Glutaminase inhibition with telaglenastat (CB-839) improves treatment response in combination with ionizing radiation in head and neck squamous cell carcinoma models

Highlights

• CAL-27, FaDu, and HN5 have glutamine dependent proliferation.

• Telaglenastat inhibits HNSCC spheroid growth.

• Telaglenastat increases IR-induced cell death.

• Telaglenastat decreases aerobic respiration.

• Combination of telaglenastat and IR reduces tumor size in CAL-27 xenograft mice.

Abstract

The efficacy of ionizing radiation (IR) for head and neck cancer squamous cell carcinoma (HNSCC) is limited by poorly understood mechanisms of adaptive radioresistance. Elevated glutaminase gene expression is linked to significantly reduced survival (p < 0.03). The glutaminase inhibitor, telaglenastat (CB-839), has been tested in Phase I/II cancer trials and is well tolerated by patients. This study investigated if telaglenastat enhances the cellular response to IR in HNSCC models. Using three human HNSCC cell lines and two xenograft mouse models, we examined telaglenastat's effects on radiation sensitivity. IR and telaglenastat combinatorial treatment reduced cell survival (p ≤ 0.05), spheroid size (p ≤ 0.0001) and tumor growth in CAL-27 xenograft bearing mice relative to vehicle (p ≤ 0.01), telaglenastat (p ≤ 0.05) or IR (p ≤ 0.01) monotherapy. Telaglenastat significantly reduced the Oxygen Consumption Rate/Extracellular Acidification Rate ratio in CAL-27 and HN5 cells in the presence of glucose and glutamine (p ≤ 0.0001). Telaglenastat increased oxidative stress and DNA damage in irradiated CAL-27 cells. These data suggest that combination treatment with IR and telaglenastat leads to an enhanced anti-tumor response. This pre-clinical data, combined with the established safety of telaglenastat justifies further investigation for the combination in HNSCC patients.

Introduction

Survival remains poor for head and neck cancer (HNC) patients. Although, 40% of HNC patients receive curative ionizing radiation (IR) therapy, failure within the radiation field approaches 50% with resultant morbidity and mortality [1,2]. Multimodality treatment including the use of concurrent chemotherapy with radiation is used to treat many HNCs. However, the use of systemic therapy lacks a clear molecular basis and is often administered based on empiric evidence, contributing to limited efficacy and increased toxicity [[3], [4], [5]]. Radiotherapy dose escalation is limited by the need to protect surrounding healthy tissues and current options for FDA-approved radiosensitizers are limited. Even though an increasing variety of intelligently designed, gene-targeted drugs are in or are entering clinical use, many are transient in their activity with the majority of patients recurring after a short period of benefit [6]. Therefore, there is increased need to understand and overcome mechanisms of radiation resistance to improve patient survival.

Radiation induces programmed cell survival and metabolic responses within the cell [7,8], representing potential mechanisms of adaptive resistance that reduce radiation efficacy and are consequently interesting targets for combination therapy. Preliminary Reverse Phase Protein Array analysis by our laboratory identified activation of metabolic pathways upon radiation treatment (data not shown). Based on these findings as well as increasing evidence that glutamine plays an important role in DNA damage repair, and the availability of a targeted inhibitor already in Phase I/II trials, we explore the role of elevated glutaminase as a potential mechanism of adaptive resistance to radiation therapy [[9], [10], [11]].

Glutaminase is the enzyme responsible for the conversion of glutamine to glutamate. Glutamate is subsequently converted to α-ketoglutarate, a key component of the Krebs cycle. Notably, glutaminase overexpression has been linked to increased metastasis [12]. If increased glutaminase is independently associated with worse tumor control, and radiation induces glutaminase levels or activity, then we hypothesize that inhibition of glutaminase reduces substrate availability for the Krebs cycle, decreases aerobic respiration, and potentially reduces cellular proliferation.

Inhibition of glutaminase by telaglenastat (Calithera Biosciences, Inc.) has been associated with reduced growth of lymphomas, breast, renal and pancreatic cancers in pre-clinical studies [12]. In Phase II clinical trials for renal cell and triple-negative breast cancer in combination with chemotherapy, telaglenastat exhibited increased biostability and bioavailability as compared to other glutaminase inhibitors, and was well tolerated by patients [12,13]. Notably, neither the role of glutamine in HNC pathogenesis nor the efficacy of telaglenastat in combination with radiation has been previously evaluated.

Using The Cancer Genome Atlas's (TCGA) transcriptome database, we identified that increased glutaminase gene expression was associated with reduced survival in HNSCC patients. As this association supports glutaminase as an important drug target in the treatment of HNSCC, we examined if the combination of glutaminase inhibitor, telaglenastat, and IR is more effective than monotherapy. Clonogenic assays revealed that combinatorial treatment decreased cell survival in CAL-27 and HN5 cell lines. Using 2 heterotopic HNSCC xenograft models, we identified that the combination of telaglenastat and IR reduced tumor volume relative to monotherapy. Telaglenastat also increased IR induced oxidative stress and DNA damage. In summary, our finding that the addition of telaglenastat significantly improves radiation treatment response in HNSCC provides preclinical data in support of future clinical trials.