Targeting REV7 exposes a mitotic vulnerability in colorectal cancer cells.

Therapeutic resistance remains a barrier to durable treatment of colorectal cancer (CRC), highlighting the need to identify vulnerabilities in resistant tumor cells. REV7 (MAD2L2) is a multifunctional genome-maintenance protein with established roles in REV3-dependent translesion synthesis (TLS) as well as mitotic progression, spindle organization, and chromosome segregation. Although perturbing REV7-dependent DNA damage tolerance has emerged as a strategy to overcome chemoresistance, whether pharmacologic targeting of REV7 exposes vulnerabilities to mitotic disruption remains poorly understood. Here, we investigated the REV7-targeting small-molecule inhibitor C10, which disrupts the REV7-REV3 interaction, and found that C10 sensitized HCT116 and HT-29 CRC cells to the microtubule-disrupting agent colchicine, resulting in markedly reduced clonogenic survival. In contrast, C10 did not substantially enhance sensitivity to cisplatin. Genetic loss of REV7 similarly increased sensitivity to colchicine, independently supporting a REV7-dependent vulnerability to microtubule disruption. Combined C10 and colchicine treatment was associated with pronounced alterations in mitotic and post-mitotic nuclear morphology, including condensed/mitotic-like and multinucleated configurations, and increased micronucleus formation. In HT-29 cells, combined treatment also increased the frequency of cells containing multiple γ-tubulin-positive foci, consistent with disrupted centrosome-associated organization. Together, these findings support a model in which REV7-dependent functions contribute to cellular tolerance and recovery from microtubule-mediated mitotic stress, whereas REV7-REV3 disruption renders CRC cells vulnerable to mitotic perturbation. This study broadens the potential consequences of targeting REV7 beyond its canonical role in TLS and suggests that combining REV7-directed inhibition with mitotic perturbation warrants further investigation as a therapeutic strategy in CRC and potentially other cancers.