The clinical and functional role of extracellular matrix molecules in multiple myeloma

Multiple myeloma (MM) is a hematological malignancy that, due to recurrent patient relapses and therapy resistance, remains largely uncurable. The first part of this study therefore aimed to identify novel potential therapeutical targets. The analysis of two whole exome sequencing datasets (Multiple Myeloma Research Foundation (MMRF) CoMMpass study cohort (n=1,192 samples from 984 patients) and an in-house validation cohort (n=67 samples from 43 patients)) revealed that genes encoding extracellular matrix (ECM) molecules, including the collagen, laminin, integrin, A Disintegrin And Metalloproteinase (ADAM) and ADAM with Thrombospondin motifs (ADAMTS) families, are recurrently mutated in MM and may be potential cancer driver mutations due to increased mutation rates compared to other protein-coding genes. To gain further insight into the role of ECM mutations in MM, the ECM mutation status was correlated with patient survival in the MMRF cohort. While ECM mutations were prognostic for a shorter progression-free survival (PFS), their presence correlated with a high tumor mutational burden (TMB), TP53 mutations and chromosome 1q gains, and multivariate survival analyses suggested that they are not independent prognostic markers. In contrast, considering mutations in individual ECM genes, ADAM18 and ADAMTS13 mutations were independent prognostic markers associated with a significantly shorter overall survival (OS). In general, however, the mutations were widely spread across the 121 ECM genes assessed and did not cluster in clear hotspots or certain protein domains. Furthermore, individual mutations only affected very few patients and may therefore not be promising therapeutical targets. In contrast, the high expression of many ECM genes was found to be associated with shorter PFS and/or OS in an RNA sequencing dataset from the MMRF study cohort (n=921 samples from 806 patients). Among the considerably expressed ECM genes ITGA7, ITGAL, ITGB1, ITGB7, LAMC1, ADAM8, ADAM9, ADAM15, ADAM22 and ADAMTS13 were the genes with the most consistent and promising prognostic value. Since high expression levels of these genes were detected in a considerable number of patients, these molecules could be interesting novel therapeutical targets. Notably, these genes contained many integrin and ADAM genes. Integrins have been the focus of extensive research while ADAMs have thus far scarcely been studied in the context of MM. Therefore, the second part of this study aimed to elucidate the clinical and functional role of the most highly expressed ADAM genes with prognostic value, ADAM8, ADAM9 and ADAM15 using two RNA sequencing datasets (MMRF and validation cohort (n=73 samples from 51 patients)) and seven human MM cell lines (HMCL). Validating the results obtained in the MMRF cohort, high expression levels of ADAM8/9/15 were verified as prognostic markers, even in multivariate survival analyses, in the validation cohort. Looking at further clinically relevant parameters, high expression levels of ADAM8, ADAM9 and ADAM15 were associated with the presence of high-risk cytogenetic abnormalities and extramedullary disease. ADAM8 and ADAM15 were additionally found to be upregulated in progressive disease compared to baseline samples from the same patient in the MMRF dataset and in relapsed/refractory MM compared to untreated samples in the validation cohort, suggesting a link between high ADAM8/9/15 expression and MM progression. To assess the functional role of ADAM8/9/15 in MM, gene set enrichment analyses (GSEA) were performed. The comparison of the gene expression profiles of patient samples with a high or low expression of ADAM8/9/15 revealed an upregulation of proliferation- and cell cycle-associated gene sets in the ADAM8/9/15high samples from both patient cohorts. Conversely, siRNA-mediated knockdown of ADAM8, ADAM9 or ADAM15 in human MM cell lines led to a downregulation of these gene sets. Virtually all commonly used proliferation markers assessed herein were upregulated in the ADAM8/9/15high samples on RNA level in both cohorts. Furthermore, high ADAM8/9/15 expression was associated with high Ki67 protein expression measured in immunohistochemical (IHC) stainings of patient tissue from the validation cohort and high ADAM8/15 expression was associated with high MYC protein expression (IHC data was available from a previous project). Considering individual signaling pathways of known relevance in MM, the PI3K/AKT/MTOR pathway was the most highly enriched gene set when comparing differentially expressed genes between samples with a high or low ADAM8 expression from the MMRF cohort. In line with that, Western blot experiments revealed that ADAM8 siRNA knockdown significantly downregulated the expression and activation of IGF1R, a receptor tyrosine kinase known to influence the PI3K/AKT/MTOR pathway, as well as the activation of pAKT in the majority of HMCL. Cell counting experiments showed that a downregulation of IGF1R by CRISPR/Cas9-mediated knockout can reduce the proliferation of HMCL, suggesting that ADAM8 might influence the proliferation of MM cells by downregulating IGF1R. Furthermore, MTORC1 signaling was enriched in the GSEA comparing MM patient samples with a high or low ADAM9 expression from both cohorts and MTOR activation was reduced in Western Blots in several HMCL after ADAM9 siRNA knockdown. MYC targets were strongly enriched in the GSEA comparing patient samples with a high or low ADAM15 expression and ADAM15 siRNA knockdown reduced MYC protein expression in one HMCL. Taken together, these data suggest that ECM mutations might be driver mutations in MM and that the high expression of several ECM genes is prognostic for shorter patient survival. Furthermore, this study revealed that the high expression of ADAM8, ADAM9 and ADAM15 is associated with MM progression and proliferation signaling, suggesting that they might be interesting novel therapeutical targets.