Genome-wide CRISPR-Cas9 screen identifies genes conferring lenvatinib resistance in hepatocellular carcinoma

Background: Tyrosine kinase inhibitors (TKIs), such as lenvatinib, sorafenib, and regorafenib, form a key component of current systemic therapy for advanced hepatocellular carcinoma (HCC). However, intrinsic and acquired resistance significantly diminish their clinical benefit. Although multiple signaling pathways have been implicated in TKI response, the molecular determinants that sustain broad TKI resistance remain incompletely defined. To uncover these mechanisms, we performed an unbiased genome-wide genetic interrogation in a mouse HCC cell line. Materials and Methods: We established a murine HCC cell line from melanocortin-4 receptor-deficient mice fed a high-fat diet. These cells exhibited pronounced intrinsic resistance to lenvatinib. To identify genes underlying this resistance, we performed a genome-wide CRISPR-Cas9 knockout screen using a pooled sgRNA library. Top candidates were prioritized by guide depletion scores and validated using siRNAs and shRNAs. Transcriptomic and proteomic analyses were conducted to define downstream pathways. To assess therapeutic relevance, we tested inhibitors targeting enzymes within this network and evaluated their combinatorial effects with lenvatinib and other TKIs across multiple cancer cell lines. Results: The CRISPR screen identified PCIF1, an mRNA methyltransferase responsible for installing m6Am modification at the 5′ cap, as a leading determinant whose loss markedly sensitized human HCC cells to lenvatinib in both in vitro assays and in vivo tumor models. Integrative multi-omics analysis revealed that PCIF1 regulates a MYBL2-driven transcriptional program that converges on nucleotide metabolic pathways. Within this downstream network, a rate-limiting enzyme in nucleic acid biosynthesis (Molecule A) emerged as a key effector. Pharmacological inhibition of Molecule A exhibited potent synergistic effects not only with lenvatinib but also with additional TKIs, enhancing drug sensitivity and suppressing proliferation across human HCC and other cancer cell lines. Expression of Molecule A was significantly associated with lenvatinib resistance, underscoring its potential relevance as a biomarker and therapeutic target beyond the PCIF1-MYBL2 regulatory axis. Conclusion: Our findings demonstrate that, even when proliferation appears unchanged, TKI exposure imposes a metabolic stress that drives a rewiring of nucleotide biosynthesis and a pronounced dependency on Molecule A. This adaptive shift is observed across distinct TKIs and cancer types, indicating that altered nucleotide metabolism represents a broadly conserved mechanism of drug resistance. Because clinically approved inhibitors of Molecule A are already available, these results provide a strong rationale for therapeutic repositioning strategies aimed at overcoming TKI resistance in hepatocellular carcinoma.