Figure 4: Glutaminase inhibition preferentially radiosensitizes KEAP1 mutant lung cancer cells.

(A) Schematic depicting potential interaction between glutaminase inhibition and ionizing radiation in KEAP1 mutant cells (images were produced and modified from Servier Medical Art, see Acknowledgements). (B) Western blot analysis for ASCT2, NFE2L2, and HMOX1. Left: parental, KEAP1^NULL, and NFE2L2^NULL H1299 cells. Right: transient siRNA knock-down of NFE2L2 in H1975 NSCLC cells (wild-type for KEAP1 and NFE2L2). (C) Gene set enrichment analysis (GSEA) of a previously defined glutamine metabolism signature using RNA-seq data from parental and KEAP1^NULL H1299 cells. (D) As in C but using RNA-seq data from the tumor biopsies of patients in the CRT and SABR cohorts, comparing samples with and without pathogenic KEAP1 mutations. (E) Oncoprint of genes recurrently mutated in NSCLC (24) in the three cell line used for the experiments with CB-839. (F) Clonogenic survival of parental and KEAP1^NULL H1299 cells in the presence or absence of CB-839 (100 nM; 24 hour pre-treatment) and 4 Gy of ionizing radiation (n=4; *P<0.01, **P<0.001). Results were normalized against untreated cells. (G) Clonogenic survival of KEAP1^NULL and parental H1299 cells (KEAP1 wildtype) in the presence or absence of CB-839 (100 nM; 24 hour pre-treatment) and 4 Gy of ionizing radiation (n=4; *P<0.01). (H) Clonogenic survival of H1437 cells (KEAP1 wildtype) with and without siRNA knock-down of KEAP1 in the presence or absence of 2 Gy and 500 nM CB-839 (n=4; *P<0.01). (I) Clonogenic survival of parental and KEAP1 transfected A549 cells (KEAP1 mutant) in the presence or absence of CB-839 (0.1 nM; 24 hour pre-treatment) and 2 Gy of ionizing radiation (n=4; *P<0.01, **P<0.001). Results for panel F through I were normalized to untreated cells. (J) Intracellular reactive oxygen species (ROS) levels measured by DCFDA intensity via FACS in parental and KEAP1^NULL H1299 cells in the presence or absence of CB-839 (100 nM; 24 hour pre-treatment) and 2 Gy of ionizing radiation (n=4; *P<0.01). (K) Glutathione (GSH) to glutathione disulfide (GSSG) ratio in parental and KEAP1^NULL H1299 cells in the presence or absence of CB-839 (1 μM; 24 hour pre-treatment; n=4; *P< 0.05, **P< 0.01). (L) Clonogenic survival of KEAP1^NULL H1299 cells treated with or without 2 Gy of ionizing radiation and/or 100 nM CB-839 in the presence or absence of NAC (1 mM) (n=4; *P<0.01). (M) Western blot analysis for γH2AX in parental and KEAP1^NULL H1299 cells in the presence or absence of CB-839 (100 nM) treated with or without 2 Gy IR.