Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of goat model for CML

ABSTRACT: To evaluate the long-term growth potential of BCR-ABL-transduced primitive human hematopoietic cells, lin- cord blood cells containing an MSCV-BCR-ABL-IRES-GFP (BCR-ABL) or control-GFP transgene (MIG) were injected IP into fetal goats at 45-55 days of gestation. Six transplant goats were born alive. One was examined three weeks after birth and showed GFP+ cells in the blood, bone marrow (BM), liver, kidney, lung, heart, and both skeletal and smooth muscle. FISH analysis also showed the liver of this goat contained BCR-ABL-GFP transgenic cells. The remaining five goats appear normal although, in some, the WBC count is elevated 3- to 5-fold. GFP+ cells, including cells identifiable by FACS as human CD34+ cells, have been detected in the blood of all these goats. The presence of BCR-ABL-GFP transgenic cells in the BM and liver was confirmed by FISH analysis, and quantitative real-time PCR analysis of genomic DNA isolated from unpurified BM cells obtained from three of the transplant goats demonstrated 3-5Ã104 copies of the transgene per microgram of DNA. Microarray transcript profiling was performed on blood and liver tissues of normal goats, BCR-ABL chimeric goats, MIG chimeric goats, and normal human samples. RNA for human genes was detected in goats transplanted with cord blood cells but not in normal goats, and the RNA abundance of some genes in BCR-ABL chimeric goat blood was similar to or greater than levels observed in MIG goat blood or normal human samples. Quantitative RT-PCR confirmed the differential expression of several genes in goats carrying the BCR-ABL vs. control transgene. These results demonstrate long-term engraftment but slow expansion in a large animal model of primitive human hematopoietic cells transduced with a BCR-ABL fusion gene and transplanted in utero. This novel xenotransplant goat model should be useful for analyzing the initial phases of development of human CML and for assessing new therapies with potential long-term benefits. Experiment Overall Design: Total RNA was extracted from liver (L) and blood (B) samples of normal goats (ng), humans (hu), chimeric goats engrafted with human cord blood stem cells containing control (mig) vector, and chimeric goats engrafted with CML (bcrabl) vector. RNA samples were profiled on Affymetrix human U133A GeneChips and examined for differentially expressed genes in CML vs control goats, filtering for signals significantly above background levels observed in normal goat to select for specific human gene expression.

ORGANISM(S): Homo sapiens

SUBMITTER: Don Baldwin

PROVIDER: E-GEOD-17588 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To evaluate the long-term growth potential of BCR-ABL-transduced primitive human hematopoietic cells, lin- cord blood cells containing an MSCV-BCR-ABL-IRES-GFP (BCR-ABL) or control-GFP transgene (MIG) were injected IP into fetal goats at 45-55 days of gestation. Six transplant goats were born alive. One was examined three weeks after birth and showed GFP+ cells in the blood, bone marrow (BM), liver, kidney, lung, heart, and both skeletal and smooth muscle. FISH analysis also showed the liver of this goat contained BCR-ABL-GFP transgenic cells. The remaining five goats appear normal although, in some, the WBC count is elevated 3- to 5-fold. GFP+ cells, including cells identifiable by FACS as human CD34+ cells, have been detected in the blood of all these goats. The presence of BCR-ABL-GFP transgenic cells in the BM and liver was confirmed by FISH analysis, and quantitative real-time PCR analysis of genomic DNA isolated from unpurified BM cells obtained from three of the transplant goats demonstrated 3-5×104 copies of the transgene per microgram of DNA. Microarray transcript profiling was performed on blood and liver tissues of normal goats, BCR-ABL chimeric goats, MIG chimeric goats, and normal human samples. RNA for human genes was detected in goats transplanted with cord blood cells but not in normal goats, and the RNA abundance of some genes in BCR-ABL chimeric goat blood was similar to or greater than levels observed in MIG goat blood or normal human samples. Quantitative RT-PCR confirmed the differential expression of several genes in goats carrying the BCR-ABL vs. control transgene. These results demonstrate long-term engraftment but slow expansion in a large animal model of primitive human hematopoietic cells transduced with a BCR-ABL fusion gene and transplanted in utero. This novel xenotransplant goat model should be useful for analyzing the initial phases of development of human CML and for assessing new therapies with potential long-term benefits.

Project description: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.

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.

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Transcription profiling of goat model for CML

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