Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality with limited effective therapies. Lenvatinib is the most widely used first-line multi-kinase inhibitor in advanced HCC, yet clinical benefit is modest. Given that lenvatinib activity relies heavily on tumor microenvironment (TME), an in vivo CRISPR screen is superior to identify synthetic target within TME.

Parallel kinome-wide CRISPR screens were conducted in lenvatinib-resistant HCC cell in vitro and in vivo to identify in vivo-specific vulnerabilities. Candidate target was validated with genetic and pharmacological inhibition in 2D/3D cultures, patient-derived organoids (PDOs), cell and patient derived xenografts (CDXs and PDXs) and immunocompetent mice. Mechanistic analyses included transcriptomics and biochemical assays. Clinical activity of the combined therapy was assessed in a phase I trial (NCT06519721). Approval for animal experiments and the clinical trial was obtained from the relevant regulatory authorities. Written informed consent was obtained from all participants where appropriate.

AURORA kinase A (AURKA) emerged as an in vivo-specific synthetic target of lenvatinib in HCC. The combination of lenvatinib with AURKA inhibitors alisertib or vic-1911 exhibited robust antitumor effects across CDXs, PDOs, PDXs, immunocompetent mouse models, and 3D but not 2D culture. Mechanistically, cancer cells cultured in vitro in 3D conditions or in vivo exhibit higher dependency on receptor tyrosine kinase (RTK) signaling, lenvatinib-mediated RTK blockade triggered compensatory activation of the DDR1-FAK-AKT survival pathway in 3D and in vivo contexts, conferring resistance, which was abrogated by AURKA inhibition. The phase I study enrolling 12 patients with advanced or recurrent HCC progressing on prior systemic therapies (including lenvatinib) demonstrated encouraging clinical responses with the combination of lenvatinib plus vic-1911, with overall response rates of 41.6% (confirmed responses) and 58.3% (including unconfirmed responses). The combination may be particularly beneficial for patients with post-liver transplant recurrence, in whom immune-checkpoint inhibitors are contra-indicated.

We identified AURKA as an in vivo-specific synthetic target of lenvatinib and combined AURKA inhibition with lenvatinib exhibited potent early clinical activity in HCC. This work provides proof-of-concept that in vivo CRISPR screen-guided target discovery can be successfully translated to early-phase clinical evaluation, establishing a promising combinatorial strategy for advanced HCC.

This work was supported by the National Natural Science Foundation of China and Dutch Cancer Society through the Oncode Institute. We thank JS InnoPharm (Shanghai) for providing the vic-1911.