Novel CXCR4-Targeted Therapy to Inhibit Multiple Myeloma Bone Dissemination
Authors: Roccaro Aldo M , Sacco Antonio , Moschetta Michele , Mishima Yuji , Maiso Patricia , Huynh Daisy , Chiarini Marco , Cardarelli Pina M. , Cohen Lewis , Kuhne Michelle , Ghobrial Irene M. 
Published: October 2019

Abstract
Background. Presence of multiple osteolytic lesions is one of the main clinical features of patients with active multiple myeloma (MM), thus suggesting the ability of clonal plasma cells to disseminate from bone to bone. The bone marrow (BM) homing process of MM cells has been shown to be supported by the activation of the CXCR4/CXCL12 axis. Nevertheless, the role of CXCR4 in mediating MM cell bone metastasis, and the role of CXCR4-targeted therapy in inhibiting MM cell dissemination from bone-to-bone has not been previously reported.
Methods. We dissected the in vivo functional relevance of CXCR4 in mediating MM cell dissemination, tumor growth and survival, by using gain- and loss-of function approaches. Tumor growth was monitored by bioluminescence imaging (BLI) and MM cell homing and engraftment within the BM niches was evaluated by performing intravital confocal microscopy. CXCR4-oeverexpressing (CXCR4+) MM cells were tested in vivo using a model of bone-to-bone dissemination. Empty vector or CXCR4+ cells were loaded into murine femurs and subsequently implanted s.q. into recipient mice. The ability of MM cells to disseminate from the implanted bone to the host bones was evaluted by flow cytometry and immunohistochemistry (IHC; CD138; hematoxylin-eosin) on the harvested host bones. Modulation of EMT-related genes (twist, snail, slug, E-cadherin, vimentin) was evaluated ex vivo by qRT-PCR. A novel anti-CXCR4 monoclonal antibody (BMS-936564) was tested both in vivo and in vitro to define the possible role in modulating MM cell dissemination and growth.
Results. CXCR4-overexpression led to changes in actin cytoskeleton reorganization of MM cells with protrusion of cell pseudopodia, compared to empty vector-transfected cells, sustained by modulation of EMT-related markers, as shown by up-regulation of Slug, Snail and Twist, together with down-regulation of E-cadherin, in CXCR4-overexpressing (CXCR4+) MM.1S cells, compared to empty vector-transfected cells. CXCR4+ MM cells presented with enhanced invasive properties, compared to control cells. These findings were next recapitulated in vivo using an in vivo model of MM cell dissemination: CXCR4+ MM cells presented with higher ability to metastasize from bone to bone compared to control cells, as confirmed on harvested host femurs by using IHC and flow cytometry. Lower expression of human (h)-E-cadherin, and higher mRNA expression of h-Twist, h-Snail, h-Slug was confirmed within the BM of the host femurs. We next tested the novel monoclonal antibody, anti-CXCR4 (BMS-936564), in vivo. BMS-936564 exerted an anti-MM activity in situ, within the s.q. implanted bones; and also reduced MM cell dissemination from the implanted bone to the host bone. We examined whether BMS-936564 might have modulated EMT-related genes in the MM cells metastasized to the host bones, and found higher mRNA expression of human (h)-E-cadherin, together with reduced mRNA expression of h-Twist, h-Snail, h-Slug, h-vimentin and h-CXCR4 in the BM cells harvested from the host bones of mice treated with BMS-936564. Similar findings were confirmed in a model of breast cancer in vivo. CXCR4 silencing in MM cells led to inhibition of MM cell homing to the bone marrow, as shown by using intra-vital confocal microscopy and inhibition of MM tumor growth in vivo, as shown by BLI. Prolonged survival was documented in those mice that were injected with CXCR4-silenced MM cells compared to control mice injected with scramble probe-infected MM cells.
Conclusion. CXCR4 enhanced the acquisition of an EMT-like phenotype in MM cells, leading to higher bone-to-bone dissemination of clonal plasma cells in vivo; while CXCR4-silencing in MM cells led to inhibited tumor growth and improved survival. BMS-936564 led to inhibited MM cell bone-to-bone dissemination, supported by suppression of the CXCR4-driven EMT-like phenotype. These studies suggest that targeting CXCR4 may represent a novel therapeutical strategy to prevent MM cell dissemination.

Disclosures
Cardarelli: BMS: Employment. Cohen:BMS: Employment. Kuhne:BMS: Employment. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium/Takeda: Membership on an entity's Board of Directors or advisory committees.