Gao, R.; Van Moer, K.; Pulido, C.; Oudin, A.; Li, C.; Poussard, M.; L Ramos, t.; Murera, D.; Bartoleni, E.; Ives, A.; Gerbe de Thore, M.; Mondini, M.; Deutsch, E.; Berchem, G.; Auclair, C.; Janji, B.

Background: Immune checkpoint blockade (ICB) therapies demonstrate low efficacy in microsatellite stable (MSS) colorectal cancer (CRC) due to an immune-desert tumor microenvironment (TME) characterized by low antigen presentation and limited tumor-infiltrating lymphocytes (TILs). Harmine, a natural small-molecule and its promising derivatives ACB1801 have shown anti-tumor potential in preclinical models; however, their potential to reprogram the TME and overcome ICB resistance in MSS CRC remains unexplored. This study investigates whether and how ACB1801 can reshape TME to sensitize MSS CRC to ICB therapies. Methods: We used the CT26 MSS colorectal cancer mouse model to evaluate the ability of the harmine derivative ACB1801 to enhance the efficacy of anti-PD-1 therapy. To characterize its mode of action, we performed immune landscape analysis and transcriptomic profiling of both CD45- and CD45+ tumor-derived cells. In parallel, mechanistic studies were conducted in vitro using mouse and human MSS CRC cell lines. Results We demonstrate that the harmine derivative ACB1801 enhances the effectiveness of anti-PD-1 therapy in an MSS CRC mouse model. Combination therapy significantly increased CD8+ T cell infiltration and reduced regulatory T-cell (Treg) density in the TME. Transcriptomic profiling of CRC cells isolated from tumors treated with either anti-PD-1 alone or in combination with ACB1801 revealed significant enrichment of metabolic pathways in the combination group, characterized by reduced glycolysis and enhanced ferroptosis signatures. These findings were supported by in vitro data showing that ACB1801 reduces tumor cell glycolytic activity and promotes ferroptotic vulnerability. Mechanistically, ACB1801 induced STAT1 signaling, promoted CXCL10 release, and enhanced major histocompatibility complex class I (MHC-I)-dependent antigen presentation on tumor cells, thereby increasing tumor susceptibility to anti-PD-1 therapy. Conclusion: Collectively, our findings indicate that combination therapy with harmine derivatives and ICBs represents a promising strategy for treating MSS CRC patients.