Methods, kits and systems for determining ar activity of cancer and methods for treating cancer based on same

Abstract

The present disclosure includes, among other things, methods, kits, and systems for determining AR activity of cancer, e.g., prostate cancer. In various embodiments, the present disclosure relates to the use of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation to measure AR activity of a cancer. In some embodiments, differential modifications and / or differential accessibility are detected and quantified at one or more genomic loci of a biological sample, e.g., in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from a subject with cancer. In various embodiments a determined AR activity is useful, e.g., in selecting treatment for and / or treating a cancer, e.g., a prostate cancer.

Description

Attorney Docket: 2014191-0045METHODS, KITS AND SYSTEMS EOR DETERMINING AR ACTIVITY OF CANCER AND METHODS FOR TREATING CANCER BASED ON SAME RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 740,889, filed on December 31, 2024 and U.S. Provisional Patent Application No. 63 / 809,960, filed on May 21, 2025, the entire contents of each are hereby incorporated by reference in their entirety.BACKGROUND

[0002] The androgen receptor (AR) is a nuclear receptor that, in healthy subjects, remains in the cytoplasm until activated by binding of an androgenic hormone (e.g., testosterone or dihydrotestosterone). Upon binding of an androgenic hormone, AR translocates to the nucleus and promotes expression of androgen regulated genes. Given its widespread expression in many cell and tissue types, the AR plays a role in a diverse range of biological functions, including development and maintenance of the reproductive, musculoskeletal, cardiovascular, immune, neural and haemopoietic systems.

[0003] Aberrant AR activity is associated with the onset and progression of prostate cancer as well as other cancers. Because of the important role that AR signaling plays in prostate cancer, therapies targeting the AR pathway have become a mainstay strategy for treating metastatic prostate cancer. The utility of these therapies has expanded with the emergence of second-generation AR antagonists, which began with the approval of enzalutamide in 2012 by the United States Food and Drug Administration (FDA). Together with apalutamide and darolutamide, which were approved in 2018 and 2019, respectively, these agents have improved the survival of patients with prostate cancer, with applications for both androgen-dependent and castration-resistant disease. Aberrant AR pathway activity has also been associated with other cancers, including, e.g., breast, bladder, liver, kidney, lung, and larynx cancer and bladder cancer. Current clinical protocols for administering AR-targeted therapies either do not take into account AR activity or expression status or only take into account AR expression status, an indirect measure of AR activity.

[0004] PSMA (Prostate-Specific Membrane Antigen), is a cell surface protein that is highly expressed in prostate cancer and is a key biomarker for diagnosis and prognosis of patients with prostate cancer. PSMA-targeted therapeutics (e.g., antibody-drug conjugates and Page 1 of 13113185130vlAttorney Docket: 2014191-0045radionuclide therapeutics) have been developed as one modality for treating patients having prostate cancer. For such therapeutics, PSMA expression level, as assessed using PET imaging, is commonly used as a criterion for determining patient eligibility for treatment. To date, no blood-based assay has been developed that can be used to assess patient eligibility for treatment with PSMA-targeted agents.SUMMARY

[0005] The present disclosure is based, at least in part, on the demonstration that AR activity in a cancer can be measured by detecting and quantifying histone modifications at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample, obtained or derived from a subject. The present disclosure also encompasses methods where chromatin accessibility, DNA methylation, and / or binding of one or more transcription factors are detected at one or more genomic loci instead of (or in addition to) histone modifications. These new assays provide minimally invasive ways of determining AR activity that are more accurate, objective, and comprehensive than current tissue-based approaches.

[0006] As discussed elsewhere in the present disclosure, blood-based assays for measuring AR activity provide significant advantages as compared to current clinical protocols, which rely on cancer staging (often without taking AR expression or activity into account) and / or AR expression status (an indirect measure of AR activity) determined using tissue samples obtained via biopsy. Methods that rely on a tissue sample are invasive and focus only on a small region at a single tumor site at a given time and therefore do not accurately capture tumor heterogeneity or receptor evolution and only partially characterize the relevant patient population. Moreover, resistance to antiandrogens frequently develop, and measurement of AR activity can provide further insight into the susceptibility of a cancer in a subject to AR-targeted therapies and better inform therapy selection. AR expression status is an insufficient proxy for AR transcriptional dependency. Methods that detect AR activity in a cell, rather than simply AR expression status would therefore provide significant advantages, including, e.g., being better able to predict patient responsiveness to treatment with AR-targeted therapies and / or better inform therapy selection.

[0007] The present disclosure includes, among other things, technologies for determiningPage l of 13113185130vlAttorney Docket: 2014191-0045AR activity and for the detection, monitoring, and / or treatment of cancer (including, e.g., prostate or breast cancer) based on AR activity. In various embodiments, the present disclosure relates to the measurement of histone modifications in a sample obtained or derived from a subject to detect and / or treat cancer (including, e.g., breast or prostate cancer) based on AR activity. The present disclosure includes, among other things, histone modification measurements in cell-free DNA (cfDNA) that are characteristic of cancer, and which in various embodiments are useful, e.g., for detecting, monitoring, selecting treatment for, and / or treating cancer (including, e.g., prostate or breast cancer) based on AR activity. In some embodiments, histone modification measurements in cfDNA can be used to detect or determine resistance of a cancer (e.g., breast or prostate cancer) to a therapy. In various embodiments, the present disclosure includes exemplary genomic loci that comprise differential epigenomic modifications depending on AR activity in a cancer (e.g., breast or prostate cancer). In various embodiments, genomic loci differentially modified in cfDNA are or include one or more enhancer regions or subregions. In various embodiments, genomic loci differentially modified in cfDNA are or include one or more promoter regions or subregions.

[0008] Among other things, the present disclosure also provides certain insights for adjusting for mechanisms of resistance to anti androgens. Among the insights provided herein is that exclusion of loci correlated with ERG expression from an AR activity score generator can provide a more generally applicable AR activity score generator. Without wishing to be bound by theory, TMPRSS2-ERG fusions have previously been shown to result in increased ERG expression and rewiring of the AR cistrome. Exclusion of loci that are correlated with ERG expression can result in a more generally applicable AR activity score generator, which can be applied to cancers comprising wild-type ERG or an ERG fusion.

[0009] The present disclosure also provides an insight that, in some embodiments, use of genomic loci that exhibit differential enhancer signal depending on AR activity and (1) that overlap with AR ChlP-seq sites having an AR motif (“AR motif sites”), or (2) that lack AR motifs but which are highly correlated with AR motif sites can provide improved results.

[0010] Among other things, the present disclosure also provides the insight that AR activity can be predictive of responsiveness to PSMA-targeted agents in patients with prostate cancer. The demonstration that AR-activity measurements can be used to identify subjects withPage 3 of 13113185130vlAttorney Docket: 2014191-0045an improved likelihood of responding to treatment with a PSMA-targeted agent represents an important contribution to the field and offers the potential to lead to improved patient outcomes, whether used as a stand-alone screening assay or used in conjunction with other assays for assessing patient suitability for treatment with a PSMA-targeted agent.

[0011] In various embodiments, a genomic locus is differentially modified if it is characterized by increased or decreased histone modification as compared to a reference (e.g, a sample from a cell line or subject without aberrant AR activity or a healthy subject). Increased or decreased histone modification can be or include, e.g, increased or decreased histone methylation (hypermethylation or hypomethylation, respectively) of one or more particular methylation marks, or a combination thereof; increased or decreased pan-methylation; increased or decreased histone acetylation (hyperacetylation or hypoacetylation, respectively) of one or more particular acetylation marks, or a combination thereof; and / or increased or decreased panacetylation (e.g, pan-H3 acetylation). In various embodiments, histone methylation can be or include histone methylation marks selected from H3K4mel, H3K4me2, H3K4me3, or a combination thereof. In various embodiments, histone methylation can be or include H3K4me3. In various embodiments, histone acetylation can be or include histone acetylation marks selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, or a combination thereof. In various embodiments, histone acetylation can be or include H3K27ac.

[0012] In various embodiments, the present disclosure relates to the measurement of DNA methylation in a sample obtained or derived from a subject to detect and / or treat cancer (including, e.g, breast or prostate cancer) based on AR activity. In some embodiments, DNA methylation measurements in cfDNA can be used to detect or determine resistance of a cancer (e.g., breast or prostate cancer) to a therapy or transformation of a cancer from one subtype to another. In various embodiments, a genomic locus is differentially modified if it is characterized by increased or decreased DNA methylation as compared to a reference (e.g, a sample from an AR-negative or healthy subject). In various embodiments, genomic loci differentially modified in cfDNA are or include one or more enhancer regions or subregions. In various embodiments, genomic loci differentially modified in cfDNA are or include one or more promoter regions or subregions.

[0013] The present disclosure further relates, in various embodiments, to thePage 4 of 13113185130vlAttorney Docket: 2014191-0045measurement of chromatin accessibility in cell-free DNA (cfDNA) to determine AR activity. The present disclosure includes, among other things, chromatin accessibility measurements in cfDNA that are associated with AR activity in cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating an AR-positive cancer. In some embodiments, chromatin accessibility measurements in cfDNA can be used to detect or determine resistance of a cancer (e.g, breast or prostate cancer) to a therapy or transformation of a cancer from one subtype to another. In various embodiments, the present disclosure includes genomic loci that are differentially accessible based on AR activity. In various embodiments, genomic loci differentially accessible in cfDNA are or include one or more enhancer regions or subregions. In various embodiments, genomic loci differentially accessible in cfDNA are or include one or more promoter regions or subregions.

[0014] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g, H3K4me3) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with chromatin accessibility.

[0015] In various embodiments, a genomic locus is differentially accessible if it is characterized by increased or decreased chromatin accessibility as compared to a reference e.g., a sample from an AR-negative or healthy subject). Increased or decreased histone modification can be or include, e.g., increased or decreased accessibility as determined by various chromatin accessibility assays known in the art.

[0016] The present disclosure further relates, in various embodiments, to the measurement of transcription factor binding in cell-free DNA (cfDNA) to determine AR activity. In some embodiments, transcription factor binding measurements in cfDNA can be used to detect or determine resistance of a cancer (e.g., breast or prostate cancer) to a therapy or transformation of a cancer from one subtype to another. In various embodiments, the present disclosure includes genomic loci that are differentially bound by transcription factors depending on AR activity in a cancer. In various embodiments, genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more enhancer regions or subregions. InPage 5 of 13113185130vlAttorney Docket: 2014191-0045various embodiments, genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more promoters.

[0017] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with transcription factor binding.

[0018] In various embodiments, a genomic locus is differentially bound by transcription factors if it is characterized by increased or decreased transcription factor binding as compared to a reference (e.g., a sample from an AR-negative or healthy subject). Increased or decreased transcription factor binding can be or include, e.g., increased or decreased transcription factor binding as determined by various transcription factor binding assays known in the art.

[0019] In some embodiments, methods provided herein can result in improved therapeutic outcomes in a subject with a cancer (e.g., prostate cancer, breast cancer, and / or an AR+ cancer), e.g., as compared to current standards of care for treating cancer with an AR-targeting agent that do not account for AR activity or expression and / or as compared to methods that comprise measuring AR expression rather than AR activity.

[0020] Among other things, the present disclosure describes a method of measuring androgen receptor (AR) activity of a cancer in a subject, the method comprising:quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject:(i) one or more histone modifications,(ii) chromatin accessibility,(iii) binding of one or more transcription factors, and / or(iv) DNA methylation.

[0021] In some embodiments, one or more histone modifications are quantified using a histone modification assay that measures H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, H3K4me3, or pan-acetylation. In some embodiments, a histone modification assay detects H3K4me3 modifications. In some embodiments, a histonePage 6 of 13113185130vlAttorney Docket: 2014191-0045modification assay detects H3K27ac modifications.

[0022] In some embodiments, a histone modification assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

[0023] In some embodiments, chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde- Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), a DNase hypersensitivity assay, and a fragmentomics assay.

[0024] In some embodiments, binding of one or more transcription factors is quantified using a transcription factor binding assay that detects binding of one or more of p300, mediator complex, cohesion complex, RNA pol II, FOXA1, ESRI, PR, MYC, EN1, FOXM1, KLF4, AP-2, RARa, orRUNXl.

[0025] In some embodiments, a transcription factor binding assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

[0026] In some embodiments, DNA methylation is quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq).

[0027] In some embodiments, two or more of the following, each at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject are quantified:(i) one or more histone modifications,(ii) chromatin accessibility,(iii) transcription factor binding, and / or(iv) DNA methylation.Page 7 of 13113185130vlAttorney Docket: 2014191-0045

[0028] In some embodiments, a method comprises quantifying:(i) H3K27ac modifications and H3K4me3 modifications;(ii) H3K27ac modifications and DNA methylation;(iii) H3K4me3 modifications and DNA methylation; or(iv) H3K27ac modifications, H3K4me3 modifications, and DNA methylation.

[0029] In some embodiments, a liquid biopsy sample is a plasma sample, serum sample, or urine sample.

[0030] In some embodiments, a method comprises quantifying:(i) one or more histone modifications at one or more regulatory regions (e.g., promoter or enhancer regions or subregions) associated with one or more of the genes listed in Table 1,(ii) chromatin accessibility at one or more of the genes listed in Table 1,(iii) binding of one or more transcription factors associated with promoting expression of one or more of the genes listed in Table 1, and / or(iv) DNA methylation at one or more of the genes listed in Table 1.

[0031] In some embodiments, a method comprises quantifying enhancer signal at one or more enhancer regions or subregions associated with one or more of the genes listed in Table 1 (e.g., quantifying enhancer signal at one or more of the loci listed in Table 1).

[0032] In some embodiments, a method comprises quantifying promoter signal at a promoter for one or more of the genes listed in Table 1.

[0033] In some embodiments, enhancer signal comprises H3K27ac modifications.

[0034] In some embodiments, promoter signal comprises H3K4me3 modifications.

[0035] Among other things, the present disclosure describes a method of measuring AR activity of a cancer (e.g., PRAD) in a subject, comprising:obtaining a biological sample comprising cell-free DNA (cfDNA), optionally a liquid biopsy sample, from the subject; anddetermining an AR activity score for the sample, wherein the AR activity score is determined by a method comprising measuring enhancer or promoter signal at one or more loci that have previously been determined to have increased enhancer or promoter signal in an AR+ cancer as compared to one or more non-cancerous samples (e.g., one or more plasma or tissuePage 8 of 13113185130vlAttorney Docket: 2014191-0045samples obtained from a subject not diagnosed with cancer).

[0036] In some embodiments, one or more loci with increased enhancer or promoter signal show no correlation with ERG expression.

[0037] In some embodiments, enhancer signal is measured at one or more loci that have previously been determined to have increased enhancer signal and (i) that overlap with AR ChlP-seq sites having an AR motif or (ii) that are highly correlated with AR motif sites.

[0038] In some embodiments, a method comprises measuring enhancer signal at two or more loci with increased enhancer or promoter signal in AR+ cancer, and combining (e.g., summing, averaging, geometric mean averaging, or taking the median of) the enhancer or promoter signal measured at the two more loci.

[0039] In some embodiments, a method for determining AR activity score comprises correcting the measured enhancer or promoter signal for ctDNA fraction (e.g., dividing enhancer or promoter signal by ctDNA fraction).

[0040] In some embodiments, one or more loci used in one or more methods described herein are within one or more enhancer regions for one or more of the genes listed in Table 1.

[0041] In some embodiments, one or more loci used in one or more methods described herein are within one or more promoter regions for one or more of the genes listed in Table 1.

[0042] In some embodiments, a method comprises measuring enhancer signal for at least 1, 5, 10, 20, 30, or 40 of the loci listed in Table 1.

[0043] In some embodiments, enhancer signal and / or promoter signal in the liquid biopsy sample is measured using a method that comprises sequencing cfDNA comprising one or more histone modifications (e.g., H3K4me3 and / or H3K27ac), e.g., using cfChlP-seq.

[0044] In some embodiments, sequence reads at each genomic loci are processed prior to combining with sequence reads at other genomic loci (e.g., quantile normalized and / or adjusted for background signal).

[0045] In some embodiments, a liquid biopsy sample is a plasma sample, serum sample, or urine sample.

[0046] In some embodiments, a cancer is determined to have elevated AR activity (e.g., as compared to AR activity in a subject not diagnosed with cancer and / or a cancer that does not have elevated AR activity) if an AR activity score determined using a method described herein isPage 9 of 13113185130vlAttorney Docket: 2014191-0045equal to or greater than an AR activity reference value.

[0047] In some embodiments, an AR activity reference value is a predetermined threshold value and / or a normalized value.

[0048] In some embodiments, an AR activity reference value is an AR activity score determined in a reference subject or population of subjects.

[0049] In some embodiments, a reference subject or population of subjects is:(i) a subject or population of subjects that have not been diagnosed with cancer, (ii) a subject or population of subjects that have been diagnosed with a cancer that does not have elevated AR expression,(iii) a subject or population of subjects that have been diagnosed with an AR+ cancer (e.g., AR+ PRAD) and which were found to not respond to treatment with an antiandrogen;(iv) a subject or population of subjects that have been diagnosed with an AR+ cancer (e g., AR+ PRAD) and which were found to respond to treatment with an antiandrogen; or (v) a subject or population of subjects having mCRPC.

[0050] In some embodiments, an AR activity reference value is an AR activity score that corresponds to the median, lower bound of the top tertile, lower bound of the top quartile, or lower bound of the top quintile of AR activity scores measured in a population of subjects that have been diagnosed with mCRPC.

[0051] In some embodiments, a method of the present disclosure comprises measuring ctDNA fraction in the biological sample.

[0052] In some embodiments, a method comprises comparing a ctDNA fraction measured in the biological sample to a ctDNA fraction reference value.

[0053] In some embodiments, a ctDNA fraction reference value is a predetermined threshold value and / or a normalized value.

[0054] In some embodiments, a ctDNA fraction reference value is a ctDNA fraction value determined in a reference subject or population of subjects. In some embodiments, a reference subject or population of subjects is:(i) a subject or population of subjects that have not been diagnosed with cancer, (ii) a subject or population of subjects that have been diagnosed with a cancer that is AR-negative, orPage 10 of 13113185130vlAttorney Docket: 2014191-0045(iii) a subject or population of subjects having mCRPC.

[0055] In some embodiments, a ctDNA fraction reference value is a ctDNA fraction that corresponds to the median, lower bound of the top tertile, lower bound of the top quartile, or lower bound of the top quintile of ctDNA fractions measured in a population of subjects that have been diagnosed with mCRPC.

[0056] In some embodiments, the present disclosure provides a method of treating a subject having a cancer, determining whether a subject having a cancer is likely to respond to a therapy, predicting whether a subject having a cancer is likely to respond to a therapy, and / or selecting a therapy for a subject having a cancer. In some embodiments, a method comprises measuring AR activity using technologies and / or methods described herein.

[0057] Among other things, the present disclosure describes a method of treating a subject having a cancer, the method comprising:(i) obtaining a biological sample comprising cell-free DNA (cfDNA), optionally a liquid biopsy sample, from the subject;(ii) measuring AR activity in the biological sample, wherein AR activity is measured using a method of the present disclosure; and(iii) administering an antiandrogen to the subject if AR activity is greater than or equal to an AR activity reference value and not administering an antiandrogen to the subject if the AR activity is less than the AR activity reference value.

[0058] In some embodiments, an AR activity reference value is an AR activity score measured using a method described herein in:(a) a healthy subject or population of healthy subjects;(b) a subject or population of subjects having a cancer previously shown to respond to treatment with the antiandrogen; or(c) a subject or population of subjects having a cancer previously shown to not respond to treatment with an antiandrogen (e.g., an AR antagonist).

[0059] In some embodiments, an AR activity reference value is an AR activity score is that is at least about 1.2, 1.4, 1.6, 1.8, 2, 2.5, 3, 4, or 5 times higher than an AR activity score measured in a healthy subject or population of healthy subjects or a subject or population of subjects that have been diagnosed with cancer that does not have elevated AR expression.Page 11 of 13113185130vlAttorney Docket: 2014191-0045

[0060] In some embodiments, a method of treating a subject having a cancer comprises:(i) obtaining a biological sample comprising cell-free DNA (cfDNA), optionally a liquid biopsy sample, from a subject;(ii) measuring AR activity in the biological sample; and(iii) administering a PSMA-targeted agent to the subject if the measured AR activity is less than an AR activity reference value and not administering a PSMA- targeted agent to the subject if measured AR activity is equal to or greater than the AR activity reference value.

[0061] In some embodiments, an AR activity reference value is an AR activity score measured in:(i) a healthy subject or population of healthy subjects;(ii) a subject or population of subjects having prostate cancer (e g., mCRPC); or (iii) a subject or population of subjects having a cancer previously shown to respond to treatment with the PSMA-targeted agent; or(iv) a subject or population of subjects having a cancer previously shown to not respond to treatment with the PSMA-targeted agent.

[0062] In some embodiments, a method of treating a subject having a cancer of the present disclosure comprises measuring ctDNA fraction in the biological sample. In some embodiments, (i) a PSMA-targeted agent is administered to a subject if AR activity measured in a sample is less than an AR activity reference value and a ctDNA fraction measured in a sample is greater than or equal to a ctDNA fraction reference value, and (ii) a PSMA-targeted agent is not administered to a subject if AR activity measured in the sample is equal to or greater than an AR activity reference value or a ctDNA fraction is less than a ctDNA fraction reference value.

[0063] In some embodiments, a subject has previously been diagnosed with cancer.

[0064] In some embodiments, a cancer is prostate cancer, breast cancer, laryngeal carcinoma, bladder cancer, or hepatocellular carcinoma (HCC), optionally wherein the cancer is prostate adenocarcinoma (PRAD) or AR+ breast cancer. In some embodiments, a cancer is an AR+ cancer. As used herein, “AR+ cancer” refers to a cancer that expresses AR.

[0065] In some embodiments, PRAD is metastatic castration resistant prostate cancer (mCRPC).Page 12 of 13113185130vlAttorney Docket: 2014191-0045

[0066] In some embodiments, AR expression status is determined using an immunohistochemistry (IHC) assay.

[0067] Among other things, the present disclosure describes a method of monitoring cancer (e.g., AR-positive cancer) in a subject, comprising measuring AR activity of a cancer using a method described herein at a first and a second time point. In some embodiments, a subject has been administered an antiandrogen therapy prior to the first time point or after the first time point and before the second time point.

[0068] Among other things the present disclosure describes a method of monitoring cancer (e.g., AR-positive cancer) in a subject comprising measuring AR activity of the cancer using a method described herein at a first and a second time point, or a subject has been administered a PSMA-targeted agent prior to or at the first time point or after the first time point and before the second time point. In some embodiments, a subject has been administered an antiandrogen therapy prior to the first time point or after the first time point and before the second time point.

[0069] Among other things, the present disclosure describes a method of determining the likelihood that a subject will respond to treatment with an antiandrogen, comprising measuring AR activity of the cancer using a method described herein at a first and a second time point. In some embodiments, a subject was administered androgen depletion therapy (ADT) before the first time point or between the first time point and the second time point. In some embodiments, a subject has an increased likelihood of responding to treatment with an antiandrogen therapy if AR activity increases between a first and a second time point. In some embodiments, a subject has been administered an antiandrogen therapy prior to a first time point or after a first time point and before a second time point. In some embodiments, a subject is less likely to respond to continued treatment with an antiandrogen therapy if AR activity increases or stays approximately the same between the first time point and the second time point.

[0070] Among other things, the present disclosure describes a method of determining the likelihood that a subject will respond to treatment with a PSMA-targeted agent, comprising measuring AR activity of the cancer at a first and a second time point. In some embodiments, a subject was administered a PSMA-targeted agent before or at the first time point or between the first time point and the second time point. In some embodiments, a subject has a decreasedPage 13 of 13113185130vlAttorney Docket: 2014191-0045likelihood of responding to treatment with a PSMA-targeted agent if AR activity increases between the first and the second time point. In some embodiments, a subject has an increased likelihood of responding to continued treatment with the PSMA-targeted agent if AR activity decreases or stays approximately the same between the first time point and the second time point.

[0071] In some embodiments, a subject was administered a PSMA-targeted agent at or before the first time point or between the first time point and the second time point. In some embodiments, a subject was administered a PSMA-targeted agent at or before the first time point or between the first time point and the second time point, and (i) if AR activity increases between the first and the second time point, the method does not comprise administering a PSMA-targeted agent at or subsequent to the second time point; and (ii) if AR activity decreases or stays the same between the first and the second time point, the method comprises administering a PSMA targeted agent at or subsequent to the second time point.

[0072] In some embodiments, a PSMA-targeted agent is administered to a subject based on the change in AR activity between the first time point and the second time point, optionally wherein the type, dose, and / or frequency of administration of the PSMA-targeted agent is adjusted based on the change in AR activity. In some embodiments, a PSMA-targeted agent is administered after the second time point if AR activity decreases or stays the same between the first time point and the second time point, optionally wherein a dose of a PSMA-targeted agent administered after the second time point is the same as a dose administered before the second time point. In some embodiments, if AR activity increases between the first time point and the second time point, the amount of PSMA-targeted agent administered to a subject is increased, a non-PSMA-targeted agent is administered to a subject, and / or a different cancer therapy is administered to a subject (e.g., a therapy that does not comprise administering a PSMA-targeted agent to a subject).

[0073] In some embodiments, a PSMA-targeted agent is not administered if AR activity increases between the first time point and the second time point.

[0074] In some embodiments, a PSMA-targeted agent is a radioligand (e g., 177Lu-PSMA-617).

[0075] Among other things, the present disclosure describes a method of treating a cancer in a subject, the method comprising measuring AR activity of the cancer using a methodPage 14 of 13113185130vlAttorney Docket: 2014191-0045described herein at a first and a second time point. In some embodiments, a subject was administered ADT before a first time point or between a first time point and a second time point. In some embodiments,(i) if AR activity increases between a first and a second time point, an antiandrogen therapy is administered to a subject; and(ii) if AR activity decreases or stays the same between a first and a second time point, an antiandrogen is not administered to a subject.

[0076] In some embodiments, a subject was administered an antiandrogen therapy prior to a first time point or after a first time point and before a second time point.

[0077] In some embodiments, an antiandrogen therapy is administered to a subject based on the change in AR activity between a first time point and a second time point, optionally wherein the type, dose, and / or frequency of administration of the antiandrogen therapy is adjusted based on the change in AR activity.

[0078] In some embodiments, an antiandrogen therapy is administered after a second time point if AR activity decreases between a first time point and the second time point, optionally wherein the dose of the antiandrogen therapy administered after the second time point is the same as the dose administered before the second time point.

[0079] In some embodiments, if AR activity in a subject increases or stays approximately the same between a first time point and a second time point, a method comprises(i) increasing the amount of antiandrogen administered to the subject,(ii) administering a different antiandrogen to the subject, and / or(iii) administering a different cancer therapy to the subject (e.g., a therapy that does not comprise administering an AR-targeted therapy to the subject).

[0080] In some embodiments, an antiandrogen therapy is not administered to a subject if AR activity increases between a first time point and a second time point.

[0081] In some embodiments, a cancer is prostate cancer, breast cancer, laryngeal carcinoma, bladder cancer, or hepatocellular carcinoma (HCC) optionally wherein the cancer is prostate adenocarcinoma (PRAD) or AR+ breast cancer.

[0082] In some embodiments, prostate cancer is de novo mCSPC, non-metastatic CRPC, mCRPC in chemo naive patients, or mCRPC which has progressed after chemotherapy.Page 15 of 13113185130vlAttorney Docket: 2014191-0045

[0083] In some embodiments, AR expression status is determined using an IHC assay.

[0084] In some embodiments, an antiandrogen is an AR antagonist (e.g., a molecule that binds the LBD of AR, including e.g., enzalutamide), an androgen synthesis inhibitor (e.g., abiraterone) or an AR degrader (e.g., an AR-targeted PROTAC), optionally wherein the androgen synthesis inhibitor is co-administered with a corticosteroid (e.g., prednisone).

[0085] Among other things, the present disclosure describes a method for testing the activity of a test compound, comprising incubating the test compound with a cell line, and measuring AR activity in the cell line subsequent to incubating the test compound with the cell line, wherein the AR activity is measured using a method described herein.

[0086] In some embodiments, a cell line has measurable AR activity (e.g., the cell line has been incubated with a composition that increases AR signaling activity prior to incubating with the test compound). In some embodiments, an activity is AR-targeting activity or PSMA-targeting activity.

[0087] Among other things, the present disclosure describes a method of screening a library of test compounds for AR-targeting activity, comprising testing the activity of each test compound using a method described herein.

[0088] Among other things, the present disclosure describes a method of screening a library of test compounds for PSMA-targeting activity, comprising testing the activity of each test compound using a method described herein.

[0089] Among other things, the present disclosure describes a compound identified by a method described herein.

[0090] Among other things, the present disclosure describes a kit comprising reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from those listed in Table 1.

[0091] In some embodiments, a kit comprises reagents for quantifying H3K27ac modifications for at least 1, 5, 10, 20, 30, 40, or 50 genomic loci listed in Table 1.

[0092] In some embodiments, a kit comprises one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K27ac-modified histones.

[0093] In some embodiments, a kit comprises reagents for isolation of cell-free DNAPage 16 of 13113185130vlAttorney Docket: 2014191-0045(cfDNA) from a liquid biopsy sample.

[0094] In some embodiments, a kit comprises reagents for library preparation for DNA sequencing.

[0095] In some embodiments, a kit comprises reagents for DNA sequencing.

[0096] Among other things, the present disclosure describes a non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method described herein.

[0097] Among other things, the present disclosure describes a computer system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform a method described herein.

[0098] Among other things, the present disclosure describes a system for quantifying AR activity of a cancer in a subject, the system comprising a sequencer configured to generate a sequencing dataset from a sample; and a non-transitory computer readable storage medium described herein and / or a computer system described herein.

[0099] In some embodiments, a sequencer is configured to generate a Whole Genome Sequencing (WGS) dataset from the sample.

[0100] In some embodiments, a system comprises a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample.

[0101] In some embodiments, a system comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.

[0102] In some embodiments, one or more genomic loci are selected from those listed Table 1.

[0103] In some embodiments, a device comprises reagents for quantifying H3K27ac modifications, e.g., reagents for quantifying H3K27ac modifications for at least 1, 5, 10, 20, 30, or 40 of the genomic loci listed in Table 1.

[0104] In some embodiments, a system comprises reagents for use in ChlP-seq, whereinPage 17 of 13113185130vlAttorney Docket: 2014191-0045the reagents include antibodies, and optionally wherein the one or more antibodies specifically bind H3K27ac-modified histones.

[0105] In some embodiments, a device comprises reagents for isolation of cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.

[0106] In some embodiments, a device comprises reagents for library preparation for sequencing.

[0107] In some embodiments, a sequencer comprises reagents for sequencing.BRIEF DESCRIPTION OF THE DRAWING

[0108] Fig. 1. Exemplary H3K27ac signal at different genomic loci. Shown is H3K27ac signal measured near the KLK3 gene. H3K27ac signal is shown for different in silico plasma (ISP) samples, generated by diluting tissue sequencing data with healthy plasma data to achieve the indicated simulated ctDNA% (0.5%, 5%, 10%, and 50%). Shown below is total H3K27ac signal (H3K27ac), H3K27ac signal at sites that are correlated with ERG expression (+ ERG agnostic), sites that overlap with an AR site (+AR overlap), and sites that are correlated with AR activity and that are robust in plasma samples (+ AR-cor & robust in plasma).

[0109] Fig. 2: Correlation between mean number of H3K27ac fragments for a sample and ctDNA fraction in in silico plasma (ISP) samples. Each line corresponds to a genomic loci identified using tissue samples. As shown, the mean number of H3K27ac fragments increases as ctDNA fraction increases.

[0110] Fig. 3. Correcting for ctDNA. The sum of H3K27ac fragments across genomic loci was divided by ctDNA fraction for each sample and plotted vs. ctDNA fraction. Results are shown pre- (left plot) and post- (right plot) refinement. As shown, dividing the sum of H3K27ac signal at each loci by estimated ctDNA fraction can over-amplify AR activity scores at low ctDNA fractions. Refining the ISP scores so as to remove the ctDNA relationship with mean and SD can address this over amplification.

[0111] Fig. 4. AR activity score performance. Shown is the relationship between calculated AR activity score (computed by dividing the mean number of H3K27ac fragments by ctDNA fraction) and ctDNA fraction.

[0112] Fig. 5. AR activity score performance breakdown by ctDNA fraction inPage 18 of 13113185130vlAttorney Docket: 2014191-0045patient in silico plasma (ISP) samples. AR activity scores were calculated at the indicated ctDNA fraction and compared to AR activity scores calculated at the highest ctDNA fraction (10% or greater). Each point corresponds to a different patient sample.

[0113] Fig. 6. AR Activity Score in NSCLC, NEPC, and PRAD. AR activity scores were determined for NSCLC, NEPC, and PRAD samples. Values are shown as a Z-score, based on PRAD samples (0 = average activity in PRAD samples, 1 = one standard deviation above average, etc.) As shown, measured AR activity scores are highest in PRAD samples, which have elevated AR activity as compared to NSCLC and NEPC, demonstrating that methods provided herein measure AR activity. AR activity score generators created using methods used herein also, in some embodiments, incorporate loci associated with regulatory regions of genes that are known to be associated with aberrant AR activity (exemplary genes shown) further demonstrating that AR activity score generators provided herein measure AR activity.

[0114] Fig. 7. Lower AR activity is prognostic for response to177Lu-PSMA-617 (PLUVICTO®). Fig. 7A provides a box and whiskers plot, showing AR pathway activity scores measured using plasma samples obtained from patients with NSCLC and NEPC (not associated with elevated AR activity) and PRAD before treatment with177Lu-PSMA-617 (PLUVICTO® cohort (mCRPC)). Fig. 7B shows a comparison of clinico-radiologic progression-free survival (CR PFS), which is clinical or radiological evidence of treatment response over time between patients having a high pathway activity score (“AR-high”, left curve, top quartile) and a low pathway activity score (“AR-low”, right curve, bottom three quartiles). Hazard ratio (HR) values represent relative risk of an AR-high group compared to an AR-low group.DETAILED DESCRIPTION

[0115] The present disclosure is based, at least in part, on the demonstration that the AR activity of a cancer in a subject can be determined by detecting and quantifying the presence of histone modifications at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from the subject. The present disclosure also encompasses methods where chromatin accessibility and / or binding of one or more transcription factors are detected at the one or more genomic loci instead of (or in addition to) histone modifications and / or DNA methylation. These new assays provide minimallyPage 19 of 13113185130vlAttorney Docket: 2014191-0045invasive ways of quantifying AR activity that are more accurate, objective, and comprehensive than current tissue-based approaches. No liquid biopsy platform to date has been able to provide actionable resolution on a transcriptionally regulated phenotype relevant for therapy such as AR activity.AR activity and cancer

[0116] AR is a member of the nuclear receptor superfamily and has a similar structure to estrogen receptor, progesterone receptor, glucocorticoid receptor, and thyroid hormone receptor. The AR gene is located on chromosome X (Xql 1-12) and consists of 8 exons coding an about 11 kDa protein. AR has four regions: an NH2 terminal transactivation domain (NTD) encoded by exon 1, a DNA-binding domain (DBD) encoded by exons 2-3, a hinge region encoded by exon 4, and a ligand binding domain (LBD) encoded by exons 5-6. The NTD has glutamine repeats (CAG repeats), which can vary between individuals (most men have 19-25 repeats). Shorter glutamine repeats are associated with high transcriptional activity of AR. Men with shorter glutamine repeats have a higher risk of prostate cancer. In contrast, patients with Kennedy disease have long CAG repeats (>40), which results in low AR-transcriptional activity with gynecomastia, erectile dysfunction, testicular atrophy, and muscular atrophy. Testosterone and dihydrotestosterone (DHT) bind to the LBD, followed by the conformational change of AR.

[0117] After ligand binding in the cytoplasm, AR translocates into the nucleus, forms a dimer, and binds to the androgen-response element of the promoter and the enhancer of targeted genes through the zinc-finger of the DBD. The NTD includes the transcriptional regulatory region, activation function-1 (AF-1), and the LBD includes activation function-2 (AF-2). Upon DNA binding, the AR dimer forms a complex with coactivator and coregulatory proteins at the AF-1 and AF-2 regions. These proteins include SRC1, SRC2, SRC3, p300 / CBP, and AEA54, among many others. AR regulates the gene expressions with diverse functions located downstream of the androgen-response element, including secreted proteins (KLK3, KLK2), fusion genes (TMPRSS2-ERG), growth stimulators (IGF1R, APP), PI3K modulation (FKBP5), transcription factors (NKX3.1, FOXP1), metabolic enzyme (CAMKK2), cell cycle regulators (UBE2C, TACC2), and glucuronidation (UGT1A1).

[0118] AR can also repress transcription. The formation of an activation complexPage 20 of 13113185130vlAttorney Docket: 2014191-0045involves AR, coactivators, and RNA polymerase IT recruitment to both the enhancer and promoter, whereas the formation of a repression complex involves factors bound only at the promoter and not at the enhancer.

[0119] Androgens are synthesized in the testis and adrenal glands. Testosterone is synthesized by Leydig cells in the testis, and androstenedione and dehydroepiandrosterone (DHEA) are synthesized in the adrenal glands. Most testosterone in the serum binds to sex hormone binding globulin, whereas the remaining 1% to 2% exists as free testosterone, which is converted into DHT by 5 -alpha-reductase in the prostate cells. DHT has a high binding affinity to AR. Adrenal androgen is converted to testosterone by 17-beta-hydroxysteroid dehydrogenase in the cytoplasm of prostate cells. In normal prostate tissue, immunohistochemical analysis shows strong AR staining in luminal cells, fibromuscular stromal cells, and endothelial cells but weak staining in basal cells. Epithelial AR acts to supply secretory proteins to the prostate gland, such as prostate-specific antigen (PSA). Stromal AR plays a role in prostate growth.

[0120] AR has been found to be expressed in almost all primary and metastatic prostate cancers, regardless of stage or grade (see, e.g., Aurilio, Gaetano, et al. "Androgen receptor signaling pathway in prostate cancer: from genetics to clinical applications." Cells 9.12 (2020): 2653). AR has also been found to be expressed in a subset of additional cancers, including breast cancer, adrenocortical carcinoma, astrocytoma, basal cell carcinoma, bladder cancer, cervical invasive carcinoma, colon adenocarcinoma, desmoid tumors, esophageal carcinoma, gastric carcinoma, glioblastoma, head and neck carcinoma, juvenile nasopharyngeal fibroma, melanoma, meningioma, non-small cell lung cancer, ovarian cancer, pancreatic adenocarcinoma, peritoneal adenocarcinoma, peritoneal mesothelioma, testicular germ cell, thymic carcinoma, thyomoma, rectal adenocarcinoma, renal cancer, salivary gland, sarcoma, uterine sarcoma, and uterine carcinoma, among others. See, e.g., Munoz, Javier, Jennifer J. Wheler, and Razelle Kurzrock. "Androgen receptors beyond prostate cancer: an old marker as a newtarget." Oncotarget 6.2 (2014): 592, the contents of which are incorporated by reference herein in their entirety. In some embodiments, an AR+ cancer is prostate cancer (e.g., PRAD and / or mCRPC). In some embodiments, an AR+ cancer is breast cancer. In some embodiments, an AR+ cancer can be non-responsive to treatment with hormone therapy. In some embodiments, an AR+ cancer can be responsive to treatment with hormone therapy.Page 21 of 13113185130vlAttorney Docket: 2014191-0045

[0121] In some embodiments, a cancer described herein is non-responsive to treatment with androgen deprivation therapy (ADT). In some embodiments, prostate cancer (e.g., PRAD or mCRPC) is responsive to ADT. In some embodiments, prostate cancer (e.g., PRAD and / or mCRPC) is not responsive to ADT.Prostate Cancer

[0122] AR plays an important role in prostate cancer, in particular, in castration-resistant prostate cancer (CRPC). In prostate cancer, AR is involved in PSA synthesis, regulation of lipid metabolism, promotion of growth, and several other functions.

[0123] Androgen deprivation therapy (ADT), also called androgen ablation therapy or androgen suppression therapy, is an antihormone therapy whose main use is in treating prostate cancer. Prostate cancer cells usually require androgen hormones, such as testosterone, to grow. ADT reduces the levels of androgen hormones, with drugs or surgery, to prevent prostate cancer cells from growing. ADT approaches include antiandrogens and chemical castration.

[0124] ADT therapy can also eliminate cancer cells by inducing androgen deprivation-induced senescence. Lowering androgen levels or preventing androgen from entering prostate cancer cells often makes prostate cancer shrink or grow more slowly for a time. However, ADT treatment should be combined with radiation therapy (RT), as ADT by itself does not eradicate cancer but simply decreases its aggressiveness.

[0125] Several studies have concluded that ADT benefits patients with metastatic disease, and as an adjunct to radiation therapy in patients with locally advanced disease, as well as those with unfavorable intermediate-risk or high-risk localized disease. However, in patients with low-risk prostate cancer, ADT has demonstrated no survival advantage, and significant harm, such as impotence, diabetes and bone loss.

[0126] Orchiectomy (surgical castration) consists of removing the testicles, the organ where androgens are synthesized, of a cancer patient. It is the easiest, least expensive, and most radical treatment for ending the production of androgens.

[0127] The synthesis of testosterone is mediated by a chain of processes that start in the brain. When the body detects a low level of testosterone, the hypothalamus starts to produce LHRH. LHRH activates the synthesis of LH (Luteinizing hormone) within the pituitary gland. LH induces testosterone synthesis within the testicles. Two different medicines, LHRH agonistsPage 22 of 13113185130vlAttorney Docket: 2014191-0045and antagonists, can be used to lower the amount of testosterone made by the testicles (also called “chemical castration”). They work by inhibiting the formation of LH in the pituitary gland. LHRH agonists produce a sudden increase in levels of testosterone followed by a huge falling, a process called flare. LHRH antagonists directly decrease the amount of testosterone. Examples of LHRH agonists and antagonists that can be used in androgen deprivation therapy include leuprorelin (leuprolide), goserelin, triptorelin, histrelin, buserelin, and degarelix. These drugs can be injected under the skin in order to achieve the same result as surgical castration.

[0128] Androgen deprivation therapy can suppress most prostate cancers, but some high-risk prostate cancers gradually progress to CRPC, which can grow under castrated levels of androgen. AR is the most frequently aberrant gene in metastatic CRPC (62.7%) (see Robinson, Dan, et al. "Integrative clinical genomics of advanced prostate cancer." Cell 161.5 (2015): 1215-1228, the contents of which are incorporated by reference herein in their entirety). Several mechanisms underlie the development of CRPC, including acquisition of AR point mutations, AR overexpression, changes in androgen biosynthesis, AR splice variants that are constitutively active without ligand binding, and changes in androgen cofactors. Studies of AR in CRPC have found that AR can still be active in CRPC, and therefore remains a potential target for treating CRPC.Breast Cancer

[0129] In the search for the development of alternative targeted therapies for breast cancer, it was discovered that AR can be detected in the majority of breast cancer types. Elevated levels of tissue concentrations of DHT were measured in breast carcinoma and ductal carcinoma in situ (DCIS) and were in the range of 110-698 pg / g tissue and 140-1593 pg / g, respectively. These concentrations of DHT are comparable to those measured in recurrent prostate cancer and are at levels that can transactivate the AR. Due to the AR’s long known importance to prostate cancer, a vast number of clinically approved therapeutic options that target AR have been developed, thereby facilitating a rapid translation of any of these treatments for clinical testing in breast cancer patients.

[0130] AR expression has been reported in the range of 53% to 99% of all breast cancer and 20-40% of TNBC, depending on the study cohort and sensitivity of detection methods. In breast cancer cells, AR may have roles in either cell proliferation (stimulatory effect) orPage 23 of 13113185130vlAttorney Docket: 2014191-0045anti proliferation (inhibitory effect), depending on the level of ERa expression and disease stages. In recent years, targeting AR in patients with TNBC has been of increasing interest in translational research and clinical trials. This interest is based on a poorer clinical outcome for TNBCs that express AR and the observation that targeting AR can reduce the growth of some subtypes of TNBC. AR antagonists, such as bicalutamide and enzalutamide, or the ablation of androgen production have been investigated in clinical trials for breast cancer (see Rahim, Bilal, and Ruth O’Regan. "AR signaling in breast cancer." Cancers 9.3 (2017): 21; and Traina, Tiffany A., et al. "Enzalutamide for the treatment of androgen receptor-expressing triple-negative breast cancer." Journal of clinical oncology 36.9 (2018): 884-890, the contents of each of which are incorporated by reference herein in their entirety).

[0131] AR-V7 mRNA has been detected in about 50% of primary breast cancer samples, with the highest expression in HER2-amplified subjects (see Hickey, Theresa E., et al."Expression of androgen receptor splice variants in clinical breast cancers." Oncotarget 6.42 (2015): 44728 (Hickey et al.)). In addition to AR-V7, the transcripts of other variants such as AR-V3, AR-V9, AR-V13, AR-V15, and AR-V18 have also been detected in primary breast cancer. Another study measured AR-V7 in about 10% of clinical samples that included primary, recurrent, and metastatic cancers (see Ferguson, Donna C., et al. "Androgen receptor splice variant-7 in breast cancer: clinical and pathologic correlations." Modern Pathology 35.3 (2022): 396-402). In Hickey et al., approximately 15% of the HER2-amplified subgroup expressed AR-V7, whereas about 18% of the TNBC subgroup expressed AR-V7. Notably, about 40% of the AR-V7-positive breast cancer samples showed an apocrine morphology.

[0132] AR variants can be detected in circulating tumor cells (CTCs). In one study, CTCs that tested positive for AR-V7 were detected in 41% of patients (9 out of 22) whose breast cancers were positive for Era (see Aceto, Nicola, et al. "AR expression in breast cancer CTCs associates with bone metastases." Molecular Cancer Research 16.4 (2018): 720-727). Another study investigating CTCs in TNBC revealed that 27% of patients tested positive for full-length AR, with AR-V7 co-expressed in 73% of these patients (Kasimir-Bauer, Sabine, et al."Circulating tumor cells expressing the prostate specific membrane antigen (PSMA) indicate worse outcome in primary, non-metastatic triple-negative breast cancer." Frontiers in oncology 10 (2020): 1658). Interestingly, patients with CTCs that expressed AR-V7 and prostate-specificPage 24 of 13113185130vlAttorney Docket: 2014191-0045membrane antigen may have worse outcomes after chemotherapy.

[0133] The prognostic value of AR in ERa-positive breast cancer has been demonstrated in many studies. In primary breast cancer tissue, the expression of AR was detected in about 73% of samples from 413 cases analyzed and was associated with expression of ERa, lower histologic grade, and smaller tumor size, thereby suggesting that the AR is a positive prognostic marker in this group of breast cancer (Park, S., et al. "Expression of androgen receptors in primary breast cancer." Annals of oncology 21.3 (2010): 488-492). Meta-analyses of published data have been employed to assess the prognostic significance of the expression of AR in breast cancer. In ERa-positive breast cancer, AR positivity (expression at both mRNA and protein levels) was significantly correlated with improved disease-free survival (DFS) and overall survival (OS), and tumors with an AR:ERot positivity ratio of >0.87 had the best outcome. This correlation between AR positivity and DFS or OS was not found in ERa-negative breast cancer (see Ricciardelli, Carmela, et al. "The magnitude of androgen receptor positivity in breast cancer is critical for reliable prediction of disease outcome." Clinical Cancer Research 24.10 (2018): 2328-2341; Bozovic-Spasojevic, Ivana, et al. "The prognostic role of androgen receptor in patients with early-stage breast cancer: a meta-analysis of clinical and gene expression data." Clinical Cancer Research 23.11 (2017): 2702-2712)). A study by Tagliaferri et al. indicated that a high expression of AR is a favorable prognostic indicator of clinical outcome for early-stage ERa-positive / PR-negative / HER2 -negative breast cancer (Tagliaferri, Barbara, et al. "Role of androgen receptor expression in early stage ER+ / PgR- / HER2-breast cancer." Therapeutic Advances in Medical Oncology 12 (2020): 1758835920958355). Their study found that ERa-positive breast cancer patients with less than 80% AR expression had a higher risk of relapse than patients with more than 80% AR expression, and these patients with higher AR expression had lower nuclear grade and lower proliferative properties e.g., lower Ki-67) measured in their tumors.AR expression status in cancer (e.g., breast cancer or prostate cancer) can be determined by immunohistochemistry (IHC) and / or by quantifying mRNA expression.Bladder Cancer

[0134] Bladder cancer is the ninth most common cancer worldwide with a striking sexbased difference in incidence. Emerging evidence indicates that the AR might promote the development, progression and recurrence of bladder cancer, contributing to the observed sexPage 25 of 13113185130vlAttorney Docket: 2014191-0045differences. Targeting androgen-AR signaling has promise as a potential therapy for bladder cancer and can help suppress progression of this disease. AR signaling can mediate resistance to cisplatin-based chemotherapy and BCG immunotherapy, providing a rationale to combine AR-targeted therapy with chemotherapy or BCG in bladder cancer. In addition, the identification of a new membrane AR and AR-regulated non-coding RNAs has important implications for bladder cancer treatment. The success of human clinical trials of targeted- AR therapies will help in the development of improved treatments for patients with bladder cancer.Antiandrogens

[0135] As used herein, “antiandrogens” refers to agents that can prevent androgens from mediating biological effects in a subject and include, e.g., AR antagonists, which can bind to AR and prevent its binding and activation by androgens, androgen synthesis inhibitors, and AR-degraders, which can bind to and promote degradation of AR.Androgen Synthesis Inhibitors

[0136] Abiraterone acetate is an androgen synthesis inhibitor derived from pregnenolone and is an irreversible inhibitor of 17, 20-lyase and 17-alpha hydroxylase, which are products of the CYP17 gene. The drug inhibits production of androgen in the testes, adrenal glands, and tumor cells and can be co-administered with steroid supplementation (e.g., corticosteroid supplementation, including, e.g., prednisone) to overcome secondary cortisol insufficiency and prevent overproduction of ACTH and mineralocorticoids.

[0137] Abiraterone acetate is currently approved by the US Food and Drug Administration (FDA) for use in newly diagnosed metastatic castration-sensitive PCa (CSPC) and metastatic CRPC.

[0138] Seviteronel (developmental codes VT-464 and INO-464) is an experimental cancer medication under development by Viamet Pharmaceuticals and Innocrin Pharmaceuticals for the treatment of prostate cancer and breast cancer. It is a nonsteroidal CYP17A1 inhibitor and works by inhibiting production of androgens and estrogens. As of July 2017, seviteronel is in phase II clinical trials for both prostate cancer and breast cancer. In January 2016, it was designated fast-track status by the United States Food and Drug Administration for prostate cancer. In April 2017, seviteronel received fast-track designation for breast cancer as well.Page 26 of 13113185130vlAttorney Docket: 2014191-0045AR Binding Agents

[0139] To date, AR agonist dihydroxytestosterone (DHT) (see Marks, L.S. “5alpha-reductase: history and clinical importance.” Rev. Urol. 6 Suppl 9 (2004): SI 1-21, and Purushottamachar, Puranik, and Vincent CO Njar. "A new simple and high-yield synthesis of 5a-dihydrotestosterone (DHT), a potent androgen receptor agonist." Steroids 77.14 (2012): 1530-1534, the contents of both of which are incorporated by reference herein in their entirety) and six AR antagonists have been approved by U.S. Food and Drug Administration (FDA) (see Clegg, Nicola J., et al. "ARN-509: a novel antiandrogen for prostate cancer treatment." Cancer research 72.6 (2012): 1494-1503; Munuganti, Ravi Shashi Nayana, et al. "Targeting the binding function 3 (BF3) site of the androgen receptor through virtual screening. 2. Development of 2-((2-phenoxyethyl) thio)-l H-benzimidazole derivatives." Journal of medicinal chemistry 56.3 (2013): 1136-1148; Guo, Chuangxing, et al. "Discovery of aryloxy tetramethylcyclobutanes as novel androgen receptor antagonists." Journal of medicinal chemistry 54.21 (2011): 7693-7704; Payen, Olivier, et al. "Synthesis and structure-activity relationships of the first ferrocenyl-aryl-hydantoin derivatives of the nonsteroidal antiandrogen nilutamide." Journal of medicinal chemistry 51.6 (2008): 1791-1799; Oksala, Riikka, et al. "Discovery and development of ODM-204: a novel nonsteroidal compound for the treatment of castration-resistant prostate cancer by blocking the androgen receptor and inhibiting CYP17A1." The Journal of steroid biochemistry and molecular biology 192 (2019): 105115, the contents of each of which are incorporated by reference herein in their entirety).

[0140] Androgen receptor antagonists (ARAs) block the androgen binding site of the AR and prevent nuclear translocation. This results in attenuation of coactivator mobilization leading to cellular apoptosis and decreased prostate tumor volume. Among them, flutamide, hydroxylflutamide, nilutamide, and bicalutamide are considered as the first-generation AR inhibitors. These four drugs demonstrated modest AR binding affinity and limited antagonist effects compounding with undesired agonist effects. Enzalutamide, apalutamide, ODM201, AZD3514, and BMS641988 are classified as second-generation AR inhibitors. These compounds afford moderate to good AR binding affinities and completely eradicate agonist effects. Among them, AR antagonists, such as enzalutamide and apalutamide, have had a major impact on thePage 27 of 13113185130vlAttorney Docket: 2014191-0045treatment of mCRPC (Tian, Xiaohong, Yang He, and Jinming Zhou. "Progress in antiandrogen design targeting hormone binding pocket to circumvent mutation based resistance." Frontiers in pharmacology 6 (2015): 57; and Sternberg, Cora N., et al. "Enzalutamide and survival in nonmetastatic, castration-resistant prostate cancer." New England Journal of Medicine 382.23 (2020): 2197-2206, the contents of each of which are incorporated by reference herein in their entirety.

[0141] Enzalutamide is a second generation nonsteroidal antiandrogen that has an increased affinity for LBD as compared to first generation nonsteroidal antiandrogen. The AFFIRM trials showed that enzalutamide prolonged the overall survival of patients with metastatic CRPC after chemotherapy by 4.8 months (see Scher H.I., et al. “Increased survival with enzalutamide in prostate cancer after chemotherapy.” N Engl J Med 367 (2012): 1187-97, the contents of which are incorporated by reference herein). Enzalutamide has been shown to be effective in patients with CRPC before and after taxane-based chemotherapy. Enzalutamide has been approved for use in treating de novo mCSPC, non-metastatic CRPC, mCRPC in chemo naive patients, and mCRPC which progressed after chemotherapy.

[0142] Apalutamide (ARN-509) is a nonsteroidal antiandrogen with greater efficacy than enzalutamide. Apalutamide binds to the LBD and in contrast to bicalutamide, lacks agonist activity. Apalutamide inhibits nuclear localization and DNA binding of AR in prostate cancer cells (see Clegg N.J., et al. “ARN-509: a novel antiandrogen for prostate cancer treatment.” Cancer Res 72 (2012): 1494-503, the contents of which are incorporated by reference herein in their entirety). The SPARTAN study showed that metastasis-free survival was significantly lengthened by 24.3 months with apalutamide versus placebo in patients with non-metastatic CRPC (see Smith M.R., et al. “Apalutamide treatment and metastasis-free survival in prostate cancer.” N Engl J Med 378 (2018): 1408-18, the contents of which are incorporated by reference herein in their entirety). The US Food and Drug Administration approved apalutamide for patients with non-metastatic CRPC in February 2018.

[0143] Because abiraterone and apalutamide act by different mechanisms, clinical trials are evaluating whether the combination provides superior outcomes. Preliminary reports from abiraterone plus apalutamide combination trial showed improved PSA response while resultsPage 28 of 13113185130vlAttorney Docket: 2014191-0045from abiraterone plus enzalutamide trial showed no improvement in OS and increased side effects.

[0144] Darolutamide (ODM-201) is a nonsteroidal antiandrogen that inhibits androgen binding to AR. Darolutamide antagonizes both overexpressed and mutated ARs (F876L), which confers resistance to enzalutamide and apalutamide. It can also inhibit AR transcriptional activity with point mutations of F877L, H875Y / T878A, F877L / T878A, and T878G (see Borgmann H., et al. “Moving towards precision urologic oncology: targeting enzalutamide-resistant prostate cancer and mutated forms of the androgen receptor using the novel inhibitor darolutamide (ODM-201).” Eur Urol 73 (2018): 4-8, the contents of which are incorporated by reference herein in their entirety). Darolutamide showed negligible penetrance of the brain-blood barrier (see Moilanen A.M., et al. “Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies.” Sci Rep 5 (2015): 12007, the contents of which are incorporated by reference herein in their entirety). A phase 1 study showed that darolutamide was well tolerated in patients with CRPC (see MatsubaraN., et al. “Phase 1 study of darolutamide (ODM-201): a new-generation androgen receptor antagonist, in Japanese patients with metastatic castration-resistant prostate cancer.” Cancer Chemother Pharmacol 80 (2017): 1063-72, the contents of which are incorporated by reference herein in their entirety). Darolutamide underwent a phase 3 trial (ARAMIS) in men with high-risk non-metastatic CRPC (NCT02200614).

[0145] Galeterone is an antiandrogen that inhibits CYP17, antagonizes the AR, and reduces AR expression in prostate cancer cells by causing an increase in the degradation of both full-length AR and ARV7 (see Kwegyir-Afful A.K., et al. “Galeterone and VNPT55 induce proteasomal degradation of AR / AR-V7, induce significant apoptosis via cytochrome c release and suppress growth of castration resistant prostate cancer xenografts in vivo.” Oncotarget 6 (2015): 27440-60, the contents of which are incorporated by reference herein in their entirety). Galeterone is also effective for prostate cancer cells with the T878A AR mutation (see Yu Z., et al. “Galeterone prevents androgen receptor binding to chromatin and enhances degradation of mutant androgen receptor.” Clin Cancer Res 20 (2014): 4075-85, the contents of which are incorporated by reference herein in their entirety). Open-label phase I and II studies (Androgen Receptor Modulation Optimized for Response- 1 [ARM0R1] and ARM0R2 part 1) showed thatPage 29 of 13113185130vlAttorney Docket: 2014191-0045a more than 30% decline in PSA was achieved by galeterone in 49% of patients with treatment-naive non-metastatic or metastatic CRPC (see Montgomery B., et al. “Androgen receptor modulation optimized for response (ARMOR) phase I and II studies: galeterone for the treatment of castration-resistant prostate cancer.” Clin Cancer Res 22 (2016): 1356-63. A study of galeterone compared to enzalutamide in men expressing AR-V7 metastatic CRPC (ARM0R3-SV) was conducted (NCT02438007).

[0146] EPI-506 is a prodrug of EPI-002 (ralaniten) that can inhibit the growth of prostate cancer in vitro and in vivo, has results in aberrant AR activity, including overexpression of coactivators, AR gain-of-function mutations, and constitutively active AR-V7 (see Yang Y.C., et al. “Targeting androgen receptor activation function-1 with EPI to overcome resistance mechanisms in castration resistant prostate cancer.” Clin Cancer Res22 (2016): 4466-77, the contents of which are incorporated by reference herein in their entirety). A phase I / II clinical trial of EPI-506 for patients with metastatic CRPC who are resistant to abiraterone and / or enzalutamide was conducted (NCT02606123).AR-Targeted PROTACs

[0147] Target knockdown by induced protein degradation via the ubiquitin proteasome system (UPS) has aroused great interest. On the basis of this approach, molecules named proteolysis-targeting chimeras (PROTACs) or specific and nongenetic inhibitor of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) have been developed. PROTACs are bifunctional molecules that hijack the UPS to achieve degradation of a disease-related target protein. PROTACs are chimeric molecules, comprising an E3 ligand and a target ligand that are conjugated to form one molecule. To date, PROTAC technology can be utilized to target various proteins, including, e.g., transcription factors, skeleton proteins, nuclear receptors, enzymes and regulatory proteins. The advantages of PROTAC technology over traditional small molecules include targeting undruggable targets, overcoming drug resistance, improving targeting selectivity, reducing toxicity, among others.

[0148] The earliest AR degraders were polypeptide-based molecules. Later AR degraders used the mouse double minute 2 (MDM2) E3 ligase ligand, inhibitor of apoptosis protein (IAP), cereblon (CRBN) or von Hippel-Lindau (VEIL) ligands (see W. Zhu et al., “Daam2 driven degradation of VHL promotes gliomagenesis,” Elife 6 (2017), e31926; C. Maniaci, et al.,Page 30 of 13113185130vlAttorney Docket: 2014191-0045“Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation,” Nat. Commun. 8 (1) (2017) 830; T.V. Nguyen, et al., “p97 / VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates,” Proc. Natl. Acad. Sci. USA 114 (14) (2017) 3565-3571; K. Okuhira et al., “Targeted degradation of proteins localized in subcellular compartments by hybrid small molecules,” Mol. Pharm. 91 (3) (2017) 159-166; and N. Shibata et al., “Development of protein degradation inducers of oncogenic BCR-ABL protein by conjugation of ABL kinase inhibitors and IAP ligands,” Cancer Sci. 108 (8) (2017) 1657-1666, the contents of each of which is incorporated by reference herein in their entirety. Further PROTACs that target AR are described in Jia, Xiaojuan, and Xin Han. "Targeting androgen receptor degradation with PROTACs from bench to bedside." Biomedicine & Pharmacotherapy 158 (2023): 114112, the contents of which are incorporated by reference herein in their entirety.

[0149] The first clinically evaluated PROTAC protein degrader is ARV- 110, an orally bioavailable PROTAC AR degrader that was developed by Arvinas, Inc. (see Neklesa, Taavi, et al. "ARV-110: An oral androgen receptor PROTAC degrader for prostate cancer." J. Clin. Oncol 37.259 (2019): 10-1200; Neklesa, Taavi, et al. "ARV-110: An androgen receptor PROTAC degrader for prostate cancer." Cancer research 78.13_Supplement (2018): 5236-5236; and Mullard, Asher. "First targeted protein degrader hits the clinic." Nature reviews. Drug discovery (2019), the contents of each of which incorporated by reference herein in their entirety). ARV-110 was shown to completely degrade AR in all tested cell lines (LNCaP, VCaP, MCF7, et al.) with DC50 value of < 1 nM in vitro and has shown good efficacy in vivo systems as well as in enzalutamide-resistant prostate cancer. ARV-110 has been administered to patients with mCRPC in Phase I and Phase II clinical trials.

[0150] In addition to ARV- 110, there are several other AR degraders that are in or have entered clinical stage, including ARV-766 from Arvinas, CC-94676 from BMS / Celgene, AC-0176 from Accutar Biotech, a compound from Oncopia / Roivant (Proteovant), HP518 from Hinova and GT20029 from Kintor.PSMA Expression Level and Disease

[0151] Prostate-specific membrane antigen (PSMA), also known as hydrolase 1Page 31 of 13113185130vlAttorney Docket: 2014191-0045(FOLHl is a transmembrane glutamate carboxypeptidase that is highly expressed on prostate cancer cells. It consists of a large extracellular domain, a small transmembrane domain, and a cytoplasmic tail. High PSMA expression is a biomarker of poor prognosis throughout the course of prostate cancer and across anatomical sites. Metastatic lesions are PSMA-positive in most patients that have metastatic castration-resistant prostate cancer, and high PSMA expression has been independently associated with reduced survival.

[0152] A PSMA PET scan is a nuclear medicine imaging technique that can be used in the diagnosis and staging of prostate cancer. It is carried out by injecting a radiopharmaceutical with a positron or gamma emitting radionuclide and a prostate-specific membrane antigen (PSMA) targeting ligand. After injection, imaging of positron emitters such as gallium-68 (68Ga), copper-64 (64Cu), and fluorine- 18 (18F) is carried out with a positron emission tomography (PET) scanner. For gamma emitters such as technetium-99m (99mTc) and indium- 111 (inIn) singlephoton emission computed tomography (SPECT) imaging is performed with a gamma camera.

[0153] As well as the diagnosis and staging of prostate cancer, PSMA imaging can also be used to assess suitability for and plan treatment with external beam radiotherapy and PSMA-targeted therapeutics (e.g., PSMA-targeted radionuclides).PSMA Targeted Therapeutics

[0154] PSMA can be highly expressed on the surface of prostate cancer cells but has low normal-tissue expression. As such, PSMA offers a useful target for selectively targeting prostate cancer cells. A common approach in designing therapeutics for use in treating prostate cancer (including, e.g., mCRPC) has been to link moieties that can bind to or associate with PSMA in a subject to moieties that can kill cells.

[0155] PSMA targeting therapies such as radionuclide therapies (e.g., lutetium- 177 (177LU)-PSMA-617 which is also called lutetium (177Lu) vipivotide tetraxetan or PLUVICTO®) can target prostate cancer cells while sparing most normal tissues in patients who have been selected with the use of imaging to confirm radionuclide binding.177Lu-PSMA-617 delivers beta-particle radiation selectively to PSMA-positive cells and the surrounding microenvironment. This radioligand therapy has been associated with encouraging biochemical and radiographic response rates, reduced pain, and low toxicity in multiple early-phase studiesPage 32 of 13113185130vlAttorney Docket: 2014191-0045involving patients with progression of metastatic castration -resistant prostate cancer after standard therapy.

[0156] Currently, PSMA expression in tumors is commonly assessed using PSMA-targeted positron emission tomography (PET), which has gained increased acceptance in diagnosing prostate cancer due to its superior accuracy in identifying metastases as compared to CT and MRI methods. Patient eligibility for177LuPSMA-617 currently requires PSMA PET imaging.PSMA Targeted Radionuclide Therapeutics

[0157] Moi eties that can bind to or associate with PSMA in a subject can be labeled with different radionuclides for therapeutic purposes. PSMA targeted radionuclides typically consist of a PSMA-binding domain, a linker, and a chelator labeled with various radionuclides. A PSMA-binding domain can be, e.g., a small molecule domain or an antibody moiety.

[0158] PSMA- targeting small molecule domains are divided into 3 types — urea-based, phosphorus-based, and thiol-based — with urea-based compounds commonly used due to their superior PSMA binding affinity. Changing linker or chelator structure can influence PSMA binding efficacy and pharmacokinetics. In addition, adding an albumin-binding domain, which effectively increases the agent’s size, has been explored to increase circulation time within the tumor vasculature and reduce healthy-organ circulation time, with the goal of mitigating on-target, off-tumor toxicities.

[0159] On accumulation of a PSMA-targeted radionuclide at a tumor site, radioactive decay of a- or fl-emitting radionuclides induces DNA strand breaks and causes cell death, a-radiation reaches a shorter range (40-100 pm) than fl-particles (50-12,000 pm) but has a linear energy transfer significantly higher than that of fl-particles (5-9 vs. 0.1-2.2 MeV), a-emitting radionuclides therefore lead to several ionizing events, resulting in DNA double-strand breaks (DSBs) in a short range. Both PSMA-targeted a-emitting and PSMA-targeted fl-emitting radionuclides are currently in clinical development.

[0160] A summary of exemplary PSMA targeted radionuclides currently in development is provided, e.g., in Sandhu et al. “Radionuclide therapy in prostate cancer: from standalone to combination PSMA theranostics.” Journal of Nuclear Medicine 62.12 (2021): 1660-1668, the contents of which are incorporated by reference herein in their entirety. Provided below is a shortPage 33 of 13113185130vlAttorney Docket: 2014191-0045list of exemplary PSMA targeted radionuclides.

[0161] 177Lu-PSMA-617 is a177Lu-conjugated small-molecule peptide that delivers beta-particle radiation to PSMA-positive cells and surrounding microenvironment. This radioligand therapy has been associated with encouraging biochemical and radiographic response rates, reduced pain, and low toxicity in multiple early-phase studies involving patients with progression of metastatic castration-resistant prostate cancer after standard therapy.177Lu-PSMA-617 is currently the most developed PSMA-targeted radioligand therapy, with Phase 3 outcome data.

[0162] The antitumor activity of a-emitting radioligand was first demonstrated by223Ra-dichloride, an a-emitting radionuclide therapy (RNT) that binds areas of increased bone turnover.223Ra-di chloride showed an overall survival (OS) benefit and a reduced time to the first symptomatic skeletal event in patients with mCRPC involving bone. Several a-emitting PSMA-targeted radionuclide therapies (RNTs), including225Ac-J591 (an antibody -based RNT),225Ac-PSMA-617 (a small molecule-based RNT), and227Th-pelgifatamab (BAY2315497 also called pelgifatamab corixetan, an antibody -based RNT) are currently in clinical development.225Ac-J591 is also described, e.g., in Tagawa et al. “Prostate-Specific Membrane Antigen-Targeting Alpha Emitter via Antibody Delivery for Metastatic Castration-Resistant Prostate Cancer: A Phase I Dose-Escalation Study of225Ac-J591.” Journal of Clinical Oncology 42.7 (2024): 842-851. The high-energy, short-range a-emissions enable pinpoint tumor targeting, which has advantages in patients with marrow infiltration due to the limited crossfire effect on surrounding bone marrow reserve but also has limitations in the setting of heterogeneous cellular PSMA expression within tumor deposits.

[0163] Antibody-based radioligands have pharmacokinetics different from those of small molecules and have been shown to have less uptake in glandular tissue and kidneys but may also have less tumor uptake. In a Phase 1 clinical trial, 22 men (55% had previously received177Lu-PSMA-617) received a single dose of225AC-J591 across 7 dose levels (13.3-93.3 kBq / kg). One patient receiving 80 kBq / kg had dose-limiting toxicities, with grade 4 thrombocytopenia and anemia in the context of prior treatment with177Lu-PSMA-617. Thirty-five percent of patients to date have had a PSA decline of more than 50%, and although PSMA uptake was not a selectionPage 34 of 13113185130vlAttorney Docket: 2014191-0045criterion, most patients had PSMA uptake with an SUVmax greater than that seen in the liver. This trial has recently begun the Phase 2a expansion.

[0164] Retrospective case series of patients treated with225Ac-PSMA-617 showed antitumor activity in 60-70%, including in some patients who had progressed on177Lu-PSMA-617. Xerostomia and weight loss were clinically significant. A recent study evaluating225Ac-PSMA-617 in mCRPC patients who had progressed on abiraterone or enzalutamide, taxane-based chemotherapy, and177Lu-PSMA-617 and demonstrated PSMA-ligand uptake on imaging reported a PSA decline of at least 50% in 17 of 26 (65%) patients. Median OS was 7.7 months. Grade 3 or 4 myelosuppression was seen in 35% of patients, grade 1 or 2 renal impairment in 19%, and grade 1 or 2 xerostomia in 100%.

[0165] Given their distinct properties and emerging evidence of antitumor activity when a- and P-emitting PSMA-targeted therapies are given sequentially, rational combination of these radioisotopes may serve a complementary role when delivered concurrently or sequentially.

[0166] Combination therapies: Purported acquired resistance mechanisms to PSMA-targeted radionuclides include heterogeneity or loss of PSMA expression or a failure to deliver a sustained lethal dose to the target. Potential strategies to improve PSMA-targeted therapies include combining PSMA-targeted therapies with agents that upregulate PSMA expression, increase tumor radiosensitivity, target different PSMA-binding sites, or exhibit complementary antitumor effects. To this end, several potential combinations are being explored in ongoing clinical studies. These include the combination of PSMA-targeting RNT with AR-targeted agents, DNA repair inhibitors, immunotherapies, chemotherapy, or a combination of different PSMA-targeting RNTs.PSMA-Targeting Antibody Drug Conjugates (ADC)

[0167] PSMA is a well-established target in the therapeutic field of prostate cancer, with a number of PSMA-directed ADC treatments in development.

[0168] Exemplary PSMA-direct ADCs include:• PSMA ADC: a fully human immunoglobulin G1 anti-PSMA monoclonal antibody conjugated to monomethylauri statin E, and which is described in, e g., Petrylak et al. “PSMA ADC monotherapy in patients with progressive metastatic castration-resistantPage 35 of 13113185130vlAttorney Docket: 2014191-0045prostate cancer following abiraterone and / or enzalutamide: Efficacy and safety in openlabel single-arm phase 2 study.” The Prostate 80.1 (2020): 99-108.• MLN2704: the PSMA-targeted monoclonal antibody MLN591 conjugated to maytansinoid-1 (an antimicrotubule agent drug). Described, e.g., in Gaisky et al. “Phase I trial of the prostate-specific membrane antigen-directed immunoconjugate MLN2704 in patients with progressive metastatic castration-resistant prostate cancer.” Journal of clinical oncology 26.13 (2008): 2147-2154.• MEDI3726: an engineered version of an anti-PSMA IgGlK antibody (J591 ), site- specifically conjugated with pyrrolobenzodiazepine (PBD) dimers (SG3199). Described, e.g., in de Bono et al. “Phase I study of MEDI3726: a prostate-specific membrane antigen-targeted antibody-drug conjugate, in patients with mCRPC after failure of abiraterone or enzalutamide.” Clinical Cancer Research 27.13 (2021): 3602-3609.• MLN2704: humanized monoclonal antibody MLN591 linked to the maytansinoid DM1.Described, e.g., in Milowsky et al. “Phase 1 / 2 multiple ascending dose trial of the prostate-specific membrane antigen-targeted antibody drug conjugate MLN2704 in metastatic castration -resistant prostate cancer.” Urologic Oncology: Seminars and Original Investigations. Vol. 34. No. 12. Elsevier, 2016.• ARX517: a humanized immunoglobulin G1 kappa (IgGlk) monoclonal antibody site- specifically conjugated to two of the microtubule-disrupting toxin amberstatin (AS269). Described, e.g., in Skidmore et al. “Preclinical characterization of ARX517, a nextgeneration anti-PSMA antibody drug conjugate for the treatment of metastatic castrationresistant prostate cancer.” Cancer Research 83.7_Supplement (2023): 3997-3997.• MLN2704: a humanized monoclonal antibody MLN591 targeting prostate-specific membrane antigen, linked to the maytansinoid DM1. Described, e.g., in Milowsky et al. “Phase 1 / 2 multiple ascending dose trial of the prostate-specific membrane antigen- targeted antibody drug conjugate MLN2704 in metastatic castration-resistant prostate cancer.” Urologic Oncology: Seminars and Original Investigations. Vol. 34. No. 12. Elsevier, 2016.PSMA-Targeting CAR-T Cell Therapy

[0169] CAR-T cell therapies have also been investigated for treatment of prostate cancer.Page 36 of 13113185130vlAttorney Docket: 2014191-0045A first-in -hum an Phase I study of PSMA CAR-T (NCT03089203) enrolled mCRPC patients and treated them with a PSMA-directed armored CAR-T cell. See Narayan et al., “PSMA- targeting TGFP-insensitive armored CAR T cells in metastatic castration-resistant prostate cancer: a phase 1 trial.” Nature Medicine 28.4 (2022): 724-734.Bi-Specific T-Cell Engagers (BiTEs)

[0170] PSMA targeted BiTEs can also be used to treat prostate cancer. One example is pasotuxizumab, which engages CD3 on T-cells and PSMA on prostate cancer cells. A Phase I trial enrolled 47 patients with mCRPC post >1 taxane and either abiraterone or enzalutamide, and escalated doses of pasotuxizumab in subcutaneous (SC) and continuous intravenous infusion (cIV) routes. See Hummel, Horst-Dieter, et al. “Pasotuxizumab, aBiTE® immune therapy for castration-resistant prostate cancer: Phase I, dose-escalation study findings.” Immunotherapy 13.2 (2021): 125-141.Subjects and Samples

[0171] A sample analyzed using methods, kits and systems provided herein can be any biological sample including any processed sample that includes circulating tumor DNA (ctDNA) derived from a biological sample. In various embodiments, a sample analyzed using methods, kits and systems provided herein can be a sample obtained from a mammalian subject. In various embodiments, a sample analyzed using methods, kits and systems provided herein can be a sample obtained from a human subject.

[0172] In various instances, a human subject is a subject diagnosed or seeking diagnosis as having, diagnosed as, or seeking diagnosis as at risk of having, and / or diagnosed as or seeking diagnosis as at immediate risk of having, an AR-positive cancer, e.g., AR-positive prostate or breast cancer, etc. In various instances, a human subject is a subject identified as needing AR expression status screening. In certain instances, a human subject is a subject identified as needing AR expression status screening by a medical practitioner.

[0173] The subject may not have undergone previous treatments for cancer, such as the treatments recited in this disclosure. In other embodiments, the subject has undergone previous treatments for cancer, such as the treatments recited in this disclosure.

[0174] In various embodiments a subject has one or more biomarkers and / or risk factorsPage 37 of 13113185130vlAttorney Docket: 2014191-0045for cancer, e.g., AR-positive cancer, e.g., AR-positive breast cancer, etc. In certain embodiments, a human subject is identified as in need of AR activity screening based on an initial cancer diagnosis, e.g., a breast cancer, etc. diagnosis. In various instances, a human subject is a subject not yet diagnosed as having, not at risk of having, not at immediate risk of having, not diagnosed as having, and / or not seeking diagnosis for a cancer. Genetic factors may also contribute to AR-positive cancer risk, as evidenced by individuals with a family history of AR-positive cancer.

[0175] In various embodiments, a sample from a subject, e.g., a human can be obtained from a liquid biopsy. In certain embodiments, a sample and / or reference is obtained from serum, plasma, or urine. In certain embodiments, the sample is serum. In certain embodiments, a sample comprises circulating tumor DNA (ctDNA). In certain embodiments, a sample is derived from about 1 mb of blood obtained from the subject. In certain embodiments, a sample is derived from about 0.5-2 mb of blood obtained from the subject, e.g., about 0.5 to 1.75 mb, about 0.5 to 1.5 mb, about 0.75 to 1.25 mb or about 0.9 to 1.1 mb of blood.

[0176] In various embodiments, a sample is a sample of cell-free DNA (cfDNA). cfDNA is typically found in human biofluids (e.g., plasma, serum, or urine) in short, double-stranded fragments. The concentration of cfDNA is typically low, but can significantly increase under particular conditions, including without limitation pregnancy, autoimmune disorders, myocardial infarction, and cancer. Circulating tumor DNA (ctDNA) is the component of cell-free DNA specifically derived from cancer cells. ctDNA can be present in human biofluids bound to leukocytes and erythrocytes or not bound to leukocytes and erythrocytes. Various tests for detection of tumor-derived ctDNA are based on detection of genetic or epigenetic modifications that are characteristic of cancer (e.g., of a relevant cancer). Genetic or epigenetic factors characteristic of cancer can include, without limitation, oncogenic or cancer-associated mutations in tumor-suppressor genes, activated oncogenes, chromosomal disorders, histone modifications (e.g., histone methylation and / or histone acetylation), chromatin accessibility, binding of one or more transcription factors and / or DNA methylation.

[0177] In various embodiments, ctDNA comprises less than 30%, less than 20%, or less than 10% of the cfDNA in the liquid biopsy sample obtained from the subject, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or less than 1% of the cfDNA in the sample. In various embodiments, ctDNA comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of thePage 38 of 13113185130vlAttorney Docket: 2014191-0045cfDNA in the liquid biopsy sample obtained from the subject. Tn some embodiments, the percentage of ctDNA in the liquid biopsy sample is assessed using ichorCNA which estimates the percentage of ctDNA in a sample probabilistically (see Adalsteinsson et al., Nat Commun (2017) 8(1): 1324 the entire contents of which are incorporated herein by reference).

[0178] cfDNA and ctDNA can provide a real-time or nearly real time metric of status of a source tissue. cfDNA and ctDNA demonstrate a half-life in blood of about 2 hours, such that a sample taken at a given time provides a relatively timely reflection of the status of a source tissue.

[0179] Various methods of isolating nucleic acids from a sample (e.g, of isolating cfDNA from blood or plasma) are known in the art. Nucleic acids can be isolated using, without limitation, standard DNA purification techniques, by direct gene capture (e.g., by clarification of a sample to remove assay-inhibiting agents and capturing a target nucleic acid, if present, from the clarified sample with a capture agent to produce a capture complex and isolating the capture complex to recover the target nucleic acid).

[0180] Reagents and protocols for obtaining and analyzing cfDNA and ctDNA, such as circulating in blood or other tissue, are commercially available as described in the Examples and well-known in the art (see, for example, Anker et al., Cancer and Metastasis Rev (1999) 18:65-73; Wua et al., Clin Chim Acta (2002) 321:77-87; Fiegl et al., Cancer Res (2005) 15:1141-1145; Pathak et al., Clin Chem (2006) 52:1833-1842; Schwarzenbach et al., Clin Cancer Res (2009) 15:1032-1038; Schwarzenbach et al., Nat Rev Cancer (2011) 11:426-437) the contents of each of which is separately incorporated herein by reference in their entirety).

[0181] In various embodiments, samples can be collected from individuals repeatedly over a period of time (e.g., once daily, weekly, monthly, annually, biannually, etc.). In various embodiments, such samples can be used to verify results from earlier detections and / or to identify an alteration in biological pattern because of, for example, disease progression, resistance to therapy, treatment, remission, and the like. For example, subject samples can be taken and monitored every month, every two months, or combinations of one, two, or three-month intervals according to the present disclosure. In various embodiments, samples can be collected for monitoring over time beginning at or at certain clinically determined stages, such as at resistance to a therapy, before radiographic progression, after radiographic progression, and / orPage 39 of 13113185130vlAttorney Docket: 2014191-0045at tissue biopsy. In addition, AR activity obtained at different points in time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject’s own values, as an internal, or personal, control for long-term monitoring.

[0182] Samples include materials prepared by processes including, without limitation, steps such as concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives, addition of calibrants, addition of protease inhibitors, addition of denaturants, desalting, concentration and / or extraction of sample nucleic acids, and / or amplification of sample nucleic acids (e.g., by PCR or other nucleic acid amplification techniques). Samples also include materials prepared by techniques that isolate, e.g., nucleosomes or transcription factors and / or nucleic acids associated with nucleosomes or transcription factors.

[0183] Removal from a sample of proteins that are not desirable for a relevant purpose or context (e.g., high abundance, uninformative, or undetectable proteins) can be achieved using high affinity reagents, high molecular weight filters, ultracentrifugation and / or electrodialysis. High affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to high abundance proteins. Sample preparation can also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques. Molecular weight filters include membranes that separate molecules based on size and molecular weight. Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration and microfiltration. Ultracentrifugation is the centrifugation of a sample at about 15,000-60,000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles.Electrodialysis is a procedure which uses an electromembrane or semipermeable membrane in a process in which ions are transported through semi-permeable membranes from one solution to another under the influence of a potential gradient. Since the membranes used in electrodialysis may have the ability to selectively transport ions having positive or negative charge, reject ions of the opposite charge, or to allow species to migrate through a semipermeable membrane based on size and charge, it renders electrodialysis useful for concentration, removal, or separation of electrolytes.Page 40 of 13113185130vlAttorney Docket: 2014191-0045

[0184] Separation and purification in the present disclosure may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography e.g., in capillary, column or on a chip). Electrophoresis is a method that can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip. Examples of gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof. A gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient. Examples of capillaries used for electrophoresis include capillaries that interface with an electrospray.

[0185] Capillary electrophoresis (CE) is preferred for separating complex hydrophilic molecules and highly charged solutes. CE technology can also be implemented on microfluidic chips. Depending on the types of capillary and buffers used, CE can be further segmented into separation techniques such as capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP) and capillary electrochromatography (CEC). An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and / or base and an organic such as an alcohol or acetonitrile.

[0186] Capillary isotachophoresis (CITP) is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities. Capillary zone electrophoresis (CZE), also known as free-solution CE (FSCE), is based on differences in the electrophoretic mobility of the analytes, determined by the charge on the analytes, and the frictional resistance the analytes encounter during migration, which is often directly proportional to the size of the analytes. Capillary isoelectric focusing (CIEF) allows weakly-ionizable amphoteric molecules, to be separated by electrophoresis in a pH gradient. CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.

[0187] Separation and purification techniques used in the present disclosure can include any chromatography procedures known in the art. Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobilePage 41 of 13113185130vlAttorney Docket: 2014191-0045and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), etc.

[0188] In some embodiments, whole blood is collected from a subject, and a plasma layer is separated by centrifugation. cfDNA may be then extracted from the plasma using methods known in the art.Histone Modifications, Chromatin Accessibility and Transcription Factor Binding

[0189] Histone methylation is understood to increase or decrease expression of associated coding sequences, depending on which histone residue is methylated. Histone methylation is an essential modification that can cause monomethylation (met), dimethylation (me2), and trimethylation (me3) of several amino acids, thus directly affecting heterochromatin formation, gene imprinting, X chromosome inactivation, and gene transcriptional regulation. Histone methyltransferases promote monomethylation, dimethylation, or trimethylation of histones while histone demethylases promote demethylation of histones. In general, lysine (Lys or K), arginine (Arg or R), and rarely histidine (His or H) are the most common histone methyl acceptors. Histone methylation only occurs at specific lysine and arginine sites of histone H3 and H4. In histone H3, lysine 4, 9, 26, 27, 36, 56, and 79 and arginine 2, 8, and 17 can be methylated. By comparison, histone H4 has fewer methylation sites, in which only lysine 5, 12, and 20 and arginine 3 can be methylated. Histone methylation is often associated with transcriptional activation or inhibition of downstream genes. The methylation of histone H3K4, R8, R17, K26, K36, K79, H4R3, and K 12 can activate gene transcription. However, the methylation of histone H3K9, K27, K56, H4K5, and K20 can inhibit gene transcription. Thus, for example, H3K4 methylation generally activates gene expression, while H3K27 methylation generally represses gene expression.

[0190] Histone acetylation occurs predominantly at lysine residues and is generally understood to increase expression of associated coding sequences. Without wishing to be bound by any theory, acetylation of lysine residues is thought to neutralize lysine’s positive charge and thereby cause histones to drift away from DNA, which has a negative charge. The released structure facilitates access to transcriptional machinery such as transcription factors and RNAPage 42 of 13113185130vlAttorney Docket: 2014191-0045polymerase II. Histone acetylation and deacetylation are generally catalyzed by histone acetyltransferases (HATs) and HDACs, respectively. Acetyl-CoA can be a source and co-factor of acetylation. In regulatory regions, HATs can acetylate histones and recruit HAT-containing complexes to activate the transcriptional process. For instance, H3K9ac and H3K27ac levels can be associated with promoter and enhancer activities. Furthermore, H3K27ac enhances not only the kinetics of transcriptional activation, but also accelerates the transition of RNA polymerase II from the initiation state to the elongation state.

[0191] Differential modification of a genomic locus (e.g., differential histone methylation and / or differential histone acetylation) can refer to, or be determined by or detected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state. Those of skill in the art will appreciate that a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.

[0192] Chromatin accessibility can refer to the degree to which nuclear macromolecules are able to physically contact DNA and is determined in part by the occupancy and modification status of nucleosomes. Modified histones can regulate chromatin accessibility through a variety of mechanisms, such as altering transcription factor (TF) binding through steric hindrance and modulating nucleosome affinity for active chromatin remodelers. The topological organization of nucleosomes across the genome is non-uniform: while histones can be densely arranged within facultative and constitutive heterochromatin, histones can be depleted at regulatory loci, including within enhancers, insulators and transcribed gene bodies. Active regulatory elements of the genome are generally accessible.

[0193] Differential accessibility of a genomic locus can refer to, or be determined by or detected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state. Those of skill in the art will appreciate that a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.

[0194] A reference can be a value or set of values that are predetermined or derived fromPage 43 of 13113185130vlAttorney Docket: 2014191-0045a sample or set of samples. A reference can be a sample or set of samples. A reference value can be a predetermined threshold value, a value that varies in accordance with circumstances e.g., according to patient subpopulation, age, weight, or other variables), or a ratio. Reference ratios can be ratios relating to the modification and / or accessibility of multiple loci within individual samples and / or references, or across or between samples and / or references. In various embodiments, a reference can have or represent a normal, non-diseased state. In some embodiments, such as for staging of disease or for evaluating the efficacy of treatment, a reference can have or represent a diseased state, e.g., a cancer, stage of cancer, or subtype of cancer, e.g, AR-positive cancer or AR-negative cancer. In some embodiments, a reference can correspond to a subject having breast cancer and / or a breast cancer subtype, e.g., AR-positive or AR-negative breast cancer.

[0195] In certain instances, a reference is a non-contemporaneous sample from the same source, e.g., a prior sample from the same source, e.g., from the same subject. In certain instances, a reference for the modification status of one or more genomic loci (e.g, one or more differentially modified genomic loci) can be the modification status of the one or more genomic loci (e.g., one or more differentially modified genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g, AR activity level). In certain instances, a reference for the accessibility status of one or more genomic loci (e.g., one or more differentially accessible genomic loci) can be the accessibility status of the one or more genomic loci (e.g, one or more differentially accessible genomic loci) in a sample (e.g, a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., an AR-positive cancer or AR-negative cancer).

[0196] In some illustrative but non-limiting embodiments of the present disclosure differential modification or differential accessibility can refer to a differential (e.g, between a sample and a reference) with an absolute log2(f old-change) that is greater than or equal to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or more, or any range in between, inclusive, e.g., as measured according to an assay provided herein.

[0197] Enhancers are genomic loci that can be differentially modified or differentially accessible in and / or between conditions, diseases, and other states. Enhancers are cis-acting DNA regulatory regions that are thought to bind trans-acting proteins that contribute toPage 44 of 13113185130vlAttorney Docket: 2014191-0045expression patterns of associated genes. Chromatin TmmunoPrecipitation sequencing (ChlP-seq) of histone modifications (e.g., acetylation) have identified millions of enhancers in mammalian genomes. The number of active enhancers in any given cell type is estimated to be in the tens of thousands. Certain transcription factors (TFs), sometimes referred to as “master” transcription factors, associate with active enhancers with important impacts on gene expression and cell function. Certain such transcription factors preferentially associate with enhancers that regulate genes required for establishing cell identity and function, including enhancer domains known as “super-enhancers”. Moreover, master TFs can participate in inter-connected auto-regulatory circuitries or “cliques” that are self-reinforcing, show marked cell selectivity, and function to maintain cell state and / or cell survival.Techniques for Detecting and Quantifying Histone Modifications and Transcription Factor Binding

[0198] Various techniques of molecular biology are well known in the art and / or disclosed in the present application for detecting and quantifying histone modifications and / or transcription factor binding. In some embodiments, the methods, kits and systems of present disclosure involve the detection and quantification of histone modifications and / or transcription factor binding in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA. Chromatin ImmunoPrecipitation (ChIP) is one technique of molecular biology useful in detecting and quantifying histone modifications and transcription factor binding in samples. CUT&RUN or CUT&Tag are other more recent techniques that can also be used to detect and quantify histone modifications and transcription factor binding sites. ChIP -chip, ChlP-exo, ChIP Re-ChIP, and ChlPmentation are other alternative techniques that could be used.

[0199] ChIP can involve various steps including one or more of fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. ChIP has become a very widely used tissue-based technique for determining the in vivo location of binding sites of various transcription factors and histones. Because the proteins are captured at the sites of their binding with DNA, ChIP helps to detect DNA-protein interactions that take place in living cells. More importantly, ChIP can be coupled to many commonly used molecular biology techniquesPage 45 of 13113185130vlAttorney Docket: 2014191-0045such as PCR and real-time PCR, PCR with single-stranded conformational polymorphism, Southern blot analysis, Western blot analysis, cloning, and microarray. The resulting versatility has increased the potential of this technique.

[0200] ChIP of tissue samples usually involves cross-linking of the chromatin-bound proteins by formaldehyde, followed by sonication or nuclease treatment to obtain small DNA fragments. Immunoprecipitation can be then carried out using specific antibodies to the DNA-binding protein of interest. The DNA can be then released from the proteins and analyzed using various methods. ChIP has also been used to study RNA-protein interactions. X-ChIP methods utilize fixed chromatin fragmented by sonication, while the N-ChIP methods utilize native chromatin, which can be unfixed and nuclease digested.

[0201] The first step of the technique can be the cross-linking of DNA and proteins. Formaldehyde is one of the most used cross-linking agents. One advantage of using formaldehyde can be the ease of reversibility of the cross-links and its ability to form bonds that span approximately 2 angstroms. This means that formaldehyde can bind molecules in close association with each other. Generally, formaldehyde can be added to the medium in the cell culture flask or plate. It enters the cells through the cell membrane and cross-links the proteins to the chromatin. Formaldehyde fixation of tumor tissues has also been done. Other cross-linking agents that have been used include chemicals such as methylene blue and acridine orange, cisplatin, dimethylarsinic acid, potassium chromate, and ultraviolet (UV) light and lasers.

[0202] Harvested chromatin can be sonicated in one or more sonication cycles. DNA can be typically broken into to 100-500 bp fragments to pinpoint the location of the DNA sequence of interest. An alternative to sonication can be nuclease digestion of the chromatin, e.g., in N-ChlP methods. Purification of chromatin can be achieved using a cesium chloride (CsCl) gradient centrifugation.

[0203] Chromatin can be immunoprecipitated using one or more antibodies that bind a target epitope. For example, an antibody used in ChIP can selectively bind a particular transcription factor or one or more particular histone modifications, such as one or more particular histone acetylation modifications or histone methylation modifications. In some embodiments, an antibody used to bind a target epitope can be a “pan” antibody (e.g., a panacetylation antibody, a pan-methylation antibody, an antibody that binds a group of histonePage 46 of 13113185130vlAttorney Docket: 2014191-0045modifications associated with increased transcription activation, and / or an antibody that binds a group of histone modifications associated with increased transcription repression). The antibody against the protein of interest is allowed to bind to the protein-DNA complex, and the complex can be then precipitated. Immunosorbants commonly used to separate the antigen-antibody complex from the lysate include salmon sperm DNA-protein A-Sepharose®, protein G, magnetic beads, and other engineered immunoprecipitation systems known to those of skill in the art.

[0204] Immunoprecipitated DNA can be eluted. Once the DNA of interest is isolated, many detection and quantification methods can be used to study the isolated gene fragments. Commonly utilized methods include PCR, real-time PCR, slot blot hybridization, microarray techniques, and deep or next-generation sequencing. ChlP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. ChlP-seq can be used to map DNA-binding proteins, e.g, transcription factor binding sites and histone modifications in a genome-wide manner.

[0205] Cell-free Chromatin ImmunoPrecipitation sequencing (cfChlP-seq) involves applying ChlP-seq to samples that include cell-free DNA, e.g., liquid biopsy samples including cfDNA such as plasma samples including cfDNA (e.g., see Sadeh et al., Nat Biotechnol (2021) 39: 586-598 and Jang et al., Life Sci Alliance (2023) 6(12):e202302003 the entire contents of each of which are incorporated herein by reference). In some embodiments, cfChlP-seq uses antibodies or antibody fragments that bind specific histone modifications (e.g., H3K4me3 and / or H3K27ac) and / or transcription factors that are coupled (covalently or non-covalently) to beads, e.g., magnetic beads such as Dynabeads® magnetic beads and incubated with a volume, e.g., about 1 mb of thawed plasma obtained from a subject. Without limitation, exemplary antibodies that bind H3K4me3 include PA5-27029 (available from Thermo Fisher Scientific in Waltham, MA) and Cl 5410003 (available from Diagenode in Denville, NJ) and exemplary antibodies that bind H3K27ac include ab21623 or ab4729 (both available from Abeam in Cambridge, UK) and Cl 5210016 (available from Diagenode in Denville, NJ).

[0206] In some embodiments, the antibodies or antibody fragments can be covalently coupled to beads, e.g., epoxy beads. In some embodiments, the antibodies or antibody fragments can be non-covalently coupled to beads, e.g., Protein A or Protein G beads such as Dynabeads® Protein A or Dynabeads® Protein G beads. After washing, a cfDNA library is then typicallyPage 47 of 13113185130vlAttorney Docket: 2014191-0045prepared from the captured cfDNA. Library preparation can be done on-bead or after releasing the captured cfDNA by digestion of bound histones, e. ., using proteinase K. The cfDNA library is then sequenced to generate reads of captured cfDNA sequences, e.g., by next-generation sequencing (NGS) as is known in the art. The reads are then analyzed, e.g., aligned and counted using standard bioinformatic techniques as is known in the art. A cfChlP-seq bioinformatic pipeline can include, e.g., alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.

[0207] CUT&Tag involves antibody-based binding of a target protein, e.g., transcription factor or histone modification of interest, where antibody incubation is directly followed by the shearing of the chromatin and library preparation (see Kaya-Okur et al., Nat Comm (2019) 10: 1930). CUT&Tag assays take advantage of a Tn5 transposase that is fused with Protein A to direct the enzyme to the antibody bound to its target on chromatin. Tn5 transposase is pre-loaded with sequencing adapters (generating the assembled pA-Tn5 adapter transposome) to carry out antibody-targeted tagmentation. In a typical CUT&Tag assay samples are incubated with an antibody immobilized on Concanavalin A-coated magnetic beads to facilitate subsequent washing steps. Cells can be incubated with a primary antibody specific for the target protein of interest followed by incubation with a secondary antibody. Samples can then be incubated with assembled transposomes, which consist of Protein A fused to the Tn5 transposase enzyme that is conjugated to NGS adapters. After incubation, unbound transposome can be washed away using stringent conditions. Tn5 is a Mg2+-dependent enzyme so Mg2+can be added to activate the reaction, which results in the chromatin being cut close to the protein binding site and simultaneous addition of the NGS adapter DNA sequences. Chromatin cleavage and library preparation can be achieved in one single step.

[0208] CUT&RUN is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing (see Skene and Henikoff, Elife (2017) 6:1-35, Skene et al., Nat Protoc (2018) 13:1006-1019). As only targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has low background levels. In an example CUT&RUN assay, a sample is incubated with an antibody or antibody fragment that binds the target protein, e.g., transcription factor or histone modification of interest. The sample is thenPage 48 of 13113185130vlAttorney Docket: 2014191-0045incubated with Protein-A-MNase after which CaCh can be added to initiate the calcium dependent nuclease activity of MNase to cleave the DNA around the target protein. The protein-A-MNase reaction can be quenched by adding chelating agents (EDTA and EGTA). Cleaved DNA fragments are then liberated, extracted, and used to construct a sequencing library.Techniques for Detecting and Quantifying Chromatin Accessibility

[0209] Various techniques of molecular biology are well known in the art and / or disclosed in the present application for detecting and quantifying chromatin accessibility. In some embodiments, the methods, kits and systems of the present disclosure involve the detection and quantification of chromatin accessibility in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA. ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and DNase hypersensitivity assays are exemplary techniques of molecular biology useful in detecting and quantifying chromatin accessibility in samples. Sono-Seq is another alternative method that could be used (see Auerbach et al., Proc Natl Acad USA (2009) 106(35): 14926-14931). Fragmentomics-based methods are yet another method that can be used to assess chromatin accessibility (see Ding, Spencer C., and YM Dennis Lo. "Cell-free DNA fragmentomics in liquid biopsy." Diagnostics 12.4 (2022): 978).

[0210] DNase hypersensitivity assays can use the non-specific DNA endonuclease Deoxyribonuclease I (DNase I), which selectively digests accessible DNA regions. DNase I hypersensitivity sites (DHS) identified by DNase-seq include open chromatin regulatory regions. A typical DNase hypersensitivity assay can include a first step in which nuclei are isolated from cells using lysis buffer, and nuclei are digested using DNase I. DNA fragment sizes are measured to identify optimal digestion using gel electrophoresis. Biotinylated linkers can be ligated to the ends of digested DNA after polishing to make blunt ends, and the DNA can then be isolated. DNA with biotinylated linker can be digested by restriction endonuclease Mmel and captured by streptavidin coated Dynabeads® to generate short tags to which a second sequencing adaptor can be ligated. A second linker can be ligated and amplified to generate a library for sequencing. APage 49 of 13113185130vlAttorney Docket: 2014191-0045DNase-seq bioinformatic pipeline can include, e.g., alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.

[0211] MNase-seq determines chromatin accessibility with micrococcal nuclease (MNase) that preferentially digests nucleosome-free, protein-unbound DNA. A typical MNase-seq assay can include a first step in which nuclei are isolated from either native or crosslinked chromatin and digested using MNase with titration. In vivo formaldehyde crosslinking step that is designed to capture the interaction between proteins and DNA. This crosslinking allows bound proteins to shield their associated DNA from digestion by MNase. Following crosslinking, samples are digested with MNase, which can be specifically activated by addition of Ca2+ to the buffer. Digestion can be halted by chelating the reaction, at which point the samples are RNase treated, crosslinks are reversed, and proteins are digested away from the chromatin. DNA can then be isolated via a phenol-chloroform extraction. Uncut DNA is purified and mononucleosome bands are isolated and excised through gel electrophoresis. Isolated DNA can be amplified by adding adapters to generate a library, and sequenced. MNase-seq primarily sequences regions of DNA bound by histones or other proteins. Therefore, it indirectly determines which regions of DNA are accessible by directly determining which regions are bound to nucleosomes or proteins.

[0212] FAIRE-seq is a method in which nucleosome-depleted regions of DNA (NDRs) are isolated from chromatin. A typical FAIRE-seq assay can include a first step in which cells are fixed using formaldehyde so that histones are crosslinked to interacting DNA. Crosslinked chromatin can then be sheared by sonication that generates protein-free DNA and protein-crosslinked DNA fragments. Protein-free DNA can be isolated using a phenol-chloroform extraction: DNA crosslinked with protein stays in organic phase, while protein-free DNA stays in aqueous phase. Highly crosslinked DNA remains in the organic phase and the non-crosslinked DNA is pulled to the aqueous phase. Non-crosslinked DNA from the aqueous phase can then be amplified and sequenced. Reads enriched in the sequencing pool tend to have lower nucleosome and transcription factor binding and are therefore inferred to come from accessible regions.

[0213] NOMe-seq is a method to identify nucleosome-depleted regions of DNA (NDRs) with M.CviPI methyltransferase that methylates cytosine in GpC dinucleotides not protected byPage 50 of 13113185130vlAttorney Docket: 2014191-0045nucleosomes or other proteins. Unlike CmpG, GpCmin the human genome does not occur naturally in most cell types. GpCmlevels at open chromatin regions can be compared to background signals and used to detect and quantify NDRs. A typical NOMe-seq protocol can include a step in which samples are treated with M.CviPI and S-adenosylhomocysteine (SAM) to methylate accessible GpC sites. M.CviPI treated DNA can be sheared using a sonicator, so that DNA fragments can be sequenced. DNA is treated with bisulfite, which converts unmethylated cytosine to uracil using sodium bisulfite, while methylated cytosine is unaffected. A library is generated using adapters and sequenced. Accessible chromatin is expected to have high levels of GpCmbut low levels of CmpG. Therefore, NOMe-seq identifies NDRs using the two separate methylation analyses that serve as independent (but opposite) measures, providing matched chromatin designations for each regulatory element.

[0214] ATAC-seq uses hyperactive Tn5 transposase that preferentially cuts accessible chromatin regions and simultaneously inserts adapters to the fragmented region (Buenrostro et al., Nat Methods (2013) 10(12): 1213-1218 the entirety of which is incorporated herein by reference). A typical ATAC-seq assay can include a first step in which samples are incubated with Tn5 transposase. DNA can then be isolated and purified. DNA fragmented and tagged by Tn5 transposase can be purified and then amplified to generate a library and sequenced for analysis.

[0215] “Fragmentomics” or a “fragmentomics assay” refers to methods that use certain size and sequence characteristics of cfDNA to gain insight into the epigenetic state of cells at the time their genomic DNA was released into the extracellular environment. Without wishing to be bound by theory, upon release of genomic DNA from a cell into the extracellular environment, nucleases rapidly cleave the genomic DNA into short fragments. The cleavage pattern and sequences of the fragments reflect the positioning of nucleosomes genome- wide at the point of cell death, and by finding nucleosomes that are consistently genomically positioned across cancer cells (i.e. many of the circulating tumor DNA fragments that map to that small region of the genome have the same start and end positions or similar fragment length characteristics) fragmentomics attempts to infer the location of stably positioned nucleosomes at regulatory sites, and thus to infer where the active regulatory sites are in a given cell type. Accordingly, analysis of cfDNA fragmentation patterns can be used to infer characteristics of the cells at the time theyPage 51 of 13113185130vlAttorney Docket: 2014191-0045released genomic DNA. Examples of metrics commonly measured in fragmentomics include fragment size, preferred ends, end motifs, single-stranded jagged ends, and nucleosomal footprints. Approaches for measuring fragmentomics metrics include, e.g., qPCR, electron microscopy, single molecule sequencing, and next-generation sequencing. A relationship between fragmentomic metrics and histone modifications (h3K4me3 and H3K27ac) has been established. See Bai, Jinyue, et al. "Histone modifications of circulating nucleosomes are associated with changes in cell-free DNA fragmentation patterns." Proceedings of the National Academy of Sciences 121.42 (2024): e2404058121; and Wang, Yadong, etal. "Cell-free epigenomes enhanced fragmentomics-based model for early detection of lung cancer." Clinical and Translational Medicine 15.2 (2025): e70225.Techniques for Detecting and Quantifying DNA Methylation

[0216] Various techniques of molecular biology are well known in the art and / or disclosed in the present application for detecting and quantifying DNA methylation. In some embodiments, the methods, kits and systems of the present disclosure involve the detection and quantification of chromatin accessibility in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA. Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq) are exemplary techniques of molecular biology useful in detecting and quantifying chromatin accessibility in samples. Reduced representation bisulfite sequencing (RRBS) is another alternative method that could be used (see Meissner et al., Nucleic Acids Res (2005) 33(18): 5868-5877). Illumina Infinium arrays could also be used to detect and quantify DNA methylation.

[0217] DNA methylation typically refers to the methylation of the 5’ position of cytosine (mC) by DNA methyltransferases (DNMT). It is a major epigenetic modification in humans and many other species. In mammals, most DNA methylations occur within the context of CpG dinucleotides. DNA methylation is thought to be a repressive chromatin modification. Aberrant methylation can lead to many diseases including cancers (Robertson, Nat Rev Genet (2005) 6:597-610 and Bergman and Cedar, Nat Struct Mol Biol (2013) 20:274-281).

[0218] Bisulfite sequencing (BS-Seq) or Whole-Genome Bisulfite Sequencing (WGBS) is aPage 52 of 13113185130vlAttorney Docket: 2014191-0045well-established protocol to detect methylated cytosines in genomic DNA. In this method, genomic DNA is treated with sodium bisulfite and then sequenced, providing single-base resolution of methylated cytosines in the genome. Upon bisulfite treatment, unmethylated cytosines are deaminated to uracils which, upon sequencing, are converted to thymidines. Simultaneously, methylated cytosines resist deamination and are read as cytosines. The location of the methylated cytosines can then be determined by comparing treated and untreated sequences.

[0219] MeDIP-seq was first reported by Weber et al., Nat Genet (2005) 37:853-862. In a typical MeDIP-seq protocol, antibody or antibody-fragment that binds 5-methylcytidine (5mC) is used to enrich methylated DNA fragments, then these fragments are sequenced and analyzed. If using 5mC-specific antibodies or antibody fragments, methylated DNA is isolated from genomic DNA via immunoprecipitation. Anti-5mC antibodies are incubated with fragmented genomic DNA and precipitated, followed by DNA purification and sequencing.

[0220] Methyl-CpG-Binding Domain sequencing (MBD-seq) is similar to MeDIP-seq except that it uses methyl binding domain (MBD) proteins instead of antibodies or antibody fragments to bind methylated DNA. In a typical MBD-seq protocol, genomic DNA is first sonicated and incubated with tagged MBD proteins that can bind methylated cytosines. The protein-DNA complex is then precipitated with antibody-conjugated beads that are specific to the MBD protein tag, followed by DNA purification and sequencing.Classifiers

[0221] In some embodiments, the present disclosure provides methods for obtaining a classifier, e.g., a classifier that can be used to determine AR expression status. In some embodiments, a subject is determined to have an epigenetic profile indicative of an AR-positive cancer based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject. In some embodiments, a cancer is determined to be AR-positive if an AR activity is detected that is above a threshold value. In some embodiments, the threshold value is a predetermined threshold and / or a normalized value. In some embodiments, the threshold value is an AR activity score determined in a reference population. In some embodiments, the reference population comprises subjects having cancer and previously found to respond to treatment with an AR-targeted therapy. In somePage 53 of 13113185130vlAttorney Docket: 2014191-0045embodiments, the reference population comprises subjects having cancer and previously found to not respond to treatment with an AR-targeted therapy, and wherein the threshold value is greater than the AR activity score determined in the reference population. In some embodiments, the reference population comprises subjects having an AR-positive cancer (e.g., as determined by IHC). In some embodiments, the reference population comprises subjects having an AR-negative cancer (e.g., as determined by IHC) or determined to be cancer free, and the threshold value is an AR activity score that is greater than the AR activity score determined in the reference population.Exemplary Genomic Loci

[0222] The present disclosure includes the identification of exemplary genes and genomic loci that are differentially modified and / or differentially accessible depending on AR activity in a cancer. Table 1 lists genes whose epigenomic modifications can change depending on AR activity, and provides exemplary loci that exhibit different enhancer signal depending on AR activity levels.

[0223] The present disclosure is not limited to methods that use the exact same chromosomal coordinates that are recited in Table 1. The present disclosure encompasses methods that use any of the genomic loci in Table 1 and also subregions thereof, i.e., references herein to methods that involve detecting and / or quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more of the genes listed in Table 1, regulatory regions thereof, and / or one or more genomic loci of Table 1 encompasses methods that detect these marks anywhere within these genomic loci including within any subregions. For example, where Table 1 references chr21:38075527-38082326 as a genomic locus for detecting and / or quantifying enhancer signal (e.g., H3K27ac modifications), this encompasses methods that detect and / or quantify H3K27ac modification at any position or sub-region of chr21:38075527-38082326, e.g., methods that detect and / or quantify H3K27ac modifications within chr21:38075627-38082226, etc. In some embodiments, a subregion may span at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 or at least 3000 contiguous base pairs that are located between the lower and upper coordinates of a genomic locus recited in Table 1. In some embodiments, a subregion mayPage 54 of 13113185130vlAttorney Docket: 2014191-0045span less than 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 00, 2000, 2500 or at least 3000 contiguous base pairs that are located between the lower and upper coordinates of a genomic locus recited in Table 1. In some embodiments, a subregion may have the same central coordinate as a genomic locus recited in Table 1. In some embodiments, a subregion may have a different central coordinate as a genomic locus recited in Table 1. It is also to be understood that the lower / upper coordinates of the genomic loci in Table 1 are approximate and that the present disclosure encompasses methods where any one or more of the genomic loci are expanded by increasing the size of the genomic locus by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or up to 50% in one or both directions. The present disclosure also encompasses methods that use genomic loci that are associated with one or more of the genes listed in Table 1, including, e.g., loci of regulatory regions e.g., promoter regions) associated with the genes recited in Table 1 that are not recited in Table 1.

[0224] In some embodiments an assay for determining AR activity is generated using a set of differentially modified and / or differentially accessible genomic loci that are correlated with activation of the AR signaling pathway. Sequence reads that fall into each selected genomic locus are analyzed and counted, e.g., as described herein including in the Examples.

[0225] In some embodiments, AR pathway activity can be determined by quantifying a single type of histone modification at one or more of the loci listed in Table 1 (e.g., quantifying H3K27ac modifications at one or more loci listed in Table 1). In some embodiments, AR pathway activity can be determined by quantifying multiple types of histone modifications (e.g., H3K4me3 and H3K27ac) at one or more of the loci listed in Table 1 and a regulatory region (e.g., promoter region) of one or more of the genes listed in Table 1 (e.g., quantifying H3K4me3 modifications at one or more loci within a regulatory region of a gene listed in Table 1 and quantifying H3K27ac modifications at one or more loci listed in Table 1).Differential H3K4me3 modification

[0226] A person of skill in the art will recognize that the methods disclosed herein do not require that a promoter region of every gene listed in Table 1 be assessed for H3K4me3 modification. Instead, a subset of promoter regions may be assessed for H3K4me3 modification. Promoter regions for a subset of the genes listed in Table 1 can be selected (e.g., for use inPage 55 of 13113185130vlAttorney Docket: 2014191-0045determining AR activity) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and / or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier. Those of skill in the art will appreciate that such subsets of promoter regions of the genes of Table 1 and loci included in such subsets, are together, individually, and / or in randomly selected subsets, at least as informative (e.g., as statistically significant and / or reliable) for uses disclosed herein, e.g., for quantifying AR pathway activity.

[0227] In various embodiments, AR pathway activity in a sample or subject from which the sample is obtained or derived, is determined by quantifying H3K4me3 modifications in a promoter region associated with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or all of the genes identified in Table 1 (or any subset thereof). In certain embodiments, AR pathway activity in cancer in a subject from which the sample is obtained or derived, is determined by quantifying H3K4me3 modifications in at least a number of promoter regions of genes identified in a Table 1 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 and an upper bound selected from 10, 15, 20, 25, 30, 35, 40, or 43. In certain particular embodiments, AR pathway activity in a sample or subject from which the sample is obtained or derived, is determined by quantifying H3K4me3 modifications in promoter regions of at least 1, 2, 3, 4, 5, 10, 20, 30, 35, 40, or 43 genes identified in Table 1 (e.g., about 1 to about 43, about 5 to about 43, about 10 to about 43, about 25 to about 43, about 5, about 10, about 20, about 25, about 30, about 35, about 40, or about 43 loci). In various embodiments AR pathway activity in a sample or subject from which the sample is obtained or derived, is determined by quantifying H3K4me3 modifications in promoter regions of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 1. In certain embodiments, AR pathway activity in a sample or subject from which the sample is obtained or derived, is determined by quantifying H3K4me3 modifications in a promoter region of a percent of genes identified in Table 1 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100%.Page 56 of 13113185130vlAttorney Docket: 2014191-0045

[0228] In various embodiments, differentially H3K4me3 modified refers to a methylation status characterized by an increase or decrease in a value measuring methylation (e.g., of read counts and / or normalized read counts for a given genomic locus), and / or a mean, median and / or mode thereof, and / or a log thereof e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16-fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16-fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6. In various embodiments, an increase or decrease in a value measuring methylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10-fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0-fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold. 1.2-fold to 4.0-fold. 1.4-fold to 4.0-fold, 1.6-fold to 4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6-fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6. Exemplary references include, e.g., measurements from samples obtained from one or more healthy subject(s), one or more samples obtained from a non-cancerous cell lines, or sample(s) obtained from one or more cell line(s) or subject(s) having a cancer that is not associated with aberrant AR activity.

[0229] Exemplary methods for identifying promoter regions associated with a gene are known in the art. In some embodiments, a promoter region is a promoter region that has previously been associated with a gene. In some embodiments, a promoter region is a sequence that is proximal to a transcription start site, e.g., a region that is within about 5,000, about 4,000,Page 57 of 13113185130vlAttorney Docket: 2014191-0045about 3,000, about 2,000, or about 1,000 nucleotides of a transcription start site. In some embodiments, a promoter region is a sequence that is within about 2,000 nucleotides of a transcription start site. In some embodiments, a promoter region associated with a gene listed in Table 1 comprises a loci that exhibits differential H3K4me3 modifications in the H3Kme3 data that is provided in Stelloo, Suzan, et al. "Integrative epigenetic taxonomy of primary prostate cancer." Nature communications 9.1 (2018): 4900, and that is proximal to a region listed in Table 1, e.g., a region that is within about 5,000, about 4,000, about 3,000, about 2,000, or about 1,000 nucleotides of a transcription start site.Differential H3K27ac modification

[0230] Exemplary genomic loci that can comprise differential H3K27ac modification depending on AR signaling activity are provided in Table 1, which shows the chromosomal coordinates of each genomic locus that can be differentially modified depending on the extent of activation of the AR signaling pathway.

[0231] A person of skill in the art will recognize that the methods disclosed herein do not require that every genomic locus listed in Table 1 be assessed for H3K27ac modification.Instead, a subset of loci may be assessed for H3K27ac modification. Subsets of the genomic loci of Table 1 can be selected (e.g., for use in quantifying AR pathway activity) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and / or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm. Those of skill in the art will appreciate that such subsets of loci of Table 1, and loci included in such subsets, are together, individually, and / or in randomly selected subsets, at least as informative (e.g., as statistically significant and / or reliable) for uses disclosed herein, e.g., for determining AR pathway activity.

[0232] In various embodiments, the AR pathway activity of a sample or subject from which the sample is obtained or derived, can be determined by quantifying H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 of the loci listed in Table 1 (or any subset thereof). In certain embodiments, AR activity in a cancer in a subject from which the sample is obtained or derived, is determined by quantifying H3K27ac modificationsPage 58 of 13113185130vlAttorney Docket: 2014191-0045for at least a number of loci identified in a Table 1 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 and an upper bound selected from 10, 15, 20, 25, 30, 35, 40, or 43. In certain particular embodiments, AR pathway activity of a sample or subject from which the sample is obtained or derived, is determined to by quantifying H3K27ac modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 loci identified in Table 1 (e.g., about 1 to about 43, about 5 to about 43, about 10 to about 43, about 25 to about 43, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, or about 43 loci). In various embodiments AR activity of a sample or subject from which the sample is obtained or derived, is determined by quantifying H3K27ac modification of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 1. In certain embodiments, AR pathway activity of a sample or subject from which the sample is obtained or derived, is determined by quantifying H3K27ac modifications using at least a percent of loci identified in Table 1 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100%.

[0233] In various embodiments, AR pathway activity in a sample or subject from which the sample is obtained or derived, is determined by quantifying H3K27ac modifications in promoter regions associated with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or all of the genes identified in Table 1 (or any subset thereof). In certain embodiments, AR pathway activity in cancer in a subject from which the sample is obtained or derived, is determined by quantifying H3K27ac modifications in an enhancer region of a number of genes identified in a Table 1 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 and an upper bound selected from 10, 15, 20, 25, 30, 35, 40, or 43. In certain particular embodiments, AR pathway activity in a sample or subject from which the sample is obtained or derived, is determined by quantifying H3K27ac modifications in enhancer regions of at least 1, 2, 3, 4, 5, 10, 20, 30, 35, 40, or 43 genes identified in Table 1 (e.g., about 1 to about 43, about 5 to about 43, about 10 to about 43, about 25 to about 43, about 5, about 10, about 20, about 25, about 30, about 35, about 40, or about 43 loci). In various embodiments AR pathway activity in a sample or subject from which the sample is obtained or derived, is determined by quantifying H3K27ac modifications in enhancer regions of at least 0.1%, 0.2%, 0.3%, 0.4%,Page 59 of 13113185130vlAttorney Docket: 2014191-00450.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 1. In certain embodiments, AR pathway activity in a sample or subject from which the sample is obtained or derived, is determined by quantifying H3K27ac modifications in an enhancer region of a percent of genes identified in Table 1 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100%.

[0234] In various embodiments, differentially H3K27ac modified refers to an acetylation status characterized by an increase or decrease in a value measuring acetylation (e.g., of read counts and / or normalized read counts for a given genomic locus), and / or a mean, median and / or mode thereof, and / or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16-fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16-fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6. In various embodiments, an increase or decrease in a value measuring acetylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10-fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0-fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold. 1.2-fold to 4.0-fold. 1.4-fold to 4.0-fold, 1.6-fold to 4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6-fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6.

[0235] Exemplary references include, e.g., measurements from samples obtained fromPage 60 of 13113185130vlAttorney Docket: 2014191-0045one or more healthy subject(s), one or more samples obtained from a non-cancerous cell lines, or sample(s) obtained from one or more cell line(s) or subject(s) having a cancer that is not associated with aberrant AR activity.

[0236] Exemplary methods for identifying enhancer regions associated with a gene are known in the art. In some embodiments, an enhancer region is an enhancer region that has previously been associated with a gene. In some embodiments, an enhancer region is a sequence that is proximal to a transcription start site, e.g., a region that is within about 200,000, about 150,000, about 100,000, or about 50,000 nucleotides of a transcription start site. In some embodiments, an enhancer region is a sequence that is within about 50,000 nucleotides of a transcription start site. In some embodiments, an enhancer region associated with a gene listed in Table 1 comprises a loci that exhibits differential H3K427ac modifications in the H3K27ac data that is provided in Stelloo, Suzan, et al. "Integrative epigenetic taxonomy of primary prostate cancer." Nature communications 9.1 (2018): 4900, and that is proximal to a region listed in Table 1, e.g., a region that is within about 200,000, about 150,000, about 100,000 or about 50,000 nucleotides of a transcription start site.Differential DNA methylation

[0237] In various embodiments, differentially DNA methylated refers to a methylation status characterized by an increase or decrease in a value measuring methylation (e.g., of read counts and / or normalized read counts for a given genomic locus), and / or a mean, median and / or mode thereof, and / or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16-fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16-fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6. In various embodiments, an increase or decrease in a value measuringPage 61 of 13113185130vlAttorney Docket: 2014191-0045methylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase of 0.1 -fold to 10-fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0-fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold. 1.2-fold to 4.0-fold. 1.4-fold to 4.0-fold, 1.6-fold to 4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6-fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6.Differential chromatin accessibility or transcription factor binding

[0238] Genomic loci provided in Table 1 can also demonstrate differential chromatin accessibility or transcription factor e.g., AR) binding in cancers depending on the extent of activation of the AR signaling pathway.

[0239] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with chromatin accessibility.

[0240] In some embodiments, without wishing to be limited to any particular scientific theory, chromatin accessibility corresponds and / or is correlated with H3K4me3 modifications. As a result, in some embodiments, AR pathway activity may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Table 1 in accordance with the section above.

[0241] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with transcriptionPage 62 of 13113185130vlAttorney Docket: 2014191-0045factor binding. Tn various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with transcription factor binding.

[0242] In some embodiments, without wishing to be limited to any particular scientific theory, binding of RNA pol II corresponds and / or is correlated with H3K4me3 modifications. As a result, in some embodiments, AR. pathway activity may be determined by detecting and quantifying binding of RNA pol II at one or more genomic loci in Table 1 in accordance with the section above.

[0243] In some embodiments, without wishing to be limited to any particular scientific theory, binding of FOXA1, ESRI, PR, MYC, EN1, FOXM1, KLF4, AP-2, RARa, or RUNX1 corresponds and / or is correlated with histone methylation (e.g., H3K4me3), histone acetylation (e.g., H3K27ac) or DNA methylation. As a result, in some embodiments, AR activity may be determined by detecting and quantifying binding of FOXA1, ESRI, PR, MYC, EN1, F0XM1, KLF4, AP-2, RARa, RUNX1 at one or more genomic loci in Table 1 in accordance with the sections above discussing exemplary genomic loci with differential histone methylation (e.g., H3K4me3) or histone acetylation (e.g., H3K27ac).Applications

[0244] Methods, kits and systems of the present disclosure include analysis of differentially modified and / or differentially accessible genomic loci to determine the AR activity of a cancer. Methods, kits and systems of the present disclosure can be used in any of a variety of applications. For example, methods, kits and systems of the present disclosure can be used in detecting and / or treating cancers based on AR activity. Methods, kits and systems of the present disclosure can also be used to detect or determine resistance of a cancer, e.g., breast or prostate cancer to a therapy or transformation from one cancer subtype to another.

[0245] In various embodiments, methods, kits and systems of the present disclosure can be applied to an asymptomatic human subject. As used herein, a subject can be referred to as “asymptomatic” if the subject does not report, and / or demonstrate by non-invasively observable indicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or cancer screening), sufficient characteristics of cancer to support a medically reasonable suspicion that the subject is likely suffering from cancer, e.g., breast orPage 63 of 13113185130vlAttorney Docket: 2014191-0045prostate cancer. Detection of early-stage cancer can be achieved using methods, kits and systems of the present disclosure, with attendant medical benefits including potential for early treatment and attendant improvement in therapeutic outcomes.

[0246] In various embodiments, methods, kits and systems of the present disclosure can be applied to a symptomatic human subject. As used herein, a subject can be referred to as “symptomatic” if the subject reports, and / or demonstrates by non-invasively observable indicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or cancer screening), sufficient characteristics of cancer to support a medically reasonable suspicion that the subject is likely suffering from cancer, e.g., breast or prostate cancer. For example, in various embodiments a sample from a subject, optionally where the subject has a cancer that is of unknown AR activity, can be assayed according to one or more embodiments of the present disclosure to determine AR activity of the cancer. In various embodiments a sample from a subject, where the subject has a cancer that is known or suspected of being AR-positive (or AR-negative), can be assayed according to one or more embodiments of the present disclosure to determine if the cancer is in fact AR-positive (or AR-negative).

[0247] In some embodiments, methods, kits and systems of the present disclosure can be used to determine that a subject has an AR-positive cancer.

[0248] In some embodiments, methods, kits and systems of the present disclosure can be used to validate or confirm a prior determination that a subject has an AR-positive cancer, optionally an AR-positive cancer based on IHC testing. In some embodiments, methods, kits and systems of the present disclosure can be used to validate or confirm a prior determination that a subject has an AR-positive cancer.

[0249] In some embodiments, methods, kits and systems of the present disclosure are used to identify and detect new AR related categories that are independent of IHC or ISH scoring. For example, methods provided herein can be used to identify subjects that are likely to respond to a particular antiandrogen therapy.

[0250] Those of skill in the art will appreciate that regular, preventative, and / or prophylactic screening to measure AR activity improves diagnosis of cancer, including and / or particularly early-stage cancer. Thus, the present disclosure provides, among other things, methods, kits and systems particularly useful for the diagnosis and treatment of early-stagePage 64 of 13113185130vlAttorney Docket: 2014191-0045cancer. Generally, and particularly in embodiments in which AR activity determination in accordance with the present disclosure is carried out annually, and / or in which a subject is asymptomatic at time of detecting, methods, kits and systems of the present disclosure are especially likely to detect early-stage AR-positive cancer. In various embodiments, detecting in accordance with methods, kits and systems of the present disclosure reduces cancer mortality, e.g., by early cancer diagnosis.

[0251] In various embodiments AR activity determination in accordance with the present disclosure is performed once for a given subject or multiple times for a given subject. In various embodiments, AR activity determination in accordance with the present disclosure is performed on a regular basis, e.g., every six months, annually, every two years, every three years, every four years, every five years, or every ten years.

[0252] In various embodiments, methods, kits and systems disclosed herein provide a determination of AR activity. In other instances, methods, kits and systems disclosed herein will be indicative of AR activity but not definitive for AR activity. In various instances in which methods, kits and systems of the present disclosure are used to determine AR activity, the same can be followed by a further confirmatory assay, which further assay can confirm, support, undermine, or reject a determination resulting from a prior determination, e.g., a determination in accordance with the present disclosure. As used herein, a confirmatory assay can be, e.g., an AR test based on mRNA transcript analysis.

[0253] In various embodiments, AR activity determination according to one or more methods, kits and / or systems disclosed herein is followed by treatment of cancer. In various embodiments, treatment of cancer includes administration of a therapeutic regimen including one or more cancer therapies provided herein, including without limitation one or more of antiandrogens, surgery, radiation, endocrine therapy, chemotherapy, and / or immunotherapy. In various embodiments, treatment of cancer includes administration of a therapeutic regimen including one or more treatments provided herein as available, appropriate, and / or preferred for a particular AR activity.

[0254] In various embodiments, methods, kits and systems can be used to determine whether a particular subject and / or cancer is likely to be and / or is characterized as responsive toPage 65 of 13113185130vlAttorney Docket: 2014191-0045AR targeted therapy. In some such embodiments, methods, kits and systems can be followed by treatment of the subject with an AR targeted therapy.

[0255] In various embodiments, methods, kits and systems can be used to determine whether a particular subject and / or cancer is likely to be and / or is characterized as resistant to, non-responsive to, or not recommended treatment with to AR targeted therapy. In some such embodiments, methods, kits and systems can be followed by treatment with one or more of surgery and / or radiation, chemotherapy and immunotherapy instead of an antiandrogen therapy.

[0256] Responsiveness can refer to the ability or likelihood of a therapy to cause a reduction in tumor size or inhibit tumor growth or metastasis. Responsiveness can refer to improvement in prognosis (e.g., increased time to cancer recurrence or increased life expectancy, e.g., overall survival, recurrence-free survival, metastasis-free survival, clinic-radiologic survival or disease-free survival). Responsiveness can refer to achievement of a treatment benefit, including e.g., improvement in one or more symptoms of cancer, e.g., breast or prostate cancer. Responsiveness can be measured quantitatively (e.g., as in the case of tumor size; as in the case of measurement of histone modification, chromatin accessibility, transcription factor binding, or DNA methylation at one or more genomic loci; or as in the calculation of clinical benefit (CBR)), or qualitatively (e.g., by measures such as “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD), or other qualitative criteria). Resistance can refer to the inability or unlikelihood of a therapy to achieve a desired therapeutic effect (e.g., a reduction in tumor size, improvement in prognosis, or other treatment benefit such as, e.g., improvement in one or more symptoms of cancer) in a subject and / or cancer. Resistance includes both acquired and natural resistance. In certain embodiments, resistance includes the extent to which one or more desired therapeutic benefits results from administration of a therapy to a subject and / or cancer is less than that expected and / or achieved in a reference (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of benefit achieved in a reference).

[0257] In various embodiments, methods, kits, and systems can be used to detect the clinical efficacy of a course of therapy for cancer, e.g., breast or prostate cancer. For example, methods and / or compositions of the present disclosure could be used to determine AR activity of a cancer in a subject over the course of treatment. Methods and / or compositions of the presentPage 66 of 13113185130vlAttorney Docket: 2014191-0045disclosure could be used in conjunction with, or confirmed by, other means of determining AR expression status and / or AR activity of a cancer including, for example measurements of tumor size or character by techniques such as CT, PET, mammogram, ultrasound, palpation, histology, caliper measurement after biopsy or surgical resection, or by various qualitative, quantitative, or semi quantitative scoring systems including without limitation based on IHC or ISH testing, residual cancer burden (Symmans et al., J Clin Oncol (2007) 25:4414-4422, incorporated by reference herein in its entirety) or Miller-Payne score (Ogston et al., Breast (2003) 12:320-327, incorporated by reference herein in its entirety) in a qualitative fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), or “clinical progressive disease” (cPD).

[0258] In some embodiments, methods, kits and systems for AR activity determination provided herein can inform treatment and / or payment (e.g, reimbursement for or reduction of cost of medical care, such as detecting or treatment) decisions and / or actions, e.g., by individuals, healthcare facilities, healthcare practitioners, health insurance providers, governmental bodies, or other parties interested in healthcare cost.

[0259] In some embodiments, methods, kits and systems for AR activity determination provided herein can inform decision making relating to whether health insurance providers reimburse a healthcare cost payer or recipient (or not), e.g., for (1) AR activity determination itself (e.g., reimbursement for detecting otherwise unavailable, available only for periodic / regular detecting, or available only for temporally- and / or incidentally- motivated detecting); and / or for (2) treatment, including initiating, maintaining, and / or altering therapy, e.g, based on the determined AR activity. For example, in some embodiments, methods, kits and systems for AR activity determination provided herein are used as the basis for, to contribute to, or support a determination as to whether a reimbursement or cost reduction will be provided to a healthcare cost payer or recipient. In some instances, a party seeking reimbursement or cost reduction can provide results of AR activity determination conducted in accordance with the present disclosure together with a request for such reimbursement or reduction of a healthcare cost. In some instances, a party making a determination as to whether or not to provide a reimbursement or reduction of a healthcare cost will reach a determination based in whole or in part upon receipt and / or review of results of AR activity determination conducted in accordancePage 67 of 13113185130vlAttorney Docket: 2014191-0045with the present disclosure.

[0260] In various embodiments, AR activity determination using methods, kits and systems disclosed herein can be used in classifying subjects, samples, and / or tumors (c. ., breast cancer subjects, samples, and / or tumors). In various embodiments, methods, kits and systems disclosed herein can be used to generate a set of subjects, samples, and / or tumors identified according to the present methods, kits and systems each classified as comprising a particular AR activity, or having an AR activity that falls within a certain range, and optionally using two or more of such classified subjects, samples, and / or tumors to identify biomarkers that distinguish the classes (z.e., distinguish the subjects, samples, and / or tumors according to their class, e.g., according to their AR activity).

[0261] For illustration purposes and without limitation, in an exemplary assay of the present disclosure, samples obtained from a subject (e.g., a liquid biopsy sample including cfDNA, e.g., a plasma sample including cfDNA) is analyzed by ChlP-seq for a histone modification (e.g., H3K4me3 and / or H3K27ac). ChlP-seq sequence reads are aligned to human genome build hgl9, e.g., using the Burrows-Wheeler Aligner (BWA). Non-uniquely mapping and redundant reads are optionally discarded. To provide one example of peak calling, MACS v2.1.1.20140616 can be used for ChlP-seq peak calling with a q-value (FDR) threshold of 0.01. ChlP-seq data quality can optionally be evaluated by any of one or more of a variety of measures, including total peak number, FRiP (fraction of reads in peak) score, number of high-confidence peaks (e.g., enriched > ten-fold over background), and percent of peak overlap with “blacklist” DHS peaks derived from the ENCODE project (Amemiya et al., Sci Rep (2019) 9( 1 ): 9354). If the ChlP-seq data quality is below a particular threshold the data may be discarded and the assay repeated. ChlP-seq peaks that overlap with selected genomic loci that are differentially modified as provided herein for the relevant histone modification (Table 1) can then be used to determine AR activity. The number of reads overlapping the selected genomic loci for the relevant histone modification are summed, e.g., in some embodiments all the genomic loci that are differentially modified with an absolute log2(fold-change) > 4.0 are selected. In some embodiments, the average number of reads in the local background of each ChlP-seq peak is subtracted to improve signal to noise. The data is then log2 -transformed and quantile normalized to match the distribution of the data used to train the classifier. ThePage 68 of 13113185130vlAttorney Docket: 2014191-0045normalized data is then used as input into a classifier that was trained using the same histone modification and selected genomic loci. The classifier then uses the inputted data to determine AR activity of the subject’s cancer. It will be appreciated that this or similar approaches can be applied to assays of the present disclosure that quantify chromatin accessibility, transcription factor binding and / or DNA methylation.

[0262] For the avoidance of any doubt, those of skill in the art will appreciate from the present disclosure that methods, kits and systems for AR activity determination of the present disclosure are at least for in vitro use. Accordingly, all aspects and embodiments of the present disclosure can be performed and / or used at least in vitro.

[0263] Those of skill in the art will also appreciate that, in certain embodiments, methods of the present disclosure can be implemented on and / or in conjunction with a computer program and computer system. In some embodiments, methods of the present disclosure can be implemented on and / or in conjunction with a non-transitory computer readable storage medium encoded with the computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method. A computer system can also store and manipulate data generated by methods of the present disclosure that comprise a plurality of genomic locus modification status and / or accessibility status changes / profiles, which data can be used by a computer system in implementing methods disclosed herein. In certain embodiments, a computer system (i) receives modification status and / or accessibility status data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate references), e.g., to determine AR activity. In certain embodiments, a computer system (i) compares the genomic locus modification and / or accessibility status to a reference; and (ii) outputs an indication of whether the modification status and / or accessibility status of the genomic locus is significantly different from the reference and / or provides a determination regarding AR activity.

[0264] Numerous types of computer systems can be used to implement methods of the present disclosure according to knowledge possessed by a skilled artisan in the bioinformatics and / or computer arts. Several software components can be loaded into memory during operation of such a computer system. The software components can comprise both software components that are standard in the art and components that are special to the present disclosure (e.g., dCHIPPage 69 of 13113185130vlAttorney Docket: 2014191-0045software described in Lin et al., Bioinformatics (2004) 20:1233-1240, incorporated herein by reference in its entirety; radial basis machine learning algorithms (RBM) known in the art). Methods of the present disclosure can also be programmed or modeled in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms. Such packages include, e.g., Matlab from Mathworks (Natick, MA), Mathematica from Wolfram Research (Champaign, IL), S-Plus from MathSoft (Seattle, WA), R from R Foundation for Statistical Computing (Vienna, Austria), Python from Python Software Foundation (Wilmington, DE), or Perl from Perl Foundation (Holland, MI). In certain embodiments, a computer system comprises a database for storage of genomic locus modification status and / or accessibility status data. Such stored profiles can be accessed and used to perform comparisons of interest at a later point in time. In addition to the exemplary program structures and computer systems described herein, other, alternative program structures and computer systems will be readily apparent to the skilled artisan.

[0265] Various algorithms can be applied to the comparison, between samples and references, of the modification status and / or accessibility status of genomic loci that are differentially modified in different AR states. In various embodiments, an algorithm can be a single learning statistical classifier system. Other suitable statistical algorithms are well known to those of skill in the art. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex datasets (e.g., a panel of genomic loci of interest) and making decisions based upon such datasets. In some embodiments, a single learning statistical classifier system such as a classification tree (e.g., random forest) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those described in the Examples and also those using inductive learning (e.g., decision / classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connect! onist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multilayer feed-forward networks, applications of neural networks, Bayesian learning in beliefPage 70 of 13113185130vlAttorney Docket: 2014191-0045networks, etc.), reinforcement learning (e.g., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (e.g., Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quantization (LVQ). In certain embodiments, methods of the present disclosure can include sending classification results to a medical practitioner, e.g., an oncologist.Formulation and Administration of Therapeutic Agents

[0266] The present disclosure includes methods where a therapeutic agent or regimen is administered to a subject based on the AR activity of a cancer (e.g., breast or prostate cancer). In general, the therapeutic agent or regimen provided herein will be available, appropriate, and / or preferred for the determined AR activity. Those of skill in the art will be aware of recommended and / or governmentally approved formulations and / or dosages for various therapeutic agents provided herein.

[0267] The present disclosure includes pharmaceutical compositions for delivery of one or more therapeutic agents to a subject. As disclosed herein, a pharmaceutical composition may be in any form known in the art, including formulations for administration according to any route known in the art. A suitable means of administration can be selected based on the age and condition of a subject.

[0268] Pharmaceutical composition forms of the present disclosure can include, e.g., liquid, semi-solid and solid dosage forms. Pharmaceutical composition forms of the present disclosure can include, e.g., liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, and liposomes. Selection or use of any particular form may depend, in part, on the intended mode of administration and therapeutic application.Accordingly, the compositions can be formulated for administration by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection) or a non-parenteral mode. As used herein, parenteral administration refers to modes of administration other than enteral andPage 71 of 13113185130vlAttorney Docket: 2014191-0045topical administration, usually by injection or infusion.

[0269] In some embodiments, the compositions provided herein are present in unit dosage form, which unit dosage form can be suitable for self-administration. Such a unit dosage form may be provided within a container, e.g., a pill, vial, cartridge, prefilled syringe, or disposable pen.

[0270] A pharmaceutical composition of the present disclosure can be in an injectable or infusible form. For example, the present disclosure includes sterile formulations for injection or infusion, which can be formulated in accordance with conventional pharmaceutical practices. Sterile solutions can be prepared by incorporating a composition described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Solutions can be formulated, e.g., using distilled water, physiological saline, or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, or sodium chloride as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, an alcohol such as ethanol and / or a polyalcohol such as propylene glycol or polyethylene glycol, and / or a nonionic surfactant such as polysorbate 80™ or HCO-50, and the like. In the case of sterile powders for the preparation of sterile injectable solutions, methods for preparation include vacuum drying and freeze-drying that yield a powder of a composition described herein plus any additional desired ingredient (see below) from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin. In particular instances, a pharmaceutical composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage, e.g., at 2-8°C (e.g., 4°C).

[0271] In various embodiments, a pharmaceutical composition of the present disclosure can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Generally, dispersions are prepared by incorporating a composition described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.Page 72 of 13113185130vlAttorney Docket: 2014191-0045

[0272] In various instances, a pharmaceutical composition can be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, without limitation, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0273] In certain embodiments, compositions can be formulated with a carrier that will protect the therapeutic agent against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known in the art. See, e. ., J. R. Robinson (1978) “Sustained and Controlled Release Drug Delivery Systems,” Marcel Dekker, Inc., New York.

[0274] Route of administration can be parenteral, for example, administration by injection. Administration by injection can be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. Administration can be systemic or local. In certain embodiments, a composition described herein can be therapeutically delivered to a subject by way of local administration. As used herein, “local administration” or “local delivery,” can refer to delivery that does not rely upon transport of the composition or therapeutic agent to its intended target tissue or site via the vascular system. For example, the composition may be delivered by injection or implantation of the composition or therapeutic agent or by injection or implantation of a device containing the composition or therapeutic agent. In certain embodiments, following local administration in the vicinity of a target tissue or site, the composition or therapeutic agent, or one or more components thereof, may diffuse to an intended target tissue or site that is not the site of administration.

[0275] A pharmaceutical composition can be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, a pharmaceutical composition can be formulated by suitably combining the therapeutic molecule with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder,Page 73 of 13113185130vlAttorney Docket: 2014191-0045followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. Examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are a buffer such as a phosphate buffer, or sodium acetate buffer, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulated injection can be packaged in a suitable ampule.

[0276] In various embodiments, subcutaneous administration can be accomplished by means of a device, such as a syringe, a prefilled syringe, an auto-injector (e. , disposable or reusable), a pen injector, a patch injector, a wearable injector, an ambulatory syringe infusion pump with subcutaneous infusion sets, or other device for combining with a therapeutic agent for subcutaneous injection.

[0277] An injection system of the present disclosure may employ a delivery pen as described in U.S. Pat. No. 5,308,341. Pen devices, most commonly used for self-delivery of insulin to patients with diabetes, are well known in the art. Such devices can include at least one injection needle, are typically pre-filled with one or more therapeutic unit doses of a solution that includes the therapeutic agent and are useful for rapidly delivering solution to a subject with as little pain as possible. One medication delivery pen includes a vial holder into which a vial of a therapeutic or other medication may be received. The pen may be an entirely mechanical device or it may be combined with electronic circuitry to accurately set and / or indicate the dosage of medication that is injected into the user. See, e.g., U.S. Pat. No. 6,192,891. In some embodiments, the needle of the pen device is disposable and the kits include one or more disposable replacement needles. Pen devices suitable for delivery of any one of the presently featured compositions are also described in, e.g., U.S. Pat. Nos. 6,277,099; 6,200,296; and 6,146,361, the disclosures of each of which are incorporated herein by reference in their entirety. A microneedle-based pen device is described in, e.g, U.S. Pat. No. 7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the Precision Pen Injector (PPI) device, MOLLY™, manufactured by Scandinavian Health Ltd.

[0278] In certain embodiments, administration of a therapeutic agent as described herein is achieved by administering to a subject a nucleic acid encoding a therapeutic agent described herein. Nucleic acids encoding a therapeutic agent described herein can be incorporated into aPage 74 of 13113185130vlAttorney Docket: 2014191-0045gene construct to be used as a part of a gene therapy protocol to deliver nucleic acids that can be used to express and produce therapeutic agent within cells. Expression constructs of such components may be administered in any therapeutically effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1 (HSV-1), or recombinant bacterial or eukaryotic plasmids. Viral vectors can transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized, polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation. Examples of suitable retroviruses include adenovirus-derived vectors, adeno-associated virus (AAV), pLJ, pZIP, pWE, and pEM which are known to those skilled in the art.

[0279] In some embodiments, a composition can be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition can be formulated for storage for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, or 2 years) at 2-8°C (e.g., 4°C). Thus, in some embodiments, the compositions described herein are stable in storage for at least 1 year at 2-8°C (e.g., 4°C).

[0280] A pharmaceutical composition can include a therapeutically effective amount of a therapeutic agent described herein. Such effective amounts can be readily determined by one of ordinary skill in the art. A therapeutically effective amount can be an amount at which any toxic or detrimental effects of the composition are outweighed by therapeutically beneficial effects. In some embodiments, a dose can also be chosen to reduce or avoid production of antibodies or other host immune responses against a therapeutic agent. Those of skill in the art will appreciate that data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. In various embodiments, the amount of active ingredient included in a pharmaceutical composition is such that a suitable dose within the designated range can be administered to subjects. The dose and method of administration can vary depending on weight, age, condition, and other characteristics of a patient, and can be suitably selected as needed by those skilled in the art.Page 75 of 13113185130vlAttorney Docket: 2014191-0045

[0281] Pharmaceutical compositions including certain therapeutic agents, e.g., therapeutic antibodies, can be administered as a fixed dose, or in a milligram per kilogram (mg / kg) dose. While in no way intended to be limiting, an exemplary single dose of certain pharmaceutical compositions described herein can include certain therapeutic agents as described herein in an amount equal to, e.g., 0.001 to 1000 mg / kg, 1-1000 mg / kg, 1-100 mg / kg, 0.5-50 mg / kg, 0.1-100 mg / kg, 0.5-25 mg / kg, 1-20 mg / kg, and 1-10 mg / kg body weight. Exemplary dosages of a composition described herein include, without limitation, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 8 mg / kg, or 20 mg / kg. The present disclosure is not limited to such ranges or dosages.

[0282] The present disclosure further includes methods of preparing pharmaceutical compositions of the present disclosure and kits including pharmaceutical compositions of the present disclosure.

[0283] In various embodiments, therapeutic agents of the present disclosure can be administered to a subject in a course of treatment that further includes administration of one or more additional therapeutic agents or therapies that are not therapeutic agents (e.g., surgery or radiation). Combination therapies of the present disclosure can include simultaneous exposure of a subject to therapeutic agents of two or more therapeutic regimens.

[0284] In certain embodiments, a therapeutic agent as described herein can be administered together with (e.g., at the same time and / or in the same composition as) an additional agent or therapy. In certain embodiments, a therapeutic agent of the present disclosure can be administered separately from an additional therapeutic agent or therapy (e.g., at a different time and / or in a different composition than the additional therapeutic agent or therapy). Dosing regimens of a therapeutic agent and one or more additional therapeutic agents with which it is administered in combination can be coordinated or independently determined. In various embodiments, an additional therapeutic agent or therapy administered in combination with a therapeutic agent as described herein can be administered at the same time as therapeutic agent, on the same day as therapeutic agent, or in the same week as therapeutic agent. In various embodiments, an additional therapeutic agent or therapy administered in combination with a therapeutic agent as described herein can be administered such that administration of the therapeutic agent and the additional therapeutic agent or therapy are separated by one or morePage 76 of 13113185130vlAttorney Docket: 2014191-0045hours before or after, one or more days before or after, one or more weeks before or after, or one or more months before or after administration of the therapeutic agent. In various embodiments, the administration frequency and / or dosage of one or more additional therapeutic agents can be the same as, similar to, or different from the administration frequency of a therapeutic agent. In some embodiments, the two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such therapeutic agents are administered in overlapping dosing regimens.

[0285] In certain embodiments, administration of a therapeutic agent can be to a subject having previously received, scheduled to receive, or in the course of a treatment regimen including an additional cancer therapy. Administration of a therapeutic agent can, in some instances, improve delivery or efficacy of another therapeutic agent or therapy with which it is administered in combination.

[0286] It is contemplated that therapeutic agent combination therapies can demonstrate synergy and / or greater-than-additive effects between a therapeutic agent and one or more additional therapeutic agents with which it is administered in combination. A therapeutic agent can be administered in any effective amount as determined independently or as determined by the joint action of therapeutic agent and any of one or more additional therapeutic agents or therapies administered. Administration of the therapeutic agent may, in some embodiments, reduce the therapeutically effective dosage, required dosage, or administered dosage of the additional therapeutic agent or therapy relative to a reference regimen for administration of additional therapeutic agent or therapy or therapy absent the therapeutic agent. In certain embodiment, a composition described herein can replace or augment other previously or currently administered therapy. For example, upon treating with therapeutic agent, administration of one or more additional therapeutic agents or therapies can cease or diminish, e.g., be administered at lower levels.Kits

[0287] The present disclosure includes kits for detecting modification and / or accessibility of one or more genomic loci. In some embodiments, the present disclosure provides kits forPage 77 of 13113185130vlAttorney Docket: 2014191-0045quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci. Kits of the present disclosure can include, e.g., reagents such as buffers and / or antibodies useful in the detection and quantification of histone modifications. In certain embodiments, a kit of the present disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, orH3K4me3, or pan acetylation. In certain embodiments, a kit of the present disclosure can include at least one antibody that selective binds H3K4me3 modifications. In certain embodiments, a kit of the present disclosure can include at least one antibody that selective binds H3K27ac modifications. A kit of the present disclosure can include instructional materials disclosing or describing the use of the kit in a method of determining AR activity and / or treatment disclosed herein. In various embodiments, a kit of the present disclosure can include one or more therapeutic agents useful in the treatment of cancer, e.g., as disclosed herein, optionally in combination with instruction materials for treatment of cancer, e.g., breast or prostate cancer based on AR activity.

[0288] In some embodiments, a kit of the present disclosure comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Table 1.

[0289] In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 5, 10, 15, 20, 25, 30, 35, 40, or 43 genomic loci in Table 1. In some embodiments, the kit comprises one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K27ac-modified histones.

[0290] In some embodiments, the kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample. In some embodiments, the kit comprises reagents for library preparation for sequencing. In some embodiments, the kit comprises reagents for sequencing. In some embodiments, the kit comprises instructions for determining AR activity of a cancer in a subject.Systems

[0291] The present disclosure includes systems for detecting modification and / orPage 78 of 13113185130vlAttorney Docket: 2014191-0045accessibility of one or more genomic loci. Tn some embodiments, the present disclosure provides systems for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci. Systems of the present disclosure can include a sequencer configured to generate a sequencing dataset from a sample; and a non-transitory computer readable storage medium and / or a computer system.

[0292] In some embodiments, the non-transitory computer readable storage medium is encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method of the present disclosure.

[0293] In some embodiments, the computer system comprises a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform a method of the present disclosure.

[0294] In some embodiments, the sequencer is configured to generate a Whole Genome Sequencing (WGS) dataset from the sample. In some embodiments, the system also includes a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample. The sample preparation device may include reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.

[0295] Systems of the present disclosure can include, e.g., reagents such as buffers and / or antibodies useful in the detection and quantification of histone modifications. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, or H3K4me3, or pan acetylation. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds H3K4me3 modifications. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds H3K27ac modifications. A system of the present disclosure can include instructional materials disclosing or describing the use of the system in a method of determining AR activity and / or treatment disclosed herein.Page 79 of 13113185130vlAttorney Docket: 2014191-0045

[0296] In some embodiments, a system of the present disclosure comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Table 1.

[0297] In some embodiments, the system comprises reagents for quantifying H3K27ac for at least 5, 10, 15, 20, 25, 30, 35, 40, or 43 genomic loci in Table 1. In some embodiments, the system comprises one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K27ac-modified histones.

[0298] In some embodiments, the system comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample. In some embodiments, the sequencer comprises reagents for library preparation for sequencing. In some embodiments, the sequencer comprises reagents for sequencing. In some embodiments, the system comprises instructions for determining AR activity of a cancer in a subject.Definitions

[0299] “A” or “An”: The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” refers to one element or more than one element.

[0300] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context, to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” can encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or within a fraction of a percent, of the referenced value.

[0301] “Accessibility Status” or “Chromatin Accessibility Status”: As used herein, “accessibility status” or “chromatin accessibility status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of accessible chromatin. Accessibility status can be determined by various assays known in the art, including without limitation ChlP-seq as one example. Where twoPage 80 of 13113185130vlAttorney Docket: 2014191-0045samples are separately analyzed by the same assay or comparable assays for detection of accessible DNA sequences, differences in chromatin accessibility status of genomic loci can be detected. Accessibility status can be compared to a standard or reference. A sample that has an accessibility status that differs in accessibility status from a standard or reference can be referred to as differentially modified. Suitable assays for determining chromatin accessibility are known in the art. Exemplary assays include ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde- Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and / or a DNase hypersensitivity assay.

[0302] Administration: As used herein, the term “administration” typically refers to the administration of a disease appropriate (e.g, appropriate for administration to a subject having a certain AR activity) treatment. In some embodiments, the disease appropriate treatment may comprise administering a composition to a subject, for example to achieve delivery of an agent that is, is included in, or is otherwise delivered by, the composition. In some embodiments, the disease appropriate treatment may comprise administering an appropriate surgical procedure or radiological procedure, optionally in combination with administration of a composition.

[0303] Agent: As used herein, the term “agent” may refer to any chemical or physical entity, including without limitation any of one or more of an atom, e.g., a radioactive atom, molecule, compound, conjugate, polypeptide, polynucleotide, polysaccharide, lipid, cell, or combination or complex thereof.

[0304] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen (e.g., a heavy chain variable domain, a light chain variable domain, and / or one or more CDRs). Thus, the term antibody includes, without limitation, human antibodies, non-human antibodies, synthetic and / or engineered antibodies, fragments thereof, and agents including the same. Antibodies can be naturally occurring immunoglobulins (e.g, generated by an organism reacting to an antigen). Synthetic, non-naturally occurring, or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.

[0305] As is well known in the art, typical human immunoglobulins are approximatelyPage 81 of 13113185130vlAttorney Docket: 2014191-0045150 kD tetrameric agents that include two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other to form a structure commonly referred to as a “Y-shaped” structure. Typically, each heavy chain includes a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain includes three CH domains: CHI, CH2 and CH3. A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL), separated from one another by another “switch.” Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). In each VH and VL, the three CDRs and four FRs are arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of a heavy and / or a light chain are typically understood to provide a binding moiety that can interact with an antigen. Constant domains can mediate binding of an antibody to various immune system cells (e.g., effector cells and / or cells that mediate cytotoxicity), receptors, and elements of the complement system. Heavy and light chains are linked to one another by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. When natural immunoglobulins fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure.

[0306] In some embodiments, an antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (e.g., a bispecific antibody). In some embodiments, an antibody includes at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer that includes two heavy chain monomers and two light chain monomers. Moreover, the term “antibody” can include (unless otherwise stated or clear from context) any art-known constructs or formats utilizing antibody structural and / or functional features including without limitation intrabodies, domain antibodies, antibodyPage 82 of 13113185130vlAttorney Docket: 2014191-0045mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti -idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), Small Modular ImmunoPharmaceuticals (SMIPs), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, KALBITOR®s, chimeric antigen receptors (CARs), engineered T-cell receptors (TCRs), and antigen-binding fragments of any of the above.

[0307] In various embodiments, an antibody includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR) or variable domain. In some embodiments, an antibody can be a covalently modified (“conjugated”) antibody (e.g., an antibody that includes a polypeptide including one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art.

[0308] An antibody including a heavy chain constant domain can be, without limitation, an antibody of any known class, including but not limited to, IgA, secretory IgA, IgG, IgE and IgM, based on heavy chain constant domain amino acid sequence (e.g., alpha (a), delta (5), epsilon (s), gamma (y) and mu (p)). IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. As used herein, a “light chain” can be of a distinct type, e.g., kappa (K) or lambda ( ), based on the amino acid sequence of the light chain constant domain. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human immunoglobulins. Naturally produced immunoglobulins are glycosylated, typically on the CH2Page 83 of 13113185130vlAttorney Docket: 2014191-0045domain. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, antibodies produced and / or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation.

[0309] In some embodiments, an antibody can be specific for a particular histone modification (e.g., an antibody can bind one histone modification, e.g., H3K27ac with a higher affinity than other histone modifications, under conditions that are commonly used in ChlP-seq experiments). In some embodiments, an antibody is specific for an H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, orH3K4me3 modification. In some embodiments, an antibody is specific for an H3K27ac modification. In some embodiments, an antibody is specific for an H3K4me3 modification.

[0310] In some embodiments, an antibody is a “pan” antibody. As used herein, the term pan antibody refers to an antibody that can bind a group of histone modifications having one or more features that are similar. In some embodiments, a pan antibody is a pan-methylation antibody (e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one methylated lysine, wherein the at least one methylated lysine can be at any one of a plurality of amino acid positions, e.g., in some embodiments, a pan-methylation antibody can bind an H3 protein comprising a methylated lysine at any position). In some embodiments, a pan antibody is a pan-acetylation antibody (e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one acetylated lysine, wherein the at least one acetylated lysine can be at any one of a plurality of amino acid positions, e.g., a pan-acetylation antibody can bind an H3 protein comprising an acetylated lysine at any position). In some embodiments, a pan antibody can bind one or more histone modifications that are associated with transcription activation. In some embodiments, a pan antibody can bind one or more histone modifications that are associated with transcription silencing.

[0311] Antibody fragment: As used herein, an “antibody fragment” refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region thereof. An antibody fragment can be produced byPage 84 of 13113185130vlAttorney Docket: 2014191-0045any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment can be recombinantly produced, i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment can be wholly or partially synthetically produced. In some embodiments, an antibody fragment (particularly an antigen-binding antibody fragment) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.

[0312] AR Motif: As used herein, the term “AR motif’ refers to a sequence in the genome that is bound by the androgen receptor (AR). The consensus sequence for the androgen receptor (AR) motif is 5'- AGAACA NNN TGTTCT-3', which consists of two 6-base pair "halfsites" (AGAACA and TGTTCT) separated by a 3-base pair spacer ("NNN"). AR motifs can also be identified using AR ChlP-seq data.

[0313] AR Pathway Activity: As used herein, the terms “AR pathway activity” and “AR activity” refer to the degree of activation of the Androgen Receptor (AR) signaling pathway (e.g., in a subject, a cancer, a tissue sample, or a cell). In some embodiments, AR pathway activity in a cancer in a subject can be determined using a biological sample obtained from the subject (e.g., a sample comprising cfDNA). While AR pathway activity is associated with AR expression, the two are not always directly correlated (e.g., as discussed elsewhere in the present disclosure). In some embodiments, methods provided herein can provide a more accurate measure of AR pathway activity than a measurement of AR expression. In some embodiments, AR activity refers to AR transcriptional activity (i.e., the degree to which genes regulated by AR are being expressed).

[0314] Associated with: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated with that of the other. For example, a particular entity (e.g., an epigenetic profile comprising one or more histone modifications at a set of genomic loci, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if theyPage 85 of 13113185130vlAttorney Docket: 2014191-0045interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, or a combination thereof.

[0315] “Between” or “From”: As used herein, the term “between” refers to content that falls between indicated upper and lower, or first and second, boundaries, inclusive of the boundaries. Similarly, the term “from”, when used in the context of a range of values, indicates that the range includes content that falls between indicated upper and lower, or first and second, boundaries, inclusive of the boundaries.

[0316] Biological Sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell) of interest, as described herein. In some embodiments, a biological source is or includes an organism, such as a human subject. In some embodiments, a biological sample is or includes a biological tissue or fluid. In some embodiments, a biological sample can be or include cells, tissue, or bodily fluid. “Bodily fluids” refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., blood, serum, plasma, Cowper’s fluid or preejaculate fluid, chyle, chyme, stool, interstitial fluid, intracellular fluid, lymph, menses, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vitreous humor, vomit). In some embodiments, a biological sample can be or include blood, blood components, cell-free DNA (cfDNA), circulating-tumor DNA (ctDNA), ascites, biopsy samples, surgical specimens, cellcontaining body fluids, sputum, saliva, feces, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, gynecological fluids, secretions, excretions, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavages such as a ductal lavages or bronchoalveolar lavages, aspirates, scrapings, or bone marrow. In some embodiments, a biological sample is a liquid biopsy sample obtained from a bodily fluid. In some embodiments, a biological sample is or includes DNA obtained from a single subject or from a plurality of subjects. A biological sample can be a “primary sample” obtained directly from a biological source or can be a “processed sample”, i.e., a sample that was derived from a primary sample, e.g., via dilution, purification, mixing with onePage 86 of 13113185130vlAttorney Docket: 2014191-0045or more reagents, or any other processing step(s) as described herein. A biological sample can also be referred to as a “sample.”

[0317] Blood component: As used herein, the term “blood component” refers to any component of whole blood, including red blood cells, white blood cells, plasma, platelets, endothelial cells, mesothelial cells, epithelial cells, cell-free DNA (cfDNA), and circulatingtumor DNA (ctDNA). Blood components also include the components of plasma, including proteins, metabolites, lipids, nucleic acids, and carbohydrates, and any other cells that can be present in blood, e.g., due to pregnancy, organ transplant, infection, injury, or disease.

[0318] Cancer: As used herein, the terms “cancer,” “malignancy,” “tumor,” and “carcinoma,” are used interchangeably to refer to a disease, disorder, or condition in which cells exhibit or exhibited relatively abnormal, uncontrolled, and / or autonomous growth, so that they display or displayed an abnormally elevated proliferation rate and / or aberrant growth phenotype. In some embodiments, a cancer can include one or more tumors. In some embodiments, a cancer can be or include cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, a cancer can be or include a solid tumor.

[0319] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to, breast cancer (e.g., an HR+ breast cancer (e.g., an AR+ breast cancer (e.g., luminal A breast cancer or luminal B breast cancer)), DCIS, and / or a metastatic or a locally advanced breast cancer)); lung cancer, including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung; bladder cancer (e.g., urothelial bladder cancer (UBC), muscle invasive bladder cancer (MIBC), and BCG-refractory non-muscle invasive bladder cancer (NMIBC)); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); cancer of the urinary tract; prostate cancer, such as castrationresistant prostate cancer (CRPC); cancer of the peritoneum; hepatocellular cancer; gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; hepatoma; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine carcinoma; salivary gland carcinoma; prostate cancer; vulval cancer; thyroid cancer; hepatic carcinoma; anal carcinoma; penile carcinoma; melanoma, including superficial spreading melanoma, lentigo maligna melanoma,Page 87 of 13113185130vlAttorney Docket: 2014191-0045acral lentiginous melanomas, and nodular melanomas; multiple myeloma and B-cell lymphoma (including low grade / follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myelogenous leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), Meigs’ syndrome, brain cancer, head and neck cancer, and associated metastases.

[0320] Combination therapy: As used herein, the term “combination therapy” refers to administration to a subject of two or more therapeutic agents or therapeutic regimens such that the two or more therapeutic agents or therapeutic regimens together treat a disease, condition, or disorder of the subject. In some embodiments, the two or more therapeutic agents or therapeutic regimens can be administered simultaneously, sequentially, or in overlapping dosing regimens. Those of skill in the art will appreciate that combination therapy includes but does not require that the two therapeutic agents or therapeutic regimens be administered together in a single composition, nor at the same time.

[0321] Corresponding to: As used herein, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of skill in the art appreciate that residues in a provided polypeptide or polynucleotide sequence are often designated (e.g., numbered or labeled) according to the scheme of a related reference sequence (even if, e.g., such designation does not reflect literal numbering of the provided sequence). By way of illustration, if a reference sequence includes a particular amino acid motif at positions 100-110, and a second related sequence includes the same motif at positions 110-120, the motif positions of the second relatedPage 88 of 13113185130vlAttorney Docket: 2014191-0045sequence can be said to “correspond to” positions 100-110 of the reference sequence. Those of skill in the art appreciate that corresponding positions can be readily identified, e.g., by alignment of sequences, and that such alignment is commonly accomplished by any of a variety of known tools, strategies, and / or algorithms, including without limitation software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GL SEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE. Two sequences can be identified as corresponding if they are identical or if they share substantial identity, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more residues. In various embodiments, a nucleic acid sequence can correspond to a sequence that is identical or substantially identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the complement of the nucleic acid sequence, e.g., over a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more nucleic acid residues.

[0322] “Diagnosing”, “Detecting”, “Determining” or “Screening for”: As used herein, “diagnosing”, “detecting”, “determining”, “screening for” the presence of a condition or disease (e.g., AR-positive cancer), or a related state (e.g., responsiveness of an AR-positive cancer to one or more AR-targeted therapies) includes the act, process, and / or outcome of determining whether, and / or the qualitative of quantitative probability that, a subject has or will develop the condition, disease, or related state. In some instances, diagnosing can include a determination relating to prognosis and / or likely response to one or more general or particular therapeutic agents or regimens.

[0323] Differentially accessible: As used herein, the term “differentially accessible” describes a genomic locus for which chromatin accessibility status differs between a first condition or sample and a second condition or sample (e.g., a standard or reference). A differentially accessible genomic locus can include a greater or smaller measured accessibility under a selected condition of interest, such as activation of the AR signaling pathway, as compared to a reference state, such as a system in which the AR signaling pathway has not been activated.Page 89 of 13113185130vlAttorney Docket: 2014191-0045

[0324] Differentially modified: As used herein, the term “differentially modified” describes a genomic locus for which histone modification status and / or DNA methylation status differs between a first condition or sample and a second condition or sample (e. ., a standard or reference). A differentially modified genomic locus can include a greater or smaller number or frequency of histone modification and / or DNA methylations under a selected condition of interest, such as activated AR signaling, as compared to a reference state, such as AR-negative state or a state in which the AR signaling pathway has not been stimulated.

[0325] Enhancer signal. As used herein, the term “enhancer signal” refers to an epigenetic modification or chromatin state in an enhancer region that is associated with increased expression of a gene regulated by the enhancer region. Examples of such enhancer signals are known in the art, and include histone acetylation (e.g., H3K27ac). In some embodiments, enhancer signal can be measured by quantifying histone acetylation (e g., H3K27ac), chromatin accessibility, and / or transcription factor binding.

[0326] Expression level, amount, or level: As used herein, the terms “expression level,” “amount,” or “level,” or used herein interchangeably, of a biomarker is a detectable level in a biological sample. “Expression” generally refers to the process by which information (e g., gene-encoded and / or epigenetic) is converted into the structures present and operating in the cell. Therefore, as used herein, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and / or polypeptide modifications (e.g., posttranslational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis. “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs). Expression levels can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of a biomarker can be used to identify / characterize a subject having a breast cancer (e.g., an HR+ breast cancer (e.g., an AR+ breast cancer (e.g., luminal A breast cancer or luminal B breast cancer)), DCIS, and / or aPage 90 of 13113185130vlAttorney Docket: 2014191-0045metastatic or a locally advanced breast cancer) who may be likely to respond to, or benefit from, a particular therapy (e.g., a therapy comprising an endocrine therapy, e.g., a SERM (e.g., a SERD), a GnRH agonist, and / or an Al). The expression level or amount of a biomarker provided herein in a subject having a breast cancer described herein can also be used to determine and / or track the benefit of an administered endocrine therapy over time.

[0327] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided sequences are known in the art. The term “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between protein and nucleic acid sequences as determined by the match between strings of such sequences. “Identity” (often referred to as “similarity”) can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M. ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W. ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M. and Griffin, H. G. eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G. ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J. eds.) Oxford University Press, NY (1992), each of which are separately incorporated by reference in their entirety. Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. For example, calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and nonidentical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionallyPage 91 of 13113185130vlAttorney Docket: 2014191-0045accounting for the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a computational algorithm, such as BLAST (basic local alignment search tool). Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp, Comp Appl Biosci (1989) 5(2): 151-153), incorporated by reference herein in its entirety, with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul et al., J Mol Biol (1990) 215:403-410); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Com put Methods Genome Res [Proc Int Symp] (1994), Meeting Date 1992, 111-120. Eds. Suhai, Sandor. Plenum, New York, NY (the contents of each of which is separately incorporated herein by reference in its entirety). Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. “Default values” will mean any set of values or parameters, which originally load with the software when first initialized.

[0328] ‘‘‘‘Improve,'’'’ "increase" "inhibit" or ‘‘‘‘reduce'1'’: As used herein, the terms “improve”, “increase”, “inhibit”, and “reduce”, and grammatical equivalents thereof, indicate qualitative or quantitative difference from a reference.

[0329] Methylation Status: As used herein, “methylation status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of DNA methylated sequences and / or the density (e.g., the measured density) of DNA methylation corresponding to the genomic locus. Methylation status can be determined by various assays known in the art, including without limitation Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or MethyLCpG-Binding Domain sequencing (MBD-seq). Where two samples are separately analyzed by the same assay or comparable assaysPage 92 of 13113185130vlAttorney Docket: 2014191-0045for detection of DNA methylated sequences, differences in methylation status of genomic loci can be detected. Methylation status can be compared to a standard or reference. A sample that has a methylation status that differs from a standard or reference can be referred to as differentially modified.

[0330] “Modification Status” or “Histone Modification Status”: As used herein, “modification status” or “histone modification status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of DNA sequences associated with histones bearing one or more histone modifications (e.g., one or more particular histone modifications) and / or the density (e.g., the measured density) of histone modifications (e.g., one or more particular histone modifications) corresponding to the genomic locus. Modification status can be determined by various assays known in the art, including without limitation ChlP-seq as one example. Other well-known assays include CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing and CUT&Tag (Cleavage Under Targets and Tagmentation). Where two samples are separately analyzed by the same assay or comparable assays for detection of DNA sequences associated with histones bearing one or more histone modifications (e.g., one or more particular histone modifications), differences in modification status of genomic loci can be detected. Modification status can be compared to a standard or reference. A sample that has a modification status that differs in modification status or histone modification status from a standard or reference can be referred to as differentially modified.

[0331] Promoter signal: As used herein, the term “promoter signal” refers to an epigenetic modification in a promoter region that is associated with increased expression of a gene regulated by the promoter region. Examples of such promoter signals are known in the art, and include, e.g., histone methylation (e.g., H3K4me3). In some embodiments, promoter signal can be measured by quantifying histone methylation (e.g., H3K4me3), chromatin accessibility, and / or transcription factor binding.

[0332] Regulatory sequence: As used herein in the context of expression of a nucleic acid coding sequence, a regulatory sequence is a nucleic acid sequence that controls expression of a coding sequence, e.g., a promoter sequence or an enhancer sequence (also referred to herein as a promoter region or an enhancer region). In some embodiments, a regulatory sequence canPage 93 of 13113185130vlAttorney Docket: 2014191-0045control or impact one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.).

[0333] Subject: As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a disease, disorder or condition e.g., AR-positive cancer, e.g., prostate cancer, etc.). In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject is not suffering from a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a subject that has been tested for a disease, disorder, or condition, and / or to whom therapy has been administered. In some instances, a human subject can be interchangeably referred to as a “patient” or “individual”.

[0334] Therapeutic agent: As used herein, the term “therapeutic agent” refers to any agent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be a population of model organisms or a human population. In some embodiments, an appropriate population can be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used for treatment of a disease, disorder, or condition (e.g., AR-positive cancer, e.g., AR-positive breast cancer, etc.). In some embodiments, a therapeutic agent is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent for which a medical prescription is required for administration to humans.

[0335] Therapeutically effective amount: As used herein, “therapeutically effective amount” refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition (e.g., AR-positive cancer, e.g., AR-positive breast cancer, etc.) in accordance with a therapeutic dosingPage 94 of 13113185130vlAttorney Docket: 2014191-0045regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0336] Treatment: As used herein, the term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result. In some embodiments, such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder, or condition and / or of a subject who exhibits only early signs of the disease, disorder, or condition e.g., cancer having high AR activity, e.g., breast cancer having a high AR activity, etc.). Alternatively, or additionally, such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, or condition. A “prophylactic treatment” includes a treatment administered to a subject who does not display signs or symptoms of a condition to be treated or displays only early signs or symptoms of the condition to be treated such that treatment is administered for the purpose ofPage 95 of 13113185130vlAttorney Docket: 2014191-0045diminishing, preventing, or decreasing the risk of developing the condition. Thus, a prophylactic treatment functions as a preventative treatment against a condition. A “therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of a condition and is administered to the subject for the purpose of reducing the severity or progression of the condition.Exemplary Embodiments

[0337] Without limitation to the foregoing description, the following is an enumerated list of non-limiting exemplary embodiments included in the present disclosure. Those of ordinary skill in the art will appreciate that one or more features discussed above may be included with or incorporated into any of the following numbered embodiments to form additional embodiments.1. A method of measuring androgen receptor (AR) activity of a cancer in a subject, the method comprising:quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject:(i) one or more histone modifications,(ii) chromatin accessibility,(iii) binding of one or more transcription factors, and / or(iv) DNA methylation.2. The method of embodiment 1, wherein the one or more histone modifications are quantified using a histone modification assay that measures H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, H3K4me3, or pan-acetylation.3. The method of embodiment 2, wherein the histone modification assay detects H3K4me3 modifications.4. The method of embodiment 2, wherein the histone modification assay detects H3K27ac modifications.5. The method of any one of embodiments 2-4, wherein the histone modification assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.Page 96 of 13113185130vlAttorney Docket: 2014191-00456. The method of any one of embodiments 1 -5, wherein chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde- Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and a DNase hypersensitivity assay.7. The method of any one of embodiments 1-6, wherein the binding of one or more transcription factors is quantified using a transcription factor binding assay that detects binding of one or more of p300, mediator complex, cohesion complex, RNA pol II, F0XA1, ESRI, PR, MYC, EN1, F0XM1, KLF4, AP-2, RARa, orRUNXl.8. The method of embodiment 7, wherein the transcription factor binding assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.9. The method of any one of embodiments 1-8, wherein DNA methylation is quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl -CpG-Binding Domain sequencing (MBD-seq).10. The method of any one of embodiments 1-9, comprising quantifying two or more of the following, each at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject:(i) one or more histone modifications,(ii) chromatin accessibility,(iii) transcription factor binding, and / or(iv) DNA methylation.11. The method of embodiment 10, comprising quantifying:(i) H3K27ac modifications and H3K4me3 modifications;(ii) H3K27ac modifications and DNA methylation;(iii) H3K4me3 modifications and DNA methylation; or(iv) H3K27ac modifications, H3K4me3 modifications, and DNA methylation.Page 97 of 13113185130vlAttorney Docket: 2014191-004512. The method of any one of embodiments 1-11, wherein the liquid biopsy sample is a plasma sample, serum sample, or urine sample.13. The method of any one of embodiments 1-12, comprising quantifying:(i) one or more histone modifications at one or more regulatory regions (e.g., promoter or enhancer regions or subregions) associated with one or more of the genes listed in Table 1,(ii) chromatin accessibility at one or more of the genes listed in Table 1,(iii) binding of one or more transcription factors associated with promoting expression of one or more of the genes listed in Table 1, and / or(iv) DNA methylation at one or more of the genes listed in Table 1.14. The method of embodiment 13, comprising quantifying enhancer signal at one or more enhancer regions or subregions associated with one or more of the genes listed in Table 1 (e g., quantifying enhancer signal at one or more of the loci listed in Table 1).15. The method of embodiment 13 or 14, comprising quantifying promoter signal at a promoter for one or more of the genes listed in Table 1.16. The method of any one of embodiments 13-15, wherein enhancer signal comprises H3K27ac modifications.17. The method of any one of embodiments 14-16, wherein promoter signal comprises H3K4me3 modifications.18. A method of measuring AR activity of a cancer (e.g., PRAD) in a subject, comprising:obtaining a biological sample comprising cell-free DNA (cfDNA), optionally a liquid biopsy sample, from the subject; anddetermining an AR activity score for the sample, wherein the AR activity score is determined by a method comprising measuring enhancer or promoter signal at one or more loci that have previously been determined to have increased enhancer or promoter signal in an AR+ cancer as compared to one or more non-cancerous samples (e.g., one or more plasma or tissue samples obtained from a subject not diagnosed with cancer).19. The method of embodiment 18, wherein the one or more loci with increased enhancer or promoter signal show no correlation with ERG expression.20. The method of embodiment 18 or 19, wherein the enhancer signal is measured at one orPage 98 of 13113185130vlAttorney Docket: 2014191-0045more loci that have previously been determined to have increased enhancer signal and (i) that overlap with AR ChlP-seq sites having an AR motif or (ii) that are highly correlated with AR motif sites.21. The method of any one of embodiments 18-20, wherein the method comprises measuring enhancer signal at two or more loci with increased enhancer or promoter signal in AR+ cancer, and combining (e.g., summing, averaging, geometric mean averaging, or taking the median of) the enhancer or promoter signal measured at the two more loci.22. The method of any one of embodiments 18-21, wherein the method for determining AR activity score comprises correcting the measured enhancer or promoter signal for ctDNA fraction (e.g., dividing enhancer or promoter signal by ctDNA fraction).23. The method of any one of embodiments 18-22, wherein the one or more loci are within one or more enhancer regions for one or more of the genes listed in Table 1.24. The method of any one of embodiments 18-23, wherein the one or more loci are within one or more promoter regions for one or more of the genes listed in Table 1.25. The method of any one of embodiments 18-24, comprising measuring enhancer signal for at least 1, 5, 10, 20, 30, or 40 of the loci listed in Table 1.26. The method of any one of embodiments 18-25, wherein enhancer signal and / or promoter signal in the liquid biopsy sample is measured using a method that comprises sequencing cfDNA comprising one or more histone modifications (e.g., H3K4me3 and / or H3K27ac), e.g., using cfChlP-seq.27. The method of embodiment 26, wherein the sequence reads at each genomic loci are processed prior to combining with sequence reads at other genomic loci (e.g., quantile normalized and / or adjusted for background signal).28. The method of any one of embodiments 18-27, wherein the liquid biopsy sample is a plasma sample, serum sample, or urine sample.29. The method of any one of embodiments 18-28, wherein the cancer is determined to have elevated AR activity (e.g., as compared to AR activity in a subject not diagnosed with cancer and / or a cancer that does not have elevated AR activity) if the AR activity score is equal to or greater than a reference value.30. The method of embodiment 29, wherein the reference value is a predetermined thresholdPage 99 of 13113185130vlAttorney Docket: 2014191-0045value and / or a normalized value.31. The method of embodiment 29 or 30, wherein the reference value is an AR activity score determined in a reference subject or a population of subjects.32. The method of any one of embodiments 29-31, where the reference subject or population of subjects is:(i) a subject or population of subjects that have not been diagnosed with cancer, (ii) a subject or population of subjects that have been diagnosed with a cancer that does not have elevated AR expression, or(iii) a subject or population of subjects that have been diagnosed with an AR+ cancer (e g., PRAD).33. A method of treating a subject having a cancer, the method comprising:(i) obtaining a biological sample comprising cell-free DNA (cfDNA), optionally a liquid biopsy sample, from the subject;(ii) measuring AR activity in the biological sample, wherein AR activity is measured using a method of any one of embodiments 18-32; and(iii) administering an antiandrogen to the subject if AR activity is greater than or equal to a reference value and not administering an antiandrogen to the subject if the AR activity is less than the reference value.34. The method of embodiment 33, wherein the reference value is an AR activity score measured in:(a) a healthy subject or population of healthy subjects;(b) a subject or population of subjects having a cancer previously shown to respond to treatment with the antiandrogen; or(c) a subject or population of subjects having a cancer previously shown to not respond to treatment with an antiandrogen (e.g., an AR antagonist).35. The method of embodiment 33 or 34, wherein the reference value is an AR activity score is that is at least about 1.2, 1.4, 1.6, 1.8, 2, 2.5, 3, 4, or 5 times higher than an AR activity score determined in a healthy subject or population of healthy subjects or a subject or population of subjects that have been diagnosed with cancer that does not have elevated AR expression.36. The method of any one of embodiments 33-35, wherein the subject has previously beenPage 100 of 13113185130vlAttorney Docket: 2014191-0045determined to have cancer.37. The method of any one of embodiments 33-36, wherein the cancer is prostate cancer, breast cancer, laryngeal carcinoma, bladder cancer, or hepatocellular carcinoma (HCC), optionally wherein the cancer is prostate adenocarcinoma (PRAD) or AR+ breast cancer.38. The method of embodiment 37, wherein the PRAD is metastatic castration resistant prostate cancer (mCRPC).39. The method of embodiment 37, wherein AR status is determined using an immunohistochemistry (IHC) assay.40. A method of monitoring cancer in a subject, the method comprising measuring AR activity in the subject using the method of any one of embodiments 1-28 at a first and a second time point.41. The method of embodiment 40, wherein the subject has been administered an antiandrogen therapy prior to the first time point or after the first time point and before the second time point.42. A method of monitoring cancer (e.g., AR-positive cancer) in a subject comprising measuring AR activity of the cancer using the method of any one of embodiments 1-28 at a first and a second time point.43. The method of embodiment 42, wherein the subject has been administered an antiandrogen therapy prior to the first time point or after the first time point and before the second time point.44. A method of determining the likelihood that a subject will respond to treatment with an antiandrogen, comprising measuring AR activity of the cancer using the method of any one of embodiments 1-28 at a first and a second time point.45. The method of embodiment 44, wherein the subject was administered androgen depletion therapy (ADT) before the first time point or between the first time point and the second time point.46. The method of embodiment 45, wherein the subject has an increased likelihood of responding to treatment with an antiandrogen therapy if AR activity increases between the first and the second time point.47. The method of embodiment 44, wherein the subject has been administered anPage 101 of 13113185130vlAttorney Docket: 2014191-0045antiandrogen therapy prior to the first time point or after the first time point and before the second time point.48. The method of embodiment 47, wherein the subject is less likely to respond to continued treatment with an antiandrogen therapy if AR activity increases or stays approximately the same between the first time point and the second time point.49. A method of treating a cancer in a subject, the method comprising measuring AR activity of the cancer using the method of any one of embodiments 1-28 at a first and a second time point.50. The method of embodiment 49, wherein the subject was administered ADT before the first time point or between the first time point and the second time point.51. The method of embodiment 49, wherein:(i) if AR activity increases between the first and the second time point, the method comprises administering an antiandrogen; and(ii) if AR activity decreases or stays the same between the first and the second time point, the method does not comprise administering an antiandrogen.52. The method of embodiment 49 or 50, wherein the subject was administered an antiandrogen therapy prior to the first time point or after the first time point and before the second time point.53. The method of embodiment 52, comprising administering an antiandrogen therapy to the subject based on the change in AR activity between the first time point and the second time point, optionally wherein the type, dose, and / or frequency of administration of the antiandrogen therapy is adjusted based on the change in AR activity.54. The method of embodiment 52, wherein the method comprises administering the antiandrogen therapy after the second time point if AR activity decreases between the first time point and the second time point, optionally wherein the dose of the antiandrogen therapy administered after the second time point is the same as the dose administered before the second time point.55. The method of embodiment 52, wherein, if AR activity increases or stays approximately the same between the first time point and the second time point, the method comprises increasing the amount of antiandrogen administered to the subject,Page 102 of 13113185130vlAttorney Docket: 2014191-0045administering a different antiandrogen to the subject, and / oradministering a different cancer therapy to the subject (e.g., a therapy that does not comprise administering an AR-targeted therapy to the subject).56. The method of embodiment 52, wherein the method does not comprise administering an antiandrogen therapy if AR activity increases between the first time point and the second time point.57. The method of any one of embodiments 1-56, wherein the cancer is prostate cancer, breast cancer, laryngeal carcinoma, bladder cancer, or hepatocellular carcinoma (HCC) optionally wherein the cancer is prostate adenocarcinoma (PRAD) or AR+ breast cancer.58. The method of embodiment 57, wherein the prostate cancer is de novo mCSPC, non-metastatic CRPC, mCRPC in chemo naive patients, or mCRPC which has progressed after chemotherapy.59. The method of embodiment 58, wherein AR status is determined using an IHC assay.60. The method of any one of embodiments 41, 43-48, 50-59, wherein the antiandrogen is an AR antagonist (e.g., a molecule that binds the LBD of AR, including e.g., enzalutamide), an androgen synthesis inhibitor (e.g., abiraterone) or an AR degrader (e.g., an AR-targeted PROTAC), optionally wherein the androgen synthesis inhibitor is coadministered with a corticosteroid (e.g., prednisone).61. A method for testing the activity of a test compound, comprising incubating the test compound with a cell line, and measuring AR activity in the cell line subsequent to incubating the test compound with the cell line, wherein the AR activity is measured using the method of any one of embodiments 1-28.62. The method of embodiment 61, where the cell line has measurable AR activity (e.g., the cell line has been incubated with a composition that increases AR signaling activity prior to incubating with the test compound).63. A method of screening a library of test compounds for AR-targeting activity, comprising testing the activity of each test compound using the method of embodiment 61 or 62.64. A compound identified by the method of any one of embodiments 61-63.65. A kit comprising reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one orPage 103 of 13113185130vlAttorney Docket: 2014191-0045more genomic loci, wherein the one or more genomic loci are selected from those listed in Table 1.66. The kit of embodiment 65, wherein the kit comprises reagents for quantifying H3K27ac modifications for at least 1, 5, 10, 20, 30, 40, or 50 genomic loci listed in Table 1.67. The kit of embodiment 65 or 66, wherein the kit comprises one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K27ac-modified histones.68. The kit of any one of embodiments 65-66, wherein the kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample.69. The kit of any one of embodiments 65-68, wherein the kit comprises reagents for library preparation for DNA sequencing.70. The kit of any one of embodiments 65-69, wherein the kit comprises reagents for DNA sequencing.71. A non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method of any one of embodiments 1-63.72. A computer system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform the method of any one of embodiments 1-6373. A system for quantifying AR activity of a cancer in a subject, the system comprising a sequencer configured to generate a sequencing dataset from a sample; and a non-transitory computer readable storage medium of embodiment 71 and / or a computer system of embodiment 72.74. The system of embodiment 73, wherein the sequencer is configured to generate a Whole Genome Sequencing (WGS) dataset from the sample.75. The system of embodiment 73 or 74, further comprising a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample.76. The system of embodiment 75, wherein the sample preparation device comprises reagentsPage 104 of 13113185130vlAttorney Docket: 2014191-0045for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.77. The system of embodiment 76, wherein the one or more genomic loci are selected from those listed Table 1.78. The system of any one of embodiments 75-77, wherein the device comprises reagents for quantifying H3K27ac modifications, e.g., reagents for quantifying H3K27ac modifications for at least 1, 5, 10, 20, 30, or 40 of the genomic loci listed in Table 1.79. The system of embodiment 78, wherein the reagents comprise one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K27ac-modified histones.80. The system of any one of embodiments 75-79 wherein the device comprises reagents for isolation of cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.81. The system of any one of embodiments 75-80, wherein the device comprises reagents for library preparation for sequencing.82. The system of any one of embodiments 75-81, wherein the sequencer comprises reagents for sequencing.EXAMPLES

[0338] The present Examples demonstrate the identification and use of differentially modified and / or differentially accessible genomic loci in tissue samples with different AR activities and / or from cfDNA in plasma samples obtained from subjects having breast cancer with different AR activities. The present Examples show that differentially modified and / or differentially accessible genomic loci of the present disclosure can be used to determine AR activity from cfDNA in plasma samples obtained from subjects with AR-positive and AR-negative cancers.Example 1: Exemplary Methods

[0339] This Example describes exemplary methods that can be used to generate sequencing data for use in measuring AR activity.Page 105 of 13113185130vlAttorney Docket: 2014191-0045MethodsChromatin immunoprecipitation (ChIP)

[0340] An exemplary protocol for performing chromatin immunoprecipitation (ChIP) for histone marks (H3K4me3 and H3K27ac) in cell lines or tissue samples is provided, e.g., in Schones et al., Cell (2008) 132(5): 887-898, which is incorporated by reference herein in its entirety. Briefly, cells are lysed and chromatin is MNase digested to generate approximately 80% mononucleosomes. Nucleosomes are then incubated with antibodies that bind H3K27ac or H3K4me3 modifications that were previously conjugated to magnetic epoxy beads (Invitrogen) with constant mild shaking overnight. The beads are then washed and rinsed. Sequencing libraries are generated from purified immunoprecipitated sample DNA and then sequenced.

[0341] Chromatin immunoprecipitation (ChIP) for histone marks (e.g., H3K27ac or H3K4me3) in plasma samples can be performed using methods similar to those previously described in Sadeh et al., Nat Biotechnol (2021) 39: 586-598 and Jang et al., Life Sci Alliance (2023) 6(12):e202302003. Briefly, about 1 mL frozen plasma is thawed and then prepared for ChIP. The thawed plasma is incubated with antibodies that bind H3K27ac or H3K4me3 modifications that were previously conjugated to magnetic epoxy beads (Invitrogen) with constant mild shaking overnight. The beads are then washed and rinsed. Sequencing libraries are generated from purified immunoprecipitated sample DNA and then sequenced.ChlP-seq and DNA methylation data analysis

[0342] ChlP-sequencing reads can be aligned to the human genome build hgl9 using the Burrows-Wheeler Aligner (BWA) version 0.7.15. Non-uniquely mapping and redundant reads can be discarded.Example 2: Measuring AR Activity

[0343] The present Example describes methods for measuring AR activity in tissue samples or samples comprising cfDNA and results from the same.Identification of genomic loci for use in an AR activity score generator

[0344] Previously published AR, H3K27ac, and H3K4me3 ChlP-seq data, collected from ~50 primary tissue samples obtained from PRAD subjects (see Stelloo, Suzan, et al. "Integrative epigenetic taxonomy of primary prostate cancer." Nature communications 9.1 (2018): 4900, thePage 106 of 13113185130vlAttorney Docket: 2014191-0045contents of which are incorporated by reference herein in their entirety) were used to identify loci for use in an algorithm for measuring AR activity using epigenetic modifications (H3K27ac modifications in the present Example).

[0345] First, in silico diluted samples were prepared by diluting tissue sample sequencing data in silico with plasma data obtained from healthy subjects. For each tissue sample, sequence data was diluted in silico with different amounts of healthy plasma data, to generate multiple in silico samples, each having a different ctDNA fraction.

[0346] Following in silico dilution, H3K27ac regions for incorporation into an AR activity score generator were prioritized on the basis of certain criteria, including:• Showing no correlation with ERG expression1; and• Overlapping with AR ChlP-seq sites having an AR motif or, for sites without AR motifs, being highly correlated with AR motif sites2.^oci correlated with ERG expression were excluded because TMPRSS2-ERG fusions can result in increased ERG expression and rewiring of the AR cistrome (i.e., exclusion of these sites results in a more generally applicable AR activity score generator, which can be applied to cancers comprising wild-type ERG or an ERG fusion).2Sites lacking AR motifs (“no-motif sites”) that were highly correlated with AR motif sites were identified by looking at the correlation of H3K27ac counts at AR motif sites vs. counts at sites that lacked AR motifs.

[0347] To identify sites that were not correlated with ERG expression, RNA-seq data from Stelloo et al. was obtained and the Pearson correlation of H3K27ac fragments at each AR site with the RNA-seq value for ERG was computed. Sites with a Pearson absolute value > 0.25 were then computed.

[0348] To identify sites without AR motifs that were correlated with AR motif sites, a proto AR activity score was generated using only sites that include an AR motif, and correlated with other sites. No-motif sites that had a spearman correlation > 0.6 were included among the list of loci that can be used to measure AR activity.

[0349] Exemplary results are provided in Fig. 1 for a KLK3 gene loci.

[0350] Following identification of genomic loci in silico, the tissue-derived regions were further refined using PRAD plasma data to select sub-regions with the highest signalPage 107 of 13113185130vlAttorney Docket: 2014191-0045proportional to ctDNA. The final list of tissue-derived and plasma-refined loci is provided in Table 1, below. Each of the 43 loci listed below were found to have robust signal. The listed loci also included many genes that are known to be associated with AR activity (including, e.g., KLK3, STEAP2, and others).Table 1: Exemplary genomic loci for use in an AR activity score generator.Page 108 of 13113185130vlAttorney Docket: 2014191-0045Measuring AR activity

[0351] Sequencing data obtained from plasma samples with a high ctDNA fraction (10% or greater, n=19) obtained from PRAD subjects was diluted in silico to generate a collection of in silico plasma samples having different ctDNA%. In brief, sequencing data from each plasma data was diluted with different amounts of sequencing data from health subjects, with a lowest ctDNA fraction of 0.25%.

[0352] Fig. 2 shows mean number of fragments detected vs. ctDNA fraction for each genomic loci identified using tissue samples. As shown, the mean number of fragments increases as ctDNA increases, as expected.

[0353] To correct for ctDNA fraction, the sum of H3K27ac fragments across loci was divided by the estimated ctDNA fraction. Results are shown in Fig. 3. As shown, at very low ctDNA levels, where fragments become sparse, simple division can over-amplify AR activity scores (see Fig. 3, left plot). To address this over-amplification, ISP scores were refined so as to remove the ctDNA relationship with mean and standard deviation (see Fig. 3, right plot). For the post-hoc ctDNA refinement, a loess fit was used to first correct for the mean AR activity score vs ctDNA so that it would always be 0, and then another loess fit was used to adjust the standard deviation of the AR activity score vs ctDNA so that it would always be 1. As a result, through ctDNA values, the mean and standard deviation were always 1 and comparable across different ctDNA values.Page 109 of 13113185130vlAttorney Docket: 2014191-0045

[0354] An AR activity score was calculated for each sample by dividing the mean number of fragments across genomic loci and dividing by ctDNA fraction.

[0355] For assessing AR activity score performance, plasma samples from subjects having PRAD and having a high concentration of ctDNA (10% or higher) were used to generate ISP samples, diluting down to an in silico ctDNA fraction of 0.25% using sequencing data from healthy plasma samples. AR activity scores determined at lower ctDNA fractions were then compared back to the undiluted ctDNA score, and fidelity was calculated by R2 (1 = perfect, 0 = no better than the average score, < 0 = worse than average). This analysis was performed in a LOOCV fashion (patient / healthy is left out, features are refined and then used to compute R2 on held-out patient across ctDNA).

[0356] Results are shown in Fig. 4. As shown, an R2 of 0.75 was achieved at -1.7% ctDNA. Fig. 5 shows predicted AR activity score vs. AR activity score at 10% or higher at each ctDNA fraction tested.

[0357] Lastly, AR activity scores were determined for samples from NSCLC, NEPC, and PRAD subjects. Results are shown in Fig. 6, which shows AR activity score expressed as Z-scores relative to PRAD measurements, where a Z-score of 0 indicates an average measurement in PRAD subjects. PRAD is associated with increased AR activity as compared to NSCLC and NEPC. As shown, measured AR activity scores were highest in PRAD samples, demonstrating that methods provided herein measure AR activity.Example 3: AR Activity Score is Predictive of Responsiveness to a PSMA-targeted agent.

[0358] The present Example provides further data demonstrating that technologies described herein can be used to measure AR activity in cancer patients. The present Example also provides clinical data showing that AR activity measured using technologies described herein can be used to provide clinically actionable insights. In particular, the present Example provides data demonstrating that AR activity can be used as a metric for assessing patient suitability for treatment with a PSMA-targeted agent (177Lu-PSMA-617 in the present Example).Background

[0359] Metastatic castration-resistant prostate cancer (mCRPC) is an advanced stage of prostate cancer with poor outcomes and limited therapeutic options. The FDA-approved targeted radionuclide therapy lutetium (177Lu) vipivotide tetraxetan (PLUVICTO®) is a radioligand that Page 110 of 13113185130vlAttorney Docket: 2014191-0045targets PSMA, offering a novel approach for mCRPC treatment. However, response to the therapy is heterogeneous, and resistance mechanisms remain poorly understood. Determining molecular signatures that are associated with different clinical outcomes would provide important insights into predicting response and resistance to therapy, and would be useful, e.g., in therapy selection and treatment of patients. The multimodal epigenomic liquid biopsy platform described in Examples 1 and 2 of the present disclosure was used to profile tumorspecific transcriptional activation and resistance pathways in plasma from mCRPC and identify features associated with therapeutic response to treatment with177Lu-PSMA-617.Methods

[0360] Baseline plasma samples were collected from patients with mCRPC at the time of PSMA PET imaging and prior to initiation of177Lu-PSMA-617 therapy. Epigenomic profiling of genome-wide signals from promoters, enhancers, and DNA methylation was performed on 1 m of plasma collected from each patient (N=85, ctDNA > 0.5%).

[0361] AR score was determined using the methods described in Example 1 and 2 of the present disclosure for all mCRPC samples that passed certain quality control criteria, and which had >1.7% ctDNA (N=70). AR score was also determined for cohorts of NSCLC and NEPC plasma samples for comparison, neither of which are known to be associated with elevated AR activity.

[0362] To test the association of AR score with clinico-radiologic progression-free survival (CR-PFS), which is a measure of patient response to treatment based on clinical and / or radiological evidence, samples having >3% ctDNA (N=70) were split into AR-high and AR-low groups using the 75th percentile of AR scores within the cohort as a threshold. A Cox proportional hazards model (CoxPH) was then fit using the AR-high vs. AR-low groups as a covariate to determine the difference in CR PFS between patients with high and low AR activity scores, respectively.Results

[0363] Measured AR scores are shown in Fig. 7A. As shown, AR activity scores were found to be higher in patients with PRAD as compared to NEPC and NSCLC. This observation is consistent with the known role that AR activity places in these three cancers, demonstrating that technologies provided herein can provide accurate measures of AR activity in cancer cells.Page lll of 13113185130vlAttorney Docket: 2014191-0045

[0364] Next, association between AR activity measurements and clinical outcomes (CR PFS) was tested. Patients were stratified into AR-high (top quartile) and AR-low (bottom three quartiles) groups, and this stratification was used to fit a CoxPH model based on CR-PFS.Results are shown in Fig. 7B. Patients in the AR-high group exhibited worse response to177Lu-PSMA-617 compared to patients in the AR-low group (HR = 1.98, P-value = 0.025). These data demonstrated that technologies described herein can be used to identify patients with a higher likelihood of responding more favorably to treatment with a PSMA-targeted agent.Conclusions

[0365] Comprehensive epigenomic profiling of plasma cfDNA enables a minimally invasive characterization of response and resistance mechanisms to177Lu-PSMA-617 in mCRPC. By providing real-time insights into tumor biology and therapeutic efficacy, this platform supports precision medicine approaches for optimizing outcomes in PSMA-targeted therapies.

[0366] The data provided in the present Example also demonstrated that lower AR pathway activity is associated with improved response to177Lu-PSMA-617, revealing a potential mechanism of response and resistance to PSMA-targeted therapies in mCRPC and also a metric that can be used to better inform therapy selection and identify patients that are more likely to exhibit superior clinical outcomes as compared to the general population of mCRPC patients.Example 4: Further Characterization of AR Activity Score

[0367] The present Example demonstrates that technologies described herein can be used to further characterize an AR Activity Score described herein. In particular, the present Example provides methods for assessing AR activity in cancer cell lines based on AR-targeting treatment.

[0368] Prostate cancer cell lines including AR-dependent cell lines (e.g., those that require sustained AR signaling for survival, growth and / or proliferation and AR-independent cell lines (e.g., those that do not depend on AR signaling) may be used. Each prostate cancer cell line used in this study is treated with (i) an AR inhibitor, (ii) an androgen deprivation therapy, or (iii) an exogenous androgen. (Different samples from a single cell line may be used with each treated with different ones of (i), (ii), and (iii).) DNA is extracted from treated cells and subjected toPage 112 of 13113185130vlAttorney Docket: 2014191-0045ChTP-seq and DNA Methylation analysis as described in Example 1. An AR Activity Score is estimated as described in Example 2.

[0369] Without wishing to be bound by any particular theory, an AR activity score may increase in AR-dependent cell lines with an exogenous androgen treatment and decrease when AR-dependent cells are treated with either an AR inhibitor or an androgen deprivation therapy. Without wishing to be bound by any particular theory, no (e.g., statistically significant) change in AR activity may be observed in AR-independent cells upon treatment with any one of an AR inhibitor, an androgen deprivation therapy, or an exogenous androgen (e.g., for one or more of the tested cell lines). Alternatively or additionally, an opposite effect may be observed in AR-independent cells, e.g., exogenous androgen treatment may result in a decrease in an AR activity score, whereas AR blockade or deprivation may result in an increase in an AR activity score (e.g., depending on cell line).

[0370] As demonstrated by the present example, AR activity scores disclosed herein may be used to characterize activity of AR in its role in promoting gene expression within an AR pathway in AR-dependent cancer cells. As demonstrated by the present examples, AR activity scores disclosed herein may be particularly useful in identifying patients that may respond to an AR-targeted treatment based on their AR activity.OTHER EMBODIMENTS

[0371] It will be appreciated that the scope of the present disclosure is to be defined by that which may be understood from the disclosure and claims rather than by the specific embodiments that have been presented by way of example. Elements described with respect to one aspect or embodiment of the present disclosure are also contemplated with respect to other aspects or embodiments of the present disclosure. For example, elements of claims that depend directly or indirectly from a certain independent claim presented herein serve as support for those elements being presented in additional dependent claims of one or more other independent claims. Throughout the description, where compositions or methods are described as having, including, or comprising specific elements, it is to be understood that compositions or methods that consist essentially of, consist of, or do not comprise the recited elements are likewise hereby disclosed. All references cited herein are hereby incorporated by reference.Page 113 of 13113185130vl

Claims

Attorney Docket: 2014191-0045CLAIMSWhat is claimed is:

1. A method of measuring androgen receptor (AR) activity of a cancer in a subject, the method comprising:quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject:(i) one or more histone modifications,(ii) chromatin accessibility,(iii) binding of one or more transcription factors, and / or(iv) DNA methylation.

2. The method of claim 1, wherein the one or more histone modifications are quantified using a histone modification assay that measures H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, H3K4me3, or pan-acetylation.

3. The method of claim 2, wherein the histone modification assay detects H3K4me3 modifications.

4. The method of claim 2, wherein the histone modification assay detects H3K27ac modifications.

5. The method of any one of claims 2-4, wherein the histone modification assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

6. The method of any one of claims 1-5, wherein chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing),Page 114 of 13113185130vlAttorney Docket: 2014191-0045FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and a DNase hypersensitivity assay.

7. The method of any one of claims 1-6, wherein the binding of one or more transcription factors is quantified using a transcription factor binding assay that detects binding of one or more of p300, mediator complex, cohesion complex, RNA pol II, F0XA1, ESRI, PR, MYC, EN1, F0XM1, KLF4, AP-2, RARa, orRUNXl.

8. The method of claim 7, wherein the transcription factor binding assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

9. The method of any one of claims 1-8, wherein DNA methylation is quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq).

10. The method of any one of claims 1-9, comprising quantifying two or more of the following, each at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject:(i) one or more histone modifications,(ii) chromatin accessibility,(iii) transcription factor binding, and / or(iv) DNA methylation.

11. The method of claim 10, comprising quantifying:(i) H3K27ac modifications and H3K4me3 modifications,(ii) H3K27ac modifications and DNA methylation,(iii) H3K4me3 modifications and DNA methylation, orPage 115 of 13113185130vlAttorney Docket: 2014191-0045(iv) H3K27ac modifications, H3K4me3 modifications, and DNA methylation.

12. The method of any one of claims 1-11, wherein the liquid biopsy sample is a plasma sample, serum sample, or urine sample.

13. The method of any one of claims 1-12, comprising quantifying:(i) one or more histone modifications at one or more regulatory regions (e.g., promoter or enhancer regions or subregions) associated with one or more of the genes listed in Table 1,(ii) chromatin accessibility at one or more of the genes listed in Table 1,(iii) binding of one or more transcription factors associated with promoting expression of one or more of the genes listed in Table 1, and / or(iv) DNA methylation at one or more of the genes listed in Table 1.

14. The method of claim 13, comprising quantifying enhancer signal at one or more enhancer regions or subregions associated with one or more of the genes listed in Table 1 (e g., quantifying enhancer signal at one or more of the loci listed in Table 1).

15. The method of claim 13 or 14, comprising quantifying promoter signal at a promoter for one or more of the genes listed in Table 1).

16. The method of any one of claims 13-15, wherein enhancer signal comprises H3K27ac modifications.

17. The method of any one of claims 14-16, wherein promoter signal comprises H3K4me3 modifications.

18. A method of measuring AR activity of a cancer (e.g., PRAD) in a subject, comprising: obtaining a biological sample comprising cell-free DNA (cfDNA), optionally a liquid biopsy sample, from the subject, andPage 116 of 13113185130vlAttorney Docket: 2014191-0045determining an AR activity score for the sample, wherein the AR activity score is determined by a method comprising measuring enhancer or promoter signal at one or more loci that have previously been determined to have increased enhancer or promoter signal in an AR+ cancer as compared to one or more non-cancerous samples (e.g., one or more plasma or tissue samples obtained from a subject not diagnosed with cancer).

19. The method of claim 18, wherein the one or more loci with increased enhancer or promoter signal show no correlation with ERG expression.

20. The method of claim 18 or 19, wherein the enhancer signal is measured at one or more loci that have previously been determined to have increased enhancer signal and (i) that overlap with AR ChlP-seq sites having an AR motif or (ii) that are highly correlated with AR motif sites.

21. The method of any one of claims 18-20, wherein the method comprises measuring enhancer signal at two or more loci with increased enhancer or promoter signal in AR+ cancer, and combining (e.g., summing, averaging, geometric mean averaging, or taking the median of) the enhancer or promoter signal measured at the two more loci.

22. The method of any one of claims 18-21, wherein the method for determining AR activity score comprises correcting the measured enhancer or promoter signal for ctDNA fraction (e.g., dividing enhancer or promoter signal by ctDNA fraction).

23. The method of any one of claims 18-22, wherein the one or more loci are within one or more enhancer regions for one or more of the genes listed in Table 1.

24. The method of any one of claims 18-23, wherein the one or more loci are within one or more promoter regions for one or more of the genes listed in Table 1.

25. The method of any one of claims 18-24, comprising measuring enhancer signal for at least 1, 5, 10, 20, 30, or 40 of the loci listed in Table 1.Page 117 of 13113185130vlAttorney Docket: 2014191-004526. The method of any one of claims 18-25, wherein enhancer signal and / or promoter signal in the liquid biopsy sample is measured using a method that comprises sequencing cfDNA comprising one or more histone modifications (e.g., H3K4me3 and / or H3K27ac), e.g., using cfChlP-seq.

27. The method of claim 26, wherein the sequence reads at each genomic loci are processed prior to combining with sequence reads at other genomic loci (e.g., quantile normalized and / or adjusted for background signal).

28. The method of any one of claims 18-27, wherein the liquid biopsy sample is a plasma sample, serum sample, or urine sample.

29. The method of any one of claims 18-28, wherein the cancer is determined to have elevated AR activity (e.g., as compared to AR activity in a subject not diagnosed with cancer and / or a cancer that does not have elevated AR activity) if the AR activity score is equal to or greater than an AR activity reference value.

30. The method of claim 29, wherein the AR activity reference value is a predetermined threshold value and / or a normalized value.

31. The method of claim 29 or 30, wherein the AR activity reference value is an AR activity score determined in a reference subject or population of subjects.

32. The method of claim 31, where the reference subject or population of subjects is:(i) a subject or population of subjects that have not been diagnosed with cancer, (ii) a subject or population of subjects that have been diagnosed with a cancer that does not have elevated AR expression,(iii) a subject or population of subjects that have been diagnosed with an AR+ cancer (e.g., PRAD) and which were found to not respond to treatment with an antiandrogen,Page 118 of 13113185130vlAttorney Docket: 2014191-0045(iv) a subject or population of subjects that have been diagnosed with an AR+ cancer (e g., PRAD) and which were found to respond to treatment with an antiandrogen, or(v) a subject or population of subjects having mCRPC.

33. The method of any one of claims 29-32, wherein the AR activity reference value is the AR activity score that corresponds to the median, lower bound of the top tertile, lower bound of the top quartile, or lower bound of the top quintile of AR activity scores measured in a population of subjects that have been diagnosed with mCRPC.

34. The method of any one of claims 1-33, wherein the method further comprises measuring ctDNA fraction in the biological sample.

35. The method of claim 34, wherein the method comprises comparing the ctDNA fraction measured in the biological sample to a ctDNA fraction reference value.

36. The method of claim 35, wherein the ctDNA fraction reference value is a predetermined threshold value and / or a normalized value.

37. The method of claim 36, wherein the ctDNA fraction reference value is a ctDNA fraction value determined in a reference subject or population of subjects.

38. The method of claim 37, where the reference subject or population of subjects is:(i) a subject or population of subjects that have not been diagnosed with cancer, (ii) a subject or population of subjects that have been diagnosed with a cancer that does not have elevated AR expression, or(iii) a subject or population of subjects having mCRPC.

39. The method of any one of claims 36-38, wherein the ctDNA fraction reference value is the ctDNA fraction that corresponds to the median, lower bound of the top tertile, lower bound of the top quartile, or lower bound of the top quintile of ctDNA fractions measured in aPage 119 of 13113185130vlAttorney Docket: 2014191-0045population of subjects that have been diagnosed with mCRPC.

40. A method of treating a subject having a cancer, determining whether a subject having a cancer is likely to respond to a therapy, predicting whether a subject having a cancer is likely to respond to a therapy, and / or selecting a therapy for a subject having a cancer, wherein the method comprises measuring AR activity using the method of any one of claims 1-39.

41. A method of treating a subject having a cancer, the method comprising:(i) obtaining a biological sample comprising cell-free DNA (cfDNA), optionally a liquid biopsy sample, from the subject,(ii) measuring AR activity in the biological sample, wherein AR activity is measured using a method of any one of claims 18-40, and(iii) administering an antiandrogen to the subject if AR activity is greater than or equal to an AR activity reference value and not administering an antiandrogen to the subject if the AR activity is less than the AR activity reference value.

42. The method of claim 41, wherein the AR activity reference value is an AR activity score measured in:(i) a healthy subject or population of healthy subjects,(ii) a subject or population of subjects having a cancer previously shown to respond to treatment with the antiandrogen, or(iii) a subject or population of subjects having a cancer previously shown to not respond to treatment with an antiandrogen (e.g., an AR antagonist).

43. The method of claim 41 or 42, wherein the AR activity reference value is an AR activity score is that is at least about 1.2, 1.4, 1.6, 1.8, 2, 2.5, 3, 4, or 5 times higher than an AR activity score measured in (i) a healthy subject or population of healthy subjects or (ii) a subject or population of subjects that have been diagnosed with cancer that does not have elevated AR expression.Page 120 of 13113185130vlAttorney Docket: 2014191-004544. A method of treating a subject having a cancer, the method comprising:(i) obtaining a biological sample comprising cell-free DNA (cfDNA), optionally a liquid biopsy sample, from the subject,(ii) measuring AR activity in the biological sample, wherein AR activity is measured using a method of any one of claims 18-39, and(iii) administering a PSMA-targeted agent to the subject if AR activity is less than an AR activity reference value and not administering a PSMA-targeted agent to the subject if the AR activity is equal to or greater than the AR activity reference value.

45. The method of claim 44, wherein the AR activity reference value is an AR activity score measured in:(i) a healthy subject or population of healthy subjects,(ii) a subject or population of subjects having prostate cancer (e g., mCRPC), (iii) a subject or population of subjects having a cancer previously shown to respond to treatment with the PSMA-targeted agent, or(iv) a subject or population of subjects having a cancer previously shown to not respond to treatment with the PSMA-targeted agent.

46. The method of claim 44 or 45, further comprising measuring ctDNA fraction in the biological sample.

47. The method of claim 46, wherein:(i) the PSMA-targeted agent is administered to the subject if the AR activity measured in the sample is less than the AR activity reference value and the ctDNA fraction measured in the sample is greater than or equal to a ctDNA fraction reference value, and (ii) the PSMA-targeted agent is not administered to the subject if the AR activity measured in the sample is equal to or greater than the AR activity reference value or the ctDNA fraction is less than the ctDNA fraction reference value.

48. The method of claim 47, wherein the AR activity reference value is an AR activity scorePage 121 of 13113185130vlAttorney Docket: 2014191-0045value measured in a population of subjects with mCRPC and wherein the ctDNA fraction reference value is a ctDNA fraction value measured in a population of subjects with mCRPC.

49. The method of claim 48, wherein:(i) the ctDNA fraction reference value is the median, lower bound of the top tertile, lower bound of the top quartile, or lower bound of the top quintile of ctDNA fraction measured in the population of subjects with mCRPC, and(ii) the AR activity reference value is the median, lower bound of the top tertile, lower bound of the top quartile, or lower bound of the top quintile of ctDNA% measured in the population of subjects with mCRPC.

50. The method of any one of claims 44-49, wherein the AR activity reference value is an AR activity score that is at least about 1.2, 1.4, 1.6, 1.8, 2, 2.5, 3, 4, or 5 times higher than an AR activity score determined in a healthy subject or population of healthy subjects or a subject or population of subjects that have been diagnosed with cancer that does not have elevated AR expression.

51. The method of any one of claims 40-50, wherein the subject has previously been diagnosed with cancer.

52. The method of claim 51, wherein the cancer is prostate cancer, breast cancer, laryngeal carcinoma, bladder cancer, or hepatocellular carcinoma (HCC), optionally wherein the cancer is prostate adenocarcinoma (PRAD) or AR+ breast cancer.

53. The method of claim 52, wherein the PRAD is metastatic castration resistant prostate cancer (mCRPC).

54. The method of claim 52 or 53, wherein AR expression status is determined using an immunohistochemistry (IHC) assay.Page 122 of 13113185130vlAttorney Docket: 2014191-004555. A method of monitoring cancer (e.g., AR+ cancer) in a subject, comprising measuring AR activity of the cancer using the method of any one of claims 1-39 at a first and a second time point.

56. The method of claim 55, wherein (i) the subject has been administered an antiandrogen therapy prior to or at the first time point or after the first time point and before the second time point, or (ii) the subject has been administered a PSMA-targeted agent prior to or at the first time point or after the first time point and before the second time point.

57. A method of determining the likelihood that a subject will respond to treatment with an antiandrogen, comprising measuring AR activity of the cancer using the method of any one of claims 1-39 at a first and a second time point.

58. The method of claim 57, wherein the subject was administered androgen depletion therapy (ADT) before the first time point or between the first time point and the second time point.

59. The method of claim 58, wherein the subject has an increased likelihood of responding to treatment with an antiandrogen therapy if AR activity increases between the first and the second time point.

60. The method of claim 57, wherein the subject has been administered an antiandrogen therapy prior to the first time point or after the first time point and before the second time point.

61. The method of claim 60, wherein the subject is less likely to respond to continued treatment with an antiandrogen therapy if AR activity increases or stays approximately the same between the first time point and the second time point.

62. A method of determining the likelihood that a subject will respond to treatment with a PSMA-targeted agent, comprising measuring AR activity of the cancer using the method of anyPage 123 of 13113185130vlAttorney Docket: 2014191-0045one of claims 1-39 at a first and a second time point.

63. The method of claim 62, wherein the subject was administered a PSMA-targeted agent before or at the first time point or between the first time point and the second time point.

64. The method of claim 62 or 63, wherein the subject has a decreased likelihood of responding to treatment with a PSMA-targeted agent if AR activity increases between the first and the second time point.

65. The method of claim 62 or 63, wherein the subject has an increased likelihood of responding to continued treatment with the PSMA-targeted agent if AR activity decreases or stays approximately the same between the first time point and the second time point.

66. A method of treating a cancer in a subject, the method comprising measuring AR activity of the cancer using the method of any one of claims 1-39 at a first and a second time point.

67. The method of claim 66, wherein the subject was administered ADT before the first time point or between the first time point and the second time point.

68. The method of claim 66, wherein:(i) if AR activity increases between the first and the second time point, the method comprises administering an antiandrogen, and(ii) if AR activity decreases or stays the same between the first and the second time point, the method does not comprise administering an antiandrogen.

69. The method of claim 66 or 67, wherein the subject was administered an antiandrogen therapy prior to the first time point or after the first time point and before the second time point.

70. The method of claim 69, comprising administering an antiandrogen therapy to the subject based on the change in AR activity between the first time point and the second time point,Page 124 of 13113185130vlAttorney Docket: 2014191-0045optionally wherein the type, dose, and / or frequency of administration of the antiandrogen therapy is adjusted based on the change in AR activity.

71. The method of claim 69, wherein the method comprises administering the antiandrogen therapy after the second time point if AR activity decreases between the first time point and the second time point, optionally wherein the dose of the antiandrogen therapy administered after the second time point is the same as the dose administered before the second time point.

72. The method of claim 69, wherein, if AR activity increases or stays approximately the same between the first time point and the second time point, the method comprises:(i) increasing the amount of antiandrogen administered to the subject,(ii) administering a different antiandrogen to the subject, and / or(iii) administering a different cancer therapy to the subject (e.g., a therapy that does not comprise administering an AR-targeted therapy to the subject).

73. The method of claim 69, wherein the method does not comprise administering an antiandrogen therapy if AR activity increases between the first time point and the second time point.

74. The method of claim 66, wherein the subject was administered a PSMA-targeted agent at or before the first time point or between the first time point and the second time point.

75. The method of claim 74, wherein:(i) if AR activity increases between the first and the second time point, the method does not comprise administering a PSMA-targeted agent at or subsequent to the second time point, and(ii) if AR activity decreases or stays the same between the first and the second time point, the method comprises administering a PSMA targeted agent at or subsequent to the second time point.Page 125 of 13113185130vlAttorney Docket: 2014191-004576. The method of claim 74 or 75, comprising administering a PSMA-targeted agent to the subject based on the change in AR activity between the first time point and the second time point, optionally wherein the type, dose, and / or frequency of administration of the PSMA-targeted agent is adjusted based on the change in AR activity.

77. The method of claim 76, wherein the method comprises administering the PSMA-targeted agent after the second time point if AR activity decreases or stays the same between the first time point and the second time point, optionally wherein the dose of the PSMA-targeted agent administered after the second time point is the same as the dose administered before the second time point.

78. The method of claim 76, wherein, if AR activity increases between the first time point and the second time point, the method comprises increasing the amount of PSMA-targeted agent administered to the subject, administering a non-PSMA-targeted agent to the subject, and / or administering a different cancer therapy to the subject (e.g., a therapy that does not comprise administering a PSMA-targeted agent to the subject).

79. The method of claim 76, wherein the method does not comprise administering a PSMA-targeted agent if AR activity increases between the first time point and the second time point.

80. The method of any one of claims 1-79, wherein the cancer is prostate cancer, breast cancer, laryngeal carcinoma, bladder cancer, or hepatocellular carcinoma (HCC) optionally wherein the cancer is prostate adenocarcinoma (PRAD) or AR+ breast cancer.

81. The method of claim 80, wherein the prostate cancer is de novo mCSPC, non-metastatic CRPC, mCRPC in chemo naive patients, or mCRPC which has progressed after chemotherapy.

82. The method of claim 81, wherein AR expression status is determined using an IHC assay.Page 126 of 13113185130vlAttorney Docket: 2014191-004583. The method of any one of claims 41-43, 56-61, 68-73, or 80-82, wherein the antiandrogen is an AR antagonist (e.g., a molecule that binds the LBD of AR, including e.g., enzalutamide), an androgen synthesis inhibitor (e.g., abiraterone) or an AR degrader (e.g., an AR-targeted PROTAC), optionally wherein the androgen synthesis inhibitor is co-administered with a corticosteroid (e.g., prednisone).

84. The method of any one of claims 44-54, 56, 62-65, 74-82, wherein the PSMA-targeted agent is a radioligand (e.g.,177Lu-PSMA-617).

85. A method for testing the activity of a test compound, comprising incubating the test compound with a cell line, and measuring AR activity in the cell line subsequent to incubating the test compound with the cell line, wherein the AR activity is measured using the method of any one of claims 1-39.

86. The method of claim 85, where the cell line has measurable AR activity (e.g., the cell line has been incubated with a composition that increases AR signaling activity prior to incubating with the test compound).

87. The method of claim 85 or 86, wherein the activity is AR-targeting activity or PSMA-targeting activity.

88. A method of screening a library of test compounds for AR-targeting activity, comprising testing the activity of each test compound using the method of any one of claims 85-87.

89. A method of screening a library of test compounds for PSMA-targeting activity, comprising testing the activity of each test compound using the method of any one of claims 62-88.

90. A compound identified by the method of any one of claims 61-89.Page 127 of 13113185130vlAttorney Docket: 2014191-004591. A kit comprising reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from those listed in Table 1.

92. The kit of claim 90, wherein the kit comprises reagents for quantifying H3K27ac modifications for at least 1, 5, 10, 20, 30, 40, or 50 genomic loci listed in Table 1.

93. The kit of claim 90 or 91, wherein the kit comprises one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K27ac -modified histones.

94. The kit of any one of claims 91-93, wherein the kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample.

95. The kit of any one of claims 91-94, wherein the kit comprises reagents for library preparation for DNA sequencing.

96. The kit of any one of claims 91-95, wherein the kit comprises reagents for DNA sequencing.

97. A non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method of any one of claims 1-89.

98. A computer system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform the method of any one of claims 1-89.Page 128 of 13113185130vlAttorney Docket: 2014191-004599. A system for quantifying AR activity of a cancer in a subject, the system comprising a sequencer configured to generate a sequencing dataset from a sample, and a non-transitory computer readable storage medium of claim 97 and / or a computer system of claim 98.

100. The system of claim 99, wherein the sequencer is configured to generate a Whole Genome Sequencing (WGS) dataset from the sample.

101. The system of claim 99 or 100, further comprising a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample.

102. The system of claim 101, wherein the sample preparation device comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.

103. The system of claim 102, wherein the one or more genomic loci are selected from those listed in Table 1.

104. The system of any one of claims 101-103, wherein the device comprises reagents for quantifying H3K27ac modifications, e.g., reagents for quantifying H3K27ac modifications for at least 1, 5, 10, 20, 30, or 40 of the genomic loci listed in Table 1.

105. The system of claim 104, wherein the reagents comprise one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K27ac-modified histones.

106. The system of any one of claims 101-105, wherein the device comprises reagents for isolation of cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.Page 129 of 13113185130vlAttorney Docket: 2014191-0045107. The system of any one of claims 101-106, wherein the device comprises reagents for library preparation for sequencing.

108. The system of any one of claims 101-107, wherein the sequencer comprises reagents for sequencing.Page 130 of 13113185130vl

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