Adhesion-Mediated Multiple Myeloma (MM) Disease Progression: Junctional Adhesion Molecule a Enhances Angiogenesis and Multiple Myeloma Dissemination and Predicts Poor Survival

Abstract

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<0.1 & P<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<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<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<0.002), angiogenesis (P<0.0001), cell dissemination and migration (P<0.002), cell survival (P<0.001) and expression of cellular-adhesion system molecules such as integrin-beta-1, fibronectin, RAC1 and RHOA (P<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.

Cancer-Associated Angiogenesis: The Endothelial Cell as a Checkpoint for Immunological Patrolling

“…Recently, we demonstrated that bone marrow endothelial cells from both newly diagnosed (NDMM) and relapsed-refractory multiple myeloma (RRMM) patients feed into a vicious cycle orchestrated by aberrant adhesion molecules on the bone marrow endothelial cells and plasma cell surface and correlate with poor clinical prognosis [ 31 , 35 , 88 ]. Based on this evidence and several pieces of data [ 89 , 90 ], increased adhesion molecules levels have been uncovered to contribute to more aggressive phenotype [ 29 , 91 ].…”

“…Consequently, using several pre-clinical models [ 30 , 88 , 102 ], blocking the adhesion system seems to halt blood vessel formation, reduce adhesion-mediated networks, and weaken neoplastic disease progression. These therapeutic effects of interfering with the adhesion system were observed in translational animal models, not in patients and, therefore, must be interpreted with caution.…”

New Insights into Diffuse Large B-Cell Lymphoma Pathobiology

“…Lymphoproliferative disorders [ 89 , 90 ] and DLBCL [ 91 ] are no exception. Accordingly, strategies combining anti-angiogenic therapy and immunotherapy seem to have the potential to tip the balance of the tumor microenvironment and improve the treatment response of lymphoid malignancies [ 21 , 22 , 92 , 93 ]. These pieces of evidence prompted an intense translational investigation aimed at targeting angiogenesis and the immune system in a coordinated fashion, based on the preclinical insights available [ 94 , 95 ].…”

A Comprehensive Biological and Clinical Perspective Can Drive a Patient-Tailored Approach to Multiple Myeloma: Bridging the Gaps between the Plasma Cell and the Neoplastic Niche

“…Additionally, in a mouse model, the use of an antiangiogenic anti-VEGFR-2 antibody in the early stage delayed tumor progression of MM; nonetheless, besides IMiDs, angiogenic-directed strategy did not show effective results in the unselected patient subgroup in patients with MM [130]. In order to achieve a patienttailored vasculogenic targeting, stringent patient stratification has been proposed by modulating from the critical step of MM evolution that can be critically dependent on vessel supply, such as smouldering phases [131] or extramedullary dissemination [132][133][134]. e next breakthrough of therapeutic strategy design is targeting the MM ecosystem together with the immune microenvironment.…”