Association of PARP inhibitor treatment on the prevalence and progression of clonal hematopoiesis in patients with advanced prostate cancer.

BACKGROUND: Poly ADP-ribose polymerase (PARP) inhibitors are approved for the treatment of some men with advanced prostate cancer. Rare but serious side effects include myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The impact of PARP inhibitors on clonal hematopoiesis (CH), a potential precursor lesion associated with MDS and AML, is incompletely understood in prostate cancer. We hypothesized that PARP inhibitors would increase CH prevalence and abundance. METHODS: We prospectively enrolled participants with advanced prostate cancer treated with PARP inhibitors. The presence of CH was assessed from leukocytes using an ultra-deep error-corrected dual unique molecular identifiers sequencing method targeting 49 genes most commonly mutated in CH and myeloid malignancies. Variant allele frequencies (VAF) of ≥0.5% were considered clinically significant. Blood samples were collected before and after PARP inhibitor treatment. RESULTS: Ten men were enrolled; mean age of 67 years. Six patients had Gleason 7 disease, and four had Gleason ≥8 disease at diagnosis. Nine had localized disease at diagnosis, and eight had prior treatment with radiation. The mean time between pre- and post-treatment blood samples was 11 months (range 2.6-31 months). Six patients (60%) had CH identified prior to PARP inhibitor treatment, three with multiple clones. Of 11 CH clones identified in follow-up, 5 (45%) appeared or increased after treatment. DNMT3A, TET2, and PPM1D were the most common CH alterations observed. The largest post-treatment increase involved the PPM1D gene. CONCLUSION: CH alterations are frequently found after treatment with PARP inhibitors in patients with prostate cancer and this may be one mechanism by which PARP inhibitors lead to increased risk of MDS/AML.

Characterization of structural, biochemical, pharmacokinetic, and pharmacodynamic properties of the LSD1 inhibitor bomedemstat in preclinical models.

INTRODUCTION: Lysine-specific demethylase 1 (LSD1) is emerging as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Neuroendocrine prostate cancer (NEPC) is increasingly recognized as an adaptive mechanism of resistance in mCRPC patients failing androgen receptor axis-targeted therapies. Safe and effective LSD1 inhibitors are necessary to determine antitumor response in prostate cancer models. For this reason, we characterize the LSD1 inhibitor bomedemstat to assess its clinical potential in NEPC as well as other mCRPC pathological subtypes. METHODS: Bomedemstat was characterized via crystallization, flavine adenine dinucleotide spectrophotometry, and enzyme kinetics. On-target effects were assessed in relevant prostate cancer cell models by measuring proliferation and H3K4 methylation using western blot analysis. In vivo, pharmacokinetic (PK) and pharmacodynamic (PD) profiles of bomedemstat are also described. RESULTS: Structural, biochemical, and PK/PD properties of bomedemstat, an irreversible, orally-bioavailable inhibitor of LSD1 are reported. Our data demonstrate bomedemstat has >2500-fold greater specificity for LSD1 over monoamine oxidase (MAO)-A and -B. Bomedemstat also demonstrates activity against several models of advanced CRPC, including NEPC patient-derived xenografts. Significant intra-tumoral accumulation of orally-administered bomedemstat is measured with micromolar levels achieved in vivo (1.2 ± 0.45 µM at the 7.5 mg/kg dose and 3.76 ± 0.43 µM at the 15 mg/kg dose). Daily oral dosing of bomedemstat at 40 mg/kg/day is well-tolerated, with on-target thrombocytopenia observed that is rapidly reversible following treatment cessation. CONCLUSIONS: Bomedemstat provides enhanced specificity against LSD1, as revealed by structural and biochemical data. PK/PD data display an overall safety profile with manageable side effects resulting from LSD1 inhibition using bomedemstat in preclinical models. Altogether, our results support clinical testing of bomedemstat in the setting of mCRPC.

Third generation quinoline-3-carboxamide transcriptional disrupter of HDAC4, HIF-1α, and MEF-2 signaling for metastatic castration-resistant prostate cancer.

BACKGROUND: The quinoline-3-carboxamide, Tasquinimod (TasQ), is orally active as a maintenance therapy with an on-target mechanism-of-action via allosteric binding to HDAC4. This prevents formation of the HDAC4/NCoR1/HDAC3 complex, disrupting HIF-1α transcriptional activation and repressing MEF-2 target genes needed for adaptive survival signaling in the compromised tumor micro environment. In phase 3 clinical testing against metastatic castration-resistant prostate cancer(mCRPC), TasQ (1 mg/day) increased time-to-progression, but not overall survival. METHODS: TasQ analogs were chemically synthesized and tested for activity compared to the parental compound. These included HDAC4 enzymatic assays, qRT-PCR and western blot analyses of gene and protein expression following treatment, in vitro and in vivo efficacy against multiple prostate cancer models including PDXs, pharmacokinetic analyses,AHR binding and agonist assays, SPR analyses of binding to HDAC4 and NCoR1, RNAseq analysis of in vivo tumors, 3D endothelial sprouting assays, and a targeted kinase screen. Genetic knockout or knockdown controls were used when appropriate. RESULTS: Here, we document that, on this regimen (1 mg/day), TasQ blood levels are 10-fold lower than the optimal concentration (≥2 µM) needed for anticancer activity, suggesting higher daily doses are needed. Unfortunately, we also demonstrate that TasQ is an arylhydrocarbon receptor (AHR) agonist, which binds with an EC50 of 1 µM to produce unwanted off-target side effects. Therefore, we screened a library of TasQ analogsto maximize on-target versus off-target activity. Using this approach, we identified ESATA-20, which has ~10-fold lower AHR agonism and 5-fold greater potency against prostate cancer patient-derived xenografts. CONCLUSION: This increased therapeuticindex nominates ESATA-20 as a lead candidate forclinical development as an orally active third generation quinoline-3-carboxamide analog thatretains its on-target ability to disrupt HDAC4/HIF-1α/MEF-2-dependent adaptive survival signaling in the compromisedtumor microenvironment found in mCRPC.

Bipolar Androgen Therapy Followed by Androgen Receptor Inhibition as Sequential Therapy for Prostate Cancer.

Inhibition of androgen receptor (AR) signaling has been the mainstay of treatment of advanced prostate cancer (PCa) for the past 80 years. Combination and sequential AR-inhibiting therapies are highly effective palliative therapy, but they are not curative. All patients eventually develop resistance to primary castrating therapy [ie, castration-resistant PCa (CRPC)]. At this point, they are treated with subsequent lines of secondary AR inhibitory therapies. However, resistance to these agents also develops and patients progress to a state we have termed complete androgen inhibition-resistant PCa. This phase of the disease is associated with poor prognosis. At this point, treatment shifts to non-hormonal cytotoxic therapies (eg, chemotherapy and radiopharmaceuticals). However, the majority of PCas remain addicted to signaling through AR throughout the course of the disease. Resistant PCa cells adaptively upregulate AR activity, despite castration and AR inhibitors, via mechanisms such as AR overexpression, gene amplification, mutation, and expression of ligand-independent variants to permit sustained liganded and non-liganded AR signaling. Studies dating back nearly 30 years indicate that high expression of AR induced by prolonged castration becomes a vulnerability of CRPC cells in vitro and in mouse xenografts to supraphysiologic androgen (SPA), which induces cell death and growth arrest in this context. Based on these studies, we developed a counterintuitive treatment called bipolar androgen therapy (BAT) for patients with CRPC, in which SPA is administered intermittently to result in cycling of serum testosterone from the polar extremes of supraphysiologic to near-castrate levels. This rapid cycling is intended to disrupt the adaptive of AR regulation associated with chronic exposure to high or low levels of testosterone, while simultaneously targeting the spectrum of AR expression present in heterogeneous CRPC tumors. We have now tested BAT in >250 patients with CRPC. Here we present a review of these clinical studies, which have demonstrated collectively that BAT can be safely given to men with CRPC, improves quality of life, and produces therapeutic responses in ~30% of patients. As expected, resistance to BAT is associated with adaptive downregulation of AR expression. Intriguingly, this downregulation is associated with restoration of sensitivity to subsequent AR inhibitor therapies.

The association of sex steroid hormone concentrations with non-alcoholic fatty liver disease and liver enzymes in US men.

BACKGROUND & AIMS: This study aimed to analyse the association of sex hormone levels with liver enzyme levels and non-alcoholic fatty liver disease (NAFLD) in a nationally representative sample of men. METHODS: A total of 919 men from the US National Health and Nutrition Examination Study (NHANES) III were included in this cross-sectional analysis of data from 1988 to 1991. We used existing data on serum total and free testosterone, total and free estradiol, androstanediol glucuronide (AAG) and sex steroid-binding globulin (SHBG), and estimated their associations with aspartate aminotransferase (AST), and alanine aminotransferase (ALT) and NAFLD, as determined using ultrasound, after adjusting for possible confounders including age, race, smoking, alcohol, physical activity, waist circumference and steroid hormones. RESULTS: Lower total testosterone (TT) and higher free estradiol were associated with higher odds of NAFLD after adjusting for confounders including the other sex hormones. Lower TT was associated with higher odds of elevated AST, but not ALT. Free testosterone, total estradiol, SHBG and AAG were not associated with NAFLD or liver enzymes. CONCLUSIONS: This study supports an inverse association between TT concentration and NAFLD in men independent of other sex hormones (SHBG, AAG and estradiol) and known risk factors, such as obesity, age and lifestyle. Exploration of whether TT might be a non-invasive marker for NAFLD diagnosis is warranted.

Reversing the effects of androgen-deprivation therapy in men with metastatic castration-resistant prostate cancer.

OBJECTIVE: To investigate whether bipolar androgen therapy (BAT), involving rapid cyclic administration of high-dose testosterone, as a novel treatment for metastatic castration-resistant prostate cancer (mCRPC) promotes improvements in body composition and associated improvements in lipid profiles and quality of life. PATIENTS AND METHODS: Men from two completed trials with computed tomography imaging at baseline and after three cycles of BAT were included. Cross-sectional areas of psoas muscle, visceral and subcutaneous fat were measured at the L3 vertebral level. Functional Assessment of Chronic Illness Therapy - Fatigue questionnaire and 36-item short-form health survey were used to assess quality of life. RESULTS: The 60 included patients lost a mean (sd) of 7.8 (8.2)% of subcutaneous fat, 9.8 (18.2)% of visceral fat, and gained 12.2 (6.7)% muscle mass. Changes in subcutaneous and visceral fat were positively correlated with each other (Spearman's correlation coefficient 0.58, 95% confidence interval 0.35-0.71) independent of the effects of age, body mass index, and duration of androgen-deprivation therapy. Energy, physical function, and measures of limitations due to physical health were all significantly improved at 3 months. The improvements in body composition were not correlated with decreases in lipid levels or observed improvements in quality of life. CONCLUSIONS: In the present study, BAT was associated with significant improvements in body composition, lipid parameters, and quality of life. This has promising implications for the long-term health of men with mCRPC.

Mipsagargin: The Beginning-Not the End-of Thapsigargin Prodrug-Based Cancer Therapeutics.

Søren Brøgger Christensen isolated and characterized the cell-penetrant sesquiterpene lactone Thapsigargin (TG) from the fruit Thapsia garganica. In the late 1980s/early 1990s, TG was supplied to multiple independent and collaborative groups. Using this TG, studies documented with a large variety of mammalian cell types that TG rapidly (i.e., within seconds to a minute) penetrates cells, resulting in an essentially irreversible binding and inhibiting (IC50~10 nM) of SERCA 2b calcium uptake pumps. If exposure to 50-100 nM TG is sustained for >24-48 h, prostate cancer cells undergo apoptotic death. TG-induced death requires changes in the cytoplasmic Ca2+, initiating a calmodulin/calcineurin/calpain-dependent signaling cascade that involves BAD-dependent opening of the mitochondrial permeability transition pore (MPTP); this releases cytochrome C into the cytoplasm, activating caspases and nucleases. Chemically unmodified TG has no therapeutic index and is poorly water soluble. A TG analog, in which the 8-acyl groups is replaced with the 12-aminododecanoyl group, afforded 12-ADT, retaining an EC50 for killing of <100 nM. Conjugation of 12-ADT to a series of 5-8 amino acid peptides was engineered so that they are efficiently hydrolyzed by only one of a series of proteases [e.g., KLK3 (also known as Prostate Specific Antigen); KLK2 (also known as hK2); Fibroblast Activation Protein Protease (FAP); or Folh1 (also known as Prostate Specific Membrane Antigen)]. The obtained conjugates have increased water solubility for systemic delivery in the blood and prevent cell penetrance and, thus, killing until the TG-prodrug is hydrolyzed by the targeting protease in the vicinity of the cancer cells. We summarize the preclinical validation of each of these TG-prodrugs with special attention to the PSMA TG-prodrug, Mipsagargin, which is in phase II clinical testing.

Extreme responses to immune checkpoint blockade following bipolar androgen therapy and enzalutamide in patients with metastatic castration resistant prostate cancer.

BACKGROUND: Immune checkpoint inhibition has been shown to have limited efficacy in patients with metastatic prostate cancer. Prostate cancers that harbor certain homologous recombination (HR) DNA repair gene mutations, inactivating CDK12 mutations or have underlying mismatch repair deficiency may be effectively treated with immunotherapy. Combination therapy may improve clinical response rates to immune checkpoint blockade. We observed profound prostate-specific antigen (PSA) and/or objective responses to immune checkpoint blockade following prior treatment with bipolar androgen therapy (BAT) and enzalutamide. METHODS: We report three cases of patients with metastatic castration resistant prostate cancer (mCRPC) undergoing therapy with anti-PD-1 inhibitors. All patients underwent both somatic molecular testing and germline genetic testing. RESULTS: Two of the three patients with mCRPC harbored an inactivating mutation in an HR DNA repair gene (BRCA2, ATM). No patient demonstrated mismatch repair deficiency, nor were CDK12 alterations present. All three patients had been treated with BAT and enzalutamide before immune checkpoint blockade, a paradoxical approach for the treatment of mCRPC developed by our group. CONCLUSIONS: These cases of mCRPC suggest that immune checkpoint blockade may have therapeutic potential in patients with prostate cancer, especially following immune activation ("priming") using BAT and enzalutamide.

A Phase I Study of Alpha-1,3-Galactosyltransferase-Expressing Allogeneic Renal Cell Carcinoma Immunotherapy in Patients with Refractory Metastatic Renal Cell Carcinoma.

LESSONS LEARNED: HyperAcute Renal immunotherapy was well tolerated and demonstrated antitumor activity in patients requiring salvage-line treatment for metastatic renal cell carcinoma (mRCC). HyperAcute Renal immunotherapy was safely administered with concomitant salvage-line treatments for mRCC, and it may be a candidate for inclusion in novel combinations for salvage treatment of mRCC because of its unique mechanism of action. BACKGROUND: HyperAcute Renal (HAR) immunotherapy exploits a naturally occurring barrier to xenotransplantation and zoonotic infections in humans to immunize patients against metastatic renal cell carcinoma (mRCC) cells. HAR consists of two allogeneic renal cancer cell lines genetically modified to express α(1,3)Gal, to which humans have an inherent pre-existing immunity. METHODS: Patients with refractory mRCC were eligible for this phase I dose-escalation trial. Concomitant treatment was permitted after the initial 2 months of HAR monotherapy. HAR was injected intradermally weekly for 4 weeks then biweekly for 20 weeks, totaling 14 immunizations. The primary endpoint was safety and determination of a maximum tolerated dose (MTD). RESULTS: Among 18 patients enrolled, two grade 3 adverse events (AEs) were attributed to HAR, lymphopenia and injection site reaction, and no grade 4/5 AEs occurred. The recommended phase II dose (RP2D) was 300 million cells. One patient had a partial response and eight patients had stable disease, for a disease control rate of 50% (9/18). Median overall survival with low-dose HAR was 14.2 months and was 25.3 months with high-dose HAR. CONCLUSION: In pretreated mRCC, HAR immunotherapy was well tolerated and demonstrated antitumor activity. HAR immunotherapy may be a candidate for inclusion in novel combinations for salvage treatment of mRCC.

Risk of development of visceral metastases subsequent to abiraterone vs placebo: An analysis of mode of radiographic progression in COU-AA-302.

BACKGROUND: Abiraterone increases survival in prostate cancer, but tumors resistant to abiraterone can exhibit a hormonally resistant, aggressive phenotype. We hypothesized that the therapeutic pressure of abiraterone is resulting in more clinically aggressive disease at progression, characterized by increased visceral metastases. Our objective was to determine whether abiraterone increased the risk of development of visceral metastases at the time of progression compared with placebo in a randomized phase III trial. METHODS: We performed a post hoc analysis of the COU-AA-302 trial of abiraterone plus prednisone vs placebo plus prednisone in patients with metastatic castration-resistant prostate cancer. The primary outcome was the development of visceral metastases. The cumulative incidences of visceral metastases were calculated by the Kaplan-Meier method and compared using log-rank testing. Multivariable Cox regression analysis assessed for the independent association of abiraterone with the development of visceral metastases. RESULTS: Eighty-four of 1088 patients developed visceral metastases during study. Log-rank testing and Cox regression showed no difference in time to visceral metastases between groups (HR 1.01 [95% confidence interval (CI), 0.65-1.56]; P = .97). Abiraterone treatment was not associated with the development of visceral metastases in multivariable analysis (HR 0.89 [95% CI, 0.57-1.40]; P = .62). The study was limited by censoring of radiographic outcomes at the time of completion of primary study therapy; longer term risks were not assessed. CONCLUSIONS: Abiraterone was not associated with increased risk of visceral metastatic disease at the time of progression compared with placebo.

A pilot study of prostate-specific membrane antigen (PSMA) dynamics in men undergoing treatment for advanced prostate cancer.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a rational target for noninvasive detection of recurrent prostate cancer (PCa) and for therapy of metastatic castration-resistant prostate cancer (mCRPC) with PSMA-targeted agents. Here we conducted serial measurements of PSMA expression on circulating tumor cells (CTCs) to evaluate patterns of longitudinal PSMA dynamics over the course of multiple sequential therapies. METHODS: A retrospective investigation of men with mCRPC undergoing evaluation at medical oncology clinics at our institution assessed the dynamics of PSMA expression in the context of different systemic treatments administered sequentially. Eligibility included patients who began systemic therapies with androgen receptor (AR)-directed agents or taxane agents for whom peripheral blood samples were tested for CTC mRNA of AR splice variant-7 (AR-V7), prostate-specific antigen (PSA), and PSMA (with >2 CTC + results) in a CLIA-accredited laboratory. RESULTS: From August 2015 to November 2017, we identified 96 eligible men. Fifteen had greater than or equal to 2 sequential therapies and evaluable CTC samples, greater than or equal to 1 expressing PSMA (PSMA+). Among the 15 patients included in this analysis, a total of 54 PSMA status evaluations were performed in the context of 48 therapies during a median follow-up of 18 months. At baseline, PSMA signal was detected ("positive") in 11 of 15 (73.3%) patients, while for 4 of 15 (26.7%) patients PSMA signal was undetectable ("negative"). In all but two patients, the baseline collection corresponded with a change in treatment. On the second assessment, PSMA increases were detected in all 4/4 (100%) PSMA-negative patients and 8 of 11 (72.7%) PSMA-positive patients. PSMA significantly decreased in a patient treated with 177 Lu-PSMA-617. Serum PSA declines were seen in 7 of 8 (88%) of the treatment periods where PSMA decreased. CONCLUSIONS: PSMA expression in CTCs is a dynamic marker. PSMA transcript declines appear to be associated with concurrent decreases in serum PSA. Sequential CTC sampling could provide a noninvasive response assessment to systemic treatment for mCRPC.

Bipolar androgen therapy in men with metastatic castration-resistant prostate cancer after progression on enzalutamide: an open-label, phase 2, multicohort study.

BACKGROUND: Prostate cancer that progresses after enzalutamide treatment is poorly responsive to further antiandrogen therapy, and paradoxically, rapid cycling between high and low serum testosterone concentrations (bipolar androgen therapy [BAT]) in this setting might induce tumour responses. We aimed to evaluate BAT in patients with metastatic castration-resistant prostate cancer that progressed after enzalutamide. METHODS: We did this single-centre, open-label, phase 2, multicohort study in the USA. We included patients aged 18 years or older who had histologically confirmed and radiographically documented metastatic castration-resistant prostate cancer, with no more than two previous second-line hormonal therapies, and a castrate concentration of testosterone. Patients were asymptomatic, with Eastern Cooperative Oncology Group performance status of 0-2, and did not have high-risk lesions for tumour flare (eg, >5 sites of visceral disease or bone lesions with impending fracture). For the cohort reported here, we required patients to have had progression on enzalutamide with a continued prostate-specific antigen (PSA) rise after enzalutamide treatment discontinuation. Patients received BAT, which consisted of intramuscular testosterone cipionate 400 mg every 28 days until progression and continued luteinising hormone-releasing hormone agonist therapy. Upon progression after BAT, men were rechallenged with oral enzalutamide 160 mg daily. The co-primary endpoints were investigator-assessed 50% decline in PSA concentration from baseline (PSA50) for BAT (for all patients who received at least one dose) and for enzalutamide rechallenge (based on intention-to-treat analysis). These data represent the final analysis for the post-enzalutamide cohort, while two additional cohorts (post-abiraterone and newly castration-resistant prostate cancer) are ongoing. The trial is registered with ClinicalTrials.gov, number NCT02090114. FINDINGS: Between Aug 28, 2014, and May 18, 2016, we accrued 30 eligible patients and treated them with BAT. Nine (30%; 95% CI 15-49; p<0·0001) of 30 patients achieved a PSA50 to BAT. 29 patients completed BAT and 21 proceeded to enzalutamide rechallenge, of whom 15 (52%; 95% CI 33-71; p<0·0001) achieved a PSA50 response. During BAT, the only grade 3-4 adverse event occurring in more than one patient was hypertension (three [10%] patients). Other grade 3 or worse adverse events occurring during BAT in one [3%] patient each were pulmonary embolism, myocardial infarction, urinary obstruction, gallstone, and sepsis. During enzalutamide retreatment, no grade 3-4 toxicities occurred in more than one patient. No treatment-related deaths were reported during either BAT or enzalutamide retreatment. INTERPRETATION: BAT is a safe therapy that resulted in responses in asymptomatic men with metastatic castration-resistant prostate cancer and also resensitisation to enzalutamide in most patients undergoing rechallenge. Further studies with BAT are needed to define the potential clinical role for BAT in the management of metastatic castration-resistant prostate cancer and the optimal strategy for sequencing between androgen and antiandrogen therapies in metastatic castration-resistant prostate cancer to maximise therapeutic benefit to patients. FUNDING: National Institutes of Health and National Cancer Institute.

PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

BACKGROUND: Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. METHODS: Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. RESULTS: Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA <0.1 ng/mL. PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. CONCLUSIONS: In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter total A2M levels, which can potentially alter levels of a variety of growth factors such as IL-6, TGF-beta, basic FGF, and PDGF. Alterations in levels of these cytokines and proteolytic degradation of small peptide hormones may have profound effect on host-cancer interaction.

Androgen Receptor Splice Variants Are Not Substrates of Nonsense-Mediated Decay.

BACKGROUND: Androgen receptor (AR) splice variants have been clinically associated with progressive cancer, castration-resistance, and resistance to AR antagonists and androgen synthesis inhibitors. AR variants can be generated by genomic alterations and alternative splicing, and their expression is androgen-regulated. There has been a suggestion that AR variants bearing premature termination codons and coding for truncated proteins should be regulated by the nonsense-mediated decay (NMD) mRNA surveillance pathway, suggesting that either the NMD pathway is dysfunctional in variant-expressing cell lines or that variants are somehow able to evade degradation by NMD. METHODS: We first used siRNA knockdown of the NMD regulator, UPF1, in an NMD reporter assay to determine if this surveillance pathway is functioning normally in AR variant-expressing cell lines. We then used UPF1 knockdown to determine if expression of the AR variants ARV3 and ARV7 is affected by inhibition of NMD. Next, we analyzed androgen regulation of UPF1 and used transcript expression analysis to determine if there is any association between UPF1 expression, resistance, and ARV3 or ARV7 expression. RESULTS: We found that the NMD pathway functions normally in the AR variant-expressing cell line 22Rv1 and that inhibition of NMD does not increase expression of ARV3 or ARV7. Furthermore, we found that expression of UPF1 is not androgen-regulated. We also found that UFP1 expression levels do not differentiate castration-sensitive from resistant cell line and that UPF1 expression does not correlate with expression of ARV3 or ARV7 in cells in which these variants are highly expressed. CONCLUSION: This study eliminates a possible mechanism of regulation of certain AR variants. Future research into the regulation of AR variants should focus on other mechanisms to better understand the origin of these variants and to possibly inhibit their expression for the resensitization of resistant cancers. Prostate 77:829-837, 2017. © 2017 Wiley Periodicals, Inc.

AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer.

BACKGROUND: The androgen-receptor isoform encoded by splice variant 7 lacks the ligand-binding domain, which is the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone. METHODS: We used a quantitative reverse-transcriptase-polymerase-chain-reaction assay to evaluate AR-V7 in circulating tumor cells from prospectively enrolled patients with metastatic castration-resistant prostate cancer who were initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status (positive vs. negative) and prostate-specific antigen (PSA) response rates (the primary end point), freedom from PSA progression (PSA progression-free survival), clinical or radiographic progression-free survival, and overall survival. RESULTS: A total of 31 enzalutamide-treated patients and 31 abiraterone-treated patients were enrolled, of whom 39% and 19%, respectively, had detectable AR-V7 in circulating tumor cells. Among men receiving enzalutamide, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 53%, P=0.004) and shorter PSA progression-free survival (median, 1.4 months vs. 6.0 months; P<0.001), clinical or radiographic progression-free survival (median, 2.1 months vs. 6.1 months; P<0.001), and overall survival (median, 5.5 months vs. not reached; P=0.002). Similarly, among men receiving abiraterone, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 68%, P=0.004) and shorter PSA progression-free survival (median, 1.3 months vs. not reached; P<0.001), clinical or radiographic progression-free survival (median, 2.3 months vs. not reached; P<0.001), and overall survival (median, 10.6 months vs. not reached, P=0.006). The association between AR-V7 detection and therapeutic resistance was maintained after adjustment for expression of full-length androgen receptor messenger RNA. CONCLUSIONS: Detection of AR-V7 in circulating tumor cells from patients with castration-resistant prostate cancer may be associated with resistance to enzalutamide and abiraterone. These findings require large-scale prospective validation. (Funded by the Prostate Cancer Foundation and others.).

Anticancer activities of emetine prodrugs that are proteolytically activated by the prostate specific antigen (PSA) and evaluation of in vivo toxicity of emetine derivatives.

Emetine is a small molecule protein synthesis inhibitor that is toxic to all cell types and therefore suitable for complete killing of all types of heterogeneous cancer cells within a tumor. It becomes significantly inactive (non-toxic) when derivatized at its N-2' secondary amine. This provides a strategy for targeting emetine to cancerous tumor without killing normal cells. In this report, PSA activatable peptide prodrugs of emetine were synthesized. To overcome steric hindrances and enhance protease specific cleavage, a 2-stage prodrug activation process was needed to release emetine in cancer cells. In this 2-stage process, emetine prodrug intermediates are coupled to PSA peptide substrate (Ac-His-Ser-Ser-Lys-Leu-Gln) to obtain the full prodrug. Both prodrug intermediates 10 (Ala-Pro-PABC-Emetine) and 14 (Ser-Leu-PABC-Emetine) were evaluated for kinetics of hydrolysis to emetine and potency [Where PABC = p-aminobenzyloxycarbonyl]. While both intermediates quantitatively liberate emetine when incubated under appropriate conditions, upon coupling of PSA substrate to give the full prodrugs, only prodrug 16, the prodrug obtained from 14 was hydrolyzable by PSA. Cytotoxicity studies in PSA producing LNCaP and CWR22Rv1 confirm the activation of the prodrug by PSA with an IC50 of 75 nM and 59 nM respectively. The cytotoxicity of 16 is significantly reduced in cell lines that do not produce PSA. Further, in vivo toxicity studies are done on these prodrugs and other derivatives of emetine. The results show the significance of conformational modulation in obtaining safe emetine prodrugs.

Iterative design of emetine-based prodrug targeting fibroblast activation protein (FAP) and dipeptidyl peptidase IV DPPIV using a tandem enzymatic activation strategy.

BACKGROUND: There is an urgent need to develop new agents for treating metastatic prostate cancer to overcome multiple drug resistance to the current standard targeted cancer therapy. Emetine is a highly cytotoxic natural product protein synthesis inhibitor, which is toxic to all cell types. Its cytotoxicity can be blocked by derivatizing its N-2' position. Thus emetine can be selectively delivered to cancer cells in the region of metastatic cancer as a prodrug that will be activated by an enzyme selectively overexpressed within the metastatic tumor microenvironment. In this work, we convert emetine to a prodrug activatable by the fibroblast activation protein (FAP), a serine protease overexpressed by the carcinoma associated fibroblasts. METHOD: By using an iterative structure-activity relationship strategy, several peptidyl emetine prodrug analogs (1-11) were synthesized by chemical derivatization of emetine at its N-2' position and tested for in-vitro activation by FAP. The lead prodrug 11 is made up of a DPPIV activatable prodrug precursor 10 (Ala-Pro-PABC-Emetine) coupled to FAP substrate (Ala-Ser-Gly-Pro-Ala-Gly-Pro). Activation assays of the prodrugs were performed in purified FAP, DPPIV, FBS, and human serum and were analyzed by LCMS. In vitro cytotoxicity assays of these prodrugs are carried out in prostate (LNCaP, PC3) and breast (MCF7 and MDA-MB-231) cancer cell lines. The prodrugs are also tested in normal immortalized human prostatic epithelial cell line (PrEC). RESULTS: The lead FAP activated emetine prodrug 11 is activated to emetine in tandem by FAP and DPPIV in about 70% conversion within 24 hr. In prostate and breast cancer cell lines treated with prodrug 11, it is found to be equipotent with emetine in the presence of FAP and DPPIV. However, in the PrEC cell line grown in serum free media, prodrug 11 is more than 200-fold less cytotoxic than emetine in the absence of FAP and DPPIV. CONCLUSION: This FAP activated prodrug of cytotoxic agent emetine further shows the crucial role of the N-2' position of emetine in controlling its cytotoxicity. Significantly reduced toxicity observed in the PrEC cell line in the absence of FAP and DPPIV shows that prodrug 11 could be systemically delivered to regions of metastatic prostate cancer or other solid tumor for activation by cancer selective enzymes within the cancer microenvironment, such as FAP that is overexpressed by the carcinoma-associated fibroblasts. The two-step tandem enzymatic activation of prodrug 11 by FAP and DPPIV is a strategy for overcoming steric hindrance.

Mutational Analysis of Prostate-Specific Antigen Defines the Intrinsic Proteolytic Activity of the proPSA Zymogen.

BACKGROUND: Prostate-specific antigen (PSA) is an important prostate cancer biomarker. It is also a protease expressed at high concentrations by the normal and malignant prostate. PSA is secreted as a zymogen (proPSA) with an inhibitory prodomain that must be removed for full activity. ProPSA variants, assumed to be inactive, are found in the blood of prostate cancer patients, and are indicative of poor clinical outcome. Despite the abundance of clinical reports, our understanding of PSA's enzymology is limited, in part due to a lack of appropriate experimental systems. We sought to develop a series of PSA-derived mutants that would help to enhance our understanding of the gene. METHODS: Sixteen rPSA variants were generated and characterized by a variety of biochemical methods. RESULTS: The wildtype cDNA (WT) provided the template for generating a panel of recombinants. These included variants that abolished removal of the prodomain (R24A), disabled its enzymatic activity (S213A), and/or facilitated a cell-based conversion to the active conformation (FR). The purified variants' proteolytic activity was examined using a fluorogenic substrate, known PSA-cleavable proteins, and physiologically relevant inhibitors. Upon demonstrating our successful generation and purification of the PSA variants, we characterized proPSA activity, describing cleavage of synthetic and biologic substrates, but not serum protease inhibitors. This finding was exploited in the development of a self-activating mutant (PSA_QY) that exhibited the greatest enzymatic activity of all the variants. CONCLUSIONS: The system described herein will prove useful for varied applications. ProPSA is partially functional with relatively high activity compared to the mature enzyme. In demonstrating the zymogen's intrinsic activity, we suggest that the proPSA in prostate cancer patient serum is not inert. This may have implications for our understanding of the disease. Prostate 76:1203-1217, 2016. © 2016 Wiley Periodicals, Inc.

Bipolar Androgen Therapy for Men With Androgen Ablation Naïve Prostate Cancer: Results From the Phase II BATMAN Study.

BACKGROUND: We have previously documented a paradoxical anti-tumor effect when castration-resistant prostate cancer patients were treated with intermittent, high-dose testosterone (i.e., Bipolar Androgen Therapy; BAT). Because, an adaptive increase in androgen receptor expression following chronic androgen deprivation therapy (ADT) may underlie this effect, we tested whether men with hormone-sensitive (HS) prostate cancer (PC) would also respond to BAT if given following a 6-month ADT lead-in. METHODS: Asymptomatic HS PC patients with low metastatic burden or non-metastatic biochemically recurrent disease were enrolled. Following 6-month of ADT, those with a PSA <4 ng/ml went on to receive alternating 3-month cycles of BAT and ADT. BAT was administered as intramuscular testosterone (T) cypionate or enanthate 400 mg on Days (D) 1, 29, and 57. ADT was continued throughout the study to allow rapid cycling from near castrate to supraphysiologic range T following T injections. The primary endpoint was the percent of patients with a PSA <4 ng/ml after 18 months. Secondary endpoints included radiographic response and quality of life (QoL). RESULTS: Twenty-nine of 33 patients received BAT following the ADT lead-in. The primary endpoint was met, with 17/29 men (59%, 90% confidence interval: 42-74%) having a PSA <4 ng/ml at 18 months. Ten patients receiving BAT had RECIST evaluable disease, and eight (80%) objective responses were observed (four complete; four partial). Three patients progressed per RECIST criteria and three had unconfirmed progression on bone scan. Men treated with 6-month of ADT had improved QoL following the first cycle of BAT as measured by the SF-36, FACT-P, and IIEF surveys. CONCLUSIONS: BAT demonstrated preliminary efficacy in men with HS PC following 6-month of ADT. BAT may improve QoL in men treated with ADT. Prostate 76:1218-1226, 2016. © 2016 Wiley Periodicals, Inc.