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MYCderegulation occurs in 67% of multiple myeloma (MM) cases and associates with progression and worse prognosis in MM. EnhancedMYCexpression is known to be driven by translocation or amplification events, but it only occurs in 40% of MM patients. Here, we describe a new mechanism ofMYCregulation, whereby epigenetic regulation ofMYCby increased accessibility of a cell-type specific enhancer leads to increasedMYCexpression. We found enhancer activity does not associate with enhancer hijacking events. We identified specific binding of c-MAF, IRF4, and SPIB transcription factors to the enhancer can activateMYC. In addition, we discovered focal amplification of this specific enhancer in approximately 4% of MM patients. Together, our findings define a new epigenetic mechanism ofMYCderegulation in MM beyond known translocations or amplifications and point to the importance of non-coding regulatory elements and their associated transcription factor networks as drivers of MM progression.

High expression ofMYCand its target genes define a subset of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) associated with poor outcomes. Half of these high-grade cases show chromosomal rearrangements between theMYClocus and heterologous enhancer-bearing loci, while focal deletions of the adjacent non-coding genePVT1are enriched inMYC-intact cases. To identify genomic drivers ofMYCactivation, we used high-throughput CRISPR-interference (CRISPRi) profiling of candidate enhancers in theMYClocus and rearrangement partner loci in GCB-DLBCL cell lines and mantle cell lymphoma (MCL) comparators that lacked common rearrangements betweenMYCand immunoglobulin (Ig) loci. Rearrangements betweenMYCand non-Ig loci were associated with unique dependencies on specific enhancer subunits within those partner loci. Notably, fitness dependency on enhancer modules within theBCL6super-enhancer (BCL6-SE) cluster regulated by a transcription factor complex of MEF2B, POU2F2, and POU2AF1 was higher in cell lines bearing a recurrentMYC::BCL6-SE rearrangement. In contrast, GCB-DLBCL cell lines withoutMYCrearrangement were highly dependent on a previously uncharacterized 3’ enhancer within theMYClocus itself (GCBME-1), that is regulated in part by the same triad of factors. GCBME-1 is evolutionarily conserved and active in normal germinal center B cells in humans and mice, suggesting a key role in normal germinal center B cell biology. Finally, we show that thePVT1promoter limitsMYCactivation by either native or heterologous enhancers and demonstrate that this limitation is bypassed by 3’ rearrangements that removePVT1from its position inciswith the rearrangedMYCgene.Key pointsCRISPR-interference screens identify a conserved germinal center B cellMYCenhancer that is essential for GCB-DLBCL lackingMYCrearrangements.Functional profiling ofMYCpartner loci reveals principles ofMYCenhancer-hijacking activation by non-immunoglobulin rearrangements.

Introduction Patients with hematological malignancies, including multiple myeloma (MM), experience sub-optimal responses to SARS-CoV-2 vaccination. Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM) are precursors to MM and exhibit altered immune cell composition and function. The SARS-CoV-2 pandemic and the subsequent population-wide vaccination represent an opportunity to study the real-life immune response to a common antigen. Here, we present updated results from the IMPACT study, a study we launched in November 2020 to characterize the effect of plasma cell premalignancy on response to SARS-CoV-2 vaccination (vx). Methods We performed: (i) ELISA for SARS-CoV-2-specific antibodies on 1,887 peripheral blood (PB) samples (237 healthy donors (HD), and 550 MGUS, 947 SMM, and 153 MM patients) drawn pre- and post-vx; (ii) single-cell RNA, T cell receptor (TCR), and B cell receptor (BCR) sequencing (10x Genomics) on 224 PB samples (26 HD, and 20 MGUS, 48 SMM, and 24 MM patients) drawn pre- and post-vx; (iii) plasma cytokine profiling (Olink) on 106 PB samples (32 HD, and 38 MGUS and 36 SMM patients) drawn pre- and post-vx; and (iv) bulk TCR sequencing (Adaptive Biotechnologies) on 8 PB samples from 4 patients (2 MGUS, 2 SMM) drawn pre- and post-vx. Results Patients with MGUS and SMM achieved comparable antibody titers to HD two months post-vx. However, patient titers waned significantly faster, and 4 months post-vx we observed significantly lower titers in both MGUS (Wilcoxon rank-sum, p=0.030) and SMM (p=0.010). These results indicate impaired humoral immune response in patients with MGUS and SMM.At baseline, the TCR repertoire was significantly less diverse in patients with SMM compared to HD (Wilcoxon rank-sum, p=0.039), while no significant difference was observed in the BCR repertoire (p=0.095). Interestingly, a significant increase in TCR repertoire diversity was observed post-vx in patients with SMM (paired t-test, p=0.014), indicating rare T cell clone recruitment in response to vaccination. In both HD and patients, recruited clones showed upregulation of genes associated with CD4+ naïve and memory T cells, suggesting preservation of the T cell response in SMM, which was confirmed by bulk TCR-sequencing in 4 patients.Lastly, by cytokine profiling, we observed a defect in IL-1β and IL-18 induction post-vx in patients with SMM compared to HD (Wilcoxon rank-sum, p=0.047 and p=0.015, respectively), two key monocyte-derived mediators of acute inflammation, suggesting an altered innate immune response as well. Conclusion Taken together, our findings highlight that despite the absence of clinical manifestations, plasma cell premalignancy is associated with defects in both innate and adaptive immune responses. Therefore, patients with plasma cell premalignancy may require adjusted vaccination strategies for optimal immunization. Citation Format: Yoshinobu Konishi, Romanos Sklavenitis-Pistofidis, Michelle P. Aranha, Ting Wu, Hong Yue, Elizabeth D. Lightbody, Mahshid Rahmat, Michael Timonian, Shohreh Varmeh, Daniel Heilpern-Mallory, Michael P. Agius, Nang K. Su, Jacqueline Perry, Erica Horowitz, Maya I. Davis, Anna V. Justis, Radosław P. Nowak, Mark Hamilton, Daniel Auclair, Catherine R. Marinac, Eric S. Fischer, Gad Getz, Irene M. Ghobrial. Altered immune response to vaccination in patients with plasma cell premalignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 796.

PURPOSE Smoldering multiple myeloma (SMM) is a precursor condition of multiple myeloma (MM) with a 10% annual risk of progression. Various prognostic models exist for risk stratification; however, those are based on solely clinical metrics. The discovery of genomic alterations that underlie disease progression to MM could improve current risk models. METHODS We used next-generation sequencing to study 214 patients with SMM. We performed whole-exome sequencing on 166 tumors, including 5 with serial samples, and deep targeted sequencing on 48 tumors. RESULTS We observed that most of the genetic alterations necessary for progression have already been acquired by the diagnosis of SMM. Particularly, we found that alterations of the mitogen-activated protein kinase pathway ( KRAS and NRAS single nucleotide variants [SNVs]), the DNA repair pathway (deletion 17p, TP53, and ATM SNVs), and MYC (translocations or copy number variations) were all independent risk factors of progression after accounting for clinical risk staging. We validated these findings in an external SMM cohort by showing that patients who have any of these three features have a higher risk of progressing to MM. Moreover, APOBEC associated mutations were enriched in patients who progressed and were associated with a shorter time to progression in our cohort. CONCLUSION SMM is a genetically mature entity whereby most driver genetic alterations have already occurred, which suggests the existence of a right-skewed model of genetic evolution from monoclonal gammopathy of undetermined significance to MM. We identified and externally validated genomic predictors of progression that could distinguish patients at high risk of progression to MM and, thus, improve on the precision of current clinical models.

Smoldering multiple myeloma (SMM) is a precursor condition of multiple myeloma (MM) with significant heterogeneity in disease progression. Existing clinical models of progression risk do not fully capture this heterogeneity. Here we integrate 42 genetic alterations from 214 SMM patients using unsupervised binary matrix factorization (BMF) clustering and identify six distinct genetic subtypes. These subtypes are differentially associated with established MM-related RNA signatures, oncogenic and immune transcriptional profiles, and evolving clinical biomarkers. Three genetic subtypes are associated with increased risk of progression to active MM in both the primary and validation cohorts, indicating they can be used to better predict high and low-risk patients within the currently used clinical risk stratification models.

Introduction: Patients with hematological malignancies exhibit inferior response to SARS-CoV2 vaccination, compared to healthy individuals, however little is known about patients with precursor hematological malignancies and the cellular underpinnings of vaccination response. Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Myeloma (SMM) are plasma cell premalignancies that precede Multiple Myeloma (MM) and exhibit signs of immune dysregulation; they affect approximately 5% of the population over 50 years of age, who remain largely undiagnosed, due to lack of screening. In November 2019, we launched the IMPACT study to characterize the immune response to SARS-CoV2 vaccination in patients with plasma cell dyscrasias and healthy individuals. Methods: We performed single-cell RNA-sequencing on 224 peripheral blood mononuclear cell samples drawn from 118 IMPACT (IRB #20-332) participants with MGUS (n=20), SMM (n=48), or MM (n=24), as well as healthy individuals (n=26). Samples were collected before vaccination and after 2 doses of BNT162b2 (Pfizer-BioNtech) (n=123), mRNA-1273 (Moderna) (n=83) or 1 dose of Ad26.COV2.S (Janssen) (n=14). Results: Overall, we sequenced 2,025,611 cells from 224 samples of 118 patients with MGUS, SMM, MM and healthy individuals pre- and post-vaccination for SARS-CoV2, and profiled 553,082 T-cells, 95,392 B-cells, 74,394 NK cells, 195,371 Monocytes, and 35,236 Dendritic cells (DC). We identified activated clusters of B-cells, NK cells and DCs expressing genes such as CD83, CD69, CXCR4, and genes related to the NF-kB and AP-1 pathways. We compared cell type abundances pre- and post-vaccination within each participant population and found that activated CD83+ cells significantly increased post-vaccination in healthy individuals and patients with MGUS (paired t-test, q < 0.1), but not in patients with SMM or overt MM. At baseline, patients with SMM and MM had significantly fewer memory B-cells and significantly more cytotoxic T-cells and NK cells, compared to healthy individuals (Wilcoxon, q < 0.1), which could partly explain the differences observed post-vaccination. Patients with MM also had significantly higher levels of tolerogenic IL-10-expressing DCs (DC10) at baseline (Wilcoxon, q < 0.1), which could be dampening antigen-specific T-cell responses. Conclusion: We identified a significant expansion of activated B-cell, NK cell and DC subpopulations expressing CD83, CD69 and CXCR4, following vaccination in healthy individuals and patients with MGUS, but less so in patients with SMM and overt MM. Our results provide insight into the cellular mechanisms of immune response to SARS-CoV2 vaccination in healthy individuals and patients with precursor plasma cell malignancies and suggest that asymptomatic individuals with SMM may exhibit inferior response to vaccination. Citation Format: Romanos Sklavenitis-Pistofidis, Yoshinobu Konishi, Michelle Aranha, Mahshid Rahmat, Ting Wu, Michael Timonian, Shohreh Varmeh, Daniel Heilpern-Mallory, Michael P. Agius, Nang K. Su, Elizabeth D. Lightbody, Jacqueline Perry, Erica M. Horowitz, Anna V. Justis, Daniel Auclair, Catherine R. Marinac, Eric S. Fischer, Gad Getz, Irene M. Ghobrial. Single-cell RNA-sequencing for immune profiling of SARS-CoV2 vaccine response in healthy individuals and patients with precursor plasma cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3582.

Precursor states of Multiple Myeloma (MM) and its native tumor microenvironment need in-depth molecular characterization to better stratify and treat patients at risk. Using single-cell RNA sequencing of bone marrow cells from precursor stages, MGUS and smoldering myeloma (SMM), to full-blown MM alongside healthy donors, we demonstrate early immune changes during patient progression. We find NK cell abundance is frequently increased in early stages, and associated with altered chemokine receptor expression. As early as SMM, we show loss of GrK(+) memory cytotoxic T-cells, and show their critical role in MM immunosurveillance in mouse models. Finally, we report MHC class II dysregulation in CD14(+) monocytes, which results in T cell suppression in vitro. These results provide a comprehensive map of immune changes at play over the evolution of pre-malignant MM, which will help develop strategies for immune-based patient stratification.

Myeloma bone disease is a devastating complication of multiple myeloma (MM) and is caused by dysregulation of bone remodeling processes in the bone marrow microenvironment. Previous studies showed that microRNA-138 (miR-138) is a negative regulator of osteogenic differentiation of mesenchymal stromal cells (MSCs) and that inhibiting its function enhances bone formation in vitro. In this study, we explored the role of miR-138 in myeloma bone disease and evaluated the potential of systemically delivered locked nucleic acid (LNA)-modified anti-miR-138 oligonucleotides in suppressing myeloma bone disease. We showed that expression of miR-138 was significantly increased in MSCs from MM patients (MM-MSCs) and myeloma cells compared to those from healthy subjects. Furthermore, inhibition of miR-138 resulted in enhanced osteogenic differentiation of MM-MSCs in vitro and increased the number of endosteal osteoblastic lineage cells (OBCs) and bone formation rate in mouse models of myeloma bone disease. RNA sequencing of the OBCs identified TRPS1 and SULF2 as potential miR-138 targets that were de-repressed in anti-miR-138-treated mice. In summary, these data indicate that inhibition of miR-138 enhances bone formation in MM and that pharmacological inhibition of miR-138 could represent a new therapeutic strategy for treatment of myeloma bone disease.

Enhanced expression of the MYC oncogene is associated with the initiation and maintenance of many human cancers, including multiple myeloma (MM). MM is a malignancy of clonal plasma cells, in which MYC deregulation is a key event in the progression from the precursor stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to symptomatic MM. Translocation and amplification of the 8q24.21 MYC locus are known mediators of MYC deregulation at premalignant stages for some patients. However, DNA and RNA sequencing of MM patients show that cases with an intact MYC locus also exhibit MYC deregulation, indicating that additional mechanisms are involved in the deregulation of MYC in MM. Here we describe a new epigenetic mechanism of transcriptional deregulation of MYC in malignant plasma cells. We show that activation of a novel non-coding regulatory region through the binding of MM-specific transcription factors (TFs) is associated with MYC dysregulation in MM. To define the MM-specific MYC epigenetic regulation mechanisms, we performed a high-throughput CRISPR interference (CRISPRi) screen in ANBL6 MM cells that harbor no MYC genetic aberrations. We infected ANBL6 cells with a library of >111,000 sgRNAs, tiling across ~1.2 Mb of sequence around MYC and induced expression of KRAB-dCas9 to epigenetically repress putative regulatory elements. We then sequenced the distribution of sgRNAs in the population before and after 14 passages of growth. Because the expression of MYC quantitatively tunes cellular growth, sgRNAs that reduce MYC expression are less abundant at passage 14. This screen identified a ~13 kb region that significantly reduced cellular proliferation when targeted with sgRNAs. We assessed the function of each enhancer region with individual sgRNAs in different MM cell lines and detected an 89% reduction in MYC mRNA levels on average 48 hours after activating KRAB-dCas9. To further characterize the new enhancer region, we performed chromatin immunoprecipitation (ChIP)- and assay for transposase-accessible chromatin (ATAC)- sequencing on MM cell lines and malignant cells obtained from the bone marrow of 13 SMM and 8 MM patients and normal plasma cells from 3 healthy donors. We found that enhancer elements were enriched for H3K27ac and showed greater chromatin accessibility in tumor cells than normal plasma cells. Motif analysis of the enhancer region recovered putative binding sites for multiple TFs, such as IRF4 and MAF, that play key roles in MM pathogenesis. ChIP-sequencing for these enhancer-associated TFs and luciferase reporter assays targeting their binding sites confirmed the binding and involvement of IRF4 and MAF in activating enhancer elements in MM cells with intact MYC loci. MYC abnormalities are well-known secondary genetic events that trigger the progression of precursor diseases to MM. To define the genetic status of the identified enhancer elements in malignant cells, we examined whole-genome sequencing (WGS) data of 906 MM patients from the MMRF CoMMpass cohort. We found focal amplification of the enhancer region in 2.8% (n = 26) of patients. Transcriptional analysis on the same patient cohort revealed a significant increase in MYC mRNA levels in enhancer-amplified patients compared to MM cases with no MYC aberrations. These results indicate that enhancer activity is required to induce MYC expression and progression of patients with intact MYC loci. Collectively, our findings reveal a novel mechanism of MYC deregulation in malignant plasma cells: selective gain of chromatin accessibility at the enhancer elements and amplification of the enhancer region allow for binding of regulatory factors IRF4 and MAF, which increase the transcription of MYC in the absence of the known MYC chromosomal abnormalities. Our results point to the importance of non-coding regulatory elements and their associated TF networks as drivers of MM progression and suggest a new approach to identify predictive biomarkers and therapeutic targets that could improve patient outcomes in MM and other cancers. Disclosures Fulco: Bristol Myers Squibb: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

Introduction: Multiple Myeloma (MM) is an incurable plasma cell malignancy commonly preceded by the asymptomatic stage smoldering multiple myeloma (SMM). MM is characterized with significant genomic heterogeneity of chromosomal gains and losses (CNVs), translocations, and point mutations (SNVs); alterations that are also observed in SMM patients. However, current SMM risk models rely solely on clinical markers and do not accurately capture progression risk. While incorporating some genomic biomarkers improves prediction, using all MM genomic features to comprehensively stratify patients may increase risk stratification precision in SMM. Methods: We obtained a total of 214 patient samples at SMM diagnosis. We performed whole-exome sequencing on 166 tumors; of these, RNA sequencing was performed on 100. Targeted capture was done on 48 additional tumors. Upon binarization of DNA features, we performed consensus non-negative matrix factorization to identify distinct molecular clusters. We then trained a random forest classifier on translocations, SNVs, and CNVs. The predicted clinical outcomes for the molecular subtypes were further validated in an independent SMM cohort of 74 patients. Results: We identified six genomic subtypes, four with hyperdiploidy (>48 chromosomes, HMC, HKR, HNT, HNF) and two with IgH translocations (FMD, CND) (Table 1). In multivariate analysis accounting for IMWG (20-2-20) clinical risk stages, high-risk (HMC, FMD, HKR) and intermediate-risk (HNT, HNF) genetic subtypes were independent predictors of progression (Hazards ratio [HR]: 3.8 and 5.5, P = 0.016 and 0.001, respectively). The low-risk, CND subtype harboring translocation (11;14) was enriched for the previously defined CD-2 MM signature defined by the B cell markers CD20 and CD79A (FDR = 0.003 ), showed upregulation of CCND1, E2F1, and E2F7 (FDR = 0.01, 0.0004, 0.08), and was enriched for G2M checkpoint, heme metabolism, and monocyte cell signature (FDR = 0.003, 0.003, 0.003, respectively). The FMD subtype with IgH translocations (4;14) and (14;16) was enriched for P53, mTORC1, unfolded protein signaling pathways and plasmacytoid dendritic cell signatures (FDR = 0.01, 0.005, 0.008, respectively). The HKR tumors were enriched for inflammatory cytokine signaling, MYC target genes, T regulatory cell signature, and the MM proliferative (PR) signatures (FDR = 0.02, 0.03, 0.007, 0.02, respectively). The APOBEC mutational signature was enriched in HMC and FMD tumors (P = 0.005), while there was no statistical difference across subtypes in the AID signature. The median follow-up for the primary cohort is 7.1 years. Median TTP for patients in HMC, FMD, and HKR was 3.8, 2.6, and 2.2 years, respectively; TTP for HNT and HNF was 4.3 and 5.2, respectively, while it was 11 years in CND patients (P = 0.007). Moreover, by analyzing the changes in MM clinical biomarkers over time, we found that patients from high-risk subgroups had higher odds of developing evolving hemoglobin and monoclonal protein levels over time (P = 0.01 and 0.002, respectively); Moreover, the absolute increase in M-protein was significantly higher in patients from the high-risk genetic subtypes at one, two, and five years from diagnosis (P = 0.001, 0.03, and 0,01, respectively). Applying the classifier to the external cohort replicated our findings where intermediate and high-risk genetic subgroups conferred increased risk of progression to MM in multivariate analysis after accounting for IMWG staging (HR: 5.5 and 9.8, P = 0.04 and 0.005, respectively). Interestingly, within the intermediate-risk clinical group in the primary cohort, patients in the high-risk genetic subgroups had increased risk of progression (HR: 5.2, 95% CI 1.5 - 17.3, P = 0.007). In the validation cohort, these patients also had an increased risk of progression to MM (HR: 6.7, 95% CI 1.2 - 38.3, P = 0.03), indicating that molecular classification improves the clinical risk-stratification models. Conclusion: We identified and validated in an independent dataset six SMM molecular subgroups with distinct DNA alterations, transcriptional profiles, dysregulated pathways, and risks of progression to active MM. Our results underscore the importance of molecular classification in addition to clinical evaluation in better identifying high-risk SMM patients. Moreover, these subgroups may be used to identify tumor vulnerabilities and target them with precision medicine efforts. Figure 1 Figure 1. Disclosures Bustoros: Janssen, Bristol Myers Squibb: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria. Casneuf: Janssen: Current Employment. Kastritis: Amgen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Genesis Pharma: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau. Davies: Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Takeda: Honoraria; Beigene: Honoraria; Janssen: Honoraria. Bergsagel: Genetech: Consultancy, Honoraria; Oncopeptides: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Patents & Royalties: human CRBN mouse; GSK: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Yong: BMS: Research Funding; Autolus: Research Funding; Takeda: Honoraria; Janssen: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; GSK: Honoraria; Amgen: Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees. Getz: IBM, Pharmacyclics: Research Funding; Scorpion Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

Despite significant advances in the treatment of multiple myeloma (MM), most patients succumb to disease progression. One of the major immunosuppressive mechanisms that is believed to play a role in myeloma progression is the expansion of regulatory T cells (Tregs). In this study, we demonstrate that myeloma cells drive Treg expansion and activation by secreting type 1 interferon (IFN). Blocking IFN α and β receptor 1 (IFNAR1) on Tregs significantly decreases both myeloma-associated Treg immunosuppressive function and myeloma progression. Using syngeneic transplantable murine myeloma models and bone marrow (BM) aspirates of MM patients, we found that Tregs were expanded and activated in the BM microenvironment at early stages of myeloma development. Selective depletion of Tregs led to a complete remission and prolonged survival in mice injected with myeloma cells. Further analysis of the interaction between myeloma cells and Tregs using gene sequencing and enrichment analysis uncovered a feedback loop, wherein myeloma-cell-secreted type 1 IFN induced proliferation and expansion of Tregs. By using IFNAR1-blocking antibody treatment and IFNAR1-knockout Tregs, we demonstrated a significant decrease in myeloma-associated Treg proliferation, which was associated with longer survival of myeloma-injected mice. Our results thus suggest that blocking type 1 IFN signaling represents a potential strategy to target immunosuppressive Treg function in MM.

B-cell development is a multistep process sustained by a highly coordinated transcriptional network under the control of a limited set of transcription factors. Epigenetic mechanisms, including DNA methylation, histone posttranslational modifications and microRNAs act in concert with transcription factors to promote lineage commitment, define and sustain cell identity and establish heritable cell-type- and stage-specific gene expression profiles. Epigenetic modifiers have recently emerged as key regulators of B-cell development and activation. Central to B-cell-mediated immunity are germinal centers, transient structures formed in secondary lymphoid organs where antigen-specific B cells undergo intense proliferation, immunoglobulin somatic hypermutation and isotype switching, to generate ultimately long-lived memory B cells and terminally differentiated plasma cells expressing high-affinity antibodies. Deregulation of one or more epigenetic axes represents a common feature of several B-cell disorders arising from germinal center B cells, including autoimmunity and lymphoma. Moreover, the hijacking of epigenetic determinants is central to the ability of the B-lymphotropic Epstein-Barr virus (EBV) to establish, via the germinal center reaction, life-long latency and occasionally contribute to malignant B-cell transformation. In the light of recent findings, this review will discuss the relevance of epigenetic deregulation in the pathogenesis of B-cell diseases. Understanding how specific epigenetic alterations contribute to the development of lymphomas, autoimmunity and EBV-associated disorders is instrumental to develop novel therapeutic interventions for the cure of these often fatal pathologies.

In multiple myeloma (MM), despite well-characterized precursor states such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), there is a lack of sufficient biomarkers to predict disease progression. Most genomic analyses have studied the malignant plasma cells, however, cancers form a complex ecosystem with the immune and stromal microenvironment. To characterize the cellular composition and transcriptional programs of each component of the tumor and microenvironment at different stages of MM progression, we employed single-cell RNA sequencing on 48K plasma and 40.8K immune microenvironmental cells from a cohort of 22 patients with varying stages of disease progression and 9 healthy donors. Expression profiles of plasma cells revealed clear tumor-specific differences in known oncogenic drivers in MM (MMSET/FGFR3, CCND1 and MAFB) as well as other clonally expressed genes (LAMP5, HIST1H1C, and AREG), distinguishing them from healthy plasma cells. We identified a subset of cycling plasma cells in malignant samples, observing a range of proliferative capacity across disease stages. Furthermore, our approach allowed a unique head-to-head comparison of gene expression changes in normal and malignant plasma cells from the same individual, revealing early alterations in genes related to immune modulation (NKBIA) or controlling transcription and differentiation (EID1). Some alterations were patient-specific, while others, such as MHC I overexpression and CD27 loss, were recurrently observed across subsets of the cohort. Analysis of the BM microenvironment demonstrated significant infiltration of natural killer cells, non-classical monocytes/macrophages, and T cells, even in the earliest stages of the disease. Further investigation revealed upregulation of MHC II expression at the mRNA level in CD14+ monocytes/macrophages and yet, intriguingly, analysis by CyTOF and immunohistochemistry revealed a shift towards intracellular localization of MHC II in these cells. Co-culture with MM cell lines was sufficient to induce the decrease of extracellular MHC II, providing strong evidence for MM-induced compromised antigen presentation by macrophages, and hinting at a mechanism of immune evasion. Together, our results provide a comprehensive view at the complex interplay of the immune and malignant cells in different stages of the disease. We demonstrate the immune response beginning in premalignant conditions to be heterogeneous, including compromised antigen presentation as well as alterations in cellular composition and signaling. Consideration of the type of immunological response may prove valuable in determination of progression risk, as well as open up potential strategies for therapy. Citation Format: Nicholas J. Haradhvala, Oksana Zavidij, Tarek H. Mouhieddine, Romanos Sklavenitis-Pistofidis, Jihye Park, Mairead Reidy, Abdallah Flaifel, Benjamin Ferland, Salomon Manier, Mark Bustoros, Daisy Huynh, Marzia Capelletti, Brianna Berrios, Mahshid Rahmat, Chia-Jen Liu, Meng Xiao He, Esteban Braggio, Rafael Fonseca, Yosef Maruvka, Jennifer Guerriero, Melissa Goldman, Eliezer Van Allen, Steven McCarroll, Jamil Azzi, Gad Getz, Irene M. Ghobrial. Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 139.

Introduction. Multiple myeloma (MM) is a complex and heterogeneous malignancy of plasma cells that has two precursor states: monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). MGUS and SMM are asymptomatic states that eventually give rise to overt MM, with some patients progressing, while others do not. Recent studies in MM pathobiology have highlighted epigenetic alterations that contribute to the onset, progression and heterogeneity of MM. Global hypomethylation of DNA, including tumor suppressor genes, and hypermethylation of B-cell specific enhancers, abnormal histone methylation patterns due to the overexpression of histone methyltransferases such as MMSET, and deregulation of non-coding RNAs along with mutations in different classes of chromatin modulators underline a potential for epigenetic biomarkers in disease prognosis and treatment. This study aimed to define epigenetic pathways that lead to the dynamic regulation of gene expression in MM pathogenesis. Methods. We performed ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and RNA-seq on 10 MM cell lines and CD138+ plasma cells isolated from bone marrow aspirates of 3 healthy donors, 9 SMM, 8 newly diagnosed MM (NDMM) and 9 relapsed (RRMM) patients. ATAC-seq reads were trimmed of adapters, aligned to hg19 using bowtie2, and filtered for mapping quality >=Q30 using the ENCODE ATAC-seq pipeline. Reads mapping to promoter regions, defined as -400 to +250 bases from a refseq transcription start site, were counted using bedtools for each sample. Promoter read counts were then normalized by the total number of reads in promoters in the sample, scaled to 1 million total reads, and converted to log10(x+1) space. Results. To characterize the epigenetic contribution to disease progression in MM, we first identified accessible promoter regions in normal plasma cells (NPC), SMM, NDMM and RRMM patients and found regions displaying differential accessibility in MM progression. Next, we intersected the list of differential accessible regions (DARs) with matched transcriptome data and observed two main clusters: genes with unaltered transcription profiles and genes in which the dynamics of open chromatin regions (OCRs) correlated with gene expression. Transcriptomic analysis revealed that a large portion of the differentially expressed (DE) genes in SMM remain DE in NDMM as compared to NPCs (882 genes out of 1642 and 1150 DE genes in SMM and NDMM, respectively). Those genes were significantly enriched for pathways like epithelial mesenchymal transition, cell cycle checkpoints and mitosis, KRAS signaling and interleukin-JAK-STAT pathways. To investigate the genes that behaved differently among the stages of disease, we looked at differential accessibility and expression in NDMM and SMM samples, and integrated them with Whole-Genome Bisulfite-Sequencing and 450K DNA-methylation data from MM patients and healthy donors (BLUEPRINT). This analysis led to the identification of novel genes in MM progression, such as the transcriptional repressor ZNF254 and IRAK3, a negative regulator of the TLR/IL1R signaling pathway. Although gene expression data for these genes showed comparable mRNA levels in SMM and NPCs, followed by a significant decrease in NDMM/ RRMM, ATAC-seq revealed a striking drop in promoter accessibility in SMM, NDMM and RRMM cases. Comparison of ATAC-seq peaks to DNA methylation and ChIP-seq data revealed that the altered OCR of IRAK3 is actually hypermethylated in MM patients and marked by H3K4me3, a marker of active promoters, in MM cell lines. Hypermethylation of IRAK3 has been described in hepatocellular carcinoma, where it is associated with poor prognosis. Together, our data suggest that the identified IRAK3 OCR may act as a bivalent domain that loses accessibility in the precursor states and gains DNA methylation in MM progression. Hence, IRAK3 methylation could be a novel prognostic marker in MM. Conclusion. We have generated a global epigenetic map of primary tumors from patients at the smoldering, newly diagnosed and relapsed/refractory stage of multiple myeloma. Integrative analysis of ATAC-seq data with DNA methylome, transcriptome and whole-genome map of active and repressive histone marks in our study led to the identification of IRAK3 as a novel epigenetic biomarker of disease progression. Disclosures Licht: Celgene: Research Funding. Ghobrial:Takeda: Consultancy; BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy.

Introduction: In multiple myeloma (MM), despite well-characterized precursor states such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), there is a lack of sufficient biomarkers to predict mechanisms of disease progression. Most genomic analyses have sought biomarkers by study of the malignant plasma cells, however, cancers form a complex ecosystem with the immune and stromal microenvironment. Thus, to characterize the cellular composition and transcriptional programs of each component of the tumor and microenvironment at different stages of MM progression, we employed a single-cell RNA sequencing on a cohort of 22 patients and 9 healthy donors. Methods: We performed 10X droplet-based single-cell RNA sequencing using CD138-expressing plasma cells and microenvironmental populations isolated from bone marrow (BM) aspirates of patients with MGUS (n=6), low-risk SMM (n=3), high-risk SMM (n=13), newly diagnosed MM (n=8) and from 9 healthy donors (NBM). We collected a total of ~88.8K cells, comprising ~48K CD138+ cells (~36.4 from MM stages) and ~40.8K CD45+/CD138- cells (~30.8 from MM stages).Raw read data was processed using the Cell Ranger pipeline to obtain a gene-by-cell expression matrix, which was used to identify cell types and transcriptional programs by clustering and non-negative matrix factorization. Results: Expression profiles of plasma cells revealed clear tumor-specific differences including known oncogenic drivers in MM (MMSET/FGFR3, CCND1 and MAFB) as well as Lysosome-associated Membrane Protein 5 (LAMP5),Histone Cluster 1 H1 Family Member C (HIST1H1C) and Amphiregulin (AREG) distinguishing them from healthy plasma cells. We identified a subset of cycling plasma cells, observing a range of proliferative activity of the malignant fraction. Furthermore, our approach allowed a unique head-to-head comparison of gene expression changes in normal and malignant plasma cells in the MGUS and SMM patients within an individual, excluding inter-individual variation. We were able to discriminate malignant from non-malignant plasma cells and identify transcriptional alterations including known drivers, genes related to immune modulation (NKBIA) or controlling transcription and differentiation (EID1).Some alterations were patient-specific, while others, such as MHC I overexpression and CD27 loss, were recurrently observed across subsets of the cohort. Analysis of BM microenvironment in several stages of MM progression demonstrated a striking shift in the composition of immune cells with significant infiltration of natural killer cells, non-classical monocytes/macrophages, and T cells, enriched even in the earliest stages of the disease. Further investigation revealed significant upregulation of HLA expression at the mRNA level in CD14+ monocytes/macrophages. Intriguingly, comparison of healthy and patient samples by CyTOF showed downregulation of surface MHC II representation in the corresponding cell type, and moreover, co-culture with MM cell lines induced a sharp decrease of extracellular MHC II. This provided strong evidence for compromised antigen presentation by macrophages in the disease setting, hinting at a mechanism of immune evasion. Additionally, expression signatures in cytotoxic T-cells indicated a substantial skewing towards either granzyme B/H- or granzyme K-expressing memory cell-like transcriptional program. In a subgroup of patients, we found a strong simultaneous enrichment of the anti-viral/anti-bacterial gene expression signature for interferon type-1 activated genes in CD14+ monocytes/macrophages and T cells. Together, our results provide a comprehensive view at the complex interplay of the immune and malignant cells in different stages of the disease. We, for the first time, demonstrate the immune response beginning in premalignant conditions to be heterogeneous, including compromised antigen presentation as well as alterations in cellular composition and signaling. Consideration of the type of immunological response may prove valuable in determination of progression risk, as well as open up potential strategies for therapy. Disclosures Bustoros: Dava Oncology: Honoraria. Ghobrial:Celgene: Consultancy; Janssen: Consultancy; BMS: Consultancy; Takeda: Consultancy.

Background This study aimed to determine the benefit of early therapeutic intervention with the combination of elotuzumab, Lenalidomide, and Dexamethasone in patients with high-risk smoldering multiple myeloma (SMM). ClinicalTrials.gov Identifier: NCT02279394. Aims The overarching objective of this trial is to determine progression free survival to symptomatic multiple myeloma (MM). Furthermore, the study examined whether genomic studies can help in determining patients who would benefit the most from this early therapeutic intervention. Methods Patients enrolled in this study met eligibility for high-risk SMM based on the newly defined criteria proposed by Rajkumar et al, Blood 2014. Patients were administered weekly elotuzumab (10 mg/kg) on days 1, 8, 15, and 22 for the first two 28-day cycles while receiving lenalidomide on days 1-21. For cycles 3-8, patients were administered elotuzumab infusions on days 1, 8, and 15. dexamethasone (40mg) was given on days 1, 8 and 15 to 40 of the 50 enrolled patients. After 8 cycles or best response, patients were given the option to mobilize with either cyclophosphamide or plerixafor and collect stem cells for future transplant. Patients were then allowed to continue on maintenance therapy where they were administered elotuzumab (20 mg/kg) on day 1, in combination with lenalidomide days 1-21 of a 28-day cycle. Bone marrow (BM) samples of 32 patients were obtained before starting therapy for baseline assessment and whole exome sequencing (WES) of plasma cells. Results In total, 50 patients were enrolled on this study from January 2015 and completed accrual in December 2016, with the participation of eight sites. The median age of enrolled patients was 62 years (range, 29-79) with 18 males (36%) and 32 females (64%). Interphase fluorescence in situ hybridization (iFISH) detected high-risk cytogenetics (defined by the presence of 17p deletion, t(4;14), and 1q gain) in 20 patients. The median time to response was 2.8 months (range, 1.8-4.6). The most common toxicities were fatigue (92%), followed by diarrhea (72%), and hyperglycemia (62%). The most common grade 3 or more adverse events were hypophosphatemia (34%), neutropenia (26%), and lymphocyte count decreased (22%). Three patients (6%) had grade 4 hypophosphatemia during treatment. Additionally, grade 4 cholecystitis, cataract, lymphocyte count increase, hyperglycemia, neutropenia, and thrombocytopenia occurred in one patient (2%). Diabetic Ketoacidosis and sepsis led to death in a patient (2%). Stem cell collection was successful in all mobilized patients to date. As of this abstract date, the overall response rate is 84% (41/49). There were 3 complete responses (6%), 18 very good partial responses (37%), 20 partial responses (41%), 5 minimal responses (10%), 3 stable disease (6%), and 2 unevaluable patients. All the study participants except for three have finished treatment and are currently under follow up. None of the patients showed progression to overt MM to date. We continue to collect data for progression free survival. WES was performed on 32 samples at the time of initiation of therapy. Recurrent mutations in the MAPK pathway (KRAS, NRAS) and tumor suppressor gene, TP53, were detected in 40% of the cases (16% and 24%, respectively), while mutations in the NF-KB and plasma cell differentiation pathways were present in 13% of patients. Somatic copy number alterations (SCNAs) were called based on WES: 1q duplication, 13q, 17p, and 1p deletions were identified in 25, 31, 12, and 7% of cases, respectively. Interestingly, in 6 patients, high-risk SCNAs (1q gain and 17p deletion) were not reported in iFISH but were detected by WES. The analysis of these 32 samples showed that patients who are harboring mutations in the DNA repair pathway genes, had modest response to treatment. Finally, we are analyzing the transcriptomic profile of CD138 negative cells, which represent the BM microenvironment cells (immune and stromal cells) to characterize the BM microenvironment at baseline and end of treatment, and thus, elucidate the role of these cells in the differential response to therapy. Conclusion The combination of elotuzumab, lenalidomide, and dexamethasone is well tolerated and demonstrates a high response rate with no progression to overt MM to date. Correlation with genomic studies can help define patients who benefit the most from this early therapeutic intervention. Disclosures Ghobrial: Takeda: Consultancy; Janssen: Consultancy; BMS: Consultancy; Celgene: Consultancy. Bustoros:Dava Oncology: Honoraria. Badros:GSK: Research Funding; Celgene: Consultancy, Research Funding; Karyopharm: Research Funding. Matous:Celgene: Consultancy, Honoraria, Speakers Bureau. Rosenblatt:Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jakubowiak:Karyopharm: Consultancy, Honoraria; SkylineDx: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Usmani:Abbvie, Amgen, Celgene, Genmab, Merck, MundiPharma, Janssen, Seattle Genetics: Consultancy; Amgen, BMS, Celgene, Janssen, Merck, Pharmacyclics,Sanofi, Seattle Genetics, Takeda: Research Funding. Zonder:Celgene: Consultancy, Honoraria; Pharmacyclics: Other: DSMC; Janssen: Honoraria; Takeda: Honoraria; Alnylam: Honoraria; Coelum: Honoraria; BMS: Research Funding. Munshi:OncoPep: Other: Board of director. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Celgene: Consultancy. Richardson:Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction The bone marrow (BM) microenvironment in multiple myeloma (MM) plays a pivotal role in tumor growth and bone destructive process. Mesenchymal stromal cells (MSCs) in MM exhibit different genomic and cytokine secretion profiles that ultimately impair their osteogenic differentiation abilities compared to normal MSCs. However, the underlying molecular mechanisms are not fully understood. In the present study, we explored the role of miR-138 in MSCs derived from MM patients (MM-MSCs) and the potential for anti-miR-138 treatment to rescue impaired osteogenic differentiation in MM, both in vitro and in vivo using a human xenograft MM model. Materials and methods Primary BM aspirates were obtained from MM patients and normal healthy donors, after obtaining informed consent in accordance with the Declaration of Helsinki. MiR-138 expression in MM-MSCs was measured by quantitative real-time PCR. Publicly available microarray data sets (GSE17306 and E-TABM-508) were analyzed for miR-138 expression in MM cells compared to normal plasma cells. To test the effect of inhibiting miR-138 function, a high-affinity 15-mer locked nucleic acid (LNA)-modified anti-miR oligonucleotide and a corresponding scramble sequence control oligonucleotide were used (In collaboration with Dr. Kauppinen, Denmark). Anti-miR-138 oligonucleotides were transfected into MM-MSCs or normal MSCs co-cultured with MM cell lines and osteogenic differentiation in MSCs was assessed by alizarin red staining. For the in vivo studies, 6-week-old female SCID-beige mice (n=6, each group) were injected intravenously with anti-miR-138 or scramble control oligonucleotides (15 mg/kg) 2 times a week. 3 weeks later, GFP+Luc+ MM.1S cells (3 × 106) were injected into mice. Anti-miR-138 or control oligonucleotides were continued until day 28 after injection of myeloma cells. At day 28, the effect of anti-miR138 was assessed by the number of osteoblastic lineage cell (OBC: Lin-/CD45-/CD31-/CD51+/Sca-1-) from hematopoietic cell-depleted, collagenase-treated crushed bones of mice by flow cytometry. Results MiR-138 expression in MSCs from MM patients (n=10) was significantly higher than MSCs from normal donors (n = 4) (P Conclusions These findings indicate that miR-138 plays an important role in impaired osteogenic differentiation in MSCs in MM. Inhibition of miR-138 promotes osteogenic differentiation of MSCs in MM and anti-miR-138 treatment holds the potential to prevent MM induced bone loss and lytic lesions. Additional studies are ongoing to further understand the connection between MM cells and MSCs mediated by miR-138. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Novartis: Honoraria; Noxxon: Honoraria; Celgene: Honoraria, Research Funding; Takeda: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding.