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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.

Introduction: Potent targeting of the androgen receptor (AR) in castration-resistant prostate cancer (CRPC) has altered the archetypal course of the disease, fueling the emergence of aggressive and incurable neuroendocrine prostate cancer (NEPC). Alarmingly, no targeted therapies exist for NEPC, which stems from our poor molecular understanding of the disease. What regulates the plasticity that allows cells to shed their dependence on the AR and re-emerge as “AR-indifferent” NEPC, especially under the pressure of modern AR pathway inhibitors (ARPIs) such as enzalutamide (ENZ)? Recent data suggest that neuroendocrine transdifferentiation is aligned with dynamic reprogramming of the epigenome by developmental regulators, like EZH2, making them attractive therapeutic targets. Method: To uncover reprogramming factors that are activated in response to AR pathway inhibition, we interrogated a panel of cell lines derived from ENZ-resistant prostate tumors by RNA-seq. Mirroring what is observed in a subset of patients who progress on ENZ, 25% of ENZ-resistant tumors and matched cell lines displayed reduction in canonical AR pathway activity and a NEPC phenotype. Gene set enrichment analysis revealed that these cells are enriched for a Polycomb/EZH2 signature and share a conserved transcriptional program with embryonic stem cells. In particular, we identified a distinct phosphorylated form of EZH2 (EZH2-T350), upregulated in AR-indifferent/NEPC cell lines and patient tumors, that was found to be indispensable for the emergence and maintenance of a stem-like state. Results: Blocking EZH2-T350 phosphorylation in AR-indifferent/NEPC cell lines yielded a marked reduction in expression of pluripotency transcription factors and stem-like features, including ALDH activity and spheroid formation capacity. Notably, EZH2-T350 was found to be sufficient and required for cells to enter a transient stem-like state, a pre-requisite for neuroendocrine transdifferentiation under the pressure of ARPIs both in vitro and in patient-derived prostate tumor xenografts. As our data revealed a strong association between EZH2 and AR in response to ENZ we performed ChIP-seq and observed extensive reprogramming of the AR cistrome, specifically at a core set of genes governing stem cell identity. EZH2 colocalized at the reprogrammed AR binding sites. Accordingly, treating AR-indifferent/NEPC cell lines with the EZH2 inhibitor GSK126 yielded a molecular subtype shift from PCS1 to AR-driven PCS2, and re-sensitized cells to ARPIs. Conclusion: Our findings establish the centrality of epigenetic reprogramming in driving the insurgence of a clinically aggressive neuroendocrine phenotype in response to AR pathway inhibition. Drugging the epigenome via EZH2 inhibition to reverse the NEPC state and re-sensitize tumors to our powerful arsenal of ARPIs has the potential to transform the treatment of prostate cancer. Citation Format: Alastair Davies, Musa Ahmed, Chiara Bostock, Anna Gleave, Kirsi Ketola, Fraser Johnson, Jennifer Bishop, Ladan Fazli, Haojie Huang, Hansen He, Amina Zoubeidi. EZH2 reprogramming confers intrinsic stem cell properties and developmental plasticity driving 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 5025. doi:10.1158/1538-7445.AM2017-5025