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Enhancers of Polycomb 1 & 2 in normal and leukaemic haematopoiesis

ABSTRACT: A knockdown screen of chromatin regulatory genes in human THP1 MLL-AF9 AML cells identified EPC1 and EPC2 as required for cell survival. Normal myeloid progenitor cells did not require EPC1 or EPC2 for their function. To determine the transcriptional consequences of EPC knockdown, normal murine granulocyte-macrophage progenitor (GMP) cells, or murine MLL-AF9 AML GMP-like cells, were FACS purified from mice and infected with lentiviral vectors targeting EPC1 or EPC2 for knockdown, or a non-tageting control. 42 hours later knockdown or control cells were FACS purified and exon array analysis was performed on the sorted populations. For normal GMP cells, three biological replicates (three separate animals) each of control cells, EPC1 knockdown cells and EPC2 knockdown cells; for MLL-AF9 AML GMP-like cells cells, three biological replicates (three separate leukaemias) each of control cells, EPC1 knockdown cells and EPC2 knockdown cells.

ORGANISM(S): Mus musculus

SUBMITTER: Tim Somervaille

PROVIDER: E-GEOD-39172 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Enhancers of Polycomb EPC1 and EPC2 sustain the oncogenic potential of MLL leukemia stem cells.

Huang X X Spencer G J GJ Lynch J T JT Ciceri F F Somerville T D D TD Somervaille T C P TC

Leukemia 20131029 5

Through a targeted knockdown (KD) screen of chromatin regulatory genes, we identified the EP400 complex components EPC1 and EPC2 as critical oncogenic cofactors in acute myeloid leukemia (AML). EPC1 and EPC2 were required for the clonogenic potential of human AML cells of multiple molecular subtypes. Focusing on MLL-mutated AML as an exemplar, Epc1 or Epc2 KD-induced apoptosis of murine MLL-AF9 AML cells and abolished leukemia stem cell potential. By contrast, normal hematopoietic stem and proge ...[more]

PMID: 24166297

Similar Datasets

Enhancers of Polycomb 1 & 2 in normal and leukaemic haematopoiesis

Project description:A knockdown screen of chromatin regulatory genes in human THP1 MLL-AF9 AML cells identified EPC1 and EPC2 as required for cell survival. Normal myeloid progenitor cells did not require EPC1 or EPC2 for their function. To determine the transcriptional consequences of EPC knockdown, normal murine granulocyte-macrophage progenitor (GMP) cells, or murine MLL-AF9 AML GMP-like cells, were FACS purified from mice and infected with lentiviral vectors targeting EPC1 or EPC2 for knockdown, or a non-tageting control. 42 hours later knockdown or control cells were FACS purified and exon array analysis was performed on the sorted populations.

2013-10-30 | GSE39172 | GEO

Epigenetic profiling of hematopoietic stem cells and leukemia stem cells

Project description:The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations that involve the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in a murine MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3 and H3K36me3. Histone methylation patterns are highly abnormal on MLL-AF9 fusion target loci, defining a distinct epigenetic lesion involving H3K79. Conditional inactivation of Dot1l leads to specific down-regulation of direct MLL-AF9 targets and an MLL-translocation associated gene expression signature, while global transcription levels remain largely unaffected. This correlated with a greater sensitivity of leukemic blasts towards loss of Dot1l compared to normal hematopoietic cells. Development of in vivo leukemia was absolutely dependent on Dot1l. Chromatin immunoprecipitation followed by Solexa sequencing for H3K4me3, H3K27me3, H3K36me3, H3K79me2 and biotinylated MLL-AF9 in HSC, GMP and LSC.

2011-09-13 | E-GEOD-29130 | biostudies-arrayexpress

The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukemia stem cells (expression data)

Project description:Using a mouse model of human MLL-AF9 leukemia, we identified the lysine-specific demethylase KDM1A (LSD1 or AOF2) as an essential regulator of leukemia stem cell (LSC) potential. KDM1A acts at genomic loci bound by MLL-AF9 to sustain expression of the associated oncogenic program, thus preventing differentiation and apoptosis. In vitro and in vivo pharmacologic targeting of KDM1A using tranylcypromine analogues active in the nanomolar range phenocopied Kdm1a knockdown in both murine and primary human AML cells exhibiting MLL translocations. By contrast, the clonogenic and repopulating potential of normal hematopoietic stem and progenitor cells was spared. Our data establish KDM1A as a key effector of the differentiation block in MLL leukemia which may be selectively targeted to therapeutic effect. To investigate the genetic program regulated by KDM1A in murine MLL-AF9 AML cells enriched for LSCs, we compared the transcriptome of KIT+Gr1+ control and Kdm1a KD cells using exon arrays soon after initiation of KD. At this time, there was no immunophenotypic evidence of differentiation in Kdm1a KD cells. Exonic expression values were condensed into gene-level expression summaries, which represent the mean expression value of the probe sets targeting the length of a given protein coding gene. Three separate samples from two different animals were used. Kdm1a KD samples were compared with control samples. Total six arrays.

2012-03-28 | E-GEOD-36347 | biostudies-arrayexpress

Cell of origin determines clinically relevant subtypes of MLL-rearranged AML

Project description:MLL-fusion proteins can induce acute myeloid leukemias (AML) from either hematopoietic stem cells (HSC) or granulocyte macrophage progenitors (GMP), but it remains unclear if the cell of origin influences the biology of the resultant leukemia. MLL-AF9 transduced single HSC or GMP could be continuously replated, but HSC-derived clones were more likely than GMP-derived clones to initiate AML in mice. Leukemia stem cells derived from either HSC or GMP had a similar immunophenotype consistent with a maturing myeloid cell (LGMP). Gene expression analyses demonstrated that LGMP inherited gene expression programs from the cell of origin including high-level Evi-1 expression in HSC derived LGMP. The gene expression signature of LGMP derived from HSC was enriched in poor prognosis human MLL-rearranged AML in three independent data sets. Moreover, global 5’-mC levels were elevated in HSC-derived leukemias as compared to GMP-derived leukemias. This mirrored a difference seen in 5-mC between MLL-rearranged human leukemias that are either EVI1-positive or EVI1-negative. Finally, HSC derived leukemias were more resistant to chemotherapy than GMP-derived leukemias. These data demonstrate that the cell of origin influences the gene expression profile, the epigenetic state, and the drug response in AML, and that these differences can account for clinical heterogeneity within a molecularly defined group of leukemias. Differential DNA methylation between of LSC isolated from murine HSC and GMP derived AMLs

2013-01-09 | E-GEOD-43333 | biostudies-arrayexpress

RNA sequencing expression analysis of murine Tet-off MLL-AF9;NRAS acute myeloid leukemia cells over-expressing Id2 and upon MLL-AF9 withdrawal

Project description:We investigated the role of the transcriptional regulator Id2 in the context of MLL-rearranged acute myeloid leukemia (AML). Using an AML mouse model driven by tet-regulated MLL-AF9 co-expressed with oncogenic NRASG12D (Tet-off MLL-AF9), we demonstrated that MLL-AF9 regulates the E protein pathway by suppressing Id2, while activating the expression of its target E2-2. Moreover, we found that Id2 over-expression in Tet-Off MLL-AF9 AML cells in vitro partially phenocopies MLL-AF9 depletion and results inhibition of leukemia growth, loss of leukemia stem cell-associated gene expression pattern and induction of differentiation. To compare gene expression changes associated with enforced Id2 expression and MLL-AF9 withdrawal, RNA sequencing analysis was performed on Tet-off MLL-AF9 cells transduced with an Id2 over-expressing or a control vector, or upon MLL-AF9 dox-inducible knock-down. Primary AMLs driven by Tet-off inducible MLL/AF9 expression linked to dsRED reporter, in association with oncogenic NRASG12D (Tet-off MLL-AF9) were generated by reconstituting lethally irradiated congenic mice with foetal liver cells co-transduced with a Tet-Off-MLL-AF9-dRED retroviral vector and a second vector co-expressing NRASG12D together with the Tet-Off responsive transcriptional activator. RNA sequencing analysis sequencing analysis was performed on Tet-Off MLL-AF9/dsRED+ AML cells treated in vitro with doxycycline (DOX) for 4 days to inactivate MLL-AF9 expression or left untreated (UT). For the Id2 over-expression experiment, Tet-Off MLL-AF9/dsRED+ AML cells were transduced in vitro with an Id2-GFP or a control-GFP retroviral vector. Viable GFP-positive cells were FACS-sorted 2 days after transduction and used for RNA sequencing analysis.

2016-06-29 | E-GEOD-68462 | biostudies-arrayexpress

Hematopoietic cell differentiation is required for initiation of acute myeloid leukemia [Microarray expression]

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.

2015-08-04 | E-GEOD-71687 | biostudies-arrayexpress

Hematopoietic cell differentiation is required for initiation of acute myeloid leukemia

2014-09-17 | E-GEOD-61468 | biostudies-arrayexpress

RNA sequencing expression analysis of murine MLL-AF9;NRAS acute myeloid leukemia cells silenced for E2-2.

Project description:We investigated the role of the transcriptional regulators Id2 and E2-2 (encoded by Tcf4) in the context of MLL-rearranged acute myeloid leukemia (AML). Using an AML mouse model driven by a Tet-off inducible MLL-AF9 allele co-expressed with oncogenic NRASG12D, we demonstrated that MLL-AF9 regulates the E protein pathway by suppressing Id2, while activating the expression of its target E2-2. Moreover, we found that Id2 over-expression in MLL-AF9 AML cells results inhibition of leukemia growth, loss of leukemia stem cell-associated gene expression pattern and induction of differentiation. E2-2 silencing phenocopies Id2 overexpression in MLL-AF9-AML cells. To study the gene expression changes associated with E2-2 depletion in the context of MLL-rearranged AML, RNA sequencing analysis was performed on MLL-AF9;NRAS AML cells transduced with vectors expressing hairpins against E2-2 (shTcf4#654 and shTcf4#3646) or a control hairpin against Renilla luciferase (shRen). Primary AMLs driven by MLL/AF9 expression linked to cherry reporter, in association with oncogenic NRASG12D (MLL/AF9;NRAS) were generated by reconstituting lethally irradiated congenic mice with fetal liver cells co-transduced with the MSCV-MLL/AF9-IRES-cherry retroviral vector and a second vector co-expressing NRASG12D together with luciferase (MSCV-luciferase-IRES-NRASG12D). RNA sequencing analysis sequencing analysis was performed on MLL-AF9;NRAS AML cells transduced in vitro with vectors expressing hairpins against E2-2 (shTcf4#654 and shTcf4#3646) or a control hairpin against Renilla luciferase (shRen) linked to the reporter GFP. Viable GFP-positive cells were FACS-sorted 2 days after transduction and used for RNA sequencing analysis. Two independent biological replicates of the experiment were used for the RNA sequencing (9-5-14 and 14-4-14).

2016-06-29 | E-GEOD-80745 | biostudies-arrayexpress

RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes

Project description:The translocation t(9;11)(p22;q23) leading to the leukemogenic fusion gene MLL-AF9 is a frequent translocation in infant acute myeloid leukemia (AML). This study aimed to identify genes and molecular processes downstream of MLL-AF9 (alias MLL-MLLT3) which could assist to develop new targeted therapies for such leukemia with unfavorable prognosis. In the AML cell line THP1 which harbors this t(9;11) translocation, endogenous MLL-AF9 was silenced via siRNA while ensuring specificity of the knockdown and its efficiency on functional protein level. The differential gene expression profile was validated for leukemia-association by gene set enrichment analysis of published gene sets from patient studies and MLL-AF9 overexpression studies and revealed 425 differentially expressed genes. Gene ontology analysis was consistent with a more differentiated state of MLL-AF9 depleted cells, with involvement of a wide range of downstream transcriptional regulators and with defined functional processes such as ribosomal biogenesis, chaperone binding, calcium homeostasis and estrogen response. Besides potential new therapeutic targets, the described transcription profile shaped by MLL-AF9 provides an information source into the molecular processes altered in MLL aberrant leukemia. 4 samples were analyzed, each corresponding to a pool of five independent replicate experiments. MLL-AF9 knockdown treatments are represented by 2 pooled samples employing two distinct siRNAs, each targeting MLL-AF9 breakpoint of THP1 cells. Control treatments are represented by 2 pooled samples employing two distinct non-targeting control siRNAs. siRNA-3 = siRNA-A in publication siRNA-4 = siRNA-B in publication

2014-02-13 | E-GEOD-36592 | biostudies-arrayexpress

Conditional modeling of MLL-AF9 induced leukemia revealed a stem cell derived aggressive disease [Microarray]

Project description:To address the impact of cellular origin on AML, we generated an inducible transgenic mouse model for MLL-AF9 driven leukemia. MLL-AF9 expression in long-term hematopoietic stem cells (LT-HSCs) in vitro resulted in unprecedented clonogenic growth and expression of genes involved in migration and invasion. In vivo, some LT-HSC-derived AMLs were particularly aggressive with extensive tissue infiltration, chemo-resistance and expression of genes related to epithelial-mesenchymal transition (EMT) in solid cancers. Knockdown of the EMT regulators Zeb1 and Tcf4 significantly reduced leukemic blast invasion. By classifying mouse and human leukemia according to Evi1/EVI1and Erg/ERG expression, reflecting aggressiveness and cell-of-origin and performing comparative transcriptomics we identified numerous EMT-related genes that were significantly associated with poor overall survival of AML patients. RNA from primary cells as well as doxycyclin treated cells from immortalized LT-HSC and GMP (Granulocyte macrophage progenitor) celllines was isolated for expression profiling on Affymetrix gene arrays.

2016-06-27 | E-GEOD-65382 | biostudies-arrayexpress

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