Identification of an Epithelial-to-Mesenchymal Transition (EMT)-like Programme in t(4;14)-Positive Multiple Myeloma Reveals Novel Targets for Therapeutic Intervention

Introduction: Prognosis for multiple myeloma (MM) patients has improved dramatically since the introduction of novel therapies. However, prognosis in the t(4;14) subgroup, characterized by expression of the histone methyltransferase MMSET, remains relatively poor due to the acquisition of a highly aggressive, motile and invasive phenotype and a generally modest response to therapy. We have previously demonstrated that MMSET promotes an epithelial-to-mesenchymal transition (EMT) in prostate cancer¹. While the term EMT is not commonly used to describe MM, we hypothesise that an EMT-like process plays a critical role in t(4;14)_­-positive MM disease pathogenesis. In this study, we conducted a comprehensive evaluation of the association between t(4;14) and an EMT-related gene expression signature in MM and identify N-cadherin as a therapeutically-targetable EMT-related gene in t(4;14)-positive MM.

Methods and results: Expression of a core EMT-related signature, comprising 169 mesenchymal genes and 49 epithelial genes, was assessed in CD138-selected MM plasma cells from newly-diagnosed MM patients in four independent microarray datasets (E-GEOD-19784 [n = 327 MM patients], E-GEOD-26863 [n = 304], E-MTAB-317 [n = 226] and E-MTAB-363 [n = 155]), accessed through ArrayExpress (EMBL-EBI). In each dataset, gene expression was compared in t(4;14)-positive and t(4;14)-negative patients using linear models for microarray data (LIMMA). Twenty six mesenchymal genes were found to be upregulated in t(4;14)-positive patients across the 4 datasets (p < 0.05, Fisher’s method). A general loss of epithelial genes was not observed, likely due to the haematopoietic origins of MM. Upregulated genes included key EMT drivers (TWIST1, SOX9, TCF4) and genes associated with the cytoskeleton (VIM), adhesion and migration (CDH2, ITGB1, NCAM1) and signalling pathways involved in EMT (BMPR1A, IL6R, TGFB2). The t(4;14)-mediated regulation of EMT-related genes including TWIST1, CDH2, BMPR1A and ITGB1 was confirmed by assessing the effects of MMSET knockdown, knockout and add-back in the t(4;14)-positive human myeloma cell line KMS-11.

We have previously demonstrated that N-cadherin (CDH2) expression is elevated in plasma cells from approximately 50% of newly diagnosed multiple myeloma (MM) patients and that elevated serum N-cadherin is associated with poor prognosis². In order to identify whether N-cadherin is a potential therapeutic target in t(4;14)-positive myeloma, the effects of an N-cadherin peptide inhibitor ADH-1 (Exherin™) or shRNA-mediated N-cadherin knock-down were assessed on MM PC adhesion and proliferation in vivo and in vitro. The role of N-cadherin in MM tumour establishment and intramedullary growth was investigated using the C57BL/KalwRijHsd mouse model of MM. In this model, intravenously injected luciferase-expressing mouse MM PC cells (5TGM1-SFG) home to the bone marrow and initiate systemic MM disease. C57BL/KalwRijHsd mice bearing 5TGM1-SFG N-cadherin knock-down cells had significantly reduced tumour burden, as assessed by bioluminescent imaging and serum paraprotein levels, after 4 weeks compared with mice bearing control 5TGM1-SFG cells. Furthermore, daily intraperitoneal ADH-1 administration (100 mg/kg/day) to 5TGM1-SFG cell-bearing mice significantly significantly decreased tumour burden when administered at the time of tumour inoculation, but had no effect on established tumours. These results suggest that N-cadherin plays a role in extravasation, homing and/or tumour establishment in vivo.

Conclusion: This study identified an extensive EMT-like gene expression signature driven by MMSET in t(4;14)-positive MM patients. This provides insight as to how the t(4;14) translocation leads poor prognostic outcomes in up to 20% of MM patients. Furthermore, these studies demonstrate a potential role for N-cadherin in MM tumour dissemination in t(4;14)-positive MM and suggest that N-cadherin represents a novel candidate for therapeutic targeting in these patients.

¹Ezponda et al. Oncogene, 2013

²Vandyke et al. Br J Haematol, 2013