Adhesion-Mediated Multiple Myeloma (MM) Disease Progression: Junctional Adhesion Molecule a Enhances Angiogenesis and Multiple Myeloma Dissemination and Predicts Poor Survival
Authors: Solimando Antonio Giovanni , Da Via' Matteo Claudio , Leone Patrizia , Croci Giorgio , Borrelli Paola , Tabares Gaviria Paula , Brandl Andreas , Di Lernia Giuseppe , Bianchi Francesco Paolo , Tafuri Silvio , Munawar Umair , Steinbrunn Torsten , Balduini Alessandra , Melaccio Assunta , Rauert-Wunderlich Hilka , Schmiedgen Katharina , Manz Hannah , Ditonno Paolo , Ria Roberto , Henke Erik , Terragna Carolina , Klapper Wolfram , Trinks Nora , Terpitz Ulrich , Corleone Giacomo , Rasche Leo , Rosenwald Andreas , Kortuem Martin , Cavo Michele , Ribatti Domenico , Racanelli Vito , Einsele Hermann , Vacca Angelo , Beilhack Andreas 
Published: February 2020

Multiple myeloma (MM) plasma cell (MMPC) interactions with the microenvironment control MMPC growth, survival, drug-resistance and intra- and extramedullary dissemination. Dissemination of MMPCs through bone marrow niches and in extra-medullary sites is an active process of invasion involving bone marrow endothelial cells, multiple adhesion molecules and chemokine receptors. Since enhanced angiogenesis characterizes MM, we investigated whether junctional adhesion molecule-A (JAM-A) mediated interactions between MM bone marrow endothelial cells (MMECs) and MMPCs impact disease progression.
To this end, we analyzed JAM-A expression levels in MMECs of 312 MM patients in two independent cohorts with flow cytometry, namely 111 newly diagnosed (NDMM) and 201 relapsed/refractory (RRMM) and compared them to 36 monoclonal gammopathy of undetermined significance (MGUS) and healthy subjects. To corroborate our data and investigate at a gene-expression level the prognostic value of deregulated genes (FDR&lt;0.1 & P&lt;0.05) we used a Cox-regression model in the CoMMpass dataset (n=326, IA13 release). The role of JAM-A was evaluated by shRNA knockdown and an anti-JAM-A blocking monoclonal antibody. Subsequently, we functionally validated the JAM-A downstream pathways related to cytoskeleton rearrangement, cell proliferation, epithelial mesenchymal transition, invasion and MM dissemination in vitro and in vivo.
Surface protein expression of JAM-A on MMECs predicted poor overall survival (OS) in NDMM (not reached (NR) vs. 78 months univariate hazards ratio-HR=9.14, 95% CI 2.8-29.76, P&lt;0.0001) and RRMM patients (NR vs. 130 months, HR=2.96, 95% CI 1.37-6.37, P=0.006) with significant impact also in the progression free survival (PFS) in the advanced stage cohort (8.3 vs. 27 months, HR=1.41, 95% CI 1.05-1.88; P=0.019). A sub-analysis on our cohort with extramedullary disease (EMD) MM revealed that the median OS decreased significantly in patients with JAM-Ahighvs. those with JAM-Alow MMEC expression: 84.1 months vs. not reached, irrespective from the EMD status (log-rank=4.19, P=0.04). Strikingly, among NDMM, these results maintained their significance also in the multivariate analysis (HR=9.11, 95% CI 2.79-29.76, P&lt;0.001); within the RRMM cohort the multivariate analyses confirmed JAM-Ahigh MMECs as a statistically significant independent risk factor for short OS (HR=2.39, 95% CI 1.09-5.28, P=0.03) in much the same way were the Revised international staging system (R-ISS) stages such as stage II (HR=5.34, 95% CI 1.24-22.97, P=0.024), stage III (HR=6.57, 95% CI 1.25-34.54, P=0.026) and chronic kidney disease (HR=2.21, 95% CI 1.06-4.62, P=0.034). Cox stratified model implemented for PFS confirmed only JAM-Ahigh MMECs as a statistically significant risk factor (HR=1.35, 95% CI 1.00-1.81, P=0.044) stratified by chronic kidney disease. Notably, also transcriptional upregulation of JAM-A (RNA-Seq data, CoMMpass) corroborated the prognostic impact (log-rank=13, P=0.0003) and revealed a unique gene-expression signature of activated epithelial-mesenchymal-transition, mTOR/PI3K and focal adhesion pathways in high-risk MM patients with EMD and JAM-Ahigh.
Ensuing functional shRNA knockdown of JAM-A reduced MM invasion (P&lt;0.002), angiogenesis (P&lt;0.0001), cell dissemination and migration (P&lt;0.002), cell survival (P&lt;0.001) and expression of cellular-adhesion system molecules such as integrin-beta-1, fibronectin, RAC1 and RHOA (P&lt;0.001). Notably, adding recombinant JAM-A to MMECs enhanced angiogenesis while this was impaired by blocking JAM-A with a specific monoclonal antibody in functional 2D and 3D chorioallantoic membrane-assay and two in vivo MM mouse models. Conclusively, in vivo experiments corroborated our findings that JAM-A blocking halted angiogenesis and reduced MM progression.
Collectively, our findings pinpoint JAM-A as a key player propagating a vicious cycle of MMECs and MMPCs interaction; the expression of JAM-A and the related adhesion pathways can prognostically stratify patients in the late disease stages impacting two main MM progression processes: angiogenesis and extra-medullary dissemination. Therefore, we propose JAM-A as a promising MM biomarker and novel therapeutic target in advanced disease.

Disclosures
Klapper: Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Rosenwald:MorphoSys: Consultancy. Cavo:celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.