Differential Activity of Brain Derived Neurotrophic Factor (BDNF) and RANKL in Promoting Osteoclast Activity in Multiple Myeloma Patients with or without Bone Disease

Purpose: Brain derived neurotrophic factor (BDNF) which plays an important role in the development of neuronal cells, has a high affinity receptor TrkB and expressed in malignant plasma cells and osteoblasts.Recent reports onage related changes in human serum levels of BDNF drew our attention to further investigate whether soluble BDNF from myeloma cells contributes for the bone related osteolytic activities in MM patients. To answer this question, we analyzed samples from three group of MM patients; (a) bone related early stage, (b) late stage and (c) non-bone related early stage MM (n=10 each).

Sample description and assays: Samples were obtained from patients who had given written informed consent and approved by the Institutional Review Board of NYU School of Medicineandserum samples of healthy volunteers were used for control. Multiplex luminex assay was performed to examine serum samples in triplicate (100 μl each) to measure the following analytes: BDNF, RANKL, NGF, FGF, IL-6, MIP-1α, VEGF, DKK1, OPG, and TGF-β. Immuno-profiling was performed using Luminex x-MAP technology (Luminex Corp, Austen Tx) or with Milliplex multi-analyte panels from EMD Millipore (Billerica, MA). Primary plasma cell culture supernatants were also analyzed for BDNF, NGF, FGF, IL-6, MIP-1α, OPG, RANKL and TGF-β. Data collected on a Luminex 200 instrument was further analyzed using xPONENT 3.1 software (Luminex) with 5-parameter logistic curve fitting. All standard curves generated had R² values calculated at or close to 1 and percent recovery was between 80-120%.

Results: More than 90% (9/10)of the serum samples from non-bone related and/or early stage MM patients showed an elevated level of BDNF (1135±7pg/ml) vs. control (217±7pg/ml, >5 fold) in comparison to that in bone related MM (870±12pg/ml). However, the nerve growth factor (NGF) level in the serum was found to be at a minimum detectable level of 30±3pg/ml in non-bone related MM. We observed elevated level of RANKL (305±9pg/ml, >6 fold compared to the control) in late stage bone related MM in contrast to a lesser amount in non-bone related disease (58±2pg/ml). As expected, our findings indicated a decrease in osteoprotegerin (150±9 and 115±11pg/ml) in the serum samples of early and bone related MM respectively. Although several factors were identified to be the cause for an elevated serum BDNF, in this study we asked the question of whether hypoxic conditions could influence the secretion of BDNF in MM cells. To answer this, primary plasma cells from human and mouse were exposed to hypoxia (1% oxygen level) for 24 h and the soluble BDNF measured. We found >2 fold increase in BDNF under hypoxic conditions (1530±2pg/ml) associated with an increase in RANKL (534±9pg/ml) and TGF-β (1300±23pg/ml) in non-bone related human MM. We found an increase in (>67%) differentiating preosteoclasts that are positive for TRAP activity in the presence of an exogenous source of BDNF. This effect was significantly blocked by a neutralizing BDNF antibody and/or RANKL inhibitor.

Conclusion: In summary, our data provide evidence for a role of BDNF in MM patients who are presented with non-bone related MM and a risk for progressive MM. Additional experiments conducted to dissect a clear mechanism reveal a potential synergy between BDNF, TrkB and RANKL in promoting osteoclast activities. Overall, our findings provide evidence on the role for BDNF and RANKL that could synergistically interact with proinflammatory cytokines in promoting preosteoclast differentiation at early stages of MM that is not involving the bone.