Myeloma Cells Block Runx2/Cbfa1 Activity in Human Bone Marrow Osteoblast Progenitors and Inhibit Osteoblast Formation and Differentiation.
Authors: Giuliani Nicola , Colla Simona , Morandi Francesca , Bonomini Sabrina , Rizzoli Vittorio 
Published: October 2019

Abstract
The decreased bone formation contributes to the development of bone lesions in multiple myeloma (MM) patients. Runx2/Cbfa1 is a transcription factor highly restricted to the osteoblastic lineage that has a critical role in the osteoblast formation and differentiation. In this study we investigated the potential effect of myeloma cells on osteoblastogenesis and the role of Runx2/Cbfa1 in the decreased bone formation in MM.
In a co-culture system performed in presence or absence of a transwell insert with human myeloma cell lines (HMCLs) (RPMI-8226, U266, XG-1, XG-6, OPM-2) and human bone marrow (BM) pre-osteoblastic cells, obtained from BM stromal cells (BMSC), we observed an inhibition of osteocalcin, alkaline phosphatase and collagen I expression at both mRNA and protein level. Consistently we found that the formation of both Colony Forming Unit Fibroblasts (CFU-F) and Colony Forming Unit Osteoblasts (CFU-OB) were suppressed in long term BM culture by several HMCLs. Moreover, by a gel mobility shift assay (EMSA) we found that Runx-2/Cbfa1 activity was significantly inhibited in BMSC/pre-osteoblastic cells after 48 hours of co-cultures with OPM-2 whereas the level of Runx2/Cbfa1 protein was not affected. The inhibition of osteoblast formation, differentiation and the block of the Runx2/Cbfa1 activity in BMSC/pre-osteoblastic was more pronounced in the cell-to-cell contact conditions as compared to those without the cellular contact.
Further we evaluated Runx2/Cbfa1 expression by immunohistochemistry in BM biopsies of 16 MM patients finding a significant reduction of the number of Runx2/Cbfa1 positive cells in osteolytic patients as compared to those without bone lesions (median %: 19.93% vs. 38%; p=0.001) that supports the in vitro results.
To identify molecules that could contribute to the inhibitory effect of myeloma cells on osteoblast formation, differentiation and Runx2/Cbfa1 activity, we screened HMCLs and fresh purified CD138+ MM cells for the expression of DKK1, IL-7, noggin, gremlin and secreted frizzled-related protein (sFRP)-2, -3, -4 also evaluating the potential involvement of these osteoblast inhibitors. DKK1 mRNA was expressed by 2 out of 5 HMCLs tested and by 8 out of 12 MM patients tested as well as by BMSC/pre-osteoblastic cells and hOB, however soluble DKK-1 at a widely range of concentration had not effect on the number of CFU-F and CFU-OB in human BM cultures and it did not inhibited Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells. Moreover, blocking anti-DKK1 Ab did not blunted the effect of HMCLs on osteoblast formation in human BM cultures. All the HMCLs and fresh CD138+ MM cells tested expressed IL-7 mRNA, as we have previously reported. IL-7 reduced Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells showing an inhibitory effect on CFU-F and CFU-OB formation in BM culture, moreover blocking anti-IL-7 Ab partially reduced the effect of HMCLs in co-cultures. Finally, we found that HMCLs and fresh CD138+ MM cells did not expressed noggin, gremlin, sFRP-2 and rarely produced sFRP-3 and sFRP-4 whereas BMSC/pre-osteoblastic cells and hOB were positive for these factors even if HMCLs had not effect on their expression in co-cultures.
In conclusion our data indicate that human myeloma cells inhibit osteoblast formation and differentiation in human BM cultures blocking Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells through the cell-to-cell contact. Soluble factors produced by myeloma cells as IL-7 could contribute to the inhibitory effect on osteoblastogenesis.