CRISPR/Cas9 screening reveals that ASB2 inhibits p53-dependent ferroptosis to mediates Lenvatinib resistance in hepatocellular carcinoma

Abstract Background Lenvatinib is in a first-line therapy for advanced hepatocellular carcinoma (HCC), but its resistance is one of the main obstacles to treatment failure. The molecular mechanism of Lenvatinib resistance has not been well explored. Methods Genome-wide CRISPR/Cas9 knockout screening system was developed and bioinformatic analysis was used to identify key genes associated with Lenvatinib resistance in HCC. Whole transcriptome sequencing including coding and non-coding RNAs has also been performed in Lenvatinib resistance and sensitive HCC cells. Co-immunoprecipitation, confocal localization, western blot, immunofluorescence and other experiments were employed to assess the role of ASB2 in Lenvatinib resistance. Results ASB2 was found to be significantly increased at the mRNA and protein levels in Lenvatinib resistant HCC cells. ASB2 knockdown inhibited HCC Lenvatinib resistance cell proliferation, invasion and migration. Mechanistically, ASB2 activated NF-κB pathway by promoting IκBα ubiquitination and inhibited ferroptosis by promoting P53 ubiquitination and then mediated Lenvatinib resistance in HCC. Interestingly, NOTCH1 was shown to transcriptionly promote ASB2 expression and regulate NF-κB as well as ferroptosis pathways to induce Lenvatinib resistance in HCC. In further clinical translation, we found that Venetoclax could bind to ASB2 through a virtual screen of protein potential binding small molecules, and confirmed that Venetoclax and Lenvatinib combined significantly inhibited the progression of HCC, and the efficacy was better than Lenvatinib alone in vitro and vivo. Conclusion This study reveals that ASB2 which was transcriptionly promoted by NOTCH1, activated NF-κB pathway by promoting IκBα ubiquitination and inhibited ferroptosis by promoting P53 ubiquitination and mediated HCC Lenvatinib-resistance based on CRISPR/Cas9 screening. Venetoclax can potentially inhibit the function of ASB2 and the combination of Venetoclax and Lenvatinib can significantly inhibit the progression of HCC, which provides new targets and specific strategies for the treatment of HCC Lenvatinib-resistance, bringing new hope and benefits to HCC patients.