TUBB4B is a novel therapeutic target in nonalcoholic fatty liver disease-associated hepatocellular carcinoma.

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 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 the TUBB4B as an essential gene for NAFLD-HCC cell growth. TUBB4B was overexpressed in NAFLD-HCC tumors as compared to adjacent normal tissues (N=17) and was associated with poor survival (P<0.01). RNA-seq 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 target 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 treatment of NAFLD-HCC. This article is protected by copyright. All rights reserved.