Comparison of Proteasome Inhibitors-Induced Cardiotoxicity (carfilzomib vs. bortezomib): Insights into the Molecular Mechanisms

Introduction: Proteasome inhibitors are widely used for the treatment of multiple myeloma (MM). Carfilzomib is an irreversible proteasome inhibitor, which has been licensed for the treatment of relapsed/refractory MM. In phase 3 trials, carfilzomib has been associated with higher heart failure and cardiotoxicity rates (grade ≥3: 4-6%) compared with the reversible inhibitor, bortezomib (grade ≥3: <2%). However, the mechanisms of heart damage by carfilzomib have not been fully identified yet. The aim of this study was to investigate the molecular mechanisms of carfilzomib-induced cardiotoxicity compared to bortezomib in vivo .

Methods: Male C57BL/6 mice, 12 weeks old, were randomized into the following groups: 1. Control group (Ν/S 0.9%, n=7); 2. Bortezomib group (1mg/kg ip, n=9); 3. Carfilzomib group (8 mg/kg ip, n=8). Drugs were administered every 48 hours for 6 days. At baseline and at the end of treatment the mice underwent echocardiographic assessment. Animals were sacrificed and blood and myocardial tissue samples were obtained for the analysis of proteasome peptidases activity, signaling pathways and molecular mechanisms. Protein kinase Akt, along with its downstream NO synthases; endothelial (eNOS) and inducible (iNOS), were identified as targets of possible endothelial dysfunction and inflammation. Moreover, the transcription factor FOXO1, downstream target of Akt and AMPKα, was identified in order to investigate possible changes in the expression of apoptotic factors. Finally, AMPKα was identified since - besides phosphorylating eNOS and FOXO1 - it functions as a regulator of autophagy. Blood samples were used to measure levels of malondialdehyde (MDA), a marker for oxidative stress (lipid peroxidation).

Results: Administration of bortezomib or carfilzomib resulted in significant reduction of the assayed chymotrypsin-like (CT-L) and caspase-like (C-L) proteasomal peptidase activities in the hearts of treated mice vs . controls. Echocardiography at day 6 demonstrated a statistically significant reduction in the fractional shortening (FS) in the carfilzomib group compared to the bortezomib and control groups (39.87±0.47% vs. 42.10±0.82% and 42.05±0.64 respectively, p <0.05). Western blot analysis in the carfilzomib group demonstrated a decrease in pAkt/tAkt (0.54 fold change of control, p<0.05), pAMPKα/tAMPKα (0.05 fold change of control, p<0.001) and peNOS/teNOS (0.41 fold change of control, p<0.05) levels and an increase in the expression of iNOS (5.16 fold change of control, p<0.01) compared to the control group. In contrast, no statistically significant differences were observed in the above mentioned signaling molecules between bortezomib and control. In the carfilzomib group a decrease in pAkt/tAkt (36.73% of bortezomib, p<0.001), pAMPKα/tAMPKα (4.07% of bortezomib, p<0.0001) and an increase in the expression of iNOS (607.06% of bortezomib, p<0.01) compared to the bortezomib group was observed. Levels of pFOXO1/tFOXO1 decreased in both carfilzomib and bortezomib groups versus the control group (0.24 and 0.26 fold change of control respectively, p<0.001). Interestingly, in the bortezomib group, reduction in body weight (median 20.10 mg; range 18.20 to 25.20 mg) versus control (median 24.80 mg; range 24.00 to 26.80 mg; p<0.01) and carfilzomib (median 23.60 mg; range 22.20 to 25.20 mg; p<0.05)) and increased levels of MDA in plasma (median 13.08 uM; range 8.09 to 17.21 uM), versus control (median 8.30 uM; range 5.30 to 10.86 uM; p<0.05) and carfilzomib (median 6.18 uM; range 4.89 to 7.79 uM; p<0.01), combined with a 43.75% increase in mortality (compared to zero mortality in carfilzomib and control groups), were observed.

Conclusion: The present study demonstrates that carfilzomib induces cardiac dysfunction via interfering with the Akt/eNOS axis and via simultaneous increase of iNOS, leading to possible endothelial dysfunction and inflammation. Importantly the decrease of phosphorylation of AMPKα observed in the carfilzomib group seems to be an essential mechanism for carfilzomib induced FS reduction. Moreover, AMPKα and Akt inhibition results in decreased FOXO1 phosphorylation with possible induction of apoptosis and autophagy. On the contrary bortezomib seems to cause premature death in the mice through a different mechanism that requires further investigation.