AbstractAbstract 2976The alkylating agent melphalan is one of the most active chemotherapeutic agents in the treatment of patients with multiple myeloma (MM). It reacts with DNA, producing mostly N-alkylpurine monoadducts, a small proportion of which goes on to form interstrand cross-links, which play a major role in cytotoxicity. In this report, we investigated the molecular mechanisms of therapeutic action and drug resistance to alkylating drugs using melphalan as a model. We studied 12 healthy volunteers (7M/5F; median age 41 years) and 32 MM patients (14M/18F; median age 59 years) who underwent high-dose melphalan (HDM) therapy with autologous stem cells transplantation (ASCT) as part of their first line therapy. These patients had measurable monoclonal protein in serum and/or urine after induction treatment so that further response after HDM could be assessed. Twenty-three patients achieved a further reduction of monoclonal protein after ACST (responders) and 9 patients did not (non responders). Blood samples were obtained from the healthy volunteers and the patients, within 1 week prior to ASCT and at least 1 month after exposure to any anti-myeloma treatment. Peripheral blood mononuclear cells (PBMCs) were isolated and treated with 10 μ g/mL of melphalan for 1 h at 37°C. Three molecular end-points (chromatin condensation, transcription activity, melphalan-induced DNA damage formation/repair) were measured in four genomic loci (beta-actin, p53, N-ras and delta-globin genes). Furthermore, accumulation of p53 protein, recovery of both total RNA and poly(A) mRNA synthesis as well as induction of apoptosis were also studied. In all subjects, beta-actin, p53 and N-ras genes were transcriptionally active. Importantly, delta-globin gene was silent in all healthy volunteers, while an induction of the transcription activity of this gene was found in 90% of MM patients (29/32). In all subjects, more relaxed chromatin structure and faster repair were observed in regions inside beta-actin, p53 and N-ras genes, compared to regions on both sides of the genes, while for delta-globin such a difference was observed only in MM patients. In all subjects, 5′- to 3′-end gradients of chromatin condensation and repair efficiency were observed along the transcribed strand of the active genes, with higher looseness of chromatin structure and faster repair at the 5′-end. Interestingly, inside all genes analyzed, repair was slower in responders relative to non-responders, the difference being greater and statistically significant at the 5′-end (p<0.003). PBMCs from all healthy volunteers showed evidence of p53 protein accumulation at doses as low as 10μ g/ml melphalan, responders at 75μ g/ml, while non-responders required doses of at least 100μ g/ml. These results suggest that cells with lower repairing activity and higher levels of residual adducts (i.e. PBMCs from healthy volunteers and responders) are more sensitive to melphalan-induced p53 accumulation than PBMCs from non-responders having higher levels of preferential repair. PBMCs from non-responders had almost fully recovered both poly(A) mRNA and total RNA synthesis to control levels by 6h following exposure to 100μ g/ml melphalan. In contrast, PBMCs from all healthy volunteers and most responders (18/23) showed a more extensive and prolonged inhibition of RNA synthesis, suggesting that, following exposure to melphalan, the ability to preferentially repair the transcribed strand of the active genes correlates with a rapid recovery of both total RNA and poly(A) mRNA synthesis. Finally, significant differences in the induction of melphalan-induced apoptosis were found between PBMCs from responders and non-responders to therapeutic treatment. Induction of apoptosis was evident following 10μ g/ml melphalan in all healthy volunteers, 75μ g/ml in most responders (18/23) and at least 100μ g/ml in non-responders, suggesting that PBMCs from healthy volunteers and responders to chemotherapy correlated with an increased susceptibility to melphalan-induced apoptosis. In conclusion, reduced repair efficiency of the transcribed strand of active genes in PBMCs from responders to chemotherapy correlated with an increased melphalan-induced cellular chemosensitivity and better response to chemotherapy for MM. This could lead to the identification of molecular markers to predict response to anti-myeloma therapies. Disclosures:No relevant conflicts of interest to declare.