Lineage-Specific Regulation of miRNA-Expression by the BCR-ABL Oncogene.

Micro RNAs (miRNA) are small non-coding RNAs that regulate gene expression by specific hybridization to complementary sequences in the 3′-untranslated region of corresponding mRNAs resulting in inhibition of mRNA translation or mRNA degradation. Aberrant expression of specific miRNAs has been described in a variety of human malignancies but its functional consequences are currently largely unknown. The miR-17- 92 cluster (encoding miR-17-5p, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92) has been shown to be over-expressed in chronic phase but not in blast crisis CML CD34+ cells in a BCR-ABL- and c-Myc-dependent manner (Venturini et al. 2007). Interestingly, the knock-out of the polcystronic miR-17-92 cluster in mice recently revealed an essential role in lung development and B-lymphopoiesis at the stage of pre-B-cells also affected in pre- B-ALL which often expresses the BCR-ABL oncogene. To study miR-17-92 expression in BCR-ABL + myeloid and lymphoid cells we performed miRNA specific quantitative RT-PCR (miR-qRT-PCR) in myeloid human K562 and murine 32D/BCR-ABL cells and in lymphoid human BV173 and IL-3-dependent murine TonB cells which can be induced to express BCR-ABL. We found reduced expression of both the miR-17-92 cluster and c-MYC in lymphoid as compared to myeloid cells. Inhibition of BCR-ABL tyrosine kinase activity by imatinib reveals that miR-17-92 expression depends on BCR-ABL in myeloid K562 and 32D/BCR-ABL but not in lymphoid BV173 and TonB cells. Similarly, RNAi against c-MYC reduces miR-17-92 expression in the myeloid but not in lymphoid cell lines demonstrating lineage-specific effects on BCR-ABL-mediated regulation of miR17-92 expression. Furthermore, preliminary chromatin immunoprecipitation analyses in the presence and absence of imatinib demonstrate BCR-ABL tyrosine kinase dependent recruitment of c-MYC to the miR-17-92 5′-regulatory region in myeloid, but not in lymphoid cells. Correspondingly, transcription of the miR-17-92 pri-miRNA is reduced by imatinib treatment in myeloid cell lines. To study the function of individual miRNAs encoded within the polycistronic miR-17-92 cluster in the presence and absence of BCR-ABL, TonB cells were lentivirally transduced with miR-17-19b, a variant of miR- 17-92 selected for efficient transgenic miRNA expression, and with individual miRNAs embedded within miR-30-derived sequences to induce stable miRNA-specific gain-offunction phenotypes (Scherr et al. 2007). Transduction rates were about 98%, and miRqRT- PCR demonstrated increased miRNA-expression between 2- and 14-fold for miR-17- 5p, miR-19b, and miR-20a, respectively. Over-expression of these three miRNAs and the miR-17-19b polycistron had no effect on cell proliferation of TonB cells under stimulation with IL-3 as compared to controls. In contrast, BCR-ABL-dependent proliferation in the absence of IL-3 was reduced by miR-17-5p by about 80% and completely by miR-17-19b in two experiments, whereas miR-19b and miR-20a had only minor inhibitory effects. Interestingly, addition of IL-3 to BCR-ABL+ TonB cells can rescue cell proliferation for all these miRNAs. These data demonstrate

• lineage-specific effects of BCR-ABL on expression of the miR-17-92 cluster and

• may suggest that miRNAs encoded within this polycistron encompass target genes whose expression is required for BCR-ABL-mediated cell transformation.