Chemoproteomic discovery and characterization of covalent ligands for the oncoprotein CIP2A, a unique vulnerability in BRCA1/2-deficient cancers - Loss-of-function BRCA1/2 mutations lead to defective homologous recombination (HR) activity, a frontline DNA damage repair pathway that accurately addresses double-strand breaks. In the absence of sufficient HR activity, BRCA1/2-deficient cancers are suggested to activate a non-canonical DNA maintenance mechanism dependent on the oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A). CIP2A colocalizes with the genome stability factor TOPBP1 at sites of DNA damage during mitosis. This complex is suggested to physically tether fragmented chromosomes and centromere-bearing counterparts to ensure proper division and cancer cell survival. After mitosis, chromosome fragments can undergo chromothripsis, a large- scale error-prone genomic rearrangement. Chromothriptic fragments also rely on CIP2A for proper division in the next cell cycle to ultimately potentiate oncogenesis. CIP2A knockdown is lethal in BRCA1/2-deficient cancers but does not harm HR-proficient cells, indicating CIP2A to be a unique therapeutic vulnerability. However, a bonafide CIP2A inhibitor has not yet been produced via standard drug discovery efforts and may require innovative strategies. Recent chemoproteomic analyses identified nucleophilic cysteine residues in CIP2A that can be covalently labeled (“liganded”) with electrophiles. While lead fragments provide starting points for further development, these scaffolds also non-selectively engage other proteins. Furthermore, the functional consequences of labeling CIP2A are unknown. The proposal will therefore achieve chemical inhibition of CIP2A using covalent degraders (e.g., PROTAC) and focuses on the following aims: (1) Identify potent and selective covalent ligands for CIP2A. (2) Develop heterobifunctional probes and evaluate proximity-dependent degradation of CIP2A. (3) Analyze chemical inhibition of CIP2A in BRCA1/2-deficient cells. Encouragingly, preliminary data indicate the proposed ligands label CIP2A in both cellular and in vitro settings. Building on these results, the first aim will synthesize a focused probe collection based on computational guidance and then assess target engagement using a state-of-the-art chemoproteomic workflow. In the second aim, selective ligands will be converted into heterobifunctional degraders and characterized for CIP2A degradation in BRCA1/2-deficient cells. The third aim will then evaluate the cellular activity of covalent degraders by tracking phenotypes indicative of CIP2A inhibition and defective mitosis. This proposal will provide valuable chemical tools to probe CIP2A function and lay the foundation for therapeutics to treat BRCA1/2-deficient cancers. During this fellowship, I am grateful to be mentored by multiple professors from the Rockefeller University: Tarun Kapoor, my advisor, a master in chemical probe design and cell division biology; Jiankun Lyu, an expert in computational chemistry; Ekaterina Vinogradova, a leader in chemoproteomics. Their mentorship will be supplemented with additional training both within Rockefeller and in the broader academic community. This fellowship represents an essential step towards my goal of performing independent research at the interface of chemical and cancer biology.
