Figure 1.
GLS gene deletion and chemical inhibition are synthetically lethal with FLT3 TKIs. (A) Schematic of the genome-wide CRISPR/Cas9 synthetic lethality screen in the FLT3ITD mutant cell line MOLM13. (B) KEGG pathways enrichment analysis of dropout genes from CRISPR/Cas9 screen sorted by combined score calculated using Enrichr software.35,36 Pathways relevant to AML biology are highlighted while the remaining are grayed out. (C) List of top 10 genes from the “metabolic pathways” gene list sorted according to average fold depletion from gRNAs. FDR, false discovery rate. (D) Bar graph depicting individual fold depletion for each gRNA targeting GLS. (E-G) Growth inhibition curves to AC220 of MOLM13 (E) and murine bone marrow cells expressing MLL/AF9-FLT3ITD (F) and MLL/AF9 (G) transduced respectively with “empty” gRNA control or 2 different gRNA targeting GLS (mean ± standard error of the mean [SEM], n = 3, P < .001 for treatment effect comparing control and both Gls knockout for panels E-F; ns, not significant for panel G; 2-way analysis of variance [ANOVA]). (H-I) Apoptosis in the FLT3ITD mutant cell lines MV411 (H) and MOLM13 (I) transduced with control scramble shRNA and GLS shRNA following treatment with AC220 0.5 nM for 48 hours (for MV411: mean ± SEM, n = 7, ****P < .0001, **P = .0086; for MOLM13: mean ± SEM, n = 3, **P = .0014; for AC220 treatments [comparison between scramble and GLS shRNA]: **P = .0050; for dimethyl sulfoxide vs AC220 comparison in the scramble shRNA; 2-way ANOVA with Bonferroni’s multiple comparisons). (J-K) Apoptosis in MV411 (J) and MOLM13 (K) following treatment with AC220 1 nM, CB839 100 nM or their combination (for MV411: mean ± SEM, n = 14, **P = .0033, ****P < .0001; for MOLM13: mean ± SEM, n = 16, *P = .0126, ***P = .0003; ANOVA with Tukey’s multiple comparisons). (L-M) Apoptosis in MV411 (L) and MOLM13 (M) grown in the presence (full media) or absence of glutamine following treatment with AC220 1 nM for 48 hours (for MV411 mean ± SEM, n = 24, for MOLM13 mean ± SEM, n = 11; ****P < .0001, ***P = .0007; 2-way ANOVA with Bonferroni’s multiple comparisons).

GLS gene deletion and chemical inhibition are synthetically lethal with FLT3 TKIs. (A) Schematic of the genome-wide CRISPR/Cas9 synthetic lethality screen in the FLT3ITD mutant cell line MOLM13. (B) KEGG pathways enrichment analysis of dropout genes from CRISPR/Cas9 screen sorted by combined score calculated using Enrichr software.35,36  Pathways relevant to AML biology are highlighted while the remaining are grayed out. (C) List of top 10 genes from the “metabolic pathways” gene list sorted according to average fold depletion from gRNAs. FDR, false discovery rate. (D) Bar graph depicting individual fold depletion for each gRNA targeting GLS. (E-G) Growth inhibition curves to AC220 of MOLM13 (E) and murine bone marrow cells expressing MLL/AF9-FLT3ITD (F) and MLL/AF9 (G) transduced respectively with “empty” gRNA control or 2 different gRNA targeting GLS (mean ± standard error of the mean [SEM], n = 3, P < .001 for treatment effect comparing control and both Gls knockout for panels E-F; ns, not significant for panel G; 2-way analysis of variance [ANOVA]). (H-I) Apoptosis in the FLT3ITD mutant cell lines MV411 (H) and MOLM13 (I) transduced with control scramble shRNA and GLS shRNA following treatment with AC220 0.5 nM for 48 hours (for MV411: mean ± SEM, n = 7, ****P < .0001, **P = .0086; for MOLM13: mean ± SEM, n = 3, **P = .0014; for AC220 treatments [comparison between scramble and GLS shRNA]: **P = .0050; for dimethyl sulfoxide vs AC220 comparison in the scramble shRNA; 2-way ANOVA with Bonferroni’s multiple comparisons). (J-K) Apoptosis in MV411 (J) and MOLM13 (K) following treatment with AC220 1 nM, CB839 100 nM or their combination (for MV411: mean ± SEM, n = 14, **P = .0033, ****P < .0001; for MOLM13: mean ± SEM, n = 16, *P = .0126, ***P = .0003; ANOVA with Tukey’s multiple comparisons). (L-M) Apoptosis in MV411 (L) and MOLM13 (M) grown in the presence (full media) or absence of glutamine following treatment with AC220 1 nM for 48 hours (for MV411 mean ± SEM, n = 24, for MOLM13 mean ± SEM, n = 11; ****P < .0001, ***P = .0007; 2-way ANOVA with Bonferroni’s multiple comparisons).

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