Discovery of small molecules targeting Polycomb Repressive Complex 1 ABSTRACT: The discovery that mutations and dysregulation of chromatin modifiers are major cancer drivers has inspired increased pharmaceutical efforts to identify specific inhibitors. Polycomb Repressive Complex 1 (PRC1) and PRC2 are chromatin regulators that mediate transcriptional silencing to maintain cellular identity. The PRC2 subunit EZH2 catalyzes histone H3 lysine 27 methylation (H3K27me1/2/3). Acting immediately downstream, canonical PRC1 (cPRC1) specifically recognizes H3K27me3 via its specific Chromobox (CBX) domain subunits and enforces repression of PRC2 target genes by chromatin compaction and 3D looping. Notably, non-canonical PRC1 (ncPRC1) which lacks CBX proteins and some cPRC1 complexes can be recruited and mediate repression independently of H3K27me3, but the mechanisms are poorly understood. EZH2 is overexpressed or hyperactive in 1-2% of all cancers and catalytic inhibitors such as Tazemetostat, aimed at blocking cPRC1-dependent gene repression, were recently FDA-approved for treatment of EZH2-mutant B- cell lymphomas and SMARCB1/INI1-mutant sarcomas. However, there is mounting evidence that some EZH2- dependent cancers are only partially dependent on its catalytic activity for cPRC1-dependent repression, which explains why inhibitors like Tazemetostat can have limited efficacy. Moreover, since EZH2 has well-known tumor suppressive roles in some tissues, there are concerns that its inhibition could cause secondary cancers. Thus, new approaches and therapeutic targets are urgently needed for treatment of EZH2-dependent cancers. We seek to directly target cPRC1 complexes, a therapeutic strategy that has been mostly unexplored. We hypothesize that small molecules targeting cPRC1 subunits that act downstream or independent of EZH2’s H3K27 methyltransferase activity will facilitate mechanistic insights intractable with conventional genetic tools and reveal new intervention points for therapy, potentially providing a means to overcome current limitations of catalytic inhibition. The overarching objective of this program is to identify diverse small molecules with activity against cPRC1 and to determine their mechanisms of action. To achieve this, we have developed a sensitive, HTS-compatible cPRC1/PRC2 luminescent reporter assay, complemented by published secondary and tertiary orthogonal assays which already yielded a confirmed hit compound with activity against cPRC1/PRC2 downstream or independent of H3K27me3. Successful identification of small molecules can catalyze mechanistic exploration and enable assessment of preclinical target validity, therefore advancing innovative basic chromatin research and leading to impactful translational studies, highly relevant for EZH2-dependent cancers. 1