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Development of an effective therapy for chronic myelogenous leukemia.

ABSTRACT: Targeted small-molecule drugs have revolutionized treatment of chronic myeloid leukemia (CML) during the last decade. These agents interrupt a constitutively active BCR-ABL, the causative agent for CML, by interfering with adenosine 5' triphosphate-dependent ABL tyrosine kinase. Although the efficacy of tyrosine kinase inhibitors (TKIs) has resulted in overall survival of greater than 90%, TKIs are not curative. Moreover, no currently approved TKIs are effective against the T315I BCR-ABL variant. However, a new generation of TKIs with activity against T315I is on the horizon. We will highlight the clinical utility of historical CML therapeutics, those used today (first- and second-generation TKIs), and discuss treatment modalities that are under development. Recent advances have illuminated the complexity of CML, especially within the marrow microenvironment. We contend that the key to curing CML will involve strategies beyond targeting BCR-ABL because primitive human CML stem cells are not dependent on BCR-ABL. Ultimately, drug combinations or exploiting synthetic lethality may transform responses into definitive cures for CML.

SUBMITTER: Woessner DW

PROVIDER: S-EPMC3251313 | biostudies-literature | 2011 Nov-Dec

REPOSITORIES: biostudies-literature

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Development of an effective therapy for chronic myelogenous leukemia.

Woessner David W DW Lim Carol S CS Deininger Michael W MW

Cancer journal (Sudbury, Mass.) 20111101 6

Targeted small-molecule drugs have revolutionized treatment of chronic myeloid leukemia (CML) during the last decade. These agents interrupt a constitutively active BCR-ABL, the causative agent for CML, by interfering with adenosine 5' triphosphate-dependent ABL tyrosine kinase. Although the efficacy of tyrosine kinase inhibitors (TKIs) has resulted in overall survival of greater than 90%, TKIs are not curative. Moreover, no currently approved TKIs are effective against the T315I BCR-ABL variant ...[more]

PMID: 22157291

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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).

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.

Dataset Information

Development of an effective therapy for chronic myelogenous leukemia.

Publications

Development of an effective therapy for chronic myelogenous leukemia.

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