Abstract B37: Targeting the energy generation triangle to achieve synthetic lethality for treatment of hepatocellular carcinoma
Authors: Xu Shili , Catapang Arthur , Braas Daniel , Stiles Linsey , Lee Jason T. , Graeber Thomas G. , Damoiseaux Robert , Shirihai Orian , Herschman Harvey R. 
Published: December 2017

Abstract
Despite increasing prevalence and lethality for hepatocellular carcinoma (HCC), the need for effective treatment remains unmet. Metabolic reprogramming during hepatocarcinogenesis promotes cancer cell growth and survival, but also suggests the possibility of identifying as yet unexplored drug targets. In this study, we developed a synthetic lethal drug combination to treat HCC through the inhibition of the energy generation triangle. Most cancers, including HCC, increase glycolysis (the Warburg effect) to ensure sufficient supplies of energy (ATP), reducing equivalents (NADPH), and biochemical building blocks for cell growth and proliferation. Unlike most tumors, which express both hexokinase 1 (HK1) and HK2, many HCC tumors express only HK2, whereas normal hepatocytes only express HK4. We investigated the role of HK2 in HCC using a doxycycline (DOX)-inducible shRNA knockdown system. The HK4-to-HK2 isoform switch during hepatocarcinogenesis results in high sensitivity to HK2 silencing-induced glycolysis inhibition and cytostasis in HCC cells, in contrast to cancer cells expressing both HK1 and HK2. We performed a high throughput screen (HTS) of 3,205 drug-like small molecules and 119 FDA-approved oncology drugs to identify synthetic lethal partners of HK2 knockdown, using the CellTiter-Glo assay for primary HTS and confirmed with the alamarBlue assay. Diphenyleneiodonium (DPI), a mitochondrial complex-I inhibitor, is the best compound in our HTS that shows synthetic lethality in combination with HK2 silencing/inhibition in HCC cells that express only HK2. Cell proliferation was measured by the MTT assay and cell counting. Cytotoxicity was measured using Trypan blue to distinguish live and dead cells. HK2 activity in xenograft tumors was monitored non-invasively using 18F-fludeoxyglucose PET/CT scans. Perhexiline (PER), a fatty acid oxidation inhibitor, further sensitizes HCC cells to the complex-I/HK2-targeted combination. Cellular ATP levels were examined by a luciferase activity assay, oxygen consumption was examined by a Seahorse respirometry assay, and cell metabolism was examined by a metabolomics analysis. The inclusion PER significantly enhances the reduction of HCC cellular energy production caused by the combination of HK2 inhibition and DPI, and the efficacy of the complex-I/HK2-targeted combination in treating HCC tumors in vivo. Our findings indicate that selective inhibition of the energy generation triangle (glycolysis, oxidative phosphorylation, and fatty acid oxidation), due to the unique presence of only the HK2 isoform, appears promising for HCC therapy.
Citation Format: Shili Xu, Arthur Catapang, Daniel Braas, Linsey Stiles, Jason T. Lee, Thomas G. Graeber, Robert Damoiseaux, Orian Shirihai, Harvey R. Herschman. Targeting the energy generation triangle to achieve synthetic lethality for treatment of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B37.