靶向药物筛选鉴定出能够加速错配修复缺陷（MMRd）以增强免疫治疗敏感性的新型化合物

Background: MMRd tumors respond exceptionally to immunotherapy, and pharmacologic MMR inactivation has the potential to improve immunosensitivity of MMR-proficient tumors. We previously showed that temozolomide plus cisplatin (TMZ+CDDP) induces MMRd through MSH2 silencing. However, clinical translation revealed limited efficacy, potentially due to delayed MMR inactivation preventing sufficient tumor mutational burden (TMB) and microsatellite instability (MSI) accumulation on treatment. We hypothesized that compounds enabling rapid MMR inactivation could overcome this limitation. Methods: Preclinical models treated with TMZ+CDDP were analyzed longitudinally for TMB and MSI. Clinical trial data (NCT04457284) combining TMZ+CDDP with nivolumab in metastatic colorectal cancer patients were assessed using serial ctDNA profiling. We performed computational screening of open-source databases to identify compounds inducing rapid MLH1 or MSH2 downregulation (≤3 days) across cancer cell lines. Candidates were studied using CT26 cells transfected with an out-of-frame luciferase-microsatellite reporter, where MSI-induced frameshift mutations restore luciferase expression, enabling real-time MSI monitoring. These repurposed compounds underwent MMR expression analyses. Results: In vivo, TMZ+CDDP induced MSH2 loss only after 4 weeks (W) of treatment, with MMRd genotype recapitulated only after 8W. Clinical trial analysis of 16 evaluable patients revealed that only 5 patients (31%) developed a MMRd-like genotype with gains in TMB and MSI at a median of 8W, which associated with improved survival. Critically, patients failing to increase TMB and/or MSI developed aneuploid gains in MMR genes on treatment suggesting a compensatory resistance mechanism to mutagenesis. These findings indicate that delayed MMR inactivation with TMZ+CDDP limits clinical efficacy. To address this limitation, our drug screen identified 6 compounds inhibiting significantly Msh2 and/or Mlh1 after short course treatment. Three compounds were confirmed to generate sustained bioluminescence increases within 1-2W compared to 4-8W with TMZ+CDDP. Two compounds were showed to completely abolish Msh2 expression by 1 or 2W, while the third reduced expression by 70%. The three other agents showed only transient Msh2 or Mlh1 downregulation without change in bioluminescence. These novel compounds demonstrate substantially accelerated MMR inactivation kinetics compared to TMZ+CDDP. Conclusions: While TMZ+CDDP can induce a MMRd genotype in patients, delayed MMR inactivation and compensatory MMR gene amplification limit therapeutic efficacy. Repurposed compounds enabling rapid MSH2 inhibition may prevent adaptive resistance mechanisms and improve clinical responses when combined with immunotherapy, warranting further preclinical/clinical development.