Abstract
Background: Gain(1q) is among the most frequent high-risk cytogenetic abnormalities in multiple myeloma (MM), closely linked with treatment resistance, disease progression, and the development of extramedullary disease (EMD). The F11R gene, encoding Junctional Adhesion Molecule-A (JAM-A), is located on chromosome 1q. Beyond adhesive function, JAM-A drives oncogenic signalling via cis-dimerization, promoting proliferation, survival, motility, and dissemination of cancer cells. This study aims to functionally validate JAM-A as a driver of MM progression and to develop a second-generation peptide inhibitor that disrupts JAM-A cis-dimerization, with the aim of impairing MM cell proliferation, adhesion to the bone marrow (BM) niche, invasion and tumour formation.
Methods: F11R expression and associations with cytogenetic risk and survival were analysed using the CoMMpass IA20 dataset. Functional studies included siRNA JAM-A knockdown in JAM-A–high KMS18 cells and JAM-A overexpression in JAM-A–low MM1S cells. JAM-A protein levels were confirmed by Western blot; proliferation was assessed using CyQuant assays. A structure-guided peptide library was designed to improve binding affinity and stability to the JAM-A cis-dimer interface. Candidates were screened for anti-proliferative activity in MM cell lines and primary CD138+ patient-derived cells. Mechanistic assays included β-galactosidase senescence, adhesion to HS5 stromal cells and invasion assays using transwell inserts to identify and validate a lead compound. Peptide specificity was confirmed by using JAM-A knockdown cells. The lead peptide was further evaluated in vivo using the chick chorioallantoic membrane (CAM) model, a rapid and cost-effective xenograft platform developed by our lab (PMID: 35267611) to rapidly screen novel therapies in vivo. This model supports the growth of “plasmacytoma like” tumours from MM cells. KMS18 cells were pre-treated with 200 µM of lead peptide prior to implantation. Tumours were grown for four days; tumour burden was quantified macroscopically and by immunohistochemistry for Ki67 and CD138.
Results: Median F11R expression was significantly (p<0.0001) higher in MM patients with gain(1q) (9.61 TPM, n=396) compared to those without (7.138 TPM, n=396 and progressively increased from healthy donors through to relapsed MM (RRMM) (mean healthy: 48.4 vs RRMM: 87.83, p=0.0425) High JAM-A expression was associated with inferior progression-free survival (JAM-A low: 1106 days vs JAM-A high: 914 days, p=0.0516) and overall survival (JAM-A low: 2922 days vs JAM-A high: 2356 days, p=0.0009). Functional studies demonstrated that JAM-A overexpression promotes proliferation: (54% increased in over-expressing cells versus empty vector controls, p=0.0152, (MM1S n=2). From the novel peptide library designed, a peptide termed P4 emerged as the most potent inhibitor of MM cell growth (KMS18 p=0.037, n=4), and reduced proliferation in patient-derived CD138+ cells (n=5). P4 both induced senescence and significantly reduced MM cell adhesion to HS5 stromal cells (untreated: 100% vs P4 treated:71.28%, p=0.0024, n=3), consistent with the adhesive role of JAM-A. Pre-treatment with P4 reduced tumour burden in the CAM model (n=2). Pre-treated tumour xenografts were macroscopically reduced in size and tumour mass (mean: untreated 1.13g vs mean: pre-treated: 0.28g p=<0.0001), with reductions in Ki67 proliferation index and CD138+ plasma cell content.
Conclusion: JAM-A is overexpressed in 1q amplified HR-MM patients and may represent a novel target for this group of patients. Our data suggests that a novel peptide targeting JAM-A may be effective in reducing MM cell proliferation and impairing adhesion to stromal cells. These findings support further preclinical development of JAM-A–targeting strategies in MM.
