Background and objective:The prevalence and clinical history of germline variants in bladder cancer and upper tract urothelial cancer (UTUC) remains incompletely defined, particularly regarding mismatch repair (MMR) and homologous recombination repair (HRR) variants. This study aims to evaluate the prevalence of germline variants in patients with bladder cancer and/or UTUC referred for germline testing, and to report the personal and family cancer histories of patients with MMR (Lynch syndrome) and HRR variants. Methods:We retrospectively analyzed 3561 urothelial cancer patients (3130 with bladder cancer only, 370 with UTUC, and 61 with both) who underwent germline testing between 1996 and 2025 at Myriad Genetics. We describe the prevalence of pathogenic/likely pathogenic germline variants in patients with UTUC and bladder cancer, and characterize the personal and family cancer histories of patients with MMR (MSH2, MSH6, MLH1, and PMS2) and HRR (BRCA1 and BRCA2, among others) variants, comparing with an institutional cohort of MMR carriers (n = 35; 2012-2025). We also assess the proportion of MMR variant carriers who would be referred for testing under existing guidelines.Key findings and limitationsIn this cohort, 702 (20%) patients had pathogenic/likely pathogenic germline variants, including 328 patients with MMR and 298 patients with HRR variants. MMR variants were more common in UTUC, with 27% (n = 101) harboring an MSH2 variant compared with 7.6% (n = 109) of bladder cancer patients (p < 0.001). However, conversely, 58% (n = 109) of patients with an MSH2 variant had bladder-only disease. Prior nonurothelial cancers occurred in 54-63% of MMR and 35-41% of HRR carriers. Conclusions and clinical implications:Lynch syndrome is common in patients with UTUC; yet, many carriers present with bladder cancer alone. Personal and family cancer histories frequently precede urothelial cancer, underscoring the need for routine germline testing in UTUC and consideration of broader testing across urothelial cancer. Patient summary:Inherited genetic variants, especially those associated with Lynch syndrome, are common in patients with ureter and renal pelvis urothelial cancer. However, many patients with these variants have urothelial cancer of the bladder only. Broader genetic testing, particularly in those with a suggestive personal or family cancer history, may help identify patients at risk who might otherwise be missed.

Prostate cancer (PCa) is the second leading cause of cancer death for men in the United States. While organ-confined disease has reasonable expectation of cure, metastatic PCa is universally fatal upon recurrence during hormone therapy, a stage termed castration-resistant prostate cancer (CRPC). Until such time as molecularly defined subtypes can be identified and targeted using precision medicine, it is necessary to investigate new therapies that may apply to the CRPC population as a whole. The administration of ascorbate, more commonly known as ascorbic acid or Vitamin C, has proved lethal to and highly selective for a variety of cancer cell types. There are several mechanisms currently under investigation to explain how ascorbate exerts anti-cancer effects. A simplified model depicts ascorbate as a pro-drug for reactive oxygen species (ROS), which accumulate intracellularly and generate DNA damage. It was therefore hypothesized that poly(ADP-ribose) polymerase (PARP) inhibitors, by inhibiting DNA damage repair, would augment the toxicity of ascorbate. Results Two distinct CRPC models were found to be sensitive to physiologically relevant doses of ascorbate. Moreover, additional studies indicate that ascorbate inhibits CRPC growth in vitro via multiple mechanisms including disruption of cellular energy dynamics and accumulation of DNA damage. Combination studies were performed in CRPC models with ascorbate in conjunction with escalating doses of three different PARP inhibitors (niraparib, olaparib, and talazoparib). The addition of ascorbate augmented the toxicity of all three PARP inhibitors and proved synergistic with olaparib in both CRPC models. Finally, the combination of olaparib and ascorbate was tested in vivo in both castrated and non-castrated models. In both cohorts, the combination treatment significantly delayed tumor growth compared to monotherapy or untreated control. Conclusions These data indicate that pharmacological ascorbate is an effective monotherapy at physiological concentrations and kills CRPC cells. Ascorbate-induced tumor cell death was associated with disruption of cellular energy dynamics and accumulation of DNA damage. The addition of PARP inhibition increased the extent of DNA damage and proved effective at slowing CRPC growth both in vitro and in vivo. These findings nominate ascorbate and PARPi as a novel therapeutic regimen that has the potential to improve CRPC patient outcomes.

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Prostate cancer (PCa) is the second most lethal cancer in men in the United States. African American (AA) men have twice the incidence and death rate from the disease than European American (EA) men. Early-stage PCa is treated with hormone deprivation therapy, although patients frequently experience relapse. Advanced stage PCa is associated with increased expression and activity of the DNA damage/repair pathway enzyme, poly (ADP-ribose) polymerase 1 (PARP1). Furthermore, PARP1 inhibitors are FDA-approved for the treatment of advanced PCa tumors that carry mutations in components of a specific DNA damage/repair pathway termed homologous recombination repair (HRR). However, PARPi also provide benefit in model systems without HRR incompetencies. A number of different PARPi have now been developed, tested and approved for use in PCa. These inhibitors utilize multiple biochemical mechanisms of action and exhibit distinct potencies and toxicity profiles. While there is emerging evidence of differences in DNA damage/repair pathway enzyme expression between EA and AA men, PARP1 itself has not been fully explored in the context of race. This study hypothesized that 1) AA and EA PCa may respond differently to PARPi and 2) different PARPi may differentially impact the transcriptome, irrespective of HRR status. To test these hypotheses, PCa patient samples from a racially diverse cohort were examined to define race-based differences in PARP activity/expression. Additionally, biologically relevant doses of five clinically relevant PARPi were established across multiple PCa lines carrying different genetic backgrounds, HRR status, and hormone therapy sensitivities. Collectively, these findings demonstrate a link between racial background and PARP1 expression/activity and define a core transcriptional response that lies downstream of all five PARPi, while simultaneously defining transcriptional programs unique to each inhibitor. These findings broaden our understanding of the effector pathways downstream of individual PARPi and provide a compelling rationale for a broader exploration of the impact of race on the response to PARPi. They may also help refine personalized recommendations for use of specific PARPi. ### Competing Interest Statement The authors have declared no competing interest.

Prostate cancer (PCa) is the second most lethal cancer in men in the US. African American (AA) men have twice the incidence and death rate of European American (EA) men. Advanced PCa shows increased expression and activity of the DNA damage/repair pathway enzyme, poly (ADP-ribose) polymerase 1 (PARP1). PARP1 inhibitors (PARPi) are FDA-approved for advanced PCa tumors with mutations in the homologous recombination repair (HRR) pathway. However, PARPi can provide benefit in model systems without HRR deficiencies. PARPi have distinct biochemical mechanisms, potencies, and toxicity profiles. While there is emerging evidence of differences in DNA damage/repair pathway enzyme expression between EA and AA men, PARP1 expression has not been fully explored in the context of race. This study hypothesized: (a) AA and EA PCa may respond differently to PARPi and (b) different PARPi may uniquely impact the transcriptome, irrespective of HRR status. Study results indicate a link between racial background and PARP1 expression/activity and define unique and overlapping transcriptional responses downstream of all five PARPi. These findings may lead to refined personalized recommendations for use of specific PARPi.

The six-transmembrane epithelial antigen of the prostate (STEAP; STEAP1 and STEAP2) metalloreductases are therapeutic targets for advanced prostate cancer, and their expression has been linked to androgen receptor (AR) signaling; however, the regulatory mechanism and functions of STEAP1 and STEAP2 in prostate cancer progression remain elusive. In this study, we explore how in vitro androgen modulation and AR inhibition influence the expression of STEAP family members in cell lines with varying reliance on androgen signaling. Our data show that in response to androgen deprivation, STEAP1 and STEAP2 exhibit elevated transcript levels, whereas STEAP4 levels are reduced, mirroring the expression profile of kallikrein-related peptidase 3 (KLK3). As STEAP1 and STEAP2 are implicated in the exocytic pathway, we evaluated expression profiles in small extracellular vesicles (sEV) released from prostate cancer cells and in circulating sEVs. STEAP1, but not STEAP2, is upregulated in sEVs from AR-negative cells, which express low cellular STEAP1, and AR-positive cells, which express high cellular STEAP1. These results indicate selective packaging of STEAP1 in prostate cancer cell-derived sEVs, irrespective of AR status and cellular STEAP1 expression levels. Finally, ex vivo analysis of circulating sEVs from genetically engineered mice carrying prostate cancer shows that STEAP1 is found in the sEV cargo and that its levels are independent of protumorigenic β1 integrin expression in the prostatic epithelium. IMPLICATIONS:Understanding how androgen dependence affects STEAP1 expression in both tumor cells and sEVs across distinct disease stages will illuminate the clinical benefit of combinatorial AR and STEAP1-directed therapies and inform the optimal placement of STEAP1 targeting within the prostate cancer disease continuum.

Abstract Prostate cancer (PCa) is the 2nd leading cause of cancer related deaths in US men. PCa is an androgen dependent disease driven by the androgen receptor (AR). Currently, androgen-deprivation therapy (ADT) is the standard of care first-line therapy for metastatic PCa. Resistance to ADT uniformly leads to lethal disease, termed castration-resistant prostate cancer (CRPC). Thus, there is an unmet clinical need to identify and develop novel strategies to treat CRPC. CBP/p300 are potent co-activators for AR. High expression of CBP/p300 is associated with locally advanced disease and castration resistant function of AR, resulting in poor patient outcomes and further highlighting the need to discern the role of CBP/p300 to potentially develop therapeutic targets for precision medicine. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300. In this study, CBP/p300 mediated bromodomain activity is targeted by CCS1477 (inobrodib), a first-in-class bromodomain inhibitor developed by Cell Centric. CCS1477 treatment demonstrated effective inhibition in growth and clonogenicity assays. Inhibition of the CBP/p300 bromodomain with CCS1477 resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression in addition to inhibition of associated factors such as c-MYC and its downstream targets in PCa cell lines as well as patient derived xenograft (PDX) models. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC patient samples. The clinical significance of CBP/p300 expression in PCa has been investigated via elucidation of CBP/p300 transcriptional reprogramming and its role in DNA damage response (DDR) pathways. Specifically, findings revealed that CBP/p300 bromodomain suppression sensitizes to AR-dependent DNA-repair. Transcriptional mapping identified CBP/p300 as regulators of cell proliferation and DNA repair processes, which were functionally confirmed across several PCa model systems. To assess relevance, exogenous challenge with radiation revealed that CBP/p300 are required for AR-mediated DNA repair, and CBP/p300 expression is linked to DNA repair capacity in the clinical setting. Molecular analyses revealed that CBP/p300 facilitate double-strand break (DSB) repair efficiency via homologous recombination (HR) mediated DDR. Congruently, CBP/p300 strongly correlated with HR gene expression in PCa patient tissue. These collective findings reveal that CBP/p300 govern repair of DNA DSBs by regulating HR, thus modulating genome integrity and promoting CRPC growth. These studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Citation Format: Sumaira Sardar, Lakshmi Ravindranath, Christopher McNair, Saswati N. Chand, Wei Yuan, Denisa Bogdan, Jonathan Welti, Adam Sharp, Matthew Schiewer, Lisa Butler, Johann de Bono, Kris Frese, Nigel Brooks, Neil Pegg, Karen Knudsen, Ayesha A. Shafi. Targeting the CBP/p300 axis in lethal prostate cancer impacts DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7116.

PARP is a nuclear enzyme with a major function in the DNA damage response. PARP inhibitors (PARPi) have been developed for treating tumors harboring homologous recombination repair defects that lead to a dependency on PARP. There are currently three PARPi approved for use in advanced prostate cancer, and several others are in clinical trials for this disease. Recent clinical trial results have reported differential efficacy based on the specific PARPi utilized as well as patient race. There is a racial disparity in prostate cancer, in which African American males are twice as likely to develop and die from the disease compared with European American males. Despite the disparity, there continues to be a lack of diversity in clinical trial cohorts for prostate cancer. In this review, PARP nuclear functions, inhibition, and clinical relevance are explored through the lens of racial differences. This review will touch on the biological variations that have been explored thus far between African American and European American males with prostate cancer to offer a rationale for investigating PARPi response in the context of race at both basic science and clinical development levels.

Castration resistant prostate cancer (CRPC) remains an incurable disease stage with ineffective treatments options. Here, the androgen receptor (AR) coactivators CBP/p300, which are histone acetyltransferases, were identified as critical mediators of DNA damage repair (DDR) to potentially enhance therapeutic targeting of CRPC. Key findings demonstrate that CBP/p300 expression increases with disease progression and selects for poor prognosis in metastatic disease. CBP/p300 bromodomain inhibition enhances response to standard of care therapeutics. Functional studies, CBP/p300 cistrome mapping, and transcriptome in CRPC revealed that CBP/p300 regulates DDR. Further mechanistic investigation showed that CBP/p300 attenuation via therapeutic targeting and genomic knockdown decreases homologous recombination (HR) factors in vitro, in vivo, and in human prostate cancer (PCa) tumors ex vivo. Similarly, CBP/p300 expression in human prostate tissue correlates with HR factors. Lastly, targeting CBP/p300 impacts HR-mediate repair and patient outcome. Collectively, these studies identify CBP/p300 as drivers of PCa tumorigenesis and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed and DDR therapies.

<p>Supplementary Figures 1-6</p>

Supplementary Table 1 - PDF file 282K, Supplemental table listing primer sequences utilized throughout study

Supplemental Figure 1: PLX8394 does not alter CRAF or c-Src phosphorylation; Supplemental Figure 2. Treatment of xenograft-derived tumor pieces in an ex vivo explant system; Supplemental Figure 3: Patient sample extended data; Supplemental Figure 4: Parallel ex vivo dosing and homodimerization of vemurafenib and PLX8394 treatments; Supplemental Figure 5: PBRT characterization; Supplemental Figure 6: Targeting AKT and PDGFRβ in PBRT cell lines does not resensitize to PLX8394; Supplemental Figure 7: BRAF V600E splice variant analysis of PBRT cell lines and signaling and growth response of second-line therapies; Supplemental Methods.

PDF file - 410K, Dose responses, transcriptional, biological, biochemical, and in vivo supplemental data

HuR silencing results in downregulation of PARG expression, which prevents efficient removal of PAR polymers with or without PARPi stress. Expression and function of other PAR removing enzymes is not affected with HuR silencing.

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.

Sigma1 is required for DHT-induced AR-mediated LD accumulation in prostate cancer cells. A, Confocal micrograph showing LD accumulation in LNCaP cells cultured in CSS containing medium for 3 days and then treated for 1, 2, 3, and 6 days of DHT (1 nmol/L). HCS LipidTOX stained LDs (red). DAPI stained nuclei (blue). Quantification of LD number per cell and average area of LD particles/cell. Data represent mean values from at least three independent determinations, and error bars represent SEM. LD particle numbers and lipid area were quantified using ImageJ. Statistical analysis was performed using ANOVA and Bonferroni after test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. B, Confocal micrograph showing LD accumulation in LNCaP transduced with nonspecific control and Sigma1 shRNA. Two distinct Sigma1 shRNA clones were tested and produced comparable results. Cells were cultured in CSS containing medium for 3 days and then treated for 3 days with DHT (1 nmol/L). LD number per cell was determined as in A, above. C, Immunoblots of whole-cell protein extracts from LNCaP cells infected with Sigma1 shRNA #4 and #5 and treated, serum starved for 3 days, and treated with 3 days of DHT. D, SRS confirming lipid content of LDs in LNCaP cells following 3 days of 1 nmol/L DHT treatment in CSS medium, conditions described above. E, LD numbers per cell in panel of AR-driven (C4-2, C4-2B), ARV-driven (22Rv1), and AR-negative, independent (PC3, DU145) prostate cancer cell lines. Data represent mean values from at least three independent determinations, and error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ns = no significance. F, Confocal micrograph of LDs in PC3 cells (endogenous AR-negative prostate cancer cell line), transfected with empty vector (pcDNA) or recombinant AR plasmid, then treated with DHT (1 nmol/L, 3 days). Quantification of the average number of LDs per cell. Right, Quantification of the mean number of particles per cell ± SE. *, P < 0.05; **, P < 0.01. G, Confocal micrograph showing that LDs accumulate only in AR-transduced PC3 cells. AR (green), LDs (red), DAPI stained nucleus (blue). H, Immunoblot further confirming transduction and expression of recombinant AR in PC3 cells. I, ARV7-induced LDs require Sigma1. LDs (red) in 22Rv1 cells transduced with nonspecific control shRNA or Sigma1 shRNA #5. Magnified inset (white boxes) shown below. LD stain (red), DAPI stain (blue). J, Immunoblot confirmation of Sigma1 shRNA KD in 22Rv1 cells. K, Control confirming that only ARV7-positive cells are also LD-positive. ARV7 immunostain (green), LD stain (red), DAPI stain (blue). L, LDs (red) in PC3 cells transduced with nonspecific control shRNA or Sigma1 shRNA #5 and subsequently transfected with ARV7. Magnified inset (white boxes) shown below. DAPI stain of nuclei (blue). Average number of LDs per cell calculated and analyzed as above. M, Immunoblot confirmation of Sigma1 shRNA KD and transfected ARV7 expression in PC3 cells.