Abstract LB023: Discovery, development and characterization of potent and selective USP11 inhibitors

Abstract

Ubiquitin-mediated events are emerging as the gatekeeper in cell proliferation, and the recognition of deubiquitinating enzymes (DUBs) as the critical regulators of these processes is rapidly growing. Several studies in the last decade confirmed that the USP11 (Ubiquitin-Specific proteases 11) had promoted tumor growth and metastasis. Indeed, pan-cancer data analysis reveals enhanced expression of USP11 in numerous malignancies, including lymphoma. Our lab has established that USP11 deubiquitinates and stabilizes the translation initiation factor eIF4B (Eukaryotic Initiation Factor 4B) to promote eIF4B-dependent oncogenic translation and to facilitate the proliferation of diffuse large B-cell lymphoma (DLBCL). Brody and colleagues screened 2000 FDA- approved compounds and reported that the anti-neoplastic agent mitoxantrone is an effective non-specific inhibitor of USP11, which was later observed to target USP15. Thus, catalytically targeting USP11 provides substantial challenges as it encompasses high homology with the functional orthologs USP4 and USP15. Addressing this major limitation, Spiliotopoulos et al. identified a unique USP11-specific allosteric site without impeding USP15 and USP4 activities. Utilizing this platform, we performed a virtual screening of more than 10 million compounds from publicly available databases and identified 307 potential unique hits. The chemical library of these hits was assessed using an in-house optimized in-cell high throughput screening assay. The primary screening revealed that 16 of the hit compounds with EC50 less than 10μM. The two most potent, structurally different, USP11 inhibitors, RBF4 and RBF11, were further evaluated. In-vitro deubiquitinase activity assay of USP11 showed minimal inhibition on treatment with compounds (as we are targeting an allosteric site), which indicates that RBF4 and 11 do not bind to the catalytic domain. Thermal shift assays revealed that RBF4 preferentially stabilizes USP11 compared to other functional paralogues USP4 and USP15, which validates the selective USP11-RBF4 interaction. Further, treatment with RBF4 and 11 hampered DLBCL proliferation in a dose-dependent manner. Significantly, treatment with selected compounds depleted USP11-dependent oncogenic expression and, thus, the colony-forming capacity of DLBCL. These results show that these novel compounds can serve as an ideal tool to increase our understanding of USP11 biology and may emerge as an example of potent and selective USP11 inhibitors. Currently, we are evaluating their anti-cancer potency in pre-clinical models. Citation Format: Forum Kayastha, Bandish Kapadia, Noah B. Herrington, Anirban Roychowdhury, Glen E. Kellogg, Ronald B. Gartenhaus. Discovery, development and characterization of potent and selective USP11 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB023.