Discovering the Epigenetic Modifications and the Alterations in Cellular DNA Damage Response Pathways That Drive Myelomagenesis; Clinical Implications Into the Biology of Multiple Myeloma

Abstract 299

The aim of this study was to evaluate epigenetic modifications and alterations in cellular DNA damage response pathways that may be implicated in the multistep transformation of myelomagenesis. Peripheral blood mononuclear cells (PBMCs) and plasma cells from bone marrow aspirates were collected from 15 patients with MGUS (8M/7F), 22 with asymptomatic MM (AMM; 10M/12F), 41 patients with symptomatic MM (16M/25F) who underwent autologous stem cell transplantation as part of their first line therapy, and 12 healthy volunteers (7M/5F; only PBMCs). Epigenetics (chromatin condensation, transcription activity) and DNA damage response pathways (melphalan-induced DNA damage formation/repair in four genomic loci including beta-actin, p53, N-ras and delta-globin genes, accumulation of p53 protein and induction of apoptosis) were evaluated.

In both PBMCs and plasma cells and in all genomic regions analyzed, significant differences in the local chromatin looseness between the different groups of patients were observed: healthy volunteers<MGUS<AMM<MM (p<0.02 for all comparisons). In PBMCs and plasma cells from all subjects, beta-actin, p53 and N-ras genes were transcriptionally active, while delta-globin gene was silent in all samples from healthy volunteers and MGUS patients. Notably, an induction of the transcription activity of delta-globin gene was found in 10/22 (45.5%) of AMM and 32/41 (78%) of symptomatic MM patients.

Following a 5-min treatment of PBMCs with 100μg/ml melphalan or plasma cells with 35μg/ml, the efficiency of DNA damage repair inside all genes analysed was in accordance with that of chromatin condensation and gene expression efficiency at the same genomic loci: healthy volunteers<MGUS<AMM<MM (p<0.04 for all comparisons). In particular, in the N-ras gene, PBMCs from healthy volunteers showed 128.6±38.6 adducts/10⁶ nucleotides, from MGUS patients 114.3±26.7 adducts/10⁶nucleotides, from AMM 96.7±20.9 adducts/10⁶nucleotides, and from symptomatic MM patients 56.6±27.2 adducts/10⁶nucleotides. Similarly, plasma cells from MGUS patients showed 95.7±25.0 adducts/10⁶nucleotides, from AMM patients 70.3±21.9 adducts/10⁶nucleotides, and from symptomatic MM patients 32.3±10.2 adducts/10⁶nucleotides. There was a strong correlation for the DNA damage repair data between PBMCs and plasma cells from the same individuals (R²=0.60, p<0.001).

Moreover, following a 5-min exposure of PBMCs and plasma cells with various doses of melphalan (0–120μg/ml), we found that PBMCs from healthy volunteers showed evidence of p53 protein accumulation at melphalan doses as low as 17.9±8.7 μg/ml, from MGUS patients at 29.7±12.5 μg/ml, from AMM patients at 65.6±23.8 μg/ml, and from symptomatic MM patients at 100.2±29.7 μg/ml. Plasma cells from MGUS patients showed evidence of p53 protein accumulation at melphalan doses as low as 20.2±8.9 μg/ml, from AMM patients at 35.2±14.3 μg/ml, while from symptomatic MM patients at 55.3±23.1 μg/ml (p<0.02 for all comparisons). Linear association for the p53 results between PBMCs and plasma cells from the same individuals was observed (R²=0.65, p<0.001).

Also, PBMCs and plasma cells were treated with various doses of melphalan (0–120 μg/ml) for 5 min, and the induction of apoptosis was measured 24h later. In accordance with the p53 data, PBMCs from healthy volunteers showed evidence of induction of apoptosis at melphalan doses as low as 13.2±6.9 μg/ml, from MGUS patients at 20.6±10.8 μg/ml, from AMM patients at 51.4±20.3 μg/ml, and from symptomatic MM patients at 89.7±25.1 μg/ml. Plasma cells from MGUS patients showed induction of apoptosis at melphalan doses as low as 9.9±2.9 μg/ml, from AMM patients at 25.3±8.5 μg/ml, and from symptomatic MM patients at 45.2±19.4 μg/ml (p<0.01 for all comparisons). For apoptosis data, a strong correlation was also found between PBMCs and plasma cells from the same individuals (R²=0.61, p<0.001).

In conclusion, our data indicate that myelomagenesis is associated with epigenetic alterations and modifications in the cellular DNA damage response pathways that can be used as novel molecular biomarkers for early diagnosis and prediction of clinical outcome in MM. Furthermore, in all end-points examined, a strong association between PBMCs and plasma cells from the same individuals was observed, suggesting that measurement of these novel molecular biomarkers can be performed in a readily accessible tissue such as PBMCs.