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Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.

Purpose: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3'UTR in CCND2 overexpression in multiple myeloma., Experimental Design: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3'UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3' rapid amplification of cDNA ends (RACE)-PCR., Results: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3'RACE experiments revealed that changes in CCND2 3'UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3'UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction., Conclusions: Overall, these results highlight the impact of CCND2 3'UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma., (©2015 American Association for Cancer Research.)

The development of resistance to therapy is unavoidable in the history of multiple myeloma patients. Therefore, the study of its characteristics and mechanisms is critical in the search for novel therapeutic approaches to overcome it. This effort is hampered by the absence of appropriate preclinical models, especially those mimicking acquired resistance. Here we present an in vivo model of acquired resistance based on the continuous treatment of mice bearing subcutaneous MM1S plasmacytomas. Xenografts acquired resistance to two generations of immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide) in combination with dexamethasone, that was reversible after a wash-out period. Furthermore, lenalidomide-dexamethasone (LD) or pomalidomide-dexamethasone (PD) did not display cross-resistance, which could be due to the differential requirements of the key target Cereblon and its substrates Aiolos and Ikaros observed in cells resistant to each combination. Differential gene expression profiles of LD and PD could also explain the absence of cross-resistance. Onset of resistance to both combinations was accompanied by upregulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway and addition of selumetinib, a small-molecule MEK inhibitor, could resensitize resistant cells. Our results provide insights into the mechanisms of acquired resistance to LD and PD combinations and offer possible therapeutic approaches to addressing IMiD resistance in the clinic.