Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of mouse leukemia (HSC) haematopoietic stem cells expressing BCR/ABL

ABSTRACT: The BCR-ABL oncogene, generated by Philadelphia chromosome, is present in about 95% human Chronic myeloid leukemia (CML) and 20~30% acute lymphoblastic leukemia (ALL). One of BCR-ABL isoforms, P210, is more often detected in CML and ALL patients. Although BCR-ABL kinase inhibitors are effective in controlling the diseases, they do not provide cure due to the development of drug resistance and the insensitivity of leukemia stem cells to these drugs. Identification of new therapeutic targets is critical. To identify potential target against leukemia stem cells, we studied gene expression in leukemia stem cells, which were identified in mice in our lab (Hu Y, Swerdlow S, Duffy TM, Weinmann R, Lee FY, Li S. 2006. Targeting multiple kinase pathways in leukemic progenitors and stem cells is essential for improved treatment of Ph+ leukemia. Proc Natl Acad Sci USA 103(45):16870-16875.). The sorted leukemia stem cells that expressed BCR-ABL were used for isolation of RNA, followed by the analysis of gene expression using the DNA microarray. The same lineage of non-BCR-ABL-expressing normal hematopoietic stem cells was used as control. We have identified some interesting genes that are up- or down-regulated by BCR-ABL in these leukemia stem cells. We are currently studying the functions of these identified genes. Experiment Overall Design: Identification of genes that are regulated by BCR/ABL in HSCs. Experiment Overall Design: Bone marrow cells are isolated from the long bones of CML mice that are untreated or treated with imatinib. BCR-ABL-expressing or non-BCR-ABL-expressing (transduced with the empty GFP vector) hematopoietic stem cells (GFP+Lin-c-Kit+Sca-1+) are stored by FACS directly into RNAlater (Ambion, Austin, TX) and are homogenized in RLT Buffer (RNeasy Micro Kit (Qiagen, Valencia, CA)). Total RNA is isolated by following the protocol for the RNeasy Micro Kit, and quality is assessed using a 2100 Bioanalyzer instrument and RNA 6000 Pico LabChip assay (Agilent Technologies, Palo Alto, CA). Utilizing the GeneChip Whole Transcript Sense Target Labeling Assay kit (Affymetrix, Santa Clara, CA.) between 100-300ng of total RNA undergoes reverse transcription with random hexamers tagged with T7 sequence. The double stranded cDNA that is generated is then amplified by T7 RNA polymerase to produce cRNA. Second cycle first strand cDNA synthesis then takes place incorporating dUTP which is later used as sites where fragmentation occurs by utilizing an uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1 enzyme mix. The fragmented cDNA is then labeled by terminal transferase attaching a biotin molecule using Affymetrix proprietary DNA labeling Reagent. Approximately 2.0Âµg of fragmented and biotin-labeled cDNA is then hybridized onto a Mouse Gene ST 1.0 Array (Affymetrix, Santa Clara, CA.) for 16 hours at 45Â°C. Post-hybridization staining and washing are performed according to manufacturer's protocols using the Fluidics Station 450 instrument (Affymetrix). Finally, the arrays are scanned with a GeneChip? Scanner 3000. Images are acquired and cel files generated which are then used for analysis.

ORGANISM(S): Mus musculus

SUBMITTER: Shaoguang Li

PROVIDER: E-GEOD-10912 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:We previously demonstrated that Alox5 deficiency impairs the function of LSCs and prevents the initiation of BCR-ABL-induced CML. To identify the pathways in which Alox5 gene regulates function of LSCs, we performed a comparative DNA microarray analysis using total RNA isolated from non-BCR-ABL-expressing Lin-Sca-1+c-Kit+, BCR-ABL-expressing wild type LSCs and BCR-ABL-expressing Alox5-/- LSCs. The result was validated by quantitative real-time PCR analysis of non-BCR-ABL-expressing Lin-Sca-1+c-Kit+, BCR-ABL-expressing wild type LSCs and BCR-ABL-expressing Alox5-/- LSCs. We have shown that Alox5 is a critical regulator of leukemia stem cells (LSCs) in a BCR-ABL-induced chronic myeloid leukemia (CML) mouse model, and we hypothesize that the Alox5 pathway represents a major molecular network that regulates LSC function. Therefore, we sought to further dissect this pathway by comparing the gene expression profiles of wild type and Alox5-/- LSCs derived from our mouse model for BCR-ABL-induced CML. DNA microarray analysis revealed a small group of candidate genes that exhibited changes in the levels of transcription in the absence of Alox5 expression. In particular, we noted that the expression of the Msr1 gene was up-regulated in Alox5-/- LSCs, suggesting that Msr1 might suppress the proliferation of LSCs. Using our CML mouse model, we show that Msr1 is down-regulated by BCR-ABL and this down-regulation is partially restored by Alox5 deletion, and that Msr1 deletion causes acceleration of CML development. Moreover, Msr1 deletion markedly increases LSC function through its effects on cell cycle progression and apoptosis. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and ?-Catenin. Together, these results demonstrate that Msr1 suppresses LSCs and CML development. The enhancement of Msr1 function may be of significance in the development of novel therapeutic strategies targeting CML. To identify genes that are regulated by BCR-ABL in LSCs and LSCs without Alox5 gene, we compared the gene profile between wild type(WT) LSCs or Alox5-/- LSCs.

Project description:MiR-142 is dynamically expressed and plays a regulatory role in hematopoiesis. Based on the simple observation that miR-142 levels are significantly lower in CD34+CD38- cells from blast crisis (BC) chronic myeloid leukemia (CML). CML patients compared with chronic phase (CP) CML patients (p=0.002), we hypothesized that miR-142 deficit plays a role in BC transformation. To test this hypothesis, we generated a miR-142 KO BCR-ABL (i.e., miR-142−/−BCR-ABL) mouse by crossing a miR-142−/− mouse with a miR-142+/+BCR-ABL mouse. While the miR-142+/+BCR-ABL mice developed and died of CP CML, the miR-142−/−BCR-ABL mice developed a BC-like phenotype in the absence of any other acquired gene mutations and died significantly sooner than miR-142+/+BCR-ABL CP controls (p=0.001). Leukemic stem cell (LSC)-enriched Lineage-Sca-1+c-Kit+ cells (LSKs) from diseased miR-142−/−BCR-ABL mice transplanted into congenic recipients, recapitulated the BC features thereby suggesting stable transformation of CP-LSCs into BC-LSCs in the miR-142 KO CML mouse. Single cell (sc) RNA-seq profiling showed that miR-142 deficit changed the cellular landscape of the miR-142−/−BCR-ABL LSKs compared with miR-142+/+BCR-ABL LSKs with expansion of myeloid-primed and loss of lymphoid-primed factions. Bulk RNA-seq analyses along with unbiased metabolomic profiling and functional metabolic assays demonstrated enhanced fatty acid β-oxidation (FAO) and oxidative phosphorylation (OxPhos) in miR-142−/−BCR-ABL LSKs vs miR-142+/+BCR-ABL LSKs. MiR-142 deficit enhanced FAO in miR-142−/−BCR-ABL LSKs by increasing the expression of CPT1A and CPT1B, that controls the cytosol-to-mitochondrial acyl-carnitine transport, a critical step in FAO. MiR-142 deficit also enhanced OxPhos in miR-142−/−BCR-ABL LSKs by increasing mitochondrial fusion and activity. As the homeostasis and activity of LSCs depend on higher levels of these oxidative metabolism processes, we then postulate that miR-142 deficit is a potentially druggable target for BC-LSCs. To this end, we developed a novel CpG-miR-142 mimic oligonucleotide (ODN; i.e., CpG-M-miR-142) that corrected the miR-142 deficit and alone or in combination with a tyrosine kinase inhibitor (TKI) significantly reduced LSC burden and prolonged survival of miR-142−/−BCR-ABL mice. The results from murine models were validated in BC CD34+CD38- primary blasts and patient-derived xenografts (PDXs). In conclusion, an acquired miR-142 deficit sufficed in transforming CP-LSCs into BC-LSCs, via enhancement of bioenergetic oxidative metabolism in absence of any additional gene mutations, and likely represent a novel therapeutic target in BC CML.

Dataset Information

Transcription profiling of mouse leukemia (HSC) haematopoietic stem cells expressing BCR/ABL

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure