Project description:Blood samples from patients with myeloid malignancies were analyzed using whole exome sequencing (WES). Data set from genotyping by microarray of the same samples has been deposited in ArrayExpress under accession number E-MTAB-1845 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1845/).
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:For many years, immortalized cell lines have been used as model systems for cancer research. Cell line panels were established for basic research and drug development, but did not cover the full spectrum of leukemia and lymphoma. Therefore, we now developed a novel panel (LL-100), 100 cell lines covering 22 entities of human leukemia and lymphoma including T-cell, B-cell and myeloid malignancies. Importantly, all cell lines are unequivocally authenticated and assigned to the correct tissue. Cell line samples were proven to be free of mycoplasma and virus contamination. Whole exome sequencing (WES) and RNA sequencing (RNA-seq) of the hundred authenticated leukemia-lymphoma cell lines were conducted with a uniform methodology to complement existing data on these publicly available cell lines. This part captures WES. This data set will be useful for understanding the function of oncogenes and tumor suppressor genes and to develop targeted therapies.
Project description:For the analysis of chromosome 11-centric human proteome, we analyzed the human brain hippocampal proteome. The samples from brain tissues were extensively off-gel fractionated by 12 fractions and analyzed by a high resolution mass spectrometer, Orbitrap XL ETD with collision induced dissociation, after LC separation using C18 column.
Project description:Alterations of the short arm of chromosome 12 (12p) occur in various hematological malignancies and ETV6 and CDKN1B, which are located on 12p, have been implicated as leukemogenic genes of interest. Design and Methods: We selected 7 patients with myeloid malignancies and small 12p deletions by FISH encompassing only the region centromeric of ETV6 and further evaluated them by SNP microarrays. Results: The minimal deleted region (MDR) contained nine genes only. These genes were subsequently analyzed by microarray expression profiling in an independent cohort of 781 cases mostly with different hematological malignancies or without any hematological malignancy: CREBL2, MANSC1, and CDKN1B were expressed in >25% of cases, while the other 6 genes were expressed in a minority of cases only. As CDKN1B is a cell cycle regulator and functions as a tumor suppressor gene, this gene was selected for further expression studies in 286 AML patients. In cases with low CDKN1B expression (expression level <1,160; 1st quartile), RUNX1-RUNX1T1 (11/83; 13.3%; vs. 5/203; 2.5%; p=0.001), PML-RARA rearrangements (11/83; 13.3%; vs. 4/203; 2.0%; p<0.001), 11q23/MLL rearrangements (6/83; 7.2%; vs. 4/203; 2.0%; p=0.038), and FLT3-TKD mutations (7/63; 11.1% vs. 6/167; 3.6%; p=0.047) were more frequently observed than in patients with intermediate/high expression (2nd-4th quartiles). Low CDKN1B expressers had longer median OS and EFS (not reached vs. 14.9 months; p=0.005 and 31.0 vs. 9.7 months; p=0.013, respectively) than intermediate/high expressers. Conclusions: CDKN1B is an interesting candidate gene as a potential biomarker for acute myeloid leukemia prognostication. Affymetrix SNP arrays were performed according to the manufacturer's directions using DNA extracted from mononuclear cells after Ficoll density centrifugation from bone marrow or peripheral blood samples.
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.
Project description:TET1/2/3 are methylcytosine dioxygenases regulating cytosine hydroxymethylation in the genome. Tet1 and Tet2 are abundantly expressed in HSC/HPCs and implicated in the pathogenesis of hematological malignancies. Tet2-deletion in mice causes myeloid malignancies, while Tet1-null mice develop B-cell lymphoma after an extended period of latency. Interestingly, TET1 and TET2 were often concomitantly down-regulated in acute B-lymphocytic leukemia. Here, we investigated the overlapping and non-redundant functions of Tet1/Tet2 in HSC maintenance and development of hematological malignancies using Tet1/2 double knockout (DKO) mice. DKO and Tet2-/- HSC/HPCs had overlapping and unique 5hmC and 5mC profiles and behaved differently. DKO mice exhibited strikingly decreased incidence and delayed onset of myeloid malignancies compared to Tet2-/- mice and in contrast developed lethal B-cell malignancies. Transcriptome analysis of DKO tumors revealed expression changes in many genes dysregulated in human B-cell malignancies, such as LMO2, BCL6 and MYC. These results highlight the critical roles of TET1 or TET2 individually and their cross-talks in the pathogenesis of hematological malignancies. Given the role of Tet proteins in 5mC oxidation, we employed a previously established chemical labeling and affinity purification method coupled with high-throughput sequencing (hMe-Seal) to profile the genome-wide distribution of 5hmC, as well as methylated DNA immunoprecipitation (MeDIP) coupled with high-throughput sequencing (MeDIP-seq) to profile 5mC using BM LK cells purified from young WT, Tet2-/- and DKO mice (6-10 wks old).