Walters, Denise K.

Hematopoietic and stromal cells within the bone marrow (BM) provide membrane-bound and/or soluble factors that are vital for the survival of plasma cells (PCs). Recent reports in murine BM demonstrated the dynamic formation and dispersion of PC clusters. To date, PC clustering in normal human BM has yet to be thoroughly examined. The goal of this study was to determine whether PC clusters are present in human BM and whether clustering changes as a function of age. Quantification of PCs and clustering in BM sections across six different age groups revealed that fewer PCs and PC clusters were observed in the youngest and oldest age groups. PC clustering increased with age until the sixth decade and then began to decrease. A positive correlation between the number of PCs and PC clusters was observed across all age groups. PC clusters were typically heterogeneous for immunoglobulin heavy- and light-chain expression. Taken together, these data demonstrate that PC clusters are present in human BM and that PC clustering increases until middle adulthood and then begins to diminish. These results suggest the spatial distribution of BM PC-supportive stromal cells changes with age:

AbstractIdentifying factors secreted by multiple myeloma (MM) cells that may contribute to MM tumor biology and progression is of the utmost importance. In this study, hepatoma-derived growth factor (HDGF) was identified as a protein present in extracellular vesicles (EVs) released from human MM cell lines (HMCLs). Investigation of the role of HDGF in MM cell biology revealed lower proliferation of HMCLs following HDGF knockdown and AKT phosphorylation following the addition of exogenous HDGF. Metabolic analysis demonstrated that HDGF enhances the already high glycolytic levels of HMCLs and significantly lowers mitochondrial respiration, indicating that HDGF may play a role in myeloma cell survival and/or act in a paracrine manner on cells in the bone marrow (BM) tumor microenvironment (ME). Indeed, HDGF polarizes macrophages to an M1-like phenotype and phenotypically alters naïve CD14+ monocytes to resemble myeloid-derived suppressor cells which are functionally suppressive. In summary, HDGF is a novel factor in MM biology and may function to both maintain MM cell viability as well as modify the tumor ME.

The differentiation of B cells into antibody secreting plasma cells (PCs) is governed by a strict regulatory network that results in expression of specific transcriptomes along the activation continuum. In vitro models yielding significant numbers of PCs phenotypically identical to the in vivo state enable investigation of pathways, metabolomes, and non-coding (ncRNAs) not previously identified. The objective of our study was to characterize ncRNA expression during human B cell activation and differentiation. To achieve this, we used an in vitro system and performed RNA-seq on resting and activated B cells and PCs. Characterization of coding gene transcripts, including immunoglobulin (Ig), validated our system and also demonstrated that memory B cells preferentially differentiated into PCs. Importantly, we identified more than 980 ncRNA transcripts that are differentially expressed across the stages of activation and differentiation, some of which are known to target transcription, proliferation, cytoskeletal, autophagy and proteasome pathways. Interestingly, ncRNAs located within Ig loci may be targeting both Ig and non-Ig-related transcripts. ncRNAs associated with B cell malignancies were also identified. Taken together, this system provides a platform to study the role of specific ncRNAs in B cell differentiation and altered expression of those ncRNAs involved in B cell malignancies.

Abstract Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.

Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated an overall response rate of 70-90% in patients with relapsed/refractory MM, the durable response rate is around 30%. Most patients lose their CART cells and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in CART cell therapy for MM. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and in the generation of an immunosuppressive microenvironment. We hypothesized that CAFs from bone marrows of patients with MM (MM-CAFs) inhibit BCMA CART cells and contribute to their failure and that targeting both the malignant plasma cells and CAFs can overcome this resistance. To test this hypothesis, we isolated MM-CAFs and studied their interaction with BCMA CART cells generated from normal donors (41BB costimulated, lentivirally transduced). Our initial findings suggest that MM-CAFs inhibit BCMA CART cell antigen specific proliferation in the presence of the BCMA+ MM cell line OPM2, and this inhibition is predominantly mediated through the secretion of TGF-β (Fig A). MM-CAFs also promoted MM tumor growth in an MM-TME xenograft model established in the laboratory (Fig B). Here, immunocompromised NOD-SCID-γ-/- (NSG) mice were engrafted with 1x106 luciferase+ BCMA+ OPM2, in combination with either 1x106 CAFs or vehicle control intraveneously (IV). Subsequent tumor burden was monitored by bioluminescent imaging of these mice. The presence of CAFs in this model significantly accelerated MM progression (Fig B). Based on these findings, we aimed to develop CART cell therapy targeting both malignant MM cells and their CAFs and to determine whether this strategy can reverse MM-CAF induced CART cell inhibition. To identify targets for these CART cells, we first verified the expression of Fibroblast Associated Protein (FAP), an established CAF target, on MM-CAFs. Flow cytometric analysis of MM-CAFs showed significantly higher expression of FAP, compared to fibroblasts derived from normal bone marrow (Fig C). In addition, our screening flow cytometric analysis identified CS1 as another protein overexpressed by MM-CAFs (Fig C). We therefore designed and generated FAP CART cells (41BB costimulated, lentivirally transduced) and CS1 CART cells (CD28 costimulated, lentivirally transduced). We also generated dual CART cells for both BCMA-FAP CART cells and BCMA-CS1 CART cells. These dual CART cells were generated through the dual transduction of two lentiviral vectors during CART manufacturing. Next, we evaluated the impact of CAFs on effector functions of BCMA CART cells compared to dual targeting CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of MM-CAFs, the antigen specific proliferation of BCMA CART cells, but not the dual targeting CART cells was significantly inhibited (Fig A). Similarly, in the presence of MM-CAFs, production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, to verify the significance of our laboratory findings, we investigated the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells were able to completely eradicate MM (Fig E). However, to determine the effect of targeting CAFs, we used our MM-TME model. Here, NSG mice were engrafted with the luciferase+ MM cell line OPM2, along with MM-CAFs, as described in Fig 1B. Mice were then imaged for engraftment and randomized to treatment with 1) untransduced control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A lower dose (1x106 IV) of CART cell was used to induce relapse post BCMA CART cells. Treatment with BCMA CART cells led to a transient antitumor activity in this MM-TME model (mice died within 2 weeks), while dual targeting CART cells resulted in durable remissions and long term survival of these mice (Fig F). In summary, we demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in multiple myeloma. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Lentigen: Research Funding; Kite/Gilead: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Tolero: Research Funding; Novartis: Patents & Royalties, Research Funding; Morphosys: Research Funding.

The ability to visualize and quantify the spatial arrangement and geographic proximity of immune cells with tumor cells provides valuable insight into the complex mechanisms underlying cancer biology and progression. Multiplexing, which involves immunofluorescence labeling and the visualization of multiple epitopes within formalin-fixed paraffin embedded tissue sections, is a methodology that is being increasingly employed. Despite the power of immunofluorescence multiplex analysis, application of this technology to bone marrow core biopsies has not yet been realized. Given our specific long term goal to identify immune cells in proximity to bone marrow malignant plasma cells in multiple myeloma patients, we describe in this study adaptation of multiplex immunofluorescence analysis to this tissue. We first identified a blocking strategy that quenched autofluorescence. We next employed a multiplex strategy that uses a simple stripping solution to remove primary and secondary antibodies prior to subsequent rounds of staining. This method was found to be highly efficient and did not significantly alter antigenicity or tissue integrity. Our studies illustrate for the first time that immunofluorescence multiplexing is achievable in bone marrow core biopsies and will provide a novel opportunity to analyze the role of the immune contexture in disease progression of the monoclonal gammopathies.

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Abstract Despite its efficacy, chimeric antigen receptor T-cell therapy (CART) is limited by the development of cytokine release syndrome (CRS) and neurotoxicity (NT). While CRS is related to extreme elevation of cytokines and massive T cell expansion, the exact mechanisms for NT have not yet been elucidated. Preliminary studies suggest that NT might be mediated by myeloid cells that cross the blood brain barrier. This is supported by correlative analysis from CART19 pivotal trials where CD14+ cell numbers were increased in the cerebrospinal fluid of patients that developed severe NT (Locke et al, ASH 2017). Therefore, we aimed to investigate the role of GM-CSF neutralization in preventing CRS and NT after CART cell therapy via monocyte control. First, we investigated the effect of GM-CSF blockade on CART cell effector functions. Here, we used the human GM-CSF neutralizing antibody (lenzilumab, Humanigen, Burlingame, California) that has been shown to be safe in phase II clinical trials. Lenzilumab (10 ug/kg) neutralizes GM-CSF when CART19 cells are stimulated with the CD19+ Luciferase+ acute lymphoblastic leukemia (ALL) cell line NALM6, but does not impair CART cell function in vitro. We have found that malignancy associated macrophages reduce CART proliferation. GM-CSF neutralization with lenzilumab results in enhanced CART cell antigen specific proliferation in the presence of monocytes. To confirm this in vivo, NOD-SCID-g-/- mice were engrafted with high disease burdens of NALM6 and treated with low doses of CART19 or control T cells (to induce tumor relapse), in combination with lenzilumab or isotype control antibody. The combination of CART19 and lenzilumab resulted in significant anti-tumor activity and overall survival benefit compared to control T cells (Fig 1A), similar to mice treated with CART19 combined with isotype control antibody, indicating that GM-CSF neutralization does not impair CART cell activity in vivo. This anti-tumor activity was validated in an ALL patient derived xenograft model. Next, we explored the impact of GM-CSF neutralization on CART cell related toxicities in a novel patient derived xenograft model. Here, NOD-SCID-g-/- mice were engrafted with leukemic blasts (1-3x106 cells) derived from patients with high risk ALL. Mice were then treated with high doses of CART19 cells (2-5x106 intravenously). Five days after CART19 treatment, mice began to develop progressive motor weakness, hunched bodies, and weight loss that correlated with massive elevation of circulating human cytokine levels. Magnetic Resonance Imaging (MRI) of the brain during this syndrome showed diffuse enhancement and edema, associated with central nervous system (CNS) infiltration of CART cells and murine activated myeloid cells. This is similar to what has been reported in CART19 clinical trials in patients with severe NT. The combination of CART19, lenzilumab (to neutralize human GM-CSF) and murine GM-CSF blocking antibody (to neutralize mouse GM-CSF) resulted in prevention of weight loss (Fig 1B), decrease in critical myeloid cytokines (Fig 1C-D), reduction of cerebral edema (Fig 1E), enhanced leukemic disease control in the brain (Fig 1F), and reduction in brain macrophages (Fig 1G). Finally, we hypothesized that disrupting GM-CSF through CRISPR/Cas9 gene editing during the process of CART cell manufacturing would result in functional CART cells with reduced secretion of GM-CSF. We designed guide RNA targeting exon 3 of the GM-CSF gene and generated GM-CSFk/o CART19 cells. Our preliminary data suggest that these CARTs produce significantly less GM-CSF upon activation but continue to exhibit similar production of other cytokines and exhibit normal effector functions in vitro (Fig 1H). Using the NALM6 high tumor burden relapse xenograft model as described above, GM-CSFk/o CART19 cells resulted in slightly enhanced disease control compared to CART19 cells (Fig 1I). Thus, modulating myeloid cell behavior through GM-CSF blockade can help control CART mediated toxicities and may reduce their immunosuppressive features to improve leukemic control. These studies illuminate a novel approach to abrogate NT and CRS through GM-CSF neutralization that also potentially enhances CART cell functions. Based on these results, we have designed a phase II clinical trial using lenzilumab as a modality to prevent CART related toxicities in patients with diffuse large B cell lymphoma. Disclosures Ahmed: Humanigen: Employment. Sahmoud:Humanigen: Employment. Durrant:Humanigen: Employment. Russell:Vyriad: Equity Ownership. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding.

Abstract Aberrant over production of monoclonal immunoglobulin is a classic feature of the plasma cell (PC) malignancy multiple myeloma (MM). At diagnosis and more frequently upon relapse, some MM patients present with increased production of monoclonal free light chain (FLC) without a corresponding increase in intact immunoglobulin. This phenomenon is commonly referred to as light chain escape (LCE). As previously described (Walters et al. 2018, Experimental Hematology, Vol 57 p42-49), we have established a monoclonal IgA kappa MM cell line, designated as MC-B11/14, from the bone marrow (BM) aspirate of a 46 year old man diagnosed with MM. This cell line is non-hyperdiploid and possesses an 11;14 chromosomal translocation. Given our long standing interest in immunoglobulin function and secretion, we used CRISPR technology to knock out IgA heavy chain production in an effort to create a model of LCE. Successful knockout (KO) of IgA expression was demonstrated by immunofluorescence, flow cytometry, and western blot. As expected, secretion of intact IgA was undetectable in the MC-B11/14IgA- cells. Notably, no significant difference in morphology, phenotypic markers, or growth patterns was observed between the MC-B11/14WT and MC-B11/14IgA- cells. Examination of drug sensitivity between the MC-B11/14WT and MC-B11/14IgA- cells revealed that the response to pomalidomide treatment was similar between the MC-B11/14WT and MC-B11/14IgA- cells; however, the MC-B11/14IgA- cells were found to be more resistant to treatment with bortezomib. Given that increased bortezomib resistance was the only significant difference detected between the MC-B11/14WT and MC-B11/14IgA- cell lines in our initial study, we decided to take a metabolomics approach to examine whether loss of IgA heavy chain expression may alter cellular metabolism. Metabolomics or the profiling of small molecules is a powerful tool to characterize the metabolome of cells/biological systems and has proven to be useful in identifying alterations that occur in malignancy. In this regard, we employed pathway-specific targeted LC-MS/MS protocols in order to examine over 330 metabolites in 35 metabolic pathways in MC-B11/14WT and MC-B11/14IgA- methanolmetabolite extracts using an Agilent 1290 UPLC-6490 QQQ-MS liquid chromatography mass-spectrometer. Of the 330 metabolites examined, 28 were found to be significantly more abundant in the MC-B11/14IgA- cells compared to MC-B11/14 WT cells while 79 were found to be significantly less abundant. In general, the majority of significant metabolic differences between the two cell lines were found to involve metabolites from various amino acid synthesis pathways. More specifically, the MC-B11/14 WT cell line was found to possess significantly more citraconic acid, anthranilic acid, and homovanillic acid. By contrast, further analysis revealed that the MC-B11/14IgA- cell line was found to possess an 11.5 fold increase of the metabolite 2-hydroxyglutarate (2-HG). 2-HG has previously been shown to act as a competitive antagonist of α-ketoglutarate (α-KG), which results in the inhibition of α-KG-dependent dioxygenases including the JmjC domain-containing histone demethylases (KDMs) and the ten-eleven translocation (TET) family of DNA hydroxylases. Notably, these proteins are known to play a role in histone and DNA demethylation, respectively. Taken together, these data suggest that MM cells that have undergone LCE may also possess an accumulation of 2-HG which could lead to altered patterns of histone and DNA methylation. Given that MM patients who relapse with LCE typically have a poorer prognosis, further investigation of metabolomics in light-chain only expressing cell lines and patients who have relapsed with LCE is warranted. Disclosures No relevant conflicts of interest to declare.

Abstract Intercellular communication between multiple myeloma (MM) cells and the normal bone marrow stroma leads to a modification of the bone marrow microenvironment, which favors tumor progression. New developments in extracellular vesicle (EV) research suggest that this diverse population of vesicles, released by cancer cells including MM cells, express transmembrane proteins and carry cargo that can modify recipient cells in myriad ways. In particular, tumor-derived EVs have been shown to create permissive microenvironments that lead to metastatic colonization by circulating tumor cells. As we have previously shown that MM EVs can enhance proliferation of recipient MM cells, we hypothesize that MM EVs can potentially play a much larger role in MM development and progression. In order to understand the scope of potential EV roles in MM, we executed a comprehensive proteomic analysis of the cargo of MM EVs. We isolated EVs from patient derived MM cell lines that represent the most common genetic variants of this tumor, and used in vitro generated plasma cells (IVPCs) as a reference population. In a first pass analysis, we selected proteins that were expressed ≥3 fold higher in MM EVs than in IVPC EVs, and this analysis identified 306 proteins. Of interest, included in the 306 differentially proteins were several involved in the regulation of cell adhesion such as members of the a disintegrin and metalloprotease domain (ADAM8, 9, 10, 13, 15 and 22) family, which are also associated with inhibition of cell proliferation. Moreover, CEACAM1, PTPRK, and CDH2, which are also linked to cellular adhesion, were also expressed at a higher level in MM EVs. Various proteins linked to myeloma cell biology were likewise found to be over-represented in MM EVs, and these include BCMA, ITGAV, ITGB5, IL6ST (gp130), CD276 (B7-H3), and CD28. As we are most interested in biologically relevant and actionable proteins present uniquely in MM derived EVs, we filtered from our data proteins that were also present in EVs from IVPCs. We further filtered out proteins known to be present in EVs from normal cells. This filtering strategy reduced the number of interesting candidates to 8, which included CD28, MET, TRKC, and ADAM15. Thus far, we have confirmed the presence of these 4 proteins in a panel of MM EVs, and we are currently in the process of validating additional candidate cargo proteins for their biological role in enhancing tumor cell proliferation and/or protection from apoptosis. In summary, EV proteomic analysis of cell lines representing MM genetic subtypes can lead to the identification of biologically relevant proteins transported systemically by EVs as well as suggest novel biomarkers that are easily detectable in plasma and may permit earlier recognition of disease progression in patients with MM. Disclosures No relevant conflicts of interest to declare.