Analysis Of Survivin Expression In BCR/ABL-transformed Cells: Its Roles And Therapeutic Strategy For Leukemia

Background: BCR/ABL can cause chronic myelogenous leukemia (CML) in part by altering the expression of specific genes with growth- and/or survival-promoting functions, which contribute to the maintenance and accumulation of leukemic cells. Recently, BCR/ABL has been shown to activate Survivin, an important regulator that is differentially expressed in cancer and intersects multiple pathways for tumor maintanence, but the precise molecular mechanisms behind its expression and consequences thereof in CML cells remain unclear.Objective: This study aimed to investigate the underlying molecular mechanisms governing Survivin expression in BCR/ABL-transformed leukemia cells, and to explore its roles as well as potential therapeutic strategy for leukemia.Methods: We first compared Survivin expression and intercellular distribution between BCR/ABL-transformed cells and its parental 32Dcl3 cells by using PCR, western blot, immunofluorescence and flow cytometry. Then the luciferase reporter plasmids containing human Survivin promoter as well as its deletion and site-directed mutation were constructed to identify the essential responsive elements for activation of Survivin promoter activity in BCR/ABL-expressing cells. Electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) were performed to confirm the binding of c-Myc to Survivin promoter. We also tested the correlation between c-Myc and Survivin expression in CML cell line and primary cells. Additionally, selective chemical inhibitors of MAPK, PI3K, and JAK2 signaling pathway were employed to determine which of the molecule signals are responsible for Survivin expression. Retrovirus-mediated Survivin expression and specific shRNA sequences against Survivin and c-Myc were used to explore the roles of c-Myc and Survivin in BCR/ABL-mediated transformation and survival. Finally, we use a small molecule c-Myc inhibitor, 10058-F4, to disrupt c-Myc activiy and to evaluate its anti-leukemic effect alone or combined with imatinib on BCR/ABL-dependent and -independent resistance to imatinib. Results: BCR/ABL promoted Survivin expression and its cytoplasmic accumulation in a cell cycle-independent fashion. The increase of Survivin was not a consequence of strengthened mRNA stability, but rather controlled at the transcriptional level through a mechanism mediated by JAK2/PI3K signal pathways that activated c-Myc, leading to transactivation of Survivin promoter by interaction with E-box elements. Moreover, the expression of Survivin is correlated with that of c-Myc. Dynamic downregulation of Survivin was a key event involved in imatinib-induced cell death while forced expression of Survivin partially counteracted the suppressive effect of imatinib and cytokine-deprivation on cell survival. Additionally, shRNA-mediated silencing of Survivin or c-Myc eradicated colony formation of K562 cells in semi-solid culture system, implying an essential role for this transcriptional network in BCR/ABL-mediated cell transformation and survival. Finally, interruption of c-Myc activity by 10058-F4 exerted an anti-leukemia effect with a synergistic interaction with imatinib and overcame the anti-apoptosis effect rescued by IL-3 supplement. Meanwhile, 10058-F4 enhanced the sensibility of K562/G01 cells to imatinib.Conclusions: we have identified JAK2/PI3K-mediated and c-Myc-dependent transactivation of Survivin as a novel pathway in the transcriptional network orchestrated by BCR/ABL. These results suggest that the interference with this circuitry might be a potential utility for CML treatment, both imatinib sensitive and resistant.