Methylation of the 5-alpha reductase 2 (SRD5a2) gene

Abstract

Provided herein are methods of treating a subject having benign prostatic hyperplasia that include, for example, identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; and administering to the identified subject a first therapy such as a combination therapy comprising a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator.

Description

[0001] METHYLATION OF THE 5-ALPHA REDUCTASE 2 (SRD5A2) GENE

[0002] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under Grant No. DK124502 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] TECHNICAL FIELD

[0005] The present disclosure relates to the treatment of benign prostatic hyperplasia, for example, treatment of benign prostatic hyperplasia in subjects having elevated levels of methylation of SRD5A2.

[0006] BACKGROUND

[0007] Benign prostatic hyperplasia (BPH) is a histologic diagnosis that refers to the proliferation of smooth muscle and epithelial cells within the prostatic transition zone that is likely the result of a multifactorial process. BPH is a significant health concern, being the most common proliferative pathology in men. Its prevalence increases with age, causing lower urinary tract symptoms (LUTS) in 50% of men by age 60 and in over 90% by age 80. BPH profoundly affects the quality of life for approximately 210 million men worldwide, decreasing the quality of life for men and imposing a financial burden of over $4 billion annually on the US healthcare system. The primary treatment options for BPH include medical therapy, mainly through steroid 5-alpha reductase (SRD5A2) inhibitors (5ARI) or alpha-adrenergic blockers, with surgical intervention reserved for severe or unresponsive cases.

[0008] Despite the general effectiveness of 5ARI therapy, about 34% of men exhibit resistance to this treatment. Identifying men who are likely to respond is crucial to prevent exposing others to the side effects of an ineffective treatment or progressing to needing surgery.

[0009] SUMMARY

[0010] Provided herein are methods of treating a subject having benign prostatic hyperplasia, the methods comprising: (a) determining whether a white blood cell in a sample obtained from the subject has an elevated level of methylation of SRD5A2 as compared to a reference level; and (b)(i) administering a first therapy (e.g., any of the first therapies described hereinjto the subject if the subject has a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; or (b)(ii) administering a therapeutically effective amount of a second therapy (e.g., any of the second therapies described herein) to the subject if the subject has a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level.

[0011] In some embodiments, provided herein are methods of treating a subject having benign prostatic hyperplasia, the methods comprising: (a) determining that a white blood cell in a sample obtained from the subject has an elevated level of methylation of SRD5A2 as compared to a reference level; and (b) administering a first therapy to the subject. In some embodiments, provided herein are methods of treating a subject having benign prostatic hyperplasia, the methods comprising: (a) determining that a white blood cell in a sample obtained from the subject does not have an elevated level of methylation of SRD5A2 as compared to a reference level; and (b) administering a second therapy to the subject.

[0012] Also provided herein are methods of treating benign prostatic hyperplasia in a subject, the method comprising: identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; and administering to the identified subject a first therapy.

[0013] Also provided herein are methods of treating benign prostatic hyperplasia in a subject, the method comprising administering a first therapy to a subject identified as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level.

[0014] Also provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising: identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; and selecting for the identified subject a first therapy.

[0015] Also provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising selecting a first therapy for a subject identified as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level.

[0016] Also provided herein are methods method of treating a subject having benign prostatic hyperplasia, the method comprising: (a) administering to the subject a 5-alpha reductase inhibitor; (b) after (a), identifying the subject as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; and (c) administering to the identified subject a first therapy.

[0017] Also provided herein are methods of treating a subject having benign prostatic hyperplasia, the method comprising: identifying a subject previously administered a 5- alpha reductase inhibitor, as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; and administering to the identified subject a first therapy.

[0018] Also provided herein are methods of treating a subject having benign prostatic hyperplasia, the method comprising administering to a subject previously administered a 5-alpha reductase inhibitor and later identified as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level, a first therapy.

[0019] Also provided herein are methods of treating benign prostatic hyperplasia in a subject, the method comprising: identifying a subject having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level; and administering to the identified subject a therapeutically effective amount of a second therapy.

[0020] Also provided herein are methods of treating benign prostatic hyperplasia in a subject, the method comprising administering a therapeutically effective amount of a second therapy to a subject identified as having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level.

[0021] Also provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising: identifying a subject having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level; and selecting for the identified subject a treatment comprising a therapeutically effective amount of a second therapy. Also provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising selecting a treatment comprising a therapeutically effective amount of a second therapy for a subject identified as having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level.

[0022] In some embodiments, the step of determining whether a white blood cell in a sample obtained from the subject has an elevated level of methylation of SRD5A2 as compared to a reference level comprises performing an assay to detect methylation of SRD5A2 in DNA from the white blood cell. In some embodiments, the step of identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level comprises performing an assay to detect methylation of SRD5A2 in DNA from the white blood cell from the subject.

[0023] In some embodiments, the step of identifying a subject having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level comprises performing an assay to detect methylation of SRD5A2 in DNA from the white blood cell from the subject.

[0024] In some embodiments, the assay comprises bisulfite conversion, differential enzymatic cleavage, or affinity capture. In some embodiments, the assay comprises sequencing.

[0025] In some embodiments, the methods provided herein further comprise obtaining a blood sample from the subject.

[0026] In some embodiments, methylation of SRD5A2 is detected in the promoter region of SRD5A2. In some embodiments, methylation of SRD5A2 is detected in one or more CpG islands in SRD5A2. In some embodiments, the one or more CpG islands comprise CpG - 72 to -42; CpG -40 to -35 / CpG -31 to -19; CpG -34 to -32 / CpG -18 to 65; or combinations thereof. In some embodiments, the one or more CpG islands comprises CpG -39 to -2. In some embodiments, the one or more CpG islands comprise CpG -28 to -23; CpG -22 to -20; CpG -7 to -4; or combinations thereof.

[0027] In some embodiments, the reference level is the average percentage of methylation of SRD5A2 in prostate tissue that expresses SRD5A2. In some embodiments, the elevated level of methylation of SRD5A2 is an increase in methylation compared to the reference level of at least about 4%, at least about 5%, or at least about 10%.

[0028] In some embodiments, the elevated level of methylation of SRD5A2 as compared to a reference level comprises an elevated level of methylation of the promoter region of SRD5A2. In some embodiments, the elevated level of methylation of SRD5A2 as compared to a reference level comprises an elevated level of methylation of one or more CpG islands in SRD5A2.

[0029] In some embodiments, the first therapy is a combination therapy comprising a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator.

[0030] In some embodiments, the second therapy is a 5-alpha reductase inhibitor (e g., a therapeutically effective amount of a 5-alpha reductase inhibitor). In some embodiments, the second therapy comprises a 5-alpha reductase inhibitor and an a-adrenergic blocker (e.g., a therapeutically effective amount of a 5-alpha reductase inhibitor and an a- adrenergic blocker).

[0031] In some embodiments, the 5-alpha reductase inhibitor is selected from the group consisting of: finasteride, dutasteride, alfatradiol, and epristeride. In some embodiments, the 5-alpha reductase inhibitor is finasteride.

[0032] In some embodiments, the selective estrogen receptor modulator is selected from the group consisting of: tamoxifen, raloxifene, toremifene, ospemifene, bazedoxifene, anordrin, broparestrol, clomifene, cyclofenil, lasofoxifene, ormeloxifene, acolbifene, afimoxifene (4-hydroxytamoxifen), elacestrant, enclomifene ((E)-clomifene), endoxifen (4-hydroxy-N-desmethyltamoxifen), and zuclomifene ((Z)-clomifene). In some embodiments, the selective estrogen receptor modulator is raloxifene.

[0033] Definitions

[0034] As used herein, the term “administration” can refer to the administration of a composition to a subject or system to achieve delivery of an active agent . Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, or topical. In some embodiments, an active agent described herein can be administered orally. Examples of compositions suitable for oral administration include capsules, sachets, granules, tablets, powders, solutions or suspensions in a liquid, oil-in-water liquid emulsions, water-in-oil liquid emulsions, packed in liposomes, and boluses. In some embodiments, an active agent may be contained in a composition such that it is suitable for oral administration, for example, by combining the active agent with an inert diluent or an assimilable edible carrier.

[0035] As used herein, the terms “effective amount” and “effective to treat” can refer to an amount of a compound utilized for a period of time (e.g., acute or chronic administration, periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome. Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen. Further, the dose to be administered can vary depending upon the age, weight, and general condition of the patient as well as the severity of the condition being treated, the judgment of the healthcare professional, and the particular mode of administration.

[0036] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification to describe an organism, typically a mammal, human or nonhuman, to whom treatment according to the methods of the present disclosure is provided. Veterinary applications are contemplated by the present disclosure. The terms include, but are not limited to, mammals, e.g., humans, other primates, pigs, hamsters, mice, rats, cows, horses, cats, dogs, sheep, and goats. In some embodiments, a subject is suffering from a relevant disease, disorder, or condition. 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 does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0038] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0039] DESCRIPTION OF DRAWINGS

[0040] FIG. 1A is a plot showing 5-year change in AUA symptom score versus SRD5A2 Expression; IRS= Immunoreactivity Score.

[0041] FIG. IB is a plot showing methylation of SRD5A2 in white blood cells versus prostate tissue.

[0042] FIGS. 2A, 2B, 2C and 2D are plots showing variable expression of SRD5A2 in human prostate. Expressers (FIGS. 2A and 2C), and non-expressers (FIGS. 2B and 2D) are demonstrated with immunohistochemistry (FIGS. 2A and 2B) and confocal microscopy (FIGS. 2C and 2D).

[0043] FIG. 3 is a schematic showing TNF-a, NF-KB, and IL-6 lead to upregulation of DNMT1 and methylation of SRD5A2 promoter and suppression of gene expression.

[0044] FIG. 4 is a plot showing CpG hotspots on the promoter region of SRD5A2 that regulated expression of SRD5A2 gene and protein. Differential methylation of the three marked spots (blue arrows s-segments 1 to 3 are the most statistically differentially methylated regions) determine expression of SRD5A2.

[0045] FIG. 5 is a plot showing a microarray gene expression analysis of human prostate tissue in samples unmethylated versus methylated at the SRD5A2 promoter region. Estrogen response, hedgehog and MYC signaling genes are among the most significantly upregulated pathways in A7 / J5d2-mcthylatcd samples.

[0046] FIG. 6A is a representative figure for protein expression by immunoblot assay. Patients #1, 2, 3 represent SRD5A2 unmethylated samples and Patients #4, 5, 6 represent SRD5A2 methylated samples.

[0047] FIG. 6B is a representative image of immunohistochemical analyses showing increased pERa and aromatase when SRD5A2 expression is absent. In absence of SRD5A2, aromatase expression in the stroma is increased. Scale = 50 pm.

[0048] FIG. 7 is a schematic showing in absence of SRD5A2 there is an androgenic to estrogenic switch in the prostate gland.

[0049] FIG. 8A is a plot showing SRD5A2 expression versus total prostate volume for the Medical Therapy of Prostatic Symptoms (MTOPS) cohort.

[0050] FIG. 8B is a plot showing SRD5A2 expression versus transition zone volume for the MTOPS cohort.

[0051] FIG. 8C is a plot showing SRD5A2 expression versus total prostate volume for the Beth Israel Deaconess Medical Center (BIDMC) cohort.

[0052] FIG. 8D is a plot showing SRD5A2 expression versus transition zone volume for the BIDMC cohort.

[0053] DETAILED DESCRIPTION

[0054] Over 90% of adult males develop lower urinary tract symptoms (LUTS) secondary to bladder outlet obstruction by age 80, rendering benign prostatic hyperplasia (BPH) the most common proliferative abnormality in humans. LUTS secondary to BPH negatively impact the quality of life of 210 million men globally accounting for significant life years lost and coting the U.S. healthcare system over $4 billion per year. Medical therapy for the management of BPH, which can include a-adrenergic blockers and 5a reductase inhibitors (5ARI), can target both stromal and epithelial cells in the prostate gland. 5ARIs reduce prostate size for the alleviation of LUTS. Utilization of 5ARIs remains ineffective in many patients, often leading to invasive therapies for those patients. Resistance to 5ARI therapy is a major factor limiting the effectiveness of these agents in the management of BPH. The present disclosure is based, at least in part, on the finding that there is a significant correlation between the level of SRD5A2 methylation in prostate and in white blood cells (WBCs). Absence of SRD5A2 has been found in about 30% of adult prostate tissue that does not express SRD5A2 (about 30%) corresponds with the percentage of patients who are unresponsive to steroid 5-alpha reductase (SRD5A2) inhibitor (5ARI) therapies. The absence of SRD5A2 may account for lack of response to the 5ARIs, and it was also found that there is a correlation between 5ARI response and SRD5A2 expression.

[0055] It was also discovered that in the absence of androgenic signaling, an androgen- estrogen switch can occur in the prostate, influencing tissue survival and growth. This finding demonstrates the prostate's reliance on both androgenic and estrogenic signaling for growth and development. However, past BPH clinical trials have not explored a combined therapy approach targeting both androgenic and estrogenic signaling Current treatment strategies predominantly target only the androgenic pathway.

[0056] Accordingly, the present disclosure advantageously provides methods of identifying a subject having benign prostatic hyperplasia and an elevated level of methylation of SRD5A2 as likely being resistant to a 5-alpha reductase inhibitor. For example, such methods can include determining whether a white blood cell from the subject has an elevated level of methylation of SRD5A2 as compared to a reference level (e.g., any of the reference levels described herein), where an elevated level of methylation of SRD5A2 as compared to a reference level identifies that the subject will likely be resistant to an 5ARI therapy.

[0057] The present disclosure also advantageously provides methods of selecting a treatment for a subject identified or determined as having an elevated level of methylation of SRD5A2 in a white blood cell from the subject as compared to a reference level (e.g., any of the reference levels described herein). Some embodiments can further include administering the selected treatment to the subject identified or determined as having an elevated level of methylation of SRD5A2. In some embodiments, the present disclosure advantageously provides methods of treating benign prostatic hyperplasia in a subject identified or determined as having an elevated level of methylation of SRD5A2 in a white blood cell from the subject as compared to a reference level (e.g., any of the reference levels described herein). Methods of Treating

[0058] Provided herein are methods of identifying a subject having benign prostatic hyperplasia as likely being resistant to a 5-alpha reductase inhibitor, the methods comprising: (a) determining whether a white blood cell in a sample obtained from the subject has an elevated level of methylation of SRD5A2 as compared to a reference level (e.g., any of the reference levels described herein); and (b)(i) if the subject has a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level, identifying the subject as likely resistant to a 5-alpha reductase inhibitor; or (b)(ii) if the subject has a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level, identifying the subject as likely not resistant to a 5-alpha reductase inhibitor.

[0059] In some embodiments, provided herein are methods of treating a subject having benign prostatic hyperplasia, the methods comprising: (a) determining that a white blood cell in a sample obtained from the subject has an elevated level of methylation of SRD5A2 as compared to a reference level; and (b) administering a combination therapy to the subject, wherein the combination therapy comprises a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator. In some embodiments, provided herein are methods of treating a subject having benign prostatic hyperplasia, the methods comprising: (a) determining that a white blood cell in a sample obtained from the subject does not have an elevated level of methylation of SRD5A2 as compared to a reference level; and (b) administering a therapeutically effective amount of a 5-alpha reductase inhibitor to the subject.

[0060] Also provided herein are methods of treating a subject having benign prostatic hyperplasia, the methods comprising: (a) determining whether a white blood cell in a sample obtained from the subject has an elevated level of methylation of SRD5A2 as compared to a reference level (e.g., any of the reference levels described herein); and (b)(i) administering a first therapy (e.g., any of the first therapies described herein) to the subject if the subject has a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; or (b)(ii) administering a therapeutically effective amount of a second therapy (e.g., any of the second therapies described herein) to the subject if the subject has a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level.

[0061] Also provided herein are methods of treating benign prostatic hyperplasia in a subject, the methods comprising: identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level (e.g., any of the reference levels described herein); and administering to the identified subject a first therapy(e.g., any of the first therapies described herein). In some embodiments, provided herein are methods of treating benign prostatic hyperplasia in a subject, the methods comprising administering a first therapy (e.g., any of the first therapies described hereinjto a subject identified as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level (e.g., any of the reference levels described herein).

[0062] Also provided herein are methods of treating a subject having benign prostatic hyperplasia, the methods comprising: (a) administering to the subject a 5-alpha reductase inhibitor; (b) after (a), identifying the subject as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level (e.g., any of the reference levels described herein); and (c) administering to the identified subject a first therapy (e.g., any of the first therapies described herein).

[0063] In some embodiments, the subject was previously treated with a 5-alpha reductase inhibitor. Accordingly, also provided herein are methods of treating a subject having benign prostatic hyperplasia, the methods comprising: identifying a subject previously administered a 5-alpha reductase inhibitor as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level (e.g., any of the reference levels described herein); and administering to the identified subject a first therapy(e.g., any of the first therapies described herein). In some embodiments, provided herein are methods of treating a subject having benign prostatic hyperplasia, the methods comprising administering a first therapy to a subject previously administered a 5-alpha reductase inhibitor and later identified as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level.

[0064] In some embodiments, following administration of a first therapy, the subject exhibits a reduction in a lower urinary tract symptom (LUTS) relative to baseline, e.g., compared to before administration of the first therapy. LUTS can include symptoms associated with storage of urine, and / or with voiding or emptying. LUTS can be measured using, for example, one or more of a urinary symptom questionnaire, a urinalysis test, a post-void residual volume test, uroflowmetry, and a urodynamic pressure flow study.

[0065] In some embodiments, lower urinary tract symptoms are measured using a validated urinary symptom questionnaire such as the AUA Urinary Symptom Score or the International Prostatism Symptom Score (IPSS). For example, for the AUA Urinary Symptom Score, a subject can rate on a scale of 1 to 5, the following: (i) Incomplete emptying: Over the past month, how often have you had a sensation of not emptying your bladder completely after you finished urinating? (ii) Frequency: Over the past month, how often have you had to urinate again less than 2 hours after you finished urinating? (iii) Intermittency: Over the past month, how often have you found that you stopped and started again several times when you urinated? (iv) Urgency: Over the past month, how often have you found it difficult to postpone urination? (v) Weak-stream: Over the past month, how often have you had a weak urinary stream? (vi) Straining: Over the past month, how often have you had to push or strain to begin urination? and (vii) Nocturia: Over the past month, how many times did you most typically get up to urinate from the time you went to bed at night until the time you got up in the morning?, where 0 is none, 1 is less than 1 time in 5, 2 is less than half the time, 3 is about half the time, 4 is more than half the time, and 5 is almost always. The AUA Urinary Symptom Score can provide a score between 0 and 35, where 0 to 7 is considered mild, 8 to 19 is considered moderate, and 20 to 35 is considered severe. See, e.g., “AUA Guideline on Management of Benign Prostatic Hyperplasia (2003). Chapter 1 : Diagnosis and Treatment Recommendations,” Journal of Urology, 170(2): 530- 547 (2003).

[0066] The AUA Urinary Symptom Score can be used to demonstrate a change from baseline, e.g., a subject can exhibit a reduction in AUA Urinary Symptom Score compared to the subject’s AUA Urinary Symptom Score prior to treatment with a first or second therapy. In some embodiments, following administration of a first therapy, the subject exhibits a reduction in AUA Urinary Symptom Score, e.g., as compared to the subject’s AUA Urinary Symptom Score prior to administration of the first therapy. In some embodiments, a reduction in AUA Urinary Symptom Score relative to baseline is about 1 to about 35. For example, the reduction in AUA Urinary Symptom Score relative to baseline can be about 5 to about 35, about 10 to about 35, about 15 to about 35, about 20 to about 35, about 30 to about 35, about 1 to about 30, about 5 to about 30, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 1 to about 25, about 5 to about 25, about 10 to about 25, about 15 to about 25, about 20 to about 25, about 1 to about 20, about 5 to about 20, about 10 to about 20, about 15 to about 20, about 1 to about 15, about 5 to about 15, about 10 to about 15, about 1 to about 10, or about 5 to about 10.

[0067] Other tests that can be used to evaluate treatment of BPH include, but are not limited to, uroflowmetry, a urodynamic pressure flow study, and post-void residual volume (PVR). For example, Uroflowmetry can measure the urinary flow rate throughout the course of urination, urodynamic pressure flow studies can measure pressure in the bladder during urination, and a post-void residual volume (PVR) study (e.g., using ultrasonography or catheterization) can measure the volume of urine left in the bladder after urination. In some embodiments, following administration of a first therapy (e.g., any of the first therapies described herein) to a subject, the subject exhibits one or more of an increase in the rate of urine flow during urination relative to baseline, e.g., compared to before administration of the first therapy, improved pressure during urination relative to baseline, e.g., compared to before administration of the first therapy, and decreased PVR relative to baseline, e g., compared to before administration of the first therapy.

[0068] Also provided herein are methods of treating benign prostatic hyperplasia in a subject, the methods comprising: identifying a subject having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level; (e.g., any of the reference levels described herein) and administering to the identified subject a therapeutically effective amount of a second therapy (e.g., any of the second therapies described herein). In some embodiments, provided herein are methods of treating benign prostatic hyperplasia in a subject, the methods comprising administering a therapeutically effective amount of a second therapy (e.g., any of the second therapies described herein)to a subject identified as having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level. In some embodiments, the standard benign prostatic hyperplasia therapy includes an alpha- adrenergic blocker and / or a 5-alpha reductase inhibitor.

[0069] In some embodiments, following administration of a second therapy, the subject exhibits a reduction in lower urinary tract symptoms (LUTS) relative to baseline, e.g., compared to before administration of the second therapy. In some embodiments, following administration of a second therapy, the subject exhibits a reduction in AUA Urinary Symptom Score, e.g., as compared to the subject’s AUA Urinary Symptom Score prior to administration of the second therapy. In some embodiments, the reduction in AUA Urinary Symptom Score relative to baseline is about 1 to about 35. For example, the reduction in AUA Urinary Symptom Score relative to baseline can be about 5 to about 35, about 10 to about 35, about 15 to about 35, about 20 to about 35, about 30 to about 35, about 1 to about 30, about 5 to about 30, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 1 to about 25, about 5 to about 25, about 10 to about 25, about 15 to about 25, about 20 to about 25, about 1 to about 20, about 5 to about 20, about 10 to about 20, about 15 to about 20, about 1 to about 15, about 5 to about 15, about 10 to about 15, about 1 to about 10, or about 5 to about 10.

[0070] In some embodiments, following administration of a second therapy, the subject exhibits an increase in the rate of urine flow during urination relative to baseline, e.g., compared to before administration of the first therapy.

[0071] Methods of Selecting / Identifying

[0072] Provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising: identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level (e.g., any of the reference levels described herein); and selecting for the identified subject a combination therapy comprising a first therapy. In some embodiments, provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the methods comprising selecting a first therapy for a subject identified as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level.

[0073] Also provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the methods comprising: identifying a subject having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level; and selecting for the identified subject a treatment comprising a second therapy. In some embodiments, provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the methods comprising selecting a treatment comprising a second therapy for a subject identified as having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level.

[0074] In some embodiments, provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising identifying a subject as having a prostate volume similar to a reference prostate volume (e.g., any of the reference prostate volumes described herein); and selecting the identified subject for testing to determine whether a subject has an elevated level of methylation of SRD5A2 as compared to a reference level. In some embodiments, the method further includes identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level (e.g., any of the reference levels described herein); and selecting for the identified subject a treatment comprising a first therapy. In some embodiments, the method further includes identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level (e.g., any of the reference levels described herein); and selecting for the identified subject a combination therapy comprising a first therapy. In some embodiments, the method further includes identifying a subject having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level; and selecting for the identified subject a treatment comprising a second therapy.

[0075] In some embodiments, provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising identifying a subject as having a prostate volume similar to a reference prostate volume (e.g., any of the reference prostate volumes described herein); and selecting for the identified subject a combination therapy comprising a first therapy.

[0076] In some embodiments, provided herein are methods of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising identifying a subject having increased prostate volume as compared to a reference prostate volume (e.g., any of the reference prostate volumes described herein); and selecting for the identified subject a treatment comprising a second therapy.

[0077] In some embodiments, the prostrate volume is the total prostate volume. In some embodiments, the prostrate volume is the peripheral zone volume. In some embodiments, the prostrate volume is the transition zone volume.

[0078] In some embodiments of any of the methods described herein, the reference prostate volume is a corresponding prostate volume (e.g., total prostrate volume, peripheral zone volume, and / or transition zone volume) in a healthy subject (e.g., a subject that is not diagnosed or identified as having BPH, does not present with a symptom of BPH, and is not considered to have an elevated risk of BPH). In some embodiments, a reference prostate volume is a corresponding prostrate volume in the subject having BPH as determined before the subject was diagnosed or identified as having BPH and / or presented with a symptom of BPH.

[0079] SRD5A2

[0080] 5a-Reductases (5-AR), also known as 3 -oxo- 5 a- steroid 4-dehydrogenases, are enzymes involved in steroid metabolism. There are three types of 5a-reductase isozymes, 5u-reductase type 1 (SRD5A1), 5u-reductase type 2 (SRD5A2, 5aR2, 5 alpha-SR2), and 5a-reductase type 3 (SRD5A3), which are encoded by three corresponding genes, SRD5A1, SRD5A2, and SRD5A3.

[0081] Although all three 5-AR enzymes have been shown to be expressed in prostate tissues, SRD5A2 is the predominant enzyme responsible for prostate development and growth. Normal prostate tissue can exhibit significant variability in SRD5A2 protein expression, with approximately 30% of samples expressing minimal or no protein. Immunohistochemical analyses have illustrated that while SRD5A2 is predominantly expressed in the prostatic stroma, a significant number of patients do not express any SRD5A2. Methylation of bases within the SRD5A2 promoter region can lead to gene silencing through loss of gene and protein expression.

[0082] Exemplary human 5 alpha-reductase type 2 protein sequences include GenBank Accession No. KAI4034100.1 and UniProtKB / Swiss-Prot Accession No. P31213.2. The SRD5A2 gene promoter has a total of 68,588 bases. An exemplary human SRD5A2 genomic DNA sequence includes NCBI Reference Sequence Accession No. NG_008365.2.

[0083] Assays and Samples

[0084] In some embodiments of any of the methods provided herein, an assay is used to determine whether a subject has an elevated level of methylation of SRD5A2 as compared to a reference level. For example, the step of determining whether a white blood cell in a sample obtained from the subject has an elevated level of methylation of SRD5A2 as compared to a reference level and / or the step of identifying a subject as having or not having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level can include performing an assay to detect methylation of SRD5A2 in DNA from the white blood cell.

[0085] Methylation levels of SRD5A2 can be determined using any appropriate methods known in the art (e.g., any of the assays described herein). For example, determining a level of methylation of SRD5A2 can include using an assay based on bisulfite conversion, differential enzymatic cleavage, or affinity capture. See, e.g., Sestakova et al. Biological Procedures Online, 21 : 19 (2019). In some embodiments, determining a level of methylation of SRD5A2 includes sequencing all or a portion of SRD5A2.

[0086] Further examples of assays that can be used to determine a level of methylation of SRD5A2 include, but are not limited to, methylation specific restriction endonucleases (MSRE) analysis, pyrosequencing, methylation specific high-resolution DNA melting (MS-HRM), quantitative methylation specific polymerase chain reaction (qMSP), combined bisulfite restriction analysis (COBRA), methylation-specific PCR (MS-PCR), melting curve methylation-specific PCR (McMS-PCR), quantitative analysis of methylated alleles (QAMA), MSRE-PCR, bisulfite conversion-specific methylationspecific PCR (BS-MSP), methylation-sensitive single-nucleotide primer extension conformation (MS-SNuPE), methylation-sensitive single-strand conformation analysis (MS-SSCA), melting curve combined bisulfite restriction analysis (McCOBRA), and enzymatic regional methylation assay (ERMA).

[0087] DNA containing SRD5A2 can be isolated using any appropriate methods known in the art. In some embodiments, DNA is isolated from the white blood cell by procedures that involve cell lysis and denaturation of the proteins contained therein. Tn some embodiments, DNase inhibitors are added to the lysis buffer. In some embodiments, the DNA is amplified by PCR-based techniques.

[0088] In some embodiments of any of the methods provided herein, the methods further include obtaining a white blood cell from the subject. For example, the white blood cell can be obtained from the subject in the form of a blood sample. The blood sample may be taken from a human, or from non-human mammals such as, mice, rats, non-human primates, canines, felines, ruminants, swine, or sheep. In some embodiments, blood samples are taken from a subject at multiple time points, for example, before treatment, during treatment, and / or after treatment. In some embodiments, the blood sample is taken from a subject having benign prostatic hyperplasia. In some embodiments, DNA is isolated from the white blood cell from the subject.

[0089] Methylation of SRD5A2 can be determined for the entire gene or a portion thereof. For example, methylation of SRD5A2 can be determined for the promoter region of SRD5A2 and / or one or more CpG islands in SRD5A2. In some embodiments, the methods provided herein include identifying a regulatory region and / or one or more CpG islands in SRD5A2. For example, regulatory regions and / or one or more CpG islands in SRD5A2 can be identified using in silico methods such as Ensembl Regulatory Built and UCSC Genome Browser. In some embodiments, the methods provided herein further include generating a DNA methylation profile of SRD5A2. In some embodiments, a DNA methylation profile of SRD5A2 (Chromosome 2: 31,522,480-31,581,067) includes 5000 bases upstream and downstream of the gene promoter region.

[0090] An elevated level of methylation of SRD5A2 can refer to an elevated level of methylation of the entire gene or a portion thereof. For example, an elevated level of methylation of SRD5A2 can be an elevated level of methylation of the promoter region and / or one or more CpG islands in SRD5A2. Non-limiting examples of CpG islands in SRD5A2 include CpG -72 to -42; CpG -40 to -35 / CpG -31 to -19; CpG -34 to -32 / CpG -18 to 65; CpG -39 to -2; CpG -28 to -23; CpG -22 to -20; and CpG -7 to -4.

[0091] In some embodiments, the level of methylation (e.g., of SRD5A2 or a portion thereof) is determined by dividing the number of methylated bases (e.g., the number of methylated bases in SRD5A2 or a portion thereof) by the total number of bases (e.g., the total number of bases of SRD5A2 or the portion thereof). For example, the level of methylation can be calculated by dividing the number of methylated sequencing reads of SRD5A2 or the portion thereof by the total number of sequencing reads of SRD5A2 or the portion thereof.

[0092] In some embodiments, an elevated level of methylation (e.g., of SRD5A2 or one or more portions thereof) is an increase in the level of methylation of at least 1%. For example, an elevated level of methylation (e.g., of SRD5A2 or one or more portions thereof) can be an increase in the level of methylation of at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as compared to a reference level (e.g., any of the reference levels described herein).

[0093] In some embodiments, an elevated level of methylation (e.g., of SRD5A2 or one or more portions thereof) is an increase of about 1% to about 100%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 30% to about 100%, about 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 40% to about 100%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 50% to about 100%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 60% to about 100%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 70% to about 100%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, about 80% to about 100%, about 80% to about 95%, about 80% to about 90%, or about 80% to about 85%, e.g., as compared to a reference level (e.g., any of the reference levels described herein).

[0094] Reference Levels

[0095] In some embodiments of any of the methods described herein, a reference level is a corresponding level of methylation of SRD5A2 in a healthy subject (e.g., a subject having prostate tissue that expresses SRD5A2 and / or a subject that is not diagnosed or identified as having BPH, does not present with a symptom of BPH, and is not considered to have an elevated risk of BPH). In some embodiments, a reference level is a corresponding level of methylation of SRD5A2 in a subject having prostate tissue that expresses SRD5A2. In some embodiments, the level of methylation of SRD5A2 in a subject having prostate tissue that expresses SRD5A2 is about 70%. In some embodiments, a reference level is a mean percentage of corresponding levels of methylation of SRD5A2 in healthy subjects (e.g., subjects having prostate tissue that expresses SRD5A2 and / or subjects that are not diagnosed or identified as having BPH, do not present with a symptom of BPH, and are not considered to have an elevated risk of BPH). In some embodiments, a reference level is a percentile value (e.g., mean value, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50%) of the corresponding levels detected in similar samples in a population of healthy subjects (e.g., subjects having prostate tissue that expresses SRD5A2 and / or subjects that are not diagnosed or identified as having BPH, do not present with a symptom of BPH, and are not considered to have an elevated risk of BPH). In some embodiments, a reference level can be a corresponding threshold level. In some embodiments, a reference level can be a threshold numerical value.

[0096] In some embodiments, the level or expression level of SRD5A2 (e.g., in prostate tissue) can be determined using methods known in the art, including but not limited to, multi-analyte profile test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescent assay, immunohistochemical assay, dot blot assay, slot blot assay, and SDS-PAGE. In some embodiments, expression of SRD5A2 in prostate tissue is determined using IHC immunoreactivity. In some embodiments, an Immunoreactivity Score (IRS) is determined. An Immunoreactivity Score relates to a combination of percent of cells that express the protein and the intensity of expression.

[0097] Therapies

[0098] In some embodiments, a first therapy includes a selective estrogen receptor modulator. In some embodiments, a first therapy is a combination therapy including a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator (SERM). In some embodiments, a first therapy does not include a 5- alpha reductase inhibitor as a monotherapy. In some embodiments, a first therapy includes subjecting the subject to a surgical procedure. In some embodiments, a first therapy includes an Optilume BPH catheter system. In some embodiments, a second therapy is a standard benign prostatic hyperplasia therapy. In some embodiments, standard benign prostatic hyperplasia therapy includes an alpha-adrenergic blocker and / or a 5-alpha reductase inhibitor.

[0099] Non-limiting examples of selective estrogen receptor modulators include tamoxifen, raloxifene, toremifene, ospemifene, bazedoxifene, anordrin, broparestrol, clomifene, cyclofenil, lasofoxifene, ormeloxifene, acolbifene, afimoxifene (4- hydroxytamoxifen), elacestrant, enclomifene ((E)-clomifene), endoxifen (4-hydroxy-N- desmethyltamoxifen), and zuclomifene ((Z)-clomifene). In some embodiments, the selective estrogen receptor modulator is raloxifene.

[0100] Non-limiting examples of 5-alpha reductase inhibitors include finasteride, dutasteride, alfatradiol, and epristeride. In some embodiments, the 5-alpha reductase inhibitor is finasteride.

[0101] Non-limiting examples of a-adrenergic blockers include doxazosin, terazosin, tamsulosin, and alfuzosin.

[0102] Non-limiting examples of surgical procedures that can be used to treat BPH include transurethral resection of the prostate (TURP), transurethral incision of the prostate (TUIP), transurethral microwave therapy, transurethral evaporization of the prostate, transurethral needle ablation, prostatic urethral lift, intraprostatic stent insertion, Aquablation (e.g., hydrodissection of prostatic tissue with high velocity saline under transrectal ultrasound guidance), Prostatic artery embolization, and Rezum (e g., a thermo-ablative strategy that relies on water vapor to deliver energy), or laser surgery. See, e.g., Christidis et al. Prostate Int. 5(2): 41-46 (2017).

[0103] In some embodiments, the first therapy is a combination therapy including a therapeutically effective amount of finasteride and a selective estrogen receptor modulator.

[0104] In some embodiments, the second therapy is finasteride. In some embodiments, the second therapy is combination of finasteride and doxazosin.

[0105] In some embodiments of any of the methods described herein, the method can further include another treatment or therapeutic agent (e.g., any BPH therapeutic agent known in the art), in addition to the first or second therapy. In some embodiments, a subject has undergone prior therapy, e.g., a 5-alpha reductase inhibitor therapy. In some embodiments, a subject having BPH has received treatment with a 5-alpha reductase inhibitor prior to treatment with a first therapy.

[0106] OTHER EMBODIMENTS

[0107] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0108] EXAMPLES

[0109] EXAMPLE 1. Methylation of SRD5A2 in the blood as a novel biomarker to predict sensitivity to 5-ARI treatment.

[0110] Inflammation-driven epigenetic modulation can result in lowered prostatic SRD5A2 expression. The mechanism of somatic suppression of SRD5A2 during adulthood is dependent on epigenetic changes in the promoter region of the SRD5A2 gene. DNA methylation is one of regions rich in cytosine / guanine dinucleotides, so-called CpG islands, which can be modified by the addition of a methyl group. DNA methylation at CpG islands within promoters alters chromatin structure, recruits methylated DNA-binding proteins, and prevents transcription factor binding, leading to gene silencing. This study presents evidence that non-invasive methods that evaluate SRD5A2 expression and methylation can be utilized as biomarkers that predict therapeutic response to finasteride.

[0111] Methods

[0112] Prostate biopsy tissue and clinical data from the MTOPS clinical trials were obtained (n=83), and SRD5A2 expression in prostate tissue was quantified by immunostaining. Patients from the MTOPS cohort were classified as good responders or poor responders based on a 3 -point improvement on the American Urological Association Symptom Score (AUASS).

[0113] Separately, blood and surgical prostate specimen were obtained from participants undergoing TURP in a clinical trial at our institution (NCT04288427) (n=l 1). SRD5A2 expression was analyzed using ELISA, immunohistochemical staining (IHC), and immune-reactive score (IRS). SRD5A2 methylation in white blood cells (WBCs) and prostate tissue was quantified. SRD5A2 promoter region methylation was quantified by DNA extraction and methylation detection viaMethylCollector Ultra kit from fresh human prostate surgical samples. Prostate tissues were immunostained for SRD5A2. Additionally, estrogen response gene products were quantified using western blotting.

[0114] Results

[0115] Using the MTOPS samples, we found that men who demonstrated good clinical response to finasteride had a higher SRD5A2 protein expression (p= 0.036), while poor clinical responders had lower SRD5A2 expression (FIG. 1A, R= -0.38, p= 0.0004). From our institutional cohort, SRD5A2 protein expression was negatively correlated with TZ prostate tissue methylation (R=-0.84, p= 0.0012). SRD5A2 methylation from transition zone (TZ) prostate tissue was directly correlated with a patient’s peripheral WBC methylation (FIG. IB, R= 0.74, p= 0.0128).

[0116] The expression of SRD5A2 is variable due to somatic methylation of SRD5A2 promoter region. Additionally, in the absence of SRD5A2, estrogenic response genes are upregulated, resulting in a picture of androgenic to estrogenic switch.

[0117] Conclusion

[0118] These data suggest that SRD5A2 can be a useful marker to predict response to finasteride therapy. Prior to initiation of therapy, assessment of peripheral WBC methylation for SRD5A2 can serve as a non-invasive technique to predict response to finasteride treatment. Furthermore, epigenetic silencing of SRD5A2 leads to an androgenic to estrogenic switch in the prostate.

[0119] EXAMPLE 2. Using methylation of SRD5A2 in white blood cells to predict sensitivity to 5-ARI treatment.

[0120] SRD5A2 expression varies in the prostatic stroma (FIGS. 2A-2D) and epithelium and that epigenetic modifications of the CpG island in the promoter region regulate the expression of the gene through a defined molecular pathway (FIG. 3). Pre-clinical studies have shown lack of SRD5A2 expression is associated with hypermethylation of the SRD5A2 promoter, and in vitro SRD5A2 promoter activity is suppressed by methylation. In previous studies, 96 human prostate samples for SRD5A2 protein expression were evaluated by IHC and for methylation status of the promoter by methylation pull-down assay. 36.5% of patients did not express the SRD5A2 protein with an associated concomitant hypermethylation of the SRD5A2 promoter, suggesting that methylation of the SRD5A2 promoter suppressed gene and protein expression of SRD5A2.

[0121] As part of an ongoing clinical trial at BIDMC, the methylation status of SRD5A2 in prostate tissues and white blood cells (WBCs) was analyzed in 11 men and a positive correlation was identified as shown in Example 1.

[0122] Patients lacking SRD5A2 protein expression may account for the majority of patients who are resistant to the therapeutic effects of finasteride. Therefore, alternative strategies may need to be considered in this subset of patients for the treatment of bladder outlet obstruction secondary to an enlarged prostate. In addition, patients with absent SRD5A2 expression may have different prostatic growth rates during adulthood, which could account for the broad range of prostatic sizes observed in adult men.

[0123] Methods

[0124] As part of an ongoing clinical trial at BIDMC, the methylation status c SRD5A2 in prostate tissues and white blood cells (WBCs) was analyzed in 11 men and a positive correlation was identified as shown in Example 1.

[0125] Men who have a new diagnosis of BPH and had no prior 5 ARI treatment will be enrolled. WBC SRD5A2 promoter region methylation will be quantified. Men will be started on 5ARI (Finasteride 5mg daily) and will be followed clinically with objective (urofl ow parameters) and subjective (AUA symptom score) measures.

[0126] Patients who are candidates to receive 5ARI therapy (finasteride) for medical management of lower urinary tract symptoms secondary to BPH will be enrolled in the study. Patients who have been on alpha-adrenergic inhibitors previously for management of their urinary symptoms greater than 2 months are candidates to participate in the study, however, to appropriately assess the effectiveness of ARI, patients will not start 5ARI and alpha-blocker therapy simultaneously. Enrollment will be limited to (1 ) patients with lower urinary tract symptoms as assessed by AUA urinary symptom score greater than 8, which is suggestive of moderate-severe LUTS, (2) absence of prostate nodule, tenderness or firmness, and (3) PSA < 10 ng / mL.

[0127] Patients will be excluded from the study if previously diagnosed with any prostatic malignancy or precancerous lesions (atypical glandular foci and prostatic intraepithelial neoplasia), if they have undergone pelvic radiation or received treatment with demethylating drugs or 5ARI. In addition, to keep our study population as homogeneous as possible without significant confounders, patients with urinary tract infection, diabetes mellitus, multiple sclerosis, Alzheimer’s, Parkinson’s, and neurologic deficits will be excluded since these diseases can have significant lower urinary tract symptoms, without any bladder outlet obstruction related to BPH. Furthermore, if patients have been treated with 5ARI treatment (finasteride or dutasteride) within 1 year of study enrollment will be excluded from the study.

[0128] Response to finasteride will be assessed by evaluating changes in lower urinary tract symptoms using a validated urinary symptom questionnaire (AUA Urinary Symptom Score). Response will be assessed at 6 months intervals and then ongoing throughout the study period (large studies have shown that it takes 6-12 months to notice any clinical improvement from 5ARI inhibitor therapy). If at any point a patient requires prostate reduction surgery for BPH, prostate tissue will be obtained for quantification of SRD5A2 expression and methylation.

[0129] Methylation Assessment . Biomarker methylation in WBCs will be evaluated overall for the entire promoter region and in critical CpG sites. Methylation of one site (or a combination of sites) may be more informative than analyzing the whole promoter region.

[0130] The SRD5A2 gene promoter has a total of 68,588 bases, and the DNA methylation profiling (Chromosome 2: 31,522,480-31,581,067) includes 5000 bases upstream and downstream of the gene promoter region. In order to perform methylation profiling of this gene, the regulatory regions from the Ensembl Regulatory Built and the CpG islands based on the CpG islands identified by the UCSC Genome Browser were the focus of the in silico design. About 2201 bases surrounding the SRD5A2 transcriptional sites will be used for in silico designs. These designs cover the regulatory components that include 2 CpG islands, 1 CTCF binding site and one open chromatin region in the SRD5A2 promoter regulatory region. The initial assessment resulted in a total of 14 in silica designs that cover the CpG sites.

[0131] DNA samples in Genomic Lysis Buffer will first be processed for direct bisulfite modification using EZ DNA Methylation Direct Kit (ZymoResearch), then performed gradient PCR at two different magnesium concentrations (1.5mM and 3.0mM), followed by capillary electrophoresis (CE) of the PCR products using the QIAxcel Advanced System (Qiagen). Libraries will be prepared using the KAPA Library Preparation Kit for Ion Torrent platforms (Cat# KK8310) and Ion Xpress™ Barcode Adapters (Thermo Fisher), followed by library molecules purification, quantification, and sequencing on the Ion S5™ sequencer using Ion 530™ sequencing chips (Thermo Fisher). Finally, FASTQ files from the Ion Torrent S5 server will be aligned to the local reference database using open-source Bismark Bisulfite Read Mapper with the Bowtie2 alignment algorithm. Methylation levels will be calculated in Bismark by dividing the number of methylated reads by the total number of reads.

[0132] Statistical Analysis and Power. For the primary endpoint (response to 5 ARI therapy at 12-month endpoint), 2-sided 95% Cis for the difference in proportions between non- expressors and expressors will be calculated. We propose to recruit 59 participants, which adjusts for potential confounders of age, obesity, hypertension, coronary artery disease.

[0133] Results

[0134] 5ARI high expressors from low expressors will be evaluated based on promoter methylation pattern(s). Promoter methylation as a whole as well as CpG segments (e.g., segment #1, #2 or #3, FIG. 4) will be evaluated for differentiating between 5 ARI high expressors and low expressors.

[0135] EXAMPLE 3. Androgen and estrogen levels are modified in human prostate tissue in decreased expression of SRD5A2.

[0136] To explore whether molecular subtypes can be found in prostate glands with methylated vs. unmethylated SRD5A2 promoter, 22 patients from our cohort were evaluated for molecular profiling. Using the Illumina Human HT-12v2 BeadChip gene array system to determine the genetic signature of our samples, the data were analyzed with Gene Set Enrichment Analysis, and three sets of genes were identified that were most significantly upregulated in prostate samples with methylated SRD5A2 promoter locus: estrogen response genes, Sonic hedgehog, and MYC family members (FIG. 5).

[0137] Androgen and estrogen levels are modified in human prostate tissue with decreased expression of SRD5A2. A cohort of 35 subjects’ prostate samples obtained by Transurethral Resection of the Prostate (TURP) was divided into two groups: SRD5A2 methylated with low SRD5A2 protein expression (designated SRD5A2low), and SRD5A2 unmethylated but with SRD5A2 protein expression (designated SRD5A2hlgh). The status of SRD5A2 expression was confirmed by ELISA, and its concentration was found to be significantly higher in the SRD5A2hlghgroup than in the SRD5A2lowgroup. The level of testosterone in the SRD5A2lowgroup was significantly higher and dihydrotestosterone (DHT) levels were significantly lower in the SRD5A2hlghgroup. This finding provided further confirmation of the microarray gene analysis, in which the level of prostatic estradiol in the SRD5A2lowgroup was dramatically elevated compared to the SRD5A2hlghgroup, suggesting preferential conversion of testosterone to estradiol and a change in the hormonal milieu of prostate glands that SRD5A2 expression is suppressed.

[0138] To further confirm the presence of an androgenic-to-estrogenic switch in prostate glands lacking SRD5A2 expression, several prostates were evaluated with and without SRD5A2 expression. Aromatase, the enzyme responsible for converting testosterone to estradiol, is preferentially upregulated in prostate tissues that lack SRD5A2 expression (FIGS. 6A and 6B). In human prostate, the action of estrogens is complex, and they can have both proliferative and inhibitory effects via estrogen receptor (ER)a and ERp.

[0139] Here, the phosphorylated form of ERa (pERa), and not ERp, was upregulated in prostate samples lacking SRD5A2 (FIG. 5). These results are in line with the published literature documenting that estrogenic signaling is plays a role in the development of BPH in animal models as well as in clinical settings.

[0140] The response to finasteride correlates with the expression of SRD5A2 in the prostate. We have obtained transition zone prostate biopsies and pertinent clinical data on 83 men in the finasteride only arm of the MTOPS trial. Immunohistochemical analyses illustrated that the expression of SRD5A2 was correlated with the clinical response to finasteride (measured with American urological association symptom score). This result supports our previous findings that highlights the role of SRD5A2 expression in the response and resistance to 5ARI.

[0141] EXAMPLE 4. The role of combination therapy (5ARI + SERM) in the treatment of BPH.

[0142] Estradiol (E2) is the major estrogen in males and is 70% derived from testosterone via aromatase conversion. In animal models, male dogs treated with androgens and estrogens had earlier and more extensive BPH and obstructive voiding. In primary cell cultures, E2 has been shown to stimulate stromal proliferation in both normal and BPH cells. These proliferative effects were antagonized by the antiestrogens fulvestran and tamoxifene. Clinically, while serum androgens decline with age, E2 levels remain relatively constant, resulting in a net picture of an increased serum E2 to T (testosterone) ratio, that is associated with the development of BPH and LUTS. Further, in the Physician's Health Study, a strong trend for increased risk of surgical intervention for BPH was found across quintiles for serum E2. Local production of E2 has been implicated in prostatic hyperplasia and loss of aromatase expression causes decreased estrogen-induced prostate proliferation.

[0143] In vitro studies have shown that selective estrogen receptor modulators (SERMs) including suppressed proliferation of normal human prostate epithelial and stromal cells. Raloxifene is FDA approved for fracture risk reduction in osteoporosis. Additionally, it was found to inhibit the growth of androgen dependent and independent prostate cancer effect on prostate cancer in a human xenograft prostate cancer model.

[0144] A previous study on transurethral resection of the prostate (TURP) prostate specimens demonstrated that androgen decreased while estrogen levels increased in the absence of SRD5A2. Specifically, the level of estradiol was dramatically elevated, concomitant with significant upregulation of estrogen response genes, in prostatic samples with methylation at the SRD5A2 promoter. The phosphorylation of estrogen receptor-a in prostatic stroma was upregulated when SRD5 A2 expression was absent. TNF-a suppressed SRD5A2 mRNA and protein expression, and simultaneously promoted expression of aromatase, the enzyme responsible for conversion of testosterone to estradiol. Concomitant suppression of SRD5A2 and treatment with TNF-a synergistically upregulated the aromatase levels.

[0145] In men with BPH, especially those who have androgenic to estrogenic switch with epigenetic silencing of SRD5A2 (and thus 5ARI resistance) SERMs can play a role in stopping the progression of BPH.

[0146] Methods

[0147] A cohort separate from that of Example 2, but with the same inclusion / exclusion criteria, will be recruited. Men who have a new diagnosis of BPH and had no prior 5 ARI therapy will be randomized into two treatment arms: 5ARI+SERM or 5ARI+placebo. Men will be followed up clinically every six months with an end goal of determining whether 5ARI+SERM is superior to 5 ARI alone. Response will be evaluated with objective (urodynamic parameters) and subjective (AUA Symptom score) measures that will be measured at baseline and biennially until the end of the study. If at any point a patient requires surgery for BPH, prostate tissue will be obtained for quantification of SRD5A2 expression and methylation.

[0148] In the same cohort, blood samples will be obtained from men at baseline and at follow up periods (6 months, 1 year, 3 years) and SRD5A2 methylation status will be quantified in a similar fashion to the methods of Example 2. If at any point a patient requires surgery for BPH, prostate tissue will be obtained for quantification of SRD5A2 expression and methylation.

[0149] Statistical Analysis and Power. For the primary endpoint (response to 5ARI+placebo vs 5ARI+SERM therapy at 12-month endpoint), 2-sided 95% Cis for the difference in proportions between non-expressors and expressors will be calculated. We propose to recruit 323 participants, which adjusts for potential confounders of age, obesity, hypertension, coronary artery disease.

[0150] Results

[0151] Preliminary data showed that the estrogen level is associated with SRD5A2 methylation in 35 prostate samples obtained via Transurethral Resection of the Prostate (TURP) from BPH patients. There is an androgenic-to-estrogenic switch in the prostate of patients who lack expression of SRD5A2 (FIG. 7).

[0152] In men who have lack SRD5A2 expression, we anticipate that 5ARI+SERM combination therapy will be superior to 5ARI+placebo therapy.

[0153] EXAMPLE 5. SRD5A2 Expression Correlates with Prostate Volume.

[0154] SRD5A2 is an essential enzyme associated with prostatic development and growth through conversion of testosterone to dihydrotestosterone (DHT), a potent androgen. The main medical therapy for BPH is 5a-reductase inhibitors, which act by inhibiting SRD5A2 and subsequently reducing prostate size. However, around 30% of adult BPH patients lack expression of SRD5A2 due to DNA methylation of the gene, which leads to drug resistance. This study investigated the relationship between SRD5A2 expression levels and prostate size.

[0155] Methods

[0156] Sixty-one prostate biopsies from the Medical Therapy of Prostatic Symptoms (MTOPS) trial were obtained from NIDDK Central Repository. Clinical information including age, BMI, total prostate volume, transitional zone volume and serum DHT levels were used for correlation studies. In addition, forty-nine prostate surgical tissues from a biorepository at Beth Israel Deaconess Medical Center (BIDMC) were obtained. DHT levels in the serum and prostate tissue were measured using ELISA kit. SRD5A2 DNA methylation in promoter region of Tissue and White Blood Cell (WBC) was measured using Methylamp DNA Modification kit. SRD5A2 protein expression was measured and quantified by IHC and immunoreactive score.

[0157] Results

[0158] In both cohorts, SRD5A2 expression showed a positive correlation with total prostate volume (FIG. 8A, MTOPS: R=0.28, p=0.03; FIG. 8C, BIDMC: R=0.60, p=0.0001) and transition zone volume (FIG. 8B, MTOPS: R=0.31, p=0.02; FIG. 8D BIDMC: R=0.73, p=0.0003). However, SRD5A2 expression was not significantly associated with DHT levels in either serum or prostate tissues. In the BIMDC cohort, SRD5A2 expression was additionally correlated with peripheral zone volume (R=0.64, p=0.002). Furthermore, the level of SRD5A2 DNA methylation in the tissue negatively correlates with SRD5A2 protein expression (R=-0.84, p=0.001), and positively correlates with SRD5A2 DNA methylation in WBC (R=0.75, p=0.01). These findings reveal a significant association between SRD5A2 expression and prostate size, particularly in the transition zone, where BPH commonly develops. Additionally, the results suggest that DNA methylation levels of SRD5A2 could serve as predictors of SRD5A2 expression in the prostate. These insights emphasize the potential for personalized BPH treatments that consider SRD5A2 DNA methylation and protein expression profiles to enhance therapeutic response.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a subject having benign prostatic hyperplasia, the method comprising:(a) determining whether a white blood cell in a sample obtained from the subject has an elevated level of methylation of SRD5A2 as compared to a reference level; and(b)(i) administering a combination therapy to the subject if the subject has a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level, wherein the combination therapy comprises a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator; or(b)(ii) administering a therapeutically effective amount of a 5-alpha reductase inhibitor to the subject if the subject has a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level.

2. A method of treating benign prostatic hyperplasia in a subject, the method comprising: identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; and administering to the identified subject a combination therapy comprising a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator.

3. A method of treating benign prostatic hyperplasia in a subject, the method comprising administering a combination therapy comprising a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator, to a subject identified as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level.

4. A method of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising:identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; and selecting for the identified subject a combination therapy comprising a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator.

5. A method of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising selecting a combination therapy comprising a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator, for a subject identified as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level.

6. A method of treating a subject having benign prostatic hyperplasia, the method comprising:(a) administering to the subject a 5-alpha reductase inhibitor;(b) after (a), identifying the subject as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; and(c) administering to the identified subject a combination therapy comprising a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator.

7. A method of treating a subject having benign prostatic hyperplasia, the method comprising: identifying a subject previously administered a 5-alpha reductase inhibitor, as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level; and administering to the identified subject a combination therapy comprising a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator.

8. A method of treating a subject having benign prostatic hyperplasia, the method comprising administering to a subject previously administered a 5-alpha reductase inhibitor and later identified as having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level, a combination therapy comprising a therapeutically effective amount of a 5-alpha reductase inhibitor and a selective estrogen receptor modulator.

9. A method of treating benign prostatic hyperplasia in a subject, the method comprising: identifying a subject having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level; and administering to the identified subject a therapeutically effective amount of a 5- alpha reductase inhibitor.

10. A method of treating benign prostatic hyperplasia in a subject, the method comprising administering a therapeutically effective amount of a 5-alpha reductase inhibitor to a subject identified as having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level.

11. A method of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising: identifying a subject having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level; and selecting for the identified subject a treatment comprising a therapeutically effective amount of a 5-alpha reductase inhibitor.

12. A method of selecting a treatment for a subject having benign prostatic hyperplasia, the method comprising selecting a treatment comprising a therapeutically effective amount of a 5-alpha reductase inhibitor, for a subject identified as having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level.

13. The method of claim 1, wherein the step of determining whether a white blood cell in a sample obtained from the subject has an elevated level of methylation of SRD5A2 as compared to a reference level comprises performing an assay to detect methylation of SRD5A2 in DNAfrom the white blood cell.

14. The method of any one of claims 2-8, wherein the step of identifying a subject having a white blood cell that has an elevated level of methylation of SRD5A2 as compared to a reference level comprises performing an assay to detect methylation of SRD5A2 in DNA from the white blood cell from the subject.

15. The method of any one of claims 9-12, wherein the step of identifying a subject having a white blood cell that does not have an elevated level of methylation of SRD5A2 as compared to a reference level comprises performing an assay to detect methylation of SRD5A2 in DNA from the white blood cell from the subject.

16. The method of any one of claims 13-15, wherein the assay comprises bisulfite conversion, differential enzymatic cleavage, or affinity capture.

17. The method of any one of claims 13-16, wherein the assay comprises sequencing.

18. The method of any one of claims 1-17, the method further comprising obtaining a blood sample from the subject.

19. The method of any one of claims 1-18, wherein methylation of SRD5A2 is detected in the promoter region of SRD5A2.

20. The method of any one of claims 1-19, wherein methylation of SRD5A2 is detected in one or more CpG islands in SRD5A2.

21. The method of claim 20, wherein the one or more CpG islands comprise CpG -72 to -42; CpG -40 to -35 / CpG -31 to -19; CpG -34 to -32 / CpG -18 to 65; or combinations thereof.

22. The method of claims 20 or 21, wherein the one or more CpG islands comprises CpG -39 to -2.

23. The method of claim 20, wherein the one or more CpG islands comprise CpG -28 to -23; CpG -22 to -20; CpG -7 to -4; or combinations thereof.

24. The method of any one of claims 1-23, wherein the reference level is the average percentage of methylation of SRD5A2 in prostate tissue that expresses SRD5A2.

25. The method of any one of claims 1-24, wherein the elevated level of methylation of SRD5A2 is an increase in methylation compared to the reference level of at least about 4%, at least about 5%, or at least about 10%.

26. The method of any one of claims 1-25, wherein the elevated level of methylation of SRD5A2 as compared to a reference level comprises an elevated level of methylation of the promoter region of SRD5A2.

27. The method of any one of claims 1-26, wherein the elevated level of methylation of SRD5A2 as compared to a reference level comprises an elevated level of methylation of one or more CpG islands in SRD5A2.

28. The method of any one of claims 1-27, wherein the 5-alpha reductase inhibitor is selected from the group consisting of: finasteride, dutasteride, alfatradiol, and epristeride.

29. The method of any one of claims 1-28, wherein the 5-alpha reductase inhibitor is finasteride.

30. The method of any one of claims 1-8, 13, 14, and 16-29, wherein the selective estrogen receptor modulator is selected from the group consisting of: tamoxifen, raloxifene, toremifene, ospemifene, bazedoxifene, anordrin, broparestrol, clomifene, cyclofenil, lasofoxifene, ormeloxifene, acolbifene, afimoxifene (4-hydroxytamoxifen),elacestrant, enclomifene ((E)-clomifene), endoxifen (4-hydroxy-N-desmethyltamoxifen), and zuclomifene ((Z)-clomifene).

31. The method of any one of claims 1-8, 13, 14, and 16-30, wherein the selective estrogen receptor modulator is raloxifene.

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