Abstract PO-094: Glutaminase inhibition increases ionizing radiation induced oxidative stress and DNA damage in head and neck squamous cell carcinoma models

Abstract

Abstract Ionizing radiation (IR) is a pillar of head and neck cancer treatment, the 6th most common cancer worldwide. However, its efficacy is limited by radioresistance, resulting in locoregional failure rates of ~50%. The mechanisms that tumor cells use to evade IR-induced cellular damage are poorly understood. Our initial proteomics analysis indicated that IR induced metabolic pathways, including those involving the Krebs cycle. Glutamate is a key substrate of the Krebs cycle. Glutaminase is the enzyme that converts glutamine to glutamate. Analysis of The Cancer Genome Atlas’s transcriptome database revealed that glutaminase overexpression (upper 25%) in patients with HNSCC was associated with reduced patient survival (p≤0.03). In this study, we therefore aimed to identify whether glutaminase inhibition with CB-839 (currently in Phase 1/2 trials) could enhance cellular response to IR. To do this, we used three representative human-derived HNSCC cell lines: CAL-27, FaDu, and HN5. First, we established that proliferation of all 3 HNSCC cell lines was glutamine dependent (20-50% increase, n=3 per line, p≤0.01). Second, a synergistic decrease in cell survival was observed in clonogenic assays with HNSCC cell lines treated with IR and CB-839 relative to either treatment alone (n=3 per line, p≤0.05). Thirdly, in 3D cell culture, which better represents the host microenvironment, combinatorial treatment significantly reduced HNSCC spheroid size (11-26% decrease, n=3 per line, p≤0.0001). The functional effect of CB-839 on cellular metabolism was examined using Seahorse MitoStress test assays. CB-839 significantly reduced spare respiratory capacity and OCR/ECAR ratio in HNSCC cell lines examined (p≤0.001, p≤2.0 × 10−6, n>3). To understand the mechanism behind this reduced cell growth and survival, we examined whether CB-839 enhanced oxidative stress and DNA damage when combined with IR using immunofluorescence. Oxidative stress marker, 8-oxoguanine, was ~2 fold higher with combined treatment as compared to CB-839 or IR alone in CAL-27 cells (p≤0.01, p≤0.05; respectively). Additionally, increased levels of y-H2AX were observed in CAL-27 cells treated with both CB-839 and IR relative to independent treatment. Combination of CB-839 with IR significantly inhibited tumor growth in CAL-27 xenograft mice relative to vehicle (p≤0.01), CB-839 (p≤0.05) or IR (p≤0.01) (p values, day 19). To summarize, successful treatment of HNSCC is impeded by adaptive resistance to IR and locoregional failure. Overall, these data suggest that the rational combination of IR and glutaminase inhibitor (CB-839) enhances cellular response and tumor control relative to independent treatment and provides pre-clinical data in support of additional investigation in patients with HNSCC. Citation Format: Christina A. Wicker, Brian G. Hunt, Sarah Palackdharry, William R. Elaban, Trisha M. Wise-Draper, Gordon B. Mills, Susan E. Waltz, Vinita Takiar. Glutaminase inhibition increases ionizing radiation induced oxidative stress and DNA damage in head and neck squamous cell carcinoma models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-094.