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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. Relapse following BCMA targeted CAR T-cell therapy is frequently observed in patients with multiple myeloma (MM). Here, by single cell transcriptome profiling on serially collected bone marrow samples, the authors report biallelic loss of BCMA as the mechanism of resistance underlying both relapse and lack of response to a second CAR T infusion in a patient with MM.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM.

Alternative cleavage and polyadenylation (ApA) is known to alter untranslated region (3ʹUTR) length but can also recognize intronic polyadenylation (IpA) signals to generate transcripts that lose part or all of the coding region. We analyzed 46 3ʹ-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. Recognition of intronic polyadenylation (IpA) signals can lead to expression of truncated proteins lacking C terminal domains. Analysis of 3ʹ -seq and RNA-seq shows that IpA is widespread in circulating immune cells, while multiple myeloma cells show loss of IpA isoforms that are normally expressed in plasma cells, impacting key genes in the disease.

In multiple myeloma, next-generation sequencing (NGS) has expanded our knowledge of genomic lesions, and highlighted a dynamic and heterogeneous composition of the tumor. Here we used NGS to characterize the genomic landscape of 418 multiple myeloma cases at diagnosis and correlate this with prognosis and classification. Translocations and copy number abnormalities (CNAs) had a preponderant contribution over gene mutations in defining the genotype and prognosis of each case. Known and novel independent prognostic markers were identified in our cohort of proteasome inhibitor and immunomodulatory drug-treated patients with long follow-up, including events with context-specific prognostic value, such as deletions of the PRDM1 gene. Taking advantage of the comprehensive genomic annotation of each case, we used innovative statistical approaches to identify potential novel myeloma subgroups. We observed clusters of patients stratified based on the overall number of mutations and number/type of CNAs, with distinct effects on survival, suggesting that extended genotype of multiple myeloma at diagnosis may lead to improved disease classification and prognostication.

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. Bone marrow stromal cells (BMSCs) are known to promote the development of drug resistance. Here, the authors investigate the chromatin remodeling and associated changes in gene expression in the multiple myeloma (MM) cells following their interactions with BMSCs, which are also observed in extramedullary disease (EMD).

Interactions between the inflammatory chemokine CCL20 and its receptor CCR6 have been associated with colorectal cancer growth and metastasis, however, a causal role for CCL20 signaling through CCR6 in promoting intestinal carcinogenesis has not been demonstrated in vivo. In this study, we aimed to determine the role of CCL20-CCR6 interactions in spontaneous intestinal tumorigenesis. CCR6-deficient mice were crossed with mice heterozygous for a mutation in the adenomatous polyposis coli (APC) gene (APCMIN/+ mice) to generate APCMIN/+ mice with CCR6 knocked out (CCR6KO-APCMIN/+ mice). CCR6KO-APCMIN/+ mice had diminished spontaneous intestinal tumorigenesis. CCR6KO-APCMIN/+ also had normal sized spleens as compared to the enlarged spleens found in APCMIN/+ mice. Decreased macrophage infiltration into intestinal adenomas and non-tumor epithelium was observed in CCR6KO-APCMIN/+ as compared to APCMIN/+ mice. CCL20 signaling through CCR6 caused increased production of CCL20 by colorectal cancer cell lines. Furthermore, CCL20 had a direct mitogenic effect on colorectal cancer cells. Thus, interactions between CCL20 and CCR6 promote intestinal carcinogenesis. Our results suggest that the intestinal tumorigenesis driven by CCL20-CCR6 interactions may be driven by macrophage recruitment into the intestine as well as proliferation of neoplastic epithelial cells. This interaction could be targeted for the treatment or prevention of malignancy.

Although long intergenic non-coding RNAs (lincRNA) role in various cancers is described, their significance in Multiple Myeloma (MM) remains poorly defined. Here we have studied the lincRNA profile and their clinical impact in MM. We performed RNA-seq on MM cells from 308 newly diagnosed and uniformly treated patients, 16 normal plasma cells and utilized RNA-seq data from 532 newly diagnosed patients from CoMMpass study to analyze for lincRNAs. We observed 869 differentially expressed lincRNAs in MM compared to normal plasma cells. We identified 14 lincRNAs associated with PFS and calculated a risk score to stratify patients. The median PFS between high vs low-risk groups was 17 months vs not-reached (NR); and OS 30 months vs NR, respectively (p < 0.0001 for both). In the independent validation dataset between high and low-risk groups, PFS was 27 vs 42 months (HR 2.06 [1.44−2.96]; p < 0.0005); and 4-year OS 62% vs 86% (HR 2.76 [1.51–5.05]; p < 0.0005) confirming significant clinical relevance of lincRNA in MM. Importantly, lincRNA signature was able to further identify patients with significant differential outcomes within each low and high-risk categories identified using standard risk categorization including cytogenetic/FISH, ISS, and MRD negative or positive. Our results suggest that lincRNAs have an independent effect on MM outcome and provide a rationale to evaluate its molecular and biological impact.

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. Multiple myeloma is characterized by high rates of drug resistance and relapse. Here the authors utilize a functional assay to assess the ex vivo drug sensitivity of single multiple myeloma cells based on measuring the mass accumulation rate of individual cells.

KDM3A is implicated in tumorigenesis; however, its biological role in multiple myeloma (MM) has not been elucidated. Here we identify KDM3A–KLF2–IRF4 axis dependence in MM. Knockdown of KDM3A is toxic to MM cells in vitro and in vivo . KDM3A maintains expression of KLF2 and IRF4 through H3K9 demethylation, and knockdown of KLF2 triggers apoptosis. Moreover, KLF2 directly activates IRF4 and IRF4 reciprocally upregulates KLF2 , forming a positive autoregulatory circuit. The interaction of MM cells with bone marrow milieu mediates survival of MM cells. Importantly, silencing of KDM3A , KLF2 or IRF4 both decreases MM cell adhesion to bone marrow stromal cells and reduces MM cell homing to the bone marrow, in association with decreased ITGB7 expression in MAF -translocated MM cell lines. Our results indicate that the KDM3A–KLF2–IRF4 pathway plays an essential role in MM cell survival and homing to the bone marrow, and therefore represents a therapeutic target. Several histone modifiers have been implicated in the survival of multiple myeloma cells. Here, the authors reveal a role for the histone demethylase KDM3A in the survival of this haematologic cancer, and show that mechanistically KDM3A removes H3K9 methylation from the promoters of KLF2 and IRF4 , genes essential for myeloma cell survival.