Plasticity In Prostate Cancer Research Articles

Single-cell analysis of treatment-resistant prostate cancer: Implications of cell state changes for cell surface antigen–targeted therapies

Targeting cell surface molecules using radioligand and antibody-based therapies has yielded considerable success across cancers. However, it remains unclear how the expression of putative lineage markers, particularly cell surface molecules, varies in the process of lineage plasticity, wherein tumor cells alter their identity and acquire new oncogenic properties. A notable example of lineage plasticity is the transformation of prostate adenocarcinoma (PRAD) to neuroendocrine prostate cancer (NEPC)-a growing resistance mechanism that results in the loss of responsiveness to androgen blockade and portends dismal patient survival. To understand how lineage markers vary across the evolution of lineage plasticity in prostate cancer, we applied single-cell analyses to 21 human prostate tumor biopsies and two genetically engineered mouse models, together with tissue microarray analysis on 131 tumor samples. Not only did we observe a higher degree of phenotypic heterogeneity in castrate-resistant PRAD and NEPC than previously anticipated but also found that the expression of molecules targeted therapeutically, namely PSMA, STEAP1, STEAP2, TROP2, CEACAM5, and DLL3, varied within a subset of gene-regulatory networks (GRNs). We also noted that NEPC and small cell lung cancer subtypes shared a set of GRNs, indicative of conserved biologic pathways that may be exploited therapeutically across tumor types. While this extreme level of transcriptional heterogeneity, particularly in cell surface marker expression, may mitigate the durability of clinical responses to current and future antigen-directed therapies, its delineation may yield signatures for patient selection in clinical trials, potentially across distinct cancer types.

Abstract 6586: ORIC-944, a potent and selective allosteric PRC2 inhibitor with best-in-class properties, demonstrates combination synergy with AR pathway inhibitors in prostate cancer models

Abstract Polycomb repressive complex 2 (PRC2) dysregulation occurs in multiple tumor types and is associated with poor prognosis in patients with prostate cancer. In patients treated with androgen receptor pathway inhibitors (ARPI), emergence of tumor cell plasticity is associated with resistance to therapy. Dysregulation of epigenetic reprogramming factors, including PRC2, creates an environment that is permissive for such lineage plasticity. Inhibition of the PRC2 complex therefore may provide an opportunity to overcome or prevent the emergence of plasticity in prostate cancer. Notably, emerging clinical trial data has demonstrated the potential of ARPI and PRC2 inhibitor combination therapy to improve outcomes in patients with metastatic prostate cancer. PRC2 tri-methylates histone H3 at lysine 27 (H3K27me3), leading to long-term transcriptional silencing, a key mechanism regulating cellular functions such as cell growth and differentiation. Three core subunits comprise PRC2: the catalytic subunit enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). EED is essential for the histone methyltransferase activity of PRC2. First-generation PRC2 inhibitors, which target EZH2, exhibit poor drug properties, drug-drug interaction liabilities and short half-life requiring twice daily dosing at high doses in patients. We developed a second generation PRC2 inhibitor, ORIC-944, an allosteric inhibitor of PRC2 that binds the EED subunit. ORIC-944 is a potent, highly selective, orally bioavailable inhibitor with best-in-class properties. ORIC-944 demonstrated single agent tumor growth inhibition in a spectrum of in vivo prostate cancer models, including AR-positive, AR-mutant, ARv7, ARPI-responsive and ARPI-resistant models. In vitro analysis of the combination of ARPIs and ORIC-944 showed synergy in multiple prostate cancer cells, utilizing BLISS and other quantitative methods. In vivo studies revealed that ORIC-944 demonstrated increased efficacy in combination with ARPI, consistent with the in vitro synergy. RNA-seq analysis of transcriptional changes induced by ORIC-944 provided mechanistic insight into the role of PRC2 in prostate cancer lineage plasticity and combination response. These results position ORIC-944 as a best-in-class PRC2 inhibitor for evaluation in combination with ARPI in patients with metastatic prostate cancer. A phase 1b trial of ORIC-944 is ongoing (NCT05413421). Citation Format: Anneleen Daemen, Natalie Yuen, Amber Wang, Aleksandr Pankov, Livia Ulicna, Chelsea Chen, Frank L. Duong, Jason Long, Matthew A. Marx, James G. Christensen, Lori S. Friedman, Melissa R. Junttila. ORIC-944, a potent and selective allosteric PRC2 inhibitor with best-in-class properties, demonstrates combination synergy with AR pathway inhibitors in prostate cancer models [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 6586.

ADORA2A-driven proline synthesis triggers epigenetic reprogramming in neuroendocrine prostate and lung cancers.

Cell lineage plasticity is one of the major causes for the failure of targeted therapies in various cancers. However, the driver and actionable drug targets in promoting cancer cell lineage plasticity are scarcely identified. Here, we found that a G protein-coupled receptor, ADORA2A, is specifically upregulated during neuroendocrine differentiation, a common form of lineage plasticity in prostate cancer and lung cancer following targeted therapies. Activation of the ADORA2A signaling rewires the proline metabolism via an ERK/MYC/PYCR cascade. Increased proline synthesis promotes deacetylases SIRT6/7-mediated deacetylation of histone H3 at lysine 27 (H3K27), and thereby biases a global transcriptional output toward a neuroendocrine lineage profile. Ablation of Adora2a in genetically engineered mouse models inhibits the development and progression of neuroendocrine prostate and lung cancers, and, intriguingly, prevents the adenocarcinoma-to-neuroendocrine phenotypic transition. Importantly, pharmacological blockade of ADORA2A profoundly represses neuroendocrine prostate and lung cancer growth in vivo. Therefore, we believe that ADORA2A can be used as a promising therapeutic target to govern the epigenetic reprogramming in neuroendocrine malignancies.

UP60 - Integrative proteomics analysis of lineage plasticity in prostate cancer using PDX models

UP60 - Integrative proteomics analysis of lineage plasticity in prostate cancer using PDX models

UP47 - Pathological activities of androgen receptor variants in cell plasticity in prostate cancer

UP47 - Pathological activities of androgen receptor variants in cell plasticity in prostate cancer

ASCL1 is activated downstream of the ROR2/CREB signaling pathway to support lineage plasticity in prostate cancer

Lineage plasticity is a form of therapy-induced drug resistance. In prostate cancer, androgen receptor (AR) pathway inhibitors potentially lead to the accretion of tumor relapse with loss of AR signaling and a shift from a luminal state to an alternate program. However, the molecular and signaling mechanisms orchestrating the development of lineage plasticity under the pressure of AR-targeted therapies are not fully understood. Here, a survey of receptor tyrosine kinases (RTKs) identifies ROR2 as the top upregulated RTK following AR pathway inhibition, which feeds into lineage plasticity by promoting stem-cell-like and neuronal networks. Mechanistically, ROR2 activates the ERK/CREB signaling pathway to modulate the expression of the lineage commitment transcription factor ASCL1. Collectively, our findings nominate ROR2 as a potential therapeutic target to reverse the ENZ-induced plastic phenotype and potentially re-sensitize tumors to AR pathway inhibitors.

Abstract 4753: LSD1 &8211mediated FOXA2/AP1 transcription program drives lineage plasticity in prostate cancer

Abstract Background: While castration-resistant PCa (CRPC) can be further treated with second-generation androgen deprivation therapies (ADTs), the tumor can quickly generate resistance through multiple mechanisms. One critical mechanism is that tumor cells can progress to AR-indifferent stem cell-like (SCL-PCa or SCLPC) or neuroendocrine-like CRPC (NE-PCa or NEPC) through lineage plasticity. However, the underlying molecular basis remains to be determined, and clinical treatment options for these aggressive CRPC subtypes are currently limited. FOXA1 and FOXA2, which are members of the FOXA (Forkhead Box A) protein family, are pioneer transcription factors. While FOXA1 is well known for its function as a critical pioneer factor of AR and pivotal for maintaining AR signaling, the molecular function of FOXA2 in PCa cells is poorly understood despite that FOXA2 is known to be overexpressed in NEPC. Moreover, since FOXA2, like FOXA1, is currently undruggable in the clinic, there is an urgent need to decipher the molecular basis for the chromatin binding of FOXA2 and identify druggable targets that regulate FOXA2 binding and activity in the aggressive FOXA2-positive CRPC. Methods: In the current study, we first examined the role of FOXA2 in tumorigenesis and metastasis by using various in vitro and in vivo assays including mouse subcutaneous injection and zebrafish embryo injection approaches. We then conducted integrated whole genomic transcriptome and cistrome analyses in SCLPC and NEPC cell line models to characterize the activity of FOXA2 on chromatin and to identify its collaborating transcription factors that may play specific functions in promoting SCLPC or NEPC using a series of biochemical and bioinformatic approaches. Results: We found that FOXA2 silencing decreased the growth and metastasis of SCLPC and NEPC cells. Importantly, we discovered that FOXA2 chromatin binding is tightly associated with binding of JUN family proteins (primarily c-Jun) and FOXA2 silencing dramatically interrupted the global chromatin binding of c-Jun and FOSL1. The transcription targets of FOXA2/AP-1 are highly enriched for neuroendocrine and plasticity associated genes and associated with poor clinical outcomes. Furthermore, we also found that FOXA2 chromatin binding is globally enhanced by an epigenetic factor LSD1, and LSD1 inhibition can repress FOXA2/AP-1 activity in multiple CRPC models. Conclusion: Overall, our data indicate that FOXA2 functions to maintain tumor growth and metastasis in SCLPC and NEPC models. Mechanistically, FOXA2 can act as a pioneer factor of AP-1 (c-Jun/FOSL1) and reprogram AP-1 transcription activity. This FOXA2/AP-1 axis is regulated by LSD1 via demethylating FOXA2 protein and LSD1 inhibition represses FOXA2-dependent CRPC tumor progression. These findings provide novel mechanistic insights into the molecular mechanisms for PCa lineage plasticity and treatment resistance. Citation Format: Zifeng Wang, Mingyu Liu, Songqi Zhang, Muqing Li, Susan Patalano, Jill A. Macoska, Dong Han, Shuai Gao, Hansen He, Changmeng Cai. LSD1 &8211mediated FOXA2/AP1 transcription program drives lineage plasticity in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4753.

FOXA2 drives lineage plasticity and KIT pathway activation in neuroendocrine prostate cancer.

Prostate cancer adeno-to-neuroendocrine lineage transition has emerged as a mechanism of targeted therapeutic resistance. Identifying the direct molecular drivers and developing pharmacological strategies using clinical-grade inhibitors to overcome lineage transition-induced therapeutic resistance are imperative. Here, using single-cell multiomics analyses, we investigate the dynamics of cellular heterogeneity, transcriptome regulation, and microenvironmental factors in 107,201 cells from genetically engineered mouse prostate cancer samples with complete time series of tumor evolution seen in patients. We identify that FOXA2 orchestrates prostate cancer adeno-to-neuroendocrine lineage transition and that Foxa2 expression is significantly induced by androgen deprivation. Moreover, Foxa2 knockdown induces the reversal of adeno-to-neuroendocrine transition. The KIT pathway is directly regulated by FOXA2 and specifically activated in neuroendocrine prostate cancer (NEPC). Pharmacologic inhibition of KIT pathway significantly suppresses mouse and human NEPC tumor growth. These findings reveal that FOXA2 drives adeno-to-neuroendocrine lineage plasticity in prostate cancer and provides a potential pharmacological strategy for castration-resistant NEPC.

5 Oral - Lineage plasticity in prostate cancer depends on FGFR and JAK/STAT inflammatory signaling

Background: The inherent plasticity of tumor cells provides a mechanism of resistance to molecularly targeted therapies, exemplified by adeno-to-neuroendocrine lineage transitions in prostate and lung cancer. Here we investigate the root cause of lineage plasticity following Trp53 and Rb1 loss in genetically engineered mouse models, murine and patient-derived organoid cultures, and patient biospecimens with castrate-resistant prostate cancer.

P27 - Androgen receptor-mediated transcriptional repression targets cell plasticity in prostate cancer

P27 - Androgen receptor-mediated transcriptional repression targets cell plasticity in prostate cancer

Cytokines drive prostate cancer lineage plasticity.

Lineage plasticity is a critical mechanism of therapeutic resistance in cancer. In a recent issue of Science, Chan and colleagues demonstrate that early lineage plasticity in prostate cancer is driven by JAK-STAT inflammatory cytokine signaling.

Homing in on a Moving Target: Androgen Receptor Cistromic Plasticity in Prostate Cancer.

The androgen receptor (AR) is the critical driver in prostate cancer and exerts its function mainly through transcriptional control. Recent advances in clinical studies and cell line models have illustrated that AR chromatin binding features are not static; rather they are highly variable yet reproducibly altered between clinical stages. Extensive genomic analyses of AR chromatin binding features in different disease stages have revealed a high degree of plasticity of AR chromatin interactions in clinical samples. Mechanistically, AR chromatin binding patterns are associated with specific somatic mutations on AR and other permutations, including mutations of AR-interacting proteins. Here we summarize the most recent studies on how the AR cistrome is dynamically altered in prostate cancer models and patient samples, and what implications this has for the identification of therapeutic targets to avoid the emergence of treatment resistance.

In Vivo Application of CRISPR/Cas9 Revealed Implication of Foxa1 and Foxp1 in Prostate Cancer Proliferation and Epithelial Plasticity.

Simple SummaryProstate cancer is diagnosed in one out of eight men, with large implications on life quality. Forkhead box proteins are often found mutated in prostate cancer but their functions are still not fully understood. In this study, we applied CRISPR to investigate the function of two Forkhead box proteins, Foxa1 and Foxp1, in the mouse prostate in combination with loss of Pten. Our results reveal that Foxp1 is a tumor suppressor in prostate cancer progression by controlling proliferation and genes regulated by the androgen receptor. Foxa1 controls cell plasticity, as loss of Foxa1 converted the prostatic luminal cells to basal cells. Hereby, this study sheds light on two distinct functions of Forkhead box proteins in prostate cancer.Prostate cancer is the most common cancer in men in the Western world and the number is rising. Prostate cancer is notoriously heterogeneous, which makes it hard to generate and study in pre-clinical models. The family of Forkhead box (FOX) transcription factors are often altered in prostate cancer with especially high mutation burden in FOXA1 and FOXP1. FOXA1 harbors loss or gain of function mutations in 8% of prostate cancer, which increases to 14% in metastatic samples. FOXP1 predominately occurs with loss of function mutations in 7% of primary tumors, and similar incidents are found in metastatic samples. Here, we applied in vivo CRISPR editing, to study the loss of functions of these two FOX transcription factors, in murine prostate in combination with loss of Pten. Deficiency of Foxp1 increased proliferation in combination with loss of Pten. In contrast, proliferation was unchanged when androgen was deprived. The expression of Tmprss2 was increased when Foxp1 was mutated in vivo, showing that Foxp1 is a repressor for this androgen-regulated target. Furthermore, analysis of FOXP1 and TMPRSS2 expression in a human prostate cancer data set revealed a negative correlation. Mutation of Foxa1 in the murine prostate induces cell plasticity to luminal cells. Here, epithelial cells with loss of Foxa1 were transdifferentiated to cells with expression of the basal markers Ck5 and p63. Interestingly, these cells were located in the lumen and did not co-express Ck8. Overall, this study reveals that loss of Foxp1 increases cell proliferation, whereas loss of Foxa1 induces epithelial plasticity in prostate cancer.

Lineage plasticity in prostate cancer: Looking beyond intrinsic alterations

Emergence of small cell prostate cancer is linked to the plasticity of tumour cells and avoidance of environmental pressures. This process is thought to be reversable, however to-date evidence of this has been demonstrated in small-cell prostate cancer. To study the plasticity of prostate tumours, we look to clinical cohorts of patients covering the spectra of malignancy subtypes and utilise in vitro and in vivo models of disease progression. Current models have assisted in the understanding of the extremities of this plasticity, elucidating internal mechanisms and adaptations to stressors through transition to altered cell states. By interrogating the tumour microenvironment and earlier time points, we are beginning to form a deeper understanding of the full spectra of tumour plasticity. It could be proffered that this deeper understanding will lead to better patient outcome, with earlier interventions more likely to reverse plasticity and prevent trans-differentiation to the aggressive, small cell phenotype.

Lineage plasticity in prostate cancer depends on JAK/STAT inflammatory signaling.

Drug resistance in cancer is often linked to changes in tumor cell state or lineage, but the molecular mechanisms driving this plasticity remain unclear. Using murine organoid and genetically engineered mouse models, we investigated the causes of lineage plasticity in prostate cancer and its relationship to antiandrogen resistance. We found that plasticity initiates in an epithelial population defined by mixed luminal-basal phenotype and that it depends on increased Janus kinase (JAK) and fibroblast growth factor receptor (FGFR) activity. Organoid cultures from patients with castration-resistant disease harboring mixed-lineage cells reproduce the dependency observed in mice by up-regulating luminal gene expression upon JAK and FGFR inhibitor treatment. Single-cell analysis confirms the presence of mixed-lineage cells with increased JAK/STAT (signal transducer and activator of transcription) and FGFR signaling in a subset of patients with metastatic disease, with implications for stratifying patients for clinical trials.

The driver role of JAK-STAT signalling in cancer stemness capabilities leading to new therapeutic strategies for therapy- and castration-resistant prostate cancer.

BackgroundLineage plasticity in prostate cancer (PCa) has emerged as an important mechanism leading to the onset of therapy‐ and castration‐resistant PCa (t‐CRPC), which is closely associated with cancer stem cell (CSC) activity. This study is to identify critical driver(s) with mechanism of action and explore new targeting strategy.MethodsVarious PCa cell lines with different genetic manipulations were subjected to in vitro prostasphere assay, cell viability assay and in vivo stemness potential. In addition, bioinformatic analyses such as Ingenuity pathway and Gene Set Enrichment Analysis were carried out to determine clinical relevance. The in vivo anti‐tumour activity of JAK or STAT1 inhibitors was examined in clinically relevant t‐CRPC model.ResultsWe demonstrated the role of interferon‐related signalling pathway in promoting PCa stemness, which correlated with significant elevation of interferon related DNA damage resistance signature genes in metastatic PCa. Inhibition of JAK‐STAT1 signalling suppresses the in vitro and in vivo CSC capabilities. Mechanistically, IFIT5, a unique downstream effector of JAK‐STAT1 pathway, can facilitate the acquisition of stemness properties in PCa by accelerating the turnover of specific microRNAs (such as miR‐128 and ‐101) that can target several CSC genes (such as BMI1, NANOG, and SOX2). Consistently, knocking down IFIT5 in t‐CRPC cell can significantly reduce in vitro prostasphere formation as well as decrease in vivo tumour initiating capability.ConclusionsThis study provides a critical role of STAT1‐IFIT5 in the acquisition of PCSC and highlights clinical translation of JAK or STAT1 inhibitors to prevent the outgrowth of t‐CRPC.

Identification of Two Non-Peptidergic Small Molecule Inhibitors of CBX2 Binding to K27 Trimethylated Oligonucleosomes.

The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.

Abstract 2200: Multilineage plasticity in prostate cancer through expansion of stem-like luminal epithelial cells with elevated inflammatory signaling

Abstract Lineage plasticity is a well-established mechanism of resistance to targeted therapies in lung and prostate cancer, where tumors transition from adenocarcinoma to small-cell or neuroendocrine carcinoma. Single-cell analysis of a cohort of late stage castration-resistant human prostate cancers (CRPC) revealed a greater degree of plasticity than previously appreciated, with multiple distinct neuroendocrine (NEPC), mesenchymal (EMT-like), and other subpopulations detected within single biopsies. To explore the steps responsible for initiation of this process, we utilized two genetically engineered mouse models of prostate cancer that recapitulate progression from adenocarcinoma to neuroendocrine disease. Time course studies reveal expansion of stem-like luminal epithelial cells (Sca1+, Psca+, called L2) that, based on trajectories, gave rise to at least 4 distinct subpopulations, NEPC (Ascl1+), POU2F3 (Pou2f3+), TFF3 (Tff3+) and EMT-like (Vim+, Ncam1+). Such populations are also seen in human prostate and small cell lung cancers. Furthermore, transformed L2-like cells express stem-like and gastrointestinal endoderm-like transcriptional programs, indicative of reemerging developmental plasticity programs, as well as elevated Jak/Stat, interferon, and FGF pathways. Strikingly pharmacologic inhibition of Jak/Stat and FGFR results in reversal of plasticity states and subsequent sensitivity to androgen receptor inhibitors (ARSIs). In sum, while the magnitude of multilineage heterogeneity, both within and across patients, raises considerable treatment challenges, the identification of highly plastic luminal cells as the likely source of this heterogeneity provides a target for more focused therapeutic intervention. Citation Format: Samir Zaidi, Jimmy Zhao, Joseph Chan, Roudier Martine, Kristine Wadosky, Anuradha Gopalan, Wouter Karthaus, Philip Watson, Lawrence True, Peter Nelson, Howard Scher, Michael Morris, Michael Haffner, David Goodrich, Dana Pe'er, Charles Sawyers. Multilineage plasticity in prostate cancer through expansion of stem-like luminal epithelial cells with elevated inflammatory signaling [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 2200.

Androgen receptor-mediated transcriptional repression targets cell plasticity in prostate cancer.

Androgen receptor (AR) signaling remains the key therapeutic target in the management of hormone‐naïve‐advanced prostate cancer (PCa) and castration‐resistant PCa (CRPC). Recently, landmark molecular features have been reported for CRPC, including the expression of constitutively active AR variants that lack the ligand‐binding domain. Besides their role in CRPC, AR variants lead to the expression of genes involved in tumor progression. However, little is known about the specificity of their mode of action compared with that of wild‐type AR (AR‐WT). We performed AR transcriptome analyses in an androgen‐dependent PCa cell line as well as cross‐analyses with publicly available RNA‐seq datasets and established that transcriptional repression capacity that was marked for AR‐WT was pathologically lost by AR variants. Functional enrichment analyses allowed us to associate AR‐WT repressive function to a panel of genes involved in cell adhesion and epithelial‐to‐mesenchymal transition. So, we postulate that a less documented AR‐WT normal function in prostate epithelial cells could be the repression of a panel of genes linked to cell plasticity and that this repressive function could be pathologically abrogated by AR variants in PCa.

RNA Splicing Factors SRRM3 and SRRM4 Distinguish Molecular Phenotypes of Castration-Resistant Neuroendocrine Prostate Cancer.

Neuroendocrine (NE) differentiation in metastatic castration-resistant prostate cancer (mCRPC) is an increasingly common clinical feature arising from cellular plasticity. We recently characterized two mCRPC phenotypes with NE features: androgen receptor (AR)-positive NE-positive amphicrine prostate cancer (AMPC) and AR-negative small cell or neuroendocrine prostate cancer (SCNPC). Here, we interrogated the regulation of RE1-silencing transcription factor (REST), a transcriptional repressor of neuronal genes, and elucidated molecular programs driving AMPC and SCNPC biology. Analysis of prostate cancer cell lines, mCRPC specimens, and LuCaP patient-derived xenograft models detected alternative splicing of REST to REST4 and attenuated REST repressor activity in AMPC and SCNPC. The REST locus was also hypermethylated and REST expression was reduced in SCNPC. While serine/arginine repetitive matrix protein 4 (SRRM4) was previously implicated in alternative splicing of REST in mCRPC, we detected SRRM3 expression in REST4-positive, SRRM4-negative AMPC, and SCNPC. In CRPC cell lines, SRRM3 induced alternative splicing of REST to REST4 and exacerbated the expression of REST-repressed genes. Furthermore, SRRM3 and SRRM4 expression defined molecular subsets of AMPC and SCNPC across species and tumor types. Two AMPC phenotypes and three SCNPC phenotypes were characterized, denoted either by REST attenuation and ASCL1 activity or by progressive activation of neuronal transcription factor programs, respectively. These results nominate SRRM3 as the principal REST splicing factor expressed in early NE differentiation and provide a framework to molecularly classify diverse NE phenotypes in mCRPC. SIGNIFICANCE: This study identifies SRRM3 as a key inducer of cellular plasticity in prostate cancer with neuroendocrine features and delineates distinct neuroendocrine phenotypes to inform therapeutic development and precision medicine applications.