Abstract B37: Assessing DNA damage response pathway proteins ATM and p53 in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein

Introduction: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of liver cancer that is prevalent in younger patients between 10-35 years of age and is usually not associated with underlying cirrhosis. Overall outcomes tend to be poor due to lack of effective chemotherapy. Recently, a gene fusion transcript has been identified in a significant proportion of FL-HCC whereby PRKACA, a cAMP-associated protein, and heat shock protein DNAJB1 fuse following a deletion of 400kb region. In our previous study, immunoblot analysis of p53, a tumor-suppressor protein, was significantly upregulated in FL-HCC samples in comparison to normal liver tissues. p53 is a downstream target of ATM, which is a key regulator of DNA damage response (DDR) pathway. Therefore, we hypothesized that increased p53 protein expression may be associated with ATM activation in FL-HCC. Methods: We analyzed 3 tumor and non-neoplastic FL-HCC patient samples containing DNAJB1-PRKACA fusion protein using immunohistochemistry (IHC) on paraffin-embedded sections with anti-ATM, anti-phosphorylated ATM (S1981), anti-p53, and anti-phospho-p53 (S15) antibodies. Immunoblot analysis was performed for cleaved PARP protein to assess the apoptotic cells in FL-HCC samples. ImageJ software was utilized to quantify the nuclear, cytoplasmic/mitochondrial localization of each protein. Results: Nuclear ATM expression was significantly increased in tumor samples compared to normal liver tissues in 3/3 FL-HCC patients (p=1.43E-04, p=1.51E-07 and 1.68E-05). Nuclear expression of activated (phosphorylated) ATM (p-ATM) was significantly upregulated in 2/3 tumor compared to the normal samples (p=9.47E-09 and p=0.00). Interestingly, the tumor sample lacking nuclear phospho-ATM was the only tumor sample with a significant expression of p-ATM in the mitochondrial/cytoplasmic compartment. In assessing the expression pattern of p-53, 2/3 patients revealed statistically significant increase in total p-53 compared to normal liver sample. While nuclear phospho-p53 expression was not significantly altered in any of the three tumor samples compared to non-neoplastic liver tissue, there was a statistically significant increase in the activated form of p53 (phospho-p53) expression in the cytoplasmic region in 2/3 FLHCC samples compared to normal. Coincidentally, the same 2 patients who demonstrated increased total nuclear ATM and phospho-ATM showed increased cytoplasmic p53 and phosphorylated p53. Immunoblot analysis for cleaved-PARP revealed no PARP activation in FL-HCC tumor samples. Conclusion: The expression pattern of total ATM and phospho-ATM in FL-HCC patients with PRKACA-DNAJB1 suggests that the DDR pathway may be activated in these tumors. However, increases in p53 protein expression and phosphorylated (S15) p53 are not associated with apoptosis given lack of increase in PARP cleavage. Further studies aimed at understanding the mechanisms preventing p53-induced apoptosis or DNA repair are therefore necessary. Citation Format: Anju Karki, Khashayar Vakili, Antonio R. Perez-Atayde. Assessing DNA damage response pathway proteins ATM and p53 in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B37.