Notch Pathway Activation Targets AML Cell Homeostasis and Differentiation: MLL-AF9 transformed LGMP
ABSTRACT: To determine role of Notch signaling in AML leukemia initiating cells we used a conditional mouse knock-in model of Notch1-IC to induce Notch1-IC expression in MLL-AF9 transformed LGMP. WT and Notch1-IC+ LGMP were analyzed to determined genes controlled by Notch signaling. 12 weeks old wt lethaly irradiated mice were transplanted with 50000 cKit+ MLL-AF9-IRES-YFP infected cells from MLL-AF9 EF1 wt/wt ROSAwt/CreERT2 or MLL-AF9 EF1 wt/lsl-N1-IC ROSAwt/CreERT2 mice + 250000 support wt total bone marrow cells. 4 weeks after transplant mice were injected 2 times with tamoxifen (0.2mg/g body weight) every other day. Mice were sacrificed and analyzed 6 days after last injection. LGMP were flow purified for RNA extraction and hybridization on Affymetrix microarrays.
Project description:To identify such targets of leukemia-related miRNAs such as miR-196b, we conducted Affymetrix gene arrays of leukemic BM samples from 24 mice including 9 primary (including 3 each of negative control, MLL-AF9, and miR-196b+MLL-AF9) and 15 secondary (including 3 negative control, 6 MLL-AF9, and 6 miR-196b+MLL-AF9) recipient mice A total of 24 mouse bone marrow samples including 9 primary (including 3 each of negative control, MLL-AF9, and miR-196b+MLL-AF9) and 15 secondary (including 3 negative control, 6 MLL-AF9, and 6 miR-196b+MLL-AF9) obtained from the in vivo mouse bone marrow reconstitution assays were analyzed by use of Affymetrix GeneChip Mouse Gene 1.0 ST Array (Affymetirx, Santa Clara, CA)
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.
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).
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 KitHI–Id2HI (BM_Kplus-Iplus), KitHI–Id2LOW(BM_Kplus-Iminus), KitLOW–Id2HI (BM_Kminus-Iplus) and KitLOW–Id2LOW (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. KitHigh–Id2High, KitHigh–Id2Low, KitLow–Id2High and KitLow–Id2Low 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).
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.
Project description:The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in MLL-AF9 AML. We evaluated the dependance on beta-catenin for KrasG12DMLL-AF9 leukemia. Lin-Kit+ bone marrow cells obtained from mice transplanted with primary MLL-AF9 leukemia cells and KRasG12DMLL-AF9 leukemia cells were assessed for gene expression in the presence or absence of beta-catenin
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.
Project description:Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific function of these pathways in AML is unclear. To elucidate the downstream functions of activated NRAS in AML, we employed a murine model of AML harboring Mll-AF9 and NRASG12V. We found that NRASG12V enforced leukemia self-renewal gene expression signatures and was required to maintain an MLL-AF9 and MYB-dependent gene expression program. In a multiplexed analysis of RAS-dependent signaling intermediates, the leukemia stem cell compartment was preferentially sensitive to RAS withdrawal. Use of RAS-pathway inhibitors showed that NRASG12V maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Mice harboring NRASG12V/Mll-AF9 AML were treated with doxycyline to abolish NRASG12V expression. Leukemia samples were harvested at 24 hour intervals after doxycyline treatment.
Project description:We studied the chromatin modification patterns induced by the presence of the MLL-AF9 fusion protein in a model of human hematopoietic stem/progenitor cells (HSPC) transduced with retrovirus expressing MLL-AF9cDNA (HSPC-MA9). Comparative ChIP-seq analysis between HSPC-MA9 and control HSPC, revealed a massive hyperacetylation of histones that was consistent with the transcriptional profile in the presence of MLL-AF9 fusion protein. Furthermore, we identified 66 MLL-AF9 targets, and found that H4ac was present along with H3K4me3 and H3K79me2 chromatin marks in over 50% of the MLL-AF9 target genes. Examination of histone aceylation and methylation changes upon expression of MLL-AF9 fusion protein in human hematopoietic stem/progenitor cells.