Genome-Scale CRISPR Screens Reveal DNA Repair Dependencies That Sensitize Hepatocellular Carcinoma to Oxaliplatin

Background: Most patients with hepatocellular carcinoma (HCC) present with advanced disease and have limited systemic treatment options. Oxaliplatin shows clinical activity in HCC but its effectiveness is frequently curtailed by intrinsic and acquired resistance. We sought to systematically identify genetic vulnerabilities that increase oxaliplatin sensitivity in HCC. Methods: Genome-scale negative-selection CRISPR-Cas9 screens were conducted in two genetically distinct HCC cell lines (Hep3B and MHCC-97H) under low-dose oxaliplatin to discover conserved determinants of sensitivity. Selected DNA damage response (DDR) hits were validated. An oxaliplatin-resistant MHCC-97H subline was generated for transcriptomic profiling to characterize resistance-associated programs. Screen results were integrated with TCGA-LIHC expression and survival data to evaluate clinical relevance. Additionally, we analyzed bulk RNA-seq data from biopsy specimens collected from 36 HCC patients prior to initiation of hepatic arterial infusion chemotherapy (HAIC), comparing expression levels of the DDR genes between patients with objective response and non-responders. Results: Screens in both cell lines converged on DDR pathways, particularly nucleotide excision repair (NER) and the Fanconi anemia/interstrand crosslink repair network; shared sensitizers included ERCC4 (XPF), FANCE and SLX4. Validation experiments showed that disruption of representative DDR factors (POLH and XPA) synergistically increased oxaliplatin efficacy at concentrations as low as 0.5 μM. Transcriptomic analysis of the resistant MHCC-97H subline revealed coordinated upregulation of DNA repair programs, G2/M checkpoint and E2F target signatures, and epithelial-mesenchymal transition features. Integration with TCGA-LIHC data demonstrated frequent overexpression of many screen-identified DDR genes in primary HCC and an association between higher expression of selected factors and poorer patient survival. In the HAIC cohort, several DDR genes, including ATR, BRCA2, CDK7, MUS81, MUTYH, PARG, POLH, POLK and XPA, were significantly lower in the objective response group. Conclusions: DDR components represent candidate biomarkers and therapeutic targets whose inhibition may enhance oxaliplatin efficacy in HCC.