Expression data from AMPK deficient MLL-AF9 leukemia initiating cells and whole leukemia cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Yusuke Saito
PROVIDER: E-GEOD-64602 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Deletion of AMPK significantly extended the onset of leukemogenesis and depleted leukemia initiating cells (LICs). To identify how AMPK regulates LICs, we performed gene expression profiling of LICs isolated from AMPK wild type leukemic mice or AMPK-deficient leukemic mice.
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Project description:Disrupting mutations of the RUNX1 gene are found in 10% of patients with myelodysplasia (MDS) and 30% of patients with acute myeloid leukemia (AML). Previous studies have revealed an increase in hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells in conditional Runx1-knockout (KO) mice, but the molecular mechanism is unresolved. We investigated the myeloid progenitor (MP) compartment in KO mice, arguing that disruptions at the HSC/MPP level may be amplified in downstream cells. We demonstrate that the MP compartment is increased more than fivefold in Runx1 KO mice, with a prominent skewing toward megakaryocyte (Meg) progenitors. Runx1- deficient granulocyte-macrophage progenitors are characterized by increased cloning capacity, impaired development into mature cells, and HSC and Meg transcription signatures. An HSC/MPP subpopulation expressing Meg markers was also increased in Runx1-deficient mice. Rescue experiments coupled with transcriptome analysis and Runx1 DNA-binding assays demonstrated that commitment is marked by Runx1 suppression of genes encoding adherence and motility proteins (Tek, Jam3, Plxnc1, Pcdh7, and Selp) that support HSCâMeg interactions with the BM niche. In vitro assays confirmed that enforced Tek expression in HSCs/MPPs increases Meg output. Interestingly, besides this key repressor function of Runx1 to control lineage decisions and cell numbers in progenitors, our study also revealed a critical activating function in erythroblast differentiation, in addition to its known importance in Meg and granulocyte/monocyte (G/M) maturation. Thus both repressor and activator functions of Runx1 at multiple hematopoietic stages and lineages likely contribute to the tumor suppressor activity in MDS and AML. GMP were isolated either from Runx1+/+-Tg(vav-Cre) and Runx1fl/fl-Tg(vav-Cre) mice or from mice transplanted with Runx1fl/fl-Tg(vav-Cre) progenitors engineered to express GFP with RUNX1-ERt2 or ERt2. RUNX1-ERt2 activity was induced by i.p. injection of tamoxifen on 3 consecutive days prior to GMP isolation.
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Project description:Recently, it has been demonstrated that transcriptionally active leukemia-associated fusion oncogenes alter self-renewal in and generate acute myeloid leukemia (AML) from committed progenitors, linking transformation and self-renewal pathways. AML is a heterogeneous disease, both genetically and biologically, and it is not known whether transformation is mediated by common or overlapping genetic programs downstream of multiple mutations or through the engagement of unique programs downstream of individual mutations. This distinction is important, as the demonstration of common pathways may identify common molecular targets for the treatment of AML. Here we demonstrate that the ability to alter self-renewal in vitro and in vivo is a more generalized property of leukemia-associated oncogenes. We further demonstrate that disparate leukemia-associated oncogenes initiate early common and overlapping transformation and self-renewal gene expression programs to mediate these effects. Furthermore, elements of these programs can be detected in established leukemia stem cells from an animal model and across a large cohort of patients with differing acute myeloid leukemia (AML) subtypes, where they strongly predict for disease biology. Finally, individual genes from the programs are demonstrated to partially phenocopy the leukemia-associated oncogenes and themselves alter self-renewal in committed murine progenitors and generate AML when expressed in murine bone marrow. The dataset comprises granulocyte monocyte precursors (GMP) transduced with either GFP alone or the fusion oncogenes MOZ-TIF2 or NUP98-HOXA9 or AML1-ETO, as labelled. Early gene expression changes (36 hours after transduction) were analysed between these replicate samples in two separate ways; either in a single two-class comparison of all oncogene transduced GMP with empty-vector transduced GMP or in three separate two-class comparisons comparing each oncogene individually with empty-vector transduced GMP. In the first analysis we identified 1082 genes/1119 probesets whose expression levels significantly differed (FDR level 0.05). There is a loss of self-renewal potential in the transition from the LSK to GMP population and to further enrich for potential self-renewal genes we compared the expression of these genes/probes in sorted normal LSK versus GMP populations. Genes which were not significantly (p<0.05) and similarly differentially expressed between LSK and GMP were excluded, allowing us to prioritize an immediate “leukemia initiation” program of 167 genes (182 probesets). Our second analysis demonstrated differential expression of 5750, 1161, and 5109 genes, when MOZ-TIF2, NUP98-HOXA9 and AML1-ETO transduced GMP were respectively compared with empty-vector transduced GMP (p<0.05). When our “leukemia initiation” signature was compared to the single comparisons, 127/167 (76%) genes overlapped. To assess the evolutionary nature of transcriptional programs critical for leukemia induction and maintenance, the gene expression profiles of replicate functionally validated leukemic stem cells from a MOZ-TIF2 model of leukemia (L-GMP) were then compared to their normal phenotypic counterpart (normal GMP). 2715 genes were differentially expressed between normal and leukemic GMP (p<0.05). Similarities were seen between our immediate pre-leukemic signatures and the gene expression profiles in the overt leukemias. The overlap with the leukemia initiation and leukemia self-renewal signatures was highly significant at 59/167 genes (35%) and 29/91 genes (32%), respectively (both p<0.0001). Gene expression levels were measured using Affymetrix (Santa Clara, CA) GeneChip Mouse Genome 430A 2.0 arrays (22,690 probesets) with hybridisation and washes as per the manufacturers specifications. The starting point for all analyses was the ''.CEL'' files from the MAS5 software. Data was analyzed using the R statistical package bioconductor18. Data quality was assessed using functions in the affy and affyPLM packages and outlier arrays were removed from subsequent analysis. The GCRMA algorithm (ver. 2.4.1) was used to obtain normalized expression estimates. Genes were selected for further analysis on the following basis: genes that had probesets for which the expression value was greater than 60 (which in our study constitute the average background reading of all probesets) and that had a present flag call in at least two of three samples. To detect significant changes in the expression levels, two-sample Welch t-tests (parametric; assuming unequal variances; Benjamini and Hochberg step-up multiple testing correction at a False Discovery Rate <0.05) was applied to the resulting genes.
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Project description:Leukemia initiating cells (LICs) of acute myeloid leukemia (AML) may arise from self-renewing hematopoietic stem cells (HSCs) and from committed progenitors. However, it remains unclear how leukemia-associated oncogenes instruct LIC formation from cells of different origins and if differentiation along the normal hematopoietic hierarchy is involved. Here, using murine models with the driver mutations MLL-AF9 or MOZ-TIF2, we found that regardless of the transformed cell types, myelomonocytic differentiation to the granulocyte macrophage progenitor (GMP) stage is critical for LIC generation. Blocking myeloid differentiation through disrupting the lineage-restricted transcription factor C/EBPa eliminates GMPs, blocks normal granulopoiesis, and prevents AML development. In contrast, restoring myeloid differentiation through inflammatory cytokines “rescues” AML transformation. Our findings identify myeloid differentiation as a critical step in LIC formation and AML development, thus guiding new therapeutic approaches. Primary KSL, CMP, and GMP cells from wildtype controls and C/Ebpa knockouts were used for RNA extraction and hybridization on Affymetrix microarrays. We also compared the microarray samples of leukemic granulocyte macrophage progenitor compartments (L-GMPs) from MLL-AF9 transformed control or cytokine rescued C/EBPa KO leukemic mouse bone marrow and their secondary recipients with those non-Leukemia KSLs and CMPs from MLL-AF9 transduecd KO recipients with no leukemia development.
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Project description:How cancer cells adapt to metabolically adverse conditions in patients and strive to proliferate is a fundamental question in cancer biology. Here we show that AMP-activated protein kinase (AMPK), a metabolic checkpoint kinase, confers metabolic stress resistance to leukemia-initiating cells (LICs) and promotes leukemogenesis. Upon dietary restriction, MLL-AF9-induced murine acute myeloid leukemia (AML) activated AMPK and maintained leukemogenic potential. AMPK deletion significantly delayed leukemogenesis and depleted LICs by reducing the expression of glucose transporter 1 (Glut1), compromising glucose flux, and increasing oxidative stress and DNA damage. LICs were particularly dependent on AMPK to suppress oxidative stress in the hypoglycemic bone marrow environment. Strikingly, AMPK inhibition synergized with physiological metabolic stress caused by dietary restriction and profoundly suppressed leukemogenesis. Our results indicate that AMPK protects LICs from metabolic stress and that combining AMPK inhibition with physiological metabolic stress potently suppresses AML by inducing oxidative stress and DNA damage. Research is published: http://www.sciencedirect.com/science/article/pii/S1934590915003744
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