Lactate dehydrogenase A is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma

Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. To uncover cell type-specific dependencies to LDH, we screened a diverse panel of 94 cancer cell lines for responsiveness to two novel LDH A/B inhibitors developed through the NCI Experimental Therapeutics Program (NExT). We found that Ewing sarcoma (EWS) cell lines were exquisitely sensitive, with IC50 values approximately ten-fold below the median IC50 of the panel. To understand the mechanism behind this sensitivity, we genetically knocked down LDHA and LDHB using siRNA, and discovered that EWS cell lines were sensitive to loss of LDHA only, which inhibited proliferation and induced apoptosis. Notably, treatment of EWS cells with the LDH inhibitors phenocopied these effects. Additionally, genetic knockdown of EWS-FLI1, the oncogenic driver of EWS, resulted in loss of LDHA, but not LDHB. Analysis of publicly available ChIP-seq data generated using shFLI1-transfected EWS cells revealed that LDHA, but not LDHB, is directly regulated by EWS-FLI1. Functional mechanistic studies of glycolytic intermediates and cellular bioenergetics in EWS cells treated with the LDH inhibitors demonstrated that loss of viability was due to impairment of glycolysis, which occurred both in vitro and in vivo, and perturbation of the NAD+/NADH ratio. The translational potential of these compounds was next evaluated using in vivo analyses of pharmacokinetics, pharmacodynamics, efficacy, and toxicity. Intravenous administration of the LDH inhibitors resulted in diminished LDH activity, reduction of the lactate-to-pyruvate ratio, tumor cell necrosis, and a decrease in tumor growth rate in aggressive xenograft models of EWS. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, our data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as EWS that exhibit oncogene-dependent expression of LDH and increased glycolytic activity. This abstract is also being presented as Poster B33. Citation Format: Choh Yeung, Anna E. Gibson, Sameer H. Issaq, Nobu Oshima, Marielle E. Yohe, Haiyan Lei, Ganesha Rai, Daniel J. Urban, Michelle S. Johnson, Gloria A. Benevides, Giuseppe L. Squadrito, Sandy Eldridge, John Hamre III, Arnulfo Mendoza, Jack F. Shern, Lee J. Helman, Murali C. Krishna, Matthew D. Hall, Victor M. Darley-Usmar, Leonard M. Neckers, Christine M. Heske. Lactate dehydrogenase A is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR08.