Somatic mtDNA mutation burden shapes metabolic plasticity in leukemogenesis

Abstract

The role of somatic mitochondrial DNA (mtDNA) mutations in leukemogenesis remains poorly characterized. To determine the impact of somatic mtDNA mutations on this process, we assessed the leukemogenic potential of hematopoietic progenitor cells (HPCs) from mtDNA mutator mice (Polg D257A) with or without NMyc overexpression. We observed a higher incidence of spontaneous leukemogenesis in recipients transplanted with heterozygous Polg HPCs and a lower incidence of NMyc-driven leukemia in those with homozygous Polg HPCs compared to controls. Although mtDNA mutations in heterozygous and homozygous HPCs caused similar baseline impairments in mitochondrial function, only heterozygous HPCs responded to and supported altered metabolic demands associated with NMyc overexpression. Homozygous HPCs showed altered glucose utilization with pyruvate dehydrogenase inhibition due to increased phosphorylation, exacerbated by NMyc overexpression. The impaired growth of NMyc-expressing homozygous HPCs was partially rescued by inhibiting pyruvate dehydrogenase kinase, highlighting a relationship between mtDNA mutation burden and metabolic plasticity in leukemogenesis.

Fig. 1.. Effect of mtDNA mutation burden on leukemogenic potential.

(A) Schematic representation of experimental setup. HPCs were isolated from BM of 2- to 5-month-old homozygous (Hom; Polg^mut/mut) and heterozygous (Het; Polg^wt/mut) mtDNA mutator mice, as well as wild-type (WT; Polg^wt/wt) littermate controls. HPCs were transduced with either MSCV-NMyc-HA-IRES-YFP (NMyc) or control MSCV-IRES-YFP (Ctrl) retrovirus before transplantation into lethally irradiated C57BL/6 syngeneic recipients for in vivo assessment or in vitro analysis. (B) Kaplan-Meier survival curves for transplanted recipients: WT-Ctrl (n = 39), WT-NMyc (n = 82), Het-Ctrl (n = 36), Het-NMyc (n = 83), Hom-Ctrl (n = 31), Hom-NMyc (n = 89). *P < 0.05; ***P < 0.005; ****P < 0.001; *****P < 0.0001 (log-rank test followed by pairwise comparison). (C) Representative hematoxylin and eosin (H&E)–stained sections show typical leukemic infiltrates in the BM, spleen, liver, and brain of a WT-NMyc recipient. (D) Levels of circulating YFP⁺ leukocytes were analyzed by flow cytometry using peripheral blood collected from recipients transplanted with NMyc-expressing HPCs as they became symptomatic. Hom-NMyc mice were separated into two groups (leukemic and nonleukemic) based on necropsy results. WT-NMyc (n = 27), Het-NMyc (n = 21), Hom-NMyc (tumor cases, n = 4; nontumor cases, n = 9); **P < 0.01; ****P < 0.001 (Wilcoxon test). (E) The Fine-Gray subdistribution hazard model was used to analyze the probability of death by various causes. Plots show cumulative incidence functions (CIFs) for leukemia-related deaths. ****P < 0.001 (Gray’s test). (F) Incidence of leukemia in recipients: WT-Ctrl (n = 1/32), Het-Ctrl (n = 6/25), Hom-Ctrl (n = 1/26), WT-NMyc (n = 68/77), Het-NMyc (n = 58/75), Hom-NMyc (n = 16/84). *P < 0.05; *****P < 0.0001; ns: no significant difference (Fisher’ exact test). (G) CIF for engraftment failure–related deaths. ****P < 0.001 (Gray’s test). (H) Increased numbers of scattered blasts (yellow circles) were identified in H&E-stained BM and spleen samples from mice transplanted with Hom HPCs (NMyc>Ctrl). WT-Ctrl (n = 0/32), Het-Ctrl (n = 0/25), Hom-Ctrl (n = 2/26), WT-NMyc (n = 0/77), Het-NMyc (n = 0/75), Hom-NMyc (n = 37/84). (I) Plots show CIFs for anemia-related deaths. *P < 0.05; *****P < 0.0001 (Gray’s test).

Fig. 2.. Analysis of mtDNA mutation burden, copy number, and common oncogenic mutations in leukemic samples.

(A) Comparison of mtDNA mutation burden in cells derived from normal and leukemia-infiltrated BM samples. Sample size: normal BM: WT (n = 4), Het (n = 2), and Hom (n = 3); leukemic BM: WT-NMyc (n = 8), Het-NMyc (n = 9), and Hom-NMyc (n = 5). (B) Analysis of variant allele fractions (VAFs) in cells derived from leukemia-infiltrated BM samples. Plots show area under the curve (AUC) values from VAFs ranging from 0.2 to 0.99. Sample sizes: WT-NMyc (n = 8), Het-NMyc (n = 9), and Hom-NMyc (n = 5); *P < 0.05. (C) Quantification of mtDNA content in YFP⁺ HPCs before transplantation and in cells from leukemia-infiltrated BM samples. The graph shows relative mtDNA copy number (mean ± SEM) calculated using ND2 as the mtDNA marker and 18S as the nDNA marker. Ratios were normalized to a control sample that was included in all experiments to allow comparison of values across experiments. Sample size: YFP⁺ HPCs before transplantation: WT-NMyc (n = 7), Het-NMyc (n = 6), and Hom-NMyc (n = 7); leukemic BM: WT-NMyc (n = 15), Het-NMyc (n = 17), and Hom-NMyc (n = 8). *P < 0.05 (ANOVA). (D) The exome sequences in leukemia-infiltrated BM samples were examined to detect secondary mutations (Pten/PI3K/Akt/Ras/P53) in different groups: WT-NMyc (n = 12), Het-NMyc (n = 12), and Hom-NMyc (n = 6). *P < 0.05 (Fisher’s exact test).

Fig. 3.. Unlike NMyc-expressing HPCs from Het mtDNA mutator mice, HPCs from Hom mice fail to compensate for the loss of respiratory complex activity.

(A) Cell numbers were assessed in serial methylcellulose culture passages (MCP#) at MCP#1, MCP#4, and MCP#6 across different groups and then normalized to the number of WT-NMyc cells at MCP#1. Bar graph shows normalized cell numbers (mean ± SEM). n = 8 to 14 biologic replicates per group. *P < 0.05; ***P < 0.005 (Student’s t test). (B) Colony numbers were assessed at MCP#6 and then normalized to the number of WT-NMyc colonies at MCP#1. Bar graph shows normalized colony numbers (mean ± SEM). n = 8 to 9 biologic replicates per group. (C) Proportion of methylcellulose cultures containing at least one colony at MCP#6 for each group: WT-Ctrl (n = 0/8), Het-Ctrl (n = 1/9), Hom-YFP (n = 0/8), WT-NMyc (n = 4/8), Het-NMyc (n = 6/9), and Hom-NMyc (n = 2/8). (D) Ratio of CIV to CS activity (“Normalized complex IV activity”) using sorted HPCs (pooled from two mice for each genotype). Results from a representative experiment (n = 4 technical replicates for each group) are shown. ***P < 0.005, ****P < 0.001 (one-factor ANOVA). (E to H) Seahorse assays were used to determine oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) in sorted HPCs. Tracings from representative OCR (E) and ECAR (G) experiments are shown (error bars were derived from technical replicates). Bar graphs show the results of basal OCR (F) and basal ECAR (H) measurements. Values were normalized to WT-Ctrl. n = 4 to 7 biological replicates per group. *P < 0.05; **P < 0.01; ***P < 0.005 (one-factor ANOVA). (I and J) CIV (I) and CS (J) activities were measured in WT-, Het-, and Hom-NMyc HPCs at MCP#0, MCP#1, and MCP#3. *P < 0.05; **P < 0.01; ****P < 0.001 (one-factor ANOVA).

Fig. 4.. NMyc expression and accumulation of somatic mtDNA mutations are associated with changes in gene expression.

(A) Heatmap and hierarchical clustering of Hallmark pathways with differential expression shows five distinct patterns of response among the six groups of HPCs (WT-Ctrl, WT-NMyc, Het-Ctrl, Het-NMyc, Hom-Ctrl, Hom-NMyc). (B) Heatmap of differentially expressed genes in the mitochondrial respiratory complex gene set (GO). CI (purple); CII (light brown); CIII (blue); CIV (red); others (black). n = 2 to 3 biologic replicates per group. Groups of genes showing relatively higher levels of expression in Hom-NMyc HPCs are highlighted with black lines at right.

Fig. 5.. Glucose tracing experiments highlight resistance of Hom (but not Het) HPCs to changes in glucose utilization induced by NMyc expression.

WT, Het, and Hom HPCs (with or without NMyc expression) were incubated with [U-¹³C] glucose, and the incorporation of ¹³C into glycolytic and TCA intermediates was analyzed. (A) Schematic representation of the glycolytic pathway, its branch to serine biosynthesis, with ¹³C-labeled isotopologues. The schematic highlights ¹³C-labeled isotopologues: glucose (Gluc) m+6, glucose-6-phosphate (G6P) m+6, serine (Ser) m+3, 3-phosphoglycerate (3-PG) m+3, and pyruvate (Pyr) m+3. Bar graphs show fractional enrichment of key ¹³C-labeled isotopologues. n = 2 to 3 biological replicates per group. (B) Schematic representation of the glycolytic pathway extending to the tricarboxylic acid (TCA) cycle with ¹³C-labeled isotopologues. The schematic highlights the following ¹³C-labeled isotopologues: lactate (Lac) m+3, citrate (Cit) m+2, succinate (Suc) m+2, fumarate (Fum) m+2, malate (Mal) m+2, aspartate (Asp) m+2, glutamate (Glu) m+2, 2-hydroxyglutarate (2-HG) m+2. Bar graphs show fractional enrichment of key ¹³C-labeled isotopologues. n = 2 to 3 biological replicates per group. The results of Student’s t test are provided in table S3. (C and D) Summary of fractional enrichment across key metabolites, providing a comprehensive overview of ¹³C incorporation in glycolysis, the TCA cycle, and amino acid synthesis. Results for WT-Ctrl, WT-NMyc, Het-Ctrl, and Het-NMyc are shown in (C); results for WT-Ctrl, WT-NMyc, Hom-Ctrl, and Hom-NMyc are shown in (D). (E) The ratio of Fum m+2/α-KG m+2 was assessed in the indicated HPCs transduced with or without NMyc. ****P < 0.001. (F) The ratio of Cit m+2/Pyr m+3 was assessed to compare pyruvate dehydrogenase (PDH) activity in the indicated HPCs transduced with or without NMyc. ***P < 0.001. (G) Immunoblot analysis using antibodies against phospho-PDHE1A and total PDHE1A was performed using lysates from sorted YFP⁺ HPCs. Representative immunoblot analysis and calculated phospho-PDHE1A/total PDHE1A ratios are shown.

Fig. 6.. Impaired growth of Hom-NMyc HPCs is partially rescued by inhibiting PDH kinase activity.

(A) Schematic representation of PDH regulation and the rationale for dichloroacetate (DCA) treatment. DCA treatment inhibits PDKs, thereby restoring PDH activity and facilitating the entry of pyruvate into the TCA cycle. (B) Schematic of in vitro experiments assessing the effects of DCA treatment on HPC growth upon serial passage in methylcellulose medium. Cells were replated once weekly for 5 weeks (MCP#1 to MCP#5). (C) Mean (± SEM) HPC count at MCP#1; WT-Ctrl (n = 6), WT-NMyc (n = 6), Hom-Ctrl (n = 4), Hom-NMyc (n = 4). **P < 0.01, ***P < 0.005 (ANOVA). (D) Mean (± SEM) Hom-NMyc HPC count at MCP#5. n = 4 per treatment; **P < 0.01 (ANOVA). (E) To determine the effects of DCA treatment on Hom-NMyc HPC growth in vivo, Hom-NMyc HPCs were injected into partially irradiated recipients (802 rads). Three weeks after transplantation, recipients were separated into two groups: treatment (5 mM DCA water, n = 20) and control (water, n = 15). Peripheral blood was collected at defined intervals. The study was terminated at 21 weeks after transplantation. (F) Peripheral blood cell counts and (G) percentage of NMyc-expressing YFP⁺ cells were assessed in recipients with (+) or without (−) DCA treatment. Linear mixed-effects (LME) models were used to examine associations between WBC, PLT, RBC, YFP+ and treatment (75). Fitted lines with 95% confidence intervals were derived from the models for DCA-treated (n = 19) and untreated (n = 13) groups. The plots show trends or slopes over time, allowing comparisons between the two groups. WBC and PLT counts increased over time, while RBC counts decreased over time in both groups. The percentage of YFP⁺ cells decreased significantly in the untreated group but was maintained in the treated group. . *P < 0.05, **P < 0.01, ****P < 0.001, *****P < 0.0005 (LME).

Fig. 7.. Burden of mtDNA mutations drives NMyc-induced metabolic rewiring.

In WT HPCs (left), NMyc drives the metabolism of glucose toward serine biosynthesis and pyruvate production in the cytoplasm through glycolysis. Once transported into the mitochondria, pyruvate is converted to acetyl-CoA by the PDH complex, generating electron carriers (e.g., NADH). Acetyl-CoA enters the TCA cycle and is further oxidized, producing more NADH. The oxidation of NADH (to NAD⁺) is coupled to adenosine triphosphate (ATP) production during mitochondrial respiration. This regeneration of NAD⁺ is necessary for glycolysis and the TCA cycle. NMyc’s ability to promote leukemic transformation depends on the proper interactions between the TCA cycle and OXPHOS in the mitochondria. In NMyc-expressing Hom HPCs (right), pyruvate metabolism is impaired before entering the TCA cycle due to decreased PDH activity, resulting from increased phosphorylation of PDH by PDKs. PDK activation in Hom HPCs, exacerbated by NMyc, may be related to the NADH accumulation that occurs when mitochondrial respiration is disrupted (e.g., by the heavy burden of mtDNA mutations). The PDK-mediated inhibition of PDH complex activity decreases the flux of glucose-derived metabolites into the TCA cycle. Thus, pyruvate is preferentially converted to lactate to regenerate NAD⁺ from NADH in the cytoplasm. This conversion maintains the NAD⁺/NADH ratio, allowing glycolysis to continue despite impaired mitochondrial function. Impaired serine biosynthesis may also be related to disruptions in the NAD⁺/NADH ratio and/or the shift toward glycolysis. In Het HPCs (not shown), a low level of mtDNA mutations might confer some metabolic plasticity. These cells can potentially adapt to minor mitochondrial dysfunctions, maintaining a balance between glycolysis and the TCA cycle. This metabolic plasticity may provide a survival advantage under varying metabolic conditions, allowing Het HPCs to better support leukemic transformation than Hom HPCs, which are overwhelmed by the high mutation burden.