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A.G. Solimando et al.
JAM-A expression on bone marrow endothelial cells is an independent prognostic factor for the survival of both patients with newly diagnosed MM and those with relapsed/refractory MM. Blocking JAM-A restricts angiogenesis in vitro, in utero and in vivo and represents a suitable druggable molecule to halt neo-angiogenesis and MM progression.
Introduction
Junctional adhesion molecule-A (JAM-A), also known as JAM-1, CD321, and F11R, belongs to the immunoglobulin superfamily.1 In healthy tissues, JAM-A regulates cell growth and differentiation, while its aberrant expression or deregulation confers a more aggressive phenotype with poor prognosis in different types of human cancers,1 includ- ing multiple myeloma (MM),2 breast, lung, brain, and head and neck cancers.3
Overactivation of JAM-A results either from upregulation or aberrant dimerization, driving the receptor in a state of constitutive signal transmission, or from excessive release of JAM-A ligands by normal and tumor cells into the microenvironment.4 Membrane-bound JAM-A and its solu- ble form (sJAM-A) can form homophilic interactions and also heterophilic interactions1 with lymphocyte function- associated antigen 1 (LFA-1), afadin (AFDN), calcium/calmodulin-dependent serine protein kinase (CASK) and tight junction protein-1 (TJP1) with high recep- tor/ligand binding affinities.5 These interactions trigger JAM-A downstream signaling pathways involved in the regulation of tumor cell survival, growth, angiogenesis and dissemination.6
JAM-A inhibition can be achieved directly by blocking the ligand-binding site on the extracellular receptor domain with monoclonal antibodies7 and indirectly with small-mol- ecule inhibitors.8 Moreover, neutralizing the sJAM-A9 released into the microenvironment can prevent JAM-A activation.10
JAM-A plays a pivotal role in endothelial cell physiology6 and pathology.2 Although the function of JAM-A in tumori- genesis has been investigated in solid tumors,3 and its angiogenic role has been shown in pancreatic islet carcino- ma,11 data on JAM-A-related angiogenesis in hematologic neoplasms remain elusive. Since bone marrow (BM) neo- vascularization favors the progression of MM,12 we investi- gated whether JAM-A can drive angiogenesis in MM,13 con- tributing to progression of the disease.2
We quantified JAM-A surface expression on BM-derived endothelial cells (MMEC) from 312 patients with MM and demonstrated that JAM-Ahigh MMEC correlate strongly with poor survival both in newly diagnosed (NDMM) and relapsed/refractory (RRMM) patients. Mechanistically, adding recombinant JAM-A protein to MM plasma cells (MM-cells) increased angiogenesis in both two-dimensional (2D) and three-dimensional (3D) models. Conversely, blocking JAM-A impaired MM-related angiogenesis. To corroborate these findings, we treated MM-bearing mice with JAM-A-blocking monoclonal antibodies and observed impaired MM progression and decreased MM vascularity.
Methods
Patients
Patients fulfilling the International Myeloma Working Group diagnostic criteria14 for NDMM (n=111), patients with RRMM15
(n=201) and subjects with monoclonal gammopathy of undeter- mined significance (MGUS) (n=35) were included in this study. The patients’ characteristics and genetic risk stratification are provided in Online Supplementary Tables S1 and S2. The study was approved by the Ethical Committees of Bari and Würzburg University Hospitals (reference numbers 5145 and 76/13), and all patients provided informed consent to participation in the study, in accordance with the Declaration of Helsinki (details are given in the Online Supplementary Methods).
Cell lines and cultures procedures
RPMI-8226, OPM-2 and human umbilical vein endothelial cells were cultured as described elsewhere.3 MM-cells were co- cultured with MMEC (4×105) at 1:1 and 1:5 cell ratios for 24 hours (h) with or without an inserted transwell (0.4mm pore size; Costar, Cambridge, MA, USA). Details are provided in the Online Supplementary Methods.
Chick chorioallantoic membrane assay
Fertilized chicken eggs were incubated at 37°C at a constant humidity. On day 8, sterilized gelatin sponges imbued with MMEC conditioned medium or medium obtained by treatment of MMEC with sJAM-A (100 ng/mL), with or without anti-JAM- A monoclonal antibodies were implanted on the top of the chick chorioallantoic membrane (CAM) as described in more detail in the Online Supplementary Methods.
Multiple myeloma xenograft mouse models
Twenty female 8- to 10-week-old NOD.CB17- Prkdcscid/NCrHsd mice (NOD-SCID; Envigo, Huntingdon, UK) were injected intratibially with 2×105 RPMI-8226 cells suspend- ed in phosphate-buffered saline. Mice were treated with the anti-JAM-A monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA, mouse monoclonal clone J10.4) recognizing the distal membrane extracellular domain of JAM-A.
Twenty female 6- to 8-week-old NOD-SCID mice were injected subcutaneously (s.c.), into the right flank, with 1×107 RPMI-8226 cells suspended in 200 mL RPMI-1640 medium and 200 mL MatrigelTM as described previously16 and detailed in the Online Supplementary Methods.
Functional in vitro assays
Wound-healing and MatrigelTM angiogenesis assays were per-
formed as previously described and detailed in the Online Supplementary Methods.
Protein expression studies and polymerase chain reaction analyses
Western blots, enzyme-linked immunosorbent assays, human angiogenesis array and real-time reverse transcriptase poly- merase chain reactions were performed according to the manu- facturers’ instructions (detailed in the Online Supplementary Methods).
Immunohistochemistry and in silico analysis
Details of the immunohistochemical studies and the in silico analysis, using the CoMMpass study dataset, are supplied in the
Online Supplementary Methods.
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haematologica | 2021; 106(7)