Chesi, Marta

Abstract H-MM affects about 40–50% of patients, and a similar proportion of MGUS/SMM. The genetic evolution of the disease and the clinical implications of the co-existence of 13q- or t(14q32) are unknown. We conducted a global survey of genetic abnormalities in H-MM in order to describe genetic evolution pathways. H-MM cases from the Mitelman cancer genetic database were combined with cases from the Mayo Clinic MM genetic database. All abnormalities (regional/whole chromosome gains/losses, breakpoints and translocations) were plotted against a 344-chromosome band profile created in a matrix format, and the number of genetic abnormalities per tumor (NAPT) logged. The abnormalities present in more than 5% of tumors were considered recurrent and each karyotype was scored for the presence or absence of each of the recurrent abnormalities. To investigate the temporal pattern of acquisition of genetic abnormalities, all tumors with a given abnormality were selected and the distributions of NAPT plotted. The modes of these distributions were used as a value of ‘lateness’, referred to as time of occurrence (TO). The impact of co-existence of 13q- and t(14q32) on progression free survival (PFS) and overall survival (OS) in H-MM was determined for MM patients entered into an Eastern Cooperative Oncology Group chemotherapy trial. Four hundred and sixty-nine H-MM patients were included in the karyotypic analysis. The median chromosome number was 53 (range 48–74) and the median NAPT was 10 (range 1–25). The distribution of NAPT revealed a normal distribution, suggesting an obligatory acquisition of a given number of genetic abnormalities. The most common abnormalities were trisomies of chromosomes 3,5,7,9,11,15,19 and 21 affecting a third or more of the patients. Deletions and abnormalities of even-numbered chromosomes occur in 20% or less of patients. None of the more than 300 specific translocations detected were recurrent. However, 5 translocation breakpoints were recurrent including 14q32, 8q24 and 11q13. TO analysis suggest that for H-MM, the trisomies are early genetic events, translocations occur midway and deletions tend to be later events. Amongst the trisomies, correlation analysis does not suggest a particular order of acquisition. Clustering analysis using principal component analysis suggests that in H-MM, IgH translocations are secondary events associated with disease evolution rather than defining unique biological entities. Consistent with this, survival of H-MM patients is not affected by 13q- status but is negatively impacted by the presence of t(14q32) especially those with unknown partners (p<0.05). The data presented suggest that H-MM fit a proposed aneuploid model of cancer due to auto-catalytic chromosome reassortment (Duesberg et al Cell Cycle2003:2:202–210). The presence of tumor specific trisomies and the apparent similar phenotype induced by various combinations of trisomies are consistent with this model.

Abstract Waldenström’s macroglobulinemia (WM) is an incurable low-grade lymphoproliferative disorder characterized by bone marrow (BM) infiltration of a clonal population of small B-lymphocytes, plasmacytoid lymphocytes and plasma cells that secrete monoclonal IgM antibody. Because it is difficult to obtain tumor metaphases for karyotype studies, few recurrent chromosomal abnormalities have been reported and the genetic basis of the disease remains poorly defined. Therefore, we performed a comprehensive high-resolution study to identify genomic abnormalities involved in WM pathogenesis. Fifty-seven WM patients were analyzed using a combination of array-based comparative genomic hybridization (aCGH)(N=42); gene expression profiling (GEP)(N=22), DNA sequencing (N=24) and cIgM-FISH (N=57). Agilent 244A and Affymetrix U133 platforms were used in the aCGH and GEP experiments, respectively. An NF-κB index was calculated from GEP data, as a surrogate marker of NF-κB transcription activity. NFKB2 sub cellular localization was determined by immunofluorescence staining. Overall, 83% of samples have aCGH defined chromosomal abnormalities, with a median of 3 abnormalities per patient (range 0 to 27). We identified 16 recurrent regions of copy number change found in >5% (3 or more patients); 10 deleted and 6 amplified regions. The most common abnormality was the entire or partial deletion of 6q, identified in 40% of cases. Four nonoverlapped minimal deleted regions were identified on 6q (MDR1 to MDR4), each of them being present in at least 14 of 17 patients with the abnormality. Gain of 6p was the second most common abnormality (17%) and its presence was always concomitant with 6q loss. Other recurrent deletions were 13q14 (10%) and 7q22, 8p, 11q22-q23, 11q23-q24 and 17p11-p13 (7% each). Copy gains were identified as partial or entire gains of chromosomes 18 (17%), 4 (12%) and 3 (10%), 8q (10%) and Xq27.1-q28 (10%). Three patients had either homozygous deletions or LOH with mutations affecting both alleles of TRAF3 on 14q32. TRAF3 inactivation was correlated with an increased NF-kB transcriptional signature and it was associated with activation of the non-canonical NFkB pathway. Additionally, we identified the inactivation of TNFAIP3, another negative regulator of the NF-kB pathways, in one of 24 patients. Finally, interstitial deletions identified in 4 patients at 13q14 identified a 1.1 Mb MDR, including MIRN15A and MIRN16-1. These findings confirm that focal 13q14 deletion is not restrict to B-CLL and that the MIRN15A/MIRN16-1 abnormalities might be common to several indolent B-cell diseases. Overall, we identified TRAF3 and TNFAIP3 inactivation in 5.3% and 2.4%, respectively. To our knowledge, this is the first study showing the inactivation of bona fide tumor suppressor genes in WM patients. Furthermore, these genes are negative regulators of NF-kB signaling pathway, highlighting its biologic importance in sustaining and limiting growth in WM. Mutational activation of this pathway, which is normally activated by ligand-receptor interactions within the BM microenvironment, suggest a therapeutic role for inhibitors of NF-KB pathway activation in the treatment of WM.

Abstract We have successfully generated the first immuno-competent transgenic mouse model of multiple myeloma (MM), Vk*MYC, in which sporadic MYC activation in the germinal center is AID dependent and mediated by the somatic hypermutation process. All of Vk*MYC mice develop monoclonal PC expansion by 50 weeks of age, that is manifested by high levels of serum IgGs and major M-spikes by SPEP. The PCs in Vk*MYC mice are fully differentiated, somatically mutated, have a very low proliferation index and are found exclusively in the BM. Associated with the long lifespan (medium survival = 661d), Vk*MYC mice develop anemia and diffuse bone disease (osteoporosis with low bone mineral density), with sporadic occurrence of lytic bone lesions and hind limb paralysis. The phenotypic fidelity of the Vk*MYC mice translates in a therapeutic fidelity, as Vk*MYC mice show response to known clinically active anti-myeloma drugs (bortezomib, melphalane, dexamethasone), but not to drugs that have shown no clinical activity as single agents (vincristine, hydroxyurea, fludarabine). Furthermore, acquired bortezomib resistance could efficiently be induced in Vk*MYC mice after only four cycles of suboptimal treatment, and studies are in progress to identify genetic events associated with bortezomib resistance. Preliminary aCGH experiments on a high resolution Agilent 244K array, using genomic DNA from six Vk*MYC CD138+ selected PCs, showed that the genomic complexity of Vk*MYC tumors is quite low, with only few unbalanced rearrangements, such as trisomy of chromosome 15 detected in two samples or trisomy 9 present in another tumor. The most common abnormalities found are small focal monoallelic deletions, six of which are present in more than one tumor. A focal homozygous deletion of several exons of a gene was identified in two tumors samples. Remarkably, a comparative analysis of the syntenic chromosomal region in human MM cell lines and primary patient samples identified similar recurrent homozygous deletion in this putative novel tumor suppressor gene. Overall, these data suggest that the genomic complexity in this inbred strain of transgenic mice is significantly less then in patients with MM, simplifying the search for critical loci involved in MM tumorigenesis and drug resistance and further validate the Vk*MYC mice as a particularly faithful model of human MM.

Abstract Constitutive activation of NF-κB signaling pathways has been identified in a variety of malignancies, however the molecular basis has remained largely unknown. Recently, we have shown mutations in 10 genes encoding positive and negative regulators of NF-κB signaling pathways in 20% of multiple myeloma (MM) patients, with TRAF3 being the most commonly affected gene. TRAF3 is a negative regulator of NF-κB signal and its inactivation results in the constitutive activation of the alternative or noncanonical NF-κB signaling pathway. With the exception of two recent studies in MM, there is no available data regarding the mechanisms that dysregulate NF-κB in other B-cell malignancies. The aim of this study was to analyze the TRAF3 status in a cohort of Waldenström’s macroglobulinemia (WM) patients. Forty-nine WM were analyzed by using a combination of array-based comparative genomic hybridization (aCGH) and cIgM-FISH. Genome sequencing from 28 of these patients was performed on all coding exons of TRAF3 and the adjacent intron-exon junctions. A NF-κB index, as a measure of NF-κB transcription activity, was calculated from 21 patients with gene expression data. The index is the mean expression level of four probe sets differentially expressed between human myeloma cell lines with identified mutations compared to those without identified NF-κB pathway mutations (CD74, IL2RG and 2x TNFAIP3). By performing aCGH we identified a small monoallelic deletion (∼460 Kb), including the TRAF3 locus in one of 22 patients. Genome sequencing identified a missense mutation (D483N) in the remaining allele. This substitution affects a highly conserved amino acid throughout vertebrate evolution, suggesting its functional relevance. We used cIgM-FISH to screen 27 patients with no available aCGH data and we found one biallelic TRAF3 deletion and one chromosome 14 monosomy. In the later case, no DNA was available to sequence the remaining allele. Both samples with biallelic inactivation of TRAF3 possess the two highest NFKB index values of our WM cohort, suggesting NF-κB transcription activity. Moreover, the case with biallelic deletion showed the lowest level of TRAF3 gene expression. Additionally, the entire TRAF3 coding region was sequenced in 28 of these 49 patients and one homozygous nonsense mutation (K286X) was identified. No GEP was available in this sample to calculate the NF-κB index. TRAF3 was only recently implicated in a malignant process, when inactivating mutations and biallelic deletions were reported in approximately 13% of MM patients, making it the most frequently inactivated tumor suppressor in MM. Our study has identified biallelic TRAF3 inactivation in at least 3 of 49 WM patients, suggesting that the constitutive activation of NF-κB signaling pathways may be involved in the pathogenesis of WM. Recent findings showing an association between TRAF3 loss of function and good response to bortezomib in MM patients suggest that proteasome inhibitor-based therapy may be an attractive option in the treatment of the WM patients with constitutive activation of NF-κB signaling pathways. Additional mutational screening of TRAF3 and other regulators of these pathways are ongoing to determine the extent of constitutive activation present in WM and other B-cell malignancies.

Abstract The availability of human myeloma cell lines (HMCLs) has drastically benefited out understanding of multiple myeloma (MM). They represent essential resources to the myeloma research community at large who use them to identify and more importantly validate oncogenic events in MM. Furthermore, they represent the front line tools in efforts to identify novel therapeutic agents for MM patients. However, the myeloma research community is not immune to problems of cell line contamination (mixed populations) or systemic errors where HMCLs are labeled incorrectly. As the community evolves towards the development of tailor medicine it is essential that our basic tools are both pure and correct. Otherwise, a potentially beneficial therapeutic agent designed for a specific patient subset maybe lost simply because of a simple laboratory mix-up at some point over the years. To that end we have developed a simple PCR based method that can be used to validate the purity and authenticity of HMCLs. First two reference collections of over 50 HMCL maintained at the NCI and Mayo Clinic Arizona were compared using microsatellite fingerprinting. This process identified several previously unnoticed discrepancies. First, in both labs several cell lines that reportedly originated from different patients had the same fingerprints. These include the following unexpected pairs; ANBL-6 and DP–6, CAG and ARP–1, KMS–11 and KMS–20, KMS–11 and KMS–21PE/BM, JIM3 and KPMM2. Second, both KP–6 and KAS6/1 showed a level of cross-contamination with ANBL–6. All of the discrepant cell lines were requested from the original contributor and those that were available were retested. After this step we had identified unique fingerprints for ANBL–6, DP–6, KMS–11, and KMS–20. Furthermore, pure populations of KP–6 and KAS6/1 were confirmed. However, CAG and ARP–1 still had an identical fingerprint. Since the use of microsatellite fingerprinting is an expensive task and the identification of cross-contamination is not very robust we set out to develop better method. We decided to use a simple qualitative PCR method, as the binomial presence or absence of a band makes the interpretation of the fingerprint extremely easy. Furthermore, the high sensitivity of a qualitative PCR can pick up a small amount of contamination in the culture that can be lost in microsatellite analysis. This method involves a multiplex PCR screen that simultaneously interrogates 10 regions of the human genome. The screen is divided into two reactions, one with a panel of highly polymorphic copy number variations (CNV) that are often completely absent in some individuals, and a second one based on known biologic events present in the cell lines. The integration of the results from the CNV and Biological Fingerprints will identify each HMCL as a unique entity. We validated the methodology by confirming that this qualitative screen could pick up the cross–contamination identified in the original KP–6 and KAS6/1 samples. As our understanding of myeloma increases and we strive to develop new therapies for specific biological entities it will be essential that groups confirm both the authenticity and purity of their test cultures. We hope that widespread use of this simple screening methodology will significantly improve the quality and reproducibility of assays carried out in labs around the world.

Abstract Events mediating transformation from the pre-malignant MGUS to MM is largely unknown. Previous studies have highlighted RAS mutation as a progression event. However, this abnormality is present in only a small subset of patients. Global gene expression differences between MGUS and MM have been reported but provided little coherent biological insights. To define progression events, we examined the differences in gene expression profiles (GEP, Affymetrix U133A genechip) between 101 MM and 22 MGUS using a powerful functional genomic tool, Gene Set Enrichment Analysis (Subramanium et al PNAS 2005), that allow extraction of biological information based on enrichment of genes associated with a phenotype within gene-sets derived experimentally or extracted from database. We found that a predominant theme relating to MM progression is the over-expression of proliferation related genes in MM highlighted by the enrichment of cell cycle related gene-sets. Another common theme is the enrichment of gene-sets relating to MYC activation. Seven of eight gene-sets related to MYC activation in the database are significantly enriched (p-value <0.05 and false detection rate <0.05). Leading edge (enriched genes) analysis of enriched MYC-related and proliferation-related gene-sets revealed a core, overlapping signature of 8 genes specific to MYC and not proliferation. These genes are further verified as MYC transcriptional targets using Metacore, a network analysis tool. In addition, 7 of the 8 genes had been validated as MYC target genes by chromatin immunoprecipitation previously (www.myccancergene.org). We validated this signature in independent publicly available cell lines and mouse gene expression experiments where this signature is present in mammary epithelial cell lines engineered to over-express MYC but not E2F3, SRC and BCAT, and mouse embryonic fibroblast over-expressing MYC but not HER2 or with RB knocked-out. In addition, analysis of proliferation index in these datasets showed that there is dissociation of proliferation from this signature. This showed conclusively that our signature is MYC specific and not related to proliferation. Of interest, the signature is also present in cell lines with activating RAS mutation, which also have high expression of MYC, suggesting that MYC activation is a downstream effect of activated RAS signaling. Indeed, in our patient cohort, most patients with RAS mutation express this signature. When applied to 2 large GEP MM patient cohorts, this signature was not present in normal plasma cells, rarely present in MGUS, and present in a significant proportion of MM and almost all the human myeloma cell lines. When analyzed in the context of other B-cell malignancies, the signature is very strong in Burkitt’s lymphoma, present in most myeloma but absent in chronic lymphocytic leukemia, Waldenstrom Macroglobulinemia, and normal B-cells. Our analysis therefore suggests that MYC activation may be a unifying pathological event in MGUS to MM progression. As MYC translocations occur in only about 13% of newly diagnosed MM patients, activating RAS mutations and other trans-mechanisms may mediate MYC activation. Our recent demonstration that sporadic MYC activation in a mouse strain prone to monoclonal gammopathy led to MM development provides important functional proof.

8007 Background: We recently described mutations in multiple genes in MM patients that lead to the activation of the non- canonical NF-KB pathway. Among the mutated genes, TRAF3 is the most commonly affected, and is inactivated in about 20% of patients. In this study, we further defined the clinical significance of TRAF3 inactivation in MM. Methods: Deletion of TRAF3 was identified by array- based comparative genomic hybridization using Agilent Human Genome CGH 44k oligonucleotide array and confirmed by fluorescent in situ hybridization. Mutations were detected by sequencing. We use a TRAF3 gene expression cut-off (derived from 127 Mayo clinic patients) as a surrogate for TRAF3 inactivation to investigate its association with Translocations and Cyclin D genetic subtypes, response rates, and survival using 2 large gene expression dataset: UAMS dataset (559 newly diagnosed patients treated with Total therapy II and III) and Millenium datasets (213 relapsed patients entered into randomized study of bortezomib versus dexamethasone). Results: A normalized expression level of less than 0.6 identified all but three of the known TRAF3 inactivated cases in the Mayo dataset (p<0.0001). Using this cut-off, TRAF3 inactivated cases were enriched for t(4;14) (26%, p=0.00002) and rarely D1 (mostly hyperdiploid) (9%, p=9.6E-10) in all 3 datasets. TRAF3 inactivation did not affect survival of patients treated with transplant-based therapy (UAMS and Mayo dataset) but was strongly associated with bortezomib response (89% TRAF3 low versus 40% TRAF3 normal, p0.6 had similar PFS with the two treatment. Conclusion: Mutations resulting in TRAF3 inactivation are common, particularly in non-hyperdiploid MM, and predict for response to bortezomib and resistance to dexamethasone suggesting that inhibition of the non-canonical NF-KB pathway may be one of the principle mechanisms of action of proteasome inhibitors in MM. No significant financial relationships to disclose.

Abstract The most common genetic abnormality in multiple myeloma is the deletion of chromosome 13, seen in almost half of newly diagnosed patients. Unlike chronic lymphocytic leukemia, where a recurrent minimally deleted region including MIR15A/MIR16-1 has been mapped, the deletions in multiple myeloma predominantly involve the entire chromosome and no specific driver gene has been identified. Additional candidate loci include RB1 and DIS3, but while biallelic deletion of RB1 is associated with disease progression, DIS3 is a common essential gene and complete inactivation is not observed. The Vk*MYC transgenic mouse model of multiple myeloma spontaneously acquires del(14), syntenic to human chromosome 13, and Rb1 complete inactivation, but not Dis3 mutations. Taking advantage of this model, we explored the role in multiple myeloma initiation and progression of two candidate loci on chromosome 13: RB1 and MIR15A/MIR16-1. Monoallelic deletion of Mir15a/Mir16-1, but not Rb1, was sufficient to accelerate the development of monoclonal gammopathy in wild-type mice and the progression of multiple myeloma in Vk*MYC mice, resulting in increased expression of Mir15a/Mir16-1 target genes and plasma cell proliferation, which was similarly observed in patients with multiple myeloma. Significance: In the absence of a defined, minimally deleted region the significance of del(13) in multiple myeloma has remained controversial. Here we show that haploinsufficiency of Mir15a/Mir16-1, but not Rb1, upregulates the cell cycle–regulatory network, inducing monoclonal gammopathy in mice and promoting multiple myeloma progression in both mice and men. See related commentary by Walker, p. 16. This article is highlighted in the In This Issue feature, p. 5

Abstract MYC expression is frequently dysregulated in multiple myeloma (MM). In one comprehensive study, MYC structural variations (SV) were found in nearly half of MM cases (Affer et al. Leukemia 2014). The prevalence was higher in hyperdiploid (HRD) tumors (65%) compared to non-hyperdiploid (NHRD) tumors (36%). The large amount of tumor DNA required for all of the genomic studies performed may have biased the samples analyzed (e.g., to those with higher tumor burdens). To validate the findings of recurrent MYC SV in another dataset, we analyzed the CoMMpass data. We analyzed long-insert whole genome sequencing (WGS) data from diagnostic samples in 420 patients from the IA7 release (dbGAP phs000748) for SV. The results of clinical data, processed WES (SNV), RNASeq (gene expression) and WGS (copy number) data from IA8 (http://research.themmrf.org) were used to calculate survival, RAS and NFKB pathway mutation (WES), TC class (RNA), NFKB index (RNA) and hyperdiploid index (WGS). MYC SVs were identified in 38% of tumors. They were present in 53% HRD and 28% NHRD, and by TC in 55% D1, 43% D2, 36% MMSET, 26% MAF, 13% CCND. Juxtaposition of an Ig enhancer (IgH, IgK, IgL) close to MYC was the most common MYC SV, representing ~40% of the MYC SV. Other enhancers identified have mostly been reported previously, with the most frequent being NSMCE2 (12% of SV) and TXNDC5 (5% of SV). Intrachromosomal SV (deletions, inversions, duplications) not associated with any known enhancer were also frequent (18% of SV). As expected, MYC expression was higher in tumors with MYC SV compared to those without (~2.4 fold, p-value<0.0001). We used Gene Set Enrichment Analysis (GSEA) to identify activated pathways that might substitute for MYC in HRD patients without MYC SV and observed significant activation of the NFkB pathway. Interestingly, examining patients with RAS/MAPK pathway (NRAS, KRAS, BRAF, FGFR3 - abbreviated RAS) mutations (identified in 50% of all patients) the same pattern was observed: in the absence of RAS mutation, there was a significantly higher NFkB index. In general, the HRD tumors seem to have either activation of RAS and/or MYC, or activation of NFkB (Figure). There was no difference in overall survival in patients with versus those without MYC SV. We developed a clinically applicable sequencing platform to identify MYC SV, which cannot be reliably identified by FISH. We sought to validate this targeted capture approach, where in addition to the coding exons of 81 interesting genes described previously (M3P), we also pulled down the region surrounding MYC (2 Mb), IgH (0.5 Mb), IgK (50 kb) and IgL (100 kb) allowing us to additionally identify SV in MYC and Ig loci. Using this approach we identified IgH translocations in 29/30 samples with translocations previously identified by FISH (97%). Moreover, we identified MYC SV in 19/22 patients with SV previously identified by mate-pair WGS (86%). Importantly, sequencing identified the precise translocation breakpoint, and identity of the enhancer dysregulating MYC, which may be important variables. In one informative patient two different MYC SV were present at diagnosis, only one of which was still present following a partial response to four cycles of lenalidomide and dexamethasone. This suggests that the two MYC SV are in different subclones, one of which was much more sensitive to the treatment. Interestingly, the enhancer dysregulating MYC in the sensitive subclone harbors 5 strong Ikaros binding sites identified by ChIPseq, suggesting one intriguing mechanism for sensitivity to lenalidomide. To summarize, we verified in a large dataset that MYC expression is frequently dysregulated by SV in MM (38%), and the RAS/MAPK (50%) and NFKB (23%) pathways are frequently activated by mutations. Surprisingly, given their generally good prognosis, nearly half of HRD tumors seem to be MYC driven, while this is true for only a quarter of NHRD tumors. HRD tumors not driven by MYC or RAS appear to be driven by NFKB. Remarkably, the pathways most commonly activated by mutation in MM: CCND, MYC, RAS, NFKB are common to many cancers and have been studied extensively individually. To understand their clinical impact in MM we have developed a comprehensive custom capture sequencing panel that identified 97% of IgH translocations, 86% of MYC SV, and as well as SNV and CNV of 81 recurrently mutated genes. It will be important to include such a comprehensive genetic analysis to complement clinical trials in the future. Figure. Figure. Disclosures Stewart: Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Takeda Oncology: Consultancy; Janssen Pharmaceuticals: Consultancy.

Abstract Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis. Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry. Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs. Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P<0.01). Checkpoint related molecules (PD-1, LAG-3 and Tim-3) on Tregs and Teffs were up-regulated at the protein level in the BM of myeloma injected mice compared to control (P<0.01). These data indicate the activation of Tregs and decrease of Teff activity, leading to suppression of anti-myeloma T cell activity in the MM BM. Under in vitro co-culture conditions, 5TGM1 cells induced a significant increase in the number of Tregs from non-Treg CD4+ T cells compared to controls (P<0.01). 5TGM1 cell-induced Tregs presented with enhanced ki-67 expression, thus suggesting the ability of MM cells to induce Treg proliferation. Additionally, the trans-well co-culture experiment showed that the major mechanism for Treg induction by 5TGM1 was by direct contact with T cells rather than secreting factors. Significant increase in survival was observed in the VK*MYC injected DEREG mice under Treg depletion compared to the DEREG mice without Treg depletion and wild type littermates under DT treatment (P<0.001). The Treg depleted DEREG mice were accompanied with an increase of Teffs (P<0.01), indicating recovery of anti-myeloma T cell activity. RNA sequencing of BM Tregs from VK*MYC injected mice showed increased expression of immune checkpoint related molecules and increased Treg effector molecules (IL-10, Granzyme B) compared to BM Tregs from control mice, indicating a more functionally active phenotype of VK*MYC associated Tregs. GSEA showed an enrichment of genes involved in type-1 interferon signaling in VK*MYC associated Tregs. Conclusions. We used 2 immunocompetent MM mouse models to study the characterization and significance of Tregs in MM progression. CyTOF analysis and RNA sequencing data indicated Treg activation in the MM BM microenvironment. The Treg in vivo depletion experiment showed that Tregs have a significant role in MM progression. These data indicate that immunotherapy targeting Tregs may represent a novel therapeutic strategy for MM. Studies are ongoing to understand the roles of Tregs in human MM patients. Disclosures No relevant conflicts of interest to declare.