Down-Regulation of RanGAP Augments Nucleus BCR-ABL Level to Potentiate the Drug Efficacy of Imatinib in Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a clonal disease of hematopoietic progenitor cells. 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 2 nd generation drugs, such as Nilotinib and Dasatinib induce serious side effects. Therefore, improving the potency of these drugs to relieve side effects is an important strategy for CML therapy. The CML oncogenic BCR-ABL kinase in cytoplasm can activate multiple proliferative and anti-apoptotic signaling pathways, causing deregulated cell growth and tumorigenesis. On the contrary, the nuclear BCR-ABL kinase is able to activate P73 apoptotic pathway. Therefore, entrapment of BCR-ABL into the nucleus can be used as a therapeutic strategy to selectively lead CML cells undergoing cell death\ Ran GTPase activating protein (RanGAP) 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 preliminary results demonstrated that RanGAP protein level in CML patients and K562 cells is much higher than that of normal volunteer group. Knockdown of RanGAP by shRNA is able to sensitize K562 cells to IM treatment, suggesting that down-regulation of RanGAP may potentiate IM efficacy to CML. Therefore, we hypothesize that reduction of RanGAP protein level would enhance the cytotoxicity of IM via entrapment of BCR-ABL in nucleus and then induction of apoptosis. Following this line, we plan a 3-year proposal and describe briefly as following: Year-1 Specific Aim: To investigate whether RanGAP affects the drug cytotoxicity of IM to CML cells via in vitro and ex vivo model. Hypothesis: RanGAP-mediated BCR-ABL cytoplasm export affects IM efficacy in CML cells. 1.1 To examine RanGAP level in normal granulocytes, K562 cells, and CML patients. 1.2 To examine whether RanGAP regulates nuclear export of BCR-ABL in K562 cells. 1.3 To test whether suppressed RanGAP expression enhances IM- induced anti-growth effects in K562 cells. 1.4 To test whether IM-treated K562 cells would undergo apoptosis efficiently from the nuclear accumulation of their own BCR-ABL oncoprotein. 1.5 To investigate whether reduction of RanGAP level enhances the cytotoxicity of IM in granulocytes of CML patients. Year-2 Specific Aim: To investigate the molecular mechanism of RanGAP in IM-induced apoptosis. Hypothesis: miRNAs and SUMOylation are involved in regulation of RanGAP expression. 1.1 To screen potential miRNAs could be implicated specifically in the the pathogenesis of CML. 1.2 To test which miRNA inhibits RanGAP expression level in K562 cells. 1.3 To test which miRNA affects transcriptional factor expression to inhibit RanGAP expression level in K562 cells. 1.4 To investigate whether SUMOylation increases RanGAP protein stability to reduce the cytotoxicity of IM in K562 cells. 1.5 To assess whether indicated-miRNA enhances the cytotoxic effect of imatinib in K562 cells. Year-3 Specific Aim: To examine whether reduced RanGAP expression by indicated-miRNA also enhances the cytotoxicity of Imatinib-associated derivatives via ex vivo model. Hypothesis: The expression level of RanGAP and indicated-miRNA are correlated with the malignancy of CML 1.1 To assess whether RanGAP expression level is correlated with CML malignancy grade. 1.2 To assess the correlation between the indicated-miRNA level and the malignancy of CML cells. 1.3 To investigate whether microRNA-mediated RanGAP level reduction enhances the cytotoxicity of Imatinib-associated derivatives in granulocytes from CML patients.