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Deregulation of transcription factors (TFs) leading to uncontrolled proliferation of tumor cells within the microenvironment represents a hallmark of cancer. However, the biological and clinical impact of transcriptional interference, particularly in multiple myeloma (MM) cells, remains poorly understood. The present study shows for the first time that MYC and JUNB, two crucial TFs implicated in MM pathogenesis, orchestrate distinct transcriptional programs. Specifically, our data revealed that expression levels of MYC, JUNB, and their respective downstream targets do not correlate and that their global chromatin-binding patterns are not significantly overlapping. Mechanistically, MYC expression was not affected by JUNB knockdown, and conversely, JUNB expression and transcriptional activity were not affected by MYC knockdown. Moreover, suppression of MYC levels in MM cells via targeting the master regulator BRD4 by either siRNA-mediated knockdown or treatment with the novel proteolysis targeting chimera (PROTAC) MZ-1 overcame bone marrow (BM) stroma cell/IL-6-induced MYC- but not MEK-dependent JUNB-upregulation and transcriptional activity. Consequently, targeting of the two non-overlapping MYC- and JUNB-transcriptoms by MZ-1 in combination with genetic or pharmacological JUNB-targeting approaches synergistically enhanced MM cell death, both in 2D and our novel dynamic 3D models of the BM milieu as well as in murine xenografts. In summary, our data emphasize the opportunity to employ MYC and JUNB dual-targeting treatment strategies in MM as another exciting approach to further improve patient outcomes., (© 2024. The Author(s).)

The natural history of multiple myeloma is characterized by its localization to the bone marrow and its interaction with bone marrow stromal cells. The bone marrow stromal cells provide growth and survival signals, thereby promoting the development of drug resistance. Here, we show that the interaction between bone marrow stromal cells and myeloma cells (using human cell lines) induces chromatin remodeling of cis-regulatory elements and is associated with changes in the expression of genes involved in the cell migration and cytokine signaling. The expression of genes involved in these stromal interactions are observed in extramedullary disease in patients with myeloma and provides the rationale for survival of myeloma cells outside of the bone marrow microenvironment. Expression of these stromal interaction genes is also observed in a subset of patients with newly diagnosed myeloma and are akin to the transcriptional program of extramedullary disease. The presence of such adverse stromal interactions in newly diagnosed myeloma is associated with accelerated disease dissemination, predicts the early development of therapeutic resistance, and is of independent prognostic significance. These stromal cell induced transcriptomic and epigenomic changes both predict long-term outcomes and identify therapeutic targets in the tumor microenvironment for the development of novel therapeutic approaches., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Bortezomib (BTZ) is a standard-of-care treatment in multiple myeloma (MM); however, adverse side effects and development of resistance limit its long term benefit. To improve target specificity, therapeutic efficacy, and overcome resistance, we designed nanoparticles that encapsulate BTZ and are surface-functionalized with BCMA antibodies (BCMA-BTZ-NPs). We confirmed efficient cellular internalization of the BCMA-BTZ-NPs only in BCMA-expressing MM cells, but not in BCMA-knockout (KO) cells. In addition, BCMA-BTZ-NPs showed target-specific cytotoxicity against MM cell lines and primary tumor cells from MM patients. The BCMA-BTZ-NPs entered the cell through receptor-mediated uptake, which escapes a mechanism of BTZ resistance based on upregulating P-glycoprotein. Furthermore, BCMA-BTZ-NPs induced cell death more efficiently than non-targeted nanoparticles or free BTZ, triggering potent mitochondrial depolarization followed by apoptosis. In BTZ-resistant cells, BCMA-BTZ-NPs inhibited proteasome activity more effectively than free BTZ or non-targeted nanoparticles. Additionally, BCMA-BTZ-NPs enhanced immunogenic cell death and activated the autophagic pathway more than free BTZ. Finally, we found that BCMA-BTZ-NPs selectively accumulated at the tumor site in a murine xenograft model, enhanced tumor reduction, and prolonged host survival. These results suggest BCMA-BTZ-NPs provide a promising therapeutic strategy for enhancing the efficacy of BTZ and establish a framework for their evaluation in a clinical setting., (© 2023. The Author(s).)

Splicing changes are common in cancer and are associated with dysregulated splicing factors. Here, we analyzed RNA-seq data from 323 newly diagnosed multiple myeloma (MM) patients and described the alternative splicing (AS) landscape. We observed a large number of splicing pattern changes in MM cells compared to normal plasma cells (NPC). The most common events were alterations of mutually exclusive exons and exon skipping. Most of these events were observed in the absence of overall changes in gene expression and often impacted the coding potential of the alternatively spliced genes. To understand the molecular mechanisms driving frequent aberrant AS, we investigated 115 splicing factors (SFs) and associated them with the AS events in MM. We observed that ~40% of SFs were dysregulated in MM cells compared to NPC and found a significant enrichment of SRSF1, SRSF9, and PCB1 binding motifs around AS events. Importantly, SRSF1 overexpression was linked with shorter survival in two independent MM datasets and was correlated with the number of AS events, impacting tumor cell proliferation. Together with the observation that MM cells are vulnerable to splicing inhibition, our results may lay the foundation for developing new therapeutic strategies for MM. We have developed a web portal that allows custom alternative splicing event queries by using gene symbols and visualizes AS events in MM and subgroups. Our portals can be accessed at http://rconnect.dfci.harvard.edu/mmsplicing/ and https://rconnect.dfci.harvard.edu/mmleafcutter/ ., (© 2022. The Author(s).)

BCMA targeting chimeric antigen receptor (CAR) T cell therapy has shown deep and durable responses in multiple myeloma. However, relapse following therapy is frequently observed, and mechanisms of resistance remain ill-defined. Here, we perform single cell genomic characterization of longitudinal samples from a patient who relapsed after initial CAR T cell treatment with lack of response to retreatment. We report selection, following initial CAR T cell infusion, of a clone with biallelic loss of BCMA acquired by deletion of one allele and a mutation that creates an early stop codon on the second allele. This loss leads to lack of CAR T cell proliferation following the second infusion and is reflected by lack of soluble BCMA in patient serum. Our analysis suggests the need for careful detection of BCMA gene alterations in multiple myeloma cells from relapse following CAR T cell therapy.

The multiple myeloma (MM) genome is heterogeneous and evolves through preclinical and post-diagnosis phases. Here we report a catalog and hierarchy of driver lesions using sequences from 67 MM genomes serially collected from 30 patients together with public exome datasets. Bayesian clustering defines at least 7 genomic subgroups with distinct sets of co-operating events. Focusing on whole genome sequencing data, complex structural events emerge as major drivers, including chromothripsis and a novel replication-based mechanism of templated insertions, which typically occur early. Hyperdiploidy also occurs early, with individual trisomies often acquired in different chronological windows during evolution, and with a preferred order of acquisition. Conversely, positively selected point mutations, whole genome duplication and chromoplexy events occur in later disease phases. Thus, initiating driver events, drawn from a limited repertoire of structural and numerical chromosomal changes, shape preferred trajectories of evolution that are biologically relevant but heterogeneous across patients.

Multiple myeloma (MM) and its precursor condition MGUS are characterized by chromosomal aberrations. Here, we comprehensively characterize the order of occurrence of these complex genomic events underlying MM development using 500 MGUS, and MM samples. We identify hyperdiploid MM (HMM) and non-HMM as genomically distinct entities with different evolution of the copy number alterations. In HMM, gains of 9,15 or 19 are the first and clonal events observed as clonal even at MGUS stage. These events are thus early and may underlie initial transformation of normal plasma cells to MGUS cells. However, CNAs may not be adequate for progression to MM except in 15% of the patients in whom the complex subclonal deletion events are observed in MM but not MGUS. In NHMM, besides the driver translocations, clonal deletion of 13 and 1q gain are early events also observed in MGUS. We combined this information to propose a timeline for copy number alteration.

We analyzed whole genomes of unique paired samples from smoldering multiple myeloma (SMM) patients progressing to multiple myeloma (MM). We report that the genomic landscape, including mutational profile and structural rearrangements at the smoldering stage is very similar to MM. Paired sample analysis shows two different patterns of progression: a "static progression model", where the subclonal architecture is retained as the disease progressed to MM suggesting that progression solely reflects the time needed to accumulate a sufficient disease burden; and a "spontaneous evolution model", where a change in the subclonal composition is observed. We also observe that activation-induced cytidine deaminase plays a major role in shaping the mutational landscape of early subclinical phases, while progression is driven by APOBEC cytidine deaminases. These results provide a unique insight into myelomagenesis with potential implications for the definition of smoldering disease and timing of treatment initiation.

Alternative cleavage and polyadenylation (ApA) is known to alter untranslated region (3'UTR) length but can also recognize intronic polyadenylation (IpA) signals to generate transcripts that lose part or all of the coding region. We analyzed 46 3'-seq and RNA-seq profiles from normal human tissues, primary immune cells, and multiple myeloma (MM) samples and created an atlas of 4927 high-confidence IpA events represented in these cell types. IpA isoforms are widely expressed in immune cells, differentially used during B-cell development or in different cellular environments, and can generate truncated proteins lacking C-terminal functional domains. This can mimic ectodomain shedding through loss of transmembrane domains or alter the binding specificity of proteins with DNA-binding or protein-protein interaction domains. MM cells display a striking loss of IpA isoforms expressed in plasma cells, associated with shorter progression-free survival and impacting key genes in MM biology and response to lenalidomide.

Multiple myeloma (MM) has benefited from significant advancements in treatment that have improved outcomes and reduced morbidity. However, the disease remains incurable and is characterized by high rates of drug resistance and relapse. Consequently, methods to select the most efficacious therapy are of great interest. Here we utilize a functional assay to assess the ex vivo drug sensitivity of single multiple myeloma cells based on measuring their mass accumulation rate (MAR). We show that MAR accurately and rapidly defines therapeutic susceptibility across human multiple myeloma cell lines to a gamut of standard-of-care therapies. Finally, we demonstrate that our MAR assay, without the need for extended culture ex vivo, correctly defines the response of nine patients to standard-of-care drugs according to their clinical diagnoses. This data highlights the MAR assay in both research and clinical applications as a promising tool for predicting therapeutic response using clinical samples.

Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3'UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.

Multiple myeloma is an incurable plasma cell malignancy with a complex and incompletely understood molecular pathogenesis. Here we use whole-exome sequencing, copy-number profiling and cytogenetics to analyse 84 myeloma samples. Most cases have a complex subclonal structure and show clusters of subclonal variants, including subclonal driver mutations. Serial sampling reveals diverse patterns of clonal evolution, including linear evolution, differential clonal response and branching evolution. Diverse processes contribute to the mutational repertoire, including kataegis and somatic hypermutation, and their relative contribution changes over time. We find heterogeneity of mutational spectrum across samples, with few recurrent genes. We identify new candidate genes, including truncations of SP140, LTB, ROBO1 and clustered missense mutations in EGR1. The myeloma genome is heterogeneous across the cohort, and exhibits diversity in clonal admixture and in dynamics of evolution, which may impact prognostic stratification, therapeutic approaches and assessment of disease response to treatment.

MicroRNAs (miRNAs) with tumor-suppressor potential might have therapeutic applications in multiple myeloma (MM) through the modulation of still undiscovered molecular pathways. Here, we investigated the effects of enforced expression of miR-29b on the apoptotic occurrence in MM and highlighted its role in the context of a new transcriptional loop that is finely tuned by the proteasome inhibitor bortezomib. In details, in vitro growth inhibition and apoptosis of MM cells was induced by either transient expression of synthetic miR-29b or its stable lentivirus-enforced expression. We identified Sp1, a transcription factor endowed with oncogenic activity, as a negative regulator of miR-29b expression in MM cells. Since Sp1 expression and functions are regulated via the 26S proteasome, we investigated the effects of bortezomib on miR-29b-Sp1 loop, showing that miR-29b levels were indeed upregulated by the drug. At the same time, the bortezomib/miR-29b combination produced significant pro-apoptotic effects. We also demonstrated that the PI3K/AKT pathway plays a major role in the regulation of miR-29b-Sp1 loop and induction of apoptosis in MM cells. Finally, MM xenografts constitutively expressing miR-29b showed significant reduction of their tumorigenic potential. Our findings indicate that miR-29b is involved in a regulatory loop amenable of pharmacologic intervention and modulates the anti-MM activity of bortezomib in MM cells.