Abstract
Prostate cancer lineage plasticity is associated with changes in DNA methylation and enhancer of zeste homolog 2 (EZH2) activity. How these epigenetic programs functionally interact to modulate transcriptional reprogramming in neuroendocrine prostate cancer (NEPC) is not well understood. In this study, we demonstrate that hypomethylated regions of DNA preferentially accumulate the repressive mark, H3K27me3. We established an NEPC mouse model with deletion of Ezh2 in the background of Pten and Rb1 loss plus human MYCN overexpression. Deletion or pharmacological inhibition of EZH2 in NEPC murine or patient-derived models leads to a genome-wide rewiring of DNA methylation, characterized by hypomethylation and upregulation of neuroendocrine-lineage genes along with hypermethylation and repression of polycomb repressive complex 2 (PRC2) targets. On the other hand, deletion of DNA methyltransferase 1 (DNMT1) results in significant changes in H3K27me3 distribution, particularly affecting bivalent promoters bearing both H3K27me3 and active H3K4me3 marks. In NEPC models, neuroendocrine-lineage genes are repressed upon DNMT1 deletion associated with increased H3K27me3. Conversely, in prostate adenocarcinoma models, DNMT1 deletion leads to de-repression of neuroendocrine lineage genes with a loss of H3K27me3 marks. Our findings reveal a functional interplay between two repressive epigenetic machineries that mediates lineage plasticity in prostate cancer.
Data availability
Data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession numbers: GSE289247 (H3K27me3 and H3K4me3 ChIP-Seq of PRN murine model and DNMT1-inhibited NEPC PDXs), GSE289248 (RNASeq of PRNE model), GSE289249 (RRBS or PRN and PRNE models, EZH2-deleted LNR cells and EZH2-inhibited MSK-PCa10), GSE286958 (RNASeq of DNMT1-deleted WCMC154 and 22Rv1) and GSE286954 (H3K27me3 and H3K4me3 CUT&RUN of DNMT1-deleted WCMC154 and 22Rv1). Methylome data from one pair of PRN NEPC and PRAD tumors were downloaded from our previous publication (GSE151581). EPIC-array-based methylation data from patient samples and LuCaP PDX samples were obtained from GSE227853. H3K27me3 ChIP-Seq data from LuCaP PDXs were obtained from GSE161948. All plasmid constructs used in this study are available upon request. Source data are provided with this paper.
Code availability
No custom code was generated or used in this study. All analyses were performed using standard software and tools, which are referenced in the Methods section.
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Acknowledgements
Some figures were created with BioRender.com. R.S. is supported by DoD PCRP Early Career Investigator Award (W81XWH2110090) and Urology Care Foundation Research Scholar Award (AUA 228082-01). V.B.V. is supported by DoD PCRP Early Career Investigator Award (W81XWH2210197), National Cancer Center Postdoctoral Fellowship Award, and Prostate Cancer Foundation Young Investigator Award (23YOUN15). N.J.B. is supported by NIH/NCI (K22CA269707), American Cancer Society (PF-20-119-01-TBE), and Weill Cornell Medicine JumpStart Research Career Development Award. D.S.R. is supported by NIH-NCI (R01CA274963, R01CA230913, WCM SPORE P50CA211024) and DoD PCRP (W81XWH-17-1-0652). H.B. is supported by the Prostate Cancer Foundation, DoD PCRP (W81XWH-17-1-0653) and NIH/NCI (R37CA241486-01A1, DF/HCC SPORE P50 CA272390-01, WCM SPORE P50 CA211024-01A1).
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Conceptualization: R.S., V.B.V., D.S.R., H.B. Methodology: R.S., V.B.V., E.I., Y.Y., M.A.B., B.D.R. Software: R.S., V.B.V., E.I., M.A.B., Validation: R.S., V.B.V., E.I., Y.Y., N.J.B., K.D., R.G., M.A.B., A.G.P., B.D.R. Formal Analysis: R.S., V.B.V., E.I., M.A.B. Investigation: R.S., V.B.V., E.I., Y.Y., N.J.B., K.D., R.G., M.A.B., A.G.P., B.D.R. Resources: R.S., V.B.V., M.C.H., L.M., M.Y.T., B.D.R., D.S.R., H.B. Data Curation: R.S., V.B.V., E.I., B. H., M.C.H., M.A.B. Writing – Original Draft: R.S., V.B.V., D.S.R., H.B. Writing – Review & Editing: R.S., V.B.V., E.I., Y.Y., N.J.B., K.D., R.G., M.A.B., A.G.P., M.Y.T., B. H., M.C.H., L.M., B.D.R., D.S.R., H.B. Visualization: R.S., V.B.V., E.I., M.A.B. Supervision: R.S., V.B.V., M.Y.T., M.C.H., L.M., D.S.R., H.B. Project administration: R.S., V.B.V., D.S.R., H.B. Funding acquisition: R.S., V.B.V., D.S.R., H.B.
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D.S.R. has received research funding (to institution) from Janssen, Eli Lilly and Chimerix. M.C.H. served as a paid consultant/received honoraria from Pfizer and Astra Zeneca and has received research funding from Merck, Novartis, Genentech, Promicell and Bristol Myers Squibb. H.B. has served as consultant/advisory board member for Janssen, Astra Zeneca, Merck, Pfizer, Amgen, Bayer, Novartis, Daiichi Sankyo, Abbvie, and has received research funding (to institution) from Janssen, Bristol Myers Squibb, Circle Pharma, Daiichi Sankyo, Novartis, Abbvie. All other authors declare no potential conflicts of interest.
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Singh, R., Venkadakrishnan, V.B., Imada, E. et al. Crosstalk between EZH2 and DNA methylation mediates neuroendocrine prostate cancer lineage plasticity. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69308-0
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DOI: https://doi.org/10.1038/s41467-026-69308-0
