Accumulation of Endogenous DNA Damage Sustain Malignant Progression and Increase Therapy Resistance in Multiple Myeloma

Introduction: Increased endogenous DNA damage pose a serious threat to cell health, since it may lead to mutagenesis, genomic instability and cellular apoptosis. Therefore, a better understanding of the molecular mechanisms implicated in the formation and repair of the endogenous DNA damage can be exploited for understanding pathogenesis and progression of cancer, as well as for new treatment opportunities. Herein, we report high levels of endogenous DNA damage in multiple myeloma (MM) and characterize its type.

Methods: Malignant bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of 16 patients with monoclonal gammopathy of undetermined significance (MGUS; 9F/7M; median age, 65 years; range, 43-87 years), 18 with smoldering myeloma (SMM; 10F/8M; median age, 63 years; range, 37-88 years) and 41 with MM (19F/22M; median age, 65 years; range, 34-79 years) were analyzed. Patients with MM were categorized according to their outcome to responders (≥PR, n=27) and non-responders (n=14) to subsequent melphalan therapy. PBMCs from 40 healthy controls (HC) were examined in parallel. In untreated cells, the endogenous DNA damage (using alkaline comet assay), the detection of critical markers of the DNA damage response and repair (DDR/R) network (using western blot), basal oxidative stress (using a luminescence-based assay) and the endogenous levels of abasic sites (AP-sites) were analyzed. Moreover, cells were ex vivo treated with 100μg/ml melphalan for 5min and critical DDR/R signals, including fundamental DNA repair mechanisms [nucleotide excision repair (NER) using Southern blot; interstrand cross-links repair (ICL/R) using quantitative-PCR; double-strand breaks repair (DSB/R) using immunofluorescence quantification of γH2AX foci by confocal microscopy] and the induction of apoptosis (using a photometric enzyme-immunoassay) were evaluated. 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: In both BMPCs and PBMCs from MGUS, SMM and MM patients, significant progressive increase in the endogenous single-strand breaks (SSBs) and double-strand breaks (DSBs) was found in MGUS, SMM and MM patients, respectively; PBMCs from HC showed the lowest DNA damage levels (all P<0.05). Interestingly, MM patients, non-responders to subsequent melphalan therapy showed significantly elevated levels of endogenous DNA damage compared with responders (P<0.001). Also, in both tissues analyzed, significant progressive increase in the induction of molecular markers associated with DSBs (γH2AX, p-53BP1) and SSBs (p-RPA32) was observed in MGUS, SMM and MM patients, respectively, confirming the progressive accumulation of both SSBs and DSBs in patients' cells. Moreover, in both BMPCs and PBMCs, significant progressive increase in the oxidative stress and the AP-sites was observed in MGUS, SMM and MM patients; PBMCs from HC showed the lowest levels of both factors analyzed (all P<0.05). Importantly, non-responders MM patients exhibited significantly higher levels of oxidative stress and AP-sites compared with responders (P<0.05). Significant correlations were observed between the endogenous DNA damage and the oxidative stress or the AP-sites in the same patients, suggesting that the endogenous DNA damage may, at least partly, result from oxidative stress and/or the induction of AP-sites. In addition, in both tissues analyzed, substantial progressive increase in the NER, ICL/R and DSB/R efficiencies were observed in MGUS, SMM and MM patients, respectively; PBMCs from HC showed the lowest repair rates (all P<0.001). Interestingly, non-responders MM patients showed higher NER, ICL/R and DSB/R rates than responders (P<0.001). Finally, in both BMPCs and PBMCs a gradual suppression of the apoptotic pathway was observed in MGUS, SMM and MM patients; PBMCs from HC showed the highest apoptosis rates (all P<0.001). Interestingly, non-responders MM patients showed significantly lower apoptosis rates compared to responders (P<0.001).

Conclusion: Increased levels of endogenously generated DNA damage, which are correlated with increased formation of DNA damage and preserved DNA repair, may be implicated in the transformation process of myelomagenesis and the outcome of anti-myeloma therapy.