Molecular Phenotype of Malignant CD34+ Hematopoietic Stem and Progenitor Cells in Chronic Myelogenous Leukemia.

Abstract

In this study, we examined gene expression profiles of immunomagnetically enriched CD34+ cells from bone marrow (BM) of 9 patients with untreated CML in chronic phase and from 8 healthy volunteers using Affymetrix GeneChips. Additionally, in 3 patients CD34+ from peripheral blood (PB) were compared with those from BM. Differential expression of 12 candidate genes was corroborated by quantitative real-time RT-PCR. Following hybridization of labelled cRNA to Affymetrix GeneChips covering 8793 genes we used the statistical scripting language “R” for data analysis. For normalization a method of variance stabilization transformations was used. To identify significantly differentially expressed genes we used the Significance Analysis of Microarrays (SAM) algorithm. The intraindividual comparison of CD34+ cells from BM and PB in CML showed no differentially expressed genes which is different to normal CD34+ cells which had distinct gene expression patterns comparing circulating and sedentary CD34+ cells (Steidl et al., Blood, 2002). Comparing malignant BM CD34+ cells from CML with normal BM CD34+ cells 792 genes were significantly differentially expressed (fold change: >1.3; q-value: <0.03). 735 genes had a higher and 57 genes a lower expression in CML. Gene expression patterns reflected BCR-ABL-induced functional alterations such as increased cell-cycle and proteasome activity as well as decreased apoptosis. Downregulation of several genes involved in DNA repair and detoxification in CML might be the basis for DNA instability and progression to blast crisis. An interesting finding was an upregulation of fetal hemoglobin (Hb) components such as Hb gamma A and G in leukemic progenitor cells whereas no difference in adult Hb expression was observed suggesting an induction of fetal Hb synthesis in CML. Looking at genes involved in stem cell maintenance we found an upregulation of GATA2 and a reduced expression of proteins from the Wnt signalling pathway suggesting an increased self-renewal of CML hematopoietic stem cells compared to the normal counterpart. Moreover, several genes playing a role in ubiquitin-dependent protein catabolism and in fatty acid biosynthesis such as fatty acid synthase (FAS) were stronger expressed in CML. The functional role of FAS for leukemic cell growth was assessed in cell culture experiments. Incubation of the leukemic cell line K562 with the FAS inhibitor cerulenin (10 μg/ml) for 3 days resulted in death of 99% of cells suggesting that survival of leukemic cells depends upon endogenous fatty acid synthesis. In an attempt to find a specific gene expression pattern associated with response to imatinib therapy we divided the patients included in this study into two groups: maximal reduction of BCR-ABL transcript level <3-log vs. >3-log (major molecular remission) during therapy. Comparing pretherapeutic gene expression profiles of both groups we could not identify a pattern predictive for major molecular response. In conclusion, malignant CD34+ cells in CML have a specific gene expression pattern which seems not to be predictive for response to imatinib therapy.