The use of molecularly defined therapeutics can dramatically improve the treatment of leukemia. In case of BCR-ABL positive chronic myeloid leukemia (CML), for example, specific tyrosine kinase inhibitors (TKIs) have dramatically improved the course of the disease and the survival of patients. In contrast, the results of TKI therapy in BCR-ABL-positive acute lymphoblastic leukemia (ALL) are significantly inferior. Approximately 30% of adult patients with ALL express BCR-ABL, defining a high-risk group. The basis for the success of TKI therapy in CML has been the understanding of the molecular pathology of the disease, and the understanding of disease-specific molecular aberrations continues to form the basis for the identification of new therapeutic targets and the development of new therapies.
Our laboratory investigates molecular aberrations in leukemias with the aim of improving therapeutic approaches or developing new ones. We use cell culture and mouse models as well as viral gene transfer for the genetic manipulation of hematopoietic cells. This includes the overexpression of genes and miRNAs as well as the specific inhibition of gene expression through RNA interference (RNAi). In addition, we use methods for genome- and proteome-wide analysis of gene expression in collaboration with several research groups within and outside the MHH.
Projects
Identification of potential target structures in leukemia cells
In previous work, gene-specific synthetic siRNAs (transient RNAi) and lentiviral-encoded shRNAs (stable RNAi) were established in cell lines and primary hematopoietic stem and progenitor cells from normal individuals and CML patients for functional gene analysis (Scherr et al., 2003a, 2003b, 2006). We were able to identify STAT5 (transcription factor), SHP-2 (protein tyrosine phosphatase) and Gab2 (adaptor protein) in CML cells as potential new target structures due to a leukemia-specific inhibition of cell growth (Scherr et al., 2005, 2006).
Microarray analyses (miCHIPs) and miRNA-specific qRT-PCR (miR-qRT-PCR) were used to determine the expression pattern of miRNAs in cell lines and primary hematopoietic stem and progenitor cells of CML patients (Venturini et al., 2007), which unravelled the miR-17~92 polycistron to be differentially regulated. To analyze the function of miRNAs, we established expression cassettes for the overexpression and inhibition of individual miRNAs (so-called antagomiRs) in cell culture systems (Scherr et al., 2007; Scherr et al., 2010).
Since miR-17~92 expression is increased in primary CML cells, we investigated the role of the miR-17~92 polycistron in BCR-ABL positive ALL in a further project. For this purpose, we examined miR-17~92 expression in primary BCR-ABL-positive and BCR-ABL-negative lymphoid ALL cells, as well as in primary normal CD34+ cells using miR-qRT-PCR. Surprisingly, (i) miR~17-92 expression was found to be decreased in B-lineage ALL cells, in contrast to BCR-ABL positive myeloid cells (Scherr et al., 2014) and (ii) overexpression of miR-17~92 in ALL cells regulates apoptosis in a BCR-ABL-dependent manner.
To identify miR-17~92-regulated target genes, we performed SILAC/LC-MS (Stable Isotope Labeling of Amino acids in Culture / Liquid Chromatography and Mass Spectroscopy) experiments with lymphoid TonB cells stably expressing the miR~17-19b polycistron in collaboration with Prof. A. Pich (Dept. of Institute of Toxicology, MHH). We were able to identify several candidate genes that play a role in the apoptosis pathway, including Bcl2. In both murine and human cells, we confirmed Bcl2 as a direct target of miR-17~92. Furthermore, knock-down of Bcl2 using Bcl2-specific RNAi showed a BCR-ABL-dependent induction of apoptosis. In addition, we could show that BCR-ABL positive ALL cells respond more sensitively to pharmacologic BCL2 inhibition with ABT-737 than BCR-ABL negative ALL cells.
To investigate the effects of BCL2 inhibition in clinically relevant models, primary BCR-ABL positive ALL cells were lentivirally transduced with a vector expressing luciferase and transplanted into NSG mice. After successful growth of leukemia cells, mice were treated with the BCL2 inhibitor ABT-737 or control (DMSO). Treatment with ABT-737 resulted in a highly efficient reduction of luciferase-expressing leukemia cells. In addition, animal survival was significantly prolonged by ABT-737 treatment indicating that inhibition of BCL2 serves a potential treatment strategy for BCR-ABL-positive ALL in vivo.
In myeloid cells, we demonstrated that overexpression of miR-125b can block G-CSF-induced differentiation in murine 32D cells in vitro. In vivo, however, transplantation of murine miR-125b-overexpressing bone marrow cells led to an increased growth of myeloid cells in the bone marrow after about 8 weeks. Using bioinformatic and experimental analyses, we were able to identify STAT3, c-Jun and JUND as miR-125b-regulated target genes in myeloid cells for the first time and confirm BAK1 as a target gene (Surdziel et al., 2011).