Abstract Objective: New therapeutic strategies are desperately needed for hepatocellular carcinoma (HCC), the fourth most common cause of cancer-related deaths worldwide. Most patients with HCC lack mutations in the p53 tumor suppressor gene. Therefore, we hypothesized that targeting MDM4 or MDM2 to reactivate p53 signaling could be effective in this disease. Methods: HCC cell lines (SK-Hep1, Huh-7, Hep3B) were profiled for gene and protein expression of MDM4 with qPCR and immunoblotting assays, respectively. The effects of MDM4 inhibitor NSC207895 on the same cell lines were analyzed with MTT assays for cytotoxicity and qPCR experiments to look at changes in expression of p53 downstream targets, in comparison to the effects of MDM2 inhibitor Nutlin-3a. Effects of MDM4 on targets other than p53, including family members p63 and p73, were examined in cells with mutant or null p53 for changes in expression with qPCR experiments. Results: Three HCC cell lines showed expression of MDM4. Inhibition of MDM4 showed cytotoxic (high dose, 0.16-15.0 μM) and anti-proliferative (low dose, 0.05-0.3 μM) effects in all three cell lines, including p53 mutant Huh-7 and p53 null Hep3B cells. The same cell lines were strongly resistant to Nutlin-3a. Gene expression analyses were conducted with cells exposed to NSC207895 to examine effects of MDM4 inhibition on p53 targets Bax, Puma, and p21. In SK-Hep1 cells, NSC207895 treatment led to 2.5-fold overexpression of Bax, 27.4-fold of Puma, and 23.1-fold of p21. Huh-7 showed 11.8-fold upregulation of Puma and 12.4-fold of p21, and Hep3B exhibited a very strong 58.4-fold increase in p21 gene expression with the same treatment. Interestingly, Hep3B showed 5.9-fold overexpression of p63 and 1.99-fold of p73 gene expression with NSC207895 exposure, suggesting novel interactions between MDM4 and p63/p73 in the absence of p53. Conclusions: Inhibition of MDM4 shows efficacy in in vitro models of HCC through upregulation of p53, p63, and p73 to activate p53 tumor suppressive signaling. Thus, targeting MDM4 may be a viable option for all HCC patients, no matter the p53 mutation status. Citation Format: Sarah E. Woodfield, Yan Shi, Roma H. Patel, Zhenghu Chen, Aryana M. Ibarra, Sanjeev A. Vasudevan. Targeting MDM4 in hepatocellular carcinoma to reactivate p53 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4127. doi:10.1158/1538-7445.AM2017-4127