Person:

Manier, Salomon

Epigenetic changes frequently occur during tumorigenesis and DNA hypermethylation may account for the inactivation of tumor suppressor genes in cancer cells. Studies in Multiple Myeloma (MM) have shown variable DNA methylation patterns with focal hypermethylation changes in clinically aggressive subtypes. We studied global methylation patterns in patients with relapsed/refractory MM and found that the majority of methylation peaks were located in the intronic and intragenic regions in MM samples. Therefore, we investigated the effect of methylation on miRNA regulation in MM. To date, the mechanism by which global miRNA suppression occurs in MM has not been fully described. In this study, we report hypermethylation of miRNAs in MM and perform confirmation in MM cell lines using bisulfite sequencing and methylation-specific PCR (MSP) in the presence or absence of the DNA demethylating agent 5-aza-2′-deoxycytidine. We further characterized the hypermethylation-dependent inhibition of miR-152, -10b-5p and -34c-3p which was shown to exert a putative tumor suppressive role in MM. These findings were corroborated by the demonstration that the same miRNAs were down-regulated in MM patients compared to healthy individuals, alongside enrichment of miR-152-, -10b-5p, and miR-34c-3p-predicted targets, as shown at the mRNA level in primary MM cells. Demethylation or gain of function studies of these specific miRNAs led to induction of apoptosis and inhibition of proliferation as well as down-regulation of putative oncogene targets of these miRNAs such as DNMT1, E2F3, BTRC and MYCBP. These findings provide the rationale for epigenetic therapeutic approaches in subgroups of MM.

MYC is a major oncogenic driver of Multiple Myeloma (MM) and yet almost no therapeutic agents exist that target MYC in MM. Here we report that the let-7 biogenesis inhibitor LIN28B correlates with MYC expression in MM and is associated with adverse outcome. We also demonstrate that the LIN28B/let-7 axis modulates the expression of MYC, itself a let-7 target. Further, perturbation of the axis regulates the proliferation of MM cells in vivo in a xenograft tumor model. RNA sequencing and gene set enrichment analyses of CRISPR-engineered cells further suggest that the LIN28/let-7 axis regulates MYC and cell cycle pathways in MM. We provide proof-of-principle for therapeutic regulation of MYC through let-7 with an LNA-GapmeR containing a let-7b mimic in vivo, demonstrating that high levels of let-7 expression repress tumor growth by regulating MYC expression. These findings reveal a novel mechanism of therapeutic targeting of MYC through the LIN28B/let-7 axis in MM that may impact other MYC dependent cancers as well.

Summary The development of sensitive and non-invasive “liquid biopsies” presents new opportunities for longitudinal monitoring of tumor dissemination and clonal evolution. The number of circulating tumor cells (CTCs) is prognostic in multiple myeloma (MM), but there is little information on their genetic features. Here, we have analyzed the genomic landscape of CTCs from 29 MM patients, including eight cases with matched/paired bone marrow (BM) tumor cells. Our results show that 100% of clonal mutations in patient BM were detected in CTCs and that 99% of clonal mutations in CTCs were present in BM MM. These include typical driver mutations in MM such as in KRAS, NRAS, or BRAF. These data suggest that BM and CTC samples have similar clonal structures, as discordances between the two were restricted to subclonal mutations. Accordingly, our results pave the way for potentially less invasive mutation screening of MM patients through characterization of CTCs.