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The fms-like tyrosine kinase 3 (Flt3) is a cell surface receptor that is expressed by various hematopoietic progenitor cells (HPC) and Flt3-activating mutations are commonly present in acute myeloid and lymphoid leukemias. These findings underscore the importance of Flt3 to steady-state and malignant hematopoiesis. In this study, the expression of Flt3 protein and Flt3 mRNA by single cells within the hematopoietic stem cell (HSC) and HPC bone marrow compartments of C57/BL6 mice was investigated using flow cytometry and the quantitative reverse transcription polymerase chain reaction. Flt3 was heterogeneously expressed by almost all of the populations studied, including long-term reconstituting HSC and short-term reconstituting HSC. The erythropoietin receptor (EpoR) and macrophage colony-stimulating factor receptor (M-CSFR) were also found to be heterogeneously expressed within the multipotent cell compartments. Co-expression of the mRNAs encoding Flt3 and EpoR rarely occurred within these compartments. Expression of both Flt3 and M-CSFR protein at the surface of single cells was more commonly observed. These results emphasize the heterogeneous nature of HSC and HPC and the new sub-populations identified are important to understanding the origin and heterogeneity of the acute myeloid leukemias.

BackgroundHypoxia-inducible factors (HIF)1 and 2 are transcription factors that regulate the homeostatic response to low oxygen conditions. Since data related to the importance of HIF1 and 2 in hematopoietic stem and progenitors is conflicting, we investigated the chromatin binding profiles of HIF1 and HIF2 and linked that to transcriptional networks and the cellular metabolic state.MethodsGenome-wide ChIPseq and ChIP-PCR experiments were performed to identify HIF1 and HIF2 binding sites in human acute myeloid leukemia (AML) cells and healthy CD34+ hematopoietic stem/progenitor cells. Transcriptome studies were performed to identify gene expression changes induced by hypoxia or by overexpression of oxygen-insensitive HIF1 and HIF2 mutants. Metabolism studies were performed by 1D-NMR, and glucose consumption and lactate production levels were determined by spectrophotometric enzyme assays. CRISPR-CAS9-mediated HIF1, HIF2, and ARNT−/− lines were generated to study the functional consequences upon loss of HIF signaling, in vitro and in vivo upon transplantation of knockout lines in xenograft mice.ResultsGenome-wide ChIP-seq and transcriptome studies revealed that overlapping HIF1- and HIF2-controlled loci were highly enriched for various processes including metabolism, particularly glucose metabolism, but also for chromatin organization, cellular response to stress and G protein-coupled receptor signaling. ChIP-qPCR validation studies confirmed that glycolysis-related genes but not genes related to the TCA cycle or glutaminolysis were controlled by both HIF1 and HIF2 in leukemic cell lines and primary AMLs, while in healthy human CD34+ cells these loci were predominantly controlled by HIF1 and not HIF2. However, and in contrast to our initial hypotheses, CRISPR/Cas9-mediated knockout of HIF signaling did not affect growth, internal metabolite concentrations, glucose consumption or lactate production under hypoxia, not even in vivo upon transplantation of knockout cells into xenograft mice.ConclusionThese data indicate that, while HIFs exert control over glycolysis but not OxPHOS gene expression in human leukemic cells, this is not critically important for their metabolic state. In contrast, inhibition of BCR-ABL did impact on glucose consumption and lactate production regardless of the presence of HIFs. These data indicate that oncogene-mediated control over glycolysis can occur independently of hypoxic signaling modules.

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Targeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, NMR-based metabolite quantifications and 13C-tracing, polysomal profiling, and ChIP-seq, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis and induced a cell cycle block. ROS levels were not increased following methionine depletion and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine.

Metabolic programs can differ substantially across genetically distinct subtypes of acute myeloid leukemia (AML). These programs are not static entities but can change swiftly as a consequence of extracellular changes or in response to pathway-inhibiting drugs. Here, we uncover that AML patients with FLT3 internal tandem duplications (FLT3-ITD+) are characterized by a high expression of succinate-CoA ligases and high activity of mitochondrial electron transport chain (ETC) complex II, thereby driving high mitochondrial respiration activity linked to the Krebs cycle. While inhibition of ETC complex II enhances apoptosis in FLT3-ITD+ AML, cells also quickly adapt by importing lactate from the extracellular microenvironment. 13C3-labelled lactate metabolic flux analyses reveal that AML cells use lactate as a fuel for mitochondrial respiration. Inhibition of lactate transport by blocking Monocarboxylic Acid Transporter 1 (MCT1) strongly enhances sensitivity to ETC complex II inhibition in vitro as well as in vivo. Our study highlights a metabolic adaptability of cancer cells that can be exploited therapeutically.

Activities of the retinoic acid receptor (RAR)α and RARγ are important to hematopoiesis. Here, we have investigated the effects of receptor selective agonists and antagonists on the primitive human hematopoietic cell lines KG1 and NB-4 and purified normal human hematopoietic stem cells (HSCs). Agonizing RARα (by AGN195183) was effective in driving neutrophil differentiation of NB-4 cells and this agonist synergized with a low amount (10 nM) of 1α,25-dihydroxyvitamin D3 to drive monocyte differentiation of NB-4 and KG1 cells. Treatment of cultures of human HSCs (supplemented with stem cell factor ± interleukin 3) with an antagonist of all RARs (AGN194310) or of RARα (AGN196996) prolonged the lifespan of cultures, up to 55 days, and increased the production of neutrophils and monocytes. Slowing down of cell differentiation was not observed, and instead, hematopoietic stem and progenitor cells had expanded in number. Antagonism of RARγ (by AGN205728) did not affect cultures of HSCs. Studies of CV-1 and LNCaP cells transfected with RAR expression vectors and a reporter vector revealed that RARγ and RARβ are activated by sub-nM all-trans retinoic acid (EC50–0.3 nM): ~50-fold more is required for activation of RARα (EC50–16 nM). These findings further support the notion that the balance of expression and activity of RARα and RARγ are important to hematopoietic stem and progenitor cell expansion and differentiation.

Acute myeloid leukemia remains difficult to treat due to strong genetic heterogeneity between and within individual patients. Here, we show that Pyruvate dehydrogenase kinase 1 (PDK1) acts as a targetable determinant of different metabolic states in acute myeloid leukemia (AML). PDK1low AMLs are OXPHOS-driven, are enriched for leukemic granulocyte-monocyte progenitor (L-GMP) signatures, and are associated with FLT3-ITD and NPM1cyt mutations. PDK1high AMLs however are OXPHOSlow, wild type for FLT3 and NPM1, and are enriched for stemness signatures. Metabolic states can even differ between genetically distinct subclones within individual patients. Loss of PDK1 activity releases glycolytic cells into an OXPHOS state associated with increased ROS levels resulting in enhanced apoptosis in leukemic but not in healthy stem/progenitor cells. This coincides with an enhanced dependency on glutamine uptake and reduced proliferation in vitro and in vivo in humanized xenograft mouse models. We show that human leukemias display distinct metabolic states and adaptation mechanisms that can serve as targets for treatment.