Reduced Repair Efficiency Correlates with Increased Cellular Chemosensitivity and Better Response to High Dose Melphalan of Patients with Multiple Myeloma

Abstract 2976

The 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.