Jun Wang ^{1 2}, Kwang-Su Park ³, Xufen Yu ³, Weida Gong ¹, H Shelton Earp ^{1 4 5}, Gang Greg Wang ^{1 2 4}, Jian Jin ³, Ling Cai ^{1 6}

¹Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA.
²Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA.
³Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁴Department of Pharmacology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA.
⁵Department of Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, 27599, USA.
⁶Department of Genetics, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
Correspondence: Gang Greg Wang, Jian Jin, Ling Cai

Enhancer of Zeste Homolog 2 (EZH2) and androgen receptor (AR) are crucial chromatin/gene regulators involved in the development and/or progression of prostate cancer, including advanced castration-resistant prostate cancer (CRPC). To sustain prostate tumorigenicity, EZH2 establishes non-canonical biochemical interaction with AR for mediating oncogene activation, in addition to its canonical role as a transcriptional repressor and enzymatic subunit of Polycomb Repressive Complex 2 (PRC2). However, the molecular basis underlying non-canonical activities of EZH2 in prostate cancer remains elusive, and a therapeutic strategy for targeting EZH2:AR-mediated oncogene activation is also lacking. Here, we report that a cryptic transactivation domain of EZH2 (EZH2^TAD) binds both AR and AR spliced variant 7 (AR-V7), a constitutively active AR variant enriched in CRPC, mediating assembly and/or recruitment of transactivation-related machineries at genomic sites that lack PRC2 binding. Such non-canonical targets of EZH2:AR/AR-V7:(co-)activators are enriched for the clinically relevant oncogenes. We also show that EZH2^TAD is required for the chromatin recruitment of EZH2 to oncogenes, for EZH2-mediated oncogene activation and for CRPC growth in vitro and in vivo. To completely block EZH2’s multifaceted oncogenic activities in prostate cancer, we employed MS177, a recently developed proteolysis-targeting chimera (PROTAC) of EZH2. Strikingly, MS177 achieved on-target depletion of both EZH2’s canonical (EZH2:PRC2) and non-canonical (EZH2^TAD:AR/AR-V7:co-activators) complexes in prostate cancer cells, eliciting far more potent antitumor effects than the catalytic inhibitors of EZH2. Overall, this study reports a previously unappreciated requirement for EZH2^TAD for mediating EZH2’s non-canonical (co-)activator recruitment and gene activation functions in prostate cancer and suggests EZH2-targeting PROTACs as a potentially attractive therapeutic for the treatment of aggressive prostate cancer that rely on the circuits wired by EZH2 and AR.