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Gene expression analysis of Thy1+LSK bone marrow cells from Nol3+/+ and Nol3-/- MPN mice

ABSTRACT: Apoptosis Repressor with Caspase Recruitment Domain (ARC) was recently found to be highly expressed in solid tumors and in blast cells of patients with acute myeloid leukemia (AML). In this study, we assessed the functional and molecular consequences of loss of ARC on the hematopoietic system. We found, unexpectedly, that deletion of Nol3 (the gene that encodes ARC) in mice leads to development of a myeloproliferative neoplasm (MPN) resembling primary myelofibrosis (PMF). Nol3-/--induced MPN is transplantable into secondary recipient mice and is characterized by anemia, thrombocytopenia, extramedullary hematopoiesis, and bone marrow fibrosis. Despite an overall reduction in bone marrow cellularity Nol3-/- MPN mice have an expanded Thy1+LSK stem cell population with increased cell cycling, in addition to a myelomonocytic differentiation bias. Molecularly, the Nol3-/--induced phenotype is, at least in part, mediated by activation of cyclin-dependent kinases 4 (CDK4) and 6 (CDK6) and MYC. Nol3-/- MPN Thy1+LSK cells show significant molecular similarities with primary CD34+ cells isolated from patients with PMF. Furthermore, we found that Nol3 is deleted in patients with human myeloid malignancies and has decreased expression in a subset of patients with de novo AML. Our results reveal a novel role of Nol3 in normal hematopoiesis and in the pathogenesis of myeloid malignancies. To obtain insight into the molecular mechanism of the Nol3-/- -induced MPN, we performed gene expression profiling on Thy1+LSK bone marrow cells from Nol3+/+ and Nol3-/- MPN mice

ORGANISM(S): Mus musculus

PROVIDER: GSE76121 | GEO | 2017/02/17

SECONDARY ACCESSION(S): PRJNA306357

REPOSITORIES: GEO

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Project description:Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). Beginning in early 2005, a number of novel mutations involving JAK2, CALR, MPL, TET2, ASXL1, DNMT3a, CBL, IDH1 and IDH2 have been described in MPNs, depicting a really complex genomic landscape for MPNs. To shed light on the genomic lesions that can contribute to disease phenotype and/or development, we integrated gene expression and copy number signals and identified several genomic abnormalities leading to a concordant alteration in gene expression levels. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus is accompanied by a coordinated transcriptional up-regulation in PMF patients. Inhibition of PAOX resulted in rapid cell death in PMF progenitor cells, but not in normal cells, suggesting that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells’ survival. Moreover, copy number loss in the chromatin modifier HMGXB4 gene correlates with a concomitant transcriptional down-regulation in PMF patients. Interestingly, silencing of HMGXB4 induces megakaryocyte differentiation, while inhibiting erythroid development, in human hematopoietic stem/progenitor cells. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system suggesting that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterize PMF patients. In this study, the authors take advantage of the integrative analysis of gene expression and copy number data to identify the concordant gain/up-regulation of the polyamine oxidase PAOX and the loss/down-regulation of the chromatin modifier HMGXB4. This work sheds light on the influence of genomic abnormalities on gene expression regulation in PMF CD34+ cells and on their contribution to specific features of PMF, such as a hyperplastic megakaryopoiesis.

Project description:TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukaemia (CMML), acute myeloid leukaemias (AML) and secondary AML (sAML). We show here that TET2 mutations associated with myeloid malignancies compromise catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5hmC versus healthy controls, but samples from patients with low 5hmC showed hypomethylation relative to controls at the majority of differentially methylated CpG sites. Our results demonstrate that Tet2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anticancer drugs. Genome wide DNA methylation profiling of patients samples with various myeloid malignancies and diffrential levels of 5hmC.The Illumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs in bone marrow samples and occasionally peripheral blood samples. Samples included 28 control healthy bone marrows, 29 patients samples with low 5hmC levels (7 patients with wild-type TET2 and 22 mutant TET2) and 24 with high levels of 5hmC (22 with wild-type TET2 and 2 mutant TET2). Bisulphite converted DNA from 81 samples was hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2

Project description:Ph-negative myeloproliferative neoplasms (MPNs) are characterized by many somatic mutations which have already been shown useful in the prognostic assessment of MPN patients. Moreover, aberrant microRNA (miRNA) expression seems to add to the molecular complexity of MPNs, as specific miRNA signatures capable of discriminating MPN cells from those of normal donors were previously reported. In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in primary myelofibrosis (PMF) cells, we obtained gene- (GEP) and miRNA expression profiles (miEP) of CD34+ cells from 31 healthy donors and 42 PMF patients using Affymetrix technology (HG-U219 and miRNA 2.0 arrays). Differentially expressed genes (DEG) and miRNAs (DEM) were sorted out by means of Partek Genomic Suite vs 6.6. Since each miRNA can target many mRNAs while a single mRNA can be targeted by multiple miRNAs, we performed Integrative Analysis (IA) by means of Ingenuity Pathway Analysis (IPA) to untangle this combinatorial complexity. In particular, IPA points out DEM-DEG pairs among experimentally validated interactions from TarBase, miRecords and Ingenuity Expert Findings as well as predicted microRNA-mRNA interactions from TargetScan. IPA microRNA Target Filter was then employed to select only the DEM-DEG pairs showing an anti-correlated expression pattern and to build regulatory networks. Finally, 3'UTR luciferase reporter assays were performed to validate IPA predicted miRNA-mRNA interactions. This study allowed the identification of different networks possibly involved in PMF onset and progression, highlighting an aberrant cross-regulation in miRNA-targets involved in malignant hematopoiesis. Furthermore, Integrative analysis was proved a powerful tool to unravel miRNA-mRNA interactions in functional networks, shedding light on the potential contribution of miRNAs to PMF pathogenesis. Gene expression profile (GEP) and miRNA expression profile (miEP) were performed starting from the same totalRNA of CD34+ cells from 42 PMF patients and 31 healthy donors (n=16 PB CD34+, n=15 BM CD34+) (1 replicate for each sample). In particular, GEP and miEP were performed on 23 PMF patients carrying the mutation JAK2V617F and 19 wild-type samples.

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Gene expression analysis of Thy1+LSK bone marrow cells from Nol3+/+ and Nol3-/- MPN mice

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