Targeting Glycolysis through Inhibition of Lactate Dehydrogenase Impairs Tumor Growth in Preclinical Models of Ewing Sarcoma

Abstract

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. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both in vitro and in vivo by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. SIGNIFICANCE: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.

Figure 1.. Ewing sarcoma (EWS) cells are sensitive to inhibition or loss of LDH activity.

A. Relative IC₅₀ values for top 12 most sensitive cell lines to LDH inhibitors NCI-737 and NC1–006, compared to median IC₅₀ for 94 cancer cell lines (striped bar) tested in the Oncolead cell panel assay. EWS cell lines MHHES1 and A673 are shown in black. B. IncuCyte live cell analysis of EWS cell lines TC71 and TC32 treated with NCI-737 at doses between 0.05 and 1 μM, 24 hours after plating. C. Western blot analysis of EWS cell lines TC32 and TC71 with and without treatment with 1 μM NCI-737 for 24 hours. D. Percent of TC71 and TC32 cells in early apoptosis (annexin V positive, PI negative) and late apoptosis (annexin V positive, PI positive) with and without treatment with 1 μM NCI-737 for 72 hours, measured by flow cytometry. Asterisks denote p-values, as compared to control, as follows: **p<0.01, ***p<0.001. E. Western blot analysis of LDHA and cleaved PARP in EWS cell lines (TC71, TC32, and EW8) 72 hours after LDHA siRNA knockdown. F. Relative viability of EWS cells (TC71, TC32, and EW8) following knockdown with siLDHA sequence #5 for 72 hours, expressed as percent of control confluence ± SEM. Asterisks (****) denote p-value < 0.0001, as compared to control.

Figure 2.. EWS-FLI1 regulates LDHA expression.

A. Western blot analysis of EWS-FLI1 and LDHA 72 hours following EWS-FLI1 siRNA knockdown with sequence #8 in EWS cell lines (TC71, TC32, RDES, and EW8). B. Representative ChIP-seq tracks for EWS-FLI1 (dark blue), ELF1 (light blue), and H3K24ac (yellow) from SK-N-MC EWS cells expressing either control shRNA (top) or EWS-FLI1 shRNA (bottom). RNA-seq tracks at the LDHA locus are shown for each cell line (green). C. Western blot analysis of LDHA protein level in SK-N-MC cells 72 hours following EWS-FLI1 siRNA knockdown with sequence #8.

Figure 3.. LDH inhibitors act through impairment of glycolysis in EWS.

A. Dose-response curves for change in LDH activity with increasing doses of NCI-737 or NCI-006 in EWS cell lines (TC71, TC32 and EW8). B. YSI analysis of glucose consumption and lactate production in EWS cells 24 hours after treatment with 187 nM NCI-737. Error bars indicate SD. Asterisks denote p-values between groups as follows: *p<0.05, **p<0.01, ***p<0.001. C. Intracellular pyruvate concentration in EWS cells 24 hours after treatment with 187 nM NCI-737. Error bars indicate SD. Asterisks denote p-values between groups as follows: **p<0.001, ****p<0.0001. D. NAD⁺/NADH ratio in EWS cells treated with various doses of NCI-737 at 13 hours. Error bars indicate SD. Asterisks denote p-values, as compared to controls, as follows: *p<0.05, ***p<0.001, ****p < 0.0001. E. Extracellular flux analysis was used to perform glycolytic stress tests of TC71 and TC32 cells treated with increasing doses of NCI-737. Glycolysis time-point indicates flux after glucose addition; glycolytic capacity time-point indicates flux after oligomycin addition. X-axis reflects NCI-737 dose in nM. Error bars indicate SD. Asterisks denote p-values, as compared to controls, as follows: *p<0.05, ***p<0.001, ****p<0.0001.

Figure 4.

LDH inhibitor sensitivity correlates with cellular bioenergetics. A-B. Relationship between degree of LDH inhibitor effect on glycolysis or glycolytic capacity and EWS cell line sensitivity. Error bars represent SEM. Relative cell line sensitivity determined based on experiments in Figures 1B and Supplemental Figure 1B. C. Pearson correlation between change in ECAR (glycolytic capacity measurement after treatment with 250 nM NCI-737) and cellular viability (as measured by percent inhibition compared to control after treatment with 500 nM NCI-737 using MTT assay). Correlation coefficient =−0.69 with p=0.038. D. Energy map of EWS cell lines (TC71, TC32, RDES, and EW8) with and without treatment with 100 nM NCI-737 for 24 hours. Error bars indicate SD. p<0.05 for comparisons of treated and untreated OCR for EW8, RDES, and TC71 (NS for TC32). p<0.01 for comparisons of treated and untreated ECAR for EW8, RDES, and TC71, p <0.05 for TC32. E. Percent ¹³C incorporation from glucose into major TCA cycle intermediates. Asterisks denote p-values as follows: *p>0.05, ***p<0.001, using t-test with Welch correction.

Figure 5.. LDH inhibitors have a dose- and time-dependent effect on tumor cell LDH activity in vivo.

A. Average intratumoral drug concentrations one-hour post-dosing of NCI-737 on day 3 in TC71-bearing xenografts (n=3 mice/group). B. Intratumoral LDH activity one-hour post-dosing of NCI-737 on day 3 in TC71-bearing xenografts (n=3 mice/group). Asterisk denotes p<0.05, compared to vehicle. C. Average intratumoral drug concentrations after one- and four-hours following treatment with NCI-737 at 40 mg/kg in TC71-bearing xenografts (n=3 mice/group). D. Percent change in intratumoral LDH activity after one- and four-hours following treatment with NCI-737 at 40 mg/kg in TC71-bearing xenografts (n=3 mice/group). Asterisk denotes p<0.05 between groups.

Figure 6.. LDH inhibition impairs glycolysis and affects cell survival in vivo.

A. Tumor growth curves for TC71-bearing xenografts treated seven days per week with IV NCI-737 at 60 mg/kg (n=5 mice/group). Error bars indicate SEM. Asterisks denote p-values, as compared to vehicle, as follows: *p<0.05, **p<0.01. B. Percent necrosis in tumor tissue of TC71- and EW8-bearing xenografts treated with IV NCI-737 on a seven day per week schedule (n= 5 mice/group). Error bars indicate SEM. Asterisk denotes p<0.05, for comparison between vehicle and treated tumors. C. Representative H&E stained whole slide digital images of entire tumor sections resected from TC71-bearing xenografts treated with vehicle (left) or NCI-737 (right). Viable tissue is shown in dark purple. Visual image analysis can be found in Supplemental Figure 12A. D. ¹³C lactate to pyruvate ratio change 30 minutes post injection of NCI-737 at 60 mg/kg in xenografted TC71 and EW8 tumors (n=3 mice/group). Lines indicate changes in each animal. Asterisk denotes p<0.05 comparing pre- and post-treatment animals.