A Small Molecule RAD51 Inhibitor Preferentially Affects Cells Expressing High Cytidine Deaminase Activity

RAD51 is a homologous recombination factor critical for DNA replication, recombination, and DNA double strand break repair. RAD51 forms nucleoprotein filaments at damaged DNA sites or corrupted DNA replication forks to mediate DNA strand exchange, culminating in recombinational repair of breaks and replication fork restart. RAD51 is essential for the repair of DNA lesions induced by agents that stall or collapse DNA replication forks. In this context, RAD51 is an important factor in resistance to DNA replication stress induced by the cellular cytidine deaminase AICDA (also known as AID). AICDA is a DNA-directed cytidine deaminase that normally acts to initiate somatic hypermutation and immunoglobulin class switching in activated B-lymphocytes. However, AICDA is often constitutively overexpressed in tumor cells in a range of cancer types. In transformed cells the cytidine deamination activity of AICDA, and the resulting DNA base pair mismatches, cause tumor cell hypermutation. As a byproduct of its mutational activity, AICDA also destabilizes DNA replication, causing a high concentration of DNA double stranded breaks in AIDCA-expressing cells. This leads to an obligate dependency on RAD51 for DSB repair and cell survival. We previously demonstrated that RAD51 depletion induces acute sensitivity to AICDA activity in transformed cells. We now describe the mechanistic characterization of a small molecule RAD51 inhibitor - CYT01A- that is preferentially cytotoxic to AICDA expressing cells. CYT01A is a potent RAD51 inhibitor (EC50 ~150nM) that promotes DNA replication catastrophe selectively in cells expressing AICDA. Exposure to CYT01A in cell culture leads to the depletion of nuclear RAD51, resulting in diminished nuclear foci formation in response to DNA damage. We demonstrate a dose-dependent cytotoxic response to CYT01A that was reflected by RAD51 foci suppression. We further demonstrate compromised RAD51 nucleoprotein filament formation, evidenced by persistence of Replication Protein A (RPA) foci following CYT01A treatment. RAD51 depletion and reduced foci formation correlates with a reduction in homologous recombination activity. Finally, we show that CYT01A treated cells undergo replication catastrophe, associated with caspase3/7 activation. Collectively, these data indicate that CYT01A prevents RAD51 transport into the nucleus and thereby inhibits DNA repair activity. This demonstrates the feasibility of targeting RAD51 to induce cell death selectively in cells with high DNA replication stress, such as AICDA overexpressing tumor cells. These findings form the basis for a new synthetic lethal therapeutic approach, targeting the AICDA/RAD51 axis in cancers that exhibit high levels of AICDA activity Disclosures Mills: Cyteir Therapeutics: Employment, Equity Ownership. Cyr: Cyteir Therapeutics: Employment, Equity Ownership. Maclay: Cyteir Therapeutics: Employment, Equity Ownership. Day: Cyteir Therapeutics: Employment, Equity Ownership. Khalil: Cyteir Therapeutics: Consultancy, Equity Ownership.