TUBB4B is a novel therapeutic target in non‐alcoholic fatty liver disease‐associated hepatocellular carcinoma

Abstract Non‐alcoholic fatty liver disease‐associated hepatocellular carcinoma (NAFLD‐HCC) is an emerging malignancy due to the rising prevalence of NAFLD. However, no drug is available to target NAFLD‐HCC. In this study, we aim to unravel novel therapeutic targets of NAFLD‐HCC utilizing a high‐throughput CRISPR/Cas9 screening strategy. We utilized the Epi‐drug CRISPR/Cas9 library consisting of single‐guide RNAs (sgRNAs) targeting over 1,000 genes representing the FDA‐approved drug targets and epigenetic regulators to perform loss‐of‐function screening in two NAFLD‐HCC cell lines (HKCI2 and HKCI10). CRISPR/Cas9 library screening unraveled TUBB4B as an essential gene for NAFLD‐HCC cell growth. TUBB4B was overexpressed in NAFLD‐HCC tumors compared with adjacent normal tissues ( N = 17) and was associated with poor survival ( p < 0.01). RNA‐sequencing and functional assays revealed that TUBB4B knockout in NAFLD‐HCC promoted cell apoptosis, cell cycle arrest, and cellular senescence, leading to suppressed NAFLD‐HCC growth in vitro and in vivo . We identified that TUBB4B inhibitor mebendazole (MBZ), an FDA‐approved drug, inhibited NAFLD‐HCC growth by inducing apoptosis and cellular senescence. Since protein expression of pro‐survival Bcl‐xL was induced in TUBB4B knockout NAFLD‐HCC cells, we examined combination of TUBB4B inhibition with navitoclax, a Bcl‐xL inhibitor that selectively targets senescent cells. Consistent with our hypothesis, either TUBB4B knockout or MBZ synergized with navitoclax to inhibit NAFLD‐HCC cell growth via the induction of intrinsic and extrinsic apoptosis pathways. In summary, TUBB4B is a novel therapeutic target in NAFLD‐HCC. Inhibition of TUBB4B with MBZ in combination with navitoclax synergistically inhibited NAFLD‐HCC cell growth, representing a promising strategy for the treatment of NAFLD‐HCC. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.