Methods of administration of tricyclic AKR1C3-dependent KARS inhibitors

Abstract

The present invention relates to a method for treating or identifying a subject for treatment with a tricyclic AKR1C3-dependent KARS inhibitor of formula (I) or a pharmaceutically acceptable salt thereof. The method may include determining in a subject's sample the level of at least one of the following biomarkers: AKR1C3, NFE2L2, KEAP1, or CUL3, wherein an elevated level of the biomarker determines that the subject is in need of treatment; and detecting in the subject's sample a somatic mutation in at least one of the following genes: 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 a method of identifying a subject for treatment or treating a subject with a tricyclic aldehyde ketoreductase 1C3 (AKR1C3)-dependent lysyl-tRNA synthase (KARS) inhibitor of Formula (I) or a pharmaceutically acceptable salt thereof. In embodiments described herein, the method may include determining the level of a biomarker, such as AKR1C3, in a subject sample, where an elevated level of the biomarker identifies the subject as being in need of treatment; or may include detecting a somatic mutation in at least one of the following genes in the subject sample: NFE2-like Bzip transcription factor 2 (NFE2L2), Kelch-like ECH-associated protein 1 (KEAP1), or Cullin3 (CUL3), where detection of the somatic mutation identifies the subject as being in need of treatment. [Background technology]

[0002] The NFE2L2 / NFE2L2-KEAP1 pathway has a strong genetic basis in cancer. TCGA's sequencing efforts reported that this pathway is altered in 34% of lung squamous cell carcinomas (Hammerman et al., (2012) Nature 489 519-525). TCGA and other groups have also reported that mutations in this pathway are important in the indications of other solid tumors, including head and neck squamous cell carcinoma and hepatocellular carcinoma. Aberrant activation of the NFE2L2 pathway can occur through gain of function genetic alterations in NFE2L2 or loss of function genetic alterations in KEAP1 or CUL3 (responsible for stabilizing NFE2L2 and increasing the expression of its target genes). Deregulated transcription of these target genes confers benefits to cancer cells, such as malignant tumors, and protects them from oxidative stress, chemotherapy, and radiation therapy (Jaramillo and Zhang (2013) Genes Dev. 27 2179-2191). Deterioration of NFE2L2 activity in tumors has been associated with poor prognosis (Shibata et al., (2008) Proc Natl Acad Sci USA 105 13568-13573). To the best of our knowledge, there are currently no approved therapies that selectively target cancers through genetic alterations in the NFE2L2 / KEAP1 pathway, representing an unmet medical need.

[0003] Aldehyde ketoreductase 1C3 (AKR1C3) is one of 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 1530-1539). AKR1C3 (also named type 2 3α(17β)-hydroxysteroid dehydrogenase) is an NADP(H)-dependent ketosteroid reductase (a member of the aldo-ketoreductase (AKR) superfamily) that plays a role in steroid hormone metabolism and signaling, as well as 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. 351, 67-77), as well as the synthetic prodrugs coumberone (Halim et al., (2008) J. Am. Chem. Soc. 130, 14123-14128), PR104 (Jamieson et al., (2014) Biochem Pharmacol. 88, 36-45), and TH3424 / OBI3424 (WO 2016 / 145092 A1). Additionally, the identity of a tricyclic ketone compound that is converted by AKR1C3 to a lysine tRNA synthetase (KARS) inhibitor in the presence of NADPH was identified.

[0004] AKR1C3-dependent KARS inhibitors offer an attractive strategy to selectively treat tumors that overexpress AKR1C3, such as NFE2L2 / KEAP1 mutant cancers, and other types of cancer that have been reported to overexpress AKR1C3 compared to normal tissues (Guise et al., (2010) Cancer Res. 70, 1573-1584), such as breast cancer (Lewis et al., (2004) BMC Cancer 4, 27) and prostate cancer (Fung et al., (2006) Endocr Relat Cancer 13, 169-180).

[0005] The compound of formula (I) and its pharmaceutically acceptable salts, as described in International Publication No. 2021 / 005586, have been identified as potent tricyclic AKR1C3-dependent KARS inhibitors. Currently, identifying patients for treatment with tricyclic AKR1C3-dependent KARS inhibitors and determining appropriate doses for treating such patients remains a challenge. There is a need to provide an accurate and reliable method for identifying and treating such patients. Summary of the Invention

[0006] AKR1C3-dependent KARS inhibitors provide therapy and treatment for patients suffering from cancers with genetic alterations in the NFE2L2 / KEAP1 pathway. The invention described herein provides a method of 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 a method of selecting a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating a subject. The invention described herein also provides a method of 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 AKR1C3 protein or the level of AKR1C3 mRNA. In some embodiments of the methods described herein, the subject sample is characterized as having an elevated level of AKR1C3, e.g., an elevated level of AKR1C3 protein or an elevated level of AKR1C3 mRNA. In some embodiments, an elevated level of AKR1C3 identifies the subject as one in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0008] In some embodiments, the methods described herein include detecting a somatic mutation in at least one of the following genes in a subject sample: NFE2L2, KEAP1, or CUL3. In some embodiments of the methods described herein, 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 identifies a subject 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 methods include 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 present invention are described herein.

[0011] In one aspect of the invention, a compound of formula (I): [ka] (In the formula: [ka] is a single or double bond; Z is [ka] is a single bond, then OH; or [ka] is a double bond, then either O; Each R 1 are independently (C1-C6) alkyl, (C1-C6) alkoxy, (C0-C4) alkylN(R8 )2, and halo; R 2a and R 2b are each independently selected from the group consisting of H, (C1-C6) alkyl, and halo; Each R 3 is independently selected from the group consisting of H and halo; R 4 is selected from the group consisting of aryl, 5-6 membered heteroaryl containing 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S; and 9-10 membered fused bicyclic heteroaryl containing 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S; any of the foregoing may be selected from the group consisting of one or more R 6 optionally substituted with; R 5 is 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 is selected from the group consisting of; Each R 6 is halo; (C1-C6) alkyl; (C1-C6) alkoxy; halo(C1-C6) alkyl; OH; aryl; 3- to 6-membered heterocycle; 5- to 6-membered heteroaryl; (C0-C4) alkylS(O) m (C1-C6)Alkyl;Halo(C1-C6)alkoxy;(C0-C4)AlkylS(O) m N(R 8 )2;(C0-C4) alkylN(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) alkyl OR 7 and (C3~C 10) cycloalkyl; each R 6 is 1 to 3 R when it is not halo, OH, or OP(O)(OH)2 9 or two adjacent R 6 together with the atom to which they are attached form a 5- to 7-membered heterocycle or a (C5-C8)cycloalkyl; Each R 7 and R 8 are independently selected from the group consisting of H or (C1-C6) alkyl, and 1 to 3 R 9 optionally substituted with; Each R 9 is halo; -OH; amino, (C1-C4) alkylamino, di(C1-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; 3-6 membered heterocycle optionally substituted with oxo(=O); (C0-C4) alkylS(O) m N(R 10 )2;(C0-C4)Alkyl(CO)R 10 ;(C0-C4) alkyl(CO)OR 10 (C0-C4) alkylNR 10 S(O) m (C1-C6) alkyl; (C0-C4) alkyl OR 10 ;(C0-C4) alkylN(R 10 )2;(C0-C4) alkylCN;(C0-C4) alkylN(R 10 )2; and (C0-C4) alkyl(CO)N(R 10 )2 independently selected from the group consisting of: Each R 10 is 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 (C1-C6) alkyl; and oxo (=O); Each R 11 is H; 1 to 4 R 124- to 6-membered heterocycle optionally substituted with 1 to 4 R 12 (C3-C6)cycloalkyl optionally substituted with; (C0-C3)alkyl(C3-C6)cycloalkyl(C1-C3)alkyl optionally substituted with halo; 1 to 3 R 12 (C-C)alkyl; (C-C)alkenyl; or (C-C)alkynyl, wherein each of the (C-C)alkyl; (C-C)alkenyl; and (C-C)alkynyl is selected from the group consisting of one or more R 13 optionally substituted with; Each R 12 are 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 are independently selected from the group consisting of halo, OH, amino, (C1-C4) alkylamino, di(C1-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. In some embodiments, the method comprises determining the level of AKR1C3 in a subject's sample. In some embodiments, an elevated level of AKR1C3 identifies the subject as a subject in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0012] In another aspect of the present invention, methods for selecting a compound of Formula (I) or a pharmaceutically acceptable salt thereof for treating a subject are described herein. In some embodiments, the method comprises determining the level of AKR1C3 in a subject sample. In some embodiments, an elevated level of AKR1C3 identifies the subject as a subject in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0013] In yet another aspect of the present invention, the present invention provides a method for treating a subject.In some embodiments, the method comprises: determining the level of AKR1C3 in a subject's sample, and identifying or determining that the subject needs treatment with the compound of formula (I) or its pharmaceutically acceptable salt due to elevated level of AKR1C3; and administering an effective amount of the compound of formula (I) or its pharmaceutically acceptable salt to the subject.

[0014] In some embodiments, the methods of treating a subject described herein comprise administering to the subject an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. For example, methods of treating a subject are described herein, 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 level of AKR1C3.

[0015] Also described herein are methods of treating a subject with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the subject's sample is characterized as having elevated levels of AKR1C3.

[0016] 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 compounds 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.

[0017] In some embodiments of the methods described herein, the subject sample is characterized by an elevated level of AKR1C3. In some embodiments of the methods described herein, the method comprises determining the level of AKR1C3 in the subject sample.

[0018] In various embodiments described herein, a sample, e.g., a subject sample, is or comprises a subject's cells, cell population, cell lysate, tissue, or bodily fluid. In some embodiments, a subject sample comprises the contents of a subject's cells, cell population, cell lysate, tissue, or bodily fluid, e.g., the mRNA or protein contents of a subject's cells, cell population, cell lysate, tissue, or bodily fluid. In some embodiments, a subject sample comprises a subject's genome, transcriptome, or proteome. In some embodiments, a subject sample comprises a subject's tumor genome, transcriptome, or proteome.

[0019] A subject sample can be obtained from a subject by any suitable means, such as by biopsy or cell or tissue extraction. Similarly, a control sample can be obtained from a control subject by any suitable means.

[0020] In some embodiments, the subject sample is or comprises cells, where the cells are cancerous cells, e.g., tumor cells, e.g., 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, or liver tumor cells. In some embodiments, the subject sample is or comprises cells, where 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, throat cells, esophageal cells, kidney cells, small intestine cells, colon cells, blood cells, red blood cells, white blood cells, platelet cells, or liver cells. In some embodiments, the subject sample is or comprises cells, where the cells are lung cells. In some embodiments, the subject sample is or comprises cells, where the cells are airway cells. For example, in some embodiments, the cell is 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 cancer cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara mucosa cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a duct cell), an interstitial connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a meningeal cell, an adipose tissue cell, or a neuron), a vascular cell, or a vascular endothelial cell. The subject sample may be or comprise a 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 megakaryocyte, a macrophage cell, a Langerhans cell, a mast cell, an eosinophilic cell, a neutrophilic cell, or a basophilic cell), 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 meningeal cell, or an endothelial progenitor cell. In some embodiments, the subject sample is or comprises a bodily fluid, wherein the bodily fluid is selected from the group consisting of blood, plasma, mucus, urine, and lymph.In some embodiments, the subject sample comprises a subject's genome, transcriptome, or proteome of any of the aforementioned cells or bodily fluids, for example, a subject's tumor genome, transcriptome, or proteome.

[0021] In some embodiments described herein, the subject has been diagnosed with, is in need of treatment, is being treated, is in remission, is at risk of developing, or is susceptible to developing a disease or disorder, such as cancer. 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 carcinoma), 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 has been 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 carcinoma), 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 has been diagnosed with a disease or disorder, such as cancer.

[0022] In some embodiments of the methods described herein, the subject's tumor genome comprises a somatic mutation associated with a disease or disorder, such as 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 a somatic mutation in the subject's tumor genome in the NFE2L2, KEAP1, or CUL3 gene sequence can indicate that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0023] In some embodiments, the level of AKR1C3 detected or characterized in the subject sample is compared to a control level of AKR1C3. In some embodiments, the level of AKR1C3 in the subject sample is elevated compared to the control level of AKR1C3. An elevated level of AKR1C3 in the subject sample compared to the control level of AKR1C3 may indicate that the subject is in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0024] In some embodiments, the control level comprises the level of AKR1C3 in a control sample or control dataset. For example, in some embodiments, the control level comprises the level of AKR1C3 in a control sample or control dataset, where the biomarker is a biomarker characterized or determined in the subject sample (e.g., AKR1C3 protein or AKR1C3 mRNA).

[0025] In various embodiments described herein, the control sample is or comprises a non-cancerous cell from the subject, a non-cancerous cell population from the subject, a non-cancerous tissue from the subject, a non-cancerous bodily fluid from the subject, a non-cancerous cell from a control subject, a non-cancerous cell population from a control subject, a non-cancerous tissue from a control subject, or a non-cancerous bodily fluid from a control subject. In some embodiments, the control sample is or comprises the cellular content, e.g., the protein or mRNA content, of a non-cancerous cell from the subject, a non-cancerous cell population from the subject, a non-cancerous tissue from the subject, a non-cancerous bodily fluid from the subject, a non-cancerous cell from the control subject, a non-cancerous cell population from the control subject, a non-cancerous tissue from the control subject, or a non-cancerous bodily fluid from the control subject. In some embodiments, the control sample comprises a non-cancerous cell genome, transcriptome, or proteome, e.g., the genome, transcriptome, or proteome of a non-cancerous cell from the subject, or the genome, transcriptome, or proteome of a non-cancerous cell from the control subject.

[0026] In some embodiments, the control dataset comprises biomarker (e.g., AKR1C3) level data from non-cancerous cells of a subject, a non-cancerous cell population of a subject, a non-cancerous tissue of a subject, a non-cancerous body 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 body fluid of a control subject, or a combination thereof. In some embodiments, the control dataset comprises biomarker (e.g., AKR1C3) level data from cellular content, such as non-cancerous cells of a subject, a non-cancerous cell population of a subject, a non-cancerous tissue of a subject, a non-cancerous body fluid of a control 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 body fluid of a control subject, or a combination thereof. In some embodiments, the control dataset includes biomarker (e.g., AKR1C3) level data derived from the genome, transcriptome, or proteome of one or more non-cancerous cells, e.g., the genome, transcriptome, or proteome of one or more non-cancerous cells of the subject, or the genome, transcriptome, or proteome of one or more non-cancerous cells of a control subject.

[0027] 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 lymphatic cell, 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 intestine cell, a colon 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 an airway cell. For example, in some embodiments, the cell is 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 cancer cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara mucosa cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a duct cell), an interstitial connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a meningeal cell, an adipose tissue cell, or a neuron), 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 megakaryocyte, In some embodiments, the control sample is or comprises a bodily fluid, wherein the bodily 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 the aforementioned cells or bodily fluids (e.g., the genome, transcriptome, or proteome of a non-cancerous cell of the subject, or the genome, transcriptome, or proteome of a non-cancerous cell of a control subject).

[0028] In some embodiments, the control dataset comprises biomarker level data from cells, wherein the cells are lung cells, bladder cells, stomach cells, prostate cells, esophageal cells, gastrointestinal cells, lymphoid cells, nervous system cells, ovarian cells, cervical cells, vaginal cells, pancreatic cells, throat cells, esophageal cells, kidney cells, small intestinal cells, colon cells, blood cells, red blood cells, white blood cells, platelet cells, or liver cells. In some embodiments, the control dataset comprises biomarker level data from cells, wherein the cells are lung cells. In some embodiments, the control dataset comprises biomarker (e.g., AKR1C3) level data from cells, wherein the cells are cells of the respiratory tract. For example, in some embodiments, the cell is 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 cancer cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara mucosa cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a duct cell), an interstitial connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a meningeal cell, an adipose tissue cell, or a neuron), a vascular cell (e.g., a vascular cell ... In some embodiments, the control dataset is or comprises a biomarker level data from a bodily fluid, wherein the bodily fluid is selected from the group consisting of blood, plasma, mucus, urine, and lymphatic fluid.In some embodiments, the control dataset includes biomarker (e.g., AKR1C3) level data from the genome, transcriptome, or proteome of one or more non-cancerous cells (e.g., the genome, transcriptome, or proteome of one or more non-cancerous cells of a subject, or the genome, transcriptome, or proteome 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 body fluids.

[0029] In some embodiments, the subject sample and the control sample comprise cells, cell populations, cell lysates, tissues, or body fluids (or their protein or mRNA content) of the same type. In some embodiments, the subject sample AKR1C3 level is determined or characterized in cells, cell populations, cell lysates, tissues, or body fluids (or their protein or mRNA content), and the control level of AKR1C3 is determined or characterized from a control dataset comprising biomarker level data from cells, cell populations, cell lysates, tissues, or body fluids (or their protein or mRNA content) of the same or equivalent 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 dataset comprises biomarker (e.g., AKR1C3) level data from lung cells. For example, in some embodiments, the subject sample comprises the genome, transcriptome, or proteome of lung cells, and the control sample comprises the genome, transcriptome, or proteome of lung cells. For example, in some embodiments, the subject sample comprises the genome, transcriptome, or proteome of a lung cell, and the control dataset comprises biomarker (eg, AKR1C3) level data from the genome, transcriptome, or proteome of a lung cell.

[0030] 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 a level of other suitable species of the biomarker.

[0031] In the embodiments described herein, the level of AKR1C3 in a subject sample relative to a control level of AKR1C3 (e.g., the level of AKR1C3 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 the subject sample is about 1.5-fold greater, about 2-fold greater, about 3-fold greater, about 4-fold greater, about 5-fold greater, about 6-fold greater, about 7-fold greater, about 8-fold greater, about 9-fold greater, about 10-fold greater, about 20-fold greater, about 30-fold greater, about 40-fold greater, about 50-fold greater, about 60-fold greater, about 70-fold greater, about 80-fold greater, about 90-fold greater, about 100-fold greater, about 200-fold greater, about 300-fold greater, about 400-fold greater, about 500-fold greater, about 600-fold greater, about 700-fold greater, about 800-fold greater, about 900-fold greater, about 1000-fold greater, about 1500-fold greater, or about 2000-fold greater than the level of AKR1C3 in the control sample or control dataset, the subject is in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0032] In some embodiments, the level of AKR1C3 in the subject sample is at least about 1.5-fold greater, at least about 2-fold greater, at least about 3-fold greater, at least about 4-fold greater, at least about 5-fold greater, at least about 6-fold greater, at least about 7-fold greater, at least about 8-fold greater, at least about 9-fold greater, at least about 10-fold greater, at least about 20-fold greater, at least about 30-fold greater, at least about 40-fold greater, at least about 50-fold greater, at least about 60-fold greater, at least about 70 times greater, at least about 80 times greater, at least about 90 times greater, at least about 100 times greater, at least about 200 times greater, at least about 300 times greater, at least about 400 times greater, at least about 500 times greater, at least about 600 times greater, at least about 700 times greater, at least about 800 times greater, at least about 900 times greater, at least about 1000 times greater, at least about 1500 times greater, or at least about 2000 times greater, then the subject is in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0033] In some embodiments, the level of AKR1C3 is determined or characterized by a specific assay. In some embodiments, determining the level of AKR1C3 comprises performing an antigen detection assay. For example, 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 certain embodiments, the antigen detection assay is an IHC assay.

[0034] In some embodiments, performing the antigen detection assay includes probing the subject sample with an AKR1C3 antibody, which can be an anti-AKR1C3 mouse monoclonal antibody, clone NP6.G6.A6 (catalog number ab49680; Abcam, Waltham, MA). Examples of AKR1C3 antibodies suitable for use in the methods described herein include anti-AKR1C3 mouse monoclonal antibody, clone NP6.G6.A6; mouse monoclonal antibody, clone 871701 (catalog number MAB7678; R&D Systems, Inc., Minneapolis, MN); anti-AKR1C3 rabbit polyclonal antibody ab84327 (catalog number ab84327; Abcam, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody 11194-1-AP (catalog number 11194-1-AP; Thermo Fisher Scientific, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody PA5-106891 (catalog number PA5-106891; Thermo Fisher Scientific, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody PA5-97446 (catalog number PA5-97446; Thermo Fisher Scientific, Waltham, MA). Scientific, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody PA5-29779 (catalog number PA5-29779; Thermo Fisher Scientific, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody PA5-76071 (catalog number PA5-76071; Thermo Fisher Scientific, Waltham, MA); anti-AKR1C3 goat polyclonal antibody PA5-18339 (catalog number PA5-18339; Thermo Fisher Scientific, Waltham, MA); anti-AKR1C3 rabbit polyclonal antibody BS-11401R (catalog number BS-11401R; Thermo Fisher Scientific, Waltham, MA); and anti-AKR1C3 rabbit polyclonal antibody NBP1-33556 (catalog number 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 sequence of an anti-AKR1C3 antibody, such as an anti-AKR1C3 mouse monoclonal antibody, such as the anti-AKR1C3 mouse monoclonal antibody clone NP6.G6.A6.

[0035] The AKR1C3 antibody used in the methods described herein can be conjugated to a moiety that allows for 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 moiety that allows for antibody detection using a detection assay. In such embodiments, performing the antigen detection assay further includes probing the subject sample with a secondary antibody, such as a secondary antibody conjugated to HRP, or a probe that can bind to the primary antibody, such as streptavidin. The presence of HRP can be detected by reaction with 3,3'-diaminobenzidine (DAB). Thus, in some embodiments (e.g., embodiments in which the primary or secondary antibody is conjugated to HRP), the antigen detection assay further includes applying 3,3'-diaminobenzidine (DAB) to the subject sample.

[0036] In some embodiments of the methods described herein, determining AKR1C3 levels, e.g., AKR1C3 levels in a subject sample or a control sample, can include generating a signal intensity score. Thus, in some embodiments involving performing an IHC assay, determining AKR1C3 levels in a subject sample further includes generating an IHC signal intensity score for the subject sample. In some embodiments involving performing an IHC assay, determining AKR1C3 levels in 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 compared to the IHC signal intensity score of the subject sample or the control sample can indicate that the subject is in need of treatment. For example, in some embodiments, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof 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 can be in the range of 0 to 3. In some embodiments, the subject is in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof 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 may be in the range of 0 to 300.

[0037] 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 certain embodiments, the antigen detection assay is an IHC assay.

[0038] In further embodiments of the methods described herein, determining the level of AKR1C3 (e.g., the level of AKR1C3 mRNA) in a sample, e.g., a subject sample or a control sample, comprises performing a polymerase chain reaction (PCR). In such embodiments, the PCR is effective to determine the level of AKR1C3 in the subject sample and / or the control sample. In some embodiments, determining the level of AKR1C3 further comprises performing a PCR effective to determine the level of a control marker 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, the PCR is quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), or reverse transcription qPCR (RT-qPCR).

[0039] 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, the subject sample is characterized as having an elevated level of AKR1C3 (e.g., an elevated AKR1C3 mRNA level) by PCR, e.g., a PCR effective to determine the level of AKR1C3 in the subject sample. In some embodiments, the subject sample is characterized as having an elevated level of AKR1C3 compared to the level of AKR1C3 in a control sample or control dataset, e.g., as determined by PCR. Thus, in some embodiments, the level of AKR1C3 is determined in the control sample by PCR effective to determine the level of AKR1C3 in the control sample. In some embodiments, the level of a control marker is determined in the subject sample by PCR effective to determine the level of the control marker in the subject sample. In some embodiments, the level of a control marker is determined in the control sample by PCR effective to determine the level of the control marker in the control sample. The control marker can be, for example, beta-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In some embodiments, the PCR is quantitative PCR (qPCR).

[0040] In another aspect of the invention, described herein are methods of 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 subject sample: NFE2L2, KEAP1, or CUL3. In some embodiments, a somatic mutation is detected in NFE2L2. In some embodiments, a somatic mutation is detected in KEAP1. In some embodiments, a somatic mutation is detected in CUL3. In some embodiments, a somatic mutation is detected in each of NFE2L2 and KEAP1. In some embodiments, a somatic mutation is detected in each of NFE2L2 and CUL3. In some embodiments, a somatic mutation is detected in each of KEAP1 and CUL3. In some embodiments, a somatic mutation is detected in each of NFE2L2, KEAP1, and CUL3.

[0041] Also described herein are methods for 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 in a subject sample: NFE2L2, KEAP1, or CUL3. In some embodiments, a somatic mutation is detected in NFE2L2. In some embodiments, a somatic mutation is detected in KEAP1. In some embodiments, a somatic mutation is detected in CUL3. In some embodiments, a somatic mutation is detected in each of NFE2L2 and KEAP1. In some embodiments, a somatic mutation is detected in each of NFE2L2 and CUL3. In some embodiments, a somatic mutation is detected in each of KEAP1 and CUL3. In some embodiments, a somatic mutation is detected in each of NFE2L2, KEAP1, and CUL3.

[0042] Also described herein are methods of treating a subject, the methods comprising detecting a somatic mutation in at least one of the following genes: NFE2L2, KEAP1, or CUL3 in a subject's sample; 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 the somatic mutation identifies a subject in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the somatic mutation is detected in NFE2L2. In some embodiments, the somatic mutation is detected in KEAP1. In some embodiments, the somatic mutation is detected in CUL3. In some embodiments, the somatic mutation is detected in each of NFE2L2 and KEAP1. In some embodiments, the somatic mutation is detected in each of NFE2L2 and CUL3. In some embodiments, the somatic mutation is detected in each of KEAP1 and CUL3. In some embodiments, somatic mutations are detected in each of NFE2L2, KEAP1, and CUL3.

[0043] Also described herein are methods of treating a subject, comprising administering an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof to the subject, 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. 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.

[0044] Also described herein are methods 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.

[0045] 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 compounds 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.

[0046] 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. The somatic mutation and disease-associated nucleotide sequence 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 an increased likelihood that an individual has a somatic mutation that is predisposed to having, developing, or developing a particular disease. For example, an individual with a somatic mutation may be more likely to have, develop, or be predisposed to developing cancer. In some embodiments, a subject tumor genome containing a somatic mutation indicates an increased likelihood that the subject has, develops, or is predisposed to developing cancer.

[0047] In addition to KEAP1, there is a significant increase in the specificity of the active ingredient in KEAP1, according to 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; 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; Science,34(4):176-88;Jin et al.,(2021)Cancer Medicine,10(23):8673-92;Kandoth et al.,(2013)Nature,502:333-339; 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.

[0048] Nucleotide sequences associated with somatic mutations and diseases related to CUL3 have been reported, 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. 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.

[0049] Nucleotide sequences associated with somatic mutations and diseases related to 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 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, 211-23, which are incorporated herein by reference.

[0050] The somatic mutation can comprise a mutation relative to the wild-type nucleotide sequence of a gene, e.g., NFE2L2, KEAP1, or CUL3. In some embodiments, 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 (e.g., a gene sequence deletion), an amplification mutation (e.g., a gene amplification), an inversion mutation, and a duplication mutation. For example, in some embodiments, the somatic mutation comprises a mutation in the wild-type nucleotide sequence of NFE2L2, KEAP1, or CUL3, wherein the mutation is a nonsense mutation, a missense mutation, a substitution mutation, a frameshift mutation, a point mutation, an insertion mutation, a deletion mutation, an inversion mutation, or a duplication mutation. In some embodiments, the somatic mutation comprises a single nucleotide polymorphism (SNP), e.g., a SNP in the NFE2L2, KEAP1, or CUL3 gene. In some embodiments, the somatic mutation is a mutation in a tumor cell genome, e.g., a target tumor cell genome.

[0051] In various embodiments described herein, a sample, e.g., a subject sample, is or comprises a subject's cells, cell population, cell lysate, tissue, or bodily fluid. In some embodiments, a subject sample is or comprises nucleic acid, e.g., genomic deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) (e.g., messenger RNA (mRNA)), of a subject's cells, cell population, cell lysate, tissue, or bodily fluid. In some embodiments, a subject sample is or comprises a cell genome or transcriptome, e.g., a tumor cell genome or transcriptome.

[0052] A subject sample can be obtained from a subject by any suitable means. Similarly, a control sample can be obtained from a control subject by any suitable means.

[0053] In some embodiments, the subject sample is or comprises a cell (or a type of nucleic acid, e.g., genomic DNA or its mRNA), and the cell is a cancerous cell, e.g., a tumor cell, e.g., 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 (or a type of nucleic acid, e.g., genomic DNA or its mRNA), and the cell is a lung cell, a bladder cell, a stomach cell, a prostate cell, an esophageal cell, a gastrointestinal cell, a lymphocyte cell, 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 intestine cell, a colon 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 (or a type of nucleic acid, e.g., genomic DNA or its mRNA), and the cell is a lung cell. In some embodiments, the subject sample is or comprises a cell (or a species of nucleic acid, e.g., genomic DNA or mRNA thereof), and the cell is a cell of the respiratory tract. For example, in some embodiments, the cell is or comprises one of the following cell types or species of nucleic acid (e.g., genomic DNA or mRNA) thereof: bronchial cells, bronchiolar cells, alveolar cells (e.g., alveolar type I cells, alveolar type II cells, or alveolar macrophages), epithelial basement membrane cells, endothelial cells, airway epithelial cells (e.g., goblet cells, ciliated cells, Clara cells, neuroendocrine cells, basal cells, intermediate cells, serous cells, brush cells, cancer cells, non-ciliated columnar cells, metaplastic cells, squamous cells, Clara mucous cells, or bronchiolar metaplastic cells), salivary gland cells (e.g., salivary gland serous cells, mucous cells, or ductal cells), or cells), interstitial connective tissue cells (e.g., smooth muscle cells, chondrocytes, fibroblasts, myofibroblasts, meningeal cells, adipose tissue cells, or nerve cells), vascular cells (e.g., endothelial cells, smooth muscle cells, fibroblasts, myofibroblasts, pericytes, lymphocytes, hematopoietic cells, lymphoid tissue cells (e.g., lymphocytes, plasma cells, megakaryocytes, macrophages, Langerhans cells, mast cells, eosinophilic cells, neutrophilic cells, or basophilic cells), pleural cells (e.g., mesothelial cells, multipotent submesothelial fibroblasts, or adipocytes), stem cells, perivascular epithelial-like cells, multipotent epithelial stem cells, meningeal cells, or endothelial progenitor cells.In some embodiments, the subject sample is or comprises a bodily fluid (or associated genetic material), wherein the bodily 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 bodily fluids. In some aspects, 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 bodily 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 bodily fluids selected from the group consisting of the aforementioned bodily fluids.

[0054] In some embodiments described herein, the subject has been diagnosed with, is in need of treatment for, is being treated for, is in remission from, is at risk of developing, or is susceptible to developing a disease or disorder, such as cancer. In some embodiments, the subject has been 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 carcinoma), 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 has been diagnosed with a disease or disorder, such as cancer.

[0055] 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 a somatic mutation in one or more of the NFE2L2, KEAP1, or CUL3 gene sequences. For example, in some embodiments, the subject tumor genome comprises a somatic mutation in: NFE2L2; KEAP1; CUL3; each of NFE2L2 and KEAP1; each of NFE2L2 and CUL3; each of KEAP1 and CUL3; or each of NFE2L2, KEAP1, and CUL3.

[0056] In some embodiments of the methods described herein, somatic mutations present in or detected in a subject sample are not present in a control sample or control dataset. For example, in some instances, a subject tumor genome comprises or is characterized by a somatic mutation in one or more of the NFE2L2, KEAP1, or CUL3 gene sequences, and the somatic mutation is not present in the control sample or control dataset, or the control sample or control dataset is characterized by the absence of a somatic mutation. In some instances, a somatic mutation in 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 dataset.

[0057] In some embodiments, a subject sample characterized by the presence of a somatic mutation or group of somatic mutations can be compared to a control sample or control dataset characterized by the absence of the somatic mutation or group of somatic mutations. Thus, in some embodiments, the somatic mutation or group of somatic mutations detected or present in the subject sample is not present in the control sample or control dataset. In some embodiments, the somatic mutation or group of somatic mutations is detected or present at a higher frequency in the subject sample compared to the control sample or control dataset. The detection, detection, or presence of a somatic mutation or group of somatic mutations in the subject sample can indicate 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, the detection, detection, or presence of a somatic mutation or group of somatic mutations in the subject sample and the absence of a somatic mutation or group of somatic mutations from the control sample or control dataset can indicate that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, detecting, detecting or having a greater frequency of a somatic mutation or group of somatic mutations in a subject sample compared to a control sample or control dataset can indicate that the subject is in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0058] In various embodiments described herein, the control sample is or comprises a non-cancerous cell from a subject, a non-cancerous cell population from a subject, a non-cancerous tissue from a subject, a non-cancerous body fluid from a subject, a non-cancerous cell from a control subject, a non-cancerous cell population from a control subject, a non-cancerous tissue from a control subject, or a non-cancerous body fluid from a control subject. In some embodiments, the control sample comprises a nucleic acid species, such as genomic DNA or mRNA, from a non-cancerous cell from a subject, a non-cancerous cell population from a subject, a non-cancerous tissue from a subject, a non-cancerous body fluid from a control subject, a non-cancerous cell from a control subject, a non-cancerous cell population from a control subject, a non-cancerous tissue from a control subject, or a non-cancerous body fluid from a control subject. For example, in some embodiments, the control sample comprises genomic sequence data from a non-cancerous cell from a subject, a non-cancerous cell population from a subject, a non-cancerous tissue from a subject, a non-cancerous body fluid from a control subject, a non-cancerous cell from a control subject, a non-cancerous cell population from a control subject, a non-cancerous tissue from a control subject, a non-cancerous body fluid from a control subject, or a combination thereof. In some embodiments, the control sample comprises a non-cancerous cell genome or transcriptome, eg, the genome or transcriptome of the subject's non-cancerous cell, or the genome or transcriptome of a control subject's non-cancerous cell.

[0059] In some embodiments, the control dataset includes nucleic acid (e.g., genomic DNA or mRNA) data from non-cancerous cells of the subject, a non-cancerous cell population of the subject, a non-cancerous tissue of the subject, a non-cancerous body fluid of the 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 body fluid of a control subject, or a combination thereof. For example, in some embodiments, the control dataset includes genomic sequence data from non-cancerous cells of the subject, a non-cancerous cell population of the subject, a non-cancerous tissue of the subject, a non-cancerous body fluid of a control 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 body fluid of a control subject, or a combination thereof. In some embodiments, the control dataset includes genomic or transcriptome data from one or more non-cancerous cell genomes or transcriptomes, e.g., the genomes or transcriptomes of one or more non-cancerous cells of the subject, or the genomes or transcriptomes of one or more non-cancerous cells of a control subject.

[0060] In some embodiments, the control sample is or comprises a cell (or genomic DNA or mRNA thereof), wherein the cell is a lung cell, bladder cell, stomach cell, prostate cell, esophageal cell, gastrointestinal cell, lymphatic cell, nervous system cell, ovarian cell, cervical cell, vaginal cell, pancreatic cell, throat cell, esophageal cell, kidney cell, small intestine cell, colon cell, blood cell, red blood cell, white blood cell, platelet cell, or liver cell. In some embodiments, the control sample is or comprises a cell (or genomic DNA or mRNA thereof), wherein the cell is a lung cell. In some embodiments, the control sample is or comprises a cell (or genomic DNA or mRNA thereof), wherein the cell is a cell of the respiratory tract. For example, in some embodiments, the cell is 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 cancer cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara mucosa cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a duct cell), an interstitial connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a meningeal cell, an adipose tissue cell, or a neuron), 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, In some embodiments, the control sample is or comprises a bodily fluid, wherein the bodily 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 aforementioned cells or fluids (e.g., the genome or transcriptome of a control non-cancerous cell or the genome or transcriptome of a control subject's non-cancerous cell).

[0061] In some embodiments, the control dataset comprises genomic DNA or mRNA data from cells, wherein the cells are lung cells, bladder cells, stomach cells, prostate cells, esophageal cells, gastrointestinal cells, lymphoid cells, nervous system cells, ovarian cells, cervical cells, vaginal cells, pancreatic cells, throat cells, esophageal cells, kidney cells, small intestinal cells, colon cells, blood cells, red blood cells, white blood cells, platelet cells, or liver cells. In some embodiments, the control dataset comprises genomic DNA or mRNA data from cells, wherein the cells are lung cells. In some embodiments, the control dataset comprises genomic DNA or mRNA data from cells, wherein the cells are cells of the respiratory tract. For example, in some embodiments, the cell is 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 cancer cell, a non-ciliated columnar cell, a metaplastic cell, a squamous cell, a Clara mucosa cell, or a bronchiolar metaplastic cell), a salivary gland cell (e.g., a salivary gland serous cell, a mucous cell, or a duct cell), an interstitial connective tissue cell (e.g., a smooth muscle cell, a chondrocyte, a fibroblast, a myofibroblast, a meningeal cell, an adipose tissue cell, or a neuron), a vascular cell (e.g., a vascular cell ... In some embodiments, the control dataset is or comprises a biomarker level data from a bodily fluid, wherein the bodily fluid is selected from the group consisting of blood, plasma, mucus, urine, and lymphatic fluid.In some embodiments, the control dataset includes genomic or transcriptomic data from one or more non-cancerous cell genomes or transcriptomes (e.g., the genome or transcriptome of one or more non-cancerous cells of the subject, or the genome or transcriptome of one or more non-cancerous cells of a control subject), where the genomic or transcriptomic data is derived from one or more of the aforementioned cell types and bodily fluids.

[0062] In some embodiments, the subject sample and the control sample comprise cells, cell populations, cell lysates, tissues, or bodily fluids (or their protein or mRNA or genomic DNA content) of the same type. In some embodiments, the subject sample genomic DNA or mRNA is characterized in cells, cell populations, cell lysates, tissues, or bodily fluids, and the control dataset genomic DNA or mRNA is characterized in cells, cell populations, cell lysates, tissues, or bodily fluids of the same or equivalent 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 dataset comprises genomic DNA or mRNA data from lung cells. For example, in some embodiments, the subject sample comprises the genome or transcriptome of lung cells, and the control sample comprises the genome or transcriptome of lung cells. For example, in some embodiments, the subject sample comprises the genome or transcriptome of lung cells, and the control dataset comprises genomic or transcriptome data from the genome or transcriptome of lung cells.

[0063] Detection of somatic mutations, e.g., detection of 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 sequencing genomic DNA from the subject sample, e.g., by PCR, e.g., 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 sequencing mRNA from the subject sample, e.g., 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 from the NFE2L2, KEAP1, and / or CUL3 gene sequences of the subject sample is sequenced. In some embodiments, NFE2L2, KEAP1, and / or CUL3 mRNA (eg, mRNA transcribed from NFE2L2, KEAP1, and / or CUL3 gene sequences) of a subject sample is sequenced.

[0064] In some embodiments of the methods described herein, detecting somatic mutations comprises sequencing genomic DNA of the subject sample and / or the control sample. In some embodiments, the method of genomic DNA sequencing is a high-throughput or next-generation sequencing method. In some embodiments, the sequencing method or method of 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, pyrosequencing, sequencing-by-synthesis, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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 method of genomic DNA sequencing is a single molecule method of sequencing. In some embodiments, the sequencing method or method of sequencing genomic DNA is or comprises performing PCR, quantitative PCR (qPCR), or Sanger sequencing. Thus, in some embodiments, detecting somatic mutations comprises sequencing 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, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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)).

[0065] Detection of a somatic mutation in a subject sample (e.g., a somatic mutation in an NFE2L2, KEAP1, or CUL3 genomic DNA or mRNA sequence) can indicate that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Thus, in some embodiments, if sequencing of the genomic DNA or RNA (e.g., mRNA) of the subject sample detects or results in the detection of a somatic mutation in the subject sample (e.g., a somatic mutation in an NFE2L2, KEAP1, or CUL3 gene sequence), the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0066] In some embodiments, the methods described herein include sequencing the control sample, e.g., 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 methods include sequencing genomic DNA of NFE2L2, KEAP1, and / or CUL3 gene sequences of the control sample. In some embodiments, the methods include 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, e.g., by RNA-Seq (e.g., mRNA-Seq), reverse transcription polymerase chain reaction (RT-PCR), reverse transcription quantitative PCR (RT-qPCR), or digital PCR. Thus, in some embodiments, the methods described herein comprise sequencing genomic DNA of a subject sample, wherein 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, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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.

[0067] The methods described herein may include comparing sequencing data (e.g., mRNA or genomic sequencing data) from a subject sample with a control sample or control dataset. The presence or detection of somatic mutations in the subject sample and the absence or absence of detection of somatic mutations in the control sample or control dataset may indicate that the subject is in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof. Thus, in some embodiments, the methods described herein include comparing sequencing data (e.g., sequencing data from genomic DNA sequencing or sequencing data from mRNA sequencing) of the subject sample with the sequencing data of the control sample or control dataset. Thus, in some embodiments, if sequencing of the genomic DNA or RNA (e.g., mRNA) of a subject sample detects or results in the detection of a somatic mutation (e.g., a somatic mutation in an NFE2L2, KEAP1, or CUL3 gene sequence) in the subject sample, and sequencing of the genomic DNA or RNA (e.g., mRNA) of a control sample does not detect or results in the detection of the somatic mutation in the control sample, then the subject requires treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0068] In some embodiments, detecting somatic mutations in a subject sample at a higher frequency compared to detecting somatic mutations in a control sample or control dataset can indicate that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Thus, in some embodiments, if sequencing of the genomic DNA or RNA (e.g., mRNA) of the subject and control sample detects or results in the detection of somatic mutations (e.g., somatic mutations in the NFE2L2, KEAP1, or CUL3 gene sequence) at a higher frequency in the subject sample compared to the control sample, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, if sequencing of the genomic DNA or RNA (e.g., mRNA) of the subject sample detects or results in the detection of somatic mutations (e.g., somatic mutations in the NFE2L2, KEAP1, or CUL3 gene sequence) at a higher frequency in the subject sample compared to the frequency or frequency of detecting somatic mutations in the control dataset, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0069] In some embodiments, a subject sample is characterized for the presence of somatic mutations. In some embodiments, a subject sample is characterized for the presence of somatic mutations by sequencing the genomic DNA of the subject sample. A sample, e.g., a subject sample or a control sample, can be characterized for the presence of somatic mutations using techniques known to those skilled in the art. For example, in some embodiments, a subject sample is characterized for 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. In some embodiments, a subject sample is characterized for the presence of somatic mutations (e.g., somatic mutations in at least one of the 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.

[0070] In some embodiments of the methods described herein, the subject sample is characterized by the presence of a somatic mutation (e.g., a somatic mutation in at least one of the NFE2L2, KEAP1, or CUL3 gene sequences) by sequencing genomic DNA from the subject sample. In some embodiments of the methods described herein, the control sample is characterized by the absence of a somatic mutation (e.g., a somatic mutation in at least one of the NFE2L2, KEAP1, or CUL3 gene sequences) by sequencing genomic DNA from the control sample. In some embodiments, the method of genomic DNA sequencing 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, pyrosequencing, sequencing by synthesis, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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 method of genomic DNA sequencing is a single molecule method of sequencing. In some embodiments, the sequencing or method of sequencing genomic DNA is or includes performing PCR, quantitative PCR (qPCR), digital PCR, or Sanger sequencing.

[0071] Thus, in some embodiments, the subject sample is characterized for the presence of a somatic mutation (e.g., a somatic mutation in at least one of the NFE2L2, KEAP1, or CUL3 gene sequences) by sequencing genomic DNA of the subject sample, and the sequencing or method of 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, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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 for the presence of a somatic mutation (e.g., a somatic mutation in at least one of the NFE2L2, KEAP1, or CUL3 gene sequences) by sequencing genomic DNA of the subject sample, and the sequencing or sequencing method is PCR.

[0072] A subject sample characterized by the presence of a somatic mutation (e.g., a somatic mutation present in the NFE2L2, KEAP1, or CUL3 gene sequence) can indicate that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Thus, in some embodiments, if a subject sample is characterized by the presence of a somatic mutation (e.g., if sequencing of the subject sample's genomic DNA or RNA (e.g., mRNA) characterizes the subject sample by the presence of a somatic mutation (e.g., a somatic mutation present in the NFE2L2, KEAP1, or CUL3 gene sequence), the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0073] In some embodiments of the methods described herein, the control sample is characterized by the absence of somatic mutations (e.g., somatic mutations in 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, for example, 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 for the absence of somatic mutations by sequencing 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, pyrosequencing, sequencing by synthesis, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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.

[0074] In some embodiments, the subject sample is characterized by the presence of a somatic mutation, and / or the control sample or control dataset is characterized by the absence of a somatic mutation, indicating that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Thus, in some embodiments, if sequencing of the subject sample (e.g., sequencing of genomic DNA or RNA (e.g., mRNA)) detects or results in the detection of a somatic mutation (e.g., a somatic mutation present in an NFE2L2, KEAP1, or CUL3 gene sequence) and sequencing of the control sample (e.g., sequencing of genomic DNA or RNA (e.g., mRNA)) does not detect or does not detect the somatic mutation, then the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0075] In some embodiments, the subject sample is characterized by the presence of a higher frequency of somatic mutations compared to the control sample or control dataset, indicating that the subject is in need of treatment 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 a higher frequency of somatic mutations compared to the control sample or control dataset, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0076] The invention described herein, in another aspect, includes the use of AKR1C3 levels to select 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 to select a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein if the subject's sample is characterized as having an elevated AKR1C3 level, the subject is treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0077] In yet another aspect, the invention described herein includes the use of somatic mutation to select a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof. Accordingly, also described herein is the use of somatic mutation to select a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein a sample from the subject is characterized for the presence of a somatic mutation, and if the somatic mutation is detected in one of the following genes: NFE2L2, KEAP1, or CUL3, the subject is treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0078] 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.

[0079] Also described herein are kits suitable for carrying out the methods described herein or for the uses described herein. For example, kits are described herein 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 for treating 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 are kits for detecting somatic mutations in at least one of the following genes: NFE2L2, KEAP1, or CUL3 in a subject sample; or characterizing a subject sample for the presence of somatic mutations in at least one of the following genes: 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, for example, the NFE2L2 gene sequence, the KEAP1 gene sequence, or the CUL3 gene sequence. The kits described herein can include components suitable for detecting somatic mutations in the NFE2L2, KEAP1, or CUL3 gene sequence, which methods include sequencing RNA (e.g., mRNA) transcribed from the NFE2L2, KEAP1, or CUL3 gene sequence. In some embodiments, the kits described herein include components suitable for determining mRNA or protein levels of a biomarker. In some embodiments, the kits described herein include components suitable for performing an antigen detection assay, such as an IHC assay. In some embodiments, the kits described herein include components suitable for sequencing genomic DNA or RNA (e.g., mRNA).

[0080] 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 for treating 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 are assays for detecting somatic mutations in at least one of the following genes: NFE2L2, KEAP1, or CUL3 in a subject sample; or characterizing a subject sample for the presence of somatic mutations in at least one of the following genes: NFE2L2, KEAP1, or CUL3. The assays described herein may include a step 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 a step suitable for detecting somatic mutations in one or more gene sequences, for example, the NFE2L2 gene sequence, the KEAP1 gene sequence, or the CUL3 gene sequence. The assays described herein may include steps suitable for detecting somatic mutations in the NFE2L2, KEAP1, or CUL3 gene sequence by sequencing RNA (e.g., mRNA) transcribed from the NFE2L2, KEAP1, or CUL3 gene sequence. In some embodiments, the assays described herein include steps suitable for determining AKR1C3 mRNA or protein levels. In some embodiments, the assays described herein include steps suitable for performing an antigen detection assay, such as an IHC assay. In some embodiments, the assays described herein include steps suitable for sequencing genomic DNA or RNA (e.g., mRNA).

[0081] A pharmaceutical composition containing a compound of Formula (I) or a pharmaceutically acceptable salt thereof may be administered in a unit dose of about 1 to 1,000 mg of the active ingredient, or about 1 to 500 mg, about 1 to 250 mg, about 1 to 150 mg, about 0.5 to 100 mg, or about 1 to 50 mg of the active ingredient for a subject weighing about 50 to 70 kg. The therapeutically effective dose of a compound of Formula (I) or a pharmaceutical composition thereof depends on the species, weight, age, and individual condition of the subject being treated, and the disorder or disease or its severity. A physician, clinician, or veterinarian of ordinary skill in the art can readily determine the effective amount of each active ingredient required to prevent, treat, or inhibit the progression of a disorder or disease.

[0082] The dosage characteristics cited above can be demonstrated using in vitro and in vivo tests, advantageously using mammals, such as 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 enterally, parenterally, advantageously intravenously, e.g., as a suspension or aqueous solution. The dosage in vitro is about 10 -3 Molar concentration ~10 -9 The therapeutically effective amount in vivo may range from about 0.1 to 500 mg / kg or from about 1 to 100 mg / kg, depending on the route of administration. [Brief explanation of the drawings]

[0083] [Figure 1] 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"). H-scores for clinical trial biopsies from tumors of NSCLC, HCC, and head and neck cancer ("H&N") patients with NFE2L2 / KEAP1 mutations are also shown. The percentage of samples and clinical trial biopsies assigned an H-score of greater than 250 out of 300 is shown. [Figure 2] FIG. 1 is a schematic diagram of the clinical trial design for a clinical trial to determine AKR1C3-dependent KARS inhibitor administration. [Figure 3]FIG. 1 is a schematic diagram showing the design of safety and efficacy assessments during screening / baseline and during treatment of a clinical trial to determine AKR1C3-dependent KARS inhibitor administration. DETAILED DESCRIPTION OF THE INVENTION

[0084] Various embodiments of the present invention are described herein, and it will be recognized that the features specified in each embodiment can be combined with other specified features to provide further embodiments of the present invention.

[0085] 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-associated diseases. Disclosed herein are Compound (I) and Compound (II), and pharmaceutical compositions thereof: [ka] The compound of formula (I), 6'-fluoro-N-(4-fluorobenzyl)-4'-oxo-3',4'-dihydro-1'H-spiro[piperidine-4,2'-quinoline]-1-carboxamide; [ka] 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 active in various assays and therapeutic models acting as a selective AKR1C3 inhibitor.

[0086] In one aspect, the present invention provides a compound of formula (I): [ka] (In the formula, [ka] is a single or double bond; Z is [ka] is a single bond, then either OH; or [ka] is a double bond, then O; Each R 1 are independently (C1-C6) alkyl, (C1-C6) alkoxy, (C0-C4) alkylN(R 8 )2 and halo; R 2a and R 2b are 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, 5-6 membered heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S, and 9-10 membered fused bicyclic heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; any of the foregoing may be selected from one or more R 6 optionally substituted with; R 5 is 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 is selected from the group consisting of; Each R 6 is halo; (C1-C6) alkyl; (C1-C6) alkoxy; halo(C1-C6) alkyl; OH; aryl; 3-6 membered heterocycle; 5-6 membered heteroaryl; (C0-C4) alkylS(O) m(C1-C6) alkyl; halo(C1-C6) alkoxy; (C0-C4) alkylS(O) m N(R 8 )2;(C0-C4)alkylN(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) alkyl OR 7 and (C3-C 10 ) cycloalkyl; each R 6 is 1 to 3 R when it is not halo, OH, or OP(O)(OH)2 9 or two adjacent R 6 together with the atom to which they are attached form a 5- to 7-membered heterocycle or a (C5-C8)cycloalkyl; Each R 7 and R 8 are independently selected from the group consisting of H or (C1-C6) alkyl, and 1 to 3 R 9 optionally substituted with; Each R 9 is halo; -OH; amino, (C1-C4) alkylamino, di(C1-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 heterocycle optionally substituted with oxo (=O); (C0-C4) alkylS(O) m N(R 10 )2;;(C0-C4)Alkyl(CO)R 10 ;(C0-C4) alkyl(CO)OR 10 (C0-C4) alkylNR 10 S(O) m (C1-C6) alkyl; (C0-C4) alkyl OR10 (C0-C4) alkylN(R 10 )2;(C0-C4)alkylCN;(C0-C4)alkylN(R 10 )2; and (C0-C4) alkyl(CO)N(R 10 )2 independently selected from the group consisting of: Each R 10 are 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); Each R 11 is H; 1 to 4 R 12 4- to 6-membered heterocycle optionally substituted with 1 to 4 R 12 (C3-C6)cycloalkyl optionally substituted with; (C0-C3)alkyl(C3-C6)cycloalkyl(C1-C3)alkyl optionally substituted with halo; 1 to 3 R 12 (C-C) alkyl; (C-C) alkenyl; or (C-C) alkynyl, wherein each of the (C-C) alkyl; (C-C) alkenyl; and (C-C) alkynyl is selected from the group consisting of one or more R 13 optionally substituted with; Each R 12 are 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 is independently selected from the group consisting of halo, OH, amino, (C1-C4)alkylamino, di(C1-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, comprising: The present invention provides a method for determining the level of AKR1C3 in a subject's sample, wherein an elevated level of AKR1C3 identifies the subject as one in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0087] Also described herein is a method for 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 subject sample, wherein an elevated level of AKR1C3 identifies the subject as one in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0088] In another aspect, the present invention provides a method of treating a subject, the method comprising determining the level of AKR1C3 in a subject's sample, wherein an elevated level of AKR1C3 identifies or determines a subject in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof; and administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to the subject.

[0089] In another aspect, the present invention provides 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 administration, the subject's sample is characterized by having elevated AKR1C3 levels.

[0090] In another aspect, the present 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 an elevated level of AKR1C3.

[0091] 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.

[0092] In some embodiments of the methods described herein, the level of AKR1C3 is the level of AKR1C3 protein. In some embodiments of the methods described herein, the level of AKR1C3 is the level of AKR1C3 nucleic acid species, for example, AKR1C3 mRNA.

[0093] In some embodiments, the subject sample comprises a cell, cell population, cell lysate, tissue, or bodily fluid of the subject. In some embodiments of the methods described herein, the cell is a cancerous cell. In some embodiments, the cancerous 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 bodily fluid is selected from the group consisting of blood, plasma, and lymph. In some embodiments, the subject sample comprises the genome, transcriptome, or proteome of a cell, e.g., a tumor cell of the subject. In some embodiments, the subject sample comprises the genome, transcriptome, or proteome of any of the aforementioned cells or bodily fluids.

[0094] 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.

[0095] In some embodiments of the methods described herein, the subject tumor genome comprises a somatic mutation in one or more of the NFE2L2, KEAP1, or CUL3 gene sequences.

[0096] In some embodiments of the methods described herein, the AKR1C3 level is elevated compared to a control level of AKR1C3. In some embodiments, the control level comprises the AKR1C3 level of a control sample or a control dataset. In some embodiments, the control sample comprises a sample selected from the group consisting of non-cancerous cells of a subject, a non-cancerous cell population of a subject, a non-cancerous tissue of a subject, a non-cancerous body 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, and a non-cancerous body fluid of a control subject. In some embodiments, the control dataset comprises AKR1C3 level data from a source selected from the group consisting of non-cancerous cells of a subject, a non-cancerous cell population of a subject, a non-cancerous tissue of a subject, a non-cancerous body fluid of a control 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 body fluid of a control subject, and combinations thereof. In some embodiments, the control sample comprises the genome, transcriptome, or proteome of a non-cancerous cell from a subject, a non-cancerous cell population from a subject, a non-cancerous tissue from a subject, a non-cancerous body fluid from a subject, a non-cancerous cell from a control subject, a non-cancerous cell population from a control subject, a non-cancerous tissue from a control subject, or a non-cancerous body fluid from a control subject. In some embodiments, the control sample comprises the genome, transcriptome, or proteome of any of the aforementioned cells or body fluids. In some embodiments, the control dataset comprises AKR1C3 level data from a source selected from the group consisting of the genome, transcriptome, or proteome of a non-cancerous cell from a subject, a non-cancerous cell population from a subject, a non-cancerous tissue from a subject, a non-cancerous body fluid from a control subject, a non-cancerous cell from a control subject, a non-cancerous cell population from a control subject, a non-cancerous tissue from a control subject, a non-cancerous body fluid from a control subject, and combinations thereof.

[0097] In some embodiments of the methods described herein, the level of AKR1C3 is the AKR1C3 protein level.

[0098] In some embodiments of the methods described herein, the level of AKR1C3 is an AKR1C3 RNA level. In some embodiments, the AKR1C3 RNA level is an AKR1C3 mRNA level.

[0099] In some embodiments of the methods described herein, if the level of AKR1C3 in the subject sample is about 1.5-fold greater, about 2-fold greater, about 3-fold greater, about 4-fold greater, about 5-fold greater, about 6-fold greater, about 7-fold greater, about 8-fold greater, about 9-fold greater, about 10-fold greater, about 20-fold greater, about 30-fold greater, about 40-fold greater, about 50-fold greater, about 60-fold greater, about 70-fold greater, about 80-fold greater, about 90-fold greater, about 100-fold greater, about 200-fold greater, about 300-fold greater, about 400-fold greater, about 500-fold greater, about 600-fold greater, about 700-fold greater, about 800-fold greater, about 900-fold greater, about 1000-fold greater, about 1500-fold greater, or about 2000-fold greater than the level of AKR1C3 in the control sample or control dataset, the subject is in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0100] In some embodiments of the methods described herein, the level of AKR1C3 in the subject sample is at least about 1.5-fold greater, at least about 2-fold greater, at least about 3-fold greater, at least about 4-fold greater, at least about 5-fold greater, at least about 6-fold greater, at least about 7-fold greater, at least about 8-fold greater, at least about 9-fold greater, at least about 10-fold greater, at least about 20-fold greater, at least about 30-fold greater, at least about 40-fold greater, at least about 50-fold greater, at least about 60-fold greater, at least about 70-fold greater, at least about 80-fold greater, at least about 90-fold greater, at least about 100-fold greater, at least about 200-fold greater, at least about 300-fold greater, at least about 400-fold greater, at least about 500-fold greater, at least about 600-fold greater, at least about 700-fold greater, at least about 800-fold greater, at least about 900-fold greater, at least about 10 ... times greater, at least about 70 times greater, at least about 80 times greater, at least about 90 times greater, at least about 100 times greater, at least about 200 times greater, at least about 300 times greater, at least about 400 times greater, at least about 500 times greater, at least about 600 times greater, at least about 700 times greater, at least about 800 times greater, at least about 900 times greater, at least about 1000 times greater, at least about 1500 times greater, or at least about 2000 times greater, then the subject is in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0101] In some embodiments of the methods described herein, determining the level of AKR1C3 in the 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.

[0102] In some embodiments, performing the antigen detection assay comprises probing the 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).

[0103] In some embodiments, performing the antigen detection assay further comprises probing the subject sample with a secondary antibody, hi some embodiments, the secondary antibody is conjugated to HRP.

[0104] In some embodiments, the antigen detection assay further comprises applying 3,3'-diaminobenzidine (DAB) to the subject sample.

[0105] 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 to 3.0, 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 to 300, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0106] 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 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.

[0107] 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).

[0108] In some embodiments, the antigen detection assay further comprises probing the subject sample with a secondary antibody, hi some embodiments, the secondary antibody is conjugated to HRP.

[0109] In some embodiments, the antigen detection assay further comprises applying 3,3'-diaminobenzidine (DAB) to the subject sample.

[0110] In some embodiments, the antigen detection assay further comprises generating an IHC signal intensity score for the subject sample. In some embodiments, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof 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 to 3.0. In some embodiments, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof 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 to 300. In some embodiments, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof if the IHC signal intensity score of the subject tissue sample is greater than the IHC signal intensity score of the control sample or control dataset. In some embodiments, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof if the IHC signal intensity score for the subject tissue sample is at least 5% higher, at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, or at least 100% higher than the IHC signal intensity score for the control sample or control dataset.

[0111] 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).

[0112] In some embodiments, determining the level of AKR1C3 in the subject sample further comprises performing a PCR effective to determine the level of AKR1C3 in a control 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 control sample. In some embodiments, the control marker is β-actin or GAPDH.

[0113] As noted above, 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 and / or control markers in the subject sample or a control sample. In some embodiments, the PCR is quantitative PCR (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.

[0114] In some embodiments of the methods described herein, the subject sample is characterized as having elevated AKR1C3 levels by PCR effective to determine AKR1C3 levels in the subject sample. In some embodiments, the level of AKR1C3 is determined in the control sample by PCR effective to determine AKR1C3 levels in the control sample. In some embodiments, the level of a control marker is determined in the subject sample by PCR effective to determine the level of the control marker in the subject sample. In some embodiments, the level of the control marker is determined in the control sample by PCR effective to determine the level of the control marker 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).

[0115] As described above, in some embodiments of the methods described herein, the level of AKR1C3 or a control marker in a subject sample or a control sample is determined by performing a polymerase chain reaction (PCR) effective to determine the level of AKR1C3 or 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.

[0116] 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: NFE2L2, KEAP1, or CUL3 in a sample from the subject.

[0117] In another aspect, the present invention provides a method for 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.

[0118] In another aspect, the present invention provides a method of treating a subject, the method comprising detecting a somatic mutation in at least one of the following genes: NFE2L2, KEAP1, or CUL3 in a subject's sample, thereby identifying, detecting, a subject 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 a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0119] In another aspect, the present invention provides 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 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.

[0120] In another aspect, the present 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.

[0121] In some embodiments of the methods described herein, 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, an amplification mutation (e.g., a gene amplification), a deletion mutation (e.g., a gene deletion), an inversion mutation, and a duplication mutation. In some embodiments, the somatic mutation is a mutation in the genome of a tumor cell.

[0122] In some embodiments of the methods described herein, the somatic mutation comprises a single nucleotide polymorphism (SNP).

[0123] In some embodiments of the methods described herein, the subject sample comprises a cell, a cell population, a cell lysate, a tissue, or a bodily fluid of the subject. In some embodiments, the subject sample comprises genomic DNA of a cell, a cell population, a cell lysate, a tissue, or a bodily fluid of the subject. In some embodiments, the cell is a cancerous cell. In some embodiments, the cancerous 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 bodily fluid is selected from the group consisting of blood, plasma, and lymph. In some embodiments, the subject sample comprises the genome or transcriptome of a cell of the subject, e.g., a tumor cell of the subject. In some embodiments, the subject sample comprises the genome or transcriptome of any of the aforementioned cells or bodily fluids of the subject.

[0124] In some embodiments of the methods described herein, the somatic mutation is not present in a control sample or a control dataset. In some embodiments, the control sample comprises a sample selected from the group consisting of a non-cancerous cell from the subject, a non-cancerous cell population from the subject, a non-cancerous tissue from the subject, a non-cancerous body fluid from the subject, a non-cancerous cell from the control subject, a non-cancerous cell population from the control subject, a non-cancerous tissue from the control subject, and a non-cancerous body fluid from the control subject. In some embodiments, the control sample comprises genomic DNA from a non-cancerous cell from the subject, a non-cancerous cell population from the subject, a non-cancerous tissue from the subject, a non-cancerous body fluid from the control subject, a non-cancerous cell from the control subject, a non-cancerous cell population from the control subject, a non-cancerous tissue from the control subject, or a non-cancerous body fluid from the control subject. In some embodiments, the control dataset comprises genomic sequence data from a non-cancerous cell from the subject, a non-cancerous cell population from the subject, a non-cancerous tissue from the subject, a non-cancerous body fluid from the control subject, a non-cancerous cell from the control subject, a non-cancerous cell population from the control subject, a non-cancerous tissue from the control subject, a non-cancerous body fluid from the control subject, and combinations thereof. In some embodiments, the control sample comprises the genome or transcriptome proteome of a non-cancerous cell of the subject, a non-cancerous cell population of the subject, a non-cancerous tissue of the subject, a non-cancerous body fluid of the 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, or a non-cancerous body fluid of a control subject. In some embodiments, the control sample comprises the genome or transcriptome of any of the foregoing cells or body fluids. In some embodiments, the control dataset comprises genome or transcriptome data from a source selected from the group consisting of the genome or transcriptome of a non-cancerous cell of the subject, a non-cancerous cell population of the subject, a non-cancerous tissue of the subject, a non-cancerous body fluid of a control 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 body fluid of a control subject, and combinations thereof.

[0125] In some embodiments of the methods described herein, the level of AKR1C3 (e.g., AKR1C3 mRNA or AKR1C3 protein) in the subject sample 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 greater than the level of AKR1C3 (e.g., AKR1C3 mRNA or AKR1C3 protein) in a control sample or control dataset.

[0126] In some embodiments of the methods described herein, the level of AKR1C3 (e.g., AKR1C3 mRNA or AKR1C3 protein) in a subject sample is compared to the level of AKR1C3 (e.g., AKR1C3 mRNA or AKR1C3 protein) in a control sample or control dataset. 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 greater than the level of AKR1C3 (mRNA or AKR1C3 protein).

[0127] 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. 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. In some embodiments, 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, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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 sequencing comprises performing exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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 sequencing comprises performing a polymerase chain reaction (PCR). In some embodiments, the PCR is selected from the group consisting of qPCR, RT-PCR, RT-qPCR, and digital PCR.

[0128] In some embodiments of the methods described herein, if the sequencing detects a somatic mutation in the subject's sample, the subject requires treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0129] In some embodiments of the methods described herein, the methods further include sequencing genomic DNA of a control sample. In some embodiments of the methods described herein, the methods further include comparing the sequencing data of the sequenced genomic DNA of the subject sample with the sequencing data of a control sample or control dataset.

[0130] Alternatively, in some embodiments of the methods described herein, the method further comprises sequencing mRNA from a control sample. In some embodiments of the methods described herein, the method further comprises comparing the sequencing data of the sequenced mRNA from the subject sample with the sequencing data of a control sample or control dataset.

[0131] In some embodiments of the methods described herein, the subject sample is characterized for the presence of somatic mutations by sequencing genomic DNA from the subject sample. In some embodiments of the methods described herein, the subject sample is characterized for the presence of somatic mutations by sequencing mRNA from the subject sample. In some embodiments, 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, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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 sequencing comprises performing exome sequencing, targeted genome sequencing, whole genome sequencing, single molecule real-time (SMRT) sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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 sequencing comprises performing a polymerase chain reaction (PCR). In some embodiments, the PCR is selected from the group consisting of qPCR, RT-PCR, RT-qPCR, and digital PCR.

[0132] In some embodiments of the methods described herein, if the sequencing detects a somatic mutation in the subject's sample, the subject requires treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0133] In some embodiments of the methods described herein, the control sample is characterized by the absence of somatic mutations by sequencing genomic DNA of the control sample. In some embodiments, the methods described herein further comprise comparing sequencing data of the sequenced genomic DNA of the subject sample with sequencing data of a control sample or control dataset.

[0134] 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 comprise comparing sequencing data of the sequenced mRNA of the subject sample with sequencing data of a control sample or control dataset.

[0135] In another aspect, the present invention provides the use of AKR1C3 levels to select a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein if the subject's sample is characterized as having an elevated AKR1C3 level, the subject is treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0136] In another aspect, the present invention provides the use of somatic mutation to select a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein a sample from the subject is characterized for the presence of a somatic mutation, and if a somatic mutation is detected in one of the following genes: NFE2L2, KEAP1 or CUL3, the subject is treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0137] 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.

[0138] definition For purposes of interpreting this specification, the following definitions shall apply unless otherwise stated, and where applicable, terms used in the singular shall also include the plural and vice versa.

[0139] As used herein, the terms "a," "an," "the," and similar terms as used in the context of the present invention (particularly in the context of the claims) are to be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0140] The term "(C1-C6) alkyl" as used herein refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing no unsaturation, having 1 to 6 carbon atoms, and attached to other molecules by a single bond. The term "(C1-C4) alkyl" should be construed accordingly. Examples of (C1-C6) alkyl include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, and 1,1-dimethylethyl (t-butyl).

[0141] The term "(C2-C6)alkenyl" as used herein refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing at least one double bond, having 2 to 6 carbon atoms, and attached to another molecule by a single bond. The term "(C2-C4)alkenyl" should be construed accordingly. Examples of (C2-C6)alkenyl include, but are not limited to, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-4-enyl, and penta-1,4-dienyl.

[0142] The term "(C2-C6)alkynyl" as used herein refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having 2 to 6 carbon atoms, and attached to another molecule by a single bond. The term "(C2-C4)alkynyl" should be construed accordingly. Examples of (C2-C6)alkynyl include, but are not limited to, ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl, and penta-1,4-diynyl.

[0143] As used herein, the term "(C1-C6)alkoxy" refers to a group of the formula -OR a where R ais a (C1-C6) alkyl group as generally defined above. Examples of (C1-C6) alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentoxy, and hexoxy.

[0144] As used herein, the term "(C1-C6)alkoxy(C1-C6)alkyl" refers to a group of the formula -R a -OR a In the formula, each R a are independently a (C1-C6)alkyl group as defined above. The oxygen atom may be bonded to any carbon atom within each alkyl group. Examples of (C1-C6)alkoxy(C1-C6)alkyl include, but are not limited to, methoxy-methyl, methoxy-ethyl, ethoxy-ethyl, 1-ethoxy-propyl, and 2-methoxy-butyl.

[0145] As used herein, the term "(C1-C4) alkylcarbonyl" refers to a group of the formula -C(=O)-R a where R a is a (C1-C4) alkyl group as defined above.

[0146] As used herein, the term "(C1-C6) alkylcarbonyl(C1-C6) alkyl" refers to a group of the formula -R a -C(=O)-R a In the formula, each R a are independently a (C1-C6) alkyl group as defined above. The carbon atom of the carbonyl group may be bonded to any carbon atom within each alkyl group.

[0147] As used herein, the term "(C1-C6)alkoxycarbonyl" refers to a group of the formula -C(=O)-OR a where R a is a (C1-C6) alkyl group as defined above.

[0148] As used herein, the term "(C1-C6)alkoxycarbonyl(C1-C6)alkyl" refers to a group of the formula -Ra -C(=O)-OR a In the formula, each R a are independently a (C1-C6) alkyl group as defined above.

[0149] As used herein, the term "(C1-C4)alkoxycarbonylamino" refers to a group having the formula -NH-C(=O)-OR a where R a is a (C1-C4) alkyl group as defined above.

[0150] The term "hydroxy(C1-C6)alkyl" as used herein refers to a (C1-C6)alkyl group as defined above, wherein C 1~6 One of the hydrogen atoms of the alkyl group is replaced by OH. Examples of hydroxy(C1-C6)alkyl include, but are not limited to, hydroxymethyl, 2-hydroxy-ethyl, 2-hydroxy-propyl, 3-hydroxy-propyl, and 5-hydroxy-pentyl.

[0151] The term "amino(C1-C6)alkyl" as used herein refers to a (C1-C6)alkyl group as defined above, wherein one of the hydrogen atoms of the (C1-C6)alkyl group is replaced by a primary amino group. Representative examples of amino(C1-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.

[0152] As used herein, the term "(C1-C4) alkylamino" refers to a group of the formula -NH-R a where R a is a (C1-C4) alkyl group as defined above.

[0153] As used herein, the term "(C1-C4) alkylamino(C1-C6) alkyl" refers to a group of the formula -R a1 -NH-R a2 where R a1is a (C1-C6) alkyl group as defined above, and R a2 is a (C1-C4) alkyl group as defined above. The nitrogen atom may be bonded to any carbon atom within each alkyl group.

[0154] As used herein, the term "di(C1-C4)alkylamino" refers to a group of the formula -N(R a )-R a In the formula, each R a are (C1-C4) alkyl groups as defined above and may be the same or different.

[0155] As used herein, the term "di(C1-C4)alkylamino(C1-C6)alkyl" refers to a group of the formula -R a1 -N(R a2 )-R a2 where R a1 is a (C1-C6) alkyl group as defined above, and each R a2 are (C1-C4) alkyl groups as defined above and may be the same or different. The nitrogen atom may be bonded to any carbon atom within any alkyl group.

[0156] As used herein, the term "aminocarbonyl" refers to a group of formula -C(=O)-NH2.

[0157] As used herein, the term "aminocarbonyl C 1~6 "Alkyl" is a group of the formula -R a refers to the group —C(═O)—NH, where R a is a (C1-C6) alkyl group as defined above.

[0158] As used herein, the term "(C1-C4) alkylaminocarbonyl" refers to a group of the formula -C(=O)-NH-R a where R a is a (C1-C4) alkyl group as defined above.

[0159] As used herein, the term "(C1-C4) alkylaminocarbonyl C1~6 "Alkyl" is a group of the formula -R a1 -C(=O)-NH-R a2 where R a1 is a (C1-C6) alkyl group as defined above, and R a2 is a (C1-C4) alkyl group as defined above.

[0160] As used herein, the term "di(C1-C4)alkylaminocarbonyl" refers to a group having the formula -C(=O)-N(R a )-R a In the formula, each R a are (C1-C4) alkyl groups as defined above and may be the same or different.

[0161] As used herein, the term "di(C1-C4) alkylaminocarbonyl C 1~6 "Alkyl" is a group of the formula -R a1 -C(=O)-N(R a2 )-R a2 where R a1 is the C defined above. 1~6 is an alkyl group, and each R a2 are (C1-C4) alkyl groups as defined above and may be the same or different.

[0162] The term "(C3-C8)cycloalkyl(C0-C6)alkyl" as used herein refers to a stable, monocyclic, saturated hydrocarbon group consisting solely of carbon and hydrogen atoms, having from 3 to 8 carbon atoms, and attached to another molecule by a single bond or by a (C1-C6)alkyl group as defined above. Examples of (C3-C8)cycloalkyl(C0-C6)alkyl include, but are not limited to, cyclopropyl, cyclopropyl-methyl, cyclobutyl, cyclobutyl-ethyl, cyclopentyl, cyclopentyl-propyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0163] The term "aryl" refers to a 6- to 10-membered aromatic carbocyclic moiety having a single ring system (e.g., phenyl) or a fused ring system (e.g., naphthalene). A typical aryl group is a phenyl group.

[0164] The term "phenyl(C0-C6)alkyl" as used herein refers to a phenyl ring attached to another molecule by a single bond or by a (C1-C6)alkyl group as defined above. Examples of phenyl(C0-C6)alkyl include, but are not limited to, phenyl and benzyl.

[0165] As used herein, the term "phenyl(C0-C6)alkylamino(C1-C6)alkyl" refers to a group of the formula -R a -NH-R b where R a is a (C1-C6) alkyl group as defined above, and R b is a phenyl (C0-C6) alkyl group as defined above.

[0166] As used herein, the term "phenyl(C0-C6)alkylamino((C1-C4)alkyl)(C1-C6)alkyl" refers to a group of the formula -R a1 -N(R a2 )-R b where R a1 is a (C1-C6) alkyl group as defined above, and R a2 is a (C1-C4) alkyl group as defined above, and R b is a phenyl (C0-C6) alkyl group as defined above.

[0167] As used herein, halo refers to bromo, chloro, fluoro, or iodo.

[0168] The term "halo(C1-C6)alkyl" as used herein refers to a (C1-C6)alkyl group as defined above substituted by one or more halo groups as defined above. Examples of halogen(C1-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.

[0169] The term "heterocyclyl" refers to a saturated or partially saturated, non-aromatic ring or ring system, including monocyclic, fused, bridged, and spirocyclic rings, where the number of ring atoms is specified. For example, heterocyclyl includes, but is not limited to, 5- to 6-membered heterocyclyl, 4- to 10-membered heterocyclyl, 4- to 14-membered heterocyclyl, and 5- to 14-membered heterocyclyl. Unless otherwise specified, a heterocyclyl contains 1 to 7, 1 to 5, 1 to 3, or 1 to 2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur as ring members, where N and S can be optionally oxidized to various oxidation states. The heterocyclic group can be attached at a heteroatom or a carbon atom. Examples of such heterocyclyls include, but are not limited to, azetidine, oxetane, piperidine, piperazine, pyrroline, pyrrolidine, imidazolidine, imidazoline, morpholine, tetrahydrofuran, tetrahydrothiophene, tetrahydrothiopyran, tetrahydropyran, 1,4-dioxane, 1,4 oxathiane, 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-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-diazabicyclo[2.2.1]heptane, 1,4-dioxa-8-aza-spiro[4.5]decane, 3-oxa-1,8-diazaspiro[4.5]decane, and octahydropyrrolo[3,2-b]pyrrole.

[0170] The term "fused heterocyclyl" refers to a heterocyclyl as defined above that is fused to an aryl (e.g., phenyl) or heteroaryl ring as defined above. Examples of such fused heterocyclyls 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-epiminonaphthalene, 2,3-dihydrobenzofuran, and 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine. The term "heterocyclyl(C0-C6)alkyl" as used herein refers to a heterocycle as defined above that is attached to another molecule by a single bond or by a (C1-C6)alkyl group as defined above.

[0171] 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, quinolinyl, isoquinolinyl, quinoxalinyl, benzopyranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, and 1H-benzo[d][1,2,3]triazolyl). The heteroaromatic moiety may consist of a single ring system or a fused ring system. A typical single heteroaryl ring is a 5- to 6-membered ring containing 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, and a typical fused heteroaryl ring system is a 9- to 10-membered ring system containing 1 to 4 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(C0-C6)alkyl" refers to a heteroaryl ring, as defined above, attached to another molecule by a single bond or by a (C1-C6)alkyl group, as defined above.

[0172] Unless otherwise specified, the term "compounds of the invention" refers to compounds of formula (I) as defined herein, and salts thereof, and all stereoisomers (including diastereomers and enantiomers), rotamers, tautomers, and isotopically labeled compounds (including deuterium substitution), and naturally formed moieties. The term "compounds of the (present) invention" refers to compounds as defined in any one of the embodiments mentioned below.

[0173] Compounds of formula (I) Embodiments of the invention described herein are, in part, directed to a compound of formula (I): or a pharmaceutically acceptable salt thereof: [ka] (In the formula, [ka] is a single or double bond; Z is [ka] is a single bond, then either OH; or [ka] is a double bond, then O; Each R 1 are independently (C1-C6) alkyl, (C1-C6) alkoxy, (C0-C4) alkylN(R 8 )2 and halo; R 2a and R 2b are 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, 5-6 membered heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S, and 9-10 membered fused bicyclic heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; any of the foregoing may be selected from one or more R 6 optionally substituted with; R 5 is 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 is selected from the group consisting of; Each R 6is halo; (C1-C6) alkyl; (C1-C6) alkoxy; halo(C1-C6) alkyl; OH; aryl; 3-6 membered heterocycle; 5-6 membered heteroaryl; (C0-C4) alkylS(O) m (C1-C6) alkyl; halo(C1-C6) alkoxy; (C0-C4) alkylS(O) m N(R 8 )2;(C0-C4)alkylN(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) alkyl OR 7 and (C3-C 10 ) cycloalkyl; each R 6 is 1 to 3 R when it is not halo, OH, or OP(O)(OH)2 9 or two adjacent R 6 together with the atom to which they are attached form a 5- to 7-membered heterocycle or a (C5-C8)cycloalkyl; Each R 7 and R 8 are independently selected from the group consisting of H or (C1-C6) alkyl, and 1 to 3 R 9 optionally substituted with; Each R 9 is halo; -OH; amino, (C1-C4) alkylamino, di(C1-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 heterocycle optionally substituted with oxo (=O); (C0-C4) alkylS(O) m N(R 10 )2;;(C0-C4)Alkyl(CO)R 10;(C0-C4) alkyl(CO)OR 10 (C0-C4) alkylNR 10 S(O) m (C1-C6) alkyl; (C0-C4) alkyl OR 10 (C0-C4) alkylN(R 10 )2;(C0-C4)alkylCN;(C0-C4)alkylN(R 10 )2; and (C0-C4) alkyl(CO)N(R 10 )2 independently selected from the group consisting of: Each R 10 are 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); Each R 11 is H; 1 to 4 R 12 4- to 6-membered heterocycle optionally substituted with 1 to 4 R 12 (C3-C6)cycloalkyl optionally substituted with; (C0-C3)alkyl(C3-C6)cycloalkyl(C1-C3)alkyl optionally substituted with halo; 1 to 3 R 12 (C-C) alkyl; (C-C) alkenyl; or (C-C) alkynyl, wherein each of the (C-C) alkyl; (C-C) alkenyl; and (C-C) alkynyl is selected from the group consisting of one or more R 13 optionally substituted with; Each R 12 are 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 is independently selected from the group consisting of halo, OH, amino, (C1-C4)alkylamino, di(C1-C4)alkylamino, (C1-C3)alkoxy; and C(O)—(C3-C8)cycloalkyl; m is 0, 1, or 2; and n is 0, 1 or 2) Regarding.

[0174] 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. Compounds of formula (I) are described in 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 WO 2021 / 005586 or a pharmaceutically acceptable salt thereof.

[0175] Pharmaceutical Composition The compounds of formula (I) or pharmaceutically acceptable salts thereof described herein may be components of pharmaceutical compositions comprising compounds of formula (I) or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers.

[0176] As used herein, the term "pharmaceutical composition" refers to a compound of Formula (I) or a pharmaceutically acceptable salt thereof, in a form suitable for oral or parenteral administration, together with at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions described herein are suitable for oral administration.

[0177] The term "pharmaceutically acceptable carrier" as used herein refers to a substance useful in the preparation or use of a pharmaceutical composition, and examples thereof include suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonicity agents, buffers, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegrants, lubricants, wetting agents, sweeteners, flavoring agents, dyes, and combinations thereof known to those skilled in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 22 nd Ed. Pharmaceutical Press, 2013, pp. 1049-1070).

[0178] The term "therapeutically effective amount" of a compound of Formula (I) refers to an amount of a compound of Formula (I) that elicits a biological or medical response in a subject, for example, reduces or inhibits enzyme or protein activity, ameliorates disease symptoms, alleviates symptoms, slows or delays disease progression, or prevents disease. In a non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the present invention that, when administered to a subject, is effective to (1) at least partially alleviate, inhibit, prevent, and / or ameliorate a condition, disorder, or disease (i) mediated by KARS, or (ii) sensitive to KARS inhibition, or (iii) characterized by KARS activity (normal or abnormal); or (2) alleviate or inhibit a disease sensitive to KARS inhibition. The present invention further provides a method for treating or preventing diseases and / or disorders associated with elevated AKR1C3 expression or sensitivity to KARS inhibition, comprising administering to a subject in need thereof a therapeutically effective amount of an AKR1C3-dependent KARS inhibitor. In some embodiments, a therapeutically effective amount of a compound of Formula (I) when administered to a subject is effective to inhibit KARS activity. In some embodiments, inhibition of KARS activity ameliorates disease symptoms, alleviates a disease state, slows or delays disease progression, or prevents a disease susceptible to KARS inhibition. In some embodiments, a therapeutically effective amount of a compound of Formula (I) is effective to reduce the number of cancer cells in a subject; reduce primary tumor size; inhibit or stop cancer cell invasion into peripheral organs; inhibit tumor metastasis; inhibit or stop tumor growth; and / or alleviate to some extent one or more symptoms associated with a disease or disorder. In vivo efficacy can be measured, for example, by assessing survival time, 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 enzyme activity or expression levels of biomarkers (e.g., mRNA levels or protein levels), such as the level of AKR1C3.In some embodiments, in vivo efficacy can be measured, for example, by assessing the level of KARS enzyme activity.

[0179] 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 another embodiment, the subject is a human. Unless otherwise indicated, as used herein, the term "subject" is interchangeable with the term "patient."

[0180] The term "inhibition" or "inhibiting" as used herein refers to the reduction or suppression of a given condition, symptom, disorder or disease or a significant decrease in the baseline activity of a biological activity or biological process.

[0181] As used herein, the terms "treat" or "treating" or "treatment" of any disease or disorder refer to alleviating or ameliorating the disease or disorder (i.e., delaying or halting the onset of the disease or at least one of its clinical symptoms); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those not discernible by the patient (e.g., reducing the level of a biomarker, e.g., AKR1C3 levels). "Treat," "treating," or "treatment" can also refer to modulating the disease or disorder either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both. More specifically, "treatment" refers to any action that results in improving or preserving the anatomical functions affected by a particular disease or disorder and / or the quality of life of a subject with the disease or disorder. As used herein, "treatment" can refer to any manner in which one or more symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of symptoms of a disease or disorder refers to any relief, whether permanent or temporary, lasting or transient, that may result from or be associated with treatment with the methods of the invention.

[0182] As used herein, the terms "prevent," "preventing," or "prevention" of any disease or disorder refer to prophylactic treatment of a disease or disorder; or delaying the onset or progression of a disease or disorder, for example, by prophylactic treatment. Prevention can include any action that prevents or delays the deterioration of function, quality of life, and / or another parameter associated with a particular disease or disorder in patients who have, and are at risk of, said deterioration.

[0183] 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.

[0184] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended solely to better clarify the invention and does not pose a limitation on the scope of the invention unless claimed.

[0185] 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, diastereoisomers, optical isomers (enantiomers), racemates or mixtures thereof.

[0186] Any resulting mixture of stereoisomers can be separated on the basis of the physical chemical differences of the components into pure or substantially pure geometric or optical isomers, diastereoisomers, racemates, for example, by chromatography and / or fractional crystallization.

[0187] Any resulting racemic forms of the final products or intermediates can be resolved into their enantiomers by known methods, for example, by separation of their diastereomeric salts obtained with optically active acids or bases, and liberating the optically active acidic or basic compounds. Thus, basic compounds can be used to resolve the compounds of the present invention into their enantiomers, in particular, 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. Racemic products can also be resolved by chiral chromatography, for example, by high-performance liquid chromatography (HPLC) using a chiral adsorbent.

[0188] In some embodiments, the compound of Formula (I) is a component of a pharmaceutical composition. For example, pharmaceutical compositions comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier are described herein. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein. The pharmaceutical composition may be formulated for a specific route of administration, such as oral administration, parenteral administration (e.g., by injection, infusion, transdermal administration, or topical administration), and rectal administration. In some embodiments, the pharmaceutical compositions described herein are formulated for oral administration. Additionally, topical administration may involve inhalation or intranasal application. The pharmaceutical compositions 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, the pharmaceutical composition is a tablet or gelatin capsule containing the active ingredient together with one or more of the following: a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants, such as silica, talc, stearic acid, its magnesium or calcium salts and / or polyethylene glycol; also for tablets, c) binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; if desired, d) disintegrants, such as starch, agar, alginic acid or its sodium salt or effervescent mixtures; and e) Absorbents, colorants, flavorings and sweeteners.

[0189] 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 by any suitable means, including direct delivery to the desired organ, cell, or tissue, oral, inhalation, intranasal, intratracheal, buccal, sublingual, intrathecal, intravenous, intramuscular, intraarticular, subcutaneous, intradermal, intraperitoneal, intraspinal, epidural, intradural, subdural, retrobulbar, intraocular, intracorneal, conjunctival, intraocular, intravitreal, parenteral, intracranial, intracerebral, intraventricular, direct delivery to the lung, and other parenteral routes of administration. Routes of administration can be combined, if desired. 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 by any suitable route, such as injection, e.g., intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. In some embodiments, administration is by the oral route. A variety of administration schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusions.

[0190] A composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof can be formulated or administered to a subject (e.g., a subject in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof) at an appropriate dosage level. For example, a therapeutically effective amount of a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof can be formulated 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 dose 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 weekly.

[0191] In some embodiments, the composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally.

[0192] In some embodiments, the compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof are formulated for oral administration.

[0193] Biomarkers Described herein are methods for identifying a subject for treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as well as methods for treating a subject with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. The methods described herein can include detecting or determining the level of a biomarker, such as an AKR1C3 protein or nucleic acid, such as an AKR1C3 mRNA transcript, in a subject or a subject sample. In some embodiments, an elevated level of AKR1C3 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 can include detecting somatic mutations in one or more genes, such as NFE2L2, KEAP1, or CUL3, in a subject or a subject sample. In some embodiments, somatic mutations in one or more genes, such as NFE2L2, KEAP1, or CUL3, are biomarkers. In some embodiments, subjects in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof are identified by detecting somatic mutations in one or more genes.

[0194] As used herein, the terms "determine," "determining," or "determining" include any means of determining, including direct and indirect determination. For example, "determining" can include any means of determining the presence or level of a biomarker, e.g., an AKR1C3 biomarker, e.g., AKR1C3 protein or mRNA level, in a subject or subject sample. As used herein, the terms "detect," "detecting," or "detection" include any means of detecting, including direct and indirect detection. For example, "detecting" can include any means of detecting the presence or level of a biomarker, e.g., an AKR1C3 biomarker, e.g., AKR1C3 protein or mRNA level, 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 quantification assays (e.g., Western blot, quantitative Western blot, immunohistochemistry, immunocytochemistry, enzyme-linked absorbance assay (ELISA), immunoprecipitation, immunoelectrophoresis or dot blot, immunostaining of cells, body fluids, tissues, or extracts or lysates thereof, and other methods of immunodetection). Methods for detecting or determining the presence or level of AKR1C3 nucleic acid biomarkers, such as mRNA, include, but are not limited to, RNA detection and quantification 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 in NFE2L2, CUL3, or KEAP1 gene sequences), e.g., DNA (e.g., genomic DNA), include, but are not limited to, methods of sequencing DNA (e.g., genomic DNA) (e.g., exome sequencing, targeted genome sequencing, whole genome sequencing, SMRT sequencing, ion semiconductor sequencing, pyrosequencing, 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).

[0195] The term "biomarker," as used herein, refers to an indicator, e.g., predictive, diagnostic, and / or prognostic, that can be detected in a sample, e.g., a particular gene (including, but not limited to, a change in gene sequence (e.g., somatic mutation) compared to a wild-type sequence or a change in gene expression level (e.g., as determined by the mRNA transcript of the gene) compared to a control sample or control data set) or a protein encoded by said gene (including, but not limited to, a change in protein expression level compared to a control sample or control data set). A biomarker may include one or more somatic mutations in a gene, e.g., NFE2L2, CUL3, or KEAP1. A biomarker may serve as an indicator of a particular disease or disorder, or a particular subtype of a disease or disorder (e.g., cancer), characterized by specific 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, the biomarker is a collection of genes or proteins (e.g., single or multiple gene and protein expression levels), or a population number of mutations / alterations in a collection of genes (e.g., somatic mutations). Biomarkers include, but are not limited to, polynucleotides, polynucleotide alterations (e.g., gene sequence mutations, e.g., somatic mutations), polypeptides, and proteins. In some embodiments described herein, the biomarker is an AKR1C3 protein level. In some embodiments described herein, the biomarker is an AKR1C3 nucleotide sequence expression level. In some embodiments described herein, the biomarker is an AKR1C3 gene expression level. In some embodiments described herein, the biomarker is an AKR1C3 mRNA transcription level. In some embodiments described herein, the biomarker is an NFE2L2, CUL3, or KEAP1 gene sequence. In some embodiments described herein, the biomarker is a somatic mutation in the NFE2L2, CUL3, or KEAP1 gene sequence.

[0196] The term "level" refers to the presence or amount of a biomarker in a sample, e.g., a subject or control sample, or in a dataset, e.g., a control dataset.

[0197] An "increased level," "elevated level," or "high level" of a biomarker refers to an increase in the level of the biomarker (e.g., a protein or mRNA biomarker) in a sample (e.g., a subject sample) compared to a control sample, e.g., one or more individuals not afflicted with a disease or disorder (e.g., cancer), or a control dataset, e.g., a dataset comprised of biomarker levels from one or more individuals not afflicted with a disease or disorder (e.g., cancer). In some embodiments, an increased level of the biomarker is detectable in a subject or subject sample.

[0198] A "decreased level," "reduced level," or "lower level" of a biomarker refers to a decrease in the level of the biomarker (e.g., a protein or mRNA biomarker) in a sample (e.g., a subject sample) compared to a control sample, e.g., one or more individuals not afflicted with a disease or disorder (e.g., cancer), or a control dataset, e.g., a dataset comprised of biomarker levels from one or more individuals not afflicted with a disease or disorder (e.g., cancer). In some embodiments, an increased level of the biomarker is detectable in a subject or subject sample.

[0199] 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 by which information (e.g., genetically encoded and / or epigenetic information) is converted into structures present and operating within a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of a transcribed polynucleotide, a translated polypeptide, or a polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered expressed, regardless of whether they are derived from a transcript generated by alternative splicing or a degraded transcript, or from post-translational processing of a polypeptide, for example, by proteolysis. "Expressed genes" also include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, as well as those that are transcribed into RNA but not translated into a polypeptide (e.g., import and ribosomal RNA).

[0200] As used herein, "amplification" generally refers to the process of generating multiple copies of a desired sequence. "Multiple copies" means at least two copies. A "copy" does not necessarily imply perfect sequence complementarity or identity to the template sequence. For example, a copy may contain nucleotide analogs such as deoxyinosine, intentional sequence variations (e.g., sequence variations introduced via primers containing sequences that are hybridizable to, but not complementary to, the template), and / or sequence errors that occur during amplification.

[0201] As used herein, the term "polymerase chain reaction" or "PCR" generally refers to a procedure for amplifying minute quantities of specific nucleic acids, RNA, and / or DNA fragments, as described, for example, in U.S. Pat. No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond must be available so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may correspond to the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987) and Erlich, ed., PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered an example, but not the only, 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), and involves using known nucleic acids (DNA or RNA) as primers and utilizing a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid, or a specific piece of nucleic acid complementary to a specific nucleic acid. In some embodiments, PCR is used to detect somatic mutations (e.g., somatic mutations in KEAP1, NFE2L2, or CUL3) or to detect or determine the expression level of a gene of interest (e.g., KEAP1, NFE2L2, or CUL3).

[0202] The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acids obtained from a single source (e.g., individual samples from a subject) using two or more primer sets to amplify two or more DNA sequences in a single reaction.

[0203] 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 generally relies on fluorescent dyes that intercalate with double-stranded DNA or fluorescently labeled sequence-specific DNA probes that can hybridize to the PCR product of interest to detect and quantify the amplification of the nucleic acid species of interest. qPCR can be quantitative or semi-quantitative.

[0204] "Reverse transcription PCR" or "RT-PCR" refers to a form of PCR in which an RNA template (e.g., an mRNA transcript) is converted into complementary DNA (cDNA) using the enzyme reverse transcriptase. The cDNA produced by this reaction is then amplified by PCR.

[0205] "Reverse transcription quantitative PCR" or "RT-qPCR" refers to a form of qPCR that is quantitative or semi-quantitative. Generally, RT-qPCR relies on the same methods as qPCR, but uses reverse transcriptase to produce cDNA from mRNA. RT-qPCR is used to monitor the amplification of target mRNA species, allowing for quantification of mRNA in a sample.

[0206] "Digital PCR" refers to a PCR method used to directly quantify and clone amplify nucleic acid strands, including DNA, cDNA, or RNA. Digital PCR, like conventional PCR, involves a single PCR reaction within a sample. However, compared to conventional PCR, digital PCR allows the sample to be split into multiple partitions (e.g., 10 of a single sample). 4 The PCR reaction is carried out separately in each partition.

[0207] 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" can refer to the identification of a particular type of cancer. "Diagnosis" can 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)).

[0208] As used herein, the term "sample" or "biological sample" refers to a composition obtained or derived from a subject and / or individual of interest, including, for example, cells, body fluids, and / or other molecular entities characterized and / or identified based on physical, biochemical, chemical, and / or physiological properties. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, 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, e.g., homogenized tissue, tumor tissue, cell extracts, and combinations thereof. In some aspects, the sample is a tissue sample, blood sample, or cell sample. In some embodiments, the sample is or comprises protein or genetic material. In some embodiments, the sample comprises a cellular genome, transcriptome, or proteome, for example, a tumor cell genome, transcriptome, or proteome.

[0209] A "tissue sample" refers to a collection of similar cells obtained from the tissue of a subject or individual. The source of a tissue or cell sample can be solid tissue from fresh, frozen, and / or preserved organs, tissue samples, biopsies, and / or aspirates; any blood component, such as blood or plasma; bodily fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, interstitial fluid, etc.; or cells from any point in the subject's pregnancy or development. A tissue sample can also be primary cells or cultured cells or cell lines. In some cases, a 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. A tissue sample can contain a mixed population of cell types (e.g., tumor and non-tumor cells, cancerous and non-cancerous cells). A tissue sample may contain compounds not naturally mixed with tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. In some instances, a tissue sample or tumor tissue sample is not a blood sample or blood component, such as plasma. In a preferred embodiment, the tissue or cell sample is a tumor sample.

[0210] As used herein, a "subject sample" can be a sample, e.g., a biological sample, from or derived from a subject, e.g., a subject in need of treatment or prevention. A subject sample can include any sample obtained from a subject of interest that is expected to contain or known to contain a characterized cell and / or molecular entity, e.g., a biomarker. In some embodiments, a subject sample includes components not directly from the subject. For example, a subject sample can be a mixture of material derived directly from the subject (e.g., a subject's tissue) or material derived from the subject (e.g., a cell line derived from the subject's tissue) with additional material (e.g., a buffer or cell culture medium). A subject sample can be composed of one or more cells, cell populations, cell lysates, tissues, or bodily fluids from the subject, e.g., one or more cells, cell populations, cell lysates, tissues, or bodily fluids derived from the subject, e.g., obtained from or provided by the subject. In some embodiments, the subject sample may include one or more cancerous cells, such as one or more tumor cells (e.g., liquid tumor cells or solid tumor cells); whole blood; crystals; serum; blood-derived cells; platelets; lymphatic fluid; urine; stool; mucus; sputum; sweat; saliva; semen; cerebrospinal fluid; bone marrow; amniotic fluid; one or more tissue samples; primary cells or cultured cells or cell lines; cell supernatant; cell lysate; vitreous fluid; synovial fluid; follicular fluid; milk; tears; tumor lysate; tissue culture medium; tissue extracts such as homogenized tissue; tumor tissue; cell extracts; and combinations thereof. In some embodiments, the subject sample is or includes 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 a subject, such as the genome, transcriptome, or proteome of tumor cells of the subject.

[0211] As used herein, "tumor cells" refers to any tumor cells present in a tumor or a sample thereof. Tumor cells can be distinguished from other cells, such as stromal cells and tumor-infiltrating immune cells, that may be present in a tumor sample using methods known in the art and / or described herein. Tumor cells can be liquid tumor cells or solid tumor cells.

[0212] As used herein, a "control sample" refers to a sample, tissue, cell, dataset, standard, or level used for comparison purposes. In one embodiment, a control sample is obtained from a healthy and / or non-diseased part of the body (e.g., tissue or cells) of the same subject or individual. For example, a control sample can be a healthy and / or non-diseased tissue or cells adjacent to a diseased tissue or cell (e.g., tissue or cells adjacent to a tumor). In another embodiment, a control sample is obtained from a healthy and / or non-diseased part of the body (e.g., tissue or cells) of an individual other than the same subject or individual. In some embodiments, a control sample is or comprises the genome, transcriptome, or proteome of a control cell, tissue, or bodily fluid.

[0213] As used herein, a "control dataset" refers to a dataset that includes one or more samples, tissues, cells, standards, or levels used for comparison purposes, e.g., for comparison with a subject sample. In one embodiment, a control dataset is composed of data obtained from healthy and / or non-diseased parts of the body (e.g., tissues or cells) of the same subject or individual. In some embodiments, a control dataset is composed of data obtained from one or more healthy and / or non-diseased individuals who are not the subject. In some embodiments, a control dataset includes data regarding the levels (e.g., protein or RNA levels) of one or more biomarkers in one or more healthy control individuals. In some embodiments, a control dataset includes data regarding the presence or prevalence of one or more biomarkers (e.g., the presence or prevalence of one or more disease-associated nucleotide sequences or somatic mutations) in one or more healthy control individuals. In some aspects, a control dataset includes genomic, transcriptomic, or proteomic data.

[0214] As used herein, the term "AKR1C3" refers to the protein encoded by the aldo-keto reductase family 1 member C3 gene sequence (also known as DD3; DDX; PGFS; HAKRB; HAKRe; HA1753; HSD17B5; or hluPGFS), e.g., as set forth in NCBI Gene ID: 8644, and its orthologs, or the nucleotide sequence (e.g., mRNA sequence) or gene sequence encoding said protein.

[0215] As used herein, the term "KEAP1" refers to the protein encoded by the Kelch-like ECH-associated protein 1 gene sequence (INFE2L2; or also known as KLHL19) as set forth, for example, in NCBI Gene ID: 9817, and its orthologs, or the nucleotide sequence (e.g., mRNA sequence) or gene sequence encoding said protein.

[0216] As used herein, the term "CUL3" refers to the protein encoded by the Cullin3 gene sequence (also known as CUL-3; PHA2E; or NEDAUS), e.g., as set forth in NCBI Gene ID: 8452, and its orthologs, or the nucleotide sequence (e.g., mRNA sequence) or gene sequence encoding said protein.

[0217] As used herein, the term "NFE2L2" refers to the protein encoded by the NFE2-like Bzip transcription factor 2 gene sequence (also known as NRF2; HEBP1; Nrf-2; or IMDDHH), e.g., as set forth in NCBI Gene ID: 4780, and its orthologs, or the nucleotide sequence (e.g., mRNA sequence) or gene sequence encoding said protein.

[0218] The term "KARS" as used herein refers to the protein encoded by the lysyl-Trna synthetase 1 gene sequence (also known as KRS; KARS; KARS2; LEPID; CMTRIB; DEAPLE; or DFNB89;) set forth, for example, in NCBI Gene ID: 3735, and its orthologs, or the nucleotide sequence (e.g., mRNA sequence) or gene sequence encoding said protein.

[0219] Genetic mutations 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 detecting somatic mutations in a subject's sample, for example, detecting somatic mutations in at least one of the following genes: NFE2L2, KEAP1, or CUL3A. Somatic mutations include gene sequences that increase the likelihood that an individual will carry a somatic mutation that will cause or be prone to develop a particular disease. For example, an individual with a somatic mutation may have a likelihood of developing cancer or a predisposition to developing cancer. Somatic mutations can be associated with disease through genomic studies, such as genome-wide association studies (GWAS).

[0220] Various types of genetic mutations are known in the art, including, for example, point mutations, single nucleotide polymorphisms (SNPs), substitutions, missense mutations, nonsense mutations, frameshift mutations, nucleotide repeat expansion mutations, inversions, insertions, deletions, copy number variations, amplifications, gene duplications, somatic, germline, homozygous, heterozygous, chromosomal rearrangements, splice site, gain-of-function, hypomorphic, and neomorphic mutations. In some embodiments, the somatic mutation is a point mutation, single nucleotide polymorphism (SNP) mutation, substitution mutation, missense mutation, nonsense mutation, frameshift mutation, nucleotide repeat expansion mutation, inversion mutation, insertion mutation, deletion mutation, copy number variation, amplification mutation, gene duplication mutation, somatic mutation, homozygous mutation, heterozygous mutation, chromosomal rearrangement mutation, splice site mutation, gain-of-function mutation, hypomorphic mutation, or neomorphic mutation in the gene sequence of at least one of the following genes: NFE2L2, KEAP1, or CUL3A. Somatic mutations can occur in any part of a gene sequence, including, for example, a protein coding region, a gene enhancer, an exon, an intron, a promoter, a splice site, a 5'UTR, or a 3'UTR. In some embodiments, the somatic mutation comprises an amplification of the NFE2L2 gene sequence or a portion thereof. In some embodiments, the somatic mutation comprises a deletion of the KEAP1 or CUL3 gene sequence or a portion thereof.

[0221] The KEAP1 activation of KEAP1 has been investigated by 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)Free Radical Biology and Medicine,177:58-71; 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 al.,(2013)Nature,502:333-339;Constantinopoulos 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; 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.

[0222] Disease-linked nucleotide sequences of CUL3 have been reported, 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. 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.

[0223] The disease-linked nucleotide sequence of NFE2L2 can be found, 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. 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. 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, 211-23, which are incorporated herein by reference.

[0224] As used herein, a "subject tumor genome" refers to the complete set of genetic information contained in the cells of a subject tumor, including coding and non-coding portions of chromosomal DNA. The subject tumor genome may contain a genetic mutation associated with a disease or disorder. For example, in some embodiments of the methods described herein, the subject tumor genome contains a somatic mutation associated with a disease or disorder, e.g., cancer. For example, in some embodiments, the subject tumor genome contains a somatic mutation in one or more of the NFE2L2, KEAP1, or CUL3 gene sequences. In some embodiments, the presence of a somatic mutation in the subject tumor genome can indicate that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the subject tumor genome can contain a genetic mutation not known to be associated with a disease or disorder, e.g., cancer, but which may indicate that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0225] Disease and Cancer Methods for treating a subject or identifying a subject for treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof are described herein. In certain embodiments, the subject has been diagnosed with, is suffering from, or is prone to developing a particular disease or disorder. The diseases and disorders described herein can include conditions that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose the subject to such a disease or disorder. In certain embodiments, the subject has been diagnosed with, is suffering from, or is prone to developing cancer.

[0226] The terms "cancer" and "cancerous" refer to or describe a physiological condition typically characterized by unregulated cell growth. This definition includes benign and malignant cancers. "Early stage cancer" or "early stage tumor" means a cancer that is not invasive or metastatic, or is classified as 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 tumor, gastrinoma, and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.Examples of cancer include 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, bone marrow sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, bone marrow sarcoma, fibrosarcoma, myxosarcoma, fibrosarcoma ... myeloid sarcoma, osteosarcoma, chondrosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, leiomyosarcoma, or rhabdomyosarcoma), prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia, or chronic lymphocytic leukemia (CLL)), lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma (NHL)), myeloma (e.g., multiple myeloma (MM)), mycosis fungoides, Merkel cell carcinoma, hematologic malignancies, hematologic Cancer of tissues, B-cell carcinoma, bronchial carcinoma, gastric cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, cancer of the uterus or endometrium, cancer of the oral cavity or pharynx, liver cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, adrenal gland cancer, adenocarcinoma, adenocarcinoma, inflammatory myofibroblastic tumor, gastrointestinal stromal tumor (GIST), colon cancer, myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), polycythemia vera, chordoma, synovium, Ewing's tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial Examples of the cancer include myeloid carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder carcinoma, 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 carcinoma, small cell carcinoma, essential thrombocythemia, amelanotic myeloid metaplasia, hypereosinophilic syndrome, systemic mastocytosis, familial eosinophilia, neuroendocrine carcinoma, and carcinoid tumor.More specific examples of such cancers include early stage I-III resectable and unresectable (stage IIIC) or metastatic (stage IV) melanoma, lung cancer including NSCLC, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer (SCLC), and adenocarcinoma of the lung and squamous cell carcinoma of the lung. In particular examples, 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 cancer of the peritoneum, 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), gastrointestinal cancer including stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatic cancer, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, Merkel cell carcinoma, mycosis fungoides, testicular cancer, esophageal cancer, tumors of the biliary tract, and head and neck cancer and hematological malignancies. In some embodiments, the subject has been diagnosed with, is suffering from, or is predisposed 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 carcinoma), 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 has been diagnosed with, is suffering from, or is predisposed to developing NSCLC. In some embodiments, the subject has been diagnosed with, is afflicted with, or is predisposed to developing the squamous cell carcinoma subtype of NSCLC, hi some embodiments, the subject has been diagnosed with, is afflicted with, or is predisposed to developing the adenocarcinoma subtype of NSCLC.

[0227] The term "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein. In some embodiments, a tumor is a solid tumor. In some embodiments, a tumor is a liquid tumor.

[0228] Subjects who can benefit from treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof include subjects who respond to a therapeutically effective amount of the compound. For example, subjects who can benefit from treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof include subjects who respond to treatment or are likely to respond to treatment, for example, subjects who are likely to experience, as a result of treatment experience, improvement or preservation of anatomical function affected by a particular disease or disorder; improvement in quality of life related to improvement of the disease state; alleviation or improvement of the disease or disorder; alleviation or improvement of at least one physical parameter or biomarker related to the disease or disorder; and / or improvement or improvement of one or more symptoms of the disease or disorder.

[0229] Methods for identifying subjects for treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof are described herein. Subjects for treatment can be identified by diagnosing the subject as suffering from or predisposed to developing a particular disease or disorder. For example, subjects for treatment can be identified by diagnosing the subject as suffering from or predisposed to developing cancer, such as NSCLC. Subjects for treatment can be identified by detecting the presence or elevated level of a biomarker. For example, subjects for treatment can be identified by detecting elevated levels of AKR1C3. For example, subjects for treatment can be identified by elevated levels of AKR1C3 in a subject sample compared to the level of AKR1C3 in a control sample, e.g., a control subject sample or control dataset. In some embodiments, subjects for treatment can be identified by detecting the presence of one or more somatic mutations in at least one of the following genes: NFE2L2, KEAP1, or CUL3 in a subject sample, e.g., a subject's tumor genome.

[0230] immunohistochemistry 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., an AKR1C3 protein level, by an antigen detection assay. In some embodiments, the antigen detection assay is an immunohistochemistry (IHC) assay. Generally, IHC relies on detecting the presence, amount, and / or relative quantity of a species of interest using an antigen detection compound, e.g., an antibody. In some embodiments described herein, the antigen IHC assay includes probing the sample (e.g., a control sample or a subject sample) with an antibody capable of binding to the biomarker, e.g., the AKR1C3 protein.

[0231] Antigen-based detection assays generally determine the presence, level, or distribution of a target molecule (e.g., a biomarker) in a sample by detecting the interaction of the target molecule with a specific binding agent, such as an antibody, which can be detected. 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 a detectable label conjugated to the antibody (direct detection) or by a detectable label conjugated to a secondary antibody that specifically binds to the primary antibody (e.g., indirect detection).

[0232] IHC utilizes antibodies or their derivatives or other proteinaceous 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 peroxidase, (iii) free aldehyde groups, (iv) immunoglobulins, and other irrelevant molecules that may 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.

[0233] In some embodiments of the methods described herein, determining a biomarker level, e.g., a biomarker level in a subject sample or a control sample, can 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 the subject sample compared to the IHC signal intensity score of the subject sample or the IHC signal intensity score of the control sample can indicate that the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof if the IHC signal intensity score for 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% greater than the IHC signal intensity score for the control sample.

[0234] In some embodiments, the IHC signal intensity score can range from 0 to 3, with a score of 0 indicating no detectable signal and a score of 3 indicating a strongly 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 staining nuclei multiplied by a factor of 3, the percentage of moderately staining nuclei multiplied by a factor of 2, and the percentage of weakly staining nuclei; this sum is divided by 100. In some embodiments, a subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof 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.

[0235] In some embodiments, the IHC signal intensity score can range from 0 to 300, with a score of 0 indicating no detectable signal and a score of 300 indicating a strongly 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 staining nuclei multiplied by a factor of 3, the percentage of moderately staining nuclei multiplied by a factor of 2, and the percentage of weakly staining nuclei. In some embodiments, a subject is in need of treatment with a compound of Formula (I) or a pharmaceutically acceptable salt thereof 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.

[0236] The IHC signal intensity score can be determined based on the quantification of the detectable signal from the IHC detection reagent. For example, the IHC signal intensity score can be determined based on the quantification of a detectable fluorescent signal or chromogenic signal (e.g., DAB, 3-amino-9-ethylcarbazole (AEC), 5-bromo-4-chloro-3-indolylphosphate:tetranitrobluetetrazolium (BCIP:TNBT), 5-bromo-4-chloro-3-indolylphosphate:p-nitrobluetetrazolium chloride (BCIP:NBT), or 3,3',5,5;-tetramethylbenzidine (TMB), fast red, permanent red), or its signal intensity. The color signal can be generated by a chemical reaction with an appropriate enzyme, such as HRP, glucose oxidase, or alkaline phosphatase. The enzyme can be conjugated to an antibody (e.g., a primary antibody or a secondary antibody) or a probe (e.g., a streptavidin probe). The fluorescent signal can be generated directly from a protein, for example, green fluorescent protein or red fluorescent protein.Fluorescent signals can also be generated using suitable quantum dot species, dyes, or fluorophores, such as 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-Cyanine7, PerCP-Cyanine5.5, tetramethylrhodamine (TRITC), Texas Red, eFluor 450, eFluor 506, eFluor 660, PE-eFluor 610, PerCP-eFluor 710, APC-eFluor Quantum dots or fluorophores may be produced from Super Bright 780, Super Bright 436, Super Bright 600, Super Bright 645, Super Bright 702, Super Bright 780, Qdot 525, Qdot 565, Qdot 605, Qdot 655, Qdot 705, or Qdot 800. The quantum dots or fluorophores may be conjugated to antibodies (e.g., primary or secondary antibodies) or probes (e.g., streptavidin probes). In some embodiments, the sample is counterstained with an appropriate agent, such as 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).

[0237] IHC signal intensity can be calculated based on images captured using a suitable microscope (e.g., bright-field, fluorescent, or confocal microscope) or slide scanner and camera. IHC signal intensity can be quantified from such images using a suitable software program, such as ImageJ Fiji, ImageScope, Ilastik, Cell Profiler, inForm Image Analysis Software, or IHC Profiler.

[0238] The inventors of the present disclosure have unexpectedly found that AKR1C3 levels can vary dramatically between individual tumor cells within a single tumor.The inventors have also found that IHC has properties that make it particularly well suited for use in connection with the method of treating a subject with a compound of formula (I) or its pharmaceutically acceptable salt, the method of identifying a subject for treatment with a compound of formula (I) or its pharmaceutically acceptable salt, and the method of selecting a compound of formula (I) or its pharmaceutically acceptable salt for treating a subject, wherein the method requires determining the level of AKR1C3 in a subject sample, and the subject sample is characterized as having an elevated level of AKR1C3, or the subject sample is characterized as having an elevated level of AKR1C3. Thus, the inventors have unexpectedly discovered that IHC is particularly advantageous for determining the level of AKR1C3 in a sample (e.g., a subject sample) in connection with methods for identifying a subject for treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof; methods for selecting a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating a subject; methods for treating a subject comprising a step of determining the level of AKR1C3 in a subject sample; methods for treating a subject by administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject, wherein the subject sample is characterized by having an elevated level of AKR1C3 prior to said administration; and methods for treating a subject with a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the subject sample is characterized by having an elevated level of AKR1C3.

[0239] In particular, IHC allows for the evaluation or scoring of the presence or level of proteins or other detectable markers in individual cells. IHC can be used to generate an overall score for the detection of proteins or other detectable markers in tissue samples (e.g., tumor samples), but offers the advantage of being based on the evaluation of individual cells. These characteristics of IHC are particularly advantageous for evaluating the expression of proteins that show highly heterogeneous levels of expression in individual cells of tumors. Therefore, because AKR1C3 levels vary dramatically between individual tumor cells, IHC is particularly well suited for determining the level of AKR1C3 in subject samples and / or generating an H score suitable for identifying subjects for treatment with the compound of formula (I) or its pharmaceutically acceptable salt.

[0240] kit Also described herein are kits suitable for carrying out the methods or uses described herein. The kits described herein can include, but are not limited to, instructions for use, one or more containers for storing the kit components, and / or components including a pharmaceutically acceptable solution formulated for oral administration. The kits described herein may further comprise components suitable for determining the level of a biomarker, such as AKR1C3, in a subject sample; characterizing the subject sample as having an elevated biomarker level (e.g., AKR1C3 biomarker level); identifying the subject for treatment 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 the subject sample: NFE2L2, KEAP1, or CUL3; characterizing the subject sample for the presence of a somatic mutation in at least one of the following genes: NFE2L2, KEAP1, or CUL3; determining the level of a biomarker, such as AKR1C3, in a sample (e.g., a test sample or a control sample); detecting a somatic mutation in one or more gene sequences, such as NFE2L2, KEAP1, or CUL3 gene sequences; or detecting a somatic mutation in one or more mRNA sequences, such as NFE2L2, KEAP1, or CUL3 mRNA sequences. In some embodiments, the kits described herein comprise a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the kits described herein comprise a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0241] In some embodiments, the kits described herein include components suitable for determining mRNA or protein levels of a biomarker, e.g., AKR1C3. In some embodiments, the kits described herein include components suitable for performing an antigen detection assay, e.g., an IHC assay. In some embodiments, the kits described herein include components suitable for sequencing genomic DNA or RNA (e.g., mRNA), e.g., NFE2L2, KEAP1, or CUL3 gene sequences. [Example]

[0242] Example 1: AKRIC3 immunohistochemistry assay for cancer tissues An AKR1C3 immunohistochemistry assay was designed to determine whether detection of AKR1C3 protein expression using an anti-AKR1C3 antibody could be used to identify cancerous tissues. Subject samples from patients diagnosed with NSCLC (including adenocarcinoma and squamous cell carcinoma subtypes; Figure 1, column labeled "NSCLC"), prostate cancer (Figure 1, column labeled "Prostate"), or hepatocellular carcinoma (Figure 1, column labeled "HCC"), as well as subject biopsies from patients with NSCLC, HCC, and head and neck (H&N) cancer (Figure 1, column labeled "Test Biopsy"), were collected and processed for immunostaining. Prostate group samples were collected from tumors with a Gleason grade score of 9–10 (Figure 1, "Gleason Grade 9–10"). Subject samples from the NSCLC, prostate cancer, and HCC groups did not have detected mutations in NFE2L2 or KEAP1 (Figure 1, "WT"). Subject samples included in the trial biopsy group contained mutations in NFE2L2 and / or KEAP1 ( Figure 1 , "NRF2 / KEAP1 Mut"). H&N subject samples included in the trial biopsy group included biopsies collected before treatment ( Figure 1 , "Screening") or on days 2 and 1 of treatment ( Figure 1 , "C2D1").

[0243] All samples were prepared for IHC by cutting 4-6 μm tissue sections from formalin-fixed, paraffin-embedded specimen blocks with a microtome. The tissue sections were mounted on SuperFrost Plus Microscope Slides (catalog no. 22-037-246; Fisher Scientific, Waltham, MA), air-dried overnight at room temperature, and incubated at 60°C for 1 hour.

[0244] Samples from the study biopsy group were stained using a Ventana Benchmark Ultra automated stainer (Ventana, Tucson, AZ) with the following bulk reagents: 10x Reaction Buffer (catalog no. 950-300; Roche Diagnostics Corporation; Indianapolis, IN) diluted 1x with deionized water, Benchmark Ultra Liquid Coverslips (LCS) (catalog no. 650-210; Roche Diagnostics Corporation), 10x EZ Prep Concentrate (catalog no. 950-102; Roche Diagnostics Corporation) diluted 1x with deionized water, and Ultra Cell Conditioning 1 (CC1) Solution (Roche, catalog no. 950-224). Immunostaining was performed using the ultraView Universal DAB procedure (v1.02.0018), which included antigen retrieval with CC1 solution at 95°C for 36 minutes. 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 for 32 minutes at 37°C and detected with the UltraView Universal DAB Detection Kit. Slides were counterstained with hematoxylin (catalog no. 760-2021; Roche Diagnostics Corporation) for 4 minutes at room temperature and stained with bluing reagent (catalog no. 760-2037; Roche Diagnostics Corporation) for 4 minutes at room temperature.

[0245] Samples from the NSCLC, prostate, and HCC groups were stained using a Ventana Discovery Ultra automated stainer (Ventana, Tucson, AZ) with the following bulk reagents: 10x Reaction Buffer (catalog no. 950-300; Roche Diagnostics Corporation) diluted 1x with deionized water, Benchmark Ultra Liquid Coverslips (LCS) (catalog no. 650-210; Roche Diagnostics Corporation), Discovery Wash Concentrate (catalog no. 950-510; Roche Diagnostics Corporation) diluted 1x with deionized water, and Ultra Cell Conditioning 1 (CC1) Solution (Roche, catalog no. 950-224). Immunostaining was performed using the RUO Discovery Universal procedure (v 0.00.0370), and antigen retrieval was performed with CC1 Solution at 95°C for 32 minutes. Endogenous peroxidase was blocked by incubation for 8 minutes in inhibitor CM, a component of the ChromoMap DAB Detection Kit (Cat. No. 760-159; Roche Diagnostics Corporation). The 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 for 60 minutes at 37°C, followed by a 4-minute incubation with Omnimap anti-mouse HRP secondary antibody (Cat. No. 760-4310; Roche Diagnostics Corporation) and detection with the ChromoMap DAB Detection Kit.Slides were counterstained with hematoxylin (catalog no. 760-2021; Roche Diagnostics Corporation) for 4 min at room temperature and stained with bluing reagent (catalog no. 760-2037; Roche Diagnostics Corporation) for 4 min at room temperature.

[0246] DAB staining of target samples using anti-AKR1C3 antibody detection and subsequent analysis demonstrated that most target samples were assigned an H-score of greater than 50 (Figure 1). Furthermore, 5 / 32 (16%) NSCLC adenocarcinoma target samples, 13 / 45 (29%) NSCLC squamous cell carcinoma target samples, 5 / 29 (17%) prostate cancer target samples, and 19 / 68 (28%) HCC target samples were assigned an H-score of greater than 250. These results demonstrate that anti-AKR1C3 antibody can be used to detect AKR1C3 expression levels and calculate H-scores for target samples from cancerous tissues.

[0247] Example 2: Clinical trial to determine the administration of AKR1C3-dependent KARS inhibitors A Phase 1, open-label, multicenter clinical trial will be conducted to characterize 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. The study will also optionally be 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 antitumor activity of Compound I will also optionally be evaluated. The study will include a dose escalation portion followed by a dose expansion portion. The escalation portion will characterize safety and tolerability. The dose expansion portion will evaluate preliminary antitumor activity in a defined patient population and further evaluate safety and tolerability at the MTD / RD.

[0248] Patients and cohorts Eligible patients for the study include those with advanced (metastatic or unresectable) non-small cell lung cancer harboring an NFE2L2 or KEAP1 or CUL3 mutation (dose escalation and dose expansion arm 1) and those with advanced (metastatic or unresectable) non-small cell lung cancer regardless of mutation status (dose expansion arm 2) who have failed standard therapy for their indication or are intolerant or ineligible to approved therapy.

[0249] Dose escalation and dose expansion cohort 1 includes patients with a histologically or cytologically confirmed diagnosis of advanced (metastatic or unresectable) NFE2L2 / KEAP1 / CUL3-mutated non-small cell lung cancer. Local data confirming NFE2L2 / KEAP1 / CUL3 mutation status in tissue are required for enrollment.

[0250] Dose expansion cohort 2 includes patients with histologically or cytologically confirmed advanced (metastatic or unresectable) non-small cell lung cancer, regardless of mutational status.

[0251] All patients had progressed after one platinum-based chemotherapy regimen and / or PD(L)-1 antibody therapy (if indicated) for stage IV non-small cell lung cancer. Patients may include those who have received prior treatment with VEGF / VEGFR-targeted agents, neoadjuvant / adjuvant therapy. Patients with non-small cell lung cancer whose tumors harbor actionable mutations have received treatment with approved targeted agents (e.g., EGFRi, ALKi, METi). All patients have at least one measurable lesion per RECIST v1.1. All patients have disease sites suitable for biopsy and are candidates for tumor biopsy according to their respective treatment institution's guidelines. Patients are willing to undergo new tumor biopsies at screening and during treatment on this study. A recent biopsy collected after their last systemic treatment and within 3 months prior to study enrollment may be submitted for screening.

[0252] Patients will not have cardiac dysfunction or clinically significant cardiac disease or risk factors at screening. Patients will not be symptomatic for CNS metastases or CNS metastases requiring local CNS-directed therapy (such as radiation therapy or surgery) or escalating doses of corticosteroids 2 weeks prior to study entry. Patients with treated symptomatic brain metastases will be neurologically stable (4 weeks after treatment and prior to study enrollment) and on a dose of prednisone or equivalent ≤ 10 mg / day for at least 2 weeks prior to administration of any study treatment. Patients will not be treated with drugs / supplements / herbs that are strong or moderate CYP3A4 inhibitors or strong or moderate CYP3A4 inducers that cannot be discontinued 7 days prior to study initiation and during the study period.

[0253] Objectives and Endpoints The efficacy of Compound I treatment is determined based on overall response rate, progression-free survival, and duration of response according to RECIST v1.1. Pharmacokinetics is assessed based on the plasma concentration versus time profiles 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).

[0254] The primary objective of the study is to characterize the safety and tolerability of Compound I in patients with NSCLC and identify the MTD and / or RD and dosing regimen for future studies. Safety will be 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 toxicities (DLTs) will be determined during the first 28 days of treatment with Compound I. Tolerability will be determined by dose interruptions, reductions, and dose intensity.

[0255] Secondary objectives of this 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-time profiles of Compound I and Compound II and the derived PK parameters (e.g., Cmax, Tmax, AUC).

[0256] Test Design Dose Escalation: Dose escalation will treat a minimum of 21 patients with advanced NSCLC harboring NFE2L2, or KEAP1 or CUL3 (NFE2L2 / KEAP1 / CUL3) mutations. Patient enrollment will be based on locally available test results for mutation status (the same archived samples used to determine mutation status locally will be requested for central confirmation, if possible, as needed).

[0257] Cohorts of 3 to 6 patients will be dosed with different doses of Compound I in 28-day cycles QD or BID (based on new PK data from dose escalation) until the oral (po) MTD and / or RD is reached.

[0258] The safety (including dose-DLT relationship) and tolerability of Compound I will also be evaluated to identify a regimen and / or MTD and / or RD for use in dose expansion. The dose and regimen for RD will be identified after reviewing all available data, including PK, safety, and preliminary antitumor activity. A Bayesian hierarchical logistic regression model (BHLRM) using escalation with excess dose control (EWOC) principles will guide dose escalation to determine the MTD and / or RD. The RD will not exceed the MTD of Compound I.

[0259] Based on emerging PK, safety, and tolerability data and / or preliminary antitumor activity, different dosing regimens (e.g., 2 weeks on / 2 weeks off, 3 weeks on / 1 week off, 1 week on / 1 week off) will also be evaluated.

[0260] Dose Expansion This study will enter dose expansion after MTD and / or RD are declared at dose escalation. Approximately 100 patients with advanced NSCLC will be treated across two dose expansion arms to evaluate the preliminary antitumor activity of Compound I. At least 10 patients with squamous cell carcinoma will be enrolled in each arm. The dose expansion arms include: Group 1 (approximately 40 patients): Patients with advanced NSCLC harboring NFE2L2 / KEAP1 / CUL3 mutations enrolled based on locally available test results of mutation status (the same archived samples used to determine mutation status locally will be requested for central confirmation, if available, as needed). Group 2 (approximately 60 patients): Patients with advanced NSCLC regardless of prior knowledge of NFE2L2 / KEAP1 / CUL3 mutation status.

[0261] The study design is shown in Figure 2.

[0262] The study will also optionally include an exploratory evaluation of the food effect ("FE") on the PK of single-agent Compound I in a separate cohort of patients with advanced NSCLC harboring NFE2L2 / KEAP1 / CUL3 mutations. For this subset of patients, the study design will consist of an FE run-in period and a treatment period.

[0263] Patients will undergo safety and efficacy assessments during screening / baseline and during treatment (Figure 3).

[0264] Compound I is administered orally in the form of capsules of 50 mg or 75 mg of the drug substance.

[0265] The starting dose of Compound I as a single agent will be set at 100 mg, administered poQD on a continuous schedule based on available preclinical safety, tolerability, and PK / PD data. The selection of the starting dose will follow the ICH S9 guidelines for selecting the starting dose for FIH studies conducted in patients with advanced cancer. The starting dose will also be supported by 4-week GLP toxicology studies conducted in rats and monkeys.

[0266] Table 1 lists the starting dose and possible dose levels evaluated during this study.

[0267] [Table 1]

[0268] Biomarkers Biomarker analysis is used to investigate the effects of Compound I as a single agent at the molecular and cellular levels and to determine how changes in markers relate to exposure and clinical outcomes. Additionally, potential predictive markers of efficacy and mechanisms of resistance to Compound I as a single agent are optionally investigated.

[0269] The exact date and time of biomarker sample collection will be entered on the appropriate eCRF and recruitment form. Detailed instructions for biomarker sample collection, processing, and shipping will be provided. Samples will be collected at defined visits / timepoints. Biomarker sample types and collections are listed in Table 2.

[0270] [Table 2]

[0271] Freshly obtained paired pre- and on-treatment tumor samples will be collected mandatorily during screening and treatment, if safe and medically feasible.

[0272] If a recent biopsy collected after the last systemic treatment and within 3 months prior to study enrollment is available, it may be submitted at screening in place of a newly obtained biopsy. If an inadequate tumor sample is received at screening after a new biopsy treatment (e.g., found to have low tumor content or insufficient tissue remaining), a recent biopsy will be requested as needed to allow for the analyses described in Tables 8-13. In either case, the recent biopsy will meet the specifications provided and those detailed in the test manual. A copy of the corresponding de-identified pathology report will also be submitted, if necessary.

[0273] If core needle biopsies are performed, 3-6 tumor biopsy passes will be required at both screening and post-treatment visits. On-treatment biopsies will be scheduled within ±3 days of C1D22, ideally 2-6 hours after treatment administration. The date and time of sampling will be recorded on the eCRF. The timing of on-treatment biopsies (C1D22) will be adjusted based on new data, if necessary. The decision regarding the timing of on-treatment tumor biopsies will be made by the investigator.

[0274] Whenever possible, tumor biopsies are collected from the same tumor lesion.

[0275] The investigator will make reasonable efforts to record the location and size of biopsied lesions (at baseline and on treatment). This information will be recorded on the eCRF.

[0276] The same archived tumor biopsy used for local testing will be submitted at screening, if available, to retrospectively confirm local data related to NFE2L2, KEAP1, and CUL3 alterations, which may support the development of potential companion diagnostic (CDx) assays. Copies of corresponding de-identified pathology reports may also be requested. A mandatory screening biopsy or a recent biopsy collected after the last systemic treatment and within 3 months prior to study enrollment may also be 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 may be optionally profiled for genetic alterations, potentially supporting the development of potential CDx assays.

[0277] A collection of newly obtained paired tumor samples will be used to directly test the PD effect of Compound I in the tumor (e.g., expression of ATF3, EGR1, DDIT3, AKR1C3, KARS) to evaluate whether AKR1C3 and / or KARS expression are potential predictors of response. Whole transcriptome analysis and expression of additional immune or cancer-related genes will also be optionally investigated. Protein expression of AKR1C3 will be used to support subgroup analysis in dose expansion cohort 2 and may be similarly tested in dose escalation and dose expansion cohort 1. Expression and localization of immune biomarkers, including, but not limited to, PD-L1 and CD8, will optionally be measured by IHC or using additional techniques as deemed appropriate.

[0278] Blood will be collected at screening and EOT to allow for sequence analysis of cfDNA, which will investigate the presence of emerging, pre-existing, and resistance mutations in tumors and cfDNA at various time points, as well as potentially tumor mutational burden, and their relationship to clinical response and the development of tumor resistance.

[0279] Additional blood samples will be collected at screening to support potential CDx assay development for genes including, but not limited to, NFE2L2, KEAP1, and CUL3.

[0280] AKR1C3 expression and antitumor activity To examine the relationship of baseline AKR1C3 expression to antitumor activity, specifically ORR, per RECIST v1.1, a logistic regression model is fitted to data from patients from dose expansion arm 2. A score (H-score) is used to quantify patient AKR1C3 exposure.

[0281] Assuming that patients with higher AKR1C3 expression levels are associated with a higher probability of response, estimates of response probability from the fitted model are presented along with their 90% confidence intervals for various AKR1C3 H-score values (e.g., H-score = 0, 50, 150, 250). Particular attention is paid to patients with the highest AKR1C3 concentrations (H-score ≥ 250).

[0282] 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 from the ascending arm and dose expansion arm 1. Subgroup analyses of specific patient groups (e.g., squamous patients) are optionally considered.

Claims

1. Compounds of formula (I): 【Chemical 1】 (In the formula, 【Chemistry 2】 is a single or double bond; Z is 【Chemistry 3】 is a single bond, then OH; or 【Chemistry 4】 is a double bond, then either O; Each R １ are independently １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, (C ０ -C ４ ) alkylN(R ８ ) ２ and halo; R ２ａ and R ２ｂ are each independently H, (C １ -C ６ ) selected from the group consisting of alkyl, and halo; Each R ３ is independently selected from the group consisting of H, and halo; R ４ is selected from the group consisting of aryl, 5-6 membered heteroaryl containing 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S; and 9-10 membered fused bicyclic heteroaryl containing 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S; any of the foregoing may be selected from the group consisting of one or more R ６ optionally substituted with; R ５ is H; (C １ -C ６ ) alkyl; (C ２ -C ６ ) alkenyl; (C ０ -C ４ ) alkyl OR ８ ; (C １ -C ４ ) alkyl(C ３ -C １０ ) cycloalkyl; halo(C １ -C ６ ) alkyl; (C ２ -C ３ ) alkynyl; (C １ -C ４ ) alkylN(R １０ ) ２ selected from the group consisting of: Each R ６ Ha, hello;; (C １ -C ６ ) alkyl; (C １ -C ６ ) alkoxy; halo(C １ -C ６ ) alkyl; OH; aryl; 3- to 6-membered heterocycle; 5- to 6-membered heteroaryl; (C ０ -C ４ ) alkylS(O) ｍ (C １ -C ６ ) alkyl; halo(C １ -C ６ ) alkoxy; (C ０ -C ４ ) alkylS(O) ｍ N (R ８ ) ２ ; (C ０ -C ４ ) alkylN(R ８ ) ２ ; (C ０ -C ４ ) alkyl(CO)OR ７ ;N(R ８ ) S (O) ｍ (C １ -C ６ ) alkyl; N(R ８ ) S (O) ｍ (C ３ -C ６ ) cycloalkyl; OP(O)(OH) ２ ; (C ０ -C ３ ) alkyl(CO)NHR １１ ; (C ０ -C ３ ) alkyl OR ７ and (C ３ -C １０ ) cycloalkyl; each R ６ is halo, OH or OP(O)(OH) ２ Otherwise, 1 to 3 R ９ or two adjacent R ６ together with the atoms to which they are attached form a 5- to 7-membered heterocycle or (C ５ -C ８ ) forming a cycloalkyl; Each R ７ and R ８ is H or (C １ -C ６ ) alkyl; and 1 to 3 R ９ optionally substituted with; Each R ９ is halo; —OH; amino, (C １ -C ４ ) alkylamino, di(C １ -C ４ ) alkylamino, OP(O)(OH) ２ ; (C １ -C ６ ) alkyl; (C １ -C ３ ) alkynyl; (C １ -C ６ ) alkoxy; halo(C １ -C ６ ) alkyl; (C ０ -C ４ ) alkylS(O) ｍ (C １ -C ６ ) alkyl; halo(C １ -C ６ ) alkoxy; 3- to 6-membered heterocycle optionally substituted with oxo (=O); (C ０ -C ４ ) alkylS(O) ｍ N (R １０ ) ２ ;; (C ０ -C ４ ) alkyl(CO)R １０ ; (C ０ -C ４ ) alkyl(CO)OR １０ ; (C ０ -C ４ ) alkylNR １０ S (O) ｍ (C １ -C ６ ) alkyl; (C ０ -C ４ ) alkyl OR １０ ; (C ０ -C ４ ) alkylN(R １０ ) ２ ; (C ０ -C ４ ) alkylCN; (C ０ -C ４ ) alkylN(R １０ ) ２ and (C ０ -C ４ ) alkyl(CO)N(R １０ ) ２ are independently selected from the group consisting of: Each R １０ is H, (C １ -C ６ ) alkyl; or a 3- to 6-membered heterocycle, wherein the 3- to 6-membered heterocycle is selected from the group consisting of one or more (C １ ~C ６ ) alkyl; and optionally substituted with oxo (=O); Each R １１ is H; 1 to 4 R １２ a 4- to 6-membered heterocycle optionally substituted with 1 to 4 R １２ (C ３ -C ６ ) cycloalkyl; optionally substituted with halo (C ０ -C ３ ) alkyl(C ３ -C ６ ) cycloalkyl (C １ -C ３ ) alkyl; ； 1 to 3 R １２ CH optionally substituted with ２ -aryl; (C １ -C ６ ) alkyl; (C ２ -C ６ ) alkenyl; or (C ２ -C ６ ) alkynyl, wherein (C １ -C ６ ) alkyl; (C ２ -C ６ ) alkenyl; and (C ２ -C ６ ) Each alkynyl may be one or more R １３ optionally substituted with; Each R １２ is OH, (C １ -C ３ ) alkoxy, NH ２ or optionally substituted with one or more OH (C １ -C ３ ) alkyl; Each R １３ is halo, OH, amino, (C １ -C ４ ) alkylamino, di(C １ -C ４ ) alkylamino, (C １ -C ３ )alkoxy; and C(O)—(C ３ -C ８ ) cycloalkyl; m is 0, 1 or 2; and n is 0, 1 or 2. or a pharmaceutically acceptable salt thereof, comprising: The method comprises determining the level of AKR1C3 in the subject sample, wherein an elevated level of AKR1C3 identifies the subject as one in need of treatment with a 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 for treating a subject, the method comprising determining the level of AKR1C3 in the subject's sample, wherein an elevated level of AKR1C3 identifies the subject as one requiring treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

3. 1. A method of treating a subject, comprising: a. determining the level of AKR1C3 in a subject sample, wherein elevated levels of AKR1C3 identify subjects in need of treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof; and b. A method comprising administering to a subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

4. 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 administration, the subject's sample is characterized as having a level of AKR1C3.

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

6. 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 according to 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 to 7, wherein the subject sample comprises cells, cell populations, cell lysates, tissues, or bodily fluids of the subject.

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

10. 10. The method of claim 9, wherein the cancerous cells are tumor cells.

11. 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 cell is a lung cell.

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

14. 14. The method of any one of claims 1 to 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 to 14, wherein the subject 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 to 15, wherein the level of AKR1C3 in the subject sample is elevated compared to a control level of AKR1C3.

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

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

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

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

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

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

23. the level of AKR1C3 in the subject sample is about 1.5-fold greater, about 2-fold greater, about 3-fold greater, about 4-fold greater, about 5-fold greater, about 6-fold greater, about 7-fold greater, about 8-fold greater, about 9-fold greater, about 10-fold greater, about 20-fold greater, about 30-fold greater, about 40-fold greater, about 50-fold greater, about 60-fold greater, about 70-fold greater, about 80-fold greater, about 90-fold greater, about 100-fold greater, about 150-fold greater, about 160-fold greater, about 170-fold greater, about 180-fold greater, about 190-fold greater, about 210-fold greater, about 220-fold greater, about 230-fold greater, about 240-fold greater, about 250-fold greater, about 260-fold greater, about 270-fold greater, about 280-fold greater, about 290-fold greater, about 300-fold greater, about 310-fold greater, about 320-fold greater, about 330-fold greater, about 340-fold greater, about 350-fold greater, about 360-fold greater, about 370-fold greater, about 380-fold greater, about 390-fold greater, about 400-fold greater, about 410-fold greater, about 420-fold greater, about 430-fold greater, about 440-fold greater, about 450-fold greater, about 460-fold greater, about 470-fold greater, about 480-fold greater, about 490-fold greater, about 500-fold greater, about 510-fold greater, about 520-fold greater, about 530-fold greater, about 540-fold greater, 23. The method of any one of claims 17-22, wherein the subject is in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof when the IL-16 expression level is about 100 times greater, about 100 times greater, about 200 times greater, about 300 times greater, about 400 times greater, about 500 times greater, about 600 times greater, about 700 times greater, about 800 times greater, about 900 times greater, about 1000 times greater, about 1500 times greater, or about 2000 times greater.

24. the level of AKR1C3 in the subject sample is at least about 1.5-fold greater, at least about 2-fold greater, at least about 3-fold greater, at least about 4-fold greater, at least about 5-fold greater, at least about 6-fold greater, at least about 7-fold greater, at least about 8-fold greater, at least about 9-fold greater, at least about 10-fold greater, at least about 20-fold greater, at least about 30-fold greater, at least about 40-fold greater, at least about 50-fold greater, at least about 60-fold greater, at least about 70-fold greater, at least about 23. The method of any one of claims 17-22, wherein the subject is in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof when the IL-16 expression level is 80 times greater, at least about 90 times greater, at least about 100 times greater, at least about 200 times greater, at least about 300 times greater, at least about 400 times greater, at least about 500 times greater, at least about 600 times greater, at least about 700 times greater, at least about 800 times greater, at least about 900 times greater, at least about 1000 times greater, at least about 1500 times greater, or at least about 2000 times greater.

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

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

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

30. 29. 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 murine monoclonal antibody, clone NP6.G6.A6.

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

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

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

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

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

36. 36. The method of claim 35, wherein the subject is in need of treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof if the IHC signal intensity score for 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 to 3.

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

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

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

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

42. 41. 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 murine monoclonal antibody, clone NP6.G6.A6.

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

44. 43. 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. 45. The method of claim 44, wherein the secondary antibody is conjugated to HRP.

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

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

48. 51. The method of claim 50, wherein the subject is in need of treatment with the compound of formula (I) or a pharmaceutically acceptable salt thereof when 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 may be in the range of 0 to 3.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

64. 64. The method of any one of claims 57 to 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 subject sample.

66. A method for 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 subject sample.

67. 1. A method of treating a subject, comprising: a. detecting a somatic mutation in at least one of the following genes in a subject sample: NFE2L2, KEAP1, or CUL3, thereby identifying a subject in need of treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof; and b. Administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. A method comprising:

68. 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 administration, the subject's sample 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 the subject sample is characterized by the presence of a somatic mutation in at least one of the following genes: NFE2L2, KEAP1, or CUL3.

70. 70. The method of any one of claims 65 to 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. 71. The method of any one of claims 65 to 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. 72. The method of any one of claims 65 to 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. 72. The method of any one of claims 65 to 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. 72. The method of any one of claims 65 to 71, wherein the somatic mutation comprises a single nucleotide polymorphism (SNP).

75. 75. The method of any one of claims 65 to 74, wherein the subject sample comprises cells, cell populations, cell lysates, tissues, or bodily fluids of the subject.

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

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

78. 78. The method of claim 77, wherein the cancerous cells are tumor cells.

79. 79. The method of claim 78, 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.

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

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

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

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

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

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

87. 76. The method of any one of claims 65 to 75, wherein the level of AKR1C3 in the subject 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 greater than the level of AKR1C3 in a control sample or control dataset.

88. 76. The method of any one of claims 65 to 75, wherein 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 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 greater than the level of AKR1C3 in the control sample or control dataset.

89. 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. 68. The method of any one of claims 65 to 67, wherein said detecting comprises sequencing genomic DNA or mRNA of said subject sample.

92. 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, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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. 92. The method of claim 91, wherein said sequencing comprises performing a polymerase chain reaction (PCR).

94. 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. 95. The method of any one of claims 65 to 94, wherein if the sequencing detects a somatic mutation in the subject sample, the subject requires treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

96. 96. The method of any one of claims 91 to 95, further comprising sequencing the genomic DNA or mRNA of the control sample.

97. 96. The method of any one of claims 91 to 95, further comprising comparing sequencing data from the sequencing of the subject sample with sequencing data from a control sample or control dataset.

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

99. 96. 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, combinatorial probe anchor synthesis (cPAS) sequencing, combinatorial 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. 96. The method of claim 95, wherein said sequencing comprises performing a polymerase chain reaction (PCR).

101. 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. 102. The method of any one of claims 98 to 101, wherein if the sequencing detects a somatic mutation in the subject sample, the subject requires treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

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

104. 103. The method of any one of claims 98 to 102, further comprising comparing the sequencing data of the sequenced genomic DNA or mRNA of the subject sample with the sequencing data of a control sample or control dataset.

105. Use of AKR1C3 levels to select 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 an elevated level of AKR1C3, the subject is treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

106. Use of somatic mutation to select a subject for treatment 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, and if the somatic mutation is detected in one of the following genes: NFE2L2, KEAP1 or CUL3, the subject is treated with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

107. 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 according to any one of claims 105 to 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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