Polycomb repressive complex 1 (PRC1) is an essential chromatin modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small molecule inhibitors with a well-defined mechanism of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary AML samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.

GAS41 is a chromatin-associated protein that belongs to the YEATS family and is involved in the recognition of acetyl-lysine in histone proteins. A unique feature of GAS41 is the presence of a C-terminal coiled-coil domain, which is responsible for protein dimerization. Here, we characterized the specificity of the GAS41 YEATS domain and found that it preferentially binds to acetylated H3K18 and H3K27 peptides. Interestingly, we found that full-length, dimeric GAS41 binds to diacetylated H3 peptides with an enhanced affinity when compared to those for monoacetylated peptides, through a bivalent binding mode. We determined the crystal structure of the GAS41 YEATS domain with H3K23acK27ac to visualize the molecular basis of diacetylated histone binding. Our results suggest a unique binding mode in which full-length GAS41 is a reader of diacetylated histones.

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ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.