The Activation of Wnt5a-Mediated Non Canonical Wnt Signaling in Human Bone Marrow Osteoprogenitor Cells Increases Osteoblastogenesis and Counterbalances the Inhibitory Effect of Myeloma Cells on Ror2/FZD5 Expression,
Authors: Bolzoni Marina , Storti Paola , Guasco Daniela , Lazzaretti Mirca , Martella Eugenia , Mancini Cristina , Agnelli Luca , Bonomini Sabrina , Neri Antonino , Donofrio Gaetano , Giuliani Nicola 
Published: October 2019

Abstract
Abstract 3928
Multiple Myeloma (MM) is characterized by the impairment of osteogenic differentiation of bone marrow (BM) mesenchymal stromal cells (BMSC) and osteoblast suppression. Canonical Wnt signal pathway is critical in the regulation of bone formation. However recent evidences suggest that non-canonical Wnt activation by Wnt5a, rather than canonical one by Wnt3a stimulates the osteogenic properties of BMSC. Non-canonical Wnt signaling, mainly activated by Wnt5a, is transduced through FZD receptor (FDZ5) and Ror2 co-receptor to several cascades such as Disheveled pathways involving the Rho family small GTPases or the Ca++ dependent pathways/PKC involving the nuclear factor of activated T cells (NFATc).
Actually, the effect of MM cells on non-canonical Wnt signaling and the role of the activation of this pathway on MM-induced osteoblastic exhaustion are still unknown and have been investigated in this study.
First we checked the expression of non-canonical Wnt related molecules by BMSC and osteoprogenitor cells (PreOB) and found that both cells expressed Wnt5a but lack of express Wnt1, Wnt3a and Wnt8. The presence of the Wnt5a receptors FZD2 and FZD5 was also detected in both cell types as well as of Ror2. Interestingly we found that osteogenic differentiation of BMSC towards preOB significantly increased Ror2 and FZD5 expression. Secondly, we performed a series of co-culture between PreOB and MM cells using either IL-6 dependent (XG-1), and independent (JJN3) human myeloma cell lines or purified primary CD138+ MM cells. We found that XG-1 and CD138+ MM cells inhibit Ror2 and FZD5 expression in PreOB and consistently the activity of NFATc1 at nuclear level.
Thereafter the activation of non-canonical WNT pathway in PreOB, checked by the intracytoplasmatic increase of Ca++influx, phospho-PKC expression and NFATc1 activity, was induced either by Wnt5a treatment or by Wnt5a overexpression through a lentivirus vector. Ror2 overexpression was also performed by lentivirus vector in PreOB. The transcriptional profiles of both PreOB overexpressing Wnt5a and Ror2 have been evaluated by GeneChip® HG-U133Plus 2.0 arrays. The raw intensity signals were extracted from CEL files and normalized using the RMA package for Bioconductor and custom GeneAnnot-based Chip Definition Files in R software. We found that Wnt5a treatment as well as Wnt5a overexpression significantly increased osteogenic differentiation and the expression of the osteogenic markers alkaline phosphatase and collagen I in PreOB. Consistently with these observations, we also demonstrated that siRNA-mediated Wnt5a silencing inhibited these osteogenic markers in the same cell type. Moreover we found that the activation of non-canonical WNT signal pathway in PreOB, blunted the inhibitory effect of MM cells on the osteogenic differentiation process in co-culture. Finally, we show that Ror2 overespression in PreOB activated non-canonical Wnt signaling, increased osteogenic differentiation and restored the osteogenic properties of PreOB in co-culture with MM cells.
In conclusion, our data indicate that activation of non-canonical Wnt5a/Ror2 pathway in BM osteoprogenitor cells increases osteogenic differentiation and counterbalances the inhibitory effect of MM cells suggesting that modulation of Wnt5/Ror2 pathway in the microenvironment could be a target for MM bone disease.

Disclosures:
Bolzoni: Celgene Italy: Research Funding. Giuliani:Celgene: Research Funding; Novartis: Research Funding.