Bergsagel, Leif

Abstract Background: The current standard of care for eligible newly diagnosed multiple myeloma (MM) patients is induction therapy with novel agents followed by high dose chemotherapy and autologous stem cell transplant (ASCT). The International Myeloma Working Group proposed the Revised International Staging System (R-ISS) based on the presence of adverse chromosomal abnormalities (CA) detected by FISH (t(4;14), t(14;16) or del17p), in combination with ISS and LDH at diagnosis. However, there are a limited number of studies that have validated this risk model in the transplant and novel agents setting. Aims: To determine whether R-ISS is an appropriate risk-model for estimating overall survival (OS) and progression free survival (PFS) for transplant-eligible MM patients. Patients and Methods: We retrospectively studied a cohort of 519 MM patients who received novel drugs in the induction and subsequently underwent ASCT at Mayo Clinic Arizona from 2005 to 2014. In all, 95 patients met the inclusion criteria: comprising complete data at diagnosis (ISS, serum LDH level, and CA by FISH). The primary endpoint was OS from SCT and the secondary end point was PFS from ASCT. R-ISS groups were defined as described by Palumbo et al. J Clin Oncol. 2015; 33(26):2863-2869. Results: There were 50 (52.6%) men and 45 (47.4%) women who underwent ASCT in this period, with a median age at the time of transplant of 66-years-old (range, 36-78). There were 27 patients (28.4%) with high-risk CA: 12 patients (12.6%) with del17p; 11 patients (11.6%) with t(4;14); and 6 (6%) with t(14;16). In addition, 8 patients (8.5%) had high LDH levels and 9 patients (9.5%) presented with renal impairment at diagnosis. The patients were staged at diagnosis according to the three R-ISS groups: 44 patients (46.3%) had stage I, 26 (27.4%) had stage II, and 25 (26.3%) had stage III. CyBorD was the preferred induction regimen which was received by 42 patients (44%). There were 14 patients (15%) who received at least 2 lines of induction. All patients were in at least partial response (PR) at the moment of transplant: 32 in complete response (CR), 32 in very good partial response (VGPR) and the remaining 31 in PR. The response achieved at day +100 after ASCT improved, with 54 patients (57%) in CR, and only 18 patients (19%) in PR. After a median follow-up of 61 months (range, 14-135), median OS from SCT was 108 months (95% CI: 85 - 132 months) and the median PFS was 45.4 months (95% CI: 31.1 - 53.8 months) in the whole series.MM patients with R-ISS III had a significantly shorter median OS compared to patients with R-ISS II or R-ISS I (32.1 months vs. 94.7 months vs. not reached, respectively, P<0.0001) (Figure). No statistically significant differences in baseline characteristics were identified among these groups to explain the differences in OS observed. PFS among these groups was not statistically significant, only showing a trend towards shorter PFS in R-ISS III compared with either R-ISS I or II: median 22.1 months vs. 35.7 months, respectively, (P=0.2). Renal impairment at diagnosis, IgA subtype, ≥ 2 lines of induction treatment, and less than CR achieved at day +100 after ASCT were also associated with significantly inferior OS. Multivariate analysis selected R-ISS as an independent predictor for OS (HR: 2.3, 95% CI: 1.1-4.8; P=0.03), as well as ≥ 2 lines before ASCT. CR at day +100 after ASCT was the most important independent factor for predicting PFS (HR: 0.4; 95% CI: 0.2-0.6; P<0.001). Conclusion: R-ISS assessed at diagnosis was an independent predictor for OS after ASCT in our series, with median OS for the different R-ISS groups comparable to those reported by Palumbo et al. in their subgroup of younger patients. Thus, this study lends further support for the R-ISS as a reliable prognostic tool for estimating OS in transplant-eligible MM patients. In addition, new treatment approaches are needed for the high-risk patients (R-ISS III) with a median OS of 2.5 years. Figure Figure. Disclosures Reeder: Millennium: Research Funding; BMS: Research Funding; Celgene: Research Funding; Novartis: Research Funding. Mikhael:Abbvie: Research Funding; Onyx: Research Funding; Sanofi: Research Funding; Celgene: Research Funding. Bergsagel:Amgen, BMS, Novartis, Incyte: Consultancy; Novartis: Research Funding. Stewart:celgene: Consultancy. Fonseca:Janssen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy; Novartis: Consultancy; Bayer: Consultancy; AMGEN: Consultancy; AMGEN: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; AMGEN: Consultancy; Millennium, a Takeda Company: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Novartis: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; Bayer: Consultancy; Celgene: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; BMS: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms.

Abstract Backround: Dysregulation of the PI3K/Akt signal transduction pathway has been implicated in the development of a number of malignancies, including multiple myeloma (MM). This cellular signaling mechanism and its downstream targets (eg mTOR) regulate cell growth, proliferation and apoptosis. SF1126 (Semafore) is a water soluble prodrug of the pan-PI3K inhibitor, LY294002, whose anti-proliferative and pro-apoptotic activity has been well described in the literature. Preclinical studies using SF1126 in a variety of malignancies including glioma, prostate, non-small cell lung cancer, and breast cancer appear promising and have demonstrated profound antiangiogenic effects mediated through VEGF inhibition. Aim: To demonstrate in vitro anti-myeloma activity of SF1126, alone and in combination with dexamethasone, bortezomib, and melphalan and evaluate their effects on downstream targets of PI3K/Akt. Methods: MM cell lines (MM.1R, MM.1S, RPMI 8226) were treated with SF1126 (1–100uM), dexamethasone (5uM), bortezomib (5nM), melphalan (10uM) alone, and in combination. Growth inhibition following treatment was measured by MTT assay at 24 and 48 hours. Apoptosis was assessed by annexin-V binding assay using flow cytometry. Immunoblot analysis was performed to measure downstream targets of Akt including: p-PDK1 and mTOR (4E-BP1). Results: A clear dose response was established with an IC50 of 8.75uM in the MM.1R and 7.5uM in the MM.1S cell lines at 48 hours. At 24 and 48 hours, 5uM SF1126 alone resulted in 80% and 64% cell viability by MTT assay, respectively, in the MM.1R cell line. The combination of 5uM SF1126 with conventional agents was then tested in the MM.1R cell line. Combination with 5uM dexamethasone enhanced the efficacy of 5uM SF1126 by 26% at 48 hours. Combination with 10uM melphalan enhanced the efficacy of 5uM SF1126 by 20% at 24 hours. The combination with 5nM bortezomib enhanced the efficacy of 5uM SF1126 by 23% at 48 hours. Given prior experience demonstrating that short exposure to bortezomib activates Akt, we tested sequential administration of bortezomib and SF1126 in the MM.1R cell line. Optimal cell death was induced with bortezomib prior to SF1126, followed by concurrent administration. Immunoblot analysis of p-PDK1, downstream mTOR target (4E-BP1) were performed on the MM.1S cell line treated with 5, 10, 20, and 50uM SF1126 at 12 and 24 hours. At the 12 hour time point, p-PDK-1 appeared to increase, but was significantly reduced by 48 hours. A similar pattern of initial upregulation followed by reduction by 24 hours was seen with the mTOR protein 4E-BP1. Conclusion: SF1126 has dose dependent, in vitro activity in several multiple myeloma cell lines both as a single agent and in combination with dexamethasone, bortezomib, and melphalan. The addition of SF1126 to dexamethasone in a dexamethasone resistant cell line results in increased cell death, possibly by overcoming resistance mechanisms. The addition of SF1126 to bortezomib and melphalan also resulted in increased growth inhibition over either agent alone. These results warrant further study of this promising new pan-PI3K/Akt inhibitor.

Abstract Abstract 576 Stat3 appears to play a critical role in sustaining and generating memory B-cells and long-lived plasma cells (PCs) the normal counterparts of myeloma cells. In contrast to the transient nature of Stat3 activation in normal cells, many hematological tumors including multiple myeloma (MM) harbor constitutive Stat3 activity. Aberrant STAT signaling is recognized as a master regulator of direct and indirect tumor processes including proliferation, apoptosis, invasion, angiogenesis and cancer inflammation and thus Stat3 represents an important therapeutic target. Using structure-based design, targeting the phosphotyrosine (pY)-SH2 domain interactions that stabilizes active Stat3 dimers, phosphopeptides have been identified that block Stat3 dimerization and DNA binding activity. Peptidic agents however suffer from limited cell permeability and metabolic lability, properties that have restricted their practical application in vivo at therapeutic doses. Here we describe the dependence of myeloma tumors on Stat3 and characterize the anti-tumor activity of BP-2-047, a novel, non-phosphorylated small molecule with reduced peptidic character. We analyzed the activity of Stat3 using a Stat3 gene expression index based on Stat3 target genes in 60 human myeloma cell lines (HMCLs). Several available myeloma cell lines (XG6, XG7, JJN3 and UTMC2) demonstrated elevated STAT3 gene expression signatures (2.3 to 14.8) with confirmed constitutive Stat3 phosphorylation by Western blot analysis. RNA interference revealed that Stat3 is essential for XG6, JJN3, and UTMC2 survival. We similarly analyzed the activity of Stat3 in publicly available gene expression profile data sets derived from newly diagnosed MM patients. Approximately a third of patients demonstrated a functionally significant level of Stat3 signature, comparable to those in the Stat3-dependent cell lines. We next evaluated the anti-myeloma activity of the BP-2-047 in cell-based assays against a panel of HMCLs with high and low STAT3 activity. BP-2-047 delivered a desirable activity profile for targeted therapy. Consistent with its predicted mechanism of action, we observed dose-dependent inhibition of Tyr705Stat3 phosphorylation in XG6, XG7, JJN3 and UTMC2 cells, presumably through the blockade of Stat3 binding to the pTyr motifs of gp130 and the prevention of de novo phosphorylation by JAKs. Using a luciferase reporter assay we confirmed inhibition of Stat3 transcriptional activity in MM cells stably expressing the Stat3-dependent luciferase reporter (pLucTKS3). Inhibition of transcription was associated with decreased expression of short half-life proteins, c-Myc and Mcl-1. BP-2-047 inhibited viability of myeloma tumors with IC50 values of 3.1–7.4 microM, in cell lines with constitutive Stat3 activity. Inhibition of Stat3 phosphorylation resulted in induction of dose-dependent apoptosis as determined by PI/annexin V staining and PARP cleavage. However, cells were resistant to BP-2-047-induced apoptosis when co-cultured with bone marrow stroma cells. Identification of factors that confer survival advantage in the presence of stroma is in progress. Exposure of MM patient derived bone marrow mononuclear cells to < 15 microM BP-2-047 preferentially induced apoptosis of CD138 + MM cells. In contrast, BP-2-047 at concentrations of 30 microM failed to inhibit normal bone marrow-derived CD34 colony formation. In vitro combination studies in cell lines demonstrate synergy with dexamethasone, bortezomib and lenalidomide. Studies assessing in vivo activity of BP-2-047 as a single agent and in combination with bortezomib against Stat3 activated myeloma tumors in xenograft mice are ongoing and will be reported. These data suggest that Stat3 may represent an important therapeutic target in a subset of myeloma patients and supports innovative drug development with a focus on Stat3. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 834 Multiple myeloma (MM) is an incurable bone marrow cancer. Events mediating transformation from the pre-malignant monoclonal gammopathy of undetermined significance (MGUS) to MM is unknown. We analyzed 2 gene expression datasets generated on the Affymetrix U133 platform. The test set consisted of 22 MGUS and 101 MM (GEO Accession GSE6477) and the validation set 50 MGUS and 351 MM (GEO Accession GSE2658 and GSE5900). The gene expression profiles of MM were compared to MGUS using gene-set enrichment analysis. Genes over-expressed in MM were enriched for cell cycle, proliferation and MYC activation gene-sets. We dissected the relationship between MYC and cell cycle, and identified a MYC activation signature dissociated from proliferation. We validated our MYC signature in publicly available mouse and human cell line gene expression dataset (GEO Accession GSE3151 and GSE3158 respectively), showing specific expression of our MYC signature in cell lines forced to expressed MYC but not when over-expressing other oncogene such as E2F, HER2. In these analyses, we noted that tumors with RAS mutation also consistently express this signature. Applying this signature to the test dataset, we showed that MYC is activated in 60% of myeloma but none of MGUS. This pattern is reproduced in an independent validation dataset. In order to validate the hypothesis that RAS mutation may also lead to the MYC activation signature, we correlated RAS mutation with MYC activation as measured by a MYC index calculated from the median expression of genes that constitute the MYC activation signature, and showed that almost all cases with RAS mutation have a high MYC index. Together with samples with very high expression of MYC mRNA corresponding to those with MYC translocations, these 2 mechanisms account for 67% of cases with MYC activation. To further confirm MYC activation, we performed immunohistochemistry using a validated MYC antibody together with CD138 double-staining. We can clearly identify CD138 positive plasma cells with and without nuclear MYC expression and found that nuclear expression of MYC, a marker of MYC activation, correlated strongly with the MYC signature, therefore providing clear evidence that MYC activation is present in majority of newly diagnosed MM but not in MGUS. MYC activation is not very well correlated with proliferation as assessed by the plasma cell labeling index. Among newly diagnosed myeloma patients with plasma cell labeling index less than 1, those with nuclear MYC expression have significantly shorter survival than those without (Median survival 77.7 months versus 37.9 months, log-rank p-value 0.04). Multiple pathways converge on MYC activation, which is a common transforming event in MM associated with poor prognosis. MYC nuclear staining by IHC can be a useful clinical surrogate. Targeting MYC can be a chemoprevention strategy. Disclosures: Fonseca: Amgen: Consultancy; Medtronic: Consultancy; Celgene: Consultancy; Bristol Mayor Squibs: Consultancy; Genzyme: Consultancy; Otsuka: Consultancy.

Abstract Abstract 573 Using high throughput RNA interference screening on 6,722 druggable genes we previously identified XPO1/CRM1 as one of the 50 most vulnerable targets in Multiple Myeloma (MM)1. XPO1 knockdown proved lethal in MM cell lines, but had no effect on human embryonic kidney (293) cells or lung cancer (A549) cells, showing that XPO1 is a specific myeloma vulnerability, and that myeloma cell survival is dependent upon XPO1 expression. XPO1 encodes the protein exportin 1, a nuclear transport protein that exports tumor suppressor proteins from the nucleus, where they are active, to the cytoplasm, where they become inactive. We next analyzed XPO1 in MM via gene expression profiling (GEP). XPO1 expression is up-regulated as the disease progresses: patients with active MM have a higher level of XPO1 compared to normal plasma cells (p<0.04) and to patients with monoclonal gammopathy of undetermined significance or smoldering MM (p<0.0001). The highest levels were in human MM cell lines. TC classification revealed highest levels in t(11;14) and lowest levels in t(4;14) disease. Selective inhibitors of nuclear export (SINE) compounds have recently been developed that irreversibly inhibit XPO1/CRM1 and its nuclear export function. One such inhibitor, KPT-276, decreased the viability of all 12 MM cell lines tested in vitro, as shown by MTT assay. After 72 hours of drug treatment, a median IC50 value of approximately 175 nM (range 30–1000 nM) was observed. No synergy with other commonly used anti-MM therapeutics was observed in vitro. In contrast, the drug had little effect in 8 solid tumor cell lines with the exception of the B cell lymphoma line Ramos. KPT-276 was also consistently active in inducing apoptosis against MM primary patient samples. Using an IC80 dose of KPT-276, drug-treated samples had a reduced population of cells in S phase (8%) compared to cells treated with DMSO (21%). Using the vkappa*myc transgenic MM model, KPT-276 reduced monoclonal spikes (by a mean of 56%) in all mice treated orally with 150 mg/kg dose three times per week for 4 weeks. Furthermore, KPT-276 significantly reduced tumor growth in a xenograft MM1.S mouse model. GEP was performed in the presence or absence of drug in two different MM cell lines. Two genes of probable relevance, cell division cycle 25 homolog A (CDC25A) and Bromodomain-containing protein 4 (BRD4), were dysregulated by SINE treatment. Both are involved in cell cycle control and have been linked to MYC. RT-PCR and western blotting confirm that MYC, CDC25A and BRD4 are down-regulated, as soon as six hours, after treatment with KPT-276. KPT-276 has shown marked anticancer activities against B cell malignancies in vitro and is active and tolerated in Phase I canine studies. KPT-330, a close analog of KPT-276, is currently in Phase 1 studies in human with advanced hematological and solid tumors. Disclosures: Schmidt: Karyopharm: Research Funding. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Abstract Abstract 194 Background: Immunomodulatory drugs (IMiDs) are key components of treatment for hematologic malignancies, especially multiple myeloma (MM). Recently, we demonstrated that CRBN expression is the major mediator of IMiD action operating at least in part via interferon regulatory factor 4 (IRF4), however the clinical significance of this finding remains unclear. Methods: To better understand clinical impact, we retrospectively reviewed 148 MM patients whose tumor cells were tested for CRBN expression by Gene Expression Profiling (GEP) prior to treatment with an IMiD containing therapy. Results: GEP studies show no significant differences in CRBN expression between MGUS, smoldering MM (SMM), untreated symptomatic MM and normal plasma cells. However, differences were observed between MM cell lines of varying drug sensitivity to IMiDs and between other B-cell Neoplasia (eg low grade lymphoma has higher levels). Within the genetic subtypes of MM, levels were significantly higher for hyperdiploid patients (median 1.26) as opposed to significantly lower levels for TC class D2 (median 0.76) and average for the 4p16 (median 0.97). We next screened the University of Arkansas Medical School (UAMS) GEP database of MM patients treated with combination therapies and did not find significant correlation between CRBN expression and survival in this multi-agent regimen. We then focused on patients that received only single agent IMiD therapy with low dose dexamethasone. Review of the twelve lowest expressing patients demonstrated a lack of response to IMiDs in any patient. Finally we analyzed 53 relapsed or relapsed/refractory patients treated homogeneously in a study with pomalidomide 2–4mg daily and weekly dexamethasone 40mg. CRBN levels were assessed prior to therapy initiation. Response rates varied significantly based on gene expression level. The percentage of patients that demonstrated at least a partial response was 0% for CRBN <0.81, 19% for CRBN expression of 0.81–0.90, and 33% for CBN expression >0.9. Significant differences in PFS (3.0 months vs. 8.9 months, p = 0.0006) and in OS (9.1 months vs. 27.2 months, p = 0.01) were observed when the lowest quartile of CRBN expression was compared to the top three quartiles. Values used for the cut offs are as follows: (25%: 0.889687; 50%: 1.026542, 75%: 1.211133). While this study demonstrates a positive correlation between CRBN expression and response to pomalidomide and dexamethasone, an important caveat with respect to PFS and OS is that CRBN mRNA level is primarily a reflection of the number of copies of CRBN. Since focal deletions are uncommon, this mostly reflects chromosome 3p copy number. Since trisomy 3 is common in hyperdiploid, good prognosis disease, CRBN not only is required for IMiD function but higher levels also serve as a surrogate for low risk disease. Optimal gene expression cut offs for survival were determined using the Contal and O'Quigley method. The optimal cut point for PFS was 1.18443 and for OS was 1.17816. Conclusion: In summary, this study demonstrates, for the first time, a clinical correlation between CRBN expression and response to IMiD and dexamethasone therapy. CRBN expression is also predictive of survival outcomes and when quantified prior to therapy could potentially be utilized as a predictive biomarker for response to an IMiD based treatment. Disclosures: Stewart: Celgene: Consultancy, Honoraria.

Abstract Abstract 832 We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Multiple myeloma (MM) is the second most common hematologic cancer, accounting for roughly 2% of all cancer deaths. Unfortunately, MM remains incurable and nearly all patients experience relapse. Recently, we have identified a novel cIAP1 antagonist, LCL161, with remarkable anti-MM effects. Interestingly, unlike in other tumor types, our previous work found that LCL161 does not directly induce apoptosis of MM cells, but rather drives activation and infiltration of macrophages to the tumor bed and phagocytosis of tumor cells. Furthermore, in vitro experiments demonstrate that the anti-MM activity of the agent relies on the presence of the tumor, with no observed increase in MM phagocytosis when macrophages are treated in isolation. However, LCL161 is a strong and indiscriminate transcriptional activator and thus, the molecular mechanisms underlying this immune response in MM remain poorly understood. In an effort to characterize molecular signatures associated with this response, we collected whole spleens from seven WT mice and eleven mice with VK12598 transplanted myeloma for single cell RNA-seq. Of the eleven tumor-bearing mice, three remained untreated, three were treated with LCL161, three with bortezomib, and three with cyclophosphamide. Additionally, one of the WT mice was treated with LCL161. Using the 10X genomics chromium platform, we measured mRNA levels in over 90,000 individual cells. We identified groups of cells with shared expression profiles, corresponding to known cell types, using graph-based clustering. Consistent with our previous work, we observed a marked reduction in tumor cells and activation of the noncanonical NFkB pathway in mice treated with LCL161. Turning our attention to cells identified as monocytes, with a particular focus on putative macrophages, we sought to identify molecular signatures associated with LCL161 treatment. Indeed, using a binomial model we identified over 500 upregulated genes in LCL161-treated macrophages. However, our previous data suggested that the efficacy of LCL161 was dependent on the presence of tumor cells. Thus, we refined these results by comparing the expression profile of macrophages treated with LCL161 in WT and tumor-bearing mice. These contrasts identified dozens of genes that were significantly upregulated in macrophages from LCL161 treated, tumor-bearing mice. Of note, we identified a small number of genes (including Spp1, Irg1, and Lipg) that were active only in macrophages from mice treated with LCL161 and bearing a transplanted tumor and unexpressed in all other cells. We believe these genes—and the pathways in which they are active—hold promise for the development of novel targeted therapies to better treat MM. Citation Format: Nicholas Banovich, Martin Boateng, Marta Chesi, Leif Bergsagel, Jonathan Keats. Single-cell RNA sequencing identifies macrophage-specific expression signatures associated with phagocytosis of multiple myeloma after treatment with cIAP antagonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3135.

A role for antigen-driven stimulation has been proposed in the pathogenesis of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) based largely on the binding properties of monoclonal Ig. However, insights into antigen binding to clonal B cell receptors and in vivo responsiveness of the malignant clone to antigen-mediated stimulation are needed to understand the role of antigenic stimulation in tumor growth. Lysolipid-reactive clonal Ig were detected in Gaucher disease (GD) and some sporadic gammopathies. Here, we show that recombinant Ig (rIg) cloned from sort-purified single tumor cells from lipid-reactive sporadic and GD-associated gammopathy specifically bound lysolipids. Liposome sedimentation and binding assays confirmed specific interaction of lipid-reactive monoclonal Ig with lysolipids. The clonal nature of lysolipid-binding Ig was validated by protein sequencing. Gene expression profiling and cytogenetic analyses from 2 patient cohorts showed enrichment of nonhyperdiploid tumors in lipid-reactive patients. In vivo antigen-mediated stimulation led to an increase in clonal Ig and plasma cells (PCs) in GD gammopathy and also reactivated previously suppressed antigenically related nonclonal PCs. These data support a model wherein antigenic stimulation mediates an initial polyclonal phase, followed by evolution of monoclonal tumors enriched in nonhyperdiploid genomes, responsive to underlying antigen. Targeting underlying antigens may therefore prevent clinical MM.

Abstract Introduction: Multiple myeloma (MM) is a malignancy of plasma cells that is still considered incurable due to the 90% relapse rate in patients. Despite the advent of new agents the majority of MM patients relapse secondary to therapy resistance. The potential of reovirus (RV) as a novel therapeutic agent for MM under in vitro, in vivo and ex vivo conditions has been demonstrated by us and a phase I clinical trial of MM with RV therapy has shown indications of efficacy. Recently we have shown that RV could synergize with several standard of care MM drugs such as bortezomib (BTZ), carfilzomib (CFZ) and dexamethasone and importantly, enhance its therapeutic potential in therapy resistant human MM cell lines in vitro. Utilizing the syngeneic Vk*MYC BTZ resistant transplantable MM mouse model, we demonstrate that mice harbouring BTZ insensitive MM tumors significantly respond to RV treatment. Our data indicate that this RV treatment sensitivity is manifested via, direct oncolysis in conjunction with immune modulatory effects. Methods: Vk*MYC myeloma cells (Vk12598) were injected via tail vein (IV) into 3 groups of C57BL/6 wt recipient mice. Seven days post tumor injection, mice were treated with PBS (vehicle) or live (LV) or dead reovirus (DV) (5×10⁁8 PFU) administered IV every 6 days in a total of 5 doses. Serum gamma globulins (M-spike) were assessed weekly by serum protein electrophoresis. Mice were sacrificed on day 35 post tumour injection and their spleens and bone marrow (BM) were harvested. Splenic and BM cells were stained for CD45+/CD45R-/CD138+ (MM tumors), CD4+/CD8+ (T cells), CD3+/NKG2D+ (NKT cells), CD11b+Ly6C+ (monocytic myeloid derived suppressor cells (Mo-MDSC) and CD11b+Ly6G+ (polymorphonuclear (PMN) - MDSCs) and analyzed by flow cytometry. Results: Mice treated with RV demonstrated highly significant (P&lt;0.001) reductions in their M-spikes at 4 and 5 weeks post treatment. RV treatment led to a significant NKT cell accumulation in splenic and BM of RV treated mice in comparison to vehicle treated mice suggestive of immune activation due to RV. Further, RV treatment led to a significant increase in BM Mo-MDSC number but not PMN-MDSC numbers. Interestingly, significant down regulation of splenic PMN-MDSCs was seen with RV treatment. Conclusions: Our results indicate reovirotherapy is successful in a validated syngeneic BTZ resistant MM model. These results have important implications for future clinical trials. Currently we are conducting further experiments to assess the immune modulatory (such as T - memory and T - regulatory cells) contributions of RV in this model. Citation Format: Chandini M. Thirukkumaran, Zhong Qiao Shi, Joanne Luider, Karen Kopciuk, Marta Chesi, Leif Bergsagel, Yuan Dong, Chunfen Zhang, Ahmed Mostafa, Kathy Gratton, Satbir Thakur, Don Morris. Successful oncolytic virotherapy in a bortezomib resistant syngeneic mouse model of multiple myeloma: Implications for translational significance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 541.