177 Lu-PSMA-617 (Pluvicto TM , Lu-177 RLT) is an FDA-approved targeted radioligand therapy (RLT) for metastatic castration-resistant prostate cancer (mCRPC), but its durability of response to this singular approach poses a challenge to the field. Chimeric antigen receptor (CAR) T cell therapy has revolutionized clinical practice for hematological malignancies, but its clinical development for solid tumors, including mCRPC, has been encumbered by antigen heterogeneity and the immunosuppressive tumor microenvironment (TME). Here, we evaluate the therapeutic combination of Lu-177 RLT and PSCA-CAR T cells to overcome these barriers. In human xenograft and mouse syngeneic prostate cancer models with homogeneous or heterogeneous antigen expression, the sequential administration of Lu-177 RLT, cyclophosphamide (Cy), and PSCA-CAR T cells improves tumor control and prolongs survival compared to monotherapies. Mechanistically, Lu-177 RLT alone or with Cy remodels the TME by promoting pro-inflammatory myeloid responses and activating endogenous T cells, while enhancing CAR T cell activation and effector function. We additionally evaluated 225 Ac-PSMA-617 RLT as an emerging approach in combination with CAR T cells and observed anti-tumor responses, supporting its potential as an alternative RLT partner. These findings support RLT as an immune priming strategy to enhance CAR T cell therapy and provide a rationale for clinical translation of this combination in mCRPC. One Sentence Summary:Combining 177 Lu-PSMA-617 radioligand therapy with PSCA-CAR T cells improves tumor control and survival in prostate cancer models by overcoming the antigen heterogeneity and reshaping the immunosuppressive tumor microenvironment.

217 Background: Neuroendocrine prostate cancers (NEPC) currently have no effective therapies. While CAR (Chimeric Antigen Receptor) T cell therapies targeting PSMA and PSCA receptors are currently in phase 1/2 studies in metastatic castrate-resistant prostate cancer (mCRPC), antigen targets for NEPC remain limited. Lewis Y antigen (LeY) is an oncofetal antigen expressed only in adult tumour cells and is considered a target for CAR T cell therapy in haematological, lung, ovarian and colon cancer. We previously found that LeY is a promising target for CAR T cell therapy in prostate cancer and that preconditioning LeY-directed CAR T cells with carboplatin further enhances tumour responses in mCRPC. We investigated if LeY can be used as a novel CAR T cell target for NEPC. Methods: The expression of the Lewis Y antigen was assessed in our patient-derived prostate cancer xenograft collection using immunohistochemistry (IHC). To assess the efficacy of LeY-CAR T cells, we used specimens established from a patient (Patient 508) with NEPC who had incurable metastatic disease and failed standard treatments. We generated CAR T cells from his peripheral blood mononuclear cells (PBMCs) to test their specificity and efficacy in vitro against established cell lines. Results: In our MURAL PDX collection of 59 prostate tumours, 81% (9/11) NEPCs expressed LeY antigen on IHC. Next, we successfully produced anti-LeY CAR T cells from the patient’s PBMCs using retroviral transduction. Flow cytometry verified efficient transduction of the CAR constructs. Previous studies found that a 1:1 ratio of CD4 + :CD8 + T cells improved outcomes following CAR T cell therapy 5,6 , so we further phenotyped patient T cells by flow cytometry. The CD4 + :CD8 + T cell ratio was nearly 1:1 (51.7% and 41.4% of total T cells, respectively). Next, we used an immunobead assay to investigate CAR-T specificity in releasing proinflammatory cytokines. Significant cytokine production was observed after a 4-hour co-culture with LeY+ human ovarian cancer cell line, OVCAR3, vs co-culture with control LeY- MDA-MB435 cells (p=0.0004), demonstrating the specificity of the CAR T cells.Lastly, we showed that patient CAR T cells effectively killed LeY+ cells (OVCAR3), but not control LeY- cells (MDA-MB435; p<0.0001). Tumour cell death was measured by the amount of released chromium following 16-hour co-culture. CAR T cells killed LeY+ target cells more effectively than their non-transduced equivalents at a range of effector-to-target ratios, with up to 15-fold increase in cell death. Conclusions: Our preclinical studies show that most NEPC tumors express the LeY antigen and that patient-derived CAR T cells specifically kill those cells. These findings identified a potential CAR T treatment target for NEPC for a phase 1/2 study.

The histone methyltransferase EZH2 is an epigenetic transcriptional repressor which mediates gene silencing through methylation of H3K27 residues. EZH2 is overexpressed in castration-resistant prostate cancer (CRPC) and has been associated with prostate cancer progression and resistance to anti-androgen therapies. We have developed a potent, selective, SAM-competitive EZH2 inhibitor, PF-06821497 (Mevrometostat). Using genetic and pharmacological approaches, we demonstrate an epigenetic dependency in CRPC to EZH2 inhibition. EZH2 was identified as a hit in a genome-wide CRISPR screen in AR-positive, but not in AR-negative, CRPC models and was further confirmed in independent CRISPR-competition assays. Consistent with genetic data, long-term PF-1497 treatment showed selective anti-tumor activity in AR-positive CRPC cell lines. Anti-tumor activity of PF-1497 was further validated in vivo with dose-dependent tumor growth inhibition observed in correlation with H3K27me3 biomarker inhibition in CRPC xenograft models. EZH2 inhibition induced cell cycle arrest concomitant with transcriptional upregulation of tumor suppressor genes. Interestingly, we found a subset of genes induced upon enzalutamide treatment are also upregulated upon PF-1497 treatment. Transcriptional and epigenetic profiling revealed that genes repressed by AR show enrichment for H3K27me3 indicating a co-repressor function for EZH2. Combination in vitro treatment of PF-1497 with enzalutamide showed synergistic upregulation of such co-repressed genes associated with robust inhibition of proliferation, survival, cell cycle and stem cell pathways. This transcriptional synergy was associated with robust in vivo anti-tumor combination activity of PF-1497 with enzalutamide inducing durable tumor regressions. Similar combination benefit of PF-1497 and enzalutamide was observed across a panel of CRPC PDXs with anti-tumor activity enriched in AR-positive prostate cancer models, including tumors from patients who were refractory to anti-androgen therapies. Taken together, these data highlight the promise of EZH2 inhibition in treating prostate cancer and provide support for ongoing clinical study of PF-06821497 in combination with enzalutamide in mCRPC. Shikhar Sharma, Jelena Petrovic, Sara Linker, Whijae Roh, Joan Cao, Sean Uryu, Zhenxiong Wang, Yong Zhang, Colin A. Flaveny, Conglin Fan, Tao Xie, Kimberly H. Kim, Xiaoli Wang, Robert A. Rollins, Gail P. Risbridger, Renea A. Taylor, Mitchell G. Lawrence, Thomas A. Paul, Pei-Pei Kung. EZH2 inhibition as a therapeutic strategy for castration resistant prostate cancer in combination with AR-antagonist enzalutamide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7182.

PURPOSE:AKT inhibitors, such as capivasertib, have shown activity in specific patients with metastatic castration-resistant prostate cancer when combined with docetaxel although none have been approved. Although PTEN loss is often linked to AKT pathway activation and response to AKT inhibitors, clinical trials show no consistent association. This study uses patient-derived tumor models to identify biomarkers associated with an effective response to AKT inhibitor plus docetaxel. EXPERIMENTAL DESIGN:Targeted DNA sequencing and immunostaining for PTEN and phosphorylated AKT (p-AKT; Ser473) were assessed in 39 patient-derived xenografts (PDX) from patients with prostate cancer, including adenocarcinoma and neuroendocrine (NE) phenotypes. Matching PDX-derived organoids were used to evaluate the functional effects of capivasertib and docetaxel on in vitro tumor growth. RESULTS:p-AKT protein expression varied widely across PDX models and showed no correlation with PTEN/PI3K/AKT mutations or PTEN protein levels. NE tumors displayed higher p-AKT expression than adenocarcinomas. Knockdown of AKT1 in NE organoids increased sensitivity to docetaxel, whereas AKT1 overexpression decreased it. In three of seven organoids tested, the combination of capivasertib and docetaxel produced a synergistic effect, resulting in greater growth inhibition than either agent alone. These responsive organoids exhibited an NE phenotype and high p-AKT expression, consistent with a predictive response. CONCLUSIONS:Our preclinical findings indicate that p-AKT protein expression, rather than PTEN, may be a more reliable predictor of response to AKT inhibition combined with docetaxel. Using p-AKT as a parameter, we uncovered the efficacy of this combination in NE prostate cancer, highlighting the potential to refine patient selection criteria for future clinical trials.

Tumour development and progression reshape the physical properties of the surrounding tumour microenvironment (TME) including its biomechanical traits. This is driven by a prominent cell type in the TME, cancer associated fibroblasts (CAFs), which increases tissue stiffness via extracellular matrix deposition and remodelling. Currently, it is unclear whether there are also physical changes to CAFs at the cellular level and, if so, how they relate to patient outcome. Here we show that CAFs have distinct morphological and biomechanical features from normal fibroblasts. We examined matched, patient-derived CAFs and non-malignant prostate fibroblasts (NPFs) from 35 patients with primary prostate cancer. Morphologically, CAFs had more aligned stress fibres, and larger and more elongated nuclei, based on quantitative image analysis of confocal microscopy images. In addition, single-cell mechanical measurements using real-time deformability cytometry showed that CAFs are larger and stiffer than NPFs. These changes were consistent across patients and validated with atomic force microscopy. A combined morphomechanical score encompassing these features was significantly associated with patient outcome. In transcriptomic analyses, the score was correlated with microtubule dynamics and a myofibroblast phenotype. Importantly, we also demonstrated that morphomechanical features of prostate fibroblasts are modified by approved treatments for prostate cancer, such as docetaxel, and other small molecular inhibitors, such as axitinib. In summary, changes in cellular morphomechanical properties are a consistent feature of CAFs and associated with patient outcome. Moreover, cellular morphomechanical properties can be therapeutically targeted, potentially providing a new strategy for manipulating the TME to control cancer progression. ### Competing Interest Statement The authors have declared no competing interest.

Ribosome biogenesis (RiBi) is a key determinant of cell growth and proliferation and is highly elevated in cancer due to the activation by oncogenes such as MYC . First-generation RiBi inhibitor CX-5461, while demonstrating clinical potential for cancer treatment, also induces DNA damage through off-target inhibition of TOP2⍺ and potentially other mechanisms, bringing into question RiBi as a target for cancer therapy. In this study, we test second-generation RiBi inhibitor, PMR-116. PMR-116 exhibits improved drug-like properties compared to first-generation RiBi inhibitors and has robust anti-tumour activity in the absence of global DNA damage signalling in a broad range of pre-clinical models of haematologic and solid cancers, particularly in malignancies where MYC is either the driver of disease or is elevated. Thus, our work demonstrates that RiBi is a genuine target for cancer therapy and highlights the potential to exploit a critical therapeutic vulnerability in high- MYC human cancers with dismal therapeutic outcomes. Statement of significance Despite the development of new cancer therapies, most advanced malignancies remain incurable. We demonstrate that PMR-116, a second-generation RiBi inhibitor, has robust therapeutic efficacy in preclinical models of cancer, offering great promise to treat a broad spectrum of human solid and haematologic malignancies, especially where MYC is a driver. ### Competing Interest Statement The authors have declared no competing interest.

Background and objective Under the selective pressure of treatment, prostate cancer cells express constitutively active androgen receptor (AR) variants. Whether AR variants mediate therapy resistance remains contested, because they are often coexpressed with abundant full-length AR. Therefore, we sought to determine how truncated variants shape AR chromatin occupancy and responses to treatments in both the presence and absence of full-length AR. Methods We used a cohort of patient-derived xenografts of metastatic prostate cancer with diverse AR alterations. Chromatin immunoprecipitation and RNA sequencing were used to compare the landscape of AR binding and transcriptomic features. We assessed responses to castration by castrating host mice and evaluated responses to bipolar androgen therapy by administering testosterone cypionate. Key findings and limitations By profiling the AR cistrome, we identified a distinct group of tumours defined by ARv567es expression, a variant arising due to structural rearrangements of the AR gene. ARv567es-positive tumours also had a distinct epigenomic profile and altered transcriptional features, including loss of canonical AR-regulated gene signatures and elevated expression of AR-repressed genes. ARv567es-positive tumours were resistant to castration and bipolar androgen therapy. In tumours that coexpress full-length AR, this involves dampened transcriptional responses and disruption of the autoregulatory loop that modulates AR levels. Study limitations include the need for additional models of AR-driven prostate cancer. Conclusions and clinical implications The emergence of ARv567es via gene rearrangements causes transcriptional reprogramming and therapy resistance. This highlights ARv567es as a potential as a marker to guide treatment decisions.

420 Background: Prostatic ductal adenocarcinoma (DAC) is an aggressive prostate cancer variant, prone to early metastasis at low PSA levels and showing poor response to androgen blockade despite androgen receptor expression. Although DACs do not have any efficacious therapies, DACs frequently harbour DNA damage repair (DDR) mutations. We investigate the efficacy of combined PARP inhibitor (PARPi) and androgen signalling inhibitor (ARSi) therapy in DDR-proficient DAC tumours. Methods: To model DAC, organoids were developed in Matrigel from patient-derived xenografts (PDXs) originating from DDR-proficient radical prostatectomy (287R, 275R) and BRCA2 heterozygous mutant metastatic (201.1) tumours, retaining key histologic and genomic features. These organoids were exposed to different PARP inhibitors (Talazoparib, Saruparib) and androgen signalling inhibitors (Enzalutamide, Apalutamide, Darolutamide) for up to 11 days. Cell viability and growth responses were assessed using PrestoBlue and CellTiter-Glo assays and automated imaging analysis. SynergyFinder software calculated synergy scores for each treatment combination. Results were further validated in vivo using DDR-proficient 287R PDXs. Mechanistic insights were explored through RNA sequencing of 20 DAC and ten acinar radical prostatectomy samples, and four matched DAC and acinar PDXs, with γH2AX immunohistochemistry to examine DNA damage response. Results: Overall, PARPi/ARSi combination treatment significantly reduced organoid viability compared to PARPi alone or ARSi in DDR-proficient and heterozygous BRCA2 -mutant DAC tumours. The levels of synergy were comparable regardless of which PARPi and ARSi agents were combined. In vivo results using DDR-proficient PDX 287R confirmed the efficacy of PARPi + ARSi combination by reducing tumour volume by 58% compared to control ( p =0.0198) and by 40% versus PARPi alone ( p = 0.0326). Mechanistically, RNA sequencing demonstrated upregulation of multiple DDR pathways in DACs compared to acinar prostate tumours, regardless of the DDR status. After treatment with PARPi + ARSI combination, there was an increase in γH2AX compared to the control in the DDR-proficient PDX 287R (mean 1.42 vs 0.5, p=0.0056), suggesting enhanced DNA damage may contribute to the efficacy of the combination treatment. Conclusions: Our results show that PARPi increases the efficacy of ARSi in DAC. This is notable given the poor response of DAC to AR-directed treatments and provides the rationale for a pre-planned phase 1/2 study.

Neuroendocrine prostate cancer (NEPC) tumours are classified by pathology into several distinct subtypes. Gene expression profiling has revealed transcriptional heterogeneity across NEPC, but this is rarely considered in the context of variation between pathologies. Diagnosis typically relies on immunohistochemical markers (CHGA, SYP, NCAM1) and genomic alterations in RB1, PTEN and TP53. We hypothesized that NEPC pathologies have unique transcriptional features. Single‐cell RNA sequencing of 18 632 tumour cells from nine patient‐derived xenograft models representing five pathologies (small‐cell and large‐cell neuroendocrine carcinomas, focal neuroendocrine differentiation (Focal NED), low‐grade neuroendocrine and amphicrine) demonstrated pathway‐specific enrichment. Focal NED and amphicrine tumours exhibited cellular subpopulations enriched for KRAS, IL2‐STAT5 and TNF signalling pathways, absent in small‐ and large‐cell carcinomas, which were instead enriched for Myc and E2F pathways. Furthermore, focal NED cells exhibited minimal clonal divergence from adjacent adenocarcinoma cells, while small cell carcinoma cells were clonally distinct. These data underscore significant transcriptional variation among NEPC pathologies, highlighting focal NED's unique biological context and its clinical implications.

158 Background: Despite a lack of up-to-date clinical trial data, many clinicians advocate the use of Carboplatin monotherapy to treat patients with advanced Castrate Resistant Prostate Cancer (CRPC) who have exhausted multiple other treatment options. The aim of this study was to determine the overall survival (OS) and response rate in patients with advanced CRPC treated with Carboplatin monotherapy after progressing on other chemotherapy agents. Methods: Retrospective multicentre study of the use of Carboplatin in advanced CRPC patients in Australia. Demographic data, PSA response rates, survival data and Carboplatin treatment protocols, such as dose and duration, were collected. Exploratory analyses on potential prognostic parameters were performed. Results: 51 patients received Carboplatin: median age 68 (range 55–86 years). Most patients (78.3%) received Carboplatin AUC 5 at 3-weekly intervals. The median number of cycles of Carboplatin was 3 (range 1-17). Median time on treatment was 63 days (range 1-441). Median overall survival was 29.4 weeks (IQR 11.7 weeks). 6 (11.8%) patients had a PSA response ≥50%. The median time to PSA progression on Carboplatin was 67 days (range 15-418). 16 patients (31%) required a dose delay or reduction and 8 patients (15.6%) ceased Carboplatin secondary to side effects/ toxicity. Conclusions: Our findings demonstrate that in heavily pre-treated CRPC, Carboplatin has a modest benefit in a minority of patients with a low rate of toxicity in the advanced prostate cancer population.

BackgroundThere are relatively few widely used models of prostate cancer compared to other common malignancies. This impedes translational prostate cancer research because the range of models does not reflect the diversity of disease seen in clinical practice. In response to this challenge, research laboratories around the world have been developing new patient-derived models of prostate cancer, including xenografts, organoids, and tumor explants.MethodsIn May 2023, we held a workshop at the Monash University Prato Campus for researchers with expertise in establishing and using a variety of patient-derived models of prostate cancer. This review summarizes our collective ideas on how patient-derived models are currently being used, the common challenges, and future opportunities for maximizing their usefulness in prostate cancer research.ResultsAn increasing number of patient-derived models for prostate cancer are being developed. Despite their individual limitations and varying success rates, these models are valuable resources for exploring new concepts in prostate cancer biology and for preclinical testing of potential treatments. Here we focus on the need for larger collections of models that represent the changing treatment landscape of prostate cancer, robust readouts for preclinical testing, improved in vitro culture conditions, and integration of the tumor microenvironment. Additional priorities include ensuring model reproducibility, standardization, and replication, and streamlining the exchange of models and data sets among research groups.ConclusionsThere are several opportunities to maximize the impact of patient-derived models on prostate cancer research. We must develop large, diverse and accessible cohorts of models and more sophisticated methods for emulating the intricacy of patient tumors. In this way, we can use the samples that are generously donated by patients to advance the outcomes of patients in the future.

Prostate cancer is primarily hormone-dependent, and medical treatments have focused on inhibiting androgen biosynthesis or signaling through various approaches. Despite significant advances with the introduction of androgen receptor signalling inhibitors (ARSIs), patients continue to progress to castration-resistant prostate cancer (CRPC), highlighting the need for targeted therapies that extend beyond hormonal blockade. Chimeric Antigen Receptor (CAR) T cells and other engineered immune cells represent a new generation of adoptive cellular therapies. While these therapies have significantly enhanced outcomes for patients with hematological malignancies, ongoing research is exploring the broader use of CAR T therapy in solid tumors, including advanced prostate cancer. In general, CAR T cell therapies are less effective against solid cancers with the immunosuppressive tumor microenvironment hindering T cell infiltration, activation and cytotoxicity following antigen recognition. In addition, inherent tumor heterogeneity exists in patients with advanced prostate cancer that may prevent durable therapeutic responses using single-target agents. These barriers must be overcome to inform clinical trial design and improve treatment efficacy. In this review, we discuss the innovative and rationally designed strategies under investigation to improve the clinical translation of cellular immunotherapy in prostate cancer and maximise therapeutic outcomes for these patients.

Abstract 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.

Background Men with neuroendocrine prostate cancer (NEPC) have a poor prognosis. NEPC is commonly diagnosed by immunohistochemical markers (CHGA, SYP and NCAM1) and genomic features (mutations in RB1, PTEN, TP53). But by pathology, NEPC tumours are variable, leading to a classification of NE subtypes such as small cell and large cell neuroendocrine carcinomas, focal neuroendocrine differentiation (Focal NED), and Amphicrine. We postulated the diversity observed in NEPC pathologies might arise from differences in transcriptional profiles and the aim of this study is to utilize single-cell RNA sequencing to define the transcriptional differences between NEPC subtype pathologies. Methods Gene expression profiles were obtained for 18,632 individual tumour cells from 9 patient-derived xenograft (PDX) models representing five distinct neuroendocrine pathologies of prostate cancer. Integration and clustering of cell-level data demarked transcriptionally distinct sub-populations of cells. Differential gene expression, gene set enrichment and transcriptional factor regulon analysis identified expression signatures unique to specific neuroendocrine pathologies. Copy-number estimated from expression data revealed the clonal structure of PDXs with mixed adenocarcinoma and neuroendocrine pathologies. Results Significant differences were observed in the transcriptional profiles of NEPC pathology subtypes. Focal NED cells maintain AR signaling, similar to the amphicrine subtype but different from small and large cell carcinomas. Cellular sub-populations enriched for expression of KRAS, IL2-STAT5 and TNF-signaling genes were found in focal NED and amphicrine pathologies, but not in small or large cell carcinomas. In contrast, sub-populations enriched for the YAP, Myc and E2F pathways were detected in small cell, large cell and amphicrine tumours, but not in focal NED cells. Each pathology showed unique patterns of master regulator activity as well, further implicating focal NED as a transcriptionally distinct entity. Based on copy number alterations within PDXs of mixed pathology, focal NED cells showed little clonal divergence from neighboring adenocarcinoma cells, whereas cells with small cell neuroendocrine pathology were clonally distinct. Conclusions Neuroendocrine prostate cancer subtypes can be identified by pathology and our study shows that transcriptional features identified by single-cell RNA-sequencing also distinguish neuroendocrine subtypes pathologies from each other. In particular, our data redefine focal neuroendocrine differentiation as a pathology expressing androgen receptors (AR), exhibiting its distinctive composition of transcriptionally unique sub-populations. These findings advocate for differences in the treatment of NEPC tumors, particularly those displaying focal NED. ### Competing Interest Statement The authors have declared no competing interest.

FOXA family proteins act as pioneer factors by remodeling compact chromatin structures. FOXA1 is crucial for the chromatin binding of the androgen receptor (AR) in both normal prostate epithelial cells and the luminal subtype of prostate cancer (PCa). Recent studies have highlighted the emergence of FOXA2 as an adaptive response to AR signaling inhibition treatments. However, the role of the FOXA1 to FOXA2 transition in regulating cancer lineage plasticity remains unclear. Our study demonstrates that FOXA2 binds to distinct classes of developmental enhancers in multiple AR-independent PCa subtypes, with its binding depending on LSD1. Moreover, we reveal that FOXA2 collaborates with JUN at chromatin and promotes transcriptional reprogramming of AP-1 in lineage-plastic cancer cells, thereby facilitating cell state transitions to multiple lineages. Overall, our findings underscore the pivotal role of FOXA2 as a pan-plasticity driver that rewires AP-1 to induce the differential transcriptional reprogramming necessary for cancer cell lineage plasticity. The role of the FOXA1 to FOXA2 switch in the regulation of the response to androgen receptor signalling inhibition and lineage plasticity in prostate cancer remains unclear. Here, the authors highlight the function of FOXA2 in rewiring AP-1 to induce differential transcriptional reprogramming and lineage plasticity.

There are diverse phenotypes of castration-resistant prostate cancer, including neuroendocrine disease, that vary in their sensitivity to drug treatment. The efficacy of BET and CBP/p300 inhibitors in prostate cancer is attributed, at least in part, to their ability to decrease androgen receptor (AR) signalling. However, the activity of BET and CBP/p300 inhibitors in prostate cancers that lack the AR is unclear. In this study, we showed that BRD4, CBP, and p300 were co-expressed in AR-positive and AR-null prostate cancer. A combined inhibitor of these three proteins, NEO2734, reduced the growth of both AR-positive and AR-null organoids, as measured by changes in viability, size, and composition. NEO2734 treatment caused consistent transcriptional downregulation of cell cycle pathways. In neuroendocrine models, NEO2734 treatment reduced ASCL1 levels and other neuroendocrine markers, and reduced tumour growth in vivo. Collectively, these results show that epigenome-targeted inhibitors cause decreased growth and phenotype-dependent disruption of lineage regulators in neuroendocrine prostate cancer, warranting further development of compounds with this activity in the clinic. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

BACKGROUND:Cyclin-dependent kinase 9 (CDK9) stimulates oncogenic transcriptional pathways in cancer and CDK9 inhibitors have emerged as promising therapeutic candidates. METHODS:The activity of an orally bioavailable CDK9 inhibitor, CDKI-73, was evaluated in prostate cancer cell lines, a xenograft mouse model, and patient-derived tumor explants and organoids. Expression of CDK9 was evaluated in clinical specimens by mining public datasets and immunohistochemistry. Effects of CDKI-73 on prostate cancer cells were determined by cell-based assays, molecular profiling and transcriptomic/epigenomic approaches. RESULTS:CDKI-73 inhibited proliferation and enhanced cell death in diverse in vitro and in vivo models of androgen receptor (AR)-driven and AR-independent models. Mechanistically, CDKI-73-mediated inhibition of RNA polymerase II serine 2 phosphorylation resulted in reduced expression of BCL-2 anti-apoptotic factors and transcriptional defects. Transcriptomic and epigenomic approaches revealed that CDKI-73 suppressed signaling pathways regulated by AR, MYC, and BRD4, key drivers of dysregulated transcription in prostate cancer, and reprogrammed cancer-associated super-enhancers. These latter findings prompted the evaluation of CDKI-73 with the BRD4 inhibitor AZD5153, a combination that was synergistic in patient-derived organoids and in vivo. CONCLUSION:Our work demonstrates that CDK9 inhibition disrupts multiple oncogenic pathways and positions CDKI-73 as a promising therapeutic agent for prostate cancer, particularly aggressive, therapy-resistant subtypes.