Outcomes In Castration-resistant Prostate Cancer Research Articles

Abstract C062: Defining the interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. Non-Hispanic Black men are 1.8 times more likely to develop PCa and 2.3 times more likely to die from PCa. Tumor suppressor retinoblastoma (RB1) is frequently deleted in PCa. RB represses the transcriptional activity of E2Fs and the phosphorylation of RB by CDK4/6 results in dissociation of the complex and a subsequent increase in E2F transcriptional activity, leading to cell cycle progression. RB loss is associated with tumor aggressiveness occurring in 10-15% of castration-resistant prostate cancer (CRPC) tumors and corresponds to a decrease in overall survival. RB depletion leads to increased expression of DNA repair proteins due to elevated transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in DNA repair. Elevated PARP-1 enzymatic activity is associated with PCa progression and correlates with poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, however the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry. Defining the interplay between PARP and RB/E2F is likely to contribute to the identification of novel treatment strategies and/or biomarker response. Canonically, E2F1-3 are transcription factors that positively regulate gene expression programs associated with pro-proliferative signals. Prior research by other groups has shown that E2F target genes expression is elevated in tumors excised from Black men compared to Asian and White men. There was an increase in germline RAD51 mutations observed. RAD51 is an E2F target gene involved in DDR, therefore attenuating PARP enzymatic activity may be a potential mode of targeting this axis. These data suggest that there may be differential DNA damage response (DDR) in African American men driven by aberrations in the RB/E2F signaling axis, however the mechanism is yet to be fully explored. Analysis of publicly available patient datasets were performed and demonstrate that RB1 deletions is more frequent than RB1/BRCA2 co-deletion in metastatic PCa. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 mRNA and PARP-1 protein are elevated upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. In hormone-depleted conditions, results suggest a differential response between RB depleted models compared to the control when challenged with PARP inhibition. Furthermore, an IC50 dose of Olaparib elicits differential PARP enzymatic response in the RB1 depleted model. RB1 loss is associated with an enrichment in E2F1 binding at the PARP-1 locus. Future studies have been designed to determine the impact of the RB/E2F pathway alterations on downstream consequences PARP enzymatic function and establish the contribution of PARP and the RB/E2F axis on racial disparity in PCa. Citation Format: Latese Evans. Defining the interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C062.

Abstract B063: Interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Retinoblastoma (RB) protein is a tumor suppressor that represses the transcriptional activity of E2Fs by forming an RB-E2F repressor complex. The phosphorylation of RB by CDK4/6 results in dissociation of the complex and subsequent E2F transcriptional activity, leading to cell cycle progression. RB loss occurs in 10-15% of castration-resistant prostate cancer (CRPC) which is associated tumor aggressiveness and poor clinical outcomes. RB loss is also associated differential treatment response to an array of treatment modalities, including hormone therapy and DNA damage inducing chemotherapy. RB depletion leads to enhanced expression of DNA repair proteins due to increased transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in multiple nuclear processes including DNA repair, transcriptional regulation, and chromatin dynamics. Increased PARP-1 activity is associated with and correlates to poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, but the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry, which may yield novel treatment strategies and/or biomarkers of response. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 protein increases upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. Results suggest a differential response between control and RB1 depleted cells when treated with Olaparib. RB1depleted cells also exhibit differential PARP activity in response to treatment with the IC50 dose of Olaparib. Future studies have been designed to evaluate the impact of RB alteration and manipulation of PARP activity on biological and molecular processes that are governed by RB/E2F and PARP. Citation Format: Latese Evans, Moriah Cunningham, Jasibel Vasquez Gonzalez, Matthew Schiewer. Interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B063.

BMX inhibition and HSD3B1-driven resistance in prostate cancer in the Maverick trial.

144 Background: Kinase inhibitors have been ineffective in prostate cancer and have no known role in androgen biosynthesis. Inheritance of the adrenal-permissive HSD3B1(1245C) allele encodes a 3βHSD1 enzyme missense that up-regulates the rate-limiting step of androgen biosynthesis from non-gonadal precursor steroids and confers poor clinical outcomes in castration-resistant prostate cancer (CRPC). About half of all men with prostate cancer inherit the adrenal-permissive HSD3B1 allele. Multiple clinical studies demonstrate that adrenal-permissive HSD3B1 allele inheritance confers more rapid progression on ADT and others also suggest worse CRPC outcomes even after treatment with abiraterone or enzalutamide. However, there is no known method to clinically block 3βHSD1. Furthermore, 3βHSD1 is not known to be phosphorylated. Methods: Mass spectrometry was used to identify protein phosphorylation sites and steroid metabolites, genetic and pharmacologic methods were used to identify the kinase required for 3βHSD1 phosphorylation and mouse xenograft studies were performed with BMX inhibition. The identified mechanism was used to design and launch a multicenter phase 2 study of the BMX inhibitor abivertinib in combination with abiraterone in men with metastatic CRPC. Results: 3βHSD1 enzyme activity requires tyrosine phosphorylation at Y344 by the BMX kinase. Androgen biosynthesis is blocked by a phosphorylation-defective 3βHSD1 344F, or BMX genetic knockdown, or BMX pharmacologic inhibition. BMX inhibition using zanubrutinib suppresses CRPC growth in the C4-2 and VCaP xenograft models by blocking intratumoral androgen synthesis and tumor androgen receptor (AR) signaling. Discovery of this mechanism provides the rationale for the phase 2 Maverick trial of abivertinib, a BMX inhibitor, combined with abiraterone, in men with CRPC with adrenal-permissive HSD3B1 allele inheritance (NCT05361915). Eligibility includes 1) presence of metastatic CRPC, 2) measurable and/or non-measurable disease, and 3) confirmed positivity of adrenal-permissive HSD3B1(1245C) allele inheritance via central testing (cap heterozygosity at 50%). Patients will be enrolled in 2 arms: 1) abiraterone naïve (n=45) and 2) abiraterone progressing (n=55). All patients will receive treatment with abivertinib 200mg twice daily with abiraterone 1000mg daily and prednisone 5mg by mouth twice daily. The primary outcome is 6-month radiographic progression-free survival. On-treatment biopsies will be used to inform mechanisms of response and resistance in patients. Conclusions: BMX is required for 3βHSD1 phosphorylation, androgen biosynthesis and CRPC progression with the adrenal-permissive HSD3B1(1245C) allele. The Maverick trial will test clinical proof-of-concept of BMX inhibition in men with adrenal-permissive HSD3B1(1245C) inheritance. Clinical trial information: NCT05361915 .

Polygodial, a Sesquiterpene Dialdehyde, Activates Apoptotic Signaling in Castration-Resistant Prostate Cancer Cell Lines by Inducing Oxidative Stress.

Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. Surgery, radiation therapy, chemotherapy, and androgen deprivation therapy are currently the standard treatment options for PCa. These have poor outcomes and result in the development of castration-resistant prostate cancer (CRPC), which is the foremost underlying cause of mortality associated with PCa. Taxanes, diterpene compounds approved to treat hormonal refractory PCa, show poor outcomes in CRPC. Polygodial (PG) is a natural sesquiterpene isolated from water pepper (Persicaria hydropiper), Dorrigo pepper (Tasmannia stipitata), and mountain pepper (Tasmannia lanceolata). Previous reports show that PG has an anticancer effect. Our results show that PG robustly inhibits the cell viability, colony formation, and migration of taxane-resistant CRPC cell lines and induces cell cycle arrest at the G0 phase. A toxicity investigation shows that PG is not toxic to primary human hepatocytes, 3T3-J2 fibroblast co-cultures, and non-cancerous BPH-1 cells, implicating that PG is innocuous to healthy cells. In addition, PG induces oxidative stress and activates apoptosis in drug-resistant PCa cell lines. Our mechanistic evaluation by a proteome profiler-human apoptotic array in PC3-TXR cells shows that PG induces upregulation of cytochrome c and caspase-3 and downregulation of antiapoptotic markers. Western blot analysis reveals that PG activates apoptotic and DNA damage markers in PCa cells. Our results suggest that PG exhibits its anticancer effect by promoting reactive oxygen species generation and induction of apoptosis in CRPC cells.

Association Between Adrenal-Restrictive HSD3B1 Inheritance and Hormone-Independent Subtypes of Endometrial and Breast Cancer.

BackgroundThe germline variant rs1047303 (HSD3B1[1245A/C]), restricting or enabling production of potent androgens and estrogens from adrenal precursors, affects outcomes of castration-resistant prostate cancer and is associated with estrogen receptor positivity in postmenopausal breast cancer. Like breast cancer, endometrial cancer is another malignancy with hormone-dependent and hormone-independent subtypes. We hypothesized that adrenal-restrictive HSD3B1 genotype would associate with hormone-independent cancer subtypes.MethodsWe employed a previously described classification of tumors in The Cancer Genome Atlas into genomic clusters. We determined HSD3B1 genotype frequencies by endometrial cancer genomic cluster and calculated the odds per adrenal-restrictive A allele for the largely hormone-independent copy-number (CN) high subtype vs other subtypes. An equivalent analysis was performed for the genomically similar, hormone-independent basal breast cancer subtype. Last, we performed survival analyses for UK Biobank participants with endometrial cancer by HSD3B1 genotype. All statistical tests were 2-sided.ResultsThe adrenal-restrictive HSD3B1(1245A) allele was associated with the CN-high endometrial cancer subtype (odds ratio [OR] = 1.63, 95% confidence interval [CI] = 1.14 to 2.32; P = .007). Similarly, HSD3B1(1245A) was associated with the basal breast cancer subtype (OR = 1.54, 95% CI = 1.13 to 2.08; P = .006). In the UK Biobank, endometrial cancer patients homozygous for HSD3B1(1245A) had worse overall (hazard ratio [HR] = 1.39, 95% CI = 1.16 to 1.68; P < .001) and cancer-specific (HR = 1.39, 95% CI = 1.14 to 1.70; P = .001) survival, consistent with the A allele being enriched in the more aggressive CN-high subtype.ConclusionsThese findings suggest roles for adrenal-restrictive vs adrenal-permissive steroidogenesis, by way of rs1047303 genotype, in the development of and/or outcomes from at least 3 commonly hormone-associated types of cancer: prostate, breast, and endometrial.

Abstract 397: Mesenchymal stem cell-derived systemic NRG-1 mediates a targetable molecular switch in metastatic prostate cancer

Abstract NRG-1 signals through epidermal growth factor receptor (EGFR) family members. Despite promising preclinical data, HER2 blockade in prostate cancer has failed to improve outcomes in clinical trials.1-4 We have discovered a novel complex systemic role of NRG-1 that drives sensitivity and resistance to androgen deprivation therapy (ADT) in castration-sensitive (CSPC) and castration resistant prostate cancer (CRPC), respectively. Significantly, sensitivity to ADT can be restored in CRPC by manipulating NRG-1 signaling. Our data posit NRG-1 as a systemic “switch” in prostate cancer. Plasma NRG-1 is elevated in our cohort of patients with metastatic CRPC (mean 4.9ng/ml) versus healthy controls (mean 3.2ng/ml, p&amp;lt;0.0001) or metastatic CSPC (mean 3.4ng/ml, p&amp;lt;0.0001). Paradoxically, high systemic NRG-1 predicted superior OS and PFS in CSPC but inferior clinical outcomes in CRPC. Consistently, opposite cell survival between LNCaP (CSPC) vs Du145 &amp; PC3 (CRPC) was observed with recombinant human NRG-1 and enzalutamide (ENZ). ENZ combined with increasing NRG-1 concentration induced LNCaP cell death in an additive manner, but NRG-1 rescued Du145 &amp; PC3 from ENZ-induced death in a dose-dependent manner. We next hypothesized that differential NRG-1 downstream signaling is mediated by engagement with distinct EGFR receptor dimers. While HER2 expression is similar, LNCaP express higher HER3 than Du145 &amp; PC3. High ratio of HER2 to HER3 in Du145 &amp; PC3 mirrors clinical reports of HER2 upregulation in CRPC5,6 and also predicts patient survival in the Stand Up to Cancer cohort.7 Significantly, we recapitulated the CRPC phenotype in LNCaP by inducing HER2 overexpression, HER3 suppression, and chemically-mediated HER2/HER3 dimerization. Conversely, HER2 knockdown sensitized Du145 cells to ENZ. Next, we queried composite single-cell whole human8 and CRPC9 transcriptomes for NRG-1 expression and discovered that the source of NRG-1 is the patients’ own mesenchymal stem cells (MSCs). Bone-derived MSC outgrowth cells produced copious NRG-1, which was increased substantially by coincubation with prostate cancer cell supernatants. Importantly, we were able to manipulate MSCs to reduce their NRG-1 production as a possible clinical translation. Similarly, patients treated with these compounds showed significant reduction in plasma NRG-1 levels after treatment. Additional clinical studies are pending. Together, our observations confirm that NRG-1 has differential signaling: HER3 homodimerization leads to prostate cancer cell death, whereas heterodimerization of HER3 with HER2 leads to prostate cancer cell survival via ADT resistance in a plasma NRG-1 dependent manner. Consequently, HER2 and HER3 receptor availability determine the molecular switch of NRG-1 in prostate cancer. Therefore, NRG-1 can be both a prognostic marker and therapeutic target in CRPC, and multiple clinical trials are pending. Citation Format: Jacob Orme, Roman Thaler, Dejie Wang, Siven Chinniah, Jacob Hirdler, Fernando Quevedo, Lance Pagliaro, Kwon Eugene, Alan Bryce, Brian Costello, Haojie Huang, Sean Park. Mesenchymal stem cell-derived systemic NRG-1 mediates a targetable molecular switch in metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 397.

Abstract 647: Association between adrenal-restrictive HSD3B1 inheritance and hormone independent subtypes of endometrial and breast cancer

Abstract Breast and endometrial cancer are both malignancies that can be classified into subtypes based on their hormone dependence, such as estrogen receptor (ER)-positive vs. ER-negative breast cancer and low-grade vs. high-grade endometrial cancer. The gene HSD3B1 encodes the steroid metabolism enzyme 3β-hydroxysteroid dehydrogenase 1, which catalyzes a critical step in conversion of adrenal precursor steroids to potent androgens and estrogens. Associations between the missense-encoding germline variant rs1047303 in HSD3B1 have been described to affect outcomes of castration-resistant prostate cancer and associated with ER positivity in postmenopausal breast cancer. The adrenal-permissive 1245C allele enables greater production of androgens and estrogens from adrenal precursors, whereas the adrenal-restrictive 1245A allele limits this reaction pathway. Using data from The Cancer Genome Atlas’s TCGA-UCEC and TCGA-BRCA projects, we interrogated whether the adrenal-restrictive rs1047303 genotype was associated with less hormone-driven genomic clusters of endometrial and breast cancer. We determined the genotype frequencies for different tumor subtypes, as classified by the genomic analysis of Sanchez-Vega, et al. (2018). The adrenal-restrictive HSD3B1(1245A) allele was associated with the copy number (CN) high cluster of endometrial cancer (OR 1.59, 95% CI 1.12-2.26, p = 0.0098), notably composed of serous and high-grade endometrioid endometrial cancers which are less estrogen-dependent than low-grade endometrioid tumors. Similarly, the adrenal-restrictive HSD3B1(1245A) was associated with the basal subtype of breast cancer (OR 1.54 [1.13-2.09], p = 0.0057), a subtype that is largely ER-negative and is known to share genomic features with serous-type endometrial cancers. Lastly, we interrogated overall survival of endometrial cancer patients in the UK Biobank and found that patients homozygous for the adrenal-restrictive HSD3B1(1245A) had worse overall survival (HR 1.39 [1.16-1.68], p = 0.00049) and cancer-specific survival (HR 1.39 [1.14-1.70], p = 0.0013), a finding consistent with the A allele being enriched in the more aggressive CN-high subtype of endometrial cancer. Taken together, these findings suggest a role for adrenal-restrictive vs. adrenal-permissive steroidogenesis, by way of rs1047303 genotype, in the development of and/or outcomes from at least three different commonly hormone-associated types of cancer: prostate, breast, and endometrial. Citation Format: Jeffrey M. McManus, Roberto Vargas, Peter Bazeley, Navin Sabharwal, Nima Sharifi. Association between adrenal-restrictive HSD3B1 inheritance and hormone independent subtypes of endometrial and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 647.

Modeling Androgen Deprivation Therapy-Induced Prostate Cancer Dormancy and Its Clinical Implications.

Treatment-induced tumor dormancy is a state in cancer progression where residual disease is present but remains asymptomatic. Dormant cancer cells are treatment-resistant and responsible for cancer recurrence and metastasis. Prostate cancer treated with androgen-deprivation therapy (ADT) often enters a dormant state. ADT-induced prostate cancer dormancy remains poorly understood due to the challenge in acquiring clinical dormant prostate cancer cells and the lack of representative models. In this study, we aimed to develop clinically relevant models for studying ADT-induced prostate cancer dormancy. Dormant prostate cancer models were established by castrating mice bearing patient-derived xenografts (PDX) of hormonal naïve or sensitive prostate cancer. Dormancy status and tumor relapse were monitored and evaluated. Paired pre- and postcastration (dormant) PDX tissues were subjected to morphologic and transcriptome profiling analyses. As a result, we established eleven ADT-induced dormant prostate cancer models that closely mimicked the clinical courses of ADT-treated prostate cancer. We identified two ADT-induced dormancy subtypes that differed in morphology, gene expression, and relapse rates. We discovered transcriptomic differences in precastration PDXs that predisposed the dormancy response to ADT. We further developed a dormancy subtype-based, predisposed gene signature that was significantly associated with ADT response in hormonal naïve prostate cancer and clinical outcome in castration-resistant prostate cancer treated with ADT or androgen-receptor pathway inhibitors.

Gene amplification of YB-1 in castration-resistant prostate cancer in association with aberrant androgen receptor expression.

Although Y‐box binding protein‐1 (YB‐1) is known to be overexpressed in prostate cancer, especially castration‐resistant prostate cancer (CRPC), the mechanism of its overexpression remains unclear. We aimed to elucidate the mechanism of YB‐1 overexpression in CRPC. Gene amplification in CRPC cells and tissues was examined by public database analysis, and digital PCR. The significance of YB‐1 amplification for the YB‐1/androgen receptor (AR) axis and prognosis was examined by public database analysis and immunohistochemistry. YB‐1 amplification was mainly observed in CRPC tissues by public database analysis and confirmed in CRPC cells and tissues by digital PCR. Expression of YB‐1 was increased in CRPC tissues compared with treatment‐naïve tissues. Furthermore, YB‐1 and phosphorylated YB‐1 levels were associated with AR and AR V7 expression levels. Finally, YB‐1 amplification was associated with poor outcomes in CRPC. Taken together, the present findings suggest that YB‐1 amplification contributes to progression to CRPC through regulation of AR and AR V7 expressions, and that YB‐1 is a promising therapeutic target in CRPC.

Morphology-Predicted Large-Scale Transition Number in Circulating Tumor Cells Identifies a Chromosomal Instability Biomarker Associated with Poor Outcome in Castration-Resistant Prostate Cancer.

Chromosomal instability (CIN) increases a tumor cell's ability to acquire chromosomal alterations, a mechanism by which tumor cells evolve, adapt, and resist therapeutics. We sought to develop a biomarker of CIN in circulating tumor cells (CTC) that are more likely to reflect the genetic diversity of patient's disease than a single-site biopsy and be assessed rapidly so as to inform treatment management decisions in real time. Large-scale transitions (LST) are genomic alterations defined as chromosomal breakages that generate chromosomal gains or losses of greater than or equal to10 Mb. Here we studied the relationship between the number of LST in an individual CTC determined by direct sequencing and morphologic features of the cells. This relationship was then used to develop a computer vision algorithm that utilizes CTC image features to predict the presence of a high (9 or more) versus low (8 or fewer) LST number in a single cell. As LSTs are a primary functional component of homologous recombination deficient cellular phenotypes, the image-based algorithm was studied prospectively on 10,240 CTCs in 367 blood samples obtained from 294 patients with progressing metastatic castration-resistant prostate cancer taken prior to starting a standard-of-care approved therapy. The resultant computer vision-based biomarker of CIN in CTCs in a pretreatment sample strongly associated with poor overall survival times in patients treated with androgen receptor signaling inhibitors and taxanes. SIGNIFICANCE: A rapidly assessable biomarker of chromosomal instability in CTC is associated with poor outcomes when detected in men with progressing mCRPC.

Plasma AR Copy Number Changes and Outcome to Abiraterone and Enzalutamide.

Introduction: Plasma androgen receptor (AR) copy number (CN) status identifies castration-resistant prostate cancer (CRPC) patients with worse outcome on abiraterone/enzalutamide. However, the impact of AR CN changes on clinical outcome in CRPC is unknown.Materials and Methods: Plasma samples from 73 patients treated with abiraterone or enzalutamide were collected at baseline and at the time of progression disease (PD). Droplet digital polymerase chain reaction was used to assess AR CN status.Results: We showed that 11 patients (15.1%) changed AR CN status from baseline to PD (9 patients from normal to gain, 2 from gain to normal). Patients changing AR CN status from normal at baseline to gain at PD had intermediate median overall survival (OS) of 20.5 months (95% CI = 8.0–44.2) between those who remained AR CN normal from baseline to PD (27.3 months [95% CI = 21.9–34.4]) and those who remained AR CN gain from baseline to PD (9.1 months [95% CI = 3.8–14.5], p < 0.0001). Patients changing AR CN from normal at baseline to gain at PD had a median progression-free survival (PFS) of 9.2 months (95% CI = 2.0–14.7), patients who remained AR CN normal had a median PFS of 9.1 months (95% CI = 7.2–10.1), and patients who remained AR CN gain had a median PFS of 5.4 (95% CI = 3.6–6.5, p = 0.0005). Both OS and PFS were not significantly different between patients with AR CN that changes from normal to gain and patients with stable AR CN normal.Conclusions: We showed that CRPC patients changing AR CN status from baseline to progression time point had intermediate OS and we suggested that AR CN evaluation at baseline could be the most informative for clinical outcome of CRPC patients treated with abiraterone or enzalutamide. Larger prospective studies are warranted.

Down-regulation of ADRB2 expression is associated with small cell neuroendocrine prostate cancer and adverse clinical outcomes in castration-resistant prostate cancer

ObjectivesThe net oncogenic effect of β2-adrenergic receptor ADRB2, whose downstream elements induce neuroendocrine differentiation and whose expression is regulated by EZH2, is unclear. ADRB2 expression and associated clinical outcomes in metastatic castration-resistant prostate cancer (mCRPC) are unknown. Methods and materialsThis was a retrospective analysis of a multi-center, prospectively enrolled cohort of mCRPC patients. Metastatic biopsies were obtained at progression, and specimens underwent laser capture microdissection and RNA-seq. ADRB2 expression was stratified by histology and clustering based on unsupervised hierarchical transcriptome analysis and correlated with EZH2 expression; an external dataset was used for validation. The association between ADRB2 expression and overall survival (OS) was assessed by log-rank test and a multivariable Cox proportional hazard model. ResultsOne hundred and twenty-seven patients with progressive mCRPC had sufficient metastatic tumor for RNA-seq. ADRB2 expression was lowest in the small cell-enriched transcriptional cluster (P < 0.01) and correlated inversely with EZH2 expression (r = -0.28, P < 0.01). These findings were validated in an external cohort enriched for neuroendocrine differentiation. Patients with tumors harboring low ADRB2 expression (lowest quartile) had a shorter median OS than those with higher (9.5 vs. 20.5 months, P = 0.02). In multivariable analysis, low ADRB2 expression was associated with a trend toward shorter OS (HR for death = 1.54, 95%CI 0.98–2.44). Conversely, higher expression of upstream transcriptional regulator EZH2 was associated with shortened OS (HR for death = 3.01, 95%CI 1.12–8.09). ConclusionsLow ADRB2 expression is associated with neuroendocrine differentiation and is associated with shortened survival. EZH2 is a potential therapeutic target for preventing neuroendocrine transdifferentiation and improving outcomes in mCRPC. Further studies of agents targeting β-adrenergic signaling are warranted.

Association of neuroendocrine (NE) mRNA expression profiling in hormone-sensitive tumors samples with adverse clinical outcome in castration-resistant prostate cancer (CRPC) patients.

165 Background: NE dedifferentiation is associated to clinical aggressiveness and resistance to androgen receptor inhibition in prostate cancer. We investigated impact of a NE expression signature in the clinical outcome of mCRPC patients treated with taxanes. Methods: This is a multicenter retrospective study. A customized panel of 45 NE-related gene signature was tested in total RNA from formalin-fixed paraffin-embedded hormone-sensitive tumor samples, by the nCounter platform (Nanostring Technologies). Patients were grouped according to their molecular profile by unsupervised clustering. Expression levels were correlated with taxanes response and clinical outcome. Independent association with survival was evaluated by multivariate Cox modeling. Results: Eighty seven patients were included in the study, 79 were treated with docetaxel and 8 with cabazitaxel. Median age was 64.8 (44-88.3) years and median follow-up was 20.7 (1.17-74.4) months. High expression of the NE signature was associated with a shorter time of CRPC development (N=60, median 12.8 vs 21.6, HR 2.4, 95%CI 1.3-4.3, P=0.003) and shorter OS from CRPC diagnosis (median 24.1 vs 41.33, HR 2.3, 95%CI 1.4-3.8, P=0.001). Moreover, according to the outcome to taxanes, high NE signature correlated with lower PSA-PFS (median 6.6 vs 10.1 mo P=0.047, HR 1.6, 95%CI 1-2.7, P=0.05) and OS (median 19 vs 22 mo, HR 1.8, 95%CI 1.1-2.8, P=0.014), and it was independently associated to a lower OS (HR 1.9, 95%CI 1.1-3.2, P=0.016). Conclusions: NE-related gene expression in hormone-sensitive tumor samples is associated with adverse clinical outcome and lower taxane benefit in metastatic CRPC patients. Thus, molecular characterization of primary tumors may be useful to guide treatment strategies in metastatic prostate cancer.

Abstract 1017: Novel inhibitors of AR-v7 nuclear import: new therapeutic opportunities for CRPC

Abstract Reactivation of androgen receptor (AR) signaling by active splice variants (AR-Vs) is one of key drivers of castration resistant prostate cancer (CRPC). AR-v7 is the most prevalent AR-V and its expression has been clinically associated with poor overall survival, resistance to the AR inhibitors enzalutamide and abiraterone, as well as taxane resistance. Given that treatment with AR inhibitors and taxanes are the only effective therapeutic modalities in CRPC, development of specific AR-v7 inhibitors is urgently needed. Mechanistically, AR-v7 re-activates AR signaling by being constitutively active in the nucleus. While taxane chemotherapy inhibits the nuclear import of AR which is significantly associated with clinical outcomes in CRPC, it has no effect on AR-v7 nuclear localization and activity. Mechanistically, AR-v7 lacks the microtubule-binding domain and—unlike AR—does not utilize the canonical importin-α/β pathway, or RanGTP for nuclear import. Using wheat germ agglutinin to block active protein nuclear uptake resulted in AR-v7 cytoplasmic sequestration, indicating a requirement for an alternative transport receptor. Further, mutation of AR-v7 dimerization domain (D-box) led to its cytoplasmic sequestration, indicating that the D-box is also required for nuclear import. As inhibition of AR nuclear import is a clinically validated therapeutic strategy, we developed a novel drug discovery platform to identify compounds that specifically inhibit AR-v7 nuclear import. Using cells stably expressing inducible AR-v7 in conjunction with an enzyme complementation assay we tested 166,000 compounds by high throughput screening (HTS). The robust HTS performance (Z&amp;gt;0.8) together with subsequent counter screens including confirmation and compound titration, cell toxicity, a tertiary imaging based screen, led to identification of lead compounds that inhibit AR-v7 nuclear import. The lead compounds share structural features, across two main chemotypes, which are amenable to structure-activity relationship studies to identify the most desirable compound for in vivo studies. Using newly synthesized compounds from each of the two chemotypes, we showed specific dose-dependent inhibition of AR-v7 nuclear import. Currently, we are testing these compounds, on inhibition of AR-v7 transcriptional activity across several cell models including enzalutamide-resistant cells as well as inhibition of tumor growth in AR-v7 xenograft models. In parallel we are testing the lead compound for potential direct binding to the AR-v7 dimerization domain or to candidate alternative nuclear transport receptors. Further development of our lead small molecules will yield novel chemotypes, with desirable pharmacological properties that target the unique AR-v7 nuclear import pathway and can be clinically combined with existing AR therapies. Note: This abstract was not presented at the meeting. Citation Format: Seaho Kim, Mohd Azrin Jamalruddin, Eiman Mukhtar, Michael Miller, Leigh Baxt, Stacia Kargman, Andrew Stamford, Peter Meinke, Paraskevi Giannakakou. Novel inhibitors of AR-v7 nuclear import: new therapeutic opportunities for CRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1017.

Prognostic significance of antihypertensive agents in men with castration-resistant prostate cancer

PurposeComorbidity with hypertension (HTN) may affect the outcome of castration-resistant prostate cancer (CRPC). In this study, we evaluated the prognostic impact of antihypertensive agents in patients with CRPC treated with androgen receptor axis-targeting (ARAT) agents or docetaxel chemotherapy. Patients and methodsThis study included 156 Japanese men with CRPC who were treated with ARAT agents (n = 85) or docetaxel (n = 71) at our hospital between 2008 and 2017. Associations between clinicopathological factors, HTN status, progression-free survival (PFS) and overall survival (OS) were evaluated by univariate and multivariate analysis. ResultsWhen adjusted for age, prostate-specific antigen levels at pretreatment, Gleason score, and clinical M-stage, comorbid HTN was significantly associated with better OS (hazards ratio, 95% confidence interval: 0.41, 0.21–0.77; P = 0.0051), but not with PFS (hazards ratio, 95% confidence interval: 0.64, 0.38–1.11; P = 0.11) in patients treated with ARAT agent. However, HTN was not associated with PFS or OS for patients treated with docetaxel. ConclusionsUse of antihypertensive agents has prognostic significance for patients with CRPC treated with ARAT agent, but not docetaxel.

Dietary Tomato, but Not Lycopene Supplementation, Impacts Molecular Outcomes of Castration-resistant Prostate Cancer in the TRAMP Model (P05-015-19)

ObjectivesThe objective was to determine whether dietary tomato or lycopene supplementation impacted the expression of biomarkers associated with aggressive tumors and poorer prognostic outcomes in castration-resistant prostate cancer (CRPC). MethodsTransgenic adenocarcinoma of the mouse prostate (TRAMP) mice were castrated at 12 weeks of age to mimic the effects of androgen deprivation therapy (ADT) leading to CRPC. Mice were separated into three dietary groups (n = 30/group) and fed powdered, modified AIN-93 G diets containing 10% tomato powder (TP), lycopene beadlets matched for lycopene content of TP (LYCO), or placebo beadlets (PLAC). Tumors were detected and monitored for growth by ultrasound scan. After 5 weeks of CRPC growth, mice were euthanized, and prostate and tumor tissues were collected. Histological analysis was used to identify presence of tumor neuroendocrine (NE) phenotype (by synaptophysin [syp] expression) and androgen receptor (AR) expression. ELISA and Western blot were used to quantify interleukin-6 (IL-6) and STAT3, and RT-PCR was used to measure downstream targets of both AR and STAT3. ResultsNo differences were observed between AR expression or presence of the NE phenotype between dietary groups. However, lower tumor weight was associated with both AR and syp expression in mice fed TP. A positive correlation (P = 0.03) was observed between reduced tumor IL-6 (P = 0.012) and reduced phosphorylation (activation) of STAT3 (P = 0.017) in tumors of mice fed TP when compared to PLAC or LYCO. RNA analysis showed reduced expression of genes related to invasion (mmp-2; P = 0.006), cell proliferation (cdk1; P = 0.01), NE phenotype (ngfr; P = 0.001), and androgen metabolism (srd5a2; P = 0.03) in tumors of TP-fed mice. ConclusionsPhysiological levels of dietary tomato, but not lycopene supplementation, reduced the expression of molecular biomarkers typically upregulated in CRPC tissues that are associated with tumor growth and metastasis. Funding SourcesThis research is supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health.

A distinct plasma lipid signature associated with poor prognosis in castration-resistant prostate cancer.

Lipids are known to influence tumour growth, inflammation and chemoresistance. However, the association of circulating lipids with the clinical outcome of metastatic castration-resistant prostate cancer (CRPC) is unknown. We investigated associations between the plasma lipidome and clinical outcome in CRPC. Lipidomic profiling by liquid chromatography-tandem mass spectrometry was performed on plasma samples from a Phase 1 discovery cohort of 96 CRPC patients. Results were validated in an independent Phase 2 cohort of 63 CRPC patients. Unsupervised analysis of lipidomic profiles (323 lipid species) classified the Phase 1 cohort into two patient subgroups with significant survival differences (HR 2.31, 95% CI 1.44-3.68, p = 0.0005). The levels of 46 lipids were individually prognostic and were predominantly sphingolipids with higher levels associated with poor prognosis. A prognostic three-lipid signature was derived (ceramide d18:1/24:1, sphingomyelin d18:2/16:0, phosphatidylcholine 16:0/16:0) and was also associated with shorter survival in the Phase 2 cohort (HR 4.8, 95% CI 2.06-11.1, p = 0.0003). The signature was an independent prognostic factor when modelled with clinicopathological factors or metabolic characteristics. The association of plasma lipids with CRPC prognosis suggests a possible role of these lipids in disease progression. Further research is required to determine if therapeutic modulation of the levels of these lipids by targeting their metabolic pathways may improve patient outcome.

The plasma lipidome in castration-resistant prostate cancer.

5055 Background: Biomarker studies of metastatic castration-resistant prostate cancer (CRPC) have mainly focused on changes in the cancer, however, the host environment and its interactions with cancer is increasingly important, especially given the increasing association of PC outcomes and obesity. We sought associations between the plasma lipidome and clinical outcome in CRPC. Methods: Plasma samples were obtained from a Phase 1 discovery cohort of 96 CRPC patients before and after the first cycle of docetaxel chemotherapy. Lipidomic profiling of the plasma samples was performed by liquid chromatography and electrospray ionisation-tandem mass spectrometry. Results were subsequently assessed in a Phase 2 validation cohort of 63 CRPC patients. Results: Lipidomic profiling detected 323 lipid species in plasma samples from the Phase 1 cohort. Patients could be classified into two subgroups with significant survival differences according to their baseline lipidomic profiles (median overall survival 13.7 vs 21.7 months; HR 2.31, 95% CI 1.44-3.68, p = 0.0005). The baseline levels of 46 lipids were individually prognostic (p &lt; 0.01) and predominantly sphingolipids. A prognostic three-lipid signature was derived (ceramide (d18:1/24:1), sphingomyelin (d18:2/16:0), phosphatidylcholine (16:0/16:0)) (11.7 versus 21.7 months, p = 0.00001; HR 2.94, 95% CI 1.80-4.81, p = 0.00002). The signature was associated with shorter overall survival in the Phase 2 cohort (HR 4.78, 95% CI 2.06-11.1, p = 0.0003), and was an independent prognostic factor when modeled with clinicopathological factors and metabolic characteristics (BMI, cholesterol and triacylglycerol). The AUC of ROC analysis of 12 month survival for a clinicopathological model (AUC 0.70, 95% CI 0.54-0.87, p = 0.03) was enhanced by the addition of the 3-lipid signature (AUC 0.73, 95% CI 0.57-0.89, p = 0.01). Conclusions: Our study is the first to comprehensively profile the plasma lipidome of men with CRPC using cutting-edge lipidomic profiling technology, to identify a plasma lipid signature that can reproducibly predict outcome in CRPC and can improve on clinicopathological models. Therapeutic modulation of the levels of these lipids by targeting their metabolic pathways may improve patient outcome.

Clinical Utility of Gallium-68 PSMA PET/CT Scan for Prostate Cancer.

Background:Prostate cancer is biologically and clinically a heterogeneous disease that makes imaging evaluation challenging. One of the important challenges in this cancer is to detect recurrent disease. Biochemical response using Prostate Specific Antigen (PSA) and Imaging using several PET tracers have poor sensitivity and specificity. Therefore, we analyse the role of Ga68-PSMA (Prostate Specific Membrane Antigen) imaging in prostate cancer, which is a new PET tracer.Methods:In this study, we evaluated PET scans of 262 patients with diagnosis of prostate cancer. These patients were scanned using Ga68-PSMA for either staging or response evaluation.Results:336 PSMA scans were performed. Ga68-PSMA scan resulted in the detection of extra-prostatic disease in 53.2% of cases when done at baseline before commencing any treatment. The sensitivity of Ga68-PSMA at baseline with histopathological diagnosis was 95% with 95% CI ranging from 86% to 98%. The positive predictive value was high at 98% with 95% CI ranging from 91% to 99%. In 26 (10%) patients who had surgical castration, Ga68-PSMA scan was able to detect disease progression / castration resistance in 100% of cases. The outcome of castration-resistant prostate cancer was compared with other cases where castration was not done. In those who did not undergo castration, there was a significantly better response by hormone therapy (p = 0.03) and radiotherapy (p = 0.01) on Ga68-PSMA. The sensitivity of Ga68-PSMA response with biochemical response was 66.7% with 95% CI ranging between 46 %- 82.7%. Ga68-PSMA response did not correlate with biochemical response.Conclusion:Ga68-PSMA has good sensitivity for diagnosis, staging, restaging, evaluation of therapy response and prognostication in prostate cancer.

Abstract 4965: Analytic validation and clinical qualification of a novel immunohistochemical assay for AR-V7 protein expression in metastatic prostate cancer

Abstract BACKGROUND: The androgen-receptor splice variant 7 (AR-V7) has been implicated in the development of castration resistant prostate cancer (CRPC) and resistance to current therapies including enzalutamide and abiraterone. AR-V7 mRNA expression in circulating tumour cells of patients with CRPC correlated with treatment resistance. However, the importance of AR-V7 has been questioned in light of low AR-V7 mRNA levels relative to the full-length androgen receptor in CRPC and it is critically important to develop validated assays that confirm AR-V7 protein levels and its clinical importance in patients with CRPC. METHODS: Following validation of a monoclonal antibody, immunohistochemical analysis of nuclear AR-V7 (alongside a nuclear AR N-terminal domain antibody; AR-NTD) was performed in a patient cohort identified with matched therapy-naive hormone-sensitive primary prostate cancer (HSPC) and CRPC. We determined the levels of nuclear AR-V7 as patients progressed from HSPC to CRPC. We also determined if AR-V7 expression levels associated with overall survival from time of CRPC biopsy. RESULTS: In our patient cohort (n = 39), nuclear AR-V7 (p = &amp;lt;0.0001) and nuclear AR-NTD (p = 0.0006) increased significantly as patients progressed from HSPC to CRPC. Lower nuclear AR-V7 expression was associated with improved overall survival from time of CRPC biopsy in patient groups divided by the 25th (18.7 vs 9.6 months; HR 0.36 [95% CI 0.17-0.62]; p = 0.002), 50th (13.0 vs 9.8 months; HR 0.60 [95% CI 0.29-1.07]; p = 0.09) or 75th (6.0 vs 11.5 months; HR 0.31 [95% CI 0.04-0.34]; p = 0.0004) percentile of AR-V7 expression. Similarly, a lower nuclear AR-V7 to nuclear AR-NTD ratio was associated with improved overall survival from time of CRPC biopsy in patient groups divided by the 25th (17.8 vs 9.1 months; HR 0.42 [95% CI 0.22-0.78], p = 0.01), 50th (14.0 vs 8.8 months; HR 0.43 [95% CI 0.17-0.69], p = 0.005) and 75th (11.5 vs 7.3 months; HR 0.39 [95% CI 0.09-0.69], p = 0.009) percentile. Nuclear AR-NTD did not associate with overall survival from CRPC biopsy. CONCLUSION: We provide first evidence that expression of nuclear AR-V7 protein not only increases with emerging treatment resistance in CRPC but also is associated with overall survival from time of CRPC biopsy. These data support AR-V7 protein being key to CRPC progression and that agents targeting AR splice variants will be important to improve patient outcome in CRPC. Citation Format: Daniel Nava Rodrigues, Jon Welti, Adam Sharp, Shihua Sun, Ruth Riisnaes, Ines Figueiredo, Zafeiris Zafeiriou, Pasquale Rescigno, Johann S. de Bono, Stephen R. Plymate. Analytic validation and clinical qualification of a novel immunohistochemical assay for AR-V7 protein expression in metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4965.