Korzhnev, D. M. | Search bioRxiv

bioRxiv2026

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 and REV3 interaction, and found that C10 sensitized HCT116 and HT29 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 HT29 cells, combined treatment also increased the frequency of cells containing multiple gamma 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 and 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.

cancer biology↗

bioRxiv2023

Activation Dynamics of Ubiquitin Specific Protease 7

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme responsible for the regulation of key human oncoproteins and tumor suppressors including Mdm2 and p53, respectively. Unlike other members of the USP family of proteases, the isolated catalytic domain of USP7 adopts an enzymatically inactive conformation that has been well characterized using X-ray crystallography. The catalytic domain also samples an active conformation, which has only been captured upon USP7 substrate-binding. Here, we utilized CPMG NMR relaxation dispersion studies to observe the dynamic motions of USP7 in solution. Our results reveal that the catalytic domain of USP7 exchanges between two distinct conformations, the inactive conformation populated at 95% and the active conformation at 5%. The largest structural changes are localized within functionally important regions of the enzyme including the active site, the ubiquitin-binding fingers, and the allosteric helix of the enzyme, suggesting that USP7 can adopt its active conformation in the absence of a substrate. Furthermore, we show that the allosteric L299A activating mutation disturbs this equilibrium, slows down the exchange, and increases the residence time of USP7 in its active conformation, thus, explaining the elevated activity of the mutant. Overall, this work shows that the isolated USP7 catalytic domain pre-samples its "invisible" active conformation in solution, which may contribute to its activation mechanism.

biophysics↗