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. 2023 Jan 23;29(7):1344–1359. doi: 10.1158/1078-0432.CCR-22-2747

Figure 6.

Figure 6. Glutaminase synthase inhibition disrupts redox balance and overcomes acquired cisplatin resistance in vitro and in vivo. A, IC50 values obtained from clonogenic survival curves showing that HN30-R8 are more sensitive to the glutaminase synthase inhibitor (IACS-6274) in vitro compared with their parental HN30-P counterparts. B, representative images of clonogenic survival assays in HN30-R8 cell lines treated with combination of CDDP and IACS-6274. C, isobolograms assessed by Chou and Talalay CI indicate strong synergism with combination of CDDP and IACS-6274 in HN30-R8 cells. The CI < 1.0 indicates synergism. Addition of IACS-6274 to CDDP resulted in CI = 0.10 at the effective dose (ED50) that killed 50% of the cells following treatment with the two drugs. D, reduced levels of GSH (***, P < 0.001) in HN30-R8 cells following treatment with IACS-6274. E, ROS levels are significantly elevated (****, P = 0.0001) after treatment with various doses of IACS-6274 in HN30-R8 compared with HN30-P cells in vitro. F, treatment with IACS-6274 increases expression of the DNA damage response marker, H2AX in HN30-R8 compared with HN30-P cells. Mice injected with HN30-R8 cells were treated with IACS-6274 as described in Methods. G, single-agent IACS-6274 activity was observed with 100 mg/kg twice a day dosing regimen (*, P = 0.03 two-way ANOVA) and was well tolerated (H). Treatment with IACS-6274 for 8 hours decreases GLU/GLN ratio indicating target engagement with the dose used (I). Nude male mice were injected orally with the HN30-R8 CDDP-resistant cells and treated with vehicle control, CDDP, IACS-6274 alone and in combination with CDDP as described in Methods. J, IACS-6274 showed single agent activity, enhanced CDDP sensitivity and decreased tumor growth. K, Proposed signaling model of acquired CDDP resistance in HNSCC. Under non-stressed conditions, KEAP1 directs ubiquitin-mediated degradation of NRF2 via proteasomes. Under oxidative stress generated by CDDP resistance, KEAP1 is mutated, NRF2 is stabilized and promotes transcription of ARE-containing genes associated with epigenetic modification of its downstream target genes. The hyperactivated NRF2 can protect the HNSCC cells from oxidative stress via suppression of ferroptotic and/or apoptotic cell death leading to enhanced cellular proliferation and accelerated tumor progression.

Glutaminase synthase inhibition disrupts redox balance and overcomes acquired cisplatin resistance in vitro and in vivo. A, IC50 values obtained from clonogenic survival curves showing that HN30-R8 are more sensitive to the glutaminase synthase inhibitor (IACS-6274) in vitro compared with their parental HN30-P counterparts. B, Representative images of clonogenic survival assays in HN30-R8 cell lines treated with combination of CDDP and IACS-6274. C, Isobolograms assessed by Chou and Talalay CI indicate strong synergism with combination of CDDP and IACS-6274 in HN30-R8 cells. The CI < 1.0 indicates synergism. Addition of IACS-6274 to CDDP resulted in CI = 0.10 at the effective dose (ED50) that killed 50% of the cells following treatment with the two drugs. D, Reduced levels of GSH (***, P < 0.001) in HN30-R8 cells following treatment with IACS-6274. E, ROS levels are significantly elevated (****, P = 0.0001) after treatment with various doses of IACS-6274 in HN30-R8 compared with HN30-P cells in vitro. F, Treatment with IACS-6274 increases expression of the DNA damage response marker, H2AX, in HN30-R8 compared with HN30-P cells. Mice injected with HN30-R8 cells were treated with IACS-6274 as described in Methods. G, Single-agent IACS-6274 activity was observed with 100 mg/kg twice a day dosing regimen (*, P = 0.03 two-way ANOVA) and was well tolerated (H). Treatment with IACS-6274 for 8 hours decreases GLU/GLN ratio indicating target engagement with the dose used (I). Nude male mice were injected orally with the HN30-R8 CDDP-resistant cells and treated with vehicle control, CDDP, IACS-6274 alone and in combination with CDDP as described in Methods. J, IACS-6274 showed single agent activity, enhanced CDDP sensitivity, and decreased tumor growth. K, Proposed signaling model of acquired CDDP resistance in HNSCC. Under non-stressed conditions, KEAP1 directs ubiquitin-mediated degradation of NRF2 via proteasomes. Under oxidative stress generated by CDDP resistance, KEAP1 is mutated, NRF2 is stabilized and promotes transcription of ARE-containing genes associated with epigenetic modification of its downstream target genes. The hyperactivated NRF2 can protect the HNSCC cells from oxidative stress via suppression of ferroptotic and/or apoptotic cell death leading to enhanced cellular proliferation and accelerated tumor progression.