Background: Hepatocellular carcinoma (HCC) is a significant contributor to global cancer-related mortality, with limited therapeutic options available for advanced-stage patients. Lenvatinib is one of the first-line drugs for unresectable HCC, but its clinical benefit remains constrained due to drug resistance emerging soon after initial treatment. Methods: Patient-derived organoids have shown promises in precision oncology by capturing parental tumor’s phenotypes and genetic traits. We applied genome-wide scale CRISPR-Cas9 dropout screening to a lenvatinib-resistant HCC organoid to define those drug resistance-associated genes and functional drivers. The human CRISPR Knockout Pooled Library (GeCKO v2) targeting 19,050 genes was used, and the screening system was also optimized to fulfill the demands of three-dimensional organoid growth and screening scale. Results: In conjunction with bioinformatic analysis and individual validations, we discovered a significant enrichment of spliceosome genes among the identified tumor essential genes. This underscores a pivotal role of alternative splicing in tumorigenesis and cancer progression. We found that one of the negatively screened splicing factors, splicing Factor 3b Subunit 4 (SF3B4), was upregulated in tumor tissues compared with its adjacent non-tumoral counterpart. We validated that loss of SF3B4 introduced by either CRISPR-Cas9 knockout or short hairpin RNA (shRNA) caused cell growth arrest in patient-derived organoids. Meanwhile, our screening identified a list of lenvatinib resistance-associated genes. Among them, several key ferroptosis regulators, including Protein Kinase AMP-Activated Catalytic Subunit Alpha 1 (PRKAA1), Glutamate-Cysteine Ligase Catalytic Subunit (GCLC), and Ferritin Light Chain (FTL), were suggested. Repressed expression of these proteins sensitizes tumor organoids to lenvatinib, highlighting the involvement of ferroptosis in lenvatinib-triggered cell death. Conclusions: In our study, we described the genome-wide screening in patient-derived HCC organoids. The identified tumor essential genes can represent potential HCC therapeutic targets, while inhibitors targeting lenvatinib resistance-associated genes might show an effect as lenvatinib combinatory partners.

Luyao Zhang

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