Figure 4.

Transcriptomic and metabolic profiling identified altered metabolic pathways in HSPCs expressing mutant JAK2. (A) Venn diagram shows number of distinct and overlapping genes expressed between VF and E12 mice as compared with WT controls (log fold change [FC] > 1.5 and false discovery rate [FDR] < 0.05; n = 3 per genotype). (B) Heatmap showing differentially expressed genes in BM MEPs as determined by messenger RNA (mRNA) sequencing. Data show FC to the mean. (C-E) Heatmap showing expression of nutrient transporters (C), glycolysis pathway enzymes (D), and regulators of glycolysis pathway genes (E) in BM MEPs as determined by mRNA sequencing. Data shown are normalized expression levels in VF and E12 vs WT cells (n = 3 per genotype). (F) Metabolic pathway enrichment analysis of significantly upregulated metabolites in BM MEPs from VF and E12 compared with WT mice as determined by MetaboloAnalyst 3.0 (n = 3 per genotype; FC > 1.5; P < .05). (G) Measurements of glycolytic rates in BM lineage-negative Sca-1⁺ and cKit⁺ (LSK) cells and MEPs. Extracellular acidification rate values were normalized to cell numbers. Data are from 3 independent experiments (n = 6 mice per genotype). (H) Measurements of oxygen consumption rate (OCR), indicative of mitochondrial oxidative phosphorylation in BM LSK cells and MEPs. OCR values were normalized to cell numbers. Data are from 3 independent experiments (n = 6 mice per genotype). (I) Glucose uptake capacity of LSK cells and MEPs in BM and spleen as measured by mean fluorescence intensity (MFI) of 2-NBDG fluorescence (n = 6 mice per genotype). (J) Number of mitochondria per cell in BM LSK and MEP cells as determined by transmission electron microscopy (TEM) (n = 3 samples per genotype and 60-100 cells per genotype). (K) Mitochondrial DNA copy number in BM long-term hematopoietic stem cells (LT-HSCs), multipotent progenitors, and MEPs as measured by quantitative polymerase chain reaction (qPCR) (n = 3 per genotype). (L) Mitochondrial DNA copy number in peripheral blood of MPN patients as measured by qPCR presented as violin density plots. The horizontal width of the plots shows the density of the data along the y-axis. All data are presented as mean ± standard error of the mean. One-way analyses of variance followed by Tukey’s multiple comparison tests were used for multiple group comparisons. See also supplemental Figures 4 and 5. *P < .05, **P < .01, ***P < .001. FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; PMF, primary myelofibrosis.