or 2L+ locally advanced or metastatic urothelial carcinoma), breast cancer, colorectal cancer (e.g., colon adenocarcinoma), ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, melanoma (e.g., cutaneous melanoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), thyroid cancer, sarcoma (e.g., soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, osteosarcoma, chondrosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, leiomyosarcoma, or rhabdomyosarcoma), prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic eosinophilic leukemia, or chronic lymphocytic leukemia (CLL)), lymphoma (e.g., Hodgkin lymphoma or non-Hodgkin lymphoma (NHL)), myeloma (e.g., multiple myeloma (MM)), mycosis fungoides, Merkel cell carcinoma, hematological malignancies, cancer of hematological tissues, B-cell cancer, bronchial cancer, gastric cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, uterine cancer, or endometrial cancer cancer, oral or pharyngeal cancer, liver cancer, testicular cancer, bile duct cancer, small intestine cancer or appendix cancer, salivary gland cancer, adrenal cancer, adenocarcinoma, inflammatory myofibroblastic tumor, gastrointestinal stromal tumor (GIST), colon cancer, myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), polycythemia vera, chordoma, synovioma, Ewing's tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma , acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinoma, thyroid cancer, small cell carcinoma, essential thrombocythemia, idiopathic myeloid metaplasia, high eosinophilic syndrome, systemic mastocytosis, common eosinophilic syndrome, neuroendocrine carcinoma or carcinoid tumor. More specific examples of such cancers include I-III early resectable and unresectable (IIIC stage) or metastatic (IV stage) melanoma, lung cancer (including NSCLC), squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer (SCLC)) and lung adenocarcinoma and lung squamous cell carcinoma.In a particular instance, the lung cancer is NSCLC, such as locally advanced or metastatic NSCLC (e.g., stage IIIB NSCLC, stage IV NSCLC, or recurrent NSCLC). Other examples include peritoneal cancer, hepatocellular carcinoma, bladder cancer (e.g., urothelial bladder cancer (e.g., transitional cell carcinoma or urothelial carcinoma, non-muscle invasive bladder cancer, muscle invasive bladder cancer, and metastatic bladder cancer) and non-urothelial bladder cancer), gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, Merkel cell carcinoma, mycosis fungoides, testicular cancer, esophageal cancer, biliary tract tumors, and head and neck cancer and hematological malignancies. In some embodiments, the subject is diagnosed with, suffers from, or is prone to developing a disease or disorder selected from the group consisting of non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer (e.g., bladder urothelial carcinoma), cervical cancer (e.g., cervical squamous cell carcinoma), uterine cancer (e.g., endometrial cancer), esophageal cancer (e.g., esophageal squamous cell carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), kidney cancer (e.g., papillary renal cell carcinoma), breast cancer, colorectal cancer, melanoma, gastric cancer, castration-resistant prostate cancer (CRPC), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and liver cancer (e.g., hepatocellular carcinoma). In specific embodiments, the subject is diagnosed with, suffers from, or is prone to developing NSCLC. In some embodiments, the subject is diagnosed with, suffers from, or is prone to developing a squamous cell carcinoma subtype of NSCLC. In some embodiments, the subject is diagnosed with, suffers from, or is prone to developing an adenocarcinoma subtype of NSCLC.

[0299] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation (whether malignant or benign), as well as all precancerous cells and tissues and cancerous cells and tissues. The terms "cancer", "cancer" and "tumor" are not mutually exclusive, as mentioned herein. In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a liquid tumor.

[0300] Subjects that may benefit from treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof include those subjects that respond to a therapeutically effective amount of the compound. For example, subjects that may benefit from treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof include those subjects that respond or may respond to treatment, such as subjects that experience or may experience the following as a result of treatment: anatomical functions affected by a particular disease or disorder are improved or maintained; quality of life associated with an improved disease state is improved; the disease or disorder is alleviated or improved; at least one physical parameter or biomarker associated with the disease or disorder is alleviated or improved; and / or one or more symptoms of the disease or disorder are improved or improved.

[0301] Described herein are methods for identifying a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof. The subject for treatment can be identified by diagnosing the subject as suffering from or tending to develop into a specific disease or disorder. For example, the subject for treatment can be identified by diagnosing the subject as suffering from or tending to develop into cancer (e.g., NSCLC). The subject for treatment can be identified by detecting the presence or elevated levels of a biomarker. For example, the subject for treatment can be identified by detecting elevated levels of AKR1C3. For example, the subject for treatment can be identified by the elevated AKR1C3 levels in the subject sample relative to the AKR1C3 levels in the control sample (e.g., a control subject sample or a control data set). In some embodiments, the subject for treatment can be identified by detecting one or more somatic mutations in at least one of the following genes in the subject sample (e.g., a subject tumor genome): NFE2L2, KEAP1, or CUL3.

[0302] Immunohistochemistry

[0303] In some embodiments of the methods described herein, a sample (e.g., a subject sample or a control sample) is characterized by a biomarker level (e.g., AKR1C3 protein level) by an antigen detection assay. In some embodiments, the antigen detection assay is an immunohistochemistry (IHC) assay. Generally speaking, IHC relies on the use of antigen detection compounds (e.g., antibodies) to detect the presence, amount, and / or relative amount of a substance of interest. In some embodiments described herein, the antigen IHC assay includes a step of probing a sample (e.g., a control sample or a subject sample) with an antibody that can bind to a biomarker (e.g., AKR1C3 protein).

[0304] Antigen-based detection assays typically determine the presence, level or distribution of a target molecule (e.g., a biomarker) in a sample by detecting the interaction of a detectable target molecule with a specific binding agent (such as an antibody). For example, the sample is contacted with an antibody (or other binding agent, such as an antibody fragment) under conditions that allow antibody-antigen binding. Antibody-antigen binding can be detected by means of a detectable label conjugated to the antibody (direct detection) or by means of a detectable label conjugated to a secondary antibody that specifically binds to a primary antibody (e.g., indirect detection).

[0305] IHC uses antibodies or their derivatives or other protein binding agents to analyze histological tissues under a microscope. IHC may include the following steps: blocking the tissue with a reagent to block endogenous sources of nonspecific staining, such as (i) enzymes, (ii) endogenous peroxidases, (iii) free aldehyde groups, (iv) immunoglobulins, and other unrelated molecules that can mimic specific staining; incubating the tissue with a permeabilization buffer to promote penetration of antibodies and other staining reagents into the tissue; incubating the tissue with one or more primary antibodies; rinsing the tissue with a wash buffer; incubating the tissue with one or more secondary antibodies that bind to one of the one or more primary antibodies; rinsing with a wash buffer; and incubating the tissue with a detection reagent. The present invention is not limited to this IHC protocol.

[0306] In some embodiments of the methods described herein, determining the level of a biomarker (e.g., the level of a biomarker in a subject sample or a control sample) may include generating a biomarker signal intensity score (e.g., a biomarker signal intensity score for AKR1C3 protein levels). An increase in the IHC signal intensity score of a subject sample or an increase in the IHC signal intensity score of a subject sample relative to the IHC signal intensity score of a control sample may indicate that the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, if the IHC signal intensity score of the subject sample is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% higher than the IHC signal intensity score of the control sample, the subject needs to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0307] In some embodiments, the IHC signal intensity score can range from 0 to 3, where a score of 0 indicates no detectable signal and a score of 3 indicates a strong detectable signal. In some embodiments, this scoring system is referred to as an H score. The H score is the sum of the percentage of strongly stained nuclei multiplied by 3, the percentage of moderately stained nuclei multiplied by 2, and the percentage of weakly stained nuclei; the sum divided by 100. In some embodiments, if the H score of the subject's sample is 0.5 or greater, 1.0 or greater, 1.5 or greater, 2 or greater, 2.5 or greater, 2.6 or greater, 2.7 or greater, 2.8 or greater, or 2.9 or greater, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0308] In some embodiments, the IHC signal intensity score can range from 0 to 300, where a score of 0 indicates no detectable signal and a score of 300 indicates a strong detectable signal. In some embodiments, this scoring system is referred to as the H score. The H score is the sum of the percentage of strongly stained nuclei multiplied by 3, the percentage of moderately stained nuclei multiplied by 2, and the percentage of weakly stained nuclei. In some embodiments, if the H score of the subject's sample is 50 or greater, 100 or greater, 150 or greater, 200 or greater, 250 or greater, 260 or greater, 270 or greater, 280 or greater, or 290 or greater, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0309] The IHC signal intensity score can be determined based on the quantitative determination of the detectable signal from the IHC detection reagent. For example, the IHC signal intensity score can be based on the quantitative determination of the detectable fluorescent signal or chromogen signal (e.g., DAB, 3-amino-9-ethylcarbazole (AEC), 5-bromo-4-chloro-3-indolyl phosphate: tetranitro tetrazolium blue (BCIP:TNBT), 5-bromo-4-chloro-3-indolyl phosphate: p-chloronitro tetrazolium blue (BCIP:NBT) or 3,3',5,5-tetramethylbenzidine (TMB), fast red, permanent red) or its signal intensity. The chromogen signal can be generated by a chemical reaction with a suitable enzyme (e.g., HRP, glucose oxidase or alkaline phosphatase). The enzyme can be conjugated with an antibody (e.g., a primary antibody or a secondary antibody) or a probe (e.g., a streptavidin probe). The fluorescent signal can be directly generated by a protein (e.g., green fluorescent protein or red fluorescent protein). Fluorescent signals can also be provided by suitable quantum dot materials, dyes or fluorophores (e.g., Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, coumarin, Cy3, Cy5, fluorescein (FITC), Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, PE-cyanidin 7, PerCP-cyanidin 5.5, tetramethylrhodamine (TRITC), Texas Red, eFluor 450, eFluor 506, eFluor The quantum dots or fluorophores can be conjugated to antibodies (e.g., primary or secondary antibodies) or probes (e.g., streptavidin probes).In some embodiments, the sample is counterstained with a suitable reagent (e.g., eosin, hematoxylin) or a suitable DNA binding agent (e.g., 4',6-diamidino-2-phenylindole (DAPI), propidium iodide, SYTO 9, SYTOX Green, or TO-PRO-3).

[0310] IHC signal intensity can be calculated based on images captured using a suitable microscope (e.g., bright field, fluorescence or confocal microscope) or a slide scanner and camera. IHC signal intensity can be quantified from such images using a suitable software program (e.g., ImageJ Fiji, ImageScope, Ilastik, Cell Profiler, inForm image analysis software or IHCProfiler).

[0311] The inventors of the disclosed invention unexpectedly discovered that AKR1C3 levels can vary significantly between individual tumor cells within a single tumor. The inventors also discovered that IHC has properties that make it particularly suitable for use in conjunction with the following methods: treating a subject with a compound of formula (I) or a pharmaceutically acceptable salt thereof, identifying a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, and selecting a compound of formula (I) or a pharmaceutically acceptable salt thereof for treatment of a subject, wherein the method requires determining the level of AKR1C3 in a sample from the subject, characterizing a sample from the subject as having elevated levels of AKR1C3, or wherein a sample from the subject is characterized as having elevated levels of AKR1C3. Thus, the inventors unexpectedly discovered that IHC is particularly advantageous for determining the level of AKR1C3 in a sample (e.g., a subject sample) in conjunction with the following methods: a method of identifying a subject for treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof; selecting a compound of Formula (I) or a pharmaceutically acceptable salt thereof to treat a subject; a method of treating a subject, the method comprising the step of determining the level of AKR1C3 in a subject sample; a method of treating a subject by administering to the subject an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein prior to said administration, the subject sample is characterized as having elevated levels of AKR1C3; and a method of treating a subject with a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein the subject sample is characterized as having elevated levels of AKR1C3.

[0312] It is noteworthy that IHC allows for the presence or level of proteins or other detectable markers in individual cells to be evaluated or scored. Although IHC can be used to generate a total score for detecting proteins or other detectable markers in tissue samples (e.g., tumor samples), it provides the advantage of basing the score on the evaluation of individual cells. These properties of IHC make it particularly useful for evaluating protein expression that shows highly heterogeneous expression levels in individual cells of a tumor. Therefore, because AKR1C3 levels vary significantly between individual tumor cells, IHC is particularly suitable for determining AKR1C3 levels in subject samples and / or generating an H score suitable for identifying subjects for treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0313] Reagent test kit

[0314] Also described herein are kits suitable for performing the methods described herein or for the uses described herein. The kits described herein may include components including, but not limited to, instructions for use, one or more containers for storing the kit components, and / or pharmaceutically acceptable solutions formulated for oral administration. The kits described herein may also include components suitable for the following items: determining the level of a biomarker (e.g., AKR1C3) in a subject sample; characterizing a subject sample as having an elevated level of a biomarker (e.g., AKR1C3 biomarker); identifying a subject to be treated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof; detecting a somatic mutation in at least one of the following genes in a subject sample: NFE2L2, KEAP1, or CUL3; characterizing the presence of a somatic mutation in at least one of the following genes in a subject sample: NFE2L2, KEAP1, or CUL3; determining the level of a biomarker (e.g., AKR1C3) in a sample (e.g., a subject sample or a control sample); detecting somatic mutations in one or more gene sequences (e.g., NFE2L2, KEAP1, or CUL3 gene sequences); or detecting somatic mutations in one or more mRNA sequences (e.g., NFE2L2, KEAP1, or CUL3 mRNA sequences). In some embodiments, the kits described herein include a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the kits described herein include a therapeutically effective amount of a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0315] In some embodiments, the kits described herein comprise components suitable for determining mRNA or protein levels of biomarkers (e.g., AKR1C3). In some embodiments, the kits described herein comprise components suitable for performing antigen detection assays (e.g., IHC assays). In some embodiments, the kits described herein comprise components suitable for sequencing genomic DNA or RNA (e.g., mRNA) (e.g., NFE2L2, KEAP1 or CUL3 gene sequences).

[0316] Examples

[0317] Example 1: Immunohistochemistry Assay for AKRIC3 in Cancer Tissues

[0318] The AKR1C3 immunohistochemistry assay was designed to determine whether the detection of AKR1C3 protein expression using anti-AKR1C3 antibodies can be used to identify cancer tissues. Figure 1 , column labeled “NSCLC”), prostate cancer ( Figure 1 , column labeled “Prostate”) or hepatocellular carcinoma ( Figure 1 , columns labeled "HCC") and NSCLC, HCC, and head and neck ( Figure 1 , H&N) cancer patient biopsies ( Figure 1 , column labeled "Test Biopsy") and processed for immunostaining. Prostate group samples were collected from tumors with a Gleason grade of 9-10 ( Figure 1 NSCLC, prostate, and HCC group subject samples did not have mutations detected in NFE2L2 or KEAP1 ( Figure 1 The test biopsy set included samples from subjects that included mutations in NFE2L2 and / or KEAP1 ( Figure 1 , "NRF2 / KEAP1 Mut"). H&N subject samples included in the trial biopsy set included biopsies collected prior to treatment ( Figure 1 , “screening”) or a biopsy collected on Day 1 of Cycle 2 of treatment ( Figure 1 , “C2D1”).

[0319] All samples for IHC were prepared by cutting 4-6 μm tissue sections from formalin-fixed, paraffin-embedded sample blocks with a microtome. Tissue sections were mounted on SuperFrost Plus microscope slides (Cat. No. 22-037-246; Fisher Scientific, Waltham, MA), air-dried overnight at room temperature, and incubated at 60° C. for 1 hour.

[0320] Samples included in the experimental biopsy set were stained using a Ventana Benchmark Ultra automated stainer (Ventana, Tucson, AZ) with the following bulk reagents: 10x reaction buffer (Cat. No. 950-300; Roche Diagnostics Corporation; Indianapolis, IN) (diluted to 1x in deionized water), Benchmark Ultra liquid coverslips (LCS) (Cat. No. 650-210; Roche Diagnostics), 10x EZ Prep concentrate (Cat. No. 950-102; Roche Diagnostics) (diluted to 1x in deionized water), and Ultra Cell Conditioning 1 (CC1) solution (Roche, Cat. No. 950-224). Immunostaining was performed using the ultraView Universal DAB program (v1.02.0018), including antigen retrieval at 95°C for 36 minutes with CC1 solution. Anti-human AKR1C3 mouse monoclonal primary antibody clone NP6.G6.A6 (Catalog No. A6229; Sigma Aldrich, St. Louis, MO) was diluted in DAKO antibody diluent (Catalog No. S0809; Agilent, Santa Clara, CA) and applied in a volume of 100 μl during the primary antibody titration step. The primary antibody was incubated at 37°C for 32 minutes and detected with the ultraView Universal DAB detection kit. The slides were counterstained with hematoxylin (Catalog No. 760-2021; Roche Diagnostics) for 4 minutes at room temperature and blued with blue back reagent (Catalog No. 760-2037; Roche Diagnostics) for 4 minutes at room temperature.

[0321] The samples included in the NSCLC, prostate, and HCC panels were stained using a Ventana Discovery Ultra autostainer (Ventana, Tucson, AZ) with the following bulk reagents: 10x reaction buffer (Cat. No. 950-300; Roche Diagnostics) (diluted to 1x in deionized water), Benchmark Ultra Liquid Coverslip (LCS) (Cat. No. 650-210; Roche Diagnostics), Discovery Wash Concentrate (Cat. No. 950-510; Roche Diagnostics) (diluted to 1x in deionized water), and Ultra Cell Conditioning 1 (CC1) Solution (Roche, Cat. No. 950-224). Immunostaining was performed using the RUO Discovery Universal program (v0.00.0370), including antigen retrieval with CC1 solution at 95°C for 32 minutes. Endogenous peroxidases were blocked by incubation in Inhibitor CM (a component of the ChromoMap DAB Detection Kit (Cat. No. 760-159; Roche Diagnostics) for 8 minutes. Anti-human AKR1C3 mouse monoclonal primary antibody clone NP6.G6.A6 (Cat. No. A6229; Sigma-Aldrich, St. Louis, MO) was diluted in DAKO Antibody Diluent (Cat. No. S0809; Agilent, Santa Clara, CA) and applied in a volume of 100 μl during the primary antibody titration step. The primary antibody was incubated at 37°C for 60 minutes, followed by incubation with Omnimap anti-mouse HRP secondary antibody (Cat. No. 760-4310; Roche Diagnostics) for 4 minutes and detected with the ChromoMap DAB Detection Kit. Slides were counterstained with hematoxylin (Cat. No. 760-2021; Roche Diagnostics) for 4 minutes at room temperature and blued with blueing reagent (Cat. No. 760-2037; Roche Diagnostics) for 4 minutes at room temperature.

[0322] DAB staining of subject samples and subsequent analysis using anti-AKR1C3 antibody detection demonstrated that the majority of subject samples were assigned an H score above 50 ( Figure 1 In addition, 5 / 32 (16%) NSCLC adenocarcinoma subject samples, 13 / 45 (29%) NSCLC squamous cell carcinoma subject samples, 5 / 29 (17%) prostate cancer subject samples, and 19 / 68 (28%) HCC subject samples were assigned an H score of 250 or greater. These results demonstrate that anti-AKR1C3 antibodies can be used to detect AKR1C3 expression levels in subject samples from cancer tissues and calculate H scores.

[0323] Example 2: Clinical trial to determine dosing of AKR1C3-dependent KARS inhibitors

[0324] For the purpose of characterizing the safety, tolerability and pharmacokinetics of compound I (6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide) in patients with non-small cell lung cancer, a phase I, open-label, multicenter clinical trial study was conducted. The study is also optionally conducted to identify the maximum tolerated dose and / or recommended dose of compound I in adult patients with advanced non-small cell lung cancer with or without NFE2L2 / KEAP1 / CUL3 mutations. The preliminary anti-tumor activity of compound I is also optionally evaluated. The study includes a dose escalation part, followed by a dose expansion part. The escalation part characterizes safety and tolerability. The dose expansion part evaluates the preliminary anti-tumor activity in the defined patient population and further evaluates the safety and tolerability of the MTD / RD.

[0325] Patients and cohorts

[0326] Eligible patients for the study include those with advanced (metastatic or unresectable) NSCLC harboring NFE2L2 or KEAP1 or CUL3 mutations (dose escalation and dose expansion cohort 1) and patients with advanced (metastatic or unresectable) NSCLC regardless of mutation status (dose expansion cohort 2) for whom standard of care therapy for their indication has failed or who are intolerant of or ineligible for approved therapies.

[0327] Dose escalation and dose expansion cohort 1 included patients with histologically or cytologically confirmed advanced (metastatic or unresectable) NFE2L2 / KEAP1 / CUL3 mutant NSCLC. Local data confirming NFE2L2 / KEAP1 / CUL3 mutation status in tissue was required for enrollment.

[0328] Dose expansion cohort 2 included patients with histologically or cytologically confirmed advanced (metastatic or unresectable) NSCLC, regardless of mutation status.

[0329] For stage IV non-small cell lung cancer, all patients have progressed after 1 platinum-based chemotherapy regimen and / or PD (L) -1 antibody therapy (if indicated). Patients can include those patients who have received previous therapy with VEGF / VEGFR targeting agents, neoadjuvant / adjuvant therapy. Patients with non-small cell lung cancer whose tumors carry actionable mutations have been treated with approved targeted drugs (e.g., EGFRi, ALKi, METi). According to RECIST v1.1, all patients have at least one measurable lesion. All patients have disease sites suitable for biopsy, and according to the guidelines of the treatment institution, each patient is a candidate for tumor biopsy. Patients are willing to accept new tumor biopsies at the time of screening, and accept again during the therapy of this study. Optionally submit a recent biopsy collected within 3 months after the last systemic treatment and before entering the study at the time of screening.

[0330] Patients had no impaired cardiac function or clinically significant heart disease or risk factors at screening. Patients had no symptoms of CNS metastasis 2 weeks before entering the study, or CNS metastasis requiring local CNS directed therapy (such as radiotherapy or surgery) or increased doses of corticosteroids. Treated patients with symptomatic brain metastases were neurologically stable (lasting 4 weeks after treatment and before entering the study), and received a dose of ≤10 mg of prednisone or equivalent daily for at least 2 weeks before any study treatment was administered. Patients did not include those who were treated with drugs / supplements / herbal medicines that were strong or moderate CYP3A4 inhibitors or strong or moderate CYP3A4 inducers and could not be discontinued 7 days before the start of the study and during the study.

[0331] Goals and End Points

[0332] The efficacy of Compound I treatment was determined based on overall response rate, progression-free survival, and duration of response according to RECIST v1.1. Pharmacokinetics were evaluated based on plasma concentration versus time curves and derived pharmacokinetic parameters (e.g., Cmax, Tmax, AUC) of Compound I and its cytotoxic metabolite Compound II ((R)-6'-fluoro-N-(4-fluorobenzyl)-4'-hydroxy-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide).

[0333] The main purpose of this study is to characterize the safety and tolerability of compound I in patients with NSCLC and identify MTD and / or RD and dosing regimens for future studies. Safety is determined by the incidence and severity of adverse events (AEs) and serious adverse events (SAEs), including changes in laboratory parameters, vital signs, and electrocardiograms (ECGs). The incidence and nature of dose-limiting toxicity (DLT) are determined during the first 28 days of treatment with compound I. Tolerance is determined by dose interruptions, reductions, and dose intensity.

[0334] The secondary objectives of the study include evaluating the preliminary antitumor activity of compound I and evaluating the PK of compound I. The preliminary antitumor activity of compound I will be determined by overall response rate (ORR), progression-free survival (PFS) and duration of response (DOR) according to RECIST v1.1. The pharmacokinetics of compound I will be determined by analyzing the plasma concentration versus time curves of compound I and compound II and derived PK parameters (e.g., Cmax, Tmax, AUC).

[0335] Study Design

[0336] Dose escalation. A minimum of 21 patients with advanced NSCLC harboring NFE2L2 or KEAP1 or CUL3 (NFE2L2 / KEAP1 / CUL3) mutations will be treated in dose escalation. Patient enrollment is based on locally available mutation status test results (optionally requesting central confirmation of the same archived samples used to determine mutation status locally, if available).

[0337] Cohorts of 3-6 patients were treated orally (po) with various doses of Compound I, either QD or BID (based on emerging PK data during dose escalation) in 28-day cycles until the MTD and / or RD was reached.

[0338] The safety (including dose-DLT relationship) and tolerability of compound I are also evaluated to identify the regimen and / or MTD and / or RD for dose expansion. The dose and regimen of RD are determined after reviewing all available data (including PK, safety and preliminary anti-tumor activity). The Bayesian hierarchical logistic regression model (BHLRM) using the escalation design (escalation with overdosecontrol, EWOC) principle of controlling overdose is used to guide the dose escalation to determine MTD and / or RD. RD does not exceed the MTD of compound I.

[0339] Different dosing schedules (e.g., 2 weeks on / 2 weeks off, 3 weeks on / 1 week off, 1 week on / 1 week off) were also evaluated based on emerging PK, safety, tolerability data and / or preliminary antitumor activity.

[0340] Dose expansion. After the MTD and / or RD is declared in dose escalation, the study enters dose expansion. Approximately 100 patients with advanced NSCLC are treated in two dose expansion groups to evaluate the preliminary antitumor activity of compound I. At least 10 patients with squamous cell carcinoma are enrolled in each group. The dose expansion groups include the following groups:

[0341] • Group 1 (approximately 40 patients): patients with advanced NSCLC harboring NFE2L2 / KEAP1 / CUL3 mutations are enrolled based on locally available mutation status test results (optionally requesting central confirmation of the same archived samples used to determine mutation status locally, if available).

[0342] Group 2 (approximately 60 patients): patients with advanced NSCLC, regardless of prior knowledge of NFE2L2 / KEAP1 / CUL3 mutation status.

[0343] The study design Figure 2 Shown in.

[0344] The study optionally also includes an exploratory assessment of the food effect ("FE") on the PK of Compound I single agent in a separate cohort of patients with advanced NSCLC harboring NFE2L2 / KEAP1 / CUL3 mutations. For this subset of patients, the study design consists of a FE run-in period and a treatment period.

[0345] Patients underwent safety and efficacy assessments during screening / baseline and during treatment ( Figure 3 ).

[0346] Compound I was administered orally in capsule form with either 50 mg or 75 mg of drug substance.

[0347] The starting dose of Compound I single agent was set at 100 mg, administered QD po on a continuous schedule based on available preclinical safety, tolerability and PK / PD data. The selection of the starting dose followed the ICH S9 guidelines for selecting the starting dose for FIH trials in patients with advanced cancer. The starting dose was also supported by a 4-week GLP toxicology study in rats and monkeys.

[0348] Table 1 describes the starting doses and potential dose levels evaluated during this trial.

[0349] Table 1: Provisional dose levels

[0350]

[0351]

[0352] Biomarkers

[0353] Biomarker analysis is used to explore the effects of Compound I as a single agent at the molecular and cellular levels and to determine how changes in markers are associated with exposure and clinical outcomes. In addition, potential predictive markers of efficacy and mechanisms of resistance to Compound I as a single agent are optionally explored.

[0354] Enter the exact date and time of collection of the biomarker sample on the appropriate eCRF and requisition form. Detailed instructions for the collection, handling, and transportation of the biomarker sample are provided. Samples were collected at defined visits / time points. Biomarker sample types and collection are described in Table 2.

[0355] Table 2. Biomarker sample collection plan

[0356]

[0357]

[0358]

[0359] Collection of newly obtained pre-treatment and on-treatment paired tumor samples is mandatory at screening and treatment, if safe and medically feasible.

[0360] If a recent biopsy collected within 3 months after the last systemic treatment and before entering the study is available, it is optionally submitted at screening instead of a newly obtained biopsy. If an inadequate tumor sample is received at screening after a new biopsy procedure (e.g., found to have low tumor content or insufficient remaining tissue), the most recent biopsy is optionally required for analysis as described in Tables 8-13. In both cases, the most recent biopsy meets the specifications provided and detailed in the laboratory manual. A copy of the corresponding de-identified pathology report is also optionally submitted.

[0361] When core needle biopsies are performed, 3-6 tumor biopsies are required at both screening and post-treatment visits. On-treatment biopsies will be scheduled within a ±3 day window of C1D22, ideally 2-6 hours after treatment administration; the date and time of sampling will be recorded in the eCRF. The timing of on-treatment biopsies (C1D22) will be optionally adjusted based on emerging data. The timing of on-treatment tumor biopsies is at the discretion of the investigator.

[0362] To the extent possible, tumor biopsies were collected from the same tumor lesion.

[0363] The investigator made a best effort to record the location and size of the lesion at the biopsy site (baseline and on treatment). This information was recorded in the eCRF.

[0364] If available, the same archived tumor biopsy used for local testing at screening is submitted to retrospectively confirm local data related to NFE2L2, KEAP1, and CUL3 alterations, which can support potential companion diagnostic (CDx) assay development. A copy of the corresponding de-identified pathology report is also optionally requested. The mandatory screening biopsy or the most recent biopsy collected after the last systemic treatment and within 3 months before entering the study is also optionally used to retrospectively confirm NFE2L2 / KEAP1 / CUL3 mutation status at a central laboratory (if archived samples are not available) and for AKR1C3 expression analysis using a central IHC assay. Archived and newly obtained tumor samples are optionally analyzed for genetic alterations and optionally support potential CDx assay development.

[0365] Newly obtained paired tumor samples are collected for testing the PD effects of Compound I directly in the tumor (e.g., expression of ATF3, EGR1, DDIT3, AKR1C3, KARS) and evaluating whether AKR1C3 and / or KARS expression is a potential predictor of response. Whole transcriptome analysis and expression of additional immune or cancer-related genes are also optionally explored. Protein expression of AKR1C3 is used to support subgroup analysis in dose expansion group 2, and is optionally also tested in dose escalation and dose expansion group 1. Expression and localization of immune biomarkers (including but not limited to PD-L1 and CD8) are optionally measured by IHC or using other techniques deemed appropriate.

[0366] Blood was collected at screening and EOT for sequence analysis of cfDNA. This analysis explored the presence of emerging, existing, and resistance mutations as well as the potential tumor mutational burden in tumor and cfDNA at different time points and explored their association with clinical response and development of tumor resistance.

[0367] Additional blood samples were collected at screening to support potential CDx assay development for genes including but not limited to NFE2L2, KEAP1, and CUL3.

[0368] AKR1C3 expression and anti-tumor activity

[0369] To explore the relationship between baseline AKR1C3 expression and antitumor activity, particularly ORR, according to RECIST v1.1, a logistic regression model was fitted to patient data from dose expansion cohort 2. A score (H-score) was used to quantify AKR1C3 exposure in patients.

[0370] Assuming that patients with high AKR1C3 expression values ​​are associated with a higher probability of response, the estimated probability of response according to the fitted model is presented for each AKR1C3 H score value (e.g., H score = 0, 50, 150, 250) and its 90% confidence interval. Particular attention is paid to patients with the highest AKR1C3 concentration (H score ≥ 250).

[0371] Additional analyses are optionally performed on all patients treated at the same dose and for whom baseline AKR1C3 expression is available, including all data from patients in the escalation portion and dose expansion cohort 1. Subgroup analyses for specific patient groups (eg, squamous patients) are optionally considered.

Claims

1. A method of identifying a subject for treatment with a compound of formula (I): in is a single bond or a double bond; Z OH when it is a single bond; or When it is a double bond, it is O; Each R 1 independently selected from the group consisting of: (C1-C6)alkyl, (C1-C6)alkoxy, (C0-C4)alkylN(R 8 )2 and halogenated; R 2a and R 2b Each independently selected from the group consisting of: H, (C1-C6)alkyl and halo; Each R 3 are independently selected from the group consisting of: H and halo; R 4 is selected from the group consisting of: aryl; 5- to 6-membered heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; and 9- to 10-membered fused bicyclic heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; wherein any of the foregoing groups is optionally replaced by one or more R 6 replace; R 5 Selected from the group consisting of: H, (C1-C6) alkyl, (C2-C6) alkenyl, (C0-C4) alkyl, OR 8 、(C1-C4)alkyl(C3-C 10 )cycloalkyl, halo(C1-C6)alkyl, (C2-C3)alkynyl, (C1-C4)alkylN(R 10 )2; Each R 6 independently selected from the group consisting of halo, (C1-C6) alkyl, (C1-C6) alkoxy, halo (C1-C6) alkyl, OH, aryl, 3- to 6-membered heterocyclic, 5- to 6-membered heteroaryl, (C0-C4) alkyl, S(O) m (C1-C6) alkyl, halogenated (C1-C6) alkoxy, (C0-C4) alkyl S(O) m N(R 8 )2、(C0-C4)alkyl N(R 8 )2、(C0-C4)alkyl(CO)OR 7 、N(R 8 )S(O) m (C1-C6) alkyl, N(R 8 )S(O) m (C3-C6)cycloalkyl, OP(O)(OH)2, (C0-C3)alkyl (CO)NHR 11 、(C0-C3)alkylOR 7 and (C3-C 10 )cycloalkyl; wherein each R 6 When not halogenated, OH or OP(O)(OH)2, it is optionally substituted with one to three R 9 Substitution; or two adjacent R 6 Together with the atoms to which they are attached, they form a 5- to 7-membered heterocyclic ring or a (C5-C8)cycloalkyl group; Each R 7 and R 8 are independently selected from the group consisting of: H or optionally with one to three R 9 Substituted (C1-C6) alkyl; Each R 9 Independently selected from the group consisting of: halo, -OH, amino, (C 1- C4) alkylamino, di(C 1- C4)alkylamino, OP(O)(OH)2, (C1-C6)alkyl, (C1-C3)alkynyl, (C1-C6)alkoxy, halo(C1-C6)alkyl, (C0-C4)alkylS(O) m (C1-C6) alkyl, halo (C1-C6) alkoxy, 3- to 6-membered heterocyclic ring optionally substituted by oxo (=O), (C0-C4) alkyl S (O) m N(R 10 )2、(C0-C4)alkyl(CO)R 10 、(C0-C4)alkyl(CO)OR 10 、(C0-C4) alkyl NR 10 S(O) m (C1-C6)alkyl, (C0-C4)alkylOR 10 、(C0-C4)alkyl N(R 10 )2, (C0-C4) alkyl CN, (C0-C4) alkyl N (R 10 )2 and (C0-C4)alkyl (CO)N(R 10 )2; Each R 10 independently selected from the group consisting of: H, (C1-C6)alkyl, or 3 to 6 membered heterocyclic ring, wherein the 3 to 6 membered heterocyclic ring is optionally substituted with one or more of: (C1-C6)alkyl and oxo (=O); Each R 11 Selected from the group consisting of: H, optionally with one to four R 12 substituted 4 to 6 membered heterocyclic ring, optionally substituted by one to four R 12 substituted (C3-C6) cycloalkyl, optionally substituted (C0-C3) alkyl, (C3-C6) cycloalkyl (C1-C3) alkyl, optionally substituted by one to three R 12 substituted CH2-aryl, (C1-C6)alkyl, (C2-C6)alkenyl or (C2-C6)alkynyl, wherein each of the (C1-C6)alkyl, (C2-C6)alkenyl and (C2-C6)alkynyl is optionally substituted with one or more R 13 replace; Each R 12 independently selected from the group consisting of OH, (C1-C3)alkoxy, NH2 or (C1-C3)alkyl optionally substituted with one or more OH; Each R 13 Independently selected from the group consisting of: halo, OH, amino, (C 1- C4) alkylamino, di(C 1- C4) alkylamino, (C1-C3) alkoxy and C(O)-(C3-C8) cycloalkyl; m is 0, 1 or 2; and n is 0, 1, or 2, or a pharmaceutically acceptable salt thereof, The method comprises determining the level of AKR1C3 in a sample from a subject, wherein elevated levels of AKR1C3 identify the subject as a subject in need of treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof.

2. A method for selecting a compound of formula (I) or a pharmaceutically acceptable salt thereof to treat a subject, the method comprising determining the level of AKR1C3 in a sample from the subject, wherein an elevated level of AKR1C3 identifies the subject as a subject in need of treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof.

3. A method of treating a subject, the method comprising: a. determining the level of AKR1C3 in a sample from a subject, wherein an elevated level of AKR1C3 identifies the subject as being in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof; as well as b. administering to the subject an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

4. A method of treating a subject, the method comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein prior to said administering, a sample from the subject is characterized as having elevated AKR1C3 levels.

5. A method of treating a subject with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein a sample from the subject is characterized as having elevated levels of AKR1C3.

6. The method of any one of claims 1 to 5, wherein the compound of formula (I) is selected from the group consisting of: 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof.

7. The method of any one of claims 1 to 6, wherein the compound of formula (I) is 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof.

8. The method of any one of claims 1-7, wherein the subject sample comprises a cell, a cell population, a cell lysate, a tissue, or a fluid of the subject.

9. The method of claim 8, wherein the cell is a cancer cell.

10. The method of claim 9, wherein the cancer cell is a tumor cell.

11. The method of claim 10, wherein the tumor cells are selected from the group consisting of lung cancer tumor cells, non-small cell lung cancer tumor cells, lung adenocarcinoma tumor cells, lung squamous cell carcinoma cells, bladder tumor cells, cervical tumor cells, esophageal tumor cells, head and neck tumor cells, kidney tumor cells, and liver tumor cells.

12. The method of claim 8, wherein the cells are lung cells.

13. The method of claim 8, wherein the fluid is selected from the group consisting of: blood, plasma, and lymph.

14. The method of any one of claims 1-13, wherein the subject is diagnosed with a disease or disorder selected from the group consisting of non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer, cervical cancer, esophageal cancer, head and neck cancer, kidney cancer, and liver cancer.

15. The method of any one of claims 1-14, wherein the subject's tumor genome comprises a somatic mutation in one or more of the NFE2L2, KEAP1, and CUL3 gene sequences.

16. The method of any one of claims 1-15, wherein the subject sample has elevated AKR1C3 levels relative to a control AKR1C3 level.

17. The method of claim 16, wherein the control level comprises the level of AKR1C3 of a control sample or a control data set.

18. The method of claim 17, wherein the control sample comprises a sample selected from the group consisting of: non-cancerous cells of the subject, a population of non-cancerous cells of the subject, a non-cancerous tissue of the subject, a non-cancerous fluid of the subject, a non-cancerous cell of a control subject, a population of non-cancerous cells of a control subject, a non-cancerous tissue of a control subject, and a non-cancerous fluid of a control subject.

19. The method of claim 17, wherein the control data set comprises biomarker level data from a source selected from the group consisting of: non-cancerous cells of the subject, a population of non-cancerous cells of the subject, a non-cancerous tissue of the subject, a non-cancerous fluid of the subject, non-cancerous cells of a control subject, a population of non-cancerous cells of a control subject, a non-cancerous tissue of a control subject, a non-cancerous fluid of a control subject, and combinations thereof.

20. The method of any one of claims 1-19, wherein the AKR1C3 level is the protein level of AKR1C3.

21. The method of any one of claims 1-19, wherein the AKR1C3 level is the RNA level of AKR1C3.

22. The method of claim 21, wherein the RNA level of AKR1C3 is the mRNA level of AKR1C3.

23. The method of any one of claims 17-22, wherein the subject is in need of treatment with the compound of Formula (I) or a pharmaceutically acceptable salt thereof if the level of AKR1C3 in the subject's sample is about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 1500 times, or about 2000 times the level of AKR1C3 in the control sample or the control dataset.

24. The method of any one of claims 17-22, wherein if the level of AKR1C3 in the subject sample is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 10 ... times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1000 times, at least about 1500 times, or at least about 2000 times, then the subject needs to be treated with the compound of Formula (I) or a pharmaceutically acceptable salt thereof.

25. The method of any one of claims 1-2, 6-20, 23 or 24, wherein said determining further comprises performing an antigen detection assay.

26. The method of claim 25, wherein the antigen detection assay is selected from the group consisting of: a Western blot assay, an enzyme-linked immunosorbent assay (ELISA), an immunohistochemistry (IHC) assay, an immunocytochemistry assay, a flow cytometry assay, an immunoprecipitation assay, an immunoelectrophoresis assay, and an immunoelectron microscopy assay.

27. The method of claim 25 or 26, wherein the antigen detection assay is an IHC assay.

28. The method of any one of claims 25-27, wherein performing the antigen detection assay comprises probing the subject sample with an AKR1C3 antibody.

29. The method of claim 28, wherein the AKR1C3 antibody is anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6.

30. The method of claim 28, wherein the AKR1C3 antibody comprises CDR sequences that share at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with the CDR sequences of the anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6.

31. The method of any one of claims 28-30, wherein the AKR1C3 antibody is conjugated to horseradish peroxidase (HRP).

32. The method of any one of claims 28-30, wherein performing the antigen detection assay further comprises probing the subject sample with a secondary antibody.

33. The method of claim 32, wherein the secondary antibody is conjugated to HRP.

34. The method of any one of claims 28-33, wherein the antigen detection assay further comprises applying 3,3'-diaminobenzidine (DAB) to the subject sample.

35. The method of any one of claims 27-34, wherein the determining further comprises generating an IHC signal intensity score for the subject's sample.

36. The method of claim 35, wherein if the IHC signal intensity score of the subject's sample is 0.5 or greater, 1.0 or greater, 1.5 or greater, 2 or greater, 2.5 or greater, 2.6 or greater, 2.7 or greater, 2.8 or greater, or 2.9 or greater, and the IHC signal intensity score can be in the range of 0-3, then the subject is in need of treatment with the compound of Formula (I) or a pharmaceutically acceptable salt thereof.

37. The method of any one of claims 4-20, 23 or 24, wherein the subject sample is characterized as having elevated levels of AKR1C3 by an antigen detection assay.

38. The method of claim 37, wherein the antigen detection assay is selected from the group consisting of: a Western blot assay, an enzyme-linked immunosorbent assay (ELISA), an immunohistochemistry (IHC) assay, an immunocytochemistry assay, a flow cytometry assay, an immunoprecipitation assay, an immunoelectrophoresis assay, and an immunoelectron microscopy assay.

39. The method of claim 37 or 38, wherein the antigen detection assay is an IHC assay.

40. The method of any one of claims 37-39, wherein the antigen detection assay comprises probing the subject sample with an AKR1C3 antibody.

41. The method of claim 40, wherein the AKR1C3 antibody is anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6.

42. The method of claim 40, wherein the AKR1C3 antibody comprises CDR sequences that share at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with the CDR sequences of the anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6.

43. The method of any one of claims 40-42, wherein the AKR1C3 antibody is conjugated to horseradish peroxidase (HRP).

44. The method of any one of claims 40-42, wherein the antigen detection assay further comprises probing the subject sample with a secondary antibody.

45. The method of claim 44, wherein the secondary antibody is conjugated to HRP.

46. ​​The method of any one of claims 40-45, wherein the antigen detection assay further comprises applying 3,3'-diaminobenzidine (DAB) to the subject sample.

47. The method of any one of claims 39-46, wherein the antigen detection assay further comprises generating an IHC signal intensity score for the subject's sample.

48. The method of claim 50, wherein if the IHC signal intensity score of the subject's sample is 0.5 or greater, 1.0 or greater, 1.5 or greater, 2 or greater, 2.5 or greater, 2.6 or greater, 2.7 or greater, 2.8 or greater, or 2.9 or greater, and the IHC signal intensity score can range from 0 to 3, then the subject is in need of treatment with the compound of Formula (I) or a pharmaceutically acceptable salt thereof.

49. The method of any one of claims 1-3, wherein the determining comprises performing a polymerase chain reaction (PCR) effective to determine the level of AKR1C3 in the subject's sample.

50. The method of claim 49, wherein the determining further comprises performing PCR effective to determine the level of a control marker in the subject sample.

51. The method of claim 49 or 50, wherein the determining further comprises performing a PCR effective to determine the level of AKR1C3 of a control sample.

52. The method of claim 51, wherein the determining further comprises performing PCR effective to determine the level of a control marker of the control sample.

53. The method of any one of claims 50-52, wherein the control marker is selected from the group consisting of: beta actin or GAPDH.

54. The method of any one of claims 49-53, wherein the PCR is quantitative PCR (qPCR).

55. The method of any one of claims 49-54, wherein the PCR is reverse transcription PCR (RT-PCR).

56. The method of any one of claims 49-55, wherein the PCR is reverse transcription qPCR (RT-qPCR).

57. The method of claim 4 or 5, wherein the subject sample is characterized as having elevated AKR1C3 levels by PCR effective to determine the AKR1C3 biomarker level of the subject sample.

58. The method of claim 57, wherein the subject sample is characterized by PCR effective to determine the level of a control marker in the subject sample.

59. The method of claim 57 or 58, wherein the control sample is characterized as having a certain AKR1C3 level by PCR effective to determine the AKR1C3 level of the control sample.

60. The method of claim 59, wherein the control sample is characterized by PCR effective to determine the level of a control marker in the control sample.

61. The method of any one of claims 58-60, wherein the control marker is selected from the group consisting of: beta actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

62. The method of any one of claims 57-61, wherein the PCR is quantitative PCR (qPCR).

63. The method of any one of claims 57-62, wherein the PCR is reverse transcription-PCR (RT-PCR).

64. The method of any one of claims 57-63, wherein the PCR is reverse transcription qPCR (RT-qPCR).

65. A method for identifying a subject in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof, the method comprising detecting a somatic mutation in at least one of the following genes: NFE2L2, KEAP1 or CUL3 in a sample from the subject.

66. A method of selecting a compound of Formula (I) or a pharmaceutically acceptable salt thereof for treating a subject, the method comprising detecting a somatic mutation in at least one of the following genes: NFE2L2, KEAP1 or CUL3 in a sample from the subject.

67. A method of treating a subject, the method comprising: a. detecting a somatic mutation in at least one of the following genes in a sample from a subject: NFE2L2, KEAP1 or CUL3, wherein the detection identifies the subject as requiring treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof; as well as b. administering to the subject an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

68. A method of treating a subject, the method comprising administering to the subject an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein prior to said administering, a sample from the subject is characterized by the presence of a somatic mutation in at least one of the following genes: NFE2L2, KEAP1, or CUL3.

69. A method of treating a subject with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein a sample from the subject is characterized by the presence of a somatic mutation in at least one of the following genes: NFE2L2, KEAP1, or CUL3.

70. The method of any one of claims 65-69, wherein the compound of formula (I) is selected from the group consisting of: 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof.

71. The method of any one of claims 65-70, wherein the compound of formula (I) is 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof.

72. The method of any one of claims 65-71, wherein the somatic mutation is selected from the group consisting of: an amplification of the NFE2L2 gene sequence or a portion thereof, a deletion of the KEAP1 gene sequence or a portion thereof, and a deletion of the CUL3 gene sequence or a portion thereof.

73. The method of any one of claims 65-71, wherein the somatic mutation comprises a mutation selected from the group consisting of a nonsense mutation, a missense mutation, a substitution mutation, a frameshift mutation, a point mutation, an insertion mutation, a deletion mutation, an inversion mutation, and a gene amplification mutation.

74. The method of any one of claims 65-71, wherein the somatic mutation comprises a single nucleotide polymorphism (SNP).

75. The method of any one of claims 65-74, wherein the subject sample comprises a cell, a cell population, a cell lysate, a tissue, or a fluid of the subject.

76. The method of claim 75, wherein the subject sample comprises genomic DNA from a cell, cell population, cell lysate, tissue, or fluid of the subject.

77. The method of claim 75 or 76, wherein the cell is a cancer cell.

78. The method of claim 77, wherein the cancer cell is a tumor cell.

79. The method of claim 78, wherein the tumor cell is selected from the group consisting of lung cancer tumor cells, non-small cell lung cancer tumor cells, lung adenocarcinoma tumor cells, lung squamous cell carcinoma cells, bladder tumor cells, cervical tumor cells, esophageal tumor cells, head and neck tumor cells, kidney tumor cells, and liver tumor cells.

80. The method of claim 75 or 76, wherein the cells are lung cells.

81. The method of claim 75 or 76, wherein the fluid is selected from the group consisting of: blood, plasma, and lymph.

82. The method of any one of claims 65-81, wherein the subject is diagnosed with a disease or disorder selected from the group consisting of non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer, cervical cancer, esophageal cancer, head and neck cancer, kidney cancer, and liver cancer.

83. The method of any one of claims 65-82, wherein the somatic mutation is not present in a control sample or a control dataset.

84. The method of claim 83, wherein the control sample comprises a sample selected from the group consisting of: non-cancerous cells of the subject, a population of non-cancerous cells of the subject, a non-cancerous tissue of the subject, a non-cancerous fluid of the subject, a non-cancerous cell of a control subject, a population of non-cancerous cells of a control subject, a non-cancerous tissue of a control subject, and a non-cancerous fluid of a control subject.

85. The method of claim 84, wherein the control sample comprises genomic DNA of non-cancer cells of the subject, a population of non-cancer cells of the subject, a non-cancerous tissue of the subject, a non-cancerous fluid of the subject, non-cancerous cells of the control subject, a population of non-cancerous cells of the control subject, a non-cancerous tissue of the control subject, or a non-cancerous fluid of the control subject.

86. The method of claim 83, wherein the control data set comprises genomic sequence data from a source selected from the group consisting of: non-cancerous cells of the subject, a population of non-cancerous cells of the subject, a non-cancerous tissue of the subject, a non-cancerous fluid of the subject, non-cancerous cells of a control subject, a population of non-cancerous cells of a control subject, a non-cancerous tissue of a control subject, a non-cancerous fluid of a control subject, and combinations thereof.

87. The method of any one of claims 65-75, wherein the level of AKR1C3 in the subject's sample is about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 1500 times, or about 2000 times the level of AKR1C3 in a control sample or a control dataset.

88. The method of any one of claims 65-75, wherein the level of AKR1C3 in the subject's sample is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1000 times, at least about 1500 times, or at least about 2000 times the level of AKR1C3 in a control sample or a control dataset.

89. The method of claim 87 or 88, wherein the level of AKR1C3 is the level of AKR1C3 mRNA.

90. The method of claim 87 or 88, wherein the level of AKR1C3 is the level of AKR1C3 protein.

91. The method of any one of claims 65-67, wherein the detection comprises sequencing genomic DNA or mRNA of the subject's sample.

92. The method of claim 91, wherein the sequencing is selected from the group consisting of: exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing, and Heliscope single molecule sequencing.

93. The method of claim 91, wherein the sequencing comprises performing a polymerase chain reaction (PCR).

94. The method of claim 93, wherein the PCR is selected from the group consisting of quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), reverse transcription qPCR (RT-qPCR), and digital PCR.

95. The method of any one of claims 65-94, wherein if the sequencing detects a somatic mutation in the subject's sample, the subject is in need of treatment with the compound of Formula (I) or a pharmaceutically acceptable salt thereof.

96. The method of any one of claims 91-95, wherein the method further comprises sequencing the genomic DNA or mRNA of a control sample.

97. The method of any one of claims 91-95, wherein the method further comprises comparing sequencing data from the sequencing of the subject's sample with sequencing data from a control sample or a control data set.

98. The method of claim 68 or 69, wherein the subject sample is characterized by the presence of the somatic mutation by sequencing the genomic DNA or mRNA of the subject sample.

99. The method of claim 95, wherein the sequencing is selected from the group consisting of: exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, combined probe anchor synthesis (cPAS) sequencing, combined probe anchor ligation technology (cPAL) sequencing, SOLiD sequencing, nanopore sequencing, Genap Sys sequencing, Sanger sequencing, Solexa sequencing, DNA nanoball sequencing, and Heliscope single molecule sequencing.

100. The method of claim 95, wherein the sequencing comprises performing a polymerase chain reaction (PCR).

101. The method of claim 100, wherein the PCR is selected from the group consisting of quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), reverse transcription qPCR (RT-qPCR), and digital PCR.

102. The method of any one of claims 98-101, wherein if the sequencing detects a somatic mutation in the subject's sample, the subject is in need of treatment with the compound of Formula (I) or a pharmaceutically acceptable salt thereof.

103. The method of any one of claims 98-102, wherein the control sample is characterized by the absence of the somatic mutation by sequencing its genomic DNA or mRNA.

104. The method of any one of claims 98-102, wherein the method further comprises comparing the sequencing data of sequencing the genomic DNA or mRNA of the subject's sample with the sequencing data of a control sample or a control data set.

105. Use of AKR1C3 levels for selecting a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein if a sample from the subject is characterized as having elevated AKR1C3 levels, the subject is treated with the compound of formula (I) or a pharmaceutically acceptable salt thereof.

106. Use of somatic mutations for selecting a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein if a sample from the subject is characterized by the presence of the somatic mutation, and wherein the somatic mutation is detected in one of the following genes, the subject is treated with the compound of formula (I) or a pharmaceutically acceptable salt thereof: NFE2L2, KEAP1 or CUL3.

107. The use of claim 105 or 106, wherein the compound of formula (I) is selected from the group consisting of: 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof.

108. The use of any one of claims 105-107, wherein the compound of formula (I) is 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; and N-(4-amino-3-fluorobenzyl)-6'-fluoro-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide, or a pharmaceutically acceptable salt thereof.

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