Cytosine Deaminase-Overexpressing hTERT-Immortalized Human Adipose Stem Cells Enhance the Inhibitory Effects of Fluorocytosine on Tumor Growth in Castration Resistant Prostate Cancer.

A promising de novo approach for the treatment of Castration-resistant prostate cancer (CRPC) exploits cell-mediated enzyme prodrug therapy comprising cytosine deaminase (CD) and fluorouracil (5-FC). The aim of this study was to determine the potential of bacterial CD-overexpressing hTERT-immortalized human adipose stem cells (hTERT-ADSC.CD) to suppress CRPC. A lentiviral vector encoding a bacterial CD gene was used to transfect and to generate the hTERT-ADSC.CD line. The ability of the cells to migrate selectively towards malignant cells was investigated in vitro. PC3 and hTERT-ADSC.CD cells were co-cultured. hTERT-ADSC.CD and 1 × 106 PC3 cells were administered to nude mice via intracardiac and subcutaneous injections, respectively, and 5-FC was given for 14 days. hTERT-ADSC.CD were successfully engineered. Enhanced in vitro hTERT-ADSC.CD cytotoxicity and suicide effect were evident following administration of 5 µM 5-FC. hTERT-ADSC.CD, together with 5-FC, augmented the numbers of PC3 cells undergoing apoptosis. In comparison to controls administered hTERT-ADSC.CD monotherapy, hTERT-ADSC.CD in combination with 5-FC demonstrated a greater suppressive effect on tumor. In CPRC-bearing mice, tumor suppression was enhanced by the combination of CD-overexpressing ADSC and the prodrug 5-FC. Stem cells exhibiting CD gene expression are a potential novel approach to treatment for CRPC.

Homologous Recombination Repair Gene Mutations in Prostate Cancer: Prevalence and Clinical Value.

Despite advances in our understanding of the molecular landscape of prostate cancer and the development of novel biomarker-driven therapies, the prognosis of patients with metastatic prostate cancer that is resistant to conventional hormonal therapy remains poor. Data suggest that a significant proportion of patients with metastatic castration-resistant prostate cancer (mCRPC) have mutations in homologous recombination repair (HRR) genes and may benefit from poly(ADP-ribose) polymerase (PARP) inhibitors. However, the adoption of HRR gene mutation testing in prostate cancer remains low, meaning there is a missed opportunity to identify patients who may benefit from targeted therapy with PARP inhibition, with or without novel hormonal agents. Here, we review the current knowledge regarding the clinical significance of HRR gene mutations in prostate cancer and discuss the efficacy of PARP inhibition in patients with mCRPC. This comprehensive overview aims to increase the clinical implementation of HRR gene mutation testing and inform future efforts in personalized treatment of prostate cancer.

Prevalence and Spectrum of AR Ligand-Binding Domain Mutations Detected in Circulating-Tumor DNA Across Disease States in Men With Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) is typically treated with agents directly or indirectly targeting the androgen receptor (AR) pathway. However, such treatment is limited by resistance mechanisms, including the development of activating mutations in the AR ligand-binding domain (AR-LBD). METHODS: This study evaluated a database of over 15,000 patients with advanced prostate cancer (PC) undergoing comprehensive circulating-tumor DNA analysis (Guardant360, Redwood City, CA) between 2014 and 2021, with associated clinical information from administrative claims (GuardantINFORM database). RESULTS: Of 15,705 patients with PC included, 54% had mCRPC at the time of their blood draw. Of those, 49% had previous treatment with an AR pathway inhibitor (ARPi). AR-LBD mutation prevalence was 15% in patients with mCRPC who were untreated with a next-generation ARPi, 22% in those after one line of ARPi therapy, and 24% in those after two lines of ARPi treatment. Next-generation ARPi treatment yielded an increase in AR L702H and T878A/S mutations after abiraterone, and an increase in AR L702H and F877L mutations after enzalutamide. AR-LBD+ patients demonstrated unique biology, including increased concurrent mutations in the cell-cycle, wingless-related integration site, homologous recombination repair, and phospho-inositide 3-kinase pathways (all P < .0005), and greater low-level (copy number <10) AR amplifications (P = .0041). AR-LBD+ patients exhibited worse overall survival (OS) relative to a matched cohort of AR-LBD- patients (50.1 v 60.7 months, unadjusted log-rank P = .013). CONCLUSION: This large database analysis demonstrates that AR-LBD mutation prevalence increases after next-generation ARPi use. AR-LBD+ tumors demonstrate unique biology (more oncogenic pathway mutations and low-level AR amplification) and reduced OS. These findings inform the development of novel therapies designed to circumvent AR-mediated therapeutic resistance.

Cyclin-dependent kinase 12 deficiency reprogrammes cellular metabolism to alleviate ferroptosis potential and promote the progression of castration-resistant prostate cancer.

BACKGROUND: Cyclin-dependent kinase 12 (CDK12)-deficient prostate cancer defines a subtype of castration-resistant prostate cancer (CRPC) with a poor prognosis. Current therapy, including PARP inhibitors, shows minimal treatment efficacy for this subtype of CRPC, and the underlying mechanism remains elusive. METHODS: Based on bioinformatics analysis, we evaluated the relationship between CDK12 deficiency and prostate cancer patient's prognosis and treatment resistance. Furthermore, we used CRISPR-Cas9 technology and mass spectrometry-based metabolomic profiling to reveal the metabolic characteristics of CDK12-deficient CRPC. To elucidate the specific mechanisms of CDK12 deficiency-mediated CRPC metabolic reprogramming, we utilized cell RNA-seq profiling and other molecular biology techniques, including cellular reactive oxygen species probes, mitochondrial function assays, ChIP-qPCR and RNA stability analyses, to clarify the role of CDK12 in regulating mitochondrial function and its contribution to ferroptosis. Finally, through in vitro drug sensitivity testing and in vivo experiments in mice, we identified the therapeutic effects of the electron transport chain (ETC) inhibitor IACS-010759 on CDK12-deficient CRPC. RESULTS: CDK12-deficient prostate cancers reprogramme cellular energy metabolism to support their aggressive progression. In particular, CDK12 deficiency enhanced the mitochondrial respiratory chain for electronic transfer and ATP synthesis to create a ferroptosis potential in CRPC cells. However, CDK12 deficiency downregulated ACSL4 expression, which counteracts the lipid oxidation stress, leading to the escape of CRPC cells from ferroptosis. Furthermore, targeting the ETC substantially inhibited the proliferation of CDK12-deficient CRPC cells in vitro and in vivo, suggesting a potential new target for the therapy of CDK12-deficient prostate cancer. CONCLUSIONS: Our findings show that energy and lipid metabolism in CDK12-deficient CRPC work together to drive CRPC progression and provide a metabolic insight into the worse prognosis of CDK12-deficient prostate cancer patients. KEY POINTS: CDK12 deficiency promotes castration-resistant prostate cancer (CRPC) progression by reprogramming cellular metabolism. CDK12 deficiency in CRPC leads to a more active mitochondrial electron transport chain (ETC), ensuring efficient cell energy supply. CDK12 phosphorylates RNA Pol II to ensure the transcription of ACSL4 to regulate ferroptosis. Mitochondrial ETC inhibitors exhibit better selectivity for CDK12-deficient CRPC cells, offering a promising new therapeutic approach for this subtype of CRPC patients.

Patient-derived castration-resistant prostate cancer model revealed CTBP2 upregulation mediated by OCT1 and androgen receptor.

BACKGROUND: Prostate cancer is dependent on androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) has proven effective in targeting prostate cancer. However, castration-resistant prostate cancer (CRPC) eventually emerges. AR signaling inhibitors (ARSI) have been also used, but resistance to these agents develops due to genetic AR alterations and epigenetic dysregulation. METHODS: In this study, we investigated the role of OCT1, a member of the OCT family, in an AR-positive CRPC patient-derived xenograft established from a patient with resistance to ARSI and chemotherapy. We conducted a genome-wide analysis chromatin immunoprecipitation followed by sequencing and bioinformatic analyses using public database. RESULTS: Genome-wide analysis of OCT1 target genes in PDX 201.1 A revealed distinct OCT1 binding sites compared to treatment-naïve cells. Bioinformatic analyses revealed that OCT1-regulated genes were associated with cell migration and immune system regulation. In particular, C-terminal Binding Protein 2 (CTBP2), an OCT1/AR target gene, was correlated with poor prognosis and immunosuppressive effects in the tumor microenvironment. Metascape revealed that CTBP2 knockdown affects genes related to the immune response to bacteria. Furthermore, TISIDB analysis suggested the relationship between CTBP2 expression and immune cell infiltration in prostate cancer, suggesting that it may contribute to immune evasion in CRPC. CONCLUSIONS: Our findings shed light on the genome-wide network of OCT1 and AR in AR-positive CRPC and highlight the potential role of CTBP2 in immune response and tumor progression. Targeting CTBP2 may represent a promising therapeutic approach for aggressive AR-positive CRPC. Further validation will be required to explore novel therapeutic strategies for CRPC management.

Aberrant Super-Enhancer Landscape in Enzalutamide-Resistant Prostate Cancer Cells.

Background: Castration-resistant prostate cancer (CRPC), which has developed resistance to next-generation antiandrogens, such as enzalutamide (Enz), is a lethal disease. Furthermore, transcriptional regulation by super enhancers (SEs) is crucial for the growth and spread of prostate cancer, as well as drug resistance. The functions of SEs, a significant class of noncoding DNA cis-regulatory elements, have been the subject of numerous recent studies in the field of cancer research. Materials and Methods: The goal of this research was to identify SEs associated with Enz resistance in C4-2B cells using chromatin immunoprecipitation sequencing and cleavage under targets and tagmentation (CUT&Tag). Using HOMER analysis to predict protein/gene-binding motifs, we identified master transcription factors (TFs) that may bind to SE sites. Using small interfering RNA, WST-1 assays, and qRT-PCR, we then confirmed the associations between TFs of SEs and Enz resistance. Results: A total of 999 SEs were screened from C4-2B EnzR cells in total. Incorporating analysis with RNA-seq data revealed 41 SEs to be strongly associated with the promotion of Enz resistance. In addition, we finally predicted that master TFs bind to SE-binding regions. Subsequently, we selected zinc finger protein 467 (ZFP467) and SMAD family member 3 to confirm the functional connections of master TFs with Enz resistance through SEs (ZNF467). Conclusions: In this study, SMAD3 and ZNF467 were found to be closely related to Enz-resistant CRPC. Our research uncovered a sizable group of SEs linked to Enz resistance in prostate cancer, dissected the mechanisms underlying SE Enz resistance, and shed light on potential clinical uses for SEs.

Prospective Study of Homologous Recombination Repair Gene Mutation Prevalence in Patients With Advanced Prostate Cancer From Latin America: Challenges and Future Approaches.

PURPOSE: The prevalence of homologous recombination repair gene mutations (HRRm) in patients with metastatic castration-resistant prostate cancer (mCRPC) in Latin America and the Caribbean (LAC) is unknown. Prevalence of homologous Recombination repair (HRR) gene mutatiOns in patientS with metastatic castration resistant ProstatE Cancer in LaTin America (PROSPECT) aimed to determine this prevalence and to describe the demographic and clinical characteristics of the participants. MATERIALS AND METHODS: This was a prospective, cross-sectional, multicenter study across 11 cancer centers in seven LAC countries. After informed consent, all eligible participants underwent genomic testing by provided blood samples for germline HRR testing; they also provided PC tissue blocks if available for somatic HRR testing. RESULTS: Between April 2021 and April 2022, 387 patients (median age, 70 years [49-89], 94.3% Eastern Cooperative Oncology Group 0-1) with mCRPC were enrolled in the study. Almost 40% of them had a family history of cancer, and the overall time from their initial PC and mCRPC diagnosis was 3 years and 1 year, respectively. The overall prevalence of germline HRRm was 4.2%. The mutations detected included the genes CHEK2 (n = 4, 1%), ATM (n = 3, 0.8%), BRCA2 (n = 3, 0.8%), BRIP1 (n = 2, 0.5%), RAD51B (n = 2, 0.5%), BRCA1 (n = 1, 0.3%), and MRE11 (n = 1, 0.3%). The prevalence of somatic HRRm could not be assessed because of high HRR testing failure rates (79%, 199/251) associated with insufficient DNA, absence of tumor cells, and poor-quality DNA. CONCLUSION: Despite the study's limitations, to our knowledge, PROSPECT was the first attempt to describe the prevalence of HRRm in patients with PC from LAC. Notably, the germline HRRm prevalence in this study was inferior to that observed in North American and European populations. The somatic HRR testing barriers identified are being addressed by several projects to improve access to HRR testing and biomarker-based therapies in LAC.

CYB561 supports the neuroendocrine phenotype in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is associated with resistance to androgen deprivation therapy, and an increase in the population of neuroendocrine (NE) differentiated cells. It is hypothesized that NE differentiated cells secrete neuropeptides that support androgen-independent tumor growth and induce aggressiveness of adjacent proliferating tumor cells through a paracrine mechanism. The cytochrome b561 (CYB561) gene, which codes for a secretory vesicle transmembrane protein, is constitutively expressed in NE cells and highly expressed in CRPC. CYB561 is involved in the α-amidation-dependent activation of neuropeptides, and contributes to regulating iron metabolism which is often dysregulated in cancer. These findings led us to hypothesize that CYB561 may be a key player in the NE differentiation process that drives the progression and maintenance of the highly aggressive NE phenotype in CRPC. In our study, we found that CYB561 expression is upregulated in metastatic and NE prostate cancer (NEPC) tumors and cell lines compared to normal prostate epithelia, and that its expression is independent of androgen regulation. Knockdown of CYB561 in androgen-deprived LNCaP cells dampened NE differentiation potential and transdifferentiation-induced increase in iron levels. In NEPC PC-3 cells, depletion of CYB561 reduced the secretion of growth-promoting factors, lowered intracellular ferrous iron concentration, and mitigated the highly aggressive nature of these cells in complementary assays for cancer hallmarks. These findings demonstrate the role of CYB561 in facilitating transdifferentiation and maintenance of NE phenotype in CRPC through its involvement in neuropeptide biosynthesis and iron metabolism pathways.

Impact of Circulating Tumor Cell-Expressed Prostate-Specific Membrane Antigen and Prostate-Specific Antigen Transcripts in Different Stages of Prostate Cancer.

PURPOSE: Prostate-specific membrane antigen (PSMA)-based images, which visually quantify PSMA expression, are used to determine prostate cancer micrometastases. This study evaluated whether a circulating tumor cell (CTC)-based transcript platform, including PSMA mRNA, could help identify potential prognostic markers in prostate cancer. EXPERIMENTAL DESIGN: We prospectively enrolled 21 healthy individuals and 247 patients with prostate cancer [localized prostate cancer (LPCa), n = 94; metastatic hormone-sensitive prostate cancer (mHSPC), n = 44; and metastatic castration-resistant prostate cancer (mCRPC), n = 109]. The mRNA expression of six transcripts [PSMA, prostate-specific antigen (PSA), AR, AR-V7, EpCAM, and KRT 19] from CTCs was measured, and their relationship with biochemical recurrence (BCR) in LPCa and mCRPC progression-free survival (PFS) rate in mHSPC was assessed. PSA-PFS and radiological-PFS were also calculated to identify potential biomarkers for predicting androgen receptor signaling inhibitor (ARSI) and taxane-based chemotherapy resistance in mCRPC. RESULTS: CTC detection rates were 75.5%, 95.3%, and 98.0% for LPCa, mHSPC, and mCRPC, respectively. In LPCa, PSMA [hazard ratio (HR), 3.35; P = 0.028) and PSA mRNA (HR, 1.42; P = 0.047] expressions were associated with BCR. Patients with mHSPC with high PSMA (HR, 4.26; P = 0.020) and PSA mRNA (HR, 3.52; P = 0.042) expressions showed significantly worse mCRPC-PFS rates than those with low expression. Increased PSA and PSMA mRNA expressions were significantly associated with shorter PSA-PFS and radiological PFS in mCPRC, indicating an association with drug resistance. CONCLUSIONS: PSMA and PSA mRNA expressions are associated with BCR in LPCa. In advanced prostate cancer, PSMA and PSA mRNA can also predict rapid progression from mHSPC to mCRPC and ARSI or taxane-based chemotherapy resistance.

Quantifying Y chromosome loss in primary and metastatic prostate cancer by chromosome painting.

Somatic Y chromosome loss in hematopoietic cells is associated with higher mortality in men. However, the status of the Y chromosome in cancer tissue is not fully known due to technical limitations, such as difficulties in labelling and sequencing DNA from the Y chromosome. We have developed a system to quantify Y chromosome gain or loss in patient-derived prostate cancer organoids. Using our system, we observed Y chromosome loss in 4 of the 13 (31%) patient-derived metastatic castration-resistant prostate cancer (mCRPC) organoids; interestingly, loss of Yq (long arm of the Y chromosome) was seen in 38% of patient-derived organoids. Additionally, potential associations were observed between mCRPC and Y chromosome nullisomy. The prevalence of Y chromosome loss was similar in primary and metastatic tissue, suggesting that Y chromosome loss is an early event in prostate cancer evolution and may not a result of drug resistance or organoid derivation. This study reports quantification of Y chromosome loss and gain in primary and metastatic prostate cancer tissue and lays the groundwork for further studies investigating the clinical relevance of Y chromosome loss or gain in mCRPC.

AR-V7 expression facilitates accelerated G2/M phase transition in castration-resistant prostate cancer.

The emergence of AR-V7, a truncated isoform of AR upon androgen deprivation therapy treatment, leads to the development of castration resistant prostate cancer (CRPC). Understanding mechanisms that regulate AR-V7 expression is critical for developing newer therapeutic strategies. In this study, we have investigated the regulation of AR-V7 during cell cycle and identified a distinct pattern of periodic fluctuation, peaking during G2/M phase. This fluctuation correlates with the expression of Cdc-2 like kinase 1 (CLK1) and phosphorylated serine/arginine-rich splicing factor 1 (p-SRSF1) during these phases, pointing towards their role in AR-V7 generation. Functional assays reveal that CLK1 knockdown prolongs the S phase, leading to altered cell cycle distribution and increased accumulation of AR-V7 and pSRSF1 in G1/S phase. Conversely, CLK1 overexpression rescues AR-V7 and p-SRSF1 levels in the G2/M phase, consistent with observed cell cycle alterations upon AR-V7 knockdown and overexpression in CRPC cells. Furthermore, overexpression of kinase-deficient CLK1 mutant leads to diminished AR-V7 levels during G2/M, underlining the essential contribution of CLK1's kinase activity in modulating AR-V7 expression. Collectively, our findings, for the first time, show periodic regulation of AR-V7 expression, its effect on cell cycle progression and the critical role of CLK1-pSRSF1 axis in modulating AR-V7 expression throughout the cell cycle.

First-line combination treatment with PARP and androgen receptor-signaling inhibitors in HRR-deficient mCRPC: Applying clinical study findings to clinical practice in the United States.

INTRODUCTION: Metastatic castration-resistant prostate cancer (mCRPC) remains incurable and develops from biochemically recurrent PC treated with androgen deprivation therapy (ADT) following definitive therapy for localized PC, or from metastatic castration-sensitive PC (mCSPC). In the mCSPC setting, treatment intensification of ADT plus androgen receptor (AR)-signaling inhibitors (ARSIs), with or without chemotherapy, improves outcomes vs ADT alone. Despite multiple phase 3 trials demonstrating a survival benefit of treatment intensification in PC, there remains high use of ADT monotherapy in real-world clinical practice. Prior studies indicate that co-inhibition of AR and poly(ADP-ribose) polymerase (PARP) may result in enhanced benefit in treating tumors regardless of alterations in DNA damage response genes involved either directly or indirectly in homologous recombination repair (HRR). Three recent phase 3 studies evaluated the combination of a PARP inhibitor (PARPi) with an ARSI as first-line treatment for mCRPC: TALAPRO-2, talazoparib plus enzalutamide; PROpel, olaparib plus abiraterone acetate and prednisone (AAP); and MAGNITUDE, niraparib plus AAP. Results from these studies have led to the recent approval in the United States of talazoparib plus enzalutamide for the treatment of mCRPC with any HRR alteration, and of both olaparib and niraparib indicated in combination with AAP for the treatment of mCRPC with BRCA alterations. SUMMARY: Here, we review the newly approved PARPi plus ARSI treatments within the context of the mCRPC treatment landscape, provide an overview of practical considerations for the combinations in clinical practice, highlight the importance of HRR testing, and discuss the benefits of treatment intensification for patients with mCRPC.

Developing a Novel Enzalutamide-Resistant Prostate Cancer Model via AR F877L Mutation in LNCaP Cells.

Prostate cancer is a leading diagnosis and major cause of cancer-related deaths in men worldwide. As a typical hormone-responsive disease, prostate cancer is commonly managed with androgen deprivation therapy (ADT) to curb its progression and potential metastasis. Unfortunately, progression to castration-resistant prostate cancer (CRPC), a notably more aggressive phase of the disease, occurs within a timeframe of 2-3 years following ADT. Enzalutamide, a recognized androgen receptor (AR) antagonist, has been employed as a standard of care for men with metastatic castration-resistant prostate cancer (mCRPC) since it was first approved in 2012, due to its ability to prolong survival. However, scientific evidence suggests that sustained treatment with AR antagonists may induce acquired AR mutations or splice variants, such as AR F877L, T878A, and H875Y, leading to drug resistance and thereby diminishing the therapeutic efficacy of these agents. Thus, the establishment of prostate cancer models incorporating these particular mutations is essential for developing new therapeutic strategies to overcome such resistance and evaluate the efficacy of next-generation AR-targeting drugs. We have developed a CRISPR (clustered regularly interspaced short palindromic repeats)-based knock-in technology to introduce an additional F877L mutation in AR into the human prostate cell line LNCaP. This article provides comprehensive descriptions of the methodologies for cellular gene editing and establishment of an in vivo model. Using these methods, we successfully identified an enzalutamide-resistant phenotype in both in vitro and in vivo models. We also assessed the efficacy of target protein degraders (TPDs), such as ARV-110 and ARV-667, in both models, and the corresponding validation data are also included here. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Generation of AR F877L-mutated LNCaP cell line using CRISPR technology Basic Protocol 2: Validation of drug resistance in AR F877L-mutated LNCaP cell line using the 2D CTG assay Support Protocol: Testing of sgRNA efficiency in HEK 293 cells Basic Protocol 3: Validation of drug resistance in AR F877L-mutated LNCaP cell line in vivo.

Androgen receptor splice variants drive castration-resistant prostate cancer metastasis by activating distinct transcriptional programs.

One critical mechanism through which prostate cancer (PCa) adapts to treatments targeting androgen receptor (AR) signaling is the emergence of ligand-binding domain-truncated and constitutively active AR splice variants, particularly AR-V7. While AR-V7 has been intensively studied, its ability to activate distinct biological functions compared with the full-length AR (AR-FL), and its role in regulating the metastatic progression of castration-resistant PCa (CRPC), remain unclear. Our study found that, under castrated conditions, AR-V7 strongly induced osteoblastic bone lesions, a response not observed with AR-FL overexpression. Through combined ChIP-seq, ATAC-seq, and RNA-seq analyses, we demonstrated that AR-V7 uniquely accesses the androgen-responsive elements in compact chromatin regions, activating a distinct transcription program. This program was highly enriched for genes involved in epithelial-mesenchymal transition and metastasis. Notably, we discovered that SOX9, a critical metastasis driver gene, was a direct target and downstream effector of AR-V7. Its protein expression was dramatically upregulated in AR-V7-induced bone lesions. Moreover, we found that Ser81 phosphorylation enhanced AR-V7's pro-metastasis function by selectively altering its specific transcription program. Blocking this phosphorylation with CDK9 inhibitors impaired the AR-V7-mediated metastasis program. Overall, our study has provided molecular insights into the role of AR splice variants in driving the metastatic progression of CRPC.

Alternative splicing in prostate cancer progression and therapeutic resistance.

Prostate cancer (CaP) remains the second leading cause of cancer deaths in western men. CaP mortality results from diverse molecular mechanisms that mediate resistance to the standard of care treatments for metastatic disease. Recently, alternative splicing has been recognized as a hallmark of CaP aggressiveness. Alternative splicing events cause treatment resistance and aggressive CaP behavior and are determinants of the emergence of the two major types of late-stage treatment-resistant CaP, namely castration-resistant CaP (CRPC) and neuroendocrine CaP (NEPC). Here, we review recent multi-omics data that are uncovering the complicated landscape of alternative splicing events during CaP progression and the impact that different gene transcript isoforms can have on CaP cell biology and behavior. We discuss renewed insights in the molecular machinery by which alternative splicing occurs and contributes to the failure of systemic CaP therapies. The potential for alternative splicing events to serve as diagnostic markers and/or therapeutic targets is explored. We conclude by considering current challenges and promises associated with splicing-modulating therapies, and their potential for clinical translation into CaP patient care.

PDIA2 has a dual function in promoting androgen deprivation therapy induced venous thrombosis events and castrate resistant prostate cancer progression.

Androgen deprivation therapy (ADT) is the first line of treatment for metastatic prostate cancer (PCa) that effectively delays the tumor progression. However, it also increases the risk of venous thrombosis event (VTE) in patients, a leading cause of mortality. How a pro-thrombotic cascade is induced by ADT remains poorly understood. Here, we report that protein disulfide isomerase A2 (PDIA2) is upregulated in PCa cells to promote VTE formation and enhance PCa cells resistant to ADT. Using various in vitro and in vivo models, we demonstrated a dual function of PDIA2 that enhances tumor-mediated pro-coagulation activity via tumor-derived extracellular vehicles (EVs). It also stimulates PCa cell proliferation, colony formation, and xenograft growth androgen-independently. Mechanistically, PDIA2 activates the tissue factor (TF) on EVs through its isomerase activity, which subsequently triggers a pro-thrombotic cascade in the blood. Additionally, TF-containing EVs can activate the Src kinase inside PCa cells to enhance the AR signaling ligand independently. Androgen deprivation does not alter PDIA2 expression in PCa cells but enhances PDIA2 translocation to the cell membrane and EVs via suppressing the clathrin-dependent endocytic process. Co-recruitment of AR and FOXA1 to the PDIA2 promoter is required for PDIA2 transcription under androgen-deprived conditions. Importantly, blocking PDIA2 isomerase activity suppresses the pro-coagulation activity of patient plasma, PCa cell, and xenograft samples as well as castrate-resistant PCa xenograft growth. These results demonstrate that PDIA2 promotes VTE and tumor progression via activating TF from tumor-derived EVs. They rationalize pharmacological inhibition of PDIA2 to suppress ADT-induced VTE and castrate-resistant tumor progression.

Genomic Correlates of Prostate-Specific Membrane Antigen Expression and Response to 177Lu-PSMA-617: A Retrospective Multicenter Cohort Study.

PURPOSE: While 177Lu-PSMA-617 (LuPSMA) is an effective therapy for many patients with metastatic castration-resistant prostate cancer (mCRPC), biomarkers associated with outcomes are not well defined. We hypothesized that prostate cancer mutational profile may associate with clinical activity of LuPSMA. We devised a study to evaluate associations between mCRPC mutational profile with LuPSMA clinical outcomes. METHODS: This was a multicenter retrospective analysis of patients with mCRPC with next-generation sequencing (NGS) who received LuPSMA. PSA50 response (ie, ≥50% decline in prostate-specific antigen [PSA]) rate, PSA progression free survival (PSA PFS), and overall survival (OS) were compared between genetically defined subgroups. RESULTS: One hundred twenty-six patients with NGS results who received at least one cycle of LuPSMA were identified. The median age was 73 (IQR, 68-78) years, 124 (98.4%) received ≥1 prior androgen receptor-signaling inhibitor, and 121 (96%) received ≥1 taxane-based chemotherapy regimen. Fifty-eight (46%) patients with a DNA damage repair gene mutation (DNA damage response group) and 59 (46.8%) with a mutation in TP53, RB1, or PTEN tumor suppressor genes (TSG group) were identified. After adjusting for relevant confounders, the presence of ≥1 TSG mutation was associated with shorter PSA PFS (hazard ratio [HR], 1.93 [95% CI, 1.05 to 3.54]; P = .034) and OS (HR, 2.65 [95% CI, 1.15 to 6.11]; P = .023). There was improved OS favoring the DNA damage response group (HR, 0.37 [95% CI, 0.14 to 0.97]; P = .044) on multivariable analysis. Univariate analysis of patients with ATM mutations had significantly higher rates of PSA50 response, PSA PFS, and OS. CONCLUSION: Outcomes on LuPSMA varied on the basis of mutational profile. Prospective studies to define the clinical activity of LuPSMA in predefined genomic subgroups are justified.

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer.

Mammalian switch/sucrose nonfermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, an orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 (ATP binding cassette subfamily B member 1) overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.

Development and Characterisation of a New Patient-Derived Xenograft Model of AR-Negative Metastatic Castration-Resistant Prostate Cancer.

As the treatment landscape for prostate cancer gradually evolves, the frequency of treatment-induced neuroendocrine prostate cancer (NEPC) and double-negative prostate cancer (DNPC) that is deficient for androgen receptor (AR) and neuroendocrine (NE) markers has increased. These prostate cancer subtypes are typically refractory to AR-directed therapies and exhibit poor clinical outcomes. Only a small range of NEPC/DNPC models exist, limiting our molecular understanding of this disease and hindering our ability to perform preclinical trials exploring novel therapies to treat NEPC/DNPC that are urgently needed in the clinic. Here, we report the development of the CU-PC01 PDX model that represents AR-negative mCRPC with PTEN/RB/PSMA loss and CTNN1B/TP53/BRCA2 genetic variants. The CU-PC01 model lacks classic NE markers, with only focal and/or weak expression of chromogranin A, INSM1 and CD56. Collectively, these findings are most consistent with a DNPC phenotype. Ex vivo and in vivo preclinical studies revealed that CU-PC01 PDX tumours are resistant to mCRPC standard-of-care treatments enzalutamide and docetaxel, mirroring the donor patient's treatment response. Furthermore, short-term CU-PC01 tumour explant cultures indicate this model is initially sensitive to PARP inhibition with olaparib. Thus, the CU-PC01 PDX model provides a valuable opportunity to study AR-negative mCRPC biology and to discover new treatment avenues for this hard-to-treat disease.

Treatment Patterns and Clinical Outcomes Among Patients With Metastatic Prostate Cancer Harboring Homologous Recombination Repair Mutations.

BACKGROUND: There is currently limited literature assessing the real-world treatment patterns and clinical outcomes of patients with metastatic castration-resistant prostate cancer (mCRPC) and homologous recombination repair (HRR) mutations. METHODS: Medical charts were abstracted for mCRPC patients with ≥ 1 of 12 HRR somatic gene alterations treated at US oncology centers participating in the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange. Treatment patterns and clinical outcomes were assessed from the initiation of first-line or later (1L+) mCRPC therapy received on or after July 1, 2014. RESULTS: Among 138 patients included in the study, the most common somatic HRR mutations were CDK12 (47.8%), BRCA2 (22.5%), and ATM (21.0%). Novel hormonal therapy and taxane chemotherapy were most commonly used in 1L; taxane use increased in later lines. Median overall survival (95% confidence interval [CI]) was 36.3 (30.7-47.8) months from initiation of 1L therapy and decreased for subsequent lines. Similarly, there was a trend of decreasing progression-free survival and prostate-specific antigen response from 1L to 4L+ therapy. CONCLUSIONS: Treatment patterns identified in this study were similar to those among patients with mCRPC regardless of tumor HRR mutation status in the literature.