Biology subjects

Kothandaraman, N.

Publications and source records attributed to Kothandaraman, N..

3 recordsLinked to original sources

bioRxiv2026

Targeting REV1 Exposes Context-Dependent Therapeutic Responses in Colorectal Cancer

Translesion synthesis (TLS) promotes DNA damage tolerance and contributes to chemotherapy resistance by enabling replication across damaged DNA templates. REV1, a central TLS regulator, is a potential therapeutic target to enhance the efficacy of genotoxic chemotherapy. Here, we investigated the therapeutic consequences of pharmacologic and genetic REV1 targeting in colorectal cancer (CRC) and evaluated the sensitivity of patient-derived CRC organoids to the REV1 inhibitor JH-RE-06. JH-RE-06 enhanced oxaliplatin sensitivity in HCT116 cells and 5-fluorouracil (5-FU) sensitivity in HT-29 cells, whereas reciprocal combinations produced limited sensitization, indicating treatment- and cellular context-dependent responses. siRNA-mediated REV1 depletion similarly enhanced chemotherapy sensitivity, supporting REV1 involvement in these responses. MNNG sensitivity assays and MLH1 expression analyses did not reveal consistent treatment-associated changes in mismatch repair function, while targeted HPRT1 sequencing identified no detectable sequence alterations in the region examined. Chemosensitization was not accompanied by consistent increases in caspase-3/7 activation under the conditions tested. REV1 inhibition also caused LC3-II accumulation and, after prolonged inhibitor exposure, reduced beta-tubulin abundance, indicating additional autophagy-associated and cytoskeletal responses. In two patient-derived CRC organoid models, JH-RE-06 exhibited single-agent growth-inhibitory activity, with mean IC50 values of 2.01 micromolar and 1.4 micromolar, respectively. Together, these findings demonstrate context-dependent chemosensitization and organoid growth inhibition following REV1 targeting, suggesting that its therapeutic consequences may extend beyond canonical TLS-mediated DNA damage tolerance.

cancer biology↗

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↗

bioRxiv2025

REV1 inhibition enhances trinucleotide repeat mutagenesis

Trinucleotide repeat (TNR) instability has been implicated in the pathogenesis of numerous neurodegenerative disorders. Because TNR instability causes mutagenesis of the underlying gene, we refer to the repeat instability phenomenon as TNR mutagenesis in this study. While germline expansions destabilize TNR to cause disease anticipation, somatic cell TNR instability drives earlier onset of symptoms and further disease progression. However, the drivers behind these repeat length changes remain unclear. Current models suggest that DNA replication slippage events and the action of genome instability pathways, such as DNA repair, cause TNR mutagenesis. Whether mutagenic polymerases from the translesion synthesis (TLS) pathway result in TNR instability is unclear. TLS polymerases are best at bypassing difficult-to-replicate DNA regions due to bulky lesions or gaps in DNA. While some effects of TLS polymerases on TNR instability have been explored in lower organisms, evidence in human cells is lacking. Using a quantitative GFP reporter with expanded CAG repeats, we show that inhibition of the TLS polymerase REV1 by its inhibitor, JH-RE-06, or siRNA knockdown increases TNR instability and the underlying mutability. These results suggest that REV1 protects Trinucleotide repeat length mutagenesis through potential continuous DNA synthesis when replicative polymerases stall ahead of repeat secondary structures. Collectively, we present evidence of the role of the TLS pathway in TNR instability, with potential implications for understanding mutability mechanisms, disease biology, and therapeutic targeting.

genetics↗