Figure 3.
Upregulation of MCT4 is essential in AML-adapted glucose metabolism. (A) An overview of glucose metabolism. (B-C) ECAR (B) and OCR (C) in MLL-AF9 mouse AML upon MCT4 KO in vitro (n = 5). (D-E) In vitro (MCT4 KO by CRISPR-Cas9; n = 3) (D) and in vivo glucose uptake (MCT4 KD by inducible shRNA; n = 6-8) (E) in MLL-AF9 AML. (F) In vitro intracellular metabolite profiling by LC-MS showing the relative levels (normalized to nontargeting gRNA control) of glycolytic and PPP metabolites, ATP/ADP and NADP+/NADPH ratios, and nucleotides in MCT4 KO AML in 20% and 2% O2 (n = 3-5). (G) In vitro cell growth of MCT4 KO AML supplemented with 100 mM of nucleosides (Nu), 10 mM of ribose (R), 2 mM of lactate (L), 0.5 mM of pyruvate (P), or combination of R and P (R+P; n = 3). (H) In vitro glucose uptake of human primary bulk AML blasts and CD34+CD38− with scrambled shRNA (control) or MCT4 KD (6-8 individual AML patient samples). (I) In vitro cell growth of MCT4 KD in primary AML samples supplemented with combination of 10 mM of R and 0.5 mM of P (5 individual AML patient samples). (J) Mitochondrial mass examined by MitoTracker staining in CD34+CD38− upon MCT4 KD (n = 6). (K) Mitochondrial ROS level examined by MitoSOX staining in CD34+CD38− upon MCT4KD (n = 6). (L) In vitro enzymatic activities (normalized to pHi of normal HSPC pH of 7.3) of HK1, PFK1, aldolase, GAPDH, PGK, PGM, PKM2, GAPDH, and PGD at different pHs (n = 3). (M) In vitro 15-minute 13C glucose flux and analysis of 13C-labeled metabolites in glycolytic and PPP metabolites and amino acids (n = 3-4). (N) In vivo 30-minute 13C glucose flux and analysis of 13C-labeled metabolites in glycolysis, PPP, and TCA cycle (n = 4). (O) NAD+/NADH in MLL-AF9 murine AML upon MCT4 KO supplemented with NAD+ (n = 3). (P) In vitro cell growth of MLL-AF9 murine AML upon MCT4 KO supplemented with NAD+ (n = 3). (Q-R) pHi (Q) and glucose uptake (R) of human AML cell lines treated with NHE1 inhibitor (HMA) in vitro (n = 3). (S) In vitro cell growth of AML treated with NHE1 inhibitor (HMA) supplemented with 10 mM of R, 0.5 mM of P, or R+P (n = 3). *P < .05, **P < .01, ***P < .001. AMP, adenosine 5′-monophosphate; MFI, mean fluorescence intensity; ns, not significant; UMP, uridine 5′-monophosphate.

Upregulation of MCT4 is essential in AML-adapted glucose metabolism. (A) An overview of glucose metabolism. (B-C) ECAR (B) and OCR (C) in MLL-AF9 mouse AML upon MCT4 KO in vitro (n = 5). (D-E) In vitro (MCT4 KO by CRISPR-Cas9; n = 3) (D) and in vivo glucose uptake (MCT4 KD by inducible shRNA; n = 6-8) (E) in MLL-AF9 AML. (F) In vitro intracellular metabolite profiling by LC-MS showing the relative levels (normalized to nontargeting gRNA control) of glycolytic and PPP metabolites, ATP/ADP and NADP+/NADPH ratios, and nucleotides in MCT4 KO AML in 20% and 2% O2 (n = 3-5). (G) In vitro cell growth of MCT4 KO AML supplemented with 100 mM of nucleosides (Nu), 10 mM of ribose (R), 2 mM of lactate (L), 0.5 mM of pyruvate (P), or combination of R and P (R+P; n = 3). (H) In vitro glucose uptake of human primary bulk AML blasts and CD34+CD38− with scrambled shRNA (control) or MCT4 KD (6-8 individual AML patient samples). (I) In vitro cell growth of MCT4 KD in primary AML samples supplemented with combination of 10 mM of R and 0.5 mM of P (5 individual AML patient samples). (J) Mitochondrial mass examined by MitoTracker staining in CD34+CD38− upon MCT4 KD (n = 6). (K) Mitochondrial ROS level examined by MitoSOX staining in CD34+CD38− upon MCT4KD (n = 6). (L) In vitro enzymatic activities (normalized to pHi of normal HSPC pH of 7.3) of HK1, PFK1, aldolase, GAPDH, PGK, PGM, PKM2, GAPDH, and PGD at different pHs (n = 3). (M) In vitro 15-minute 13C glucose flux and analysis of 13C-labeled metabolites in glycolytic and PPP metabolites and amino acids (n = 3-4). (N) In vivo 30-minute 13C glucose flux and analysis of 13C-labeled metabolites in glycolysis, PPP, and TCA cycle (n = 4). (O) NAD+/NADH in MLL-AF9 murine AML upon MCT4 KO supplemented with NAD+ (n = 3). (P) In vitro cell growth of MLL-AF9 murine AML upon MCT4 KO supplemented with NAD+ (n = 3). (Q-R) pHi (Q) and glucose uptake (R) of human AML cell lines treated with NHE1 inhibitor (HMA) in vitro (n = 3). (S) In vitro cell growth of AML treated with NHE1 inhibitor (HMA) supplemented with 10 mM of R, 0.5 mM of P, or R+P (n = 3). *P < .05, **P < .01, ***P < .001. AMP, adenosine 5′-monophosphate; MFI, mean fluorescence intensity; ns, not significant; UMP, uridine 5′-monophosphate.

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