Telaglenastat as an alternative to cisplatin as a radiosensitizer in the treatment of head and neck squamous cell carcinoma

Abstract

The efficacy of radiation treatment (RT) of head and neck squamous cell carcinoma (HNSCC) is limited by radioresistance and the toxicity of FDA approved radiosensitizers. In extension to our previous research where we demonstrated that telaglenastat (CB839) increased efficacy of RT in in vitro and in vivo HNSCC models, here, we examine the radiosensitizing effects of telaglenastat in comparison to cisplatin's, as cisplatin is currently the standard of care for concurrent therapy. Combination of telaglenastat with RT reduced tumor volume in a HNSCC patient derived xenograft mouse model. The efficacy of telaglenastat with RT in reducing cell survival and increasing apoptosis was similar if not greater than that of cisplatin with RT in Cal27 and HN5 HNSCC cells. The addition of telaglenastat increased reactive oxygen species and reduced the antioxidant glutathione in both Cal27 and HN5 cells. Reverse Phase Protein Array analyses revealed alterations in cell death and DNA damage response proteins. This study provides the scientific underpinnings for the use of telaglenastat as a radiosensitizer in the treatment of HNSCC either as an alternative to cisplatin or in cisplatin-ineligible patients.

Keywords: Cisplatin; Glutaminase inhibition; Head and neck squamous cell carcinoma; Radiosensitizer; Telaglenastat.

Fig. 1.. Effect of telaglenastat and radiation on PDX tumor growth and apoptotic and proliferative markers.

(A) Telaglenastat with radiation significantly decreased average tumor volume in HNSCC PDX mice as compared to vehicle treatment on day 12 (p<0.05). On days 4 through 8, tumors were treated with 0 or 2 Gy RT (vehicle n=8, other groups n=10, error bars represent SEM) (B) TUNEL and Ki67 IHC of mouse PDX tumors that were harvested on day 22. Scale bars represent 200 μm. Telaglenastat (Tel.), Radiation (RT).

Fig. 2.. Telaglenastat, cisplatin, and RT restrict HNSCC spheroid growth.

(A) Cal27 and (B) HN5 cells were seeded on Matrigel and treated with telaglenastat (1 μM; tel.) and cisplatin (Cal27: 0.25 μM; HN5: 1 μM; cis.) for 24 hours then irradiated at 4 Gy. Measurements taken 7 days post-RT (SEM, one-way ANOVA, n=4, *p<0.05, **p<0.01).

Fig. 3.. (A) Telaglenastat, cisplatin, and radiation reduce colony formation and induce pro-apoptotic signaling in HNSCC cells .

Cal27 and HN5 cells were treated with telaglenastat (tel.) and cisplatin (cis.) for 24 hours then irradiated at 4 Gy. Cells grew for 8–10 days after irradiation and cell survival was measured by counting colonies. (n=3) (B) Activation of caspases 3/7 were assessed in Cal27 and HN5 cells. Cells were treated overnight with vehicle or drug prior to RT treatment. Twenty-four hours post-RT cells were imaged. SEM, one-way ANOVA, n≥3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Fig. 4.. Pro-oxidant effects of telaglenastat, cisplatin and RT in HNSCC cell lines.

(A) ROS production assessment in Cal27 and HN5 cells using DCFH-DA flow cytometry. (B) Total GSH per mg of protein determined using spectrophotometry. Cal27 and HN5 cells were treated with combinations of vehicle, 10 μM telaglenastat (tel.) and cisplatin (0.5 μM for DCFH-DA assay; 0.2 μM for GSH assay; cis.) overnight prior to treatment with 0 or 4 Gy RT. Lysates were collected 24 hours post-irradiation (SEM, n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Fig. 5.. Telaglenastat, cisplatin and RT increase yH2AX signal in HNSCC cell lines.

The DNA damaging effects of telaglenastat, cisplatin and RT were assessed indirectly using immunofluorescence detection of yH2AX foci in Cal27 and HN5 cells. Cells were pre-treated overnight with combinations of vehicle, 10 μM telaglenastat (tel.), and cisplatin (cis, Cal27 0.25 μM, HN5 1 μM) prior to irradiation with 0 or 2 Gy. Cells were fixed and processed for yH2AX staining 4 hours after irradiation. SEM, n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Representative 40X images are shown. Scale bar represents 100 μm.

Fig. 6.. Global proteomic changes induced by telaglenastat, cisplatin and radiation in Cal27 and HN5 HNSCC Cells.

Heat map of global proteomic changes in cell growth, cell death, DNA damage and, cell migration and invasion pathways in Cal27 and HN5 HNSCC cells. Vehicle (veh.), Radiation (RT), Telaglenastat (Tel.), Cisplatin (Cis.). Heatmap represents average levels by treatment and centered. Heat map color correspondence: Decreased and increased protein expression run blue to red, respectively. Accompanying statistics are in Supplemental Table 1.