ABSTRACT: Next Generation Sequencing Facilitates Quantitative Analysis of Bone Marrow Macrophages and Splenic Macrophages Transcriptomes in Mouse MLL-AF9 AML Leukemia
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).
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:Leptin is enriched in bone marrow adipose tissue, but its impact on therapeutic response in acute myeloid leukemia (AML) remains incompletely elucidated. Here, we report that in MLL-AF9 mouse model, exogenous leptin significantly compromised the cytotoxicity of Ara-C, whereas a high-affinity leptin receptor antagonist, Allo-aca, restored chemosensitivity without altering basal leukemia growth, accompanied by marked alterations in mitochondria-associated proteins.
Project description:In leukemogenesis Notch signaling can be up- and down-regulated in a context-dependent manner. Here we report that deletion of hairy and enhancer of split-1 (Hes1) promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. Subsequently, the FMS-like tyrosine kinase 3 (FLT3) was up-regulated in mouse cells of a Hes1- or RBP-J-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development by down-regulating FLT3 expression. 4 samples are analyzed, two pairs of MLL-AF9/Hes1-/- and MLL-AF9/Hes1+/+ leukemic bone marrows.
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: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:Investigation of the transcriptional profile of MLL-rearranged AML in response to DHODH inhibition by AG636. Chemo-refractory syngeneic murine AML model driven by doxycycline-inducible expression of MLL-AF9 and constitutive expression of oncogenic Nras (MN) was transplanted into Ptprca recipients. Mice bearing MN tumors were treated with doxycycline or AG636 for one or four days. Leukemic stem cells (cKit high; CD11b low) from bone marrow and spleen were isolated and RNA sequencing performed.
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.
Project description:Through targeted metabolomics analysis of bulk leukemia cells and leukemia stem cells (LSCs) derived from the MLL-AF9-driven acute myeloid leukemia (AML) model, in comparison with normal granulocyte-monocyte progenitor (GMP) cells and whole bone marrow (WBM) cells from normal mice, we identified enhanced purine metabolism in AML LSCs. Pharmacological targeting of the purine metabolism using mycophenolate mofetil (MMF) resulted in decreased levels of purine metabolites crucial for nucleolus rRNA synthesis. Importantly, inhibition of purine metabolism or disruption of the nucleolus rRNA synthesis, achieved by inhibiting pol I activity with CX-5461, triggered myeloid differentiation in the MLL-AF9-driven AML LSCs. These findings unveil a regulatory axis involving purine metabolism and nucleolar rRNA synthesis in maintaining AML LSC activity.
Project description:To explore oncogene addiction programs in a genetically defined leukemia context we developed an AML mouse model driven by a conditional MLL-AF9 allele together with oncogenic Ras, which enabled us to examine the consequences of MLL-AF9 inhibition in established disease. In order to produce a tightly regulated system that was easy to monitor, we constructed two retroviral vectors containing dsRed-linked MLL-AF9 under control of a tetracycline response element promoter, and KrasG12D or NrasG12D linked to the “Tet-off” tet-transactivator, which activates TRE expression in a doxycycline repressible manner. Leukemias were generated by retroviral cotransduction of both vectors into hematopoietic stem and progenitor cells, which were transplanted into syngeneic mice. Cells harboring both constructs induced aggressive myelomonocytic leukemia. Five independent primary leukemia cell lines were established from bone marrow of terminal mice. Treatment of these lines with doxycycline rapidly turned off MLL-AF9 expression, and induced terminal myeloid differentiation and complete disease remission in vivo. To identify molecular mechanisms underlying addiction to MLL-AF9, we analyzed global gene expression changes following doxycycline-induced suppression of MLL-AF9.