368. Differential Sensitivity of Normal and CML-Derived CD34+Cells to Inhibition of SHP2, Gab2 and Stat5 Gene Expression by RNA Interference (RNAi)

Abstract

RNA interference has rapidly become an efficient tool for functional genomics in a variety of organisms. Stable expression of shRNA driven by pol III promoters upon lentiviral gene transfer can induce long-term gene silencing in mammalian cells. We recently demonstrated that lentivirus-mediated anti bcr-abl RNAi can specifically silence bcr-abl gene expression, inhibit oncogene driven cell proliferation, and eradicate leukemic cells depending on the dose of lentivirus-mediated shRNA expression. However, since effective depletion requires a threshold of lentiviral integrations into target cell genomes, the risk of insertional mutagenesis may limit the therapeutic value of this approach. To search for new potential therapeutic targets in CML, we studied the function of several signaling molecules in purified normal and CML CD34+cells from chronic phase CML patients harvested at initial diagnosis. We selected SHP2, Gab2, and Stat5 based on their constitutive tyrosine phosphorylation in bcr-abl+ cell lines. Several shRNAs for each target gene were evaluated using a bicistronic EGFP-encoding reporter plasmid system as described earlier. Effective shRNA expression cassettes were cloned into lentiviral plasmids encoding RFP to track lentiviral transduction. Lentivirus-mediated RNAi targeting SHP2, Gab2, or Stat5 results in a reduction of mRNA and protein by more than 90 % and induces a rapid depletion of transduced bcr-abl+ and negative cell lines. In addition, RNAi against all three targets enhances imatinib induced depletion of bcr-abl+K562 cells. We next transduced normal and CML-derived primary CD34+cells with control and anti-SHP2, Gab2, and Stat5 lentiviruses, and analysed colony-formation of transduced, i.e. RFP+progenitor cells in methylcellulose cultures. To eliminate effects of different transduction rates we plated CD34+cells for each transfection in the presence of high (GM-CSF: 20 ng/ml; IL-3: 10 ng/ml) or low (GM-CSF: 0.2 ng/ml; IL-3: 0.1 ng/ml) cytokine concentrations indicating the functional relevance of the respective RNAi-target for CFU-colony formation. Whereas anti-SHP2, Gab2, and Stat5 RNAi did not reduce the proliferation of normal transduced CFU (n=5), proliferation of transduced CFU from CML patients was specifically reduced between 50 to 85 % under low cytokine concentration (n=9-13). Furthermore, co-expression of both anti-bcr-abl and anti-SHP2 shRNA from a single lentiviral vector strongly cooperates to inhibit colony formation of CML-progenitors. This study establishes lentivirus-mediated RNAi as a valuable tool for functional genomics in primary hematopoietic cells and demonstrates that normal CFUs are more resistant to inhibition of SHP2, Gab2, and Stat5 gene expression than CML-progenitors. Furthermore, the cooperative effects of RNAi targeting different genes simultaneously and the combined effects of protein and gene expression antagonists suggest that co-treatment with a leukemia-specific and a non-specific inhibitor may harbor significant therapeutic potential in CML.