Abstract 1467: Revealing therapeutically relevant states of cohesin through dominant-negative mutational mapping of Smc1 as a synthetic lethal anti-cancer approach

Abstract Leveraging synthetic lethal interactions is an approach to selectively kill tumor cells. A SL interaction between two genes occurs when the disruption of either gene alone is viable, but the disruption of both genes simultaneously results in cell death. Although targeting SL interactions holds immense therapeutic potential, only PARP inhibitors have translated to the clinic. PARP inhibitors are unique in that they cause a gain-of-function (GoF) change in the PARP protein resulting in DNA trapping and selective cytotoxicity in BRCA1/2-mutated tumors. This study employs a novel method of dominant SL screening that aims to identify GoF protein states, modelled by missense mutations, to guide inhibitor design. Here, we evaluate the potential of the cohesin complex as a drug target. Cohesin is composed of four core subunits (SMC1, SMC3, RAD21, and STAG2) and plays roles in sister chromatid cohesion, DNA damage repair, and chromatin organization. This study shows that mutations in the C-terminal ATPase domain cause dominant effects and cluster in a druggable domain, suggesting this state could be phenocopied therapeutically. Using S. Cerevisiae as a model, dominant SL screening of a random mutagenesis library was used to identify states of the target protein, Smc1, that cause dominant lethality in a cohesin-compromised background. This revealed 17 key residues, all residing in the C-terminal ATPase domain, with specific clustering in the signature motif. Directed SL testing of 11 mutants was performed with a panel of cancer-like backgrounds. These data showed that expression of the mutants cause dominant SL in backgrounds with altered cohesin regulation, mitotic defects and replication deficiencies. Further, cell cycle analysis was performed on select mutants, showing that mutations are causing dominant G2 arrest, leading to SL cell death in a cohesin-compromised background. To test if these mutants rely on cohesin complex formation, we tested reliance on scMcd1(hsRAD21) and Smc3. Overexpressing the cohesin ring rate-limiting factor Mcd1 in conjunction with mutant protein shows worsened phenotypes, while introducing a secondary mutation that disrupts the Smc1-Smc3 interface abolishes dominant lethality. These data suggest that the mutant Smc1 proteins rely on the creation of complexes to elicit their dominant negative properties. Importantly, structural mapping reveals that the hits from the screen are conserved and map to ATP interacting residues on the human structure, revealing a potentially druggable domain. The performed mutational mapping can guide in silico molecular docking to design small molecules that can then be tested for phenocopying of the dominant state for better clinical translation. Citation Format: Elizabeth Stephens, Nigel O’Neil, Peter Stirling, Philip Hieter. Revealing therapeutically relevant states of cohesin through dominant-negative mutational mapping of Smc1 as a synthetic lethal anti-cancer approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1467.

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