Cytotoxic Effect of Proteasome Inhibitor Bortezomib in Combination with Purine Nucleoside Analogues on Chronic Lymphocytic Leukemia Cells in Vitro.
Authors: Smolewski Piotr , Duechler Markus , Linke Anna , Cebula Barbara , Schwarzmeier Josef D. , Robak Tadeusz 
Published: April 2020

Abstract
Background: Bortezomib (Velcade, PS-341), a selective inhibitor of the 26S proteasome which was recently approved for the treatment of patients with refractory multiple myeloma, showed also anti-leukemic activity in several studies. Bortezomid alone was found to induce apoptosis of chronic lymphocytic leukemia (CLL) cells in vitro. So far, there was no data on effects of its combination with other drugs routinely used in this disease.
Aim: Cytotoxic activity of bortezomib in combination with purine nucleoside analogues, fludarabine (FA) and cladribine (2-CdA) was tested in CLL cells in vitro.
Methods: The study was performed on CLL lymphocytes derived from 26 patients. Cell viability was assessed by propidium iodide staining. Apoptosis was evaluated by both Annexin-V and caspase activation flow cytometry assays. Additionally, expression of the apoptosis-regulating proteins Bax, Bak, Bid, Bcl-w, Bcl-2, XIAP and Mcl-1 was evaluated in CLL cells.
Results: Bortezomib alone induced significant, dose-dependent cytotoxicity, inducing apoptosis of CLL cells. The viability of CLL cells treated with bortezomib decreased from 73.4%±14.9 (at 1.25nM) to 39.1%±15.4% (at 10nM). The mean apoptotic index estimated after 24h of treatment was in a range from 29.9%±18.0% at the dose of 1.25nM to 66.1%±21.8% at 10nM of bortezomib. Combination of this agent with FA or 2-CdA resulted in an increased cytotoxicity, comparing single drugs. It was especially evident when 5nM of bortezomib was combined with suboptimal doses of FA or 2-CdA. Cytotoxic effect of 5nM bortezomib +FA was significantly higher than 5nM bortezomib (p=0.0004) or FA (p&lt;0.0001) alone. Similarly, cytotoxicity of 5nM bortezomib +2-CdA combination was significantly higher than 5nM bortezomib or 2-CdA used as a single agents (p=0.001 and p&lt;0.0001, respectively). The combination index (CI) was 0.82 for bortezomib+FA and 0.87 for bortezomib+2-CdA, indicating the additive effect of these combinations. Corresponding to enhanced apoptosis, the expression of some apoptosis-regulating proteins was altered. Namely, XIAP protein was moderately downregulated by 2-CdA and FA, but not changed by bortezomib alone. However, combination of bortezomib with 2-CdA or FA significantly decreased XIAP expression (0.42-fold and 0.53-fold decrease, respectively; p=0.0007 and p=0.003, respectively). Bortezomib alone did not influence Bid levels, but its combination with 2-CdA induced a significant, 1.58-fold increase of Bid expression, comparing to the untreated control (p=0.028). Combination with FA also upregulated Bid protein (mean increase 1.39-fold vs. control), however, this difference was not statistically significant (p=0.582). The level of Mcl-1 protein was moderately elevated in the cells treated with bortezomib alone (1.41-fold increase; p=0.501). 2-CdA or FA only slightly decreased Mcl-1expression, but both purine nucleoside analogues prevented the Mcl-1 upregulation when combined with bortezomib.
Conclusion: This study provides the strong rationale for the use of bortezomib in combination with purine nucleoside analogues for the treatment of CLL. This combination downregulates the expression of apoptosis inhibitors XIAP and Mcl-1 and upregulates proapoptotic Bid protein in CLL cells.