Panobinostat Potentiates Melphalan-Induced Apoptosis of Myeloma Cells and Minimizes Untoward Side Effects in Multiple Myeloma

Introduction: Studies demonstrating the dynamic interplay between protein acetylation status, cell cycle progression and apoptosis in multiple myeloma (MM) cells have provided a strong rationale for the use of the nonselective histone deacetylase inhibitor (HDACi) panobinostat, as a therapeutic option for MM. However, despite the promising preclinical data, the clinical responses achieved after treatment of MM patients with single-agent panobinostat have been disappointing. On the other hand, this HDACi has been shown to function synergistically with a range of structurally and functionally diverse chemical compounds, including genotoxic drugs, biologically active polypeptides, and novel immune therapies. Since a better understanding of the molecular pathways targeted by panobinostat in MM cells may provide a better rationale for the selection of new drug combinations with synergistic potential, herein we investigate the mechanistic basis for the action of the combination treatment of panobinostat and melphalan in MM.

Methods: Malignant bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of 26 unselected newly diagnosed MM patients (12F/14M; median age 60 years, range 42-66) were analyzed. PBMCs from 25 apparently healthy controls were examined in parallel. Cells were pre-treated or not with 5nM panobinostat for 24h, then treated with 100μg/ml melphalan for 5min and critical DNA damage response and repair (DDR/R) signals, including fundamental DNA repair mechanisms [nucleotide excision repair (NER) using Southern blot analysis; interstrand cross-links repair (ICL/R) using quantitative-PCR; double-strand breaks repair (DSB/R) using immunofluorescence quantification of γH2AX foci by confocal microscopy], cells' viability, cytotoxicity and apoptosis rates were evaluated. Moreover, in untreated cells, chromatin organization (using micrococcal nuclease digestion-based analysis) and the levels of certain DNA repair proteins associated with NER and DSB/R mechanisms (using western blot) were also analyzed. The study was approved by the Institutional review board of Alexandra Hospital and all subjects provided informed consent. The study was conducted according to the Declaration of Helsinki.

Results: Following ex vivo panobinostat-only treatment, both BMPCs and PBMCs showed acetylation of histone H4 and significant relaxation of chromatin structure. On the other hand, only BMPCs showed a dramatic panobinostat-induced decrease in the levels of critical DNA repair proteins associated with NER (XPC, ERCC3, DDB2) and DSB/R (RAD50, MRE11, KU70, KU80, DNA-PK) mechanisms. No significant effect of panobinostat treatment on the DNA repair proteins was observed in PBMCs from the same ΜΜ patients and in PBMCs from healthy controls. In BMPCs from MM patients, panobinostat potentiates melphalan cytotoxicity, through the inhibition of critical DNA repair mechanisms and the resulting intensification of melphalan-induced DNA damage. Indeed, combined treatment of BMPCs with panobinostat and melphalan significantly reduced the NER, ICL/R and DSB/R capacity of the cells, resulting in significantly increased DNA damage burden compared with melphalan-only treatment (all P<0.01). Moreover, co-treatment of BMPCs with panobinostat and melphalan resulted in significant enhancement of the cytotoxicity and apoptosis rates of the cells, while it significantly reduced BMPCs' viability compared with melphalan-only treatment (P<0.001). In contrast to the BMPCs results, panobinostat pre-treatment of PBMCs from the same patients reduced melphalan-induced toxicity through amelioration of the DNA repair capacity of these cells and the resulting lower melphalan-induced DNA damage burden. Indeed, co-treatment of PBMCs with panobinostat and melphalan significantly increased the NER, ICL/R and DSBs/R capacity of the cells, decreased DNA damage burden, reduced cytotoxicity and apoptosis rates, while it significantly increased PBMCs' viability compared with melphalan-only treatment (all P<0.05).

Conclusion: Our data demonstrate that panobinostat potentiates melphalan-induced apoptosis of myeloma cells, and in parallel it helps minimize untoward side effects, thus paving the way to combination therapies with lower side effects and improved anti-myeloma efficacy.