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Mitochondrial Dysfunction in Human Leukemic Stem/Progenitor Cells upon Loss of RAC2.

ABSTRACT: Leukemic stem cells (LSCs) reside within bone marrow niches that maintain their relatively quiescent state and convey resistance to conventional treatment. Many of the microenvironmental signals converge on RAC GTPases. Although it has become clear that RAC proteins fulfill important roles in the hematopoietic compartment, little has been revealed about the downstream effectors and molecular mechanisms. We observed that in BCR-ABL-transduced human hematopoietic stem/progenitor cells (HSPCs) depletion of RAC2 but not RAC1 induced a marked and immediate decrease in proliferation, progenitor frequency, cobblestone formation and replating capacity, indicative for reduced self-renewal. Cell cycle analyses showed reduced cell cycle activity in RAC2-depleted BCR-ABL leukemic cobblestones coinciding with an increased apoptosis. Moreover, a decrease in mitochondrial membrane potential was observed upon RAC2 downregulation, paralleled by severe mitochondrial ultrastructural malformations as determined by automated electron microscopy. Proteome analysis revealed that RAC2 specifically interacted with a set of mitochondrial proteins including mitochondrial transport proteins SAM50 and Metaxin 1, and interactions were confirmed in independent co-immunoprecipitation studies. Downregulation of SAM50 also impaired the proliferation and replating capacity of BCR-ABL-expressing cells, again associated with a decreased mitochondrial membrane potential. Taken together, these data suggest an important role for RAC2 in maintaining mitochondrial integrity.

SUBMITTER: Capala ME

PROVIDER: S-EPMC4446344 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Mitochondrial Dysfunction in Human Leukemic Stem/Progenitor Cells upon Loss of RAC2.

Capala Marta E ME Maat Henny H Bonardi Francesco F van den Boom Vincent V Kuipers Jeroen J Vellenga Edo E Giepmans Ben N G BN Schuringa Jan Jacob JJ

PloS one 20150527 5

Leukemic stem cells (LSCs) reside within bone marrow niches that maintain their relatively quiescent state and convey resistance to conventional treatment. Many of the microenvironmental signals converge on RAC GTPases. Although it has become clear that RAC proteins fulfill important roles in the hematopoietic compartment, little has been revealed about the downstream effectors and molecular mechanisms. We observed that in BCR-ABL-transduced human hematopoietic stem/progenitor cells (HSPCs) depl ...[more]

PMID: 26016997

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Project description:Background:Hypoxia-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. Methods:Genome-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. Results:Genome-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. Conclusion:These 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.

Dataset Information

Mitochondrial Dysfunction in Human Leukemic Stem/Progenitor Cells upon Loss of RAC2.

Publications

Mitochondrial Dysfunction in Human Leukemic Stem/Progenitor Cells upon Loss of RAC2.

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