Bidirectional Notch Signaling Between Multiple Myeloma (MM) Cells and Osteocytes As a Potential Target to Inhibit Tumor Growth and Osteoclast Recruitment in MM

Multiple myeloma (MM) is characterized by monoclonal plasma cells that induce devastating bone disease, by markedly increasing osteoclastic bone destruction, and severely suppressing bone formation. Although the contributions of bone marrow stromal cells, immune cells, and osteoclasts to tumor growth and  bone disease in MM have been extensively studied, little information is available on the role of osteocytes (Ots) in MM, which are the primary regulators of bone homeostasis and comprise 95% of bone cells. We recently showed that MM cells and Ots physically interact in vivo through the osteocytic lacunar-canalicular network. This network allows direct cell-to-cell communication and distributes Ot-secreted molecules among cells in bone and the bone marrow to regulate osteoblast and osteoclast activity. Further, apoptotic Ots recruit osteoclast precursors to specific areas of bone for localized bone resorption.

In the current study we report that Ot-MM cell interactions activate bidirectional Notch signaling between Ots and MM cells which induce: 1) Ot apoptosis, that is maintained by MM-derived TNFα; 2) stimulate Ot and MM cells Rankl production, and 3) markedly increase MM cell growth. For these studies we used co-cultures of murine Ot-like MLO-A5 cells with murine and human MM cell lines or primary patient MM cells in vitro,  6wk-old female SCID mice injected intratibially with human JJN3 MM cells or saline in vivo, and a novel ex vivo calvarial organ culture system with murine 5TGM1 MM cells to provide a more realistic bone microenvironment with an intact osteocytic network.

We found that the prevalence of Rankl-expressing Ots was significantly higher in JJN3-injected mice compared to controls. Similarly, increased Rankl expression in osteocytic cells was observed in co-cultures of Ots with JJN3, 5TGM1 or MM patient cells. Further, inhibition of MM-induced Ot apoptosis with DEVD significantly reduced Rankl levels as did anti-TNFα treatment. We next determined the effects of Ot apoptosis on osteoclast precursor recruitment. Conditioned media (CM) from JJN3 and MLO-A5 cells co-cultured in direct contact enhanced osteoclast precursor chemotaxis by 50% compared to CM from MLO-A5 or JJN3 cells cultured alone. This effect was inhibited by blocking Ot apoptosis with DEVD or by a combination of the Notch inhibitor GSIXX and anti-TNFα. Taken together, these results show that MM-induced Ot apoptosis increases Ot Rankl expression and potentiates Ot-mediated recruitment of osteoclast precursors.

Importantly, we found that the Ot-MM Notch signaling was bidirectional. Direct contact of MM cells with Ot-like cells increased the expression of the Notch target genes Hes1/Hey1 in MM patient and 5TGM1 cells, which was abolished by the Notch inhibitor GSIXX. Moreover, expression of Rankl was increased in 5TGM1 and MM patient cells cultured in direct contact with MLO-A5 cells. Further, MM-Ot-like cell-to-cell contact specifically increased Notch receptor (R) 3 expression in patient MM cells, and Notch R1-3 as well as induced R4 in 5TGM1 cells.  Finally, cell-to-cell contact between MM patient cells or 5TGM1 cells with MLO-A5 cells increased MM cell proliferation that was blocked by GSIXX. Culture of 5TGM1 cells in an ex vivo bone organ system containing authentic murine Ots also activated MM cell Notch signaling, with increased expression of Hey1, and increased the expression of Notch R3 and Rankl. All these effects were inhibited by the addition of Notch inhibitor GSIXX to the ex vivo cultures.

These results indicate that bidirectional Notch signaling between MM cells and Ots increases MM tumor growth, Ots apoptosis and the expression of molecules that regulate osteoclast activity and chemotaxis. They further show that Ots can regulate tumor growth and osteoclast activity in MM and suggest that development of pharmacologic agents that selectively target NotchR3-mediated signaling in primary MM cells should decrease tumor growth and avoid the serious side-effects associated with generalized Notch inhibition.