Abstract 2983: BUB1B is an inactive kinase required for survival of chromosomally unstable cells

Abstract Chromosome instability (CIN) is a prevalent hallmark of aggressive cancers that is largely absent in normal cells. Targeting tumors with CIN is a therapeutic strategy that is rapidly advancing. Volastra Therapeutics has undertaken multiple orthogonal approaches to target CIN as a cancer vulnerability. One such approach that relies on the Cancer Dependency Map (DepMap) identified BUB1B (BUB1 Mitotic Checkpoint Serine/Threonine Kinase B) as a top scoring gene required for survival of cell lines with increased genomic instability. BUB1B, a core component of the spindle assembly checkpoint that functions in cell cycle progression and chromosome segregation, was also highlighted in published screens as a major cancer vulnerability in cells with aneuploidy and whole-genome duplication. To corroborate the dependency of chromosomally unstable cancer cells on BUB1B, we used three independent cell models: 1) an isogenic CIN system based on modulating kinetochore-microtubule dynamics, 2) a Cas9-engineered system to induce dicentric chromosomes through telomere deprotection and 3) a non-isogenic cell line model based on differential levels of aneuploidy and micronuclei. Data in these three orthogonal systems confirmed that chromosomally unstable cells rely heavily on BUB1B for survival. We next explored the development of BUB1B small molecule inhibitors. The C-terminus of BUB1B is responsible for chromosome congression and contains the putative kinase domain, suggesting that catalytic activity may be responsible for CIN suppression. Whether BUB1B possesses enzymatic activity has been controversial, with some groups reporting low but inhibitable BUB1B kinase activity, while others indicate that BUB1B lacks key kinase residues and is catalytically inactive. To probe the function of BUB1B, we generated full-length and kinase domain recombinant proteins. Both constructs showed very weak ATPase activity comparable to published and commercially available proteins. Efforts to boost the kinase activity through improved substrate specificity or enhanced phosphorylation were not successful, and additional purification resulted in further loss of kinase activity. Furthermore, modeling of the BUB1B kinase domain suggests that ATP binding to the ATP cleft is physically hindered by bulky residues. Finally, substrate sequence mapping did not match sequences predicted for BUB1B. Together the data suggest that BUB1B is not enzymatically active and that the measured low ATPase activity may be attributable to a co-purifying kinase. Our work confirms chromosomally unstable cells have greater dependency on BUB1B. However, the protein lacks catalytic activity and thus cannot be targeted with a kinase inhibitor strategy. BUB1B’s function as a protein interaction hub with specific partners could permit alternative therapeutic strategies that may result in effective treatments for patients with chromosomally unstable cancers. Citation Format: Nazario Bosco, Rafael S. Depetris, Jasmina Prabhakara, Akanksha Verma, Jared L. Johnson, Lewis C. Cantley, Scott Drutman, Shinsan M. Su, Rumin Zhang, Derek A. Cogan, Christina H. Eng. BUB1B is an inactive kinase required for survival of chromosomally unstable cells [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 2983.