Id2 and E proteins orchestrate the initiation and maintenance of MLL-rearranged acute myeloid leukemia
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Margherita Ghisi
PROVIDER: E-GEOD-68463 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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
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
Project description:We investigated the role of the transcriptional regulator Id2 in the context of MLL-rearranged acute myeloid leukemia (AML). Using an Id2/GFP-reporter mouse model of MLL-AF9-driven AML, we showed that Id2 is expressed heterogeneously across the leukemic population. Moreover, differential expression of Id2 and the stemness marker Kit defines subsets of AML cells with different leukemogenic properties with lower levels of Id2 associated with enrichment in leukemia stem cell potential. To define gene expression patterns associated with distinct endogenous levels of Id2 and higher LSC potential, RNA sequencing analysis was performed on FACS-sorted KitHIâId2HI (BM_Kplus-Iplus), KitHIâId2LOW(BM_Kplus-Iminus), KitLOWâId2HI (BM_Kminus-Iplus) and KitLOWâId2LOW (BM_Kminus-Iminus) MLL-AF9-cherry+ AML cells obtained from bone marrow of terminally sick mice. Primary MLL-AF9+ AMLs were generated by reconstituting lethally irradiated congenic mice with foetal liver cells obtained from Id2-GFP reporter mice and transduced with a retroviral vector co-expressing MLL-AF9 and the cherry reporter protein. KitHighâId2High, KitHighâId2Low, KitLowâId2High and KitLowâId2Low MLL-AF9/cherry+ AML cells obtained from bone marrow of terminally sick primary recipients were FACS-sorted and used for RNA sequencing analysis (3 samples/subset).
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Project description:MLL-fusions may induce leukemogenic gene expression programs by recruiting the histone H3K79 methyltransferase to MLL-target promoters. We evaluated gene expression changes after cre-mediated loss of Dot1l in leukemia cells obtained from mice injected with MLL-9 transformed lineage negative bone marrow cells. MLL-AF9 murine leukemia cells carrying two conditional Dot1l alleles were retrovirally transduced with Cre or empty control vector, and gene expression changes were monitored on day 3, 5, and 7 after transduction.
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Project description:Next generation DNA sequencing of acute myeloid leukemia (AML) patient samples has revealed novel recurrent mutations while at the same time highlighting the genetic heterogeneity of the disease. These observations suggest that an extraordinarily large number of combinations of mutations can contribute to leukemogenesis. In order to address the question of the contribution of patient genetic background to AML we have developed a model system to generate multiple human leukemias in a single donorâs genetic background. Stepwise RNA-seq data from this model shows that in the context of AML driven by the MLL-AF9 (MA9) oncogene, the genetic background of the donor does not have a detectable effect. Comparison of these model leukemias from multiple single donors to AML patient samples containing MA9 translocations revealed conserved gene expression patterns not previously highlighted in this genetic sub-type. We further demonstrate that the expression of one of these genes, RET, is essential both in vivo and in vitro growth of MA9 AMLs . study of methylome during the development of MLL-AF9 AML
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Project description:miR-101 has been reported as a tumor suppressor in several types of cancer. However, its role in AML is still unknown. In this study, we restored the expression of miR-101 in MLL-AF9 transduced LSK cells by introducing a retroviral miR-101 expression vector. To identify miR-101-regulated gene expression, we performed genome-wide gene expression analysis on GFP+ MLL-AF9-transduced LSK cells overexpressing miR-101 versus control vector. Our gene expression analysis and subsequent functional studies demonstrate that enforced expression of miR-101 has antitumor effects on the development of MLL-rearranged AML. Each group has 3 samples.
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Project description:HOXA9 and MEIS1 are essential downstream effectors of the MLL-AF9 oncoprotein during leukemia induction. Leukemia derived from MLL-AF9-transduced LSK cells has a more aggressive phenotype than that derived from HOXA9/MEIS1-transduced LSK cells. To determine differential miRNA expression that contributes to increased aggressiveness in MLL-AF9-induced leukemia, miRCURY LNA microRNA Array was performed on LSK cells transduced with MLL-AF9 versus HOXA/MEIS1 oncogenes.
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Project description:HOXA9 and MEIS1 are essential downstream effectors of the MLL-AF9 oncoprotein during leukaemia induction. Leukaemia derived from MLL-AF9-transduced LSK cells has a more aggressive phenotype than that derived from HOXA9/MEIS1-transduced LSK cells. To determine differential gene expression that contributes to increased aggressiveness in MLL-AF9-induced leukaemia, microarray was performed on LSK cells transduced with MLL-AF9 versus HOXA/MEIS1 oncogenes.
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2015-08-04 | E-GEOD-71691 | biostudies-arrayexpress
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.
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