Chromatin Histone Modifying and DNA Repair Inhibition Enhances the Anti-Myeloma Activity of Melphalan

Repair of DNA and, thus, preservation of the genetic code are critical for normal cellular function. However, tumor cells use DNA repair pathways to develop resistance to chemotherapy. Therefore, inhibiting DNA repair may override this drug resistance. Herein, we further investigate the mechanistic basis of the link between DNA repair efficiency and response to anti-myeloma therapy and provide evidence that histone modifiers might prove efficacious when used in combination with DNA-damaging agents.

We studied clonal bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of 26 unselected newly diagnosed multiple myeloma (MM) patients (12F/14M; median age 60 years, range 42-66) who responded (n=17) or did not respond (n=9) to subsequent melphalan therapy. PBMCs from healthy controls (n=25) were also included in the study. Cells were ex vivotreated with melphalan alone or in the presence of histone deacetylase inhibitor HDACi) vorinostat or the Non-Homologous End-Joining (NHEJ) inhibitor SCR7 and the extent of the DNA damage formation/repair (monoadducts by using Southern blot analysis; interstrand crosslinks by using quantitative-PCR; double-strand breaks repair using immunofluorescence quantification of γH2AX foci by confocal microscopy), the induction of the apoptotic pathway (using a photometric enzyme-immunoassay) and the local chromatin condensation were evaluated. Finally, the expression of a focused panel of 84 genes involved in DNA damage response pathways (ATM/ATR signaling, DNA repair pathways, cell cycle regulation, apoptosis) was also examined.

Both BMPCs and PBMCs from responders to melphalan therapy showed more condensed chromatin structure and slower rates of DNA repair activity (P<0.0022) compared to non-responders. Moreover, apoptosis rates of both BMPCs and PBMCs were inversely correlated with individual DNA repair efficiency, being higher in responders' cells compared to those of non-responders (P=0.0011). PBMCs from MM patients showed higher looseness of chromatin structure, increased DNA repair activity and higher apoptosis rates compared to healthy controls. Microarray analyses of untreated PBMCs consistently point to an altered expression of several DNA damage response-related genes in MM patients' responders to subsequent therapy compared to non-responders. Particularly, responders' PBMCs showed upregulation of ATR, BLM, DDB1, EXO1, FANCA, MRE11A, MSH2, PCNA, RAD50, RAD51, RPA1, XPA, XPC, XRCC1 and TP53 genes and downregulation of ATM, CDC25C, CDKN1A, CHEK2, ERCC1, MPG, PNKP and UNG genes compared to non-responders (in all cases, fold changes between groups were >2, P<0.001), suggesting that perturbation in the molecular components of DNA damage response pathways plays an important role in the therapeutic action of the genotoxic drugs. Interestingly, co-treatment of cells with melphalan and the NHEJ inhibitor SCR7 significantly reduced the rates of DNA damage repair and increased melphalan sensitivity of both BMPCs and PBMCs. Moreover, co-treatment of BMPCs with melphalan and the HDACi vorinostat resulted in hyperacetylation of histone H4, increased DNA damage burden and higher apoptosis rates of these cells. On the other hand, co-treatment of PBMCs with melphalan in the presence of vorinostat did not significantly alter the melphalan-induced DNA damage burden and the apoptosis rates of these cells. Interestingly, using Western blot analysis, we found that vorinostat decreased the levels of critical DNA repair proteins (Rad50, Mre11, Ku70, Ku86, DNA-PKcs) in malignant BMPCs, while it did not suppressed these DNA proteins in PBMCs, a finding that can explain, in part, the selectivity of vorinostat in causing cell death in malignant cells at concentrations that cause little or no normal cell death.

Collectively, responders to melphalan therapy are characterized by slower rates of DNA repair mechanisms, resulting in higher accumulation of the cytotoxic DNA lesions, which in turn triggers the induction of the apoptotic pathway, a priority for successful clinical outcome. The enhancement of melphalan cytotoxicity by DNA repair modifiers offers a promising strategy toward treatment of MM and improvement of existing regimens.