Abstract B16: Evaluation of LDH inhibition as a treatment strategy in Ewing sarcoma
Authors: Heske Christine M. , Gibson Anna E. , Baumgart Josh T. , Yeung Choh , Issaq Sameer H. , Mendoza A , Johnson Michelle S. , Squadrito Guiseppe L. , Culp Lillian , Darley-Usmar Victor M. , Neckers Len M. 
Published: February 2019

Long-term outcomes for patients with relapsed, recurrent, or metastatic Ewing sarcoma (ES) remain poor despite advances in multimodal chemotherapy and local tumor control. The discovery of new targets and novel therapies is therefore critical to improving care for these patients. Recent insights into the metabolic landscape of ES have revealed that the EWS/FLI1 fusion oncoprotein regulates metabolic pathways in this disease, including shifting glucose consumption away from oxidative metabolism and towards glycolysis, a pathway that relies on LDH. Targeting this increased dependence on glycolysis presents an opportunity to inhibit the growth of ES cells through a novel therapeutic approach, while potentially limiting the toxicity delivered to normal cells. We therefore sought to evaluate the activity of NCGC-737 and NCGC-006, two novel LDHA/B inhibitors identified and validated as part of the Experimental Therapeutics Program of the National Cancer Institute (NCI-NExT), in ES.

For in vitro studies, proliferation of ES cells lines was assessed after inhibition of LDHA/B by each agent using IncuCyte and MTS assays. Protein expression of phospho- and total LDH was evaluated by Western blot. LDH activity was assessed using the pyruvate-dependent oxidation of NADH. NAD/NADH levels were determined using NAD/NADH-Glo. Analysis of glycolytic profiles was performed using the Agilent Extracellular Flux Analyzer. For in vivo studies, female SCID mice underwent orthotopic injection of ES cells from established cell lines. When tumors reached a desired size, mice were randomized and then treated on a variety of dosing schedules. Toxicity assessments included evaluation of overall appearance, weekly weights, blood sampling, and full necropsies on selected mice. Tumors were measured twice per week for assessment of efficacy. Tumors were harvested at midpoints and at study endpoint for assessments of drug level, target inhibition, and biology.

ES cell lines displayed varying sensitivity to NCGC-737 and NCGC-006, with IC-50 values ranging from 50 nM to 500 nM. While protein expression of phospho-LDH, total LDH-A, and total LDH-B were not correlated with sensitivity to either agent, glycolytic profiles were predictive of sensitivity. Cell lines that underwent a greater reduction in glycolytic capacity (the change in ECAR measured before and after oligomycin treatment) after LDHA/B inhibition experienced a greater antiproliferative effect, while cell lines that were able to maintain glycolytic capacity despite LDHA/B inhibition exhibited less of an effect on growth. In vivo studies to describe the toxicity of these agents demonstrated that hemolysis was the primary dose-limiting toxicity, and was dose dependent. Additional toxicity studies of specific tissues are ongoing and will be reported. Preliminary in vivo studies to optimize dosing regimen established that compared to oral dosing, intravenous dosing resulted in higher and more consistent tumor drug levels and improved target inhibition, with up to 93% of intratumoral LDH activity inhibited. Efficacy studies are ongoing and will be reported.

Preclinical data suggest that inhibition of LDHA/B may represent a potentially novel therapeutic strategy in the treatment of ES.

Citation Format: Christine M. Heske, Anna E. Gibson, Josh T. Baumgart, Choh Yeung, Sameer H. Issaq, A Mendoza, Michelle S. Johnson, Guiseppe L. Squadrito, Lillian Culp, Victor M. Darley-Usmar, Len M. Neckers. Evaluation of LDH inhibition as a treatment strategy in Ewing sarcoma [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 B16.