Identification of Roles of microRNAs in Drug Resistance Development of Chronic Myeloid Leukemia

Chronic myelogenous leukemia (CML) is a clonal disease of hematopoietic progenitor cells. The CML oncogenic BCR-ABL kinase in cytoplasm can activate multiple proliferative and anti-apoptotic signaling pathways, causing deregulated cell growth and tumorigenesis. Several mutations have been identified in BCR-ABL gene and result in drug resistance formation. Resistance can be classified as BCR-ABL dependent (primary resistance, e.g., mutation in the BCR-ABL gene) and BCR-ABL independent (secondary resistance, e.g., multidrug resistance gene overexpression). Imatinib mesylate (IM) is currently considered the first line of treatment for patients with CML and the annual expenditure is about NT$1200 million in Taiwan. However, not only IM but also the 2nd generation drugs, such as Nilotinib and Dasatinib induce drug resistance. Therefore, improving the potency of these drugs to relieve drug resistance is an important strategy for CML therapy. Ran GTPase activating protein 1 (RanGAP1) locates mainly in nuclear membrane and once it hydrolyze Ran-bound GTP to GDP will facilitates proteins export through the nuclear pore complex (NPC) to cytosol. Our previous results demonstrated that reduction of RanGAP1 protein level would enhance the cytotoxicity of IM via entrapment of BCR-ABL in nucleus and then induction of apoptosis. However, the mechanisms for RanGAP1 gene regulation are still unclear. MicroRNAs (miRNAs), belonging to non-coding small RNAs, are well-known regulators in mediating cellular physiology and disease formation. Several miRNAs have been developed as drugs for cancer therapy. However, few studies reported the drug resistance-related miRNAs and their functions in CML therapy. Furthermore, whether the RanGAP1 could also be regulated by miRNAs still needs more elucidation. Following these investigations, we will try to identify the drug resistance-related miRNAs, and explore the effects of these miRNAs on the cytotoxicity of drug resistant CML cells. We plan a 3-year proposal and describe briefly as following: Year-1 Specific Aim: Identification of miRNA signatures in drug resistant CML cells Hypothesis: aberrant miRNA expressions affect drug resistance formation in CML cells 1.1 To establish the Imatinib, Nilotinib and Dasatinib-resistant K562 and KU812 cells. 1.2 To explore the downregulated miRNAs in drug-resistant K562 and KU812 cells. 1.3 To identify the MDR-related genes as target genes of candidate miRNAs. 1.4 To test the effects of candidate miRNAs on target genes expressions and singling pathways. 1.5 To test the effects of candidate miRNAs on drug sensitivity of CML cells. Year-2 Specific Aim: Exploring the drug resistance-related miRNAs-mediated networks in CML cells. Hypothesis: miRNAs-mediated unknown target genes involve in CML drug resistance. 2.1 To identify drug resistance-related miRNAs-mediated gene profiles in CML cells. 2.2 To identify the effects of miRNAs on cellular physiology. 2.3 To identify the unknown genes as target genes of overexpressed miRNAs. 2.4 To test the effects of overexpressed miRNAs on target genes expressions and singling pathways. 2.5 To test the effects of overexpressed miRNAs on drug sensitivity of CML cells. Year-3 Specific Aim: The combined treatment effects of miRNAs and drugs in vitro cell model and ex vivo clinical samples. Hypothesis: The expression level of miRNAs and target genes are correlated with the malignancy of CML. Target to miRNAs could enhance drug sensitivity in CML cells. 3.1 To test the combined treatment effects of miRNAs and Imatinib-associated derivatives in CML cells. 3.2 To test the combined treatment effects of miRNAs and Imatinib-associated derivatives in granulocytes form CML patients. 3.3 To assess the relationship between downregulated miRNAs and MDR-related levels or overexpressed miRNAs and unknown target genes levels is correlated with CML malignancy grade.