ABSTRACT: Expression data from murine hematopoietic cells expressing BCR-ABL alone, NUP98-HOXA9 alone, BCR-ABL and NUP98-HOXA9, or null for both oncogenes
Project description:Leukemia is a complex malignancy with hundreds of distinct mutations associated with disease development. Studies have shown that oncogenes cooperate to promote leukemia transformation, however, the downstream effectors of this cooperation are largely unknown. Using a genetically defined mouse model of acute leuekmia, we investigated the regulated of genes downstream of the cooperative oncogenic interaction between BCR-ABL and NUP98-HOXA9 and identified a unique gene signature abberrantly expression in leukemia. Total RNA was isolated from hematopoietic cells transduced with BCR-ABL and Nup98-HOXA9 retroviruses and transplanted into recipient mice. Bone marrow cells were purified by GFP (BCR-ABL) and YFP (NUP98-HOXA9) using FACS.
Project description:To investigate the function of NOX2 (CYBB) in leukemia stem cells, we created leukemia by tranforming primitive hematopoetic cells isolated from wild type and NOX2-KO mice with NUP98/HOXA9 and BCR/ABL oncogenes, isolated mRNA, and performed RNA-seq experiments to profile transcriptional changes upon NOX2 loss.
Project description:Leukemia is a complex malignancy with hundreds of distinct mutations associated with disease development. Studies have shown that oncogenes cooperate to promote leukemia transformation, however, the downstream effectors of this cooperation are largely unknown. Using a genetically defined mouse model of acute leuekmia, we investigated the regulated of genes downstream of the cooperative oncogenic interaction between BCR-ABL and NUP98-HOXA9 and identified a unique gene signature abberrantly expression in leukemia.
Project description:Analysis of gene expression of leukemia stem cells (LSCs) in different tissues. The hypothesis is that the distinct niche in different tissues affects the gene expression of LSCs. Total RNA from LSCs in different tissues including bone marrow (BM), spleen (SPL), peripheral blood (BL), gonadal adipose tissue (AT) as well as normal counterparts for LSCs from normal BM (NBM) was isolated and subjected to RNA-seq. LSCs were derived from a blast crisis chronic myeloid leukemia (bcCML) murine model induced by co-expression of human leukemic oncogenes BCR-ABL and NUP98-HOXA9. Triplicates were generated for LSCs from each tissues.
Project description:RNA-seq of mouse hematopoietic stem and progenitor cells expressing NUP98-HOXA9 (NHX9) show upregulation of HOX and other NUP98 fusion target genes, but expression changes are dampened or lost with the introduction of mutations in NUP98's FG repeats or HOXA9's DNA binding domain
Project description:Development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human hematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains nucleoporin’s IDR, tandemly dispersed phenylalanine-andglycine (FG) repeats1-3. However, it remains largely elusive how unstructured IDRs contribute to oncogenesis. We here show that IDR or FG repeats harbored within NUP98-HOXA9, a homeodomain-containing transcription factor (TF) chimera recurrently detected in acute leukemia patients1,4,5, is essential for establishing nuclear liquid-liquid phase separation (LLPS) puncta and for inducing leukemic transformation of primary hematopoietic cells in vitro and in vivo. Strikingly, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera TF oncoproteins but is also required for formation of a broad, ‘super-enhancer’-like binding pattern, typically seen at a battery of leukemia-related loci exemplified by HOX, MEIS and PBX genes, potentiating their transcriptional activation. An artificial HOX chimera, created by replacing NUP98’s FG repeats with an unrelated LLPSforming IDR of FUS6,7, had similar enhancement effects on chimera’s chromatin binding and target gene activation. Via Hi-C mapping, we further demonstrated that the phase-separated NUP98-HOXA9 protein assembly is able to induce formation of CTCF-independent chromatin looping enriched at leukemic oncogenes. Together, this report describes a proof-of-principle example wherein cancer acquires mutation to establish condensates of oncogenic TFs via a phase separation mechanism, which simultaneously enhances their chromatin targeting and induces organization of aberrant three-dimensional chromatin structure during tumorous transformation. As a range of LLPS-competent molecules are implicated in various human cancers, this mechanism can potentially be generalized to many malignant and diseased settings.
Project description:To explore the molecular mechanisms induced by the leukemic fusion protein NUP98-HOXA9, we performed gene expression analysis of human hematopoietic progenitors and primary samples of patients that express NUP98-HOXA9. By combining these data with ChIP-seq results, we observed that the fusion protein is able to both activate and repress the expression of its target genes.
Project description:Msi2 is a critical regulatior of myeoid leukemia, and these data identify genes that are changed following Msi2 deletion in bcCML and de novo AML stem cells. Leukemic stem cells were extracted from wild-type and Msi2 mutant MLL-AF9 driven AML and BCR-ABL/NUP98-HOXA9 driven bcCML, in triplicate. RNA was extracted and hybridized on Affymetrix microarrays. The mouse strain was a genetic trap Msi2 mutant made by Center for Animal Resources and Development (CARD) of Kumamoto University. They call their strain B6;CB-Msi2Gt(pU-21T)2Imeg, see also http://cardb.cc.kumamoto-u.ac.jp/transgenic/strainsDetailAction.do?strainId=834
Project description:To investigate the effect of NUP98-HOXA9 on global gene expression, we established FLAG-NUP98-HOXA9 expressing mouse embryonic stem cell lines.
Project description:Oncogenic tyrosine kinases, such as BCR-ABL, TEL-ABL, TEL-PDGF-beta-R and FLT3-ITD, play a major role in the development of hematopoietic malignancy. They activate many of the same signal transduction pathways. To identify the critical target genes required for transformation in hematopoietic cells, we used a comparative gene expression strategy in which selective small molecules were applied to 32Dcl3 cells that had been transformed to factor-independent growth by these respective oncogenic alleles. Experiment Overall Design: In our microarray study, we have total 22 samples from four different cell lines expressing BCR-ABL, TEL-ABL, FLT3-ITD or TEL-PDGF-betaR. Each cell line was treated with specific small molecule inhibitors for 4 hours, then RNA was extracted and cRNA was hybridized to Affymetrix U74 oligonucleotide arrays. Untreated cells or cells treated with unrelated inhibitors were used as controls or references. We have three replicates and six references obtained from three independent experiments for cells expressing BCR-ABL or FLT3-ITD. We have one treated and one untreated sample for each cell line expressing TEL-ABL or TEL-PDGF-beta-R.
