A COMBINATION OF BORTEZOMIB WITH CX-4945, A CASEIN KINASE 2 (CK2) INHIBITOR, HAS SYNERGISTIC CYTOTOXIC EFFECTS IN ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) CELL LINES

Introduction. The proteasome inhibitor bortezomib is a new treatment option for patients with refractory or relapsed ALL, particularly when used in combination with conventional chemotherapy or other targeted agents. Indeed, a limited efficacy of bortezomib alone in ALL patients has been reported. A terminal pro-apoptotic endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is one of the several mechanisms of bortezomib-induced apoptosis. CX-4945, a potent CK2 inhibitor, has been found to induce apoptotic cell death in T-ALL preclinical models, via perturbation of ER/UPR pathway. Here, we have explored the cytotoxic effects of a bortezomib/CX-4945 combination in a panel of B- and T-ALL cell lines. Methods. B- (KOPN-8, NALM-6, RS4;11) and T- (MOLT-4, Jurkat, CEM-R) ALL cell lines were pretreated with bortezomib (Selleck Chemicals, Houston TX, USA), for six hours and then with CX-4945 (Selleck Chemicals, Houston TX, USA), for 16/24 hours. MTT assays were performed to analyze cell viability. Apoptosis induction was evaluated by Annexin V/PI staining and flow cytometric analysis. Protein expression was studied by western blot. Results. Cells were cultured in the presence of bortezomib or CX-4945, either alone or in combination at a fixed ratio. The combined treatment was more effective in reducing cell viability in all B-ALL cell lines and in MOLT-4 cells. Annexin V/PI staining was performed; interestingly, no synergism was detected if the two drugs were administered together since the beginning of treatment. In response to treatment with 2.5 nM bortezomib followed by 5 μM CX-4945, we detected an increase in apoptosis in B-ALL cell lines and in MOLT-4 cells after 24 h. Western blot analysis for the cleaved forms of caspase-3 and PARP, confirmed a higher apoptosis induction by the combined treatment. A reduction in anti-apoptotic Bcl2 concomitant with an increase in proapoptotic Bax, suggested that bortezomib/CX-4945 treatment caused a mitochondrial apoptosis. IRE1a and CHOP (established markers of ER stress/UPR-mediated apoptosis) levels increased in response to the combined treatment, in contrast the expression of GRP78/BIP (a marker of UPR activation) decreased, suggesting that a potential mechanism by which the drug combination induced cell death, involved ER stress induction by bortezomib and the inability to respond by adequate activation of the UPR signaling which was blocked by CX-4945. Conclusions. Here, we demonstrated that the proteasome inhibitor bortezomib and the CK2 inhibitor CX-4945 interact in a synergistic manner to induce apoptosis both in B- and in T-ALL cell lines. Drug cytotoxicity was associated with modulation of the ER stress/UPR signaling pathway. Importantly, the synergism was observed only when bortezomib treatment preceded CX-4945 administration. Therefore, our findings support clinical application of bortezomib in combination with CX- 4945 in B- and T-ALL treatment.