Figure 2.
Genetic inhibition of MCT4 reverted aberrant alkaline pHi and suppressed cellular growth in AML. (A-B) pHi (A) and intracellular lactate (B) in mouse MLL-AF9 AML with MCT4 KO in vitro (n = 4). (C) Ten-minute 10-mM 13C lactate flux and analysis of 13C-labeled metabolites in MLL-AF9 AML in vitro with MCT4 KO by gas chromatography–mass spectrometry (GC-MS; n = 4). (D) In vitro growth of MLL-AF9 AML upon MCT4 KO (n = 4). (E) In vitro apoptosis assay of MLL-AF9 AML upon MCT4 KO (n = 4). (F) BrdU incorporation assay in MCT4 KO AML in vitro (n = 4). (G) Distribution of cell populations in G1, S, and G2/M phase in cell cycle upon MCT4 KO in MLL-AF9 AML in vitro (n = 4). (H) Western blot of cell cycle–related proteins in MCT4 KO AML. (I) In vivo pHi of MLL-AF9 AML with scrambled shRNA control (n = 70) and MCT4 knockdown (KD; n = 70; from 3 mice) imaged by multiphoton fluorescence microscope was determined based on the calibrated standard cell (supplemental Figure 1C). (J) Representative images of multiphoton fluorescence microscope showing in vivo pHi from pH reporter expressing AML 2 days after in vivo–induced MCT4 KD by shRNA. (K) Thirty-minute 25-mg/kg 13C lactate flux and analysis of 13C-labeled metabolites in MLL-AF9 AML in vivo with MCT4 KO by GC-MS (n = 4). (L) Kaplan-Meier survival analysis of mice transplanted with MLL-AF9 AML upon in vivo–induced MCT4 (n = 8 of 9) or scrambled shRNA (n = 8). Red area indicates doxycycline induction. (M) Representative FACS plots showing the proportion of host leukocytes (CD45.2) and AML (CD45.1) in recipient BM at 60 days after doxycycline withdrawal and 24 weeks after secondary transplantation. (N) Serial colony-forming assay of mouse cKit+ MLL-AF9 AML with MCT4 KO (n = 3; triplicate wells for each experiment). (O) FACS analysis of MCT4 protein expression in primary human AML samples (n = 6) with MCT4 KD by shRNA and scrambled shRNA control. (P-Q) pHi (P) and intracellular lactate (Q) in primary human AML with MCT4 KD (n = 4) ex vivo. (R) In vivo engraftment of primary human AML with MCT4 KD by shRNA or scrambled shRNA in NSG (9 individual AML patient samples; 1-2 mice per each sample). (S) pHi in human CD34+CD38− primary AML with MCT4 KD (n = 6) ex vivo. (T) Serial colony-forming assay of human CD34+CD38− primary AML with MCT4 KO (5 individual AML patient samples; triplicate wells for each experiment). *P < .05, **P < .01, ***P < .001. CFU, colony-forming unit; MFI, mean fluorescence intensity; ns, not significant.

Genetic inhibition of MCT4 reverted aberrant alkaline pHi and suppressed cellular growth in AML. (A-B) pHi (A) and intracellular lactate (B) in mouse MLL-AF9 AML with MCT4 KO in vitro (n = 4). (C) Ten-minute 10-mM 13C lactate flux and analysis of 13C-labeled metabolites in MLL-AF9 AML in vitro with MCT4 KO by gas chromatography–mass spectrometry (GC-MS; n = 4). (D) In vitro growth of MLL-AF9 AML upon MCT4 KO (n = 4). (E) In vitro apoptosis assay of MLL-AF9 AML upon MCT4 KO (n = 4). (F) BrdU incorporation assay in MCT4 KO AML in vitro (n = 4). (G) Distribution of cell populations in G1, S, and G2/M phase in cell cycle upon MCT4 KO in MLL-AF9 AML in vitro (n = 4). (H) Western blot of cell cycle–related proteins in MCT4 KO AML. (I) In vivo pHi of MLL-AF9 AML with scrambled shRNA control (n = 70) and MCT4 knockdown (KD; n = 70; from 3 mice) imaged by multiphoton fluorescence microscope was determined based on the calibrated standard cell (supplemental Figure 1C). (J) Representative images of multiphoton fluorescence microscope showing in vivo pHi from pH reporter expressing AML 2 days after in vivo–induced MCT4 KD by shRNA. (K) Thirty-minute 25-mg/kg 13C lactate flux and analysis of 13C-labeled metabolites in MLL-AF9 AML in vivo with MCT4 KO by GC-MS (n = 4). (L) Kaplan-Meier survival analysis of mice transplanted with MLL-AF9 AML upon in vivo–induced MCT4 (n = 8 of 9) or scrambled shRNA (n = 8). Red area indicates doxycycline induction. (M) Representative FACS plots showing the proportion of host leukocytes (CD45.2) and AML (CD45.1) in recipient BM at 60 days after doxycycline withdrawal and 24 weeks after secondary transplantation. (N) Serial colony-forming assay of mouse cKit+ MLL-AF9 AML with MCT4 KO (n = 3; triplicate wells for each experiment). (O) FACS analysis of MCT4 protein expression in primary human AML samples (n = 6) with MCT4 KD by shRNA and scrambled shRNA control. (P-Q) pHi (P) and intracellular lactate (Q) in primary human AML with MCT4 KD (n = 4) ex vivo. (R) In vivo engraftment of primary human AML with MCT4 KD by shRNA or scrambled shRNA in NSG (9 individual AML patient samples; 1-2 mice per each sample). (S) pHi in human CD34+CD38− primary AML with MCT4 KD (n = 6) ex vivo. (T) Serial colony-forming assay of human CD34+CD38− primary AML with MCT4 KO (5 individual AML patient samples; triplicate wells for each experiment). *P < .05, **P < .01, ***P < .001. CFU, colony-forming unit; MFI, mean fluorescence intensity; ns, not significant.

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