P-372: Transcriptomic expression of BMP-2, BMP-6 and Smad6 genes in newly diagnosed and relapsed multiple myeloma patients

Introduction: Extensive research has been conducted to elucidate the molecular mechanisms underlying the pathophysiology of Multiple Myeloma. The role of BMPs (Bone Morphogenetic Proteins) and their downstream gene expression regulators, such as Smad (Small Mother Against Decapentaplegic) transcription factors in the molecular-level imbalance of bone metabolism remains uncertain. Most studies were conducted in preclinical models which did not account the effect of the human microenvironment. In our study, we investigated the transcriptional levels of BMP-2, BMP-6 and Smad6 in human bone marrow aspirates.

Methods: Our study included 39 patients (19 newly diagnosed patients, 20 refractory/relapsed patients) and 10 controls (patients with other non-Hodgkin lymphomas without bone marrow infiltration). RNA isolation of bone marrow mononuclear cells was conducted using the Omega Biotek® EZNA Total RNA kit, and RNA quantification by using a Nanodrop2000® spectrophotometer. cDNA synthesis was performed using the QuantiTect® Reverse Transcription Kit (50), Qiagen®. The primers for BMP-2, BMP-6, and Smad6 were designed by Qiagen®. B2-microglobulin was the reference gene. The RT-PCR was conducted using the QuantiNova SYBR Green PCR kit from Qiagen®. We validated our PCR products using gel electrophoresis and melt curves. PCR reactions were conducted in duplicate. Differential expression was calculated using the ΔΔCt method and graphs were created using the GraphPad Prism program.

Results: There was no significant difference in gene expression of BMP-2 between controls and newly diagnosed patients (p=ns), and relapsed patients (p=ns). On the other hand, BMP-6 was found to be increased by 4.8 times in newly diagnosed patients (p = 0.0004) and 2.56 times in relapse patients (p = 0.0226). Finally, for Smad6, no statistically significant difference was found between controls and newly diagnosed patients (p = 0.2861), but it decreased by 1.5 times in relapsed patients (p = 0.012).

Conclusions: BMPs and, to a lesser extent, Smads have been investigated in the past as potent regulators of bone metabolism in Multiple Myeloma, mostly in preclinical setting. Our findings in bone marrow aspirates of myeloma patients suggest that BMP-2 did not show a significant difference between controls and patients, indicating that it may not have a significant impact on bone metabolism. Conversely, the increased BMP-6 levels may indicate either increased fixation in the matrix and an inability to act through a positive feedback mechanism or involvement in the induction of genes that inhibit apoptosis. Smad6 expression is reduced in R/R patients, suggesting that MM cells have developed mechanisms of dependence on BMPs and induces Smad6 repression through alternative signaling pathways or inhibitors. The role of BMPs and Smads still appears unclear; further experiments may elucidate their role on signaling pathways in MM.