Semaphorin-4D and Plexin-B1 Are Elevated in Multiple Myeloma Microenvironment and Possibly Contribute in the Development of Lytic Bone Disease
Authors: Terpos Evangelos , Kastritis Efstathios , Bagratuni Tina , Christoulas Dimitrios , Papatheodorou Athanasios , Kanellias Nikolaos , Gkotzamanidou Maria , Eleutherakis-Papaiakovou Evangelos , Gavriatopoulou Maria , Dimopoulos Meletios A 
Published: October 2019

Abstract
Abstract 1819
Multiple myeloma (MM) is characterized by the presence of osteolytic lesions due to increased bone resorption and reduced osteoblast function. It has been recently reported that, during bone remodeling, osteoclasts express semaphorin 4D (Sema4D), previously shown to be an axon guidance molecule, which potently inhibits bone formation through the suppression of insulin-like growth factor-1 signaling and the modulation of osteoblast motility. Sema4D acts through its binding to its receptor plexin-B1. There is no information in the literature for the role of Sema4D/plexin-B1 signaling in myeloma-related bone disease.
To address this issue we evaluated Sema4D and plexin-B1 in the supernatants of six myeloma cell lines (LR5, MR20, L363, U261, H929, and JJN3) and four ovarian cancer cell lines (A2780, C30, OVCA3 and SKOV3) before and after incubation for 24h and 48h with stromal cell line HS5. Furthermore, we measured Sema4D and plexin-B1 in the bone marrow plasma and serum of 72 newly diagnosed patients with MM (37M/35F, median age 70 years) before the administration of any kind of therapy, in 5 newly diagnosed patients with systemic amyloidosis (AL) and in healthy controls (bone marrow plasma, n=5; serum, n=20), gender- and age-matched. Sema4D and plexin-B1 levels were evaluated using ELISA methodology (USCN Life Science Inc., Wuhan, China). In all patients and controls, we also measured serum C-telopeptide of collagen type-I (a bone resorption marker) and bone-specific alkaline phosphatase (a bone formation marker). Evidence of bone involvement in all patients was documented using plain radiographs.
Only one myeloma cell line produced high Sema4D (MR20: 104.45 ng/ml) compared to all others (mean±SD: 1.6±1.4 ng/ml), while there were undetectable Sema4D levels in the supernatants of all ovarian cancer cell lines. Regarding plexin-B1, two myeloma cells lines (H929: 25.3 ng/ml and JJN3: 30.8 ng/ml) and two ovarian cancer cell lines (OVCA3: 5125 ng/ml and SKOV3: 3516 ng/ml) produced high levels compared to the other myeloma (4±2.5 ng/ml) and ovarian cancer cell lines (27.6±3.8 ng/ml). The levels of Sema4D and plexin-B1 in the RPMI+FBS were not detectable. The plexin-B1 levels in the supernatants of the myeloma cell lines (76±140 ng/ml) were decreased compared to the respective levels of the ovarian cancer cell lines (963±1440 ng/ml, p=0.008). After incubation for 24h and 48h with stromal cell line HS5 (Sema4D and plexin-B1 level in the HS5 supernatant was not detectable and 464.4 ng/ml, respectively), there was no alteration in the Sema4D levels of the supernatants of both the myeloma and the ovarian cancer cell lines, while there was a 5- to 8- fold increase in the plexin-B1 levels in all studied cell lines.
The mean Sema4D levels of the bone marrow plasma of the MM patients were 149 ng/ml (±112 ng/ml) and of the Al patients 232 ng/ml (±113 ng/ml), respectively; both dramatically elevated compared to controls (23±12 ng/ml, p<0.01 and p=0.03, respectively). Similarly, the circulating Sema4D concentrations of MM (71±110 ng/ml) and AL patients (75±94 ng/ml) were increased compared to controls (18±10.9 ng/ml; p<0.001 and p=0.045, respectively). There was a strong correlation of Sema4D levels in the serum and in the bone marrow plasma (r=0.628, p<0.001). Sema4D in MM patients correlated with serum calcium (r=0.628, p<0.001) and ISS stage (p=0.037). There was a trend for higher semaphorin levels in patients with osteolysis in plain radiographs compared to patients with no bone disease (p=0.1). Regarding plexin-B1, its bone marrow plasma and serum levels were increased in myeloma patients compared to controls (44±29 ng/ml vs. 3.4±0.8 ng/ml, p<0.01 and 11±20 ng/ml vs. 2.6±2.7 ng/ml, p=0.01, respectively).
Our data suggest that there are increased levels of Sema4D and its receptor plexin-B1 in both the bone marrow plasma and the serum of patients with symptomatic, newly-diagnosed MM. These high sema4D correlated with hypercalcemia and ISS stage and possibly contribute to osteolytic bone disease related to myeloma. Further studies with higher number of patients will reveal the exact role of Sema4D/plexin-B pathway in MM.

Disclosures:
No relevant conflicts of interest to declare.