Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from bone marrow derived granulocyte monocyte precursors (GMPs) from wt -, Gfi1 knock-out -, human-Gfi1-SNP36N knock in and human wt-Gfi1 knock in mice

ABSTRACT: The incidence of cancer is not only influenced by exposure to environmental factors such as toxic substances and chemotherapy, but is also a result of individual predispositions. Single Nucleotide Polymorphisms (SNPs) represent one such individual predisposition to cancer1. Gfi136N is a SNP in the coding region of the gene “Growth factor independence 1 (Gfi1)” predisposing to development of Acute myeloid leukemia (AML). However, the mechanisms how this polymorphism is associated with AML are unknown. Here we present a mouse model in which the presence of Gfi136N leads to altered epigenetic modifications in the myeloid progenitor fraction “Granulocytic Monocytic Progenitor (GMP)” and demonstrates how this might predispose to AML. Presence of Gfi136N leads to a different epigenetic programming of the GMPs resulting in higher levels of the leukemogenic transcription factor Hoxa9. To our knowledge, this is the first mouse model of a SNP leading to epigenetic changes. Thus, SNPs not only result in an altered structure or expression level of certain proteins but here we show that SNPs might also lead to different cancer related epigenetic modifications. We used microarrays to detail the changes in the program of gene expression of GMPs induced by the knock in of human Gfi1 variants in the mouse Gfi1 locus Bone marrow derived granulocyte monocyte precursors (GMPs) from wt.-,Gfi1 knock-out.-, human Gfi1-knock in and human Gfi1-SNP36N knock in mice were collected by fluorescence activated cell sorting (FACS) for total RNA extraction and gene expression analysis on affymetrix mouse Gene 1.0 ST arrays. We aimed to identify differentially regulated target genes of the human Gfi1-SNP36N variant in a mouse model system which mit contribute to the development of acute myeloid leukemia (AML).

ORGANISM(S): Mus musculus

SUBMITTER: Tarik Moroy

PROVIDER: E-GEOD-25551 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:MDS is characterized by a disturbed function of the myeloid lineage of the hematopoietic system that may transform to AML, a malignant disease of the myeloid compartment. Epigenetic and genetic aberrations contribute to the initiation and progression of MDS/AML. GFI1 is a transcriptional repressor, which regulates expression of its target genes by, among other approaches, recruiting HDACs to its target genes to remove histone 3 lysine 9 (H3K9) acetylation, a marker for active gene expression. Low levels of GFI1 expression and deletion of one GFI1 allele contribute to MDS/AML development in human patients and are associated with a specific gene expression signature and inferior prognosis. To explore the mechanism behind this, we used a mouse strain, which expresses GFI1 only at 5-10% of the normal level (GFI1-Knock-down (KD)). Knock-down of GFI1 or loss of one murine Gfi1 allele reduced latency and increased incidence of AML in different murine models of human MDS/AML development. On the epigenetic level, KD of Gfi1 lead to increased amount of H3K9 acetylation, resulting in increased expression of genes involved in AML development. On a translational level both murine as well as human AML cells with low expression of GFI1 are resistant to standard epigenetic therapy. We show that treatment with histone acetyltransferase inhibitors might be a novel treatment approach for low Gfi1-expressing blast cells. GFI1 has a dose dependent role in myeloid malignancies and is a biomarker for therapeutic intervention. We used microarrays to detail the global programme of gene expression in bone marrow cells of Nup98HoxD13 leucemic mice and identified distinct classes of up-regulated genes during this process. Bone marrow cells of leukemic mice (from KI & KD Nup98HoxD13 mice) were collected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of leukemic cell population, so we took samples of mice which had the same age.

Project description:Transcriptional repressor Growth factor independence 1 (GFI1) is a key regulator of haematopoiesis. We previously established that the germline variant GFI1-36N promotes acute myeloid leukemia (AML) development, however the mechanism is not full elucidated. Here using multi-omics approach, we show GFI1-36N expression impedes DNA repair in leukemic cells. We demonstrate the presence of GFI1-36N is associated with increased frequency of chromosomal aberrations and mutational burden in murine and human AML cells. In particular, GFI1-36N modulates DNA repair pathways, O6-methylguanine-DNA-methyltransferase (MGMT) and homologous recombination repair (HR). Mechanistically, GFI1-36N exhibits impaired binding to Ndrg1 promoter element compared to GFI1-36S (wild type), causing decreased NDRG1 levels consequently leading to suppression of MGMT expression, imprinted at the transcriptome and proteome, thus leaving the AML cells vulnerable to DNA damaging agents. Targeting MGMT via temozolomide and HR via olaparib caused specifically extensive lethality in in vitro and ex vivo human and AML samples expressing GFI1-36N. Whereas the effects were insignificant on non-malignant GFI1-36S or GFI1-36N cells. Further, mice transplanted with GFI1-36N leukemic cells treated with combination of temozolomide and olaparib had a significantly longer AML-free survival than mice transplanted with GFI1-36S leukemic cells. In summary, we show that GFI1-36N disturbs DNA repair activity via NDRG1-MGMT axis and thus provides critical insights into novel therapeutic option for AML presented with GFI1-36N variant. Key Points Presence of GFI1-36N impedes Homologous DNA and MGMT DNA repair selectively in AML cells via the NDRG1-MGMT axis. Use of temozolomide and olaparib allows selectively targeting GFI1-36N leukemic cells. Introduction Gfi1 is a transcription factor which regulates the development of haematopoietic cells as well as neuronal and intestinal epithelial cells 1-5. We reported that a variant of GFI1, denominated GFI1-36N (characterized by an exchange of serine to asparagine at position 36), has a prevalence of 5-7% in a healthy control population but is found at an increased frequency of 10-15% among MDS and AML patients 6,7. The expression of germline variant GFI1-36N predisposes the carriers to develop de novo AML and MDS and correlates with a poor prognosis 6,7. Recently, we and other showed that malignant cells with GFI1-36N variant have increased H3K9-acetylation at target genes resulting in higher expression of genes required for cell survival and proliferation 8. GFI1 exerts its repressive role by recruiting histone-modifying enzymes (deacetylases HDAC1-3, demethylase LSD1, methyl transferase G9a) and regulates the accessibility of DNA to its target genes such as Hoxa9, Pbx1, Meis1, CSF1 and CSFR1 9-15. We also showed that GFI1 regulates apoptosis through its regulation of p53 in lymphoblastic leukemia 16 and we have demonstrated that GFI1 facilitates DNA repair 17. However, it is not known how these activities are affected in the GFI1-36N variant and whether the ability of GFI1 to regulate DNA repair pathways is maintained and how this might affect the development of myeloid malignancies. In this study, we leveraged multi-omics profiling to gain mechanistic insights into the molecular architecture that drives leukemia in the presence of GFI1-36N. We provide evidence that GFI1-36N interferes with DNA in leukemic myeloid cells, which leads to a higher frequency of genetic aberrations in MDS/AML cells. We also show that GFI1-36N myeloid leukemic cells are more sensitive to targeting MGMT and HR repair deficient cells, which opens a new selective therapeutic window to treat AML/MDS.

Project description:GFI1 is a transcriptional repressor protein that plays an essential role in HSCs development, lymphoid and myeloid differentiation and Acute Myeloid Leukaemic (AML) pathogenesis. Low expression levels of GFI1 is associated with a poor prognosis in AML development. In addition, a single nucleotide polymorphism (SNP) variant of GFI1 results in the generation of GFI1 protein with asparagine (N) instead of serine (S) at the 36th amino acid position, known as GFI136N. Expression of the GFI1-36N allele leads as well to poor prognosis and promotes AML development. In this study, we demonstrated with the help of RNAseq transcriptomic analysis that the presence of GFI1-36N is associated with increased frequency of chromosomal aberrations and mutational burden in murine and human AML cells. In particular, GFI1-36N modulates DNA repair pathways, O6-methylguanine-DNA-methyltransferase (MGMT)-mediated repair and homologous recombination repair (HR). Mechanistically, GFI1-36N exhibits impaired binding to Ndrg1 promoter element compared to GFI1-36S (wild type), causing decreased NDRG1 levels, consequently leading to suppression of MGMT expression, imprinted at the transcriptome and proteome, thus leaving the AML cells vulnerable to DNA damaging agents. Furthermore, we showed that a low expression level of GFI1 in leukemic cells is associated with high OXPHOS and enhanced glutamine metabolism. However, we hypothesise that the observed metabolic phenotype is mediated through FOXO1 protein. RNAseq transcriptomic analysis revealed higher Foxo1 mRNA expression levels with lower GFI1 expression, providing the first hint of Foxo1 as a potential target gene of GFI1 protein. The mRNA and protein levels of high Foxo1 with reduced GFI1 expression was confirmed by RT-PCR and western blot, respectively. In addition, CHIPseq and ATACseq analysis further proved that Foxo1 is a potential target gene of GFI1. In summary, we show that GFI1 plays a role during DNA repair and metabolism and thus provides critical insights into a novel therapeutic option for AML patients carrying the GFI1-36N variant or having a low expression level of GFI1.

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Expression data from bone marrow derived granulocyte monocyte precursors (GMPs) from wt -, Gfi1 knock-out -, human-Gfi1-SNP36N knock in and human wt-Gfi1 knock in mice

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