The MASTL/YBX1/PAK4 axis regulated by stress-activated STK24 triggers lenvatinib resistance and tumor progression in hepatocellular carcinoma

Background and Aims: Many patients with HCC present inadequate responses to lenvatinib therapy. Therefore, it is important to elucidate the underlying mechanisms of resistance and to formulate effective reversal strategies. Approach and Results: We conducted transcriptome and proteome sequencing analyses of lenvatinib-resistant cell lines and patient-derived tissues, identifying microtubule-associated serine/threonine kinase-like (MASTL) as a critical factor associated with lenvatinib resistance in HCC. Then, we utilized subcutaneous mouse models, half maximal inhibitory concentration (IC 50 ) measurements, and colony formation assays to determine the biological function of MASTL in promoting tumor growth and mediating resistance to lenvatinib. To further elucidate the underlying mechanisms, we performed co-immunoprecipitation and mass spectrometry analyses, revealing that MASTL facilitates the phosphorylation of Y-box binding protein-1 (YBX1). Using chromatin immunoprecipitation assays, we subsequently confirmed that phosphorylated YBX1 transcriptionally activates PAK4, identifying PAK4 as a downstream effector of the MASTL pathway. Moreover, mass spectrometry and phosphorylation analysis indicated that serine/threonine protein kinase 24 (STK24), a stress-responsive kinase, can activate MASTL in HCC under lenvatinib exposure. Notably, disruption of the MASTL/YBX1/PAK4 signaling axis restored HCC sensitivity to lenvatinib. Conclusions: We propose that the MASTL/YBX1/PAK4 axis, which is activated by stress-induced STK24, plays a crucial role in lenvatinib resistance. Inhibiting this axis by targeting MASTL effectively overcomes lenvatinib resistance in HCC.