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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.

Introduction We have shown that in myeloma (MM) the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide, in a CRBN dependent fashion, inhibit thioredoxin reductase and thus increase intracellular oxidative stress as a consequence of peroxide accumulation (Sebastian et al. 2017). Accordingly, we have also shown that CRBN expression alone does not fully correlate with lenalidomide sensitivity, as the cells ability to decompose H2O2 is important - MM cells with lower H2O2 decomposition capacity are more sensitive to IMiDs. We hypothezised that the specific metabolic pathways used by cells for biomass production would influence IMiD sensitivity since they also influence this cellular oxidative capacity. Cellular proteins are key elements of this biomass, and required for the proliferation of MM cells and production of immunoglobulin proteins. Methods We cultured MM cells under various nutrients conditions to understand its effect on cell proliferation and drug responses. Cell viability was assessed by MTT assay, the Luminescent Assay, and direct cell counting. MM cells glycolytic rate was measured using the Seahorse XF analyzer. Western blots were performed to quantify various protein expression levels in MM cells cultured under different nutrients conditions. CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and a lentivirus system was used to over-express CRBN in MM cells. Results MM cells resistant to lenalidomide, but still expressing functional CRBN, show a higher glycolytic pathway use than sensitive cells. We found that the lenalidomide sensitive cell line MM1.S is highly dependent on extracellular glutamine for cell proliferation. In contrast, JJN3, a lenalidomide resistant cells line with wild type CRBN, preferentially consumes glucose. We found that glutamine depletion from culture media completely abolished lenalidomide sensitivity in MM1.S cell line. Western blot analysis revealed that antibody production by MM1.S was primarily dependent on glutamine availability, and that JJN3 antibody production was dependent upon glucose availability. While glutamine depletion from culture medium completely abolished lenalidomide sensitivity in MM1.S cell line, IKZF1, and IKZF3 degradation was unchanged. We further studied the role of CRBN in glutamine dependent cellular biomass protein production using isogenic cell lines (+/- wtCRBN). We found that wtCRBN expressing cells proliferate more and show higher antibody production in the presence of glutamine, over CRBN negative isogenic cell lines. Lenalidomide treatment further increased glutamine dependent antibody production in wtCRBN expressing cells. One possibile explanation is that activation of glutamine catabolism can facilitate protein demethylation via the supplementation α-ketoglutarate (α-KG). Therefore, we tested whether lenalidomide treatment could induce protein demethylation in sensitive cell lines. Western blot probed with mono-methyl lysine antibody showed lenalidomide induced protein demethylation. We further confirmed that protein demethylation, and lenalidomide sensitivity is a consequence of elevated α-KG by treating cells with cell-permeable 5-octyl- α-ketoglutarate which act as a substrate of the lysine demethylases. 5-Octyl-α-ketoglutarate treatment inhibited cell proliferation preferentially in wtCRBN expressing cells, and also enhanced lenalidomide induced sensitivity. Protein demethylation is associated with protein ubiquitination and proteasomal degradation. We thus hypothesize that lenalidomide induced protein demethylation also likely increases proteasome inhibitors sensitivity in MM. Conclusion MM cells with preferential glutamine consumption are likely to be more sensitive to lenalidomide, and extracellular glutamine depletion can induce lenalidomide resistance. MM cells expressing CRBN, but not dependent on glutamine for protein biomass production are more likely to be resistant to lenalidomide. Our study postulates that CRBN protein requirement in lenalidomide sensitivity cells is restricted to high glutamine dependency, and that it is quite likely CRBN has a role in glutamine metabolism. Moreover, MM cells that consume more glutamine are under higher oxidative stress and exhibit less H2O2 decomposition capacity and therefore increasing sensitivity to IMiDs. References Sebastian , S., et al., Blood 2017 129:991-1007 Disclosures Stewart: Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy. Bergsagel:Janssen Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy; Celgene: Consultancy. Fonseca:AbbVie, Amgen, Bayer, Celgene, Kite, Janssen, Juno, Merck, Pharmacylics, Sanofi, Takeda: Other: Consultant/Advisor; Prognosticatin of MM based on Genetic Categorization by FISH: Patents & Royalties; Adaptive Biotechnologies: Other: Scientific Advisory Board.

Conventional therapies to multiple myeloma (MM) are not aimed at specific molecular targets leading ultimately to treatment resistance. Recent reports have shown that iron is instrumental in cancer development and progression and that high intracellular iron levels are associated with poor prognosis. We have demonstrated that MM cells exhibit dysregulated iron homeostasis and that limitation of cytosolic iron inhibits MM cell growth both in vitro and in vivo. The potential therapeutic role of iron should be further investigated to better understand how targeting high-iron MM cells could prevent or delay MM development and recurrence. Our study will provide crucial insights into the iron biology of MM pathogenesis and may lead to novel MM therapy. In this study, two mouse models, young Vk*MYC and old KaLwRij mice, were injected with iron dextran (1.25 mg/kg, IP, once a week). Tumor burden was monitored by serial Serum Protein Electrophoresis (SPEP) tests, flow cytometry, and immunohistochemistry. In vitro co-culturing of ARP1 MM cells with macrophages was employed to determine iron transfer. To determine iron's roles in MM evolution, we injected iron dextran into Vk*MYC mice at 8-week age. Vk*MYC mice develop MGUS around 40-50 weeks with plasma cell (PC) bone marrow infiltration and kidney damage etc. Iron-dextran was used because it is primarily taken up by macrophages. After 14-16 weeks of iron injection, M spike was detected in the injected Vk*MYC mice. The percentage of bone marrow plasma cells (CD138+) were significantly increased to 9% in the Vk*MYC mice injected with iron compared to control mice injected with vehicle by flow cytometry and immunohistochemistry. The acceleration of disease progression via iron injection was also tested in KaLwRij mice, which also spontaneously develops MGUS in old age. M protein was detected in 12 of 15 mice (80%) injected with iron dextran for 10 weeks and 1 of 5 KaLwRij (20%) control mice at 18-months of age. CD138+ B220- plasma cells were determined by flow cytometry. A significant increase of CD138+B220- plasma cells in iron treated mice (4% versus 2%) was observed compared to vehicle control mice. Deparaffined sections of bone marrow from the above mice were stained with Prussian blue and confirmed positive staining of macrophages from iron administrated mice. These results indicate that iron accelerates MGUS development in vivo. We next evaluated whether MM cells accumulate iron from the microenvironment. ARP1 MM cells were co-cultured with primary macrophages derived from mouse bone marrow to mimic disease environment in vitro. Under these conditions, MM cells induced macrophage polarization from M0 to M1 and M2. Furthermore, increased macrophage polarization was confirmed in vivo from the KaLwRij mice injected with 5TGM1 MM cells. To confirm that MM cells uptake iron from macrophages, increased intracellular ferritin levels were observed by western blot in ARP1 MM cells following co-culture with iron-loaded macrophages. We observed that this increase in intracellular ferritin was mediated via the transferrin receptor. This iron mobilization was prevented by iron chelation. Additionally, we confirmed that ferritin levels were higher in CD138+ primary human MM cells compared to CD138- non-MM cells by western blot. Our data indicate that MM cells promote macrophage polarization resulting in the transferring of iron into MM cells. The blockade of iron trafficking between MM cells and macrophages might hold a promise for the prevention and therapy in MM. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Zhan:BIPHARM LLC: Consultancy, Other: % Allocation of Profit.

Abstract Background and Aims. Despite the fact that the myeloma mutational landscape was elucidated several years ago, there are yet no approved precision therapies targeting specific driver mutations. The role of common, but somewhat enigmatic, mutational events remains elusive, for example, the loss-of-function mutation of the RNA-processing exosome ribonuclease, DIS3, the third most-common mutation after KRAS and NRAS mutations. Furthermore, complex clonal architectural patterns (such as subclonal BRAF mutations nested within RAS-mutant clones) complicate the design of targeted therapy trials. To generate precision in vivo models that recapitulate the complex clonal and mutational architecture of myeloma, we integrated two powerful technological platforms: the first RAS-driven myeloma in vivo model (VQ model) created through a collaboration between the Zhang and Asimakopoulos labs coupled with flexible avian leukosis virus (ALV)-mediated gene transfer that permits myeloma genome editing in a temporally- and spatially- controlled fashion (Prdm1: TVb-mRFP model)(Asimakopoulos and Varmus, J Virol, 2009; 83(10):4835-43). VQ is based on the state-of-art Vk*MYC model (Chesi et al., Cancer Cell 2008;13(2):167-80). VQ was generated by conditionally activating an NrasQ61R allele in Vk*MYC mice. The resultant VQ model recapitulates an advanced, proliferative and drug-resistant phase of disease characterized by RAS mutations. The transducibility of VQ restored the functionality of the ALV-gene transfer technology. This technology exploits the fact that avian retroviruses cannot transduce mammalian cells unless they are engineered to ectopically express an avian retroviral receptor (in this case, the receptor TVb, driven by a plasma-cell-specific regulatory elements). Recently, the ALV gene transfer technology was adapted to achieve CRISPR-Cas9 mediated gene editing in the tumor niche in vivo (Oldrini et al., Nat Comm 2018;9(1):1466). Approach. We generated TVb-expressing VQ lines (CD138+ B220neg) that home to the bone marrow upon transplantation into Prdm1:TVb-mRFP fully immunocompetent syngeneic recipients in the C57BL6/J background. These VQ cells were engineered to express the nuclease Cas9. To determine the efficiency of gene transfer through ALV vectors (as a prelude to sgRNA delivery), pilot experiments were conducted using two types of ALV vector carrying a fluorescent reporter, GFP (green fluorescent protein): a) an RCAS-type vector that drives reporter expression from the native ALV LTR and b) an RCAN-type vector that drives reporter expression from an internal CMV promoter. Results. ALV titers achieved were routinely in excess of 107-108 infectious units/mL. Parental Vk*MYC cells expressing the avian receptor TVb were poorly transducible with ALV vectors. By contrast, VQ cells expressing TVb were readily transducible. At multiplicity of infection (MOI)=2, 68% of VQ myeloma cells expressed reporter fluorescence after a single round of transduction. At MOI=5 the transduction rate rose to 80%. The mean fluorescence intensity (MFI) positively correlated with MOI, consistent with higher-copy proviral integration at higher MOI. At equivalent MOI, GFP fluorescence from RCAS vectors was more intense than GFP fluorescence driven by an internal CMV promoter in RCAN vectors, suggesting that the ALV LTR is a stronger promoter than CMV in VQ myeloma cells. 100% transduced VQ cells were CD138+B220neg at all conditions tested. Conclusions and Current Focus. We are using ALV-CRISPR technology to generate precision myeloma models to support the next wave of personalized therapy and immunotherapy for this recalcitrant and incurable cancer. Progress on three in vivo models under construction will be presented at the meeting:Subclonal Braf mutation nested within clonal Nras mutation. These models will be treated with next-generation RAF inhibitors (Karoulia Z et al.Nat Rev Cancer 2017;17(11):676-91).Dis3 loss-of function mutation in constellation with co-clonal Ras mutation. The pathogenic consequences of Dis3 disruption remain poorly understood as no parallels exist in other cancers.The murine homolog of the t(4;14) translocation. We are targeting breakpoint cluster regions within perfectly syntenic sequences on mouse chr. 5 (containing Fgfr3 and Mmset loci) and mouse chr.12 (containing the murine Igh locus). Disclosures No relevant conflicts of interest to declare.

Abstract Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,770 in 2018, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency, suggesting that additional genetic mutations are required for the malignant MM progression. Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre or NrasLSL Q61R/+; IgG1-Cre. To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly aggressive MM, which was characterized by high proliferative index, hyperactivation of AKT and ERK pathways, and disease hallmarks (e.g. osteolytic lesions, anemia, and kidney injury). VQ myeloma was readily transplantable into serial syngeneic recipients. In our preliminary study, combined Bortezomib and AZD6244 treatment attenuated MM phenotypes and prolonged the survival of VQ recipients. To facilitate easy molecular or genetic manipulations of VQ MM cells, we established two cell lines from primary cells. These cells could be easily infected (see another abstract from our co-author, Dr. Asimakopoulos's group). The cultured MM cells also express high levels of CD155, a ligand of immune checkpoint TIGIT, which has been recently reported to mediate a significant immune checkpoint blockade in MM patients. We further investigated the transcriptional signature of VQ MM cells (from bone marrow [BM] and from lymph nodes [LN]) vs wild-type (WT) plasma cells using the Illumina Bio-Rad's Single-Cell Sequencing platform. Illumina's Sure Cell app was used to de-multiplex samples, process barcodes and perform single-cell 3' gene counting. The processed data were then analyzed using Seurat R package to yield cluster information and their associated enriched genes. Our results revealed distinct clusters of CD138+ MM and WT cells, and also showed high similarity & overlap between BM & LN MM samples. Consistent with the genetic changes introduced to VQ MM cells, both Myc and Nras expression levels were significantly elevated in MM clusters compared to WT clusters. We also found that transcriptional levels of several potential therapeutic targets were significantly elevated in MM clusters, including Integrin alpha 4 and metabolic enzymes. Currently we are validating the scRNA-Seq results. Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. The VQ mouse model represents an important innovation that will serve as a platform to investigate pathogenesis of resistant and refractory multiple myeloma and allow for testing the efficacy of novel therapeutic agents. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction: Multiple Myeloma (MM) is consistently preceded by pre-malignant asymptomatic monoclonal gammopathies (AMG). To date, our understanding of the pathogenesis of progression to MM remains incomplete. Genetic analyses of AMG cells compared to MM-derived plasma cells (PCs) have found few differences, suggesting that progression may be mediated in part by tumour-extrinsic mechanisms. To comprehensively examine the cellular and molecular complexities of MM pathogenesis, we performed an unbiased single cell RNA-sequencing (scRNA-seq) analysis of tumour cells as well as immune cells from the tumour microenvironment (TME) derived from transgenic mice transitioning from AMG to MM. Methods: We employed the Vk*MYC immune-competent mouse model of MM (C57BL/6/KaLwRij), which is a clinically and biologically faithful model of untreated disease that similarly progresses from AMG to MM with age. We established an age-based cohort of Vk*MYC mice to recapitulate a range of MM disease stages, generated single-cell suspensions from flushed bone marrow and subjected these cells to scRNA-seq profiling (10x Genomics). Results: Across 12 samples profiled to date, our scRNA-seq dataset contains 82,853 high-quality cells, expressing 17,922 genes. We employed dimensionality reduction and unsupervised graph-based clustering to visualize and group transcriptionally-similar cell populations, which revealed 42 clusters. Expression of known marker genes and computed correlation scores with bulk gene expression reference datasets enabled annotation of cell types, revealing both malignant cells and non-malignant immune cell populations. We first focused on single cell T/NK profiles in our data given the emerging utility of immune checkpoint inhibitors that target these populations. Although we did not observe numerical differences in the proportion of CD8+ T cells across disease stages, analysis of immune checkpoint receptor genes revealed increased expression of Pdcd1 (PD-1) and Lag3 in CD8+ T cells from mice with disease. Co-expression of LAG3 and PD-1 proteins was also confirmed using a Vk*MYC transplantable model, with a positive correlation between disease burden (%CD138+/B220- cells) and %PD1+LAG3+ CD8+ T cells by flow cytometry. Consistent with reports of PD-1 and LAG3 co-expression on non-functional exhausted T cells, CD8+ T cells from diseased mice demonstrated elevated T cell exhaustion scores in our scRNA-seq dataset. These observations suggest that T cell exhaustion may be mediated by multiple immune checkpoint receptors during disease evolution. Although combinatorial treatment with PD-1 and LAG3 antibodies failed to induce tumour regression in mice with established disease, the addition of cyclophosphamide (Cy) to these antibodies resulted in marked improvement in survival of the mice compared to Cy alone, presumably by promoting immunogenic cell death. Studies exploring the combination of LAG3 and PD-1 antibodies as a strategy to inhibit transition from AMG to MM in the Vk*MYC mice are ongoing and will be reported. We also performed subclustering analysis of 5,228 Sdc1+ (CD138) PCs in our scRNA-seq dataset revealing 11 distinct clusters, with evidence of inter- and intra-tumoural heterogeneity across all Vk*MYC mice. Differential gene expression analysis revealed a non-malignant PC (nPC) cluster as supported by lower Myc transgene and Ccnd2 expression. Moreover, this cluster was predominantly comprised of cells from age-matched control mice or mice with earlier disease. Single-cell chromosomal copy number analysis revealed loss of Chr5 in the majority of tumour cells from MM mice, but not in the nPC cluster. Loss of Chr5 was observed in tumor subclones from all AMG mice suggesting that it is an early and potentially unifying event in Vk*MYC mice during disease progression. Further, the data support the establishment of intratumoural heterogeneity early in disease evolution. Conclusions: Our approach of using scRNA-seq to characterize the pathogenesis of disease evolution in MM has enabled simultaneous measurement of intratumoural heterogeneity and immune cell phenotypes in the TME. In turn, this has provided insights into mechanisms that may contribute to transition from AMG to MM, including induction of T cell exhaustion and loss of mouse Chr5. Ongoing and future work aims to evaluate whether these mechanisms can be exploited therapeutically in pre-malignant AMG. Disclosures Sebag: Amgen Canada: Membership on an entity's Board of Directors or advisory committees; Janssen Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene Canada: Membership on an entity's Board of Directors or advisory committees; Takeda Canada: Membership on an entity's Board of Directors or advisory committees. Pugh:Prosigna: Honoraria; N/A: Patents & Royalties: Hybrid-capture sequencing for determining immune cell clonality; N/A: Patents & Royalties: Combined hybrid-capture DNA sequencing for disease detection; Boehringer Ingelheim: Research Funding; Chrysalis Biomedical Advisors: Honoraria; Merck: Honoraria; DynaCare: Consultancy.

Abstract Autologous stem cell transplantation (ASCT) remains an important consolidation treatment for multiple myeloma (MM) patients, even in the era of novel agents. The prolongation of plateau-phase induced by ASCT is generally attributed to intensive cytoreduction. However, ASCT generates inflammation and profound lymphodepletion, which may result in hitherto unexpected immunological effects. To investigate potential immunological contributions to myeloma control after ASCT, we developed preclinical models of transplantation for MM using Vk*MYC myeloma that generates bony lytic lesions, a serum M band and marrow plasmacytosis that are hallmarks of clinical disease. Myeloma-bearing B6 recipients underwent myeloablative conditioning and were transplanted with naïve B6 bone marrow (BM) grafts with or without T cells from donors that were myeloma-naïve (SCT) or had low M bands at the time of harvest to mimic ASCT. Surprisingly, we demonstrate the broad induction of T cell-dependent myeloma control with enhanced median survival in recipients of grafts containing T cells compared to T cell depleted (TCD) BM alone (SCT= 91 days and ASCT > 100 days post-transplant vs TCD BM alone= 44 days; p<0.0001). Myeloma was most efficiently controlled when recipients were transplanted with memory T cells (CD44+) from autologous grafts (median survival: ASCT-CD44+ T cells >90 days post-transplant vs. CD44─ T cells = 50 days; p = 0.0006). Importantly, T cells adoptively transferred from recipients surviving > 120 days (MM-primed) protected secondary recipients compared to T cells from naïve donors (median survival: MM-primed > 120 days post-transplant vs 65 days naïve T cells; p = 0.0003). Furthermore, MM-primed CD8 T cells were restricted in TCR repertoire and provided protection in a myeloma clone-specific fashion, indicative of a tumor-specific T cell response. Despite this immune-mediated control of myeloma after SCT, progression still occurred in the majority of recipients. We phenotyped CD8+ T cells from the BM of MM-relapsed, MM-controlled and MM-free (that had never seen myeloma) mice 8 weeks after SCT. Expression of the inhibitory receptors, programmed cell death protein 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) on BM CD8+ T-cells strongly correlated with myeloma cell number (r = 0.729, p<0.0001 and r = 0.796, p<0.0001 respectively). Additionally, the co-stimulatory/adhesion receptor CD226 (DNAM-1) was markedly downregulated as myeloma progressed (r = - 0.865, p<0.0001), as was interferon-γ secretion (r = - 0.76, p = 0.0022). t-SNE analysis confirmed an irreversible exhaustion signature at myeloma progression, characterized by the absence of DNAM-1 and co-expression of PD-1, TIM-3, TIGIT together with CD101 and CD38. Immune-checkpoint inhibition (CPI) early post-SCT, using antibodies against PD-1 or TIGIT facilitated long-term myeloma control (median survival in both treatment arms > 120 days post-SCT vs. 60 and 68 days respectively; p <0.05). Furthermore, TIGIT blockade limited CD8+ T cell exhaustion, increased CD107a and IFNγ secretion and expanded a memory CD8+ T cell population in the BM. Genetic deletion of either IFNγ or the IFNγ receptor from the donor graft resulted in dramatic myeloma progression after SCT. Consequently, treatment with a CD137 (4-IBB) agonist early after SCT profoundly augmented CD8+IFNγ+GranzymeB+ T-cell expansion in the BM, such that majority of treated animals eliminated myeloma and survived long-term. These data provide insights into an unappreciated mechanism of action of ASCT whereby myeloma immune-equilibrium is established and suggest that combination with immunotherapeutic strategies is a rational approach to generate long term disease control. Disclosures Smyth: Bristol Myers Squibb: Other: Research agreement; Tizona Therapeutics: Research Funding.

Abstract We have previously proposed the dysregulation of the D type cyclins by chromosome translocations to the IgH locus or by not fully characterized mechanisms in hyperdyploid patients as a unifying genetic event in the pathogenesis of Monoclonal Gammopathy of Uncertain Significance (MGUS) and multiple myeloma (MM). However, no experimental data are available to evaluate the direct consequences of cyclin Ds overexpression in plasma cells. Similarly, activating mutations of KRAS have been identified in 15% of MM patients, but never in MGUS, and have been proposed to contribute to the progression of MGUS to MM, but this hypothesis has never been experimentally validated. We evaluated the contribution of CyclinD1 and KRAS to the development and progression of monoclonal gammopathy (MG) that develops spontaneously in C57BL/6 mice. We aged a cohort of EuCCND1 mice, and lslKRASV12 mice crossed to three different CRE expressing mice: IgG1CRE, CD19CRE or Vk* CRE (KRAS/CRE). A cohort of Vk* MYC mice, Vk* MYC x EuCCND1 (MYC/CD1) and triple transgenic mice (CD1/KRAS/CRE) was also analyzed. Mice were bled every 10 weeks beginning at 30 weeks, and the presence of MG was evaluated by serum protein electrophoresis. The median Time To M-Spike (mTTS) detection was 97 weeks in C57Bl/6, 63w in EuCCND1, 54w in KRAS/CRE, 54w in Vk* MYC, 38w in CD1/KRAS/CRE and 36w in MYC/CD1 mice. This indicates that acceleration of the development of MG occurs in all these strain of mice. However, we noticed that the spontaneous MG in C57BL/6 wt, EuCCND1 or KRAS/CRE rarely progresses (as determined by detection of a M-spike >7 g/L), while in the other mouse strains it frequently progresses, with a mTTS >7 g/L around 65w. A clear progression of MGUS to MM was observed only in Vk* MYC mice, and was further accelerated in MYC/CD1 mice, which are characterized by extra-medullary disease and shorter overall survival. In conclusion, these data support our hypothesis that Cyclin Ds dysregulation in plasma cells is the initial transformation step leading to MGUS. Further oncogenic events, like activating mutations of KRAS are required to expand the monoclonal plasma cells. Among the oncogenes examined MYC provides the greatest acceleration of the MG in wildtype and EuCCND1 mice, and results in a disease with the malignant features of MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor’s protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.