Project description:Tyrosine kinase inhibitors (TKI) are highly effective in treatment of chronic myeloid leukemia (CML) but do not eliminate leukemia stem cells (LSC), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSC, suggesting that additional kinase-independent mechanisms contribute to LSC preservation. We investigated whether signals from the bone marrow (BM) microenvironment protect CML LSC from TKI treatment. Coculture with human BM mesenchymal stromal cells (MSC) significantly inhibited apoptosis and preserved CML stem/progenitor cells following TKI exposure, maintaining colony forming ability and engraftment potential in immunodeficient mice. We found that the N-Cadherin receptor plays an important role in MSC-mediated protection of CML progenitors from TKI. N-Cadherin-mediated adhesion to MSC was associated with increased cytoplasmic N-Cadherin-M-NM-2-catenin complex formation, as well as enhanced M-NM-2-catenin nuclear translocation and transcriptional activity. Increased exogenous Wnt-mediated M-NM-2-catenin signaling played an important role in MSC-mediated protection of CML progenitors from TKI treatment. Our results reveal a close interplay between N-Cadherin and the Wnt-M-NM-2-catenin pathway in protecting CML LSC during TKI treatment. Importantly, these results reveal novel mechanisms of resistance of CML LSC to TKI treatment, and suggest new targets for treatment designed to eradicate residual LSC in CML patients. RNA was obtained from CML CD34+ cells treated with or without IM (5M-NM-<M) and MSC for 96 hours, amplified, labeled and hybridized to GeneChip 1.0 arrays (Affymetrix, Santa Clara, CA). Microarray data analysis was performed using R (version 2.9) with genomic analysis packages from Bioconductor (version 2.4). The 33297 probes represented on the microarray were filtered by cross-sample mean, and for standard deviation of greater than the 25% quantile, yielding 18624 probes representing 12553 genes. Linear regression was used to model the gene expression with the consideration of a 2x2 factorial design and matched samples. Differentially expressed genes were identified by calculating empirical Bayes moderated t-statistic, and p-values were adjusted by FDR using the M-bM-^@M-^\LIMMAM-bM-^@M-^] package. Gene Set Enrichment Analysis (GSEA) was performed using GSEA software version 2.04 to detect enrichment of predetermined gene sets using t-scores from all genes for 1263 gene sets in the C2 (curated gene sets) category from the Molecular Signature Database (MsigDB).

Project description:We investigated the ability of HDAC inhibitors (HDACi) to target CML stem cells. Treatment with HDACi combined with IM effectively induced apoptosis in quiescent CML progenitors resistant to elimination by IM alone, and eliminated CML stem cells capable of engrafting immunodeficient mice. In vivo administration of HDACi with IM markedly diminished LSC in a transgenic mouse model of CML. The interaction of IM and HDACi inhibited genes regulating hematopoietic stem cell maintenance and survival. HDACi treatment represents a novel and effective strategy to target LSC in CML patients receiving tyrosine kinase inhibitors. CML CD34+CD38- cells were selected using flow cytometry sorting and treated with IM, LBH and the combination of IM and LBH or cultured without exposure to drugs (controls) for 24 hours (n=3 each). Total RNA from 5000 cells was extracted using the RNeasy kit (Qiagen), amplified and labeled using GeneChip Two-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, CA). 15 Âµg cRNA from each sample was hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Microarray data analyses were performed using R (version 2.9) with genomic analysis packages from Bioconductor (version 2.4). Expression data were normalized using the robust multiarray average (RMA) algorithm, with background adjustment, quantile normalization and median polish summarization. Probesets with low expression levels or low variability across samples were filtered. For genes with multiple probesets, the gene level expression was set to be the median of the probesets. Linear regression was used to model the gene expression with the consideration of 2x2 factorial design and matched samples. Differentially expressed genes were identified by calculating empirical Bayes moderated t-statistic, and p-values were adjusted by FDR using the âLIMMAâ package. Gene Set Enrichment Analyses (GSEA) was performed using GSEA software version 2.04 [http://www.broadinstitute.org/gsea/] to detect enrichment of predetermined gene sets using t-scores and gene sets in C2 (curated gene sets) category from the Molecular Signature Database (MsigDB). Gene sets representing common functional categories were categorized and grouped. We also analyzed enrichment of gene sets with common transcription factor binding sites (586 sets) from MsigDB.

Project description:We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin- CD34-) hematopoietic stem cells (HSCs) from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular caryotyping and quantitative analysis of BCR/ABL transcript demonstrated that about one third of CD34- was leukemic. CML CD34- cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures and cytokines induced CD34 expression on some HSCs, cell cycling, acquisition of clonogenic activity and increased expression of BCR/ABL transcript. CML CD34- cells showed an engraftment rate in immunodeficient mice similar to that of CD34+ cells. Gene expression profiling revealed the down-regulation of cell cycle arrest genes together with genes involved in antigen presentation and processing, while the expression of angiogenic factors was strongly up-regulated when compared to normal counterparts. Flow cytometry analysis confirmed the significant down-regulation of HLA class I and II molecules in CML CD34-cells. Increasing doses of imatinib mesilate (IM) did not affect fusion transcript levels, BCR-ABL kinase activity and the clonogenic efficiency of CML CD34- cells as compared to leukemic CD34+cells. Thus, we identified in CML a novel CD34- leukemic stem cell subset with peculiar molecular and functional characteristics which may be a potential target for CML therapeutics. Leukemic cells were obtained from 12 chronic phase Ph+ CML patients at diagnosis and before treatment. Normal samples were leukapheresis products from 12 healthy stem cell donors receiving recombinant human granulocyte colony-stimulating factor (G-CSF; Lenograstim, Sanofi-Aventis, Milan, Italy). The protocol was approved by the ethical committee of the University Hospital and each patient/donor gave written informed consent. Hemopoietic stem/progenitor cell purification and phenotypic analyses were performed as previously described (Lemoli et al, Br J Haematol, 2003; Lemoli RM et al., Blood, 1997). Aliquots of sorted Lin-CD34-, Lin-CD34+ and Lin+CD34+ were reanalyzed by FacScan (Becton Dickinson, Franklin Lakes, NJ) to assess their purities. Total cellular RNA was extracted from 0.5x105 cells of each sample using RNeasy Micro kit (Qiagen, Valencia, CA) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the concentration and purity/integrity of RNA samples using Agilent 2100 Bioanalyzer. RNAs originating from 12 normal donors or from 12 CML patients were pooled in order to obtain at least 2 mg per sample. One-cycle target labeling assays, as well as the Affymetrix Human HG-U95Av2 GeneChip arrays hybridization, staining, and scanning, were performed, using Affymetrix standard protocols (Affymetrix, Santa Clara,CA).

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Homo sapiens

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