Abstract: Hepatocellular carcinoma (HCC), the most common cancer of the liver, is the third leading cause of
cancer death worldwide. In the U.S., the overall 5-year survival rate for HCC is only 17% and only 3% for distantly
metastasized HCC. Surgical resection and liver transplantation are the current standard of care for HCC, but
only ~5% qualify for resection due to cirrhosis. Limited organ availability also severely restricts the number of
transplants. New targeted agents for HCC including kinase inhibitors and immunotherapies show promise, but
patients using targeted agents will inevitably develop drug resistance. Effective new therapies for HCC are
urgently needed. We propose a new, re-engineered p53 tumor suppressor-apoptotic protein hybrid for gene
therapy of HCC called p53-Bad*. Wild-type p53 was approved for gene therapy as Gendicine® for cancers in
China with limited success. Limited efficacy may be related to the dominant negative effect that mutant p53
exerts over wt p53. As nuclear p53 activity is dependent on homo-tetramerization, mutant p53 in cancer cells
can also tetramerize with wt p53 to cause its inactivation. Mutant p53 can also have gain-of-function and other
activities that can counter the effects of wt p53. To overcome this limitation, a potent p53 fusion protein with
novel mechanisms of action is proposed here that overcomes dominant negative inhibition and causes apoptosis
regardless of the p53 status of cancer cells. By fusing p53 to an apoptotic BH3 protein with an inherent
mitochondrial targeting signal, such as Bad, this increases apoptosis further by combining the apoptotic effects
of mitochondrial p53 with those of pro-apoptotic BH3 proteins. p53-Bad* (a mutant version of Bad with increased
mitochondrial localization) is our top cancer cell-killing construct. We hypothesize that the combination of p53
and BH3 proteins at the mitochondria, with liver cancer specificity driven by a modified alpha fetoprotein (AFP)
promoter, will potently induce apoptosis in liver cancer both in vitro (regardless of p53 status), and in vivo in
zebrafish and mouse models. Zebrafish are an ideal model system for studying human cancer, with “histologic
and genetic similarities between zebrafish and human tumors” while an orthotopic xenograft mouse model is an
important step toward translation. The following aims are proposed: Aim 1: a. p53-BH3 fusion (p53-Bad*) will
localize to the mitochondria and cause potent apoptosis in relevant hepatocellular carcinoma (HCC) cell lines
regardless of cellular p53 status; b. the use of modified/combination AFP promoters will confer liver cancer cell
specificity and kill HCC cells and not normal human cells. Aim 2: Expression of p53-Bad* under the liver specific
promoter fabp10a in zebrafish, crossed with a liver cancer zebrafish model will produce offspring with reduced
expression of liver cancer specific phenotypes. Aim 3: p53-Bad* (expressed in adenovirus with the optimized
HCC specific promoter) will shrink liver tumors in an orthotopic xenograft mouse model of HCC (using AFP-
expressing human HCC liver cells, Hep3B). The ultimate goal of this study is the development of a new, effective
therapy for treatment of HCC patients worldwide.