![Figure 6. Combined effects of AC220 and CB839 in FLT3ITD primary samples and in vivo. (A) Extracellular acidification rate (ECAR) of primary FLT3ITD mutated AML samples treated with vehicle control or AC220 2.5 nM measured using a Seahorse analyzer (mean ± SEM, n = 4, maximal glycolytic capacity; ****P < .0001; 2-way ANOVA with Bonferroni’s multiple comparisons). (B) Oxygen consumption rate (OCR) of primary FLT3ITD mutated AML samples treated with vehicle control, AC220 2.5 nM, CB839 100 nM, or their combination measured using a Seahorse analyzer. Real-time basal and maximal respiration are shown and in the inset a bar charts for the basal respiration in the 4 different conditions is shown (mean ± SEM, n = 4; **P = .0034 between AC220 and AC220 + CB839; ANOVA with Tukey’s multiple comparisons). (C) Relative viability in primary FLT3ITD mutated AML samples treated with vehicle control, AC220 2.5 nM, CB839 100 nM, or their combination. Far left panel shows a summary plot for all 5 patients (mean ± SEM, n = 5; **P = .0176 between AC220 and AC220 + CB839; ANOVA with Tukey’s multiple comparisons). The other panels show data for each individual patient (PT) with VAF (variant allele frequency) for FLT3ITD. Note in PT5, AC220 was used at 5 nM given the low variant allele frequency for FLT3ITD. (D) Survival curve of mice transplanted respectively with MV411 transduced with control scramble shRNA (n = 9) and GLS shRNA (n = 8) after treatment with AC220 (P = .0030 by log-rank test). (E-F) Percentage of RFP-positive cells, measured by flow cytometry, within 45 positive human cells from the bone marrow (E) and spleen (F) of mice transplanted respectively with MV411 transduced with control scramble shRNA (n = 9) and GLS shRNA (n = 8) after treatment with AC220 (box and whiskers showing minimum to maximum range for bone marrow [*P = .0201] and spleen [**** P < .0001]; unpaired Student t test). (G) Schematic model showing the action mechanism of combined GLS and FLT3 TK inhibition. FLT3ITD mutant cells use both glucose and glutamine to support their metabolism (left). FLT3-TKI treatment (AC220) blocks glucose uptake and mostly glycolysis, rendering the cells dependent on glutamine metabolism (middle). GLS gene silencing, chemical inhibition (CB839), or glutamine starvation enhances the efficacy of FLT3-TKI by blocking glutamine metabolism and its ability to support both TCA cycle/mitochondrial function and GSH synthesis/redox metabolism (right).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/131/15/10.1182_blood-2017-12-820035/4/blood820035f6.jpeg?Expires=1735835691&Signature=z9nz7BCToLnhFPkdITZSBQMBgaQO8JgHBfmj~pnqP~KJYor33gFBSsdlx6myb0emE5FpneAJrJln7sQj19EsoH7XycB9eAsV6EKZNon8k11NrV524dr1QF3DqognIGk2xaiWqM-zYPUreWIpzgQ2LHAYdeHlSJkdKMQo~I5v8GMF~QfqtLiA5ABCi2EPT-9nXeutbVCrox7DapsbakFRh8XsZBdPuL-eAhiSw3ywi5OHIe~dfRqDNb37Ggkl8sG42DLie~wvO3IYt0vfHJgeu3~f4R02XgIIVatXPlkv5XII1fJJWcTq6p1YLurubs-OBlA5HKyIFjOIKkw5Pt6UTQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Combined effects of AC220 and CB839 in FLT3ITDprimary samples and in vivo. (A) Extracellular acidification rate (ECAR) of primary FLT3ITD mutated AML samples treated with vehicle control or AC220 2.5 nM measured using a Seahorse analyzer (mean ± SEM, n = 4, maximal glycolytic capacity; ****P < .0001; 2-way ANOVA with Bonferroni’s multiple comparisons). (B) Oxygen consumption rate (OCR) of primary FLT3ITD mutated AML samples treated with vehicle control, AC220 2.5 nM, CB839 100 nM, or their combination measured using a Seahorse analyzer. Real-time basal and maximal respiration are shown and in the inset a bar charts for the basal respiration in the 4 different conditions is shown (mean ± SEM, n = 4; **P = .0034 between AC220 and AC220 + CB839; ANOVA with Tukey’s multiple comparisons). (C) Relative viability in primary FLT3ITD mutated AML samples treated with vehicle control, AC220 2.5 nM, CB839 100 nM, or their combination. Far left panel shows a summary plot for all 5 patients (mean ± SEM, n = 5; **P = .0176 between AC220 and AC220 + CB839; ANOVA with Tukey’s multiple comparisons). The other panels show data for each individual patient (PT) with VAF (variant allele frequency) for FLT3ITD. Note in PT5, AC220 was used at 5 nM given the low variant allele frequency for FLT3ITD. (D) Survival curve of mice transplanted respectively with MV411 transduced with control scramble shRNA (n = 9) and GLS shRNA (n = 8) after treatment with AC220 (P = .0030 by log-rank test). (E-F) Percentage of RFP-positive cells, measured by flow cytometry, within 45 positive human cells from the bone marrow (E) and spleen (F) of mice transplanted respectively with MV411 transduced with control scramble shRNA (n = 9) and GLS shRNA (n = 8) after treatment with AC220 (box and whiskers showing minimum to maximum range for bone marrow [*P = .0201] and spleen [**** P < .0001]; unpaired Student t test). (G) Schematic model showing the action mechanism of combined GLS and FLT3 TK inhibition. FLT3ITD mutant cells use both glucose and glutamine to support their metabolism (left). FLT3-TKI treatment (AC220) blocks glucose uptake and mostly glycolysis, rendering the cells dependent on glutamine metabolism (middle). GLS gene silencing, chemical inhibition (CB839), or glutamine starvation enhances the efficacy of FLT3-TKI by blocking glutamine metabolism and its ability to support both TCA cycle/mitochondrial function and GSH synthesis/redox metabolism (right).
