Abstract

Irinotecan resistance remains a significant challenge in metastatic colorectal cancer (mCRC) therapy. To address this, we identified USP1 as a synthetic lethal partner of Irinotecan through genome-wide CRISPR/Cas9 screening in HCT-116 cells. Combining the USP1 inhibitor I-138 with Irinotecan in HCT-116, HT-29, and SW620 cell lines significantly reduced IC 50 , suppressed proliferation, and diminished colony formation compared to monotherapy, demonstrating a synergistic effect (combination index CI<1). The synergistic therapeutic efficacy was further validated in the xenograft mouse model. Mechanistic studies revealed that I-138 significantly upregulated pCREB (Ser133), concurrently dynamically regulating the activity of USP1, FANCD2/FANCI, and PCNA upon DNA damage response and repair. RNA sequencing further highlighted the enrichment of cAMP, PI3K-AKT, and Wnt pathways, which are all linked to CREB activity in the combination group. These findings establish USP1 inhibition as a promising strategy to overcome Irinotecan resistance through the combination strategy, providing a novel therapeutic avenue for CRC.