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Abstract Multiple myeloma (MM) develops from well-defined precursor stages; however, invasive bone marrow (BM) biopsy limits screening and monitoring strategies for patients. We enumerated circulating tumor cells (CTC) from 261 patients (84 monoclonal gammopathy of undetermined significance, 155 smoldering multiple myeloma, and 22 MM), with neoplastic cells detected in 84%. We developed a novel approach, MinimuMM-seq, which enables the detection of translocations and copy-number abnormalities through whole-genome sequencing of highly pure CTCs. Application to CTCs in a cohort of 51 patients, 24 with paired BM, was able to detect 100% of clinically reported BM biopsy events and could replace molecular cytogenetics for diagnostic yield and risk classification. Longitudinal sampling of CTCs in 8 patients revealed major clones could be tracked in the blood, with clonal evolution and shifting dynamics of subclones over time. Our findings provide proof of concept that CTC detection and genomic profiling could be used clinically for monitoring and managing disease in MM. Significance: In this study, we established an approach enabling the enumeration and sequencing of CTCs to replace standard molecular cytogenetics. CTCs harbored the same pathognomonic MM abnormalities as BM plasma cells. Longitudinal sampling of serial CTCs was able to track clonal dynamics over time and detect the emergence of high-risk genetic subclones.

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.

Supplementary data contains additional Materials and Methods used to generate results presented only in the supplementary figures and tables, as well as more detailed bioinformatics methods. Figure S1 shows correlation between clinical measures of disease pathology, survival, and circulating tumor cells enumeration. Figure S2 shows characteristics of isolated circulating tumor cells from peripheral blood of precursor disease patients. Figure S3 shows cohort-level genomic characterization of tumor in MM precursor stages with CTCs. Figure S4 shows longitudinal and tissue-matched genomic characterization of driver mutations. Figure S5 shows comparison of mutational processes between BMPCs and CTCs assigned to most likely PCAWG composite reference signature. Table S1 shows clinical characteristics and sampling of participants in this study. Table S2 shows whole-genome sequencing coverage and library metrics. Table S3 shows clinical BM FISH results and cells recovered for cohort with matched samples. Table S4 shows comparison of BCR sequence between BMPCs and CTCs. Table S5 shows clinical BM FISH results of peripheral blood only cohort and CTCs recovered. Table S6 shows enumeration of single nucleotide variants and short insertions and deletions discovered from WGS of CTCs. Table S7 shows enumeration of structural variants reconstructed from WGS of CTCs.

Multiple myeloma (MM) develops from well-defined precursor stages; however, invasive bone marrow (BM) biopsy limits screening and monitoring strategies for patients. We enumerated circulating tumor cells (CTC) from 261 patients (84 monoclonal gammopathy of undetermined significance, 155 smoldering multiple myeloma, and 22 MM), with neoplastic cells detected in 84%. We developed a novel approach, MinimuMM-seq, which enables the detection of translocations and copy-number abnormalities through whole-genome sequencing of highly pure CTCs. Application to CTCs in a cohort of 51 patients, 24 with paired BM, was able to detect 100% of clinically reported BM biopsy events and could replace molecular cytogenetics for diagnostic yield and risk classification. Longitudinal sampling of CTCs in 8 patients revealed major clones could be tracked in the blood, with clonal evolution and shifting dynamics of subclones over time. Our findings provide proof of concept that CTC detection and genomic profiling could be used clinically for monitoring and managing disease in MM.Significance:In this study, we established an approach enabling the enumeration and sequencing of CTCs to replace standard molecular cytogenetics. CTCs harbored the same pathognomonic MM abnormalities as BM plasma cells. Longitudinal sampling of serial CTCs was able to track clonal dynamics over time and detect the emergence of high-risk genetic subclones.This article is highlighted in the In This Issue feature, p. 247

B cell maturation antigen (BCMA) target loss is considered to be a rare event that mediates multiple myeloma (MM) resistance to anti-BCMA chimeric antigen receptor T cell (CAR T) or bispecific T cell engager (TCE) therapies. Emerging data report that downregulation of G protein coupled receptor family C group 5 member D (GPRC5D) protein often occurs at relapse after anti-GPRC5D CAR T. To examine the tumor intrinsic factors that promote MM antigen escape, we performed combined bulk and single cell whole genome sequencing/ copy number variation analysis of 25 patients treated with anti-BCMA and/ or -GPRC5D CAR T/ TCE. In two cases, MM relapse post TCE/CAR T was driven by BCMA negative clones harboring focal biallelic deletions at the TNFRSF17 locus at relapse or by selective expansion of pre-existing subclones with biallelic TNFRSF17 loss. In another three cases of MM relapse, we identified non-truncating missense mutations or in-frame deletions in the extracellular domain of BCMA which negates the efficacies of anti-BCMA TCEs, despite detectable surface BCMA protein expression. With respect to GPRC5D, we report the first four cases of MM relapse with biallelic mutations of GPRC5D following anti-GPRC5D TCE, including two cases with convergent evolution where multiple subclones lost GPRC5D through different somatic events. Our data support that immunoselection of BCMA or GPRC5D negative or mutant clones post CAR T/ TCE therapies may be more prevalent than currently perceived. The engineering and selection of immunotherapies in MM should account for target antigen structural and extracellular domain mutations.

International audience; The International Myeloma Working Group recently fully incorporated 18F-FDG PET into multiple myeloma (MM) diagnosis and response evaluation. Moreover, a few studies demonstrated the prognostic value of several biomarkers extracted from this imaging at baseline. Before these 18F-FDG PET biomarkers could be fully endorsed as risk classifiers by the hematologist community, further characterization of underlying molecular aspects was necessary. Methods: Reported prognostic biomarkers (18F-FDG avidity, SUVmax, number of focal lesions, presence of paramedullary disease [PMD] or extramedullary disease) were extracted from 18F-FDG PET imaging at baseline in a group of 139 patients from CASSIOPET, a companion study of the CASSIOPEIA cohort (ClinicalTrials.gov identifier NCT02541383). Transcriptomic analyses using RNA sequencing were realized on sorted bone marrow plasma cells from the same patients. An association with a high-risk gene expression signature (IFM15), molecular classification, progression-free survival, a stringent clinical response, and minimal residual disease negativity were explored. Results:18F-FDG PET results were positive in 79.4% of patients; 14% and 11% of them had PMD and extramedullary disease, respectively. Negative 18F-FDG PET results were associated with lower levels of expression of hexokinase 2 (HK2) (fold change, 2.1; adjusted P = 0.04) and showed enrichment for a subgroup of patients with a low level of bone disease. Positive 18F-FDG PET results displayed 2 distinct signatures: either high levels of expression of proliferation genes or high levels of expression of GLUT5 and lymphocyte antigens. PMD and IFM15 were independently associated with a lower level of progression-free survival, and the presence of both biomarkers defined a group of "double-positive" patients at very high risk of progression. PMD and IFM15 were related neither to minimal residual disease assessment nor to a stringent clinical response. Conclusion: Our study confirmed and extended the association between imaging biomarkers and transcriptomic programs in MM. The combined prognostic value of PMD and a high-risk IFM15 signature may help define MM patients with a very high risk of progression.

Multiple myeloma (MM) is a haematological malignancy of plasma cells characterized by substantial intraclonal genetic heterogeneity. Although therapeutic advances made in the past few years have led to improved outcomes and longer survival, MM remains largely incurable. Over the past decade, genomic analyses of patient samples have demonstrated that MM is not a single disease but rather a spectrum of haematological entities that all share similar clinical symptoms. Moreover, analyses of samples from monoclonal gammopathy of undetermined significance and smouldering MM have also shown the existence of genetic heterogeneity in precursor stages, in some cases remarkably similar to that of MM. This heterogeneity highlights the need for a greater dissection of underlying disease biology, especially the clonal diversity and molecular events underpinning MM at each stage to enable the stratification of individuals with a high risk of progression. Emerging single-cell sequencing technologies present a superlative solution to delineate the complexity of monoclonal gammopathy of undetermined significance, smouldering MM and MM. In this Review, we discuss how genomics has revealed novel insights into clonal evolution patterns of MM and provide examples from single-cell studies that are beginning to unravel the mutational and phenotypic characteristics of individual cells within the bone marrow tumour, immune microenvironment and peripheral blood. We also address future perspectives on clinical application, proposing that multi-omics single-cell profiling can guide early patient diagnosis, risk stratification and treatment strategies.

Prevalence estimates for monoclonal gammopathy of undetermined significance (MGUS) are based on predominantly White study populations screened by serum protein electrophoresis supplemented with immunofixation electrophoresis. A prevalence of 3% is reported for MGUS in the general population of European ancestry aged 50 years or older. MGUS prevalence is two times higher in individuals of African descent or with a family history of conditions related to multiple myeloma. We aimed to evaluate the prevalence and clinical implications of monoclonal gammopathies in a high-risk US population screened by quantitative mass spectrometry.We used quantitative matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and EXENT-iQ software to screen for and quantify monoclonal gammopathies in serum from 7622 individuals who consented to the PROMISE screening study between Feb 26, 2019, and Nov 4, 2021, and the Mass General Brigham Biobank (MGBB) between July 28, 2010, and July 1, 2021. M-protein concentrations at the monoclonal gammopathy of indeterminate potential (MGIP) level were confirmed by liquid chromatography mass spectrometry testing. 6305 (83%; 2211 from PROMISE, 4094 from MGBB) of 7622 participants in the cohorts were at high risk for developing a monoclonal gammopathy on the basis of Black race or a family history of haematological malignancies and fell within the eligible high-risk age range (30 years or older for PROMISE cohort and 18 years or older for MGBB cohort); those over 18 years were also eligible if they had two or more family members with a blood cancer (PROMISE cohort). Participants with a plasma cell malignancy diagnosed before screening were excluded. Longitudinal clinical data were available for MGBB participants with a median follow-up time from serum sample screening of 4·5 years (IQR 2·4-6·7). The PROMISE study is registered with ClinicalTrials.gov, NCT03689595.The median age at time of screening was 56·0 years (IQR 46·8-64·1). 5013 (66%) of 7622 participants were female, 2570 (34%) male, and 39 (1%) unknown. 2439 (32%) self-identified as Black, 4986 (65%) as White, 119 (2%) as other, and 78 (1%) unknown. Using serum protein electrophoresis with immunofixation electrophoresis, the MGUS prevalence was 6% (101 of 1714) in high-risk individuals aged 50 years or older. Using mass spectrometry, we observed a total prevalence of monoclonal gammopathies of 43% (1788 of 4207) in this group. We termed monoclonal gammopathies below the clinical immunofixation electrophoresis detection level (0·2 g/L) MGIPs, to differentiate them from those with higher concentrations, termed mass-spectrometry MGUS, which had a 13% (592 of 4207) prevalence by mass spectrometry in high-risk individuals aged 50 years or older. MGIP was predominantly of immunoglobulin M isotype, and its prevalence increased with age (19% [488 of 2564] for individuals aged50 years, 29% [1464 of 5058] for those aged ≥50 years, and 37% [347 of 946] for those aged ≥70 years). Mass-spectrometry MGUS prevalence increased with age (5% [127 of 2564] for individuals aged50 years, 13% [678 of 5058] for those aged ≥50 years, and 18% [173 of 946] for those aged ≥70 years) and was higher in men (314 [12%] of 2570) compared with women (485 [10%] 5013; p=0·0002), whereas MGIP prevalence did not differ significantly by gender. In those aged 50 years or older, the prevalence of mass spectrometry was significantly higher in Black participants (224 [17%] of 1356) compared with the controls (p=0·0012) but not in those with family history (368 [13%] of 2851) compared with the controls (p=0·1008). Screen-detected monoclonal gammopathies correlated with increased all-cause mortality in MGBB participants (hazard ratio 1·55, 95% CI 1·16-2·08; p=0·0035). All monoclonal gammopathies were associated with an increased likelihood of comorbidities, including myocardial infarction (odds ratio 1·60, 95% CI 1·26-2·02; p=0·00016 for MGIP-high and 1·39, 1·07-1·80; p=0·015 for mass-spectrometry MGUS).We detected a high prevalence of monoclonal gammopathies, including age-associated MGIP, and made more precise estimates of mass-spectrometry MGUS compared with conventional gel-based methods. The use of mass spectrometry also highlighted the potential hidden clinical significance of MGIP. Our study suggests the association of monoclonal gammopathies with a variety of clinical phenotypes and decreased overall survival.Stand Up To Cancer Dream Team, the Multiple Myeloma Research Foundation, and National Institutes of Health.

Glucocorticoids (GC) effects occur through binding to the GC receptor (GR) which, once translocated to the nucleus, binds to GC response elements (GREs) to activate or repress target genes. Among GCs, dexamethasone (Dex) is widely used in treatment of multiple myeloma (MM), mainly in combination regimens. However, despite a definite benefit, all patients relapse. Moreover, while GC efficacy can be largely attributed to lymphocyte-specific apoptosis, its molecular basis remains elusive.To determine the functional role of GR binding in myeloma cells, we generated bulk and single cell multi-omic data and high-resolution contact maps of active enhancers and target genes. We show that a minority (6%) of GR binding sites are associated with enhancer activity gains and increased interaction loops. We find that enhancers contribute to regulate gene activity through combinatorial assembly of large stretches of enhancers and/or enhancer cliques. Furthermore, one enhancer, proximal to GR-responsive genes, is predominantly associated with increased chromatin accessibility and higher H3K27ac occupancy. Finally, we show that Dex exposure leads to co-accessibility changes between predominant enhancer and other regulatory regions of the interaction network. Notably, these epigenomic changes are associated with cell-to-cell transcriptional heterogeneity. As consequences, BIM critical for GR-induced apoptosis and CXCR4 protective from chemotherapy-induced apoptosis are rather upregulated in different cells.In summary, our work provides new insights into the molecular mechanisms involved in Dex escape.

The International Myeloma Working Group recently fully incorporated (18)F-FDG PET into multiple myeloma (MM) diagnosis and response evaluation. Moreover, a few studies demonstrated the prognostic value of several biomarkers extracted from this imaging at baseline. Before these (18)F-FDG PET biomarkers could be fully endorsed as risk classifiers by the hematologist community, further characterization of underlying molecular aspects was necessary. Methods: Reported prognostic biomarkers ((18)F-FDG avidity, SUV(max), number of focal lesions, presence of paramedullary disease [PMD] or extramedullary disease) were extracted from (18)F-FDG PET imaging at baseline in a group of 139 patients from CASSIOPET, a companion study of the CASSIOPEIA cohort (ClinicalTrials.gov identifier NCT02541383). Transcriptomic analyses using RNA sequencing were realized on sorted bone marrow plasma cells from the same patients. An association with a high-risk gene expression signature (IFM15), molecular classification, progression-free survival, a stringent clinical response, and minimal residual disease negativity were explored. Results: (18)F-FDG PET results were positive in 79.4% of patients; 14% and 11% of them had PMD and extramedullary disease, respectively. Negative (18)F-FDG PET results were associated with lower levels of expression of hexokinase 2 (HK2) (fold change, 2.1; adjusted P = 0.04) and showed enrichment for a subgroup of patients with a low level of bone disease. Positive (18)F-FDG PET results displayed 2 distinct signatures: either high levels of expression of proliferation genes or high levels of expression of GLUT5 and lymphocyte antigens. PMD and IFM15 were independently associated with a lower level of progression-free survival, and the presence of both biomarkers defined a group of “double-positive” patients at very high risk of progression. PMD and IFM15 were related neither to minimal residual disease assessment nor to a stringent clinical response. Conclusion: Our study confirmed and extended the association between imaging biomarkers and transcriptomic programs in MM. The combined prognostic value of PMD and a high-risk IFM15 signature may help define MM patients with a very high risk of progression.

Background: Multiple Myeloma (MM) is a hematological malignancy characterized by abnormal proliferation of terminally differentiated plasma cells (PCs) in the bone marrow (BM). MM is almost always preceded by the precursor stage smoldering multiple myeloma (SMM). BM biopsies are useful to monitor disease progression, but they are invasive and not routinely collected from patients for disease monitoring during precursor stages. Profiling circulating tumor cells (CTCs) from peripheral blood (PB) could aid early detection, disease monitoring, and biomarker identification to predict patients at high risk of progression that may benefit from early therapeutic intervention. Methods: Paired PB and BM aspirates were collected from 40 SMM patients enrolled in the PCROWD study (IRB #14-174) at Dana-Farber Cancer Institute. Malignant PCs were enriched by magnetic bead-based methods and underwent 5’ single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics). Results: We analyzed 105,246 BM PCs and 33,234 PB PCs from 15 patients. To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements, assessed by concurrent scBCR-seq. A total of 86,986 BM tumor cells and 8,718 CTCs were captured. A median of 5, 26, and 47 CTCs were present per mL of blood from low, intermediate, and high-risk SMM patients as defined by the International Myeloma Working Group (IMWG) “20/2/20” criteria, suggesting sequencing-based CTC enumeration corresponds to prognosis. High levels of driver genes commonly upregulated in patients with specific translocations, including CCND1 and MAF, were detected in both BM tumor and CTC clusters in 3 patients with t(11;14) and t(14;16) confirmed by fluorescence in situ hybridization (FISH) clinical testing, and 2 additional patients with inconclusive FISH results (Wilcoxon, q Conclusions: This study highlights the utility of scRNA-seq for molecular profiling of CTCs, even in asymptomatic low tumor burden disease. Additional analyses are ongoing in the expanded cohort of 40 patients with paired samples to help gain further insight into CTC heterogeneity. Overall, this study will help enable the design of new molecular liquid biopsy-based approaches to diagnosis, disease monitoring, and biological insights to improve treatment strategies for precursor myeloma patients. Citation Format: Elizabeth D. Lightbody, Danielle T. Firer, Romanos Sklavenitis-Pistofidis, Michael Agius, Ankit K. Dutta, Michelle Aranha, Jean-Baptiste Alberge, Laura Hevenor, Nang Kham Su, Cody Boehner, Erica Horowitz, Jacqueline Perry, Anna Cowan, Hadley Barr, Anna Justis, Daniel Auclair, Catherine R. Marinac, Gad Getz, Irene Ghobrial. Single-cell RNA sequencing of rare circulating tumor cells in precursor myeloma patients reveals molecular underpinnings of tumor cell circulation [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 641.

Introduction: Multiple myeloma (MM) develops from indolent stages monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Precursor conditions are incidentally diagnosed and require invasive bone marrow (BM) biopsies for complete characterization, highlighting the urgent need for improved early detection methods. Minimally invasive blood biopsies to measure circulating multiple myeloma cells (CMMCs) as markers of MM disease development are a promising solution to this unmet need. Here, we present our novel method CatchTheFISH, for whole genome sequencing (WGS) of CMMCs that enables genomic profiling and WGS based cytogenetic analyses from enriched liquid biopsy samples. Application of WGS in a cohort of 20 patients, revealed CMMCs were of tumor origin and able to faithfully match BM sequencing results and detect 100% of clinically reported events. Methods: Peripheral blood from 110 SMM patients from the PCROWD observational study (Dana-Farber Cancer Institute IRB #14-174) was collected and processed on CellSearch system (Menarini Silicon Biosystems), with enrichment and enumeration of CMMCs based on CD138+38+CD45-19- immunophenotype. In 20 patients, CMMCs and white blood cells were sorted for library construction, quantification and WGS on Illumina NovaSeq6000. Mutation analyses were performed with the cancer genome analysis pipelines of the Broad Institute. Results: CMMCs were detected in 84% of SMM patients enrolled in the study, with a median count of 13 CMMCs (range 0 to 43836). We first demonstrated the concordance of WGS results obtained from CMMCs with BM. In 100% of patients tested with paired BM and CMMCs (n = 8), we observed full agreement in structural event calling between our samples and clinical reports, including translocations and CNAs (trisomies, tetrasomy, monosomy 13 and 1q gain/amplification). Next, we showed WGS of CMMCs provided increased diagnostic yield compared to BM biopsy for the detection of structural events and MM-associated driver mutations. In 7 patients (88%) we detected additional aberrations not found by FISH. Unknown translocation events of IGH-MYC and t(14;20) were distinguished in two patients. Additionally, our method enabled detection of MM driver mutations. Three patients (38%) were found to harbor RAS mutations (KRAS and NRAS) in BM samples, which were also validated in matched CMMCs. Finally, we assessed a validation cohort of 12 SMM patients with CMMCs only. WGS detected comprehensive mutation data across all scales including clinically relevant translocations, trisomies, CNAs and mutations. Conclusion: Our findings provide proof of principle that capture and genomic profiling of CMMCs could be a robust surrogate to BM biopsy, allowing minimally invasive detection and monitoring of disease, unlocking the clinical potential of liquid biopsies for MM diagnostics. Citation Format: Ankit K. Dutta, Jean-Baptiste Alberge, Elizabeth D. Lightbody, Romanos Sklavenitis-Pistofidis, Cody J. Boehner, Tarek H. Mouhieddine, Anna Cowan, Nang Kham Su, Erica M. Horowitz, Andrew Dunford, Chip Stewart, Ziao Lin, Laura Hevenor, Hadley Barr, Amanda Cao, Ornkleaw Zepp, Thai Bui, Steve Gross, Daniel Auclair, Gad Getz, Irene M. Ghobrial. Genome sequencing of circulating multiple myeloma cells for minimally invasive molecular characterization of precursor disease pathology [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 640.

Background: Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant condition of multiple myeloma (MM) traditionally identified by serum protein electrophoresis (SPEP) and immunofixation (IFX). Recently, novel, ultra-sensitive mass spectrometry (MS)-based approaches have allowed for monoclonal (M)-protein detection at concentrations below SPEP levels. MGUS has only few known risk factors implicated in its development and progression. Thus, we analyze MS results of the PROMISE nationwide U.S. screening study to evaluate risk factors for (1) MGUS at traditional SPEP/IFX levels of detection and (2) low-level monoclonal gammopathies, which bear uncertain etiology and clinical significance. Methods: PROMISE enrolled individuals age ≥40 who are Black and/or have a first-degree relative with a blood cancer or MM precursor condition. Those with ≥2 first-degree relatives were eligible at age ≥18. Participants were screened for monoclonal gammopathy by MALDI-TOF MS and provided a survey querying metabolic comorbidities and lifestyle. M-protein concentrations ≥0.02 g/dL were considered traditionally-defined MGUS, whereas M-proteins Results: 1,893 screened participants completed the survey. MGUS and MGIP were detected in 13.4% and 22.0% of Blacks and 8.6% and 26.7% of individuals with family history. Adjusting for sex, age at screening, income, and education, obesity or BMI of ≥30 was associated with MGUS (OR, 1.55; 95% CI, 1.08-2.21), compared to BMI 6 hours/day. Physical activity (metabolic equivalents/week), smoking status (current, past, never), alcohol consumption (g/day) had no associations with MGUS. No risk factor associations were found for MGIP. Conclusion: In screening a high-risk population by mass spectrometry, we found associations of both traditionally established (obesity) and novel risk factors (diabetes, hypertension, short sleep) with MGUS. None of these exposures were associated with MGIP despite finding a high prevalence of MGIP in Blacks and individuals with family history, suggesting that these risk factors may not be etiologically involved in MGIP development but possibly its clonal expansion to more advanced stages. Citation Format: David J. Lee, Habib El-Khoury, Jean-Baptiste Alberge, D.J. Sakrikar, David Barnidge, Mark C. Perkins, Stephen Harding, Jacqueline Perry, Maya I. Davis, Julia Amstutz, Erica Horowitz, Timothy R. Rebbeck, Irene M. Ghobrial, Catherine R. Marinac. Obesity, metabolic comorbidities, and lifestyle factors and their association with monoclonal gammopathies in a high-risk screened population: Results of the PROMISE study [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 3651.

Background Multiple myeloma (MM) is a heterogeneous plasma cell malignancy that remains challenging to cure. Global hypomethylation correlates with an aggressive phenotype of the disease, while hypermethylation is observed at particular regions of myeloma such as B cell-specific enhancers. The recently discovered active epigenetic mark 5-hydroxymethylCytosine (5hmC) may also play a role in tumor biology; however, little is known about its level and distribution in myeloma. In this study, we investigated the global level and the genomic localization of 5hmC in myeloma cells from 40 newly diagnosed patients, including paired relapses, and of control individuals. Results Compared to normal plasma cells, we found global 5hmC levels to be lower in myeloma (P

Additional file 3. Methods: This file describes normal plasma cells purification, myeloma cells purification, 5mC and 5hmC dosage by mass spectrometry, bioinformatics methods and statistical analysis.

Additional file 2. Table S1: Patients characteristics, survival and mass spectrometry quantification of 5hmC and 5mC. Table S2: Scoring of 1816 5hmC-enriched domains across NDMM samples. Table S3: Motif analysis of core regulatory circuitries. Table S4: Scoring of groups-specific 5hmC-enriched domains. Table S5: Differential 5hmC-enriched domains between at diagnosis and relapse (DiffBind analysis).

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.

Our understanding of disease progression in multiple myeloma (MM) and its precursor conditions, monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), is classically founded on bulk analysis studies. Low disease burden at the precursor stages has precluded comprehensive analyses of the transcriptomic events underlying malignant transformation. Here, we use single-cell RNA sequencing data from 29,387 bone marrow plasma cells from 26 patients with MGUS, SMM, or MM and 9 healthy controls to characterize the transcriptional transformation at each step of progression. Due to varying disease burdens, many samples contained a mixture of healthy and neoplastic plasma cells. We leveraged this impurity to perform a patient-specific characterization of the disease, comparing each patient's neoplastic and healthy plasma cells. This approach isolated the disease phenotype in each patient, which is usually confounded by biological and technical variability when comparing tumors to samples from healthy donors. We found that neoplastic cells from patients with MGUS and SMM already exhibit phenotypic changes similar to those of advanced myeloma. We observed upregulation of genes corresponding to known MM subtypes, such as CCND1 in patients with t(11;14) translocations and other known driver genes such as HIST1H1C. We also found universal downregulation of certain genes such as CD27, a member of the tumor necrosis factor receptor family associated with the differentiation of B cells into plasma cells, which may signify a common loss of a normal plasma phenotype across samples with different driver events and stages. Pathway analysis of differentially expressed genes further revealed that biological pathways related to myeloma were altered as early as MGUS. We observed that SMM patients with hyperdiploidy exhibit upregulation of ribosomal proteins, as reported in advanced disease. Upregulated genes in select MGUS and SMM samples were enriched for the eukaryotic translation initiation factor 3 (eIF3) complex, which plays important roles in translation, as well as proteasome activity, a function central to the survival of MM and targeted by therapies such as bortezomib. We observed enrichment of the E2F family of transcription factors in MGUS and SMM samples; these are master regulators of proliferation that have been suggested as therapeutic targets in myeloma. Five samples were enriched for genes associated with extracellular exosomes, which has been reported to play an important role in cancer cell signaling and to contribute to osteolysis and drug resistance in MM. Pathway enrichment of genes downregulated in neoplastic cell populations revealed weakened response to endoplasmic reticulum and oxidative stress, presumably allowing myeloma cells to tolerate high volumes of abnormal protein production without apoptosing. To further identify shared gene expression patterns across samples, we employed a Bayesian Non-Negative Matrix Factorization method to decompose our data into 31 gene signatures that capture its variability. In addition to recovering signatures corresponding to known MM subtypes, demonstrating that our method captures cohesive transcriptional networks, we find signatures that capture disease biology shared across subtypes. Most notably, we identified a signature that is active in healthy plasma cells across disease stages and dramatically lost in MM and precursor cells. The top genes in this signature include CD27 and CD79A, which are associated with the B cell lineage and whose downregulation may signify dedifferentiation of premalignant cells as early as MGUS, and JSRP1, CTSH, and HCST, genes as of yet unreported to be involved in plasma and MM cell biology. This phenotype would be obscured at early disease stages by bulk analysis. We validated the discovery and behavior of this signature in an external single-cell dataset from Ledergor et al. (Nature Medicine 2018). In summary, using single-cell RNA sequencing, we discovered that canonical MM pathways are altered as early as MGUS and identified a signature of genes which distinguishes healthy and neoplastic cells even at early disease stages. Our identification of patient-specific transcriptional changes as early as MGUS paves the way for future work exploring personalized treatment approaches prior to malignant disease. Disclosures Haradhvala: Constellation Pharmaceuticals a MorphoSys Company: Consultancy. Zavidij: Constellation Pharmaceuticals: Current Employment. Sontag: curai health: Current holder of individual stocks in a privately-held company; Takeda Pharmaceuticals: Research Funding; Genentech: Research Funding; IBM: Research Funding. 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. 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.

Introduction: Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by the abnormal growth of clonal plasma cells in the bone marrow (BM). In most cases MM develops from early, asymptomatic disease stages known as Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Despite effective new therapies, most MM patients inevitably relapse and require further treatment, highlighting the need for better early detection methods for precursor patients and targeted interventions to prevent early disease from progressing. The initial diagnosis of MGUS/SMM remains an incidental process following the identification of increased clonal immunoglobulin in the blood. BM biopsy is the gold standard for diagnosis and monitoring of MM progression, but is intrusive, painful, and comes with possible secondary complications for patients. Consequently, repeated assessment is not a feasible option for MGUS and SMM patients who are asymptomatic. Here we tested the utility of circulating multiple myeloma cells (CMMCs) from non-invasive blood biopsy to accompany BM as a method to monitor disease development, by enumerating CMMCs from MGUS/SMM patients. Methods: Peripheral blood from 185 precursor patients (75 MGUS and 110 SMM) from the Dana-Farber Cancer Institute observational PCROWD study (IRB #14-174) was collected in CellRescue TM Preservative Tubes and processed on the CellSearch CellTracks Autoprep system using the CMMC assay kit using 4mL of blood. This assay employs the enrichment of CMMCs through the immunophenotype of CD138 +CD45 -19 -, and leukocyte exclusion based on CD45 +CD19 +. Nucleated cells were identified using DAPI staining. The CellTracks Analyzer II fluorescence microscope system was subsequently used to scan captured CMMC cartridges, with software allowing the automated scoring and enumeration of CMMCs. Additional molecular analyses were carried out on SMM patients. Briefly, minipools of CMMCs were sorted by DEPArray and underwent whole genome amplification using Ampli1 kit, PCR-free library construction, quantification and low pass whole genome sequencing (~0.5x) on the Illumina HiSeq2500. To assess whether molecular analyses can be performed to detect hyperdiploidy as a genomic biomarker of MM disease, ichorCNA analyses was performed to determine copy number variant (CNV) events and infer tumour fraction. Results: CMMCs were detected in 27% of MGUS patients collected, with a median count of 2 CMMCs (range 0 to 1328). Comparably, CMMCs were detected in 57% of SMM patients, with a median enumeration of 13 CMMCs (range 0 to 43836). Enumeration of CMMCs illustrated a correlation with clinical measure of disease including the International Myeloma Working Group 2/20/20 risk stratification model. A higher CMMC count was associated with increasing risk group based on the 3-risk factor model, with a median of 5, 29 and 59 CMMCs detected at low, intermediate, and high-risk SMM groups, respectively. CMMC counts were significantly increased at intermediate (P = 5.0 x 10 -4) and high-risk stages (P = 3.7 x 10 -3) compared to low-risk. While enumeration provides a correlative measure of CMMCs that may be of tumor origin, downstream molecular characterization can confirm MM-associated genetic alterations. At the precursor stages, a low tumour burden is evident clinically, thus both normal and malignant plasma cells are present. Therefore, to determine the concordance between bone marrow and peripheral blood CMMCs, we performed genomic analyses to identify arm level gain or loss events. Molecular analyses of CMMCs was carried out in patients who had matched BM and clinical fluorescent in situ hybridization (FISH) results. We showed that CMMCs can capture 100% of clinically annotated BM FISH CNV events. Furthermore, CMMC samples identified additional yield, with further CNVs identified that were not observed by FISH. In cases that did not have BM biopsy results, sequencing of CMMCs revealed the existence of genetic aberrations. Conclusion: Our results demonstrate clinical correlation and molecular characterization of CMMCs from MGUS/SMM patients. This study provides a foundation for non-invasive detection, enumeration and genomic interrogation of rare CMMCs from the peripheral blood of MGUS/SMM, illustrating the clinical potential of using liquid biopsies for monitoring and managing disease in the precursor setting of MM. Disclosures 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.

Background: In order to gain a deeper understanding of the clinical, molecular and immune parameters involved in multiple myeloma (MM) disease initiation, progression and response to treatment, the Multiple Myleoma Research Foundation (MMRF) and its partners launched the CureCloud Research Initiative (NCT03657251), a Direct-to-Patient (DTP) effort aimed at enrolling 5,000 individuals from whom comprehensive molecular and immune analyses are generated from blood specimens and the resulting data aggregated with the correlating clinical information. Methods: To support the molecular characterization of liquid biopsies for CureCloud, a set of myeloma-specific blood-based approaches were developed. The first of such assays is a hybrid selection panel that captures 70 commonly altered MM and Clonal Hematopoiesis of Indeterminate Potential (CHIP) genes detecting somatic variants present in a patient's circulating-free DNA (cfDNA). This MM-70 assay was thoroughly validated to >90% sensitivity for events at 1% variant allele frequency with a specificity of Results: 580 subjects were enrolled to the study at abstract submission, including 127 smolderers, 173 newly diagnosed and 265 relapsed participants. 430 cases have been analyzed on the MM-70 assay. A mean cfDNA amount of 31 ng was recovered for input. Mean raw coverage depth of 176,000x was attained, with a mean duplex depth of 1271 (517-2406). 417 cases passed the minimum CLIA validation thresholds. Some of the most frequently mutated genes observed were in the MAPK, DNA repair, epigenetic, cell cycle, and NF-kappaB pathways in agreement with MM retrospective genomic research studies performed on bone marrow aspirates. Of interest, CHIP mutations were detected in a large proportion of cases (174) with their prevalence increasing with age (Figure 1A). 117 of patients had only one CHIP mutation while 57 had two or more with the same genomic loci were recurrently mutated in DNMT3A, TP53, ASXL1, and PPM1D. No significant differences in CHIP were observed between smoldering and overt myeloma subjects (Figure 1B). Interestingly, a greater (>0.1) CHIP Variant Allelic Fraction (VAF) was seen in individuals with higher risk genomic alterations (Figure 1C). Conclusion: This genomic characterization of one of the largest cohorts to date of individuals with myeloma and precursor conditions using a cfDNA-based liquid biopsy assay was able to recapitulate known myeloma-specific mutations and reinforced the importance of CHIP alterations in MM. CHIP mutations are indeed frequent in myeloma, increase with age, and higher burden might be related to disease aggressiveness. Figure 1 Figure 1. Disclosures 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.

Background Multiple myeloma (MM) evolves from monoclonal gammopathy of undetermined significance (MGUS), a clinically detectable but asymptomatic premalignant phase seen in ~3% of the general population 50 years of age or older. The prevalence of MGUS has not been described in a population at high risk of developing MM, specifically Black/African American (AA) individuals or first-degree relatives of patients with hematologic malignancies (HM). In 2019, we launched the first nationwide US screening study for individuals at high risk of MM to help better identify what population would benefit most from screening and early intervention for precursor MM stages. We aim to assess the prevalence of MGUS in a population at high risk of MM and characterize clinical variables of individuals who screen positive. Here, we report interim screening data on the first 2,960 participants. Methods Individuals aged 40 or older with an additional MM risk factor are eligible to be screened in the PROMISE Study. High-risk individuals include Black/AAs and those with a first-degree relative diagnosed with a hematologic malignancy or a precursor condition to MM. Blood from all participants was analyzed via serum protein electrophoresis, immunofixation, and Optilite® to measure the serum free light chains (sFLC), IgG, IgA and IgM. Results were returned to all participants, and those who tested positive for a monoclonal gammopathy (MGUS/SMM) were referred to a hematologist for clinical follow-up and invited to periodically complete epidemiologic exposure and psychosocial questionaries, including a 4-item cancer worry questionnaire and the RAND 36-item Short Form Survey (SF-36). To investigate the use of the higher-sensitivity matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with the Optilite® IgG, IgA, IgM and sFLC results as a screening test for all participants, we rescreened 1,092 samples from PROMISE. The Binding Site Group proprietary software was used for the analysis of the combined MS/Optilite® results, allowing for the detection and quantification of M-protein. Heavy-Chain MGUS (HC-MGUS) was defined by the presence of one or more paired heavy and light chain monoclonal peaks detected by MS. Pairing was based on mass to charge ratio of identified peaks. To enrich the PROMISE cohort with Black/AA individuals, we identified and screened 1,868 Black/AA additional individuals from the Mass General Brigham (MGB) Biobank who met the PROMISE enrollment criteria. Screening was performed by MS/Optilite®, and results have not been returned to participants. Results We screened 2,960 participants with the combined MS/Optilite® approach. We report here the prevalence of HC-MGUS and plan on presenting the estimated rate of light chain MGUS in our cohort, at the meeting. We detected HC-MGUS in 9.6% (95% CI: 8.6-11%) of our cohort, with a prevalence of 10% (95% CI: 8.3-12%) in the PROMISE cohort and 9.4% (95% CI: 8.1-11%) in the MGB cohort (Table 1 and Figure 1). HC-MGUS prevalence increased with age in high-risk individuals from 4.9% (CI: 3.3-6.9%) for participants aged 40-49 to 13% (CI: 10-17%) in the 70-79 range (P < 1.2E-5). Among monoclonal HC-MGUS, we found 65% IgG, 18% IgM, and 18% IgA. M-spike was quantified in 97% of samples. Median M-spike concentration was, 0.058g/dL (max. 2.6g/dL) for IgG, 0.0043g/dL (max. 0.6g/dL) for IgM, and 0.067g/dL (max. 0.8g/dL) for IgA. In the Promise cohort, no significant change in cancer worry was observed across the pre- and post-screening interval among participants who screened positive (P = 0.52). Health-related quality of life, as measured by the SF-36, was not significantly different in screen-positive vs. screen-negative individuals for any of the eight subscales (all P > 0.20). Conclusions We present the largest dataset on monoclonal gammopathy prevalence and screening in individuals at high risk for MM, and more specifically the largest cohort of Black/AA, using a novel high-sensitivity testing approach. Our results confirm that older adults who are Black/AA or have a first-degree relative with an HM have a high prevalence MGUS and may benefit from precision screening approaches to allow for early detection and clinical intervention. Preliminary data on cancer worry and quality of life indicates that the psychosocial burden of screening in this population is likely minimal. Figure 1 Figure 1. Disclosures Sakrikar: The Binding Site: Current Employment. Krause: The Binding Site: Current Employment. Barnidge: The Binding Site: Current Employment. Bustoros: Takeda: Consultancy, Honoraria; Janssen, Bristol Myers Squibb: Honoraria, Speakers Bureau. Perkins: The Binding Site: Current Employment. Harding: The Binding Site: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kapoor: Ichnos Sciences: Research Funding; Amgen: Research Funding; Pharmacyclics: Consultancy; Takeda: Research Funding; Sanofi: Consultancy; Regeneron Pharmaceuticals: Research Funding; AbbVie: Research Funding; BeiGene: Consultancy; Sanofi: Research Funding; Karyopharm: Research Funding; Glaxo SmithKline: Research Funding; Karyopharm: Consultancy; Cellectar: Consultancy. Mo: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy; Eli Lilly: Consultancy; Janssen: Honoraria; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees. Murray: The Binding Site: Patents & Royalties: Potential Royalties for use of mass spectrometry in M-protein detection. Getz: Scorpion Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; IBM, Pharmacyclics: Research Funding. Marinac: JBF Legal: Consultancy; GRAIL Inc: Research Funding. 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.

Multiple myeloma (MM) is a plasma cell malignancy that remains challenging to cure despite a substantially improving median survival. During the last decade, DNA copy number variation and gene expression studies have described the pathology and its heterogeneity among patients. Epigenetic modifications play important roles in MM, but they are rarely associated with clinical aspects of the disease. In this epigenomics study, we produced quantifications of genomic 5-methylcytosine (5mC) and of 5-hydroxymethylcytosine (5hmC) as well as genome-wide maps of hydroxymethylation to analyse myeloma cells taken from a cohort of 40 newly diagnosed and homogeneously treated patients. We found 5hmC to be globally depleted in MM compared to normal plasma cells, as well as being reduced in advanced clinical stages of the disease. From the hydroxymethylome data, we observed that remaining 5hmC is organised in large peak clusters and is associated with well-known disease-related genes. Based on their signal correlation, these 5hmC peak clusters can be gathered in 2 regulation networks involving core transcription factors such as IRF4, MYC, PRDM1 and TCF3. By performing paired hydroxymethylomes at diagnosis and at relapse, we found the disease progression to be heterogeneous and patient-specific. We found that the location of 5hmC at tumor suppressor TP53INP1 is associated with better outcome while global high level of 5hmC tends to be associated with better overall survival. Together, our study suggests that 5hmC provides new biological insights of the disease severity and progression, and can be used for retrospective studies.Key Points5hmC is globally depleted in MM and even more in advanced stages of the disease.5hmC is locally detected at transcriptionally active regions of MM where its presence can be associated with survival.

Background: Targeting the anti-apoptotic BCL2 protein in haematological malignancies has demonstrated significant anti-tumoral activity in a subset of multiple myeloma patients harbouring rearrangements involving the CCND1 and the immunoglobulin heavy chain enhancers (Eμ and α1/2). The mechanisms underlying the dependency of this subgroup of MM patients on BCL2 remains to be elucidated as well as the mechanisms of resistance to BCL2 inhibition with BH3 mimetic venetoclax. Methods and Results: Sorted bone marrow plasma cells from a cohort of t(11;14) myeloma patients treated with venetoclax were profiled through multi-omics single cell mRNA expression (scRNAseq), copy number profiling (scCNVseq) as well as chromatin accessibility with single cell ATAC-seq. Sequenced reads were aligned to hg38 reference genome. Samples were processed with CellRanger suite v3.0 and downstream analyses were realized with Seurat, Monocle, Signac, and Cicero R packages. Single plasma cells exhibited differential chromatin accessibility landscapes within and across individual patients as well as pre- and post-venetoclax with enrichment of MYC:MAX, RELA, IRF family, RUNX1/3 and ETS motifs. Integration of mRNA and ATAC data revealed a dynamic change of regulatory motifs across individual cell clusters with evidence of selective pressures driven by venetoclax treatment. Similarly mRNA profiling of the apoptotic genes pre- and post-venetoclax exposure showed loss of BCL2 and upregulation of MCL1 and/or BCL2L1 as well as loss of the BH3-only pro-apoptotic genes PMAIP1 and BCC3 in single cell clusters. mRNA levels mirrored open chromatin at the gene bodies and their respective promoter loci consistent with a direct transcriptional regulation. In a patient with several fold upregulation of the BCL2L1 transcript in the post-venetoclax sample (Figure A-B), scATACseq identified a gain in the chromatin accessibility mapping to a genomic region centromeric to BCL2L1 locus on chromosome 20 (chr20:31,617,200-31,619,900). Single cell CNV analysis identified a 5q loss (chr5:142,400,001-156,240,000) mapping to NR3C1 locus explaining with the clinical resistance to dexamethasone. Importantly scCNV also revealed a copy number gain mapping to the same locus with the newly acquired chromatin accessibility on chromosome 20. Mate-pair analysis of the sequencing reads identified the potent IGLL5 B-cell enhancer on chromosome 22 (chr22:22,960,001-22,980,000) as the mate partner juxtaposed the BCL2L1 locus (Figure C). This finding explains the robust upregulation of BCL2L1 mRNA observed in this patient and the shift in BCL2 dependency detected by ex vivo BH3 sensitivity profiling. Of note, while scCNV analysis also depicted a gain in 1q21 (chr1:149,940,001-169,980,001) MCL1 locus at the time of venetoclax resistance the acquisition of t(20,22) shifted the plasma cells dependency to BCL-xL rather than MCL1. This finding was corroborated by the plasma cells ex vivo resistance to dual BCL2 and MCL1 inhibition. Conclusion: Dynamic single cell epigenome and transcriptome profiling of pre- and post-venetoclax of primary plasma cells identified a de novo translocation driving BCL-xL transcription with the IGLL5 B-cell enhancer. This demonstrates that in addition to canonical TF-promoter regulation, restructuring of immunoglobulin regulatory sequences (i.e., enhancers) can also drive aberrant malignant circuitry endowing resistance to anti-BCL2 agents. Figure. Disclosures Neri: Celgene, Janssen: Consultancy, Honoraria, Research Funding. Bahlis:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

Background Positron emission tomography (PET) using 18Fluorodeoxyglucose (FDG) provides independent prognostic informations in newly diagnosed multiple myeloma (NDMM) patients (Moreau et al, ASH 2019; Moreau et al, JCO 2017; Zamagni et al, Blood 2011). At baseline, FDG-PET/CT characteristics such as maximum standardized uptake value (SUVmax), presence of extramedullary disease (EMD), and paramedullary disease (PMD) define high-risk NDMM patients. Similarly, the presence of negative FDG-PET/CT at baseline has been associated with favorable outcome in NDMM patients (Abe et al, EJNMMI 2019; Moreau et al, ASH 2019). The aim of the present study was to identify MM molecular features associated with these functional imaging biomarkers. Methods A group of 136 patients from CASSIOPET, a companion study of the CASSIOPEIA cohort (ClinicalTrials.gov, number NCT02541383) were subjected to whole genome expression profiling using RNA sequencing (RNA-seq) on sorted bone marrow plasma cells in addition to FDG-PET/CT imaging at baseline. RNA-seq reads were aligned to hg38 reference genome with STAR and subjected to differential expression testing with DESeq2 with sample purity treated as a model covariate. High risk group with the GEP70 signature and classification from the seven molecular subgroups (CD-1, CD-2, HY, LB, MF, MS, and PR) were determined by weighted mean value of gene expression (Zhan et al, Blood 2006). Special attention was paid to genes associated with glucose metabolism and related to plasma cells proliferation. On FDG-PET/CT, SUVmax of areas of focally increased tracer uptake on bone was determined and the presence of EMD or PMD identified. Results FDG-PET/CT was positive in 108 patients out of 136 (79,4%), with 19 (14%) and 15 (11%) of them presenting PMD and EMD disease respectively. Expression level of glucose transporter GLUT1 was independent of these three imaging biomarkers (FDG-PET/CT positivity, EMD and PMD), while HK2 was downregulated in negative scans only (Fold Change = 2.1, padj=0.02). GLUT5 expression was associated with positive FDG-PET/CT (Fold Change = 3.5, padj = 8E-4). Both GLUT1 and HK2 weakly correlated with SUVmax (r=0.26 and 0.36, respectively). Of note, negative FDG-PET/CT were enriched for the LB group of patients, consistent with the lower incidence of MRI-defined bone lesions reported in this subgroup, and it remained independent of the GEP70 signature. Furthermore, high risk GEP70 signature was associated with a SUVmax ≥ 4, and correlated with the presence of PMD (OR=3.2, CI=[0.95-10.6], p=0.03), but not with EMD (p=0.7).Conversely, there was no patient from the LB group with detected PMD on imaging, but 25% (2/8) showed EMD, suggesting that different biological features support both disease patterns. Finally, positive PET/CT profiles seemed to display two distinct signatures with either high expression of proliferation genes (MKI67, PCNA, TOP2A, STMN1), or high expression of GLUT5 and lymphocyte antigens (CD19, CD30L, and CCR2), suggesting a different phenotype for this subgroup. This finding was independent of a high SUVmax. Conclusion Our study confirmed that negative FDG-PET/CT at baseline is associated with low HK2 expression while positive exams showed increased GLUT5 expression and proliferation markers. We describe a strong correlation between two imaging biomarkers (baseline SUVmax and PMD) and high risk signature and molecular subgroup with highly proliferative disease. On the contrary, EMD appeared independent of high risk signature or molecular subgroups. Additional studies will confirm and extend the correlation between imaging and clinical features of the disease and molecular characteristics of malignant plasma cells. Disclosures Touzeau: Sanofi: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; GlaxoSmithKline: Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses. Moreau:Amgen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Honoraria.

Background: Selective BCL2 inhibition with venetoclax, induces deep responses in relapsed MM. However, acquired resistance to venetoclax frequently occurs. Importantly, in the Bellini trial, while the combination of venetoclax and bortezomib doubled the median progression free survival compared to control, it resulted in shorter overall survival. These results highlight the challenges secondary resistance poses and the need to investigate the mechanisms mediating the emerged resistance to venetoclax. Methods & Results: Bone marrow (BM) aspirates were collected from patients (n=8) treated with venetoclax prior to initiation of therapy and at disease progression. Mononuclear cell fractions were isolated through ficoll coupled with magnetic sorting of CD138+cells. Ex-vivo functional profiling of venetoclax sensitivity was performed on CD138+cells. Unbiased mRNA and DNA profiling was conducted by single-cell RNA-sequencing (scRNA-seq), single cell ATAC-seq and single cell copy number analysis (scCNV) using the GemCode system (10x Genomics). Cell Ranger, Seurat, Signac and Monocle were used for data analysis. Ex-vivo apoptosis studies revealed a dynamic shift of the IC50s of venetoclax from a median of 100 nM in pretreatment sample to >1000 nM at disease progression. Single cell CNV profiling identified a significant expansion of a pre-treatment subclonal (70%) at the time of relapse. The scale of the 1q CNV gain varied from 3 Mb to a very focal gain of 100 kb encompassing the MCL1 gene locus. Of note copy number gain was also identified in the BCL2L1 gene, but no copy number losses were detected in the BAX or BAK gene loci. Single cell RNA profiling of the same samples confirmed the gain in the MCL1 mRNA transcript with downregulation of BCL2 at the time of relapse. Consistent with a branching evolutionary model, scATAC-seq revealed increased chromatin accessability studies at the MCL1 locus in subclones of relapsed samples. Cell trajectory analysis and pseudotime ordering of cells (Monocle) revealed the emergence of a highly proliferative clone and of an MCL1 dependent clone as the disease evolved from its original BCL2 dependent cluster at pseudotime (t0) (Figure). Furthermore, ex-vivo apoptosis profiling revealed a shift in the sensitivity of the CD138+cells with an acquired sensitivity to MCL1 inhibitor (S63845). These findings suggest that the 1q21 copy number gain with MCL1 upregulation is likely mediating the acquired venetoclax resistance. In order to functionally confirm whether the gain in MCL1 is sufficient to shift BCL2 to MCL1 dependency and induce resistant to venetoclax, we stably overexpressed MCL1 in the KMS12PE BCL2-dependent MM cell line (KMS12PE_MCL1) and examined its sensitivity to venetoclax and/or S63845 relative to control vector (KMS12PE_EV). Of interest, co-immunoprecipitation (co-IP) of BIM in KMS12PE_MCL1 demonstrated a shift in BIM loading and co-IP with MCL1 and BCL2 while it was restricted to BCL2 in KMS12PE_EV cells. Importantly, venetoclax IC50 of KMS12PE_EV increased by 200 folds in KMS12PE_MCL1 cells with acquired sensitivity to S63845. scCNV analysis of one patient did not identify any gain in the 1q locus at the time of disease progression to venetoclax. Mutation analysis however identified a de novo BCL2 mutation [NM_000633.2:c.332A>C, p.(Asp111Ala)]. To determine whether the Asp111Ala mutation alone is sufficient to confer resistance to venetoclax, the mutant was overexpressed in KMS12PE cells. While BIM binding to BCL2 was unaffected, Aps111Ala largely abrogated venetoclax-induced BIM displacement from BCL2 and reduced KMS12PE cells sensitivity to venetoclax by ~7.5 folds. Structure modeling also demonstrated the inability of venotoclax to tightly bind mutant BCL2_Asp111Ala hydrophobic groove. Conclusion: We have discovered and functionally characterized a novel BCL2 mutation that confers in vitro and in vivo resistance to venetoclax-treated MM patients. In addition we have identified through single cell profiling an enrichment of MM clones with MCL1 locus copy number gain at the time of acquired venetoclax resistance. Early detection and dynamic monitoring of these abnormalities (BCL2 mutant or 1q gain) with early therapeutic interventions targeting these clones may enhance venetoclax efficacy and improve patients' survival. Figure Disclosures Neri: Celgene, Janssen: Consultancy, Honoraria, Research Funding. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Bahlis:Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.