USP1 driven mitotic dysregulation and PLK1 stabilization confer Lenvatinib resistance in hepatocellular carcinoma

Lenvatinib is a first-line therapeutic option for advanced hepatocellular carcinoma (HCC), while acquired resistance severely limits its clinical efficacy. The precise molecular targets reversing Lenvatinib resistance remain inadequately explored. This study aims to elucidate the role of ubiquitin-specific peptidase 1 (USP1) in mediating resistance and identify potential therapeutic targets to improve treatment outcomes in HCC. Comprehensive analyses employing genetic modulation (USP1 knockdown and overexpression), pharmacological inhibition, and a series of in vitro and in vivo assays were conducted to assess the effects of USP1 on HCC cell sensitivity to Lenvatinib. Mass spectrometry-based proteomics, integrated bioinformatics analysis, and subsequent molecular validation techniques were utilized to systematically identify key USP1 substrates and interacting E3 ubiquitin ligases. Additionally, virtual screening was conducted using the ChemDiv library to identify potential inhibitors, followed by validation of candidate compounds through in vitro and in vivo experiments. Depletion of USP1 markedly enhanced sensitivity to Lenvatinib in HCC cells, while its overexpression induced resistance. Notably, USP1 knockdown led to obvious chromosome misalignment during metaphase in the presence of Lenvatinib. Mechanistically, Polo-like kinase 1 (PLK1) was identified as a critical substrate stabilized by USP1-mediated deubiquitination, essential for maintaining chromosome alignment and facilitating drug resistance. Additionally, we discovered that the E3 ubiquitin ligase STIP1 homology and U-box-containing protein 1 (STUB1) antagonized with USP1 to regulate PLK1 stability, further modulating resistance of HCC cells. c-Myc was identified as a transcriptional regulator of USP1, establishing a positive feedback loop as USP1/ PLK1/ c-Myc axis. Importantly, NTUZLB-001, a novel compound identified via in silico screening, effectively overcame resistance by promoting PLK1 destabilization. Our findings reveal a novel mechanism wherein USP1 promotes Lenvatinib resistance in HCC by regulating chromosome alignment through PLK1 deubiquitination. Targeting the USP1/PLK1 axis with NTUZLB-001 represents a promising therapeutic strategy to overcome drug resistance and enhance the clinical efficacy of Lenvatinib in HCC treatment.