Search

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8 + T cells with reduced immunosuppressive regulatory T (T reg ) cells, while late MYC acquisition in slow progressors was associated with lower CD8 + T cell infiltration and more abundant T reg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8 + T cells versus T reg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8 + T/T reg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8 + T cell cytotoxicity or depleting T reg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials., (© 2023. The Author(s).)

Cereblon is the direct binding target of the immunomodulatory drugs (IMiDs) that are commonly used to treat multiple myeloma (MM), the second most frequent hematologic malignancy. Patients respond well to initial treatment with IMiDs, but virtually all patients develop drug resistance over time, and the underlying mechanisms are poorly understood. We identified an as yet undescribed DNA hypermethylation in an active intronic CRBN enhancer. Differential hypermethylation in this region was found to be increased in healthy plasma cells, but was more pronounced in IMiD-refractory MM. Methylation significantly correlated with decreased CRBN expression levels. DNA methyltransferase inhibitor (DNTMi) in vitro experiments induced CRBN enhancer demethylation, and sensitizing effects on lenalidomide treatment were observed in 2 MM cell lines. Thus, we provide first evidence that aberrant CRBN DNA methylation is a novel mechanism of IMiD resistance in MM and may predict IMiD response prior to treatment., (© 2021 by The American Society of Hematology.)

Clinical and genetic risk factors are currently used in multiple myeloma (MM) to stratify patients and to design specific therapies. However, these systems do not capture the heterogeneity of the disease supporting the development of new prognostic factors. In this study, we identified active promoters and alternative active promoters in 6 different B cell subpopulations, including bone-marrow plasma cells, and 32 MM patient samples, using RNA-seq data. We find that expression initiated at both regular and alternative promoters was specific of each B cell subpopulation or MM plasma cells, showing a remarkable level of consistency with chromatin-based promoter definition. Interestingly, using 595 MM patient samples from the CoMMpass dataset, we observed that the expression derived from some alternative promoters was associated with lower progression-free and overall survival in MM patients independently of genetic alterations. Altogether, our results define cancer-specific alternative active promoters as new transcriptomic features that can provide a new avenue for prognostic stratification possibilities in patients with MM., (© 2021. The Author(s).)

Multiple myeloma (MM) is an incurable disease, whose clinical heterogeneity makes its management challenging, highlighting the need for biological features to guide improved therapies. Deregulation of specific long non-coding RNAs (lncRNAs) has been shown in MM, nevertheless, the complete lncRNA transcriptome has not yet been elucidated. In this work, we identified 40,511 novel lncRNAs in MM samples. lncRNAs accounted for 82% of the MM transcriptome and were more heterogeneously expressed than coding genes. A total of 10,351 overexpressed and 9,535 downregulated lncRNAs were identified in MM patients when compared with normal bone-marrow plasma cells. Transcriptional dynamics study of lncRNAs in the context of normal B-cell maturation revealed 989 lncRNAs with exclusive expression in MM, among which 89 showed de novo epigenomic activation. Knockdown studies on one of these lncRNAs, SMILO (specific myeloma intergenic long non-coding RNA), resulted in reduced proliferation and induction of apoptosis of MM cells, and activation of the interferon pathway. We also showed that the expression of lncRNAs, together with clinical and genetic risk alterations, stratified MM patients into several progression-free survival and overall survival groups. In summary, our global analysis of the lncRNAs transcriptome reveals the presence of specific lncRNAs associated with the biological and clinical behavior of the disease.

Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD.

Multiple myeloma (MM) patients undergo repetitive bone marrow (BM) aspirates for genetic characterization. Circulating tumor cells (CTCs) are detectable in peripheral blood (PB) of virtually all MM cases and are prognostic, but their applicability for noninvasive screening has been poorly investigated. Here, we used next-generation flow (NGF) cytometry to isolate matched CTCs and BM tumor cells from 53 patients and compared their genetic profile. In eight cases, tumor cells from extramedullary (EM) plasmacytomas were also sorted and whole-exome sequencing was performed in the three spatially distributed tumor samples. CTCs were detectable by NGF in the PB of all patients with MM. Based on the cancer cell fraction of clonal and subclonal mutations, we found that ~22% of CTCs egressed from a BM (or EM) site distant from the matched BM aspirate. Concordance between BM tumor cells and CTCs was high for chromosome arm-level copy number alterations (≥95%) though not for translocations (39%). All high-risk genetic abnormalities except one t(4;14) were detected in CTCs whenever present in BM tumor cells. Noteworthy, ≥82% mutations present in BM and EM clones were detectable in CTCs. Altogether, these results support CTCs for noninvasive risk-stratification of MM patients based on their numbers and genetic profile.

The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment promotes MM cell retention, survival, and resistance to different anti-MM agents, including proteasome inhibitors (PIs) such as bortezomib (BTZ). The α4β1 integrin is a main adhesion receptor mediating MM cell-stroma interactions and MM cell survival, and its expression and function are downregulated by BTZ, leading to inhibition of cell adhesion-mediated drug resistance (CAM-DR) and MM cell apoptosis. Whether decreased α4β1 expression and activity are maintained or recovered upon development of resistance to BTZ represents an important question, as a potential rescue of α4β1 function could boost MM cell survival and disease progression. Using BTZ-resistant MM cells, we found that they not only rescue their α4β1 expression, but its levels were higher than in parental cells. Increased α4β1 expression in resistant cells correlated with enhanced α4β1-mediated cell lodging in the BM, and with disease progression. BTZ-resistant MM cells displayed enhanced NF-κB pathway activation relative to parental counterparts, which contributed to upregulated α4 expression and to α4β1-dependent MM cell adhesion. These data emphasize the upregulation of α4β1 expression and function as a key event during resistance to BTZ in MM, which might indirectly contribute to stabilize this resistance, as stronger MM cell attachment to BM stroma will regain CAM-DR and MM cell growth and survival. Finally, we found a strong correlation between high ITGB1 (integrin β1) expression in MM and poor progression-free survival (PFS) and overall survival (OS) during treatment of MM patients with BTZ and IMIDs, and combination of high ITGB1 levels and presence of the high-risk genetic factor amp1q causes low PFS and OS. These results unravel a novel prognostic value for ITGB1 in myeloma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin ( TXN ), a major regulator of cellular redox status and, in addition, identified PRDM5 as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype., (© 2020 Ordoñez et al.; Published by Cold Spring Harbor Laboratory Press.)

Granulocytic myeloid-derived suppressor cells (G-MDSCs) promote tumor growth and immunosuppression in multiple myeloma (MM). However, their phenotype is not well established for accurate monitoring or clinical translation. We aimed to provide the phenotypic profile of G-MDSCs based on their prognostic significance in MM, immunosuppressive potential, and molecular program. The preestablished phenotype of G-MDSCs was evaluated in bone marrow samples from controls and MM patients using multidimensional flow cytometry; surprisingly, we found that CD11b+CD14-CD15+CD33+HLADR- cells overlapped with common eosinophils and neutrophils, which were not expanded in MM patients. Therefore, we relied on automated clustering to unbiasedly identify all granulocytic subsets in the tumor microenvironment: basophils, eosinophils, and immature, intermediate, and mature neutrophils. In a series of 267 newly diagnosed MM patients (GEM2012MENOS65 trial), only the frequency of mature neutrophils at diagnosis was significantly associated with patient outcome, and a high mature neutrophil/T-cell ratio resulted in inferior progression-free survival (P < .001). Upon fluorescence-activated cell sorting of each neutrophil subset, T-cell proliferation decreased in the presence of mature neutrophils (0.5-fold; P = .016), and the cytotoxic potential of T cells engaged by a BCMA×CD3-bispecific antibody increased notably with the depletion of mature neutrophils (fourfold; P = .0007). Most interestingly, RNA sequencing of the 3 subsets revealed that G-MDSC-related genes were specifically upregulated in mature neutrophils from MM patients vs controls because of differential chromatin accessibility. Taken together, our results establish a correlation between the clinical significance, immunosuppressive potential, and transcriptional network of well-defined neutrophil subsets, providing for the first time a set of optimal markers (CD11b/CD13/CD16) for accurate monitoring of G-MDSCs in MM., (© 2020 by The American Society of Hematology.)

Cell identity relies on the cross-talk between genetics and epigenetics and their impact on gene expression. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) is the first step of an active DNA demethylation process occurring mainly at enhancers and gene bodies and, as such, participates in processes governing cell identity in normal and pathological conditions. Although genetic alterations are well documented in multiple myeloma (MM), epigenetic alterations associated with this disease have not yet been thoroughly analyzed. To gain insight into the biology of MM, genome-wide 5hmC profiles were obtained and showed that regions enriched in this modified base overlap with MM enhancers and super enhancers and are close to highly expressed genes. Through the definition of a MM-specific 5hmC signature, we identified FAM72D as a poor prognostic gene located on 1q21, a region amplified in high risk myeloma. We further uncovered that FAM72D functions as part of the FOXM1 transcription factor network controlling cell proliferation and survival and we evidenced an increased sensitivity of cells expressing high levels of FOXM1 and FAM72 to epigenetic drugs targeting histone deacetylases and DNA methyltransferases., (Copyright© 2020 Ferrata Storti Foundation.)

The reason why a few myeloma cells egress from the bone marrow (BM) into peripheral blood (PB) remains unknown. Here, we investigated molecular hallmarks of circulating tumor cells (CTCs) to identify the events leading to myeloma trafficking into the bloodstream. After using next-generation flow to isolate matched CTCs and BM tumor cells from 32 patients, we found high correlation in gene expression at single-cell and bulk levels (r ≥ 0.94, P = 10 -16 ), with only 55 genes differentially expressed between CTCs and BM tumor cells. CTCs overexpressed genes involved in inflammation, hypoxia, or epithelial-mesenchymal transition, whereas genes related with proliferation were downregulated in CTCs. The cancer stem cell marker CD44 was overexpressed in CTCs, and its knockdown significantly reduced migration of MM cells towards SDF1-α and their adhesion to fibronectin. Approximately half (29/55) of genes differentially expressed in CTCs were prognostic in patients with newly-diagnosed myeloma (n = 553; CoMMpass). In a multivariate analysis including the R-ISS, overexpression of CENPF and LGALS1 was significantly associated with inferior survival. Altogether, these results help understanding the presence of CTCs in PB and suggest that hypoxic BM niches together with a pro-inflammatory microenvironment induce an arrest in proliferation, forcing tumor cells to circulate in PB and seek other BM niches to continue growing.

The notion that plasma cells (PCs) are terminally differentiated has prevented intensive research in multiple myeloma (MM) about their phenotypic plasticity and differentiation. Here, we demonstrated in healthy individuals (n=20) that the CD19-CD81 expression axis identifies three bone marrow (BM)PC subsets with distinct age-prevalence, proliferation, replication-history, immunoglobulin-production, and phenotype, consistent with progressively increased differentiation from CD19+CD81+ into CD19-CD81+ and CD19-CD81- BMPCs. Afterwards, we demonstrated in 225 newly diagnosed MM patients that, comparing to normal BMPC counterparts, 59% had fully differentiated (CD19-CD81-) clones, 38% intermediate-differentiated (CD19-CD81+) and 3% less-differentiated (CD19+CD81+) clones. The latter patients had dismal outcome, and PC differentiation emerged as an independent prognostic marker for progression-free (HR: 1.7; P=0.005) and overall survival (HR: 2.1; P=0.006). Longitudinal comparison of diagnostic vs minimal-residual-disease samples (n=40) unraveled that in 20% of patients, less-differentiated PCs subclones become enriched after therapy-induced pressure. We also revealed that CD81 expression is epigenetically regulated, that less-differentiated clonal PCs retain high expression of genes related to preceding B-cell stages (for example: PAX5), and show distinct mutation profile vs fully differentiated PC clones within individual patients. Together, we shed new light into PC plasticity and demonstrated that MM patients harbouring less-differentiated PCs have dismal survival, which might be related to higher chemoresistant potential plus different molecular and genomic profiles.

While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM., (© 2015 Agirre et al.; Published by Cold Spring Harbor Laboratory Press.)

Currently, multiple myeloma (MM) patients are broadly grouped into a non-hyperdiploid (nh-MM) group, highly enriched for IgH translocations, or into a hyperdiploid (h-MM) group, which is typically characterized by trisomies of some odd-numbered chromosomes. We compared the micro RNA (miRNA) expression profiles of these two groups and we identified 16 miRNAs that were downregulated in the h-MM group, relative to the nh-MM group. We found that target genes of the most differentially expressed miRNAs are directly involved in the pathogenesis of MM; specifically, the inhibition of hsa-miR-425, hsa-miR-152 and hsa-miR-24, which are all downregulated in h-MM, leads to the overexpression of CCND1, TACC3, MAFB, FGFR3 and MYC, which are the also the oncogenes upregulated by the most frequent IgH chromosomal translocations occurring in nh-MM. Importantly, we showed that the downregulation of these specific miRNAs and the upregulation of their targets also occur simultaneously in primary cases of h-MM. These data provide further evidence on the unifying role of cyclin D pathways deregulation as the key mechanism involved in the development of both groups of MM. Finally, they establish the importance of miRNA deregulation in the context of MM, thereby opening up the potential for future therapeutic approaches based on this molecular mechanism.