Abstract 4392: Pre-mRNA splicing factors promote cellular plasticity in castration-resistant prostate cancer

Androgen receptor (AR) signaling pathway inhibition (ARPI) is the primary treatment for locally advanced, recurrent, or metastatic prostate cancer. While ARPI is effective in short term, a fatal relapse is inevitable. The AKT signaling pathway inhibition has been investigated under multiple ongoing clinical trials as a co-therapeutic target with ARPI. The AKT signaling pathway is targeted due to its role in tumorigenesis, prevalent over-activation, and reciprocal activation upon ARPI in prostate cancer. However, this novel combination therapy may in fact facilitate PCa to progress into one of the most lethal subtypes called neuroendocrine (NE) prostate cancer (NEPC). Specifically, evidence has shown that ARPI contributes to NEPC progression at least in part by down-regulating the expression of RE-1 Silencing Transcription Factor (REST). Loss of REST is a hallmark of NEPC progression because REST functions as a negative master regulator in neurogenesis by suppressing genes required for neural differentiation. Similar to ARPI, our studies found that AKT inhibition reduced REST protein expression and increased NEPC markers in multiple prostate cancer cell lines. We also showed that the loss of REST upon AKT inhibition was through protein degradation mediated by an E3-ubiquitin ligase TRCP, which recognizes a phosphorylated REST degron region to recruit ubiquitins. In vivo ubiquitination assays confirmed the elevated REST ubiquitination after the treatment of PI3K inhibitors. Furthermore, mutations within the REST degron region reversed the TRCP-mediated REST ubiquitination and degradation. Finally, co-treatment of AKT pathway inhibition and ARPI showed aggravated REST depletion and increased NE markers in the prostate cancer cell line LNCaP. Collectively, these findings indicate that AKT pathway inhibition can induce NE phenotype in prostate cancer cells via REST protein degradation. This study may provide a caution to the ARPI/AKT co-target strategy as this strategy can potentially facilitate NEPC development. Citation Format: RuiQi Chen, Yinan Li, Xuesen Dong. AKT inhibition induces neuroendocrine phenotype in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1517. doi:10.1158/1538-7445.AM2017-1517

<div>Abstract<p>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.</p>Significance:<p>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.</p></div>

<div>Abstract<p>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.</p>Significance:<p>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.</p></div>

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.

Androgen deprivation therapy (ADT) remains the clinical paradigm for the management of prostate cancer (PCa) patients. However, most of the cases inevitably become resistant to ADT, leading to a more aggressive, hormone-refractory stage known as castrate-resistant prostate cancer (CRPC). A subset of CRPC patients undergoing ADT develop tumors with low AR-signaling dependence where the luminal prostate cancer cells consequently acquire alternative lineage programs, resulting in the development of neuroendocrine prostate cancer (NEPC). In the past decade, several molecular features have been associated with progression of the adenocarcinoma to NEPC, including loss of RE1-silencing transcription factor (REST), N-MYC proto-oncogene amplification, upregulation of Aurora kinase A, neural transcription factor BRN2, and reprogramming factor SRY (sex determining region Y)-box 2 (SOX2). Nonetheless, the underlying molecular mechanism involved in lineage plasticity in neuroendocrine (NE) transdifferentaition is poorly understood and remains a matter of speculation. Overexpression of Serine Peptidase Inhibitor, Kazal type 1 (SPINK1) represents the second major molecular PCa subtype (~15-20% of the cases) associated with aggressive stage and poor patient outcome. Oncogenic role of SPINK1 in PCa has already been established, and recently SPINK1 produced in the tumor stroma was found to act as a senescence-associated secretory factor and contributes to chemoresistance in a paracrine manner. Here, we show that androgen receptor (AR) along with its corepressor REST functions as a direct transcriptional repressor of SPINK1, and blocking AR signaling using anti-androgens relieve this repression, leading to increase in SPINK1 expression. We also show that the lineage reprogramming factor SOX2 binds to the SPINK1 promoter and positively regulate its expression in androgen deprivation induced NE-transdifferentiated PCa cells. Conversely, silencing SPINK1 in the NE-transdifferentiated PCa cells result in reduced expression of epithelial-mesenchymal transition (EMT) and neuroendocrine markers accompanied with a concomitant decrease in the neurite-like projections. Moreover, castration-resistant mice xenografts treated with anti-androgens show increase in SPINK1 levels as well as EMT and neuroendocrine markers. Likewise, higher SPINK1 expression was observed in NE patient-derived organoids and NEPC clinical specimens, indicating its plausible role in cellular plasticity and NE progression. Since, REST along with AR negatively regulates SPINK1 expression, and Casein kinase 1 elicits ubiquitin-mediated proteasomal degradation of REST. Therefore, we treated SPINK1-positive PCa cells with Casein kinase 1 inhibitor (iCK1), which results in restoration of the REST expression, leading to transcriptional repression of SPINK1 as well as decrease in SPINK1-mediated oncogenesis. Taken together, we highlight the clinical complications associated with high SPINK1 levels and discovered its possible role in maintaining cellular plasticity in prostate cancer cells. Notably, stabilization of REST levels using iCK1 suggests a novel therapeutic strategy for the management of SPINK1-positive subtype and also open new avenues for the treatment modalities for CRPC patients. Citation Format: Nishat Manzar, Ritika Tiwari, Vipul Bhatia, Anjali Yadav, Shannon Carskadon, Nilesh Gupta, Amina Zoubeidi, Matti Poutanen, Himisha Beltran, Nallasivam Palanisamy, Bushra Ateeq. Reprogramming transcription factors SOX2 and REST modulates SPINK1 expression in governing cellular plasticity in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C128. doi:10.1158/1535-7163.TARG-19-C128

Prostate-specific membrane antigen (PSMA) is overexpressed in most prostate adenocarcinoma (AdPC) cells and acts as a target for molecular imaging. However, some case reports indicate that PSMA-targeted imaging could be ineffectual for delineation of neuroendocrine (NE) prostate cancer (NEPC) lesions due to the suppression of the PSMA gene (FOLH1). These same reports suggest that targeting somatostatin receptor type 2 (SSTR2) could be an alternative diagnostic target for NEPC patients. This study evaluates the correlation between expression of FOLH1, NEPC marker genes and SSTR2. We evaluated the transcript abundance for FOLH1 and SSTR2 genes as well as NE markers across 909 tumors. A significant suppression of FOLH1 in NEPC patient samples and AdPC samples with high expression of NE marker genes was observed. We also investigated protein alterations of PSMA and SSTR2 in an NE-induced cell line derived by hormone depletion and lineage plasticity by loss of p53. PSMA is suppressed following NE induction and cellular plasticity in p53-deficient NEPC model. The PSMA-suppressed cells have more colony formation ability and resistance to enzalutamide treatment. Conversely, SSTR2 was only elevated following hormone depletion. In 18 NEPC patient-derived xenograft (PDX) models we find a significant suppression of FOLH1 and amplification of SSTR2 expression. Due to the observed FOLH1-supressed signature of NEPC, this study cautions on the reliability of using PMSA as a target for molecular imaging of NEPC. The observed elevation of SSTR2 in NEPC supports the possible ability of SSTR2-targeted imaging for follow-up imaging of low PSMA patients and monitoring for NEPC development.

197 Background: NEPC is an aggressive variant of prostate cancer that most commonly arises in later stages of mCRPC, especially with visceral metastases in the setting of a low PSA. There are no reliable serum markers to identify patients that are transforming to NEPC and incidence of CTCs in these patients is unknown. Detection of NEPC has clinical implications as these patients are often treated with platinum chemotherapy rather than with androgen receptor (AR) targeted therapies such as abiraterone or enzalutamide. We sought to determine if phenotypic characterization of CTCs could differentiate NEPC from mCRPC. Methods: 24 pts (16 pts with tissue confirmed NEPC and 8 pts with mCRPC) had blood collection for CTC analysis utilizing the Epic Sciences platform. Epic analysis included identification of traditional CTCs (CK+, CD45- cells, with intact nuclei, morph distinct), CK- CTCs (CK-, CD45-, intact nuclei, morph distinct), small CTCs (CK+, CD45-, intact nuclei, small cell size), and CTC clusters. We examined CTCs at the single cell level for CTC size, shape, epithelial and AR expression. Advanced digital pathology algorithms measured size and shape measurements of CTCs. Results: CTCs from NEPC patients demonstrated strong statistical differentiation from mCRPC patients, with unique CTC morphology and protein chemistry that was not seen in mCRPC CTCs. NEPC CTCs had increase prevalence of unique CTC phenotypes including: small size, AR negativity, and presence of nucleoli. Unique cell types enabled a multivariate biomarker that is strongly associated with CTCs exclusive to tissue confirmed NEPC. Conclusions: CTCs from NEPC have unique phenotypes compared with those from mCRPC. If confirmed, the utilization of a liquid biopsy to diagnose NEPC may enable earlier identification of patients prone to visceral metastasis, and intervention to cytotoxic therapeutics. Additionally, identification of patients with NEPC could help for patient selection for AR vs. NEPC targeted therapeutics. [Table: see text]

Background: Widespread and long-term use of first-and second-line androgen-deprivation therapy (ADT) is changing the molecular and phenotypic landscapes of prostate cancer. Observations made through our longstanding rapid autopsy and patient-derived xenograft (PDX) programs at the University of Washington support a shift in metastatic castration-resistant prostate cancer (mCRPC) towards androgen receptor (AR)-null phenotypes, such as neuroendocrine (NEPC) and double negative (DNPC). Currently, there are no effective therapies for AR-null mCRPC. We showed previously that DNPC (AR-null, NE-null) bypasses AR-dependence through fibroblast growth factor (FGF) signaling. However, the role of the FGF pathway in other molecular mCRPC subtypes remains to be determined. Methods and Results: Here, we define four mCRPC subtypes that can be categorized by the presence or absence of functioning AR or RE1-silencing transcription factor (REST). Transcriptomic analysis of mCRPC specimens showed that AR and REST activity define four emerging mCRPC phenotypes: adenocarcinoma (AR+/REST+), amphicrine (tumor cells that co-express AR and NE markers, AR+/REST-), DNPC (AR-/REST+) and NEPC (AR-/REST-). Immunohistochemistry of mCRPC and PDX models for AR, prostate specific antigen, synaptophysin, chromogranin, and other clinically relevant markers accurately reflected the AR/REST transcriptomic signature classifications. Furthermore, loss of REST activity can, at least in part, be attributed to alternative splicing of REST mRNA by serine/arginine repetitive matrix protein 4 (SRRM4), leading to the translation of a truncated REST protein. PCR analysis of mCRPC identified the REST splice variant exclusively in amphicrine and NEPC specimens. RNA sequencing/GSEA, qPCR and immunoblot analyses determined that overexpression of SRRM4 or siRNA knockdown of REST in C4-2B (AR+) and PC-3 (AR-) prostate cancer cells promotes expression of neuroendocrine markers. Finally, we are conducting preclinical testing of the FGFR inhibitor CH5183284 in multiple PDX models representing the four mCRPC subtypes described above to delineate the impact of FGF pathway inhibition in all mCRPC subtypes. Conclusions: Our data highlight the importance of AR and REST transcriptional programs in maintaining phenotypic stability in mCRPC and explain the phenotypic heterogeneity of mCRPC in the post-abiraterone/enzalutamide era. Understanding the mCRPC subtypes that depend on the FGF pathway for survival and proliferation will inform treatment and lead to the development of novel therapies for advanced disease. Citation Format: Mark P. Labrecque, Lisha G. Brown, Ilsa M. Coleman, Lawrence D. True, Lori Kollath, Bryce Lakely, Yu C. Yang, Holly M. Nguyen, Eva Corey, Peter S. Nelson, Colm Morrissey. Defining the molecular phenotypes of metastatic castration-resistant prostate cancer sensitive to FGF pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1092.

SRRM4 Drives Neuroendocrine Transdifferentiation of Prostate Adenocarcinoma Under Androgen Receptor Pathway Inhibition

<div>Abstract<p>Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer arising mostly from adenocarcinoma via neuroendocrine transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathologic feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. <i>HP1α</i> knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor and RE1 silencing transcription factor and enriched the repressive trimethylated histone H3 at Lys9 mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management.</p><p><b>Significance:</b> Heterochromatin proteins play a fundamental role in NEPC, illuminating new therapeutic targets for this aggressive disease. <i>Cancer Res; 78(10); 2691–704. ©2018 AACR</i>.</p></div>

<div>Abstract<p>Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer arising mostly from adenocarcinoma via neuroendocrine transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathologic feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. <i>HP1α</i> knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor and RE1 silencing transcription factor and enriched the repressive trimethylated histone H3 at Lys9 mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management.</p><p><b>Significance:</b> Heterochromatin proteins play a fundamental role in NEPC, illuminating new therapeutic targets for this aggressive disease. <i>Cancer Res; 78(10); 2691–704. ©2018 AACR</i>.</p></div>

Purpose: In this study, we aimed to study whether 5-hydroxymethylcytosine sequencing (5hmC-seq) of circulating cell-free DNA (cfDNA) predicts gene expression in tumor tissue and can distinguish tumor subtypes defined in tissue in metastatic castration-resistant prostate cancer (mCRPC). Methods: We performed 5hmC-seq on cfDNA samples from 86 mCRPC patients with matched tumor tissue profiled with 5hmC-seq (N=49) and RNA-sequencing (N=86) and we compared cfDNA 5hmC levels with matched tissue 5hmC levels and tissue gene expression. We developed a 5hmC-seq-based circulating tumor-DNA fraction (ctDNA-fraction) classifier and assessed if gene-level and pathway-level differences between mCRPC subtypes could be detected in cfDNA using a differential 5hmC-analysis adjusting for ctDNA-fraction. Results: Patients with androgen receptor (AR)-positive prostate cancer (ARPC) exhibited lower ctDNA-fraction, whereas patients with neuroendocrine (NE) prostate cancer (NEPC) and double-negative prostate cancer (DNPC) tumors displayed higher ctDNA-fraction (median ctDNA-fraction across subtypes 0.09 [95% confidence interval (CI), 0.04-0.14] (ARPC), 0.41 [95% CI, 0.18-0.64] (NEPC) and 0.37 [95% CI, 0.30-0.44] (DNPC), pairwise t-test P = 5.5 x 10-2 (ARPC vs. NEPC) and P = 8.8 x 10-4 (ARPC vs. DNPC) respectively). Nearly 30% of all protein-coding genes showed significant concordance between cfDNA 5hmC and tissue RNA-seq, after adjusting for ctDNA-fraction (adjusted p<0.05 in linear model), which were enriched in the androgen response, EGFR, and ERBB signaling pathways. Compared to ARPC, NEPC displayed upregulated 5hmC enrichment in NE-related genes, and downregulated androgen response and MYC target pathways in cfDNA. As expected, an NE pathway score calculated from cfDNA 5hmC was significantly different between tissue-confirmed ARPC and NEPC (p=0.03). Double-negative prostate cancer (DNPC) showed downregulated androgen response and MYC targets and upregulated epithelial cell pathways compared to ARPC in cfDNA. Furthermore, DNPC indicated an aggressive phenotype with cell proliferation pathways even more upregulated when compared to NEPC. Conclusions: We created a cohort of 86 matched tissue and cfDNA samples and demonstrated concordance for a significant number of transcribed protein-coding genes. Expected biological differences previously seen in tissue between NEPC and ARPC were readily detected via 5hmC profiles in cfDNA. Furthermore, DNPC showed a clear downregulation of androgen response signaling in cfDNA, indicating the possibility to identify a group of patients in addition to classical NEPC that may have reduced response to standard androgen-targeting agents. Future work will aim to develop single-sample multi-class subtype classifiers and evaluate differences in prognosis and treatment response based on cfDNA-based subtyping. Citation Format: Rensheng Wan, Raunak Shrestha, Gulfem Guler, Yuhong Ning, Aishwarya Subramanian, Adam Foye, Meng Zhang, Xiaolin Zhu, Thaidy Moreno-Rodriguez, Haolong Li, Shuang G. Zhao, SU2C/PCF West Coast Prostate Cancer Dream Team, Joshi J. Alumkal, Rahul Aggarwal, Alexander W. Wyatt, David Quigley, Samuel Levy, Eric Small, Felix Feng, Martin Sjöström. 5hmC-sequencing of matched cfDNA and tissue from men with mCRPC is concordant and identifies loss of AR signaling in NEPC and DNPC [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 1985.

Phenotypic switching is an emerging driver of cancer treatment resistance, yet early signals regulating this process remain unclear. Here, using longitudinal single-cell RNA sequencing, we mapped differentiation trajectories in the LTL331 prostate adenocarcinoma patient-derived xenograft (PDX) model undergoing neuroendocrine prostate cancer (NEPC) transformation post castration. Our analyses identified a key differentiation node marked by epithelial-mesenchymal transition (EMT) and repressor element-1 silencing transcription factor (REST) downregulation driven by the CXCR4-LASP1-G9a-SNAIL axis. Mechanistically, CXCR4 activation promotes nuclear translocation of LASP1 that links G9a and SNAIL via SH3/proline-rich motif and LIM/SNAG domain interactions, enabling SNAIL-mediated REST repression via promoter E-box motifs. Inhibition of CXCR4 or G9a reversed LTL331R NEPC cells toward a luminal androgen receptor-active phenotype. CXCR4-targeted radioligands enabled both imaging and inhibition of NEPC tumors in vivo. These findings highlight the CXCR4-LASP1-G9a-SNAIL axis as a key regulator of epigenetic and transcriptional reprogramming in NEPC transdifferentiation and support its therapeutic targeting in aggressive NEPC.

Background: The development of neuroendocrine prostate cancer (NEPC) is one mechanism of treatment resistance to androgen receptor (AR)-targeted therapies for a subset of patients with advanced prostate cancer. This is associated with transition from a prostate adenocarcinoma to small cell/NEPC histology, low AR signaling signaling, and expression of neuroendocrine markers as Chromogranin A (CGHA), Synaphophysin (SYP) and CD56). Patient derived preclinical models recapitulating the NEPC phenotype may be used to address NEPC pathogenesis and test emerging therapeutic targets. Methods: Tumor organoids were developed according to protocols previously described (Gao et al, Cell 2015). Briefly the tissue biopsies (liver and bone biopsy) were washed, enzymatically digested and then seeded in Matrigel (BD) droplets. Organoids were characterized at genomic (WES), RNA and protein level (IHC) to confirm the expression of specific markers. Lentiviral infections were performed using shRNAs against EZH2 to knock down EZH2 in organoids. Organoids were also subcutaneously injected in NSG mice to generate patient derived xenografts (PDXs) for drug treatment in vivo. Results: We developed and characterized two NEPC tumor organoids from tumor biopsies (liver and bone) of two patients both in vitro and in vivo (as PDXs). NEPC tumor organoid models retained the molecular and histological characteristic of their matched patient samples. We successfully manipulated the activity of the histone methyltransferase EZH2 by using a catalytic inhibitor and its expression by infecting organoids with shEZH2. We showed that the absence of EZH2 affects the expression of neuroendocrine-associated programs as stem cell and neuronal pathway. Moreover treatment with EZH2 inhibitor decreased tumor organoids viability and PDXs tumor volume. Drug screening approaches on NEPC organoids were used to discovery novel drug targets and combinations that could potentially benefit NEPC patients. Top single agent hits included previously identified targets such as EZH2, AURKA, as well as novel synergies. Conclusions NEPC patient tumor organoids are clinically relevant tumor models to study the NEPC phenotype in advanced prostate cancer and may be used to elucidate novel drug targets. Citation Format: Loredana Puca, Rohan Bareja, Reid Shaw, Wouter Karthaus, Dong Gao, Chantal Pauli, Juan Miguel Mosquera, Joanna Cyrta, Rachele Rosati, Rema Rao, Andrea Sboner, Carla Grandori, Giorgio Inghirami, Yu Chen, Mark A. Rubin, Himisha Beltran. Patient-derived tumor organoids of neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 992. doi:10.1158/1538-7445.AM2017-992

Background: Potent targeting of the androgen receptor (AR) in castration-resistant prostate cancer has altered the archetypal course of the disease, fueling the emergence of aggressive and incurable neuroendocrine prostate cancer (NEPC). Recent evidence suggests that these tumors can arise from non-neuroendocrine cells in response to AR pathway inhibitors (ARPIs), such as enzalutamide (ENZ), an observation consistent with lineage plasticity. What regulates this plasticity that allows cells to shed their dependence on the AR and re-emerge as “AR-indifferent” NEPC? Sequencing studies have uncovered that the evolution toward a NEPC phenotype is aligned with dynamic epigenetic reprogramming, but the molecular basis underlying this phenomenon remains poorly understood. Methods: We developed an in vivo model of acquired ENZ resistance to (a) identify reprogramming factors that facilitate lineage plasticity, and (b) determine how to best capitalize on therapeutic strategies aimed at blocking or reversing lineage transformation. Cell lines derived from ENZ-resistant tumors were profiled by RNA-seq and ChIP-seq, and functionally assessed for stem cell-associated properties. Our findings were validated across NEPC cell lines (NCI-H660), genetically engineered mouse models (PBCre4:Ptenf/f:Rb1f/f), and patient tumors and organoids. CRISPR/Cas9-mediated genomic editing allowed us to assess the effect of knocking out reprogramming factors on therapy-induced neuroendocrine transdifferentiation. Results: AR-indifferent ENZ-resistant tumors were enriched for a Polycomb/EZH2 signature; in particular, we identified EZH2 to be phosphorylated at threonine-350 (pEZH2-T350) by CDK1 in NEPC cell lines, mouse models, and patient tumors. Accordingly, RB1 loss was sufficient to enhance pEZH2-T350, which was required for prostate cancer cells to convert to a metastable stem-like state and, in turn, acquire neuroendocrine features under the pressure of ARPIs both in vitro and in patient-derived xenografts. This therapy-induced NEPC transdifferentation was associated with a marked redistribution of EZH2 and H3K27me3, specifically to a core set of genes governing lineage identity. AR colocalized at the reprogrammed EZH2 binding sites, and was found to be part of the same complex with EZH2. Treating AR-indifferent/NEPC cell lines with clinically relevant EZH2 inhibitors reversed the lineage switch and mitigated ENZ resistance. Conclusions: This research establishes the centrality of epigenetic reprogramming in driving the insurgence of a neuroendocrine phenotype in response to ARPIs, and posits that drugging the epigenome via EZH2 inhibition may reverse or delay lineage transformation to extend the durability of clinically beneficial ARPIs. Citation Format: Alastair Davies, Chiara Bostock, Musaddeque Ahmed, Yen-Yi Lin, Fraser Johnson, Ka Mun Nip, Kirsi Ketola, Jennifer Bishop, Ladan Fazli, David Goodrich, Faraz Hach, Hansen He, Himisha Beltran, Amina Zoubeidi. Identity fraud: Lineage plasticity as a mechanism of antiandrogen resistance and target for therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A033.