Modulation of Multiple Myeloma Apoptosis and Adhesion by Seliciclib, A Selective Inhibitor of Cyclin Dependent Kinase (CDK)

Abstract 3971

Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cells that eventually develop resistance to chemotherapy. Novel agents such as Thalidomid, Bortezomib and Lenalidomide improve response rate and prolong progression free and overall survival. Drug resistance, differentiation block and increased survival of the MM tumor cells result from genomic alterations including high levels of cyclin D. In addition, MM displays a high genomic instability. The most common translocations in MM lead to dysregulation of cyclin D (CCND), a regulatory protein that governs the activation of a key cell cycle regulator – cyclin dependent kinase (CDK). Cell-proliferation is tightly regulated by activity of CDKs at checkpoints located at the boundaries of cell cycle stages. The association of CDK4/6 with CCND is critical for G1 phase progression, whereas CDK2-cycllin E is necessary for S phase initiation. Deviation from the orderly control may lead to malignant transformation. Furthermore, genomic instability was reported to be affected by over expression of cyclin E. Cyclin E deregulation induces genomic instability as an early event in tumorgenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore, sought to investigate the role of cyclin E in MM. We first examined the expression profile of CCNE1 in various MM cell lines (hMMCLs) including ArP-1, ARH77, CAG, NCIH929, RPMI8226 and U266 by immunoblotting utilizing CCNE1 Abs and by qRT-PCR. CCNE1 expression was found to be heterogeneous being high in U266 and NCIH292, intermediate in ARH77, CAG and PRMI8226 and low in ArP-1 MM cell lines, respectively. A direct correlation between the level of protein and the transcript was observed. We then evaluated the effect of Seliciclib (Roscovitine, CYC202), a selective inhibitor of CDK's on hMMCLs viability, using the MTT assay as hMMCLs have been reported to be sensitive to CDK inhibition. The various hMMCLs were incubated with increasing concentration of Seliciclib (0-100 μM) for 3 days. Seliciclib inhibition of CDK resulted in dose-dependent cytotoxicity with IC50 of 20–80 μM with PRMI8226 being the most sensitive, while ARH77 hMMCL the most resistant. Cell cycle study demonstrated increased apoptosis (based on sub G1 and annexin V analysis) in the sensitive hMMCLs, with no effect on the resistant cell lines. Annexin V binding was observed within 5h and reached maximal level 12h flowing Seliciclib (50 μM) exposure. We next assessed the effect Seliciclib on MCL1, an anti-apoptotic BCL2 family member that is consistitutively expressed in MM cells. Seliciclib significantly down-regulated MCL1 expression in a time and dose dependant manner. Assessing the effect of Seliciclib on cell cycle regulators including CCND, CCNE and P27kip1 indicated a very significant reduction in CCND2 and P27 expression levels in most of the hMMCLs in a dose and time dependant manner, while CCNE1 expression levels increased. Transfection of the low expressing hMMCL RPMI8226 with CCNE1, inducing over expression of cyclin E, resulted in reduced sensitivity of the MM tumor cells to the Seliciclib induced cell death in comparison to the paternal cell line. Lastly, we investigated the effect of Seliciclib on MM cell adhesion to fibronectin (FN), as cell-adhesion-mediated-drug-resistance (CAM-DR) is a major importance protecting MM tumor cells from cytotoxic drugs induced apoptosis. hMMCLs were pre-incubated with increasing concentration of Seliciclib for 1h, plated onto 40 μg/ml FN coated plates for 1h, washed and stained by crystal violet. Seliciclib resulted in a direct dose dependant inhibition of adhesion. The adhesive protective property was evalued by CAM-DR assay, whereas FN-adhesion protected the MM tumor cells against doxorubicin mediated cytotoxicity. There was no difference between the cytotoxic effects of Seliciclib on FN-MM adherent vs. suspended tumor cells. In summary incubation of MM tumor cells with Seliciclib, a selective inhibitor of CDK's, results in apoptosis which is mediated by down regulation of MCL1 and is accompanied by reduced adhesion to FN. Its ability to prevent adhesion – mediated – drug resistance of MM tumor cells may turn Seliciclib as essential component of modern anti MM drug combination therapy. Further experiments should test its synergism with anti MM novel agents.