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ORIGINAL ARTICLE Molecular Signature of CD34+ Hematopoietic

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ORIGINAL ARTICLE Molecular Signature of CD34+ Hematopoietic Leukemia (2007) 21, 494–504 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE Molecular signature of CD34 þ hematopoietic stem and progenitor cells of patients with CML in chronic phase E Diaz-Blanco1,6, I Bruns1,6, F Neumann1, JC Fischer2, T Graef1, M Rosskopf3, B Brors4, S Pechtel1, S Bork1, A Koch1, A Baer1, U-P Rohr1, G Kobbe1, A von Haeseler5, N Gattermann1, R Haas1 and R Kronenwett1,7 1Department of Hematology, Oncology and Clinical Immunology, University of Duesseldorf, Duesseldorf, Germany; 2Institute of Transplantation Diagnostics and Cell Therapeutics, University of Duesseldorf, Duesseldorf, Germany; 3Institute of Bioinformatics, University of Duesseldorf, Duesseldorf, Germany; 4Theoretical Bioinformatics, German Cancer Research Center, Heidelberg, Germany and 5Max F. Perutz Laboratories, Center for Integrative Bioinformatics; University of Vienna; Medical University of Vienna; University of Veterinary Medicine Vienna, Vienna, Austria In this study, we provide a molecular signature of highly BCR-ABL oncoprotein.3 It constitutively activates mitogenic enriched CD34 þ cells from bone marrow of untreated patients signaling pathways such as rat sarcoma viral oncogene homolog with chronic myelogenous leukemia (CML) in chronic phase in (RAS)/mitogen-activated protein kinase (MAPK) pathways, the comparison with normal CD34 þ cells using microarrays covering 8746 genes. Expression data reflected several BCR- janus kinase (JAK)/signal transducer and activator of transcrip- ABL-induced effects in primary CML progenitors, such as tion (STAT) pathway, phosphoinositide-3 (PI3) kinase pathway transcriptional activation of the classical mitogen-activated and the MYC pathway. In addition, BCR-ABL induces adhesive protein kinase pathway and the phosphoinositide-3 kinase/AKT and cytoskeletal abnormalities resulting in deregulation of pathway as well as downregulation of the proapoptotic gene proliferation, apoptosis and release of progenitors from bone IRF8. Moreover, novel transcriptional changes in comparison marrow (BM).4,5 Several studies suggest that the expansion of with normal CD34 þ cells were identified. These include upregulation of genes involved in the transforming growth malignant cells in CML is not directly driven by the neoplastic 6 factorb pathway, fetal hemoglobin genes, leptin receptor, stem cell but by lineage-committed progenitor cells. Interest- sorcin, tissue inhibitor of metalloproteinase 1, the neuroepithe- ingly, treatment of patients with the tyrosine kinase inhibitor lial cell transforming gene 1 and downregulation of selenopro- imatinib results in the persistence of BCR-ABL-positive cells in tein P. Additionally, genes associated with early hematopoietic most of the patients in complete cytogenetic response, suggest- stem cells (HSC) and leukemogenesis such as HoxA9 and ing that blocking of BCR-ABL induces an inhibition of progenitor MEIS1 were transcriptionally activated. Differential expression 7 of differentiation-associated genes suggested an altered com- cells, whereas primitive leukemic stem cells are spared. position of the CD34 þ cell population in CML. This was A limitation of the existing data about BCR-ABL-mediated confirmed by subset analyses of chronic phase CML CD34 þ effects is that they are predominantly derived from human and cells showing an increase of the proportion of megakaryocyte- murine BCR-ABL-transformed cell lines and mouse models,3 erythroid progenitors, whereas the proportion of HSC and which might not be representative for chronic phase CML. The granulocyte–macrophage progenitors was decreased in CML. In conclusion, our results give novel insights into the biology of cell population for studying the pathophysiology of CML are CML and could provide the basis for identification of new certainly primary hematopoietic stem and progenitor cells from therapeutic targets. patients with CML. Leukemia (2007) 21, 494–504. doi:10.1038/sj.leu.2404549; The assessment of primary CML cells by means of gene published online 25 January 2007 expression analyses comparing BCR-ABL-positive cells with Keywords: CML; hematopoietic stem cells; CD34 þ cell subsets; normal cells,8–10 resulted in expression profiles associated with gene expression profiling; BCR-ABL signaling response to therapy11–15 or progression to advanced phases of disease.16,17 In a previous gene expression study using an array covering 1185 genes, we could confirm some previously Introduction described functional effects of BCR-ABL in highly enriched CD34 þ hematopoietic stem and progenitor cells.8 Although Chronic myelogenous leukemia (CML) is a malignant disorder our study gave novel insights into BCR-ABL-induced effects in of the hematopoietic stem cell (HSC) characterized by primary CML cells, no study exists to date, which provides a a reciprocal translocation between chromosomes 9 and 22 large-scale expression analysis of chronic phase CML CD34 þ (t(9;22)(q34;q11)).1 The translocation results in formation of the stem and progenitor cells in comparison with the normal BCR-ABL fusion oncogene encoding a protein with constitutive counterparts. tyrosine kinase activation, which plays a central role in Therefore, we examined highly enriched CD34 þ cells from the pathogenesis of the disease.2 Numerous mechanisms are BM of untreated patients with CML in first chronic phase using involved in the malignant transformation orchestrated by the microarrays covering 8746 genes and refined the molecular signature of hematopoietic stem and progenitor cells in CML. Correspondence: Dr R Kronenwett, Department of Hematology, Oncology and Clinical Immunology; University of Du¨sseldorf, Moorenstr. 5, 40225 Du¨sseldorf, Germany. E-mail: [email protected] Materials and methods 6These authors contributed equally to this work. 7Current address: Bayer HealthCare AG, Diagnostics Research, Leverkusen, Germany. Patients and cells Received 1 November 2006; accepted 16 November 2006; published After informed consent BM mononuclear cells were obtained by online 25 January 2007 density centrifugation as previously described18 from eight Molecular signature of CML CD34 þ cells D-B Elena et al 495 healthy volunteers and from nine patients with untreated newly Optical 96-well Reaction Plate (Applera) according to the diagnosed Ph þ CML in chronic phase for gene expression instructions of the manufacturer using commercially available analyses as well as from three healthy volunteers and from three assays-on-demand. GAPDH mRNA served as external control patients for subset analyses (patients characteristics: Supple- for relative quantification. The following Assay-on-Demand mentary Table 1). From three of the nine patients (patient Gene Expression Products (Applera) were used: GATA1 numbers 1–3), we also obtained peripheral blood (PB) mono- (Hs00231112_m1), leptin receptor (LEPR) (Hs00174497_m1), nuclear cells. Samples were processed within 2 h following TAL1 (Hs00268434_m1), tissue inhibitor of metalloproteinase 1 puncture. CD34 þ cells were positively selected using two (TIMP1) (Hs00171558_m1), ACSL (acyl-CoA synthetase long- rounds of midiMACS immunomagnetic separation (Miltenyi chain) 5 (Hs00212106_m1), CRHBP (Hs00181810_m1), seleno- Biotec, Bergisch Gladbach, Germany) following Ficoll density protein P (SEPP1) (Hs00193657_m1), ELA2 (Hs00236952_m1) centrifugation as described previously.18 Purities of CD34 þ and GAPDH (Hs99999905_m1). CT values were calculated by cells for expression analyses as assessed by flow cytometry the ABI PRISM software and relative gene expression levels were varied between 98.9 and 99.9%. expressed as the difference of CT values of target gene and GAPDH (DCT). Given a PCR efficacy of 2 a DCT value of 1 reflects a fold-change of 2. RNA isolation, cRNA labeling and hybridization to microarrays Total RNA was isolated from CD34 þ cells using the RNeasy Immunofluorescence staining and flow cytometry Mini-Kit (Qiagen, Hilden, Germany) according to the manu- Flow cytometric analysis of CD34 þ subsets (stem cells and facturer’s instructions. The amount of extracted RNA was myeloid progenitors) was performed as described previously.22 quantified using the NanoDrop spectrophotometer (NanoDrop In brief, following immunomagnetical selection, CD34 þ cells Technologies, Welmongton, DE, USA). Total RNA (median: were stained with fluorescein isothiocyanate-conjugated anti- 7.0 mg; range: 0.7–20 mg) was used to generate biotin-labeled bodies specific for lineage markers CD3 (clone S4.1), CD4 cRNA (median: 25 mg; range: 2.1–68 mg) by means of Enzo (S3.5), CD7 (CD7-6B7), CD8 (385), CD10 (5-1B4), CD14 BioArray HighYield RNA Transcript Labeling Kit (Affymetrix Ltd, (TUK4), CD19 (SJ25-C1) (Caltag, South San Francisco, CA, UK). Following fragmentation, labelled cRNA was hybridized to USA), CD2 (RPA-2.10), CD11b (ICRF44), CD20 (2H7), CD56 Affymetrix HG-Focus GeneChips stained according to the (B159), GPA (GA-R2) (Becton Dickinson-PharMingen, San manufacturer’s instructions. The quality of extracted RNA and Diego, CA, USA), and ECD-conjugated anti-CD45RA (MEM56) of cRNA was controlled using an Agilent Bioanalyzer 2100 (Beckman Coulter, Miami, FL, USA), phycoerythrin-conjugated (Agilent Technologies, Waldbronn, Germany). anti-interleukin (IL)-3Ralpha (9F5) (Becton Dickinson-Phar- Mingen), allophycocyanin conjugated anti-CD38 (HIT2) (Caltag) and PerCP-conjugated anti-CD34 (HPCA-2) (Becton Quantification, normalization and statistical analysis Dickinson-PharMingen). Dead cells were excluded by
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