Roscovitine Sensitizes Leukemia and Lymphoma Cells to TRAIL-Induced Apoptosis and Enhances Cell-Mediated Cytotoxicity

Abstract 596

Roscovitine is a selective inhibitor of cyclin-dependent kinases (CDK) and it is under evaluation in several clinical trials in the treatment of diverse cancers. TNF-related apoptosis inducing ligand (TRAIL) is a death ligand important for tumor immunosurveillance with selective antitumor activity and minimal toxicity toward tissues. Soluble TRAIL is also under evaluation in several clinical trials. Unfortunatelly, many cancers are resistant to TRAIL. To circumvent TRAIL resistance, there is effort to combinate TRAIL with other cytotoxic agents. By measuring apoptosis and proliferation, we demonstrated that combination of low dose roscovitine and low dose TRAIL (low dose= up to 30% of apoptotic cells after 24h treatment) is synergistic in 20 of 21 tested hematologic cell lines including TRAIL resistant cell lines. Moreover, this combination was tested on primary cells from 9 patients with hematologic malignancies with synergism in 4 of 8 samples from patients with acute myeloid leukemia (AML) and 1 sample from patient with mantle cell lymphoma. Remaining 4 AML samples showed additive effect. Based on these results, we decided to explore molecular mechanisms responsible for the synergism between roscovitine and TRAIL using TRAIL-resistant K562 cells. Despite decreased mRNA, the surface expression of TRAIL receptors remained unaffected after 24h roscovitine treatment. Immunoprecipitation of death-inducing signaling complex (DISC) revealed distinct proapoptotic changes (enhanced CASP8 and 10, reduced FLIP at 12 and 24h). These proapoptotic changes suggested that roscovitine might synergize with other death ligands acting through the DISC, namely TNF and FASLG. Indeed, roscovitine significantly sensitized diverse cell lines (K562, DOHH2, RAMOS) to TNF or FASLG-induced apoptosis. We subsequently proved that pretreatment of the cells (K562, DOHH2, RAMOS) with roscovitine increased by approx. 20% the level of cell-mediated cytotoxicity (peripheral blood mononuclear cells from a healthy volunteer marked with carboxyfluorescein succinimidyl ester). Thus, proapoptotic changes of the DISC seem to play essential role in mediating roscovitine-induced sensitization to TRAIL. Despite detected alterations of the DISC, we decided to unveil additional potential changes in the protein levels of key apoptotic regulators by western blotting at 1.5, 3, 6, 12 and 24h timepoints. Like Ortiz-Ferron et al. we detected gradual downregulation of MCL1 that peaked at 12h, followed, however, by substantial upregulation at 24h. We proved that even at this point, i.e. at 24h exposure to roscovitine, the cells were sensitized to TRAIL-induced apoptosis. The role of MCL1 in mediating the proapoptotic change thus remains elusive. BCL-XL showed similar kinetics as MCL1. Several proapoptotic proteins were overexpressed (BAK and BAD at 1.5h, and PUMA at 1.5h and 24h). Gene-expression profiling unveiled additional changes that might contribute to sensitization to TRAIL, e.g. upregulation of proapoptotic death inducer-obliterator 1 (DIDO1) and downregulation of antiapoptotic DNA-damage-inducible transcript 4 (DDIT4). In contrast to TRAIL (and the other death ligands) roscovitine showed only additive effect or even antagonism with the tested genotoxic agents (cytarabine, doxorubicin, fludarabine, etoposide, cisplatin) probably due to the inhibition of CDK2 by roscovitine (Yu et al., Yanjun et al.). We demonstrated that combination of roscovitine and TRAIL is synergistic in hematologic cell lines and primary cells. In addition, roscovitine was shown to have potent immunostimulatory effect by increasing cell-mediated cytotoxicity. Based on our results we suggest that roscovitine-induced sensitization to TRAIL-triggered apoptosis was mediated by proapoptotic changes of the DISC with potential contribution of the proapoptotic changes in the protein expression of the apoptotic regulators (MCL1, BCL-XL, PUMA, BAK, BAD). We also suggest that roscovitine-induced increase in cell-mediated cytotoxicity, known to be mediated in part through death ligands, was also a consequence of the proapoptotic alteration of the DISC. Roscovitine, as a single agent, or in combination with TRAIL, might have a role in the experimental treatment of selected hematologic malignancies.

Financial Support: LC 06044, MSM 0021620806, MSM 0021620808, GAUK 259211/110709, SVV-2010-254260507, IGA MZ NS/10287-3