Abstract 4565: Reckless mitotic entry as a chemosensitization approach for alkylating agents

Abstract The cell cycle is tightly regulated by checkpoints, playing a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anti-cancer treatments, allowing for DNA repair prior to cell division. Therefore, manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies such as alkylating agents and radiation. In this study, we conducted a forward genome scale CRISPR/Cas9 chemogenomic screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. Our findings revealed that canonical DNA repair pathways, including ATM/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of the membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase, known as Myt1 (encoded by PKMYT1), in ensuring cell survival. Analysis on patient cohort demonstrated that PKMYT1 is not only highly presented in malignancy, but also predisposes an unfavorable disease outcome. Genetic depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between TMZ and other clinically used alkylating agents, such as carmustine, busulfan, and dacarbazine, when combined with a novel Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage, as indicated by a substantial increase in γH2AX-positive cells in mitosis. Moreover, RP-6306 dramatically altered cell cycle timing, reducing the threshold time for mitotic entry. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities, including unaligned and partially condensed chromosomes, lagging chromosomes, chromosome bridges, and multipolar spindles. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Importantly, preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes. Citation Format: Fengchao Lang, James A. Cornwell, Karambir Kaur, Mirit I. Aladjem, Michael Aregger, Steven D. Cappell, Chunzhang Yang. Reckless mitotic entry as a chemosensitization approach for alkylating agents abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4565.