Abstract: Epigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, is an antioxidant with chemopreventive and chemotherapeutic actions. Based on its ability to modulate growth factor-mediated cell proliferation, we evaluated its efficacy in multiple myeloma (MM). EGCG induced both dose- and time-dependent growth arrest and subsequent apoptotic cell death in MM cell lines including IL-6-dependent cells and primary patient cells, without significant effect on the growth of peripheral blood mononuclear cells (PBMCs) and normal fibroblasts. Treatment with EGCG also led to significant apoptosis in human myeloma cells grown as tumors in SCID mice. EGCG interacts with the 67-kDa laminin receptor 1 (LR1), which is significantly elevated in myeloma cell lines and patient samples relative to normal PBMCs. RNAi-mediated inhibition of LR1 resulted in abrogation of EGCG-induced apoptosis in myeloma cells, indicating that LR1 plays an important role in mediating EGCG activity in MM while sparing PBMCs. Evaluation of changes in gene expression profile indicates that EGCG treatment activates distinct pathways of growth arrest and apoptosis in MM cells by inducing the expression of death-associated protein kinase 2, the initiators and mediators of death receptor-dependent apoptosis (Fas ligand, Fas, and caspase 4), p53-like proteins (p73, p63), positive regulators of apoptosis and NF-κB activation (CARD10, CARD14), and cyclin-dependent kinase inhibitors (p16 and p18). Expression of related genes at the protein level were also confirmed by Western blot analysis. These data demonstrate potent and specific antimyeloma activity of EGCG and provide the rationale for its clinical evaluation.

Abstract: Gene expression and proteomics studies have advanced our understanding of Waldenstrom’s macroglobulinemia (WM) and identified potential therapeutic targets, however, WM remains incurable. Therefore there is an urgent need for the development of novel chemotherapeutic agents targeting deregulated signaling pathways specifically present in WM. Based on role of transcription factor Sp1 in myeloma, we evaluated its molecular and functional role in WM. Our loss of function and Gain of function studies have highlighted a potential oncogenic role of Sp1 in WM. Reduction in Sp1 protein level following transient transfection of WM cells with Sp1 siRNA led to decreased WM cell viability. Conversely, overexpression of Sp1 promoted cell growth and increased IgM production in BCWM1 cell line, associated with an increased level of Sp1 dependent genes. These results demonstrate the role of Sp1 in WM cell growth and survival and provide rationale to therapeutically target Sp1 in WM using small molecule inhibitors of Sp1. We therefore evaluated the activity of Terameprocol (TMP), a small molecule with ability to inhibit Sp1-mediated transactivation by competing for binding to specific Sp1-domains within gene promoter regions. Treatment with TMP caused inhibition of WM and IgM-secreting low-grade lymphoma cell lines, as well as purified primary patient WM cell growth in a dose and time dependent fashion. Sp1 physically interacts with other TFs, influencing their activity. To identify TFs whose activity is controlled by Sp1 in WM cells, we analyzed the activation of 47 transcription factors in nuclear extracts from BCWM1 and MWCL1 cells that were siRNA-depleted for Sp1 or treated with TMP using a transcription factor profiling assay. Both depletion of Sp1 and TMP treatment decreased the activity of TFs, including STAT1, STAT3, and NF-κB, whereas other factors, such as p53, were not affected. As NF-κB and STAT-3 pathways have been shown to be constitutively activated in WM and to play a pivotal role in regulating growth and survival of WM cells, we have focused our further analysis on these TFs in an attempt to understand the molecular mechanism underlying the activity of Sp1 and its inhibition in WM. Enforced expression of Sp1 significantly induced NF-κB p65 (RelA) activity, and TMP was able to overcome this effect. Inhibition of Sp1 activity impairs basal and TNFα-stimulated NF-κB transcriptional activity as well as IL-6-induced STA3 activation in WM cells. Recent studies have reported the high frequency of the MYD88 L265P somatic mutation in patients with WM. To investigate the impact of MYD88 on the sensitivity of WM cells to Sp1 inhibition, we first analyzed effect of TMP on MYD88-silenced cells. MYD88 knockdown significantly inhibits BCWM1 cell growth compared with scrambled cells and the antitumor effect was more pronounced upon treatment with TMP. These results provided the rationale to investigate the activity of combination treatment between TMP and inhibitors known to impede the MYD88 pathway signaling. BCWM1 and MWCL1 WM cells were cultured in the absence or presence of a direct kinase inhibitor of IRAK 1 and 4 or the BTK inhibitor PCI32765. The combination treatment resulted in significant and synergistic dose-dependent antiproliferative effect and inhibition of NFkB p65 activity in MYD88 L265P–expressing WM cells suggesting that MYD88 and Sp1 pathways are both functional in WM but independent from each other. In summary, these results demonstrate Sp1 as an important transcription factor that regulates proliferation and survival of WM cells as well as IgM secreting low-grade lymphoma cells and provides preclinical rationale for clinical development of TMP in WM alone or in combination with inhibitors of MYD88 pathway. Disclosures: Anderson: oncopep: Equity Ownership; celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; acetylon: Equity Ownership. Treon:Millennium: Consultancy.

Abstract: Activating mutations in MYD88 (MYD88 Mut) are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of diffuse B-cell lymphoma (ABC DLBCL). MYD88 is a component of the Toll-like receptor (TLR) pathway. We and others previously showed that MYD88 Mut triggers assembly of a "Myddosome" complex that leads to downstream pro-survival signaling that includes IRAK4/IRAK1 and BTK triggered NF-κB (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013) and HCK mediated BTK/NF-κB, PI3K/AKT, and MAPK/ERK signaling (Yang et al, Blood 2016; Liu et al Blood Adv. 2020). The activation of the B-cell receptor (BCR) signaling component SYK has also been observed in MYD88 Mut WM (Argyropoulos et al, Leukemia 2016). In ABC DLBCL, chronic active BCR signaling underlies SYK activation that is triggered by the SRC family member LYN (Davis et al, Nature 2010). These observations led us to explore potential drivers of BCR/SYK activation in WM. We previously reported that MYD88 Mut triggered activation of SYK in WM and ABC DLBCL cells (Munshi et al, BCJ 2020). Herein, we investigated if HCK, a SRC family member that is transcriptionally upregulated and activated by MYD88 Mut could trigger the BCR pathway through SYK activation. Since LYN is an integral part of BCR signaling, we first examined its expression and activation state in MYD88 Mut WM and ABC DLBCL cells. While MYD88 Mut TMD8, HBL-1 and OCI-Ly3 ABC DLBCL cells showed strong expression of p-LYN, such expression was absent or low in MYD88 Mut BCWM.1 and MWCL-1 cells, as well as CD19-selected bone marrow derived primary lymphoplasmacytic cells (LPCs) from WM patients. In view of the above findings, we next interrogated a direct role for HCK in mediating SYK activation. We over-expressed wild-type HCK (HCK WT) or gatekeeper mutated HCK (HCK T333M) in MYD88 Mut BCWM.1 and MWCL-1 WM cell lines, and TMD8 ABC DLBCL cells. In all these cell lines, over-expression of HCK WT or HCK T333M triggered a robust increase in phosphorylation of SYK Y525/Y526 in comparison to vector only transduced cells. Moreover, using an inducible vector system, knockdown of HCK showed a marked reduction in phosphorylation of SYK Y525/Y526 in MYD88 Mut BCWM.1 WM and TMD8 ABC DLBCL cells. We next sought to clarify if HCK and activated SYK were present in the same signaling complex. We performed co-immunoprecipitation experiments using an HCK antibody in MYD88 Mut BCWM.1, TMD8 and wild-type MYD88 (MYD88 WT) Ramos cells. The HCK antibody effectively pulled down p-SYK in MYD88 Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. To confirm whether SYK activation was a result of HCK kinase activity, we next performed rescue experiments with the HCK inhibitors A419259 and KIN-8194 in MYD88 Mut BCWM.1 and MWCL-1 WM and TMD8 ABC DLBCL cells expressing either HCK WT or the HCK T333M protein that abrogated the activity of these inhibitors against HCK. Expression of the HCK T333M protein produced marked resistance to A419259 as well as KIN-8194 versus vector or HCK WT transduced BCWM.1 and MWCL-1 cells. By PhosFlow analysis, we observed that expression of HCK T333M but not HCK WT led to persistent activation of HCK and SYK in the presence of A419259 or KIN-8194 in BCWM.1 and MWCL-1 WM cells, and TMD8 ABC DLBCL cells. Consistent with these observations, treatment of primary MYD88 mutated WM LPCs cells with either A419259 or KIN-8194 also showed marked reduction in both p-HCK and p-SYK expression by PhosFlow analysis. Taken together, our findings show that SYK is activated by HCK in MYD88 Mut B-cell lymphomas cells; broaden the pro-survival signaling generated by aberrant HCK expression in response to MYD88 Mut; and help further establish HCK as an important therapeutic target in MYD88 Mut B-cell lymphomas. Disclosures Palomba: Juno: Patents & Royalties; Rheos: Honoraria; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Notch: Honoraria, Other: Stock; Kite: Consultancy; Novartis: Consultancy; BeiGene: Consultancy; Priothera: Honoraria; Nektar: Honoraria; PCYC: Consultancy; Wolters Kluwer: Patents & Royalties; WindMIL: Honoraria; Magenta: Honoraria; Pluto: Honoraria; Lygenesis: Honoraria; Ceramedix: Honoraria. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Gray: Syros, C4, Allorion, Jengu, B2S, Inception, EoCys, Larkspur (board member) and Soltego (board member: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield and Sanofi: Research Funding. Munshi: Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy. Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.

Abstract: We developed a novel in vivo multiple myeloma (MM) model by engrafting the interleukin 6 (IL-6)-dependent human MM cell line INA-6 into severe combined immunodeficiency (SCID) mice previously given implants of a human fetal bone chip (SCID-hu mice). INA-6 cells require either exogenous human IL-6 (huIL-6) or bone marrow stromal cells (BMSCs) to proliferate in vitro. In this model, we monitored the in vivo growth of INA-6 cells stably transduced with a green fluorescent protein (GFP) gene (INA-6GFP+ cells). INA-6 MM cells engrafted in SCID-hu mice but not in SCID mice that had not been given implants of human fetal bone. The level of soluble human IL-6 receptor (shuIL-6R) in murine serum and fluorescence imaging of host animals were sensitive indicators of tumor growth. Dexamethasone as well as experimental drugs, such as Atiprimod and B-B4-DM1, were used to confirm the utility of the model for evaluation of anti-MM agents. We report that this model is highly reproducible and allows for evaluation of investigational drugs targeting IL-6-dependent MM cells in the human bone marrow (huBM) milieu. (Blood. 2005;106:713-716)

Abstract: Abstract 326 The key nuclear export protein CRM1 (chromosome region maintenance 1, Exportin 1, XPO1) may directly contribute to the pathophysiology of human multiple myeloma (MM). Here, we characterized the role of CRM1 in MM biology and defined molecular mechanisms whereby novel oral, irreversible, selective inhibitors of nuclear export (SINE) targeting CRM1 mediate anti-MM activity. CRM1 gene expression is increased with disease progression, since it is significantly elevated in active MM and plasma cell leukemia (PCL) vs. normal/MGUS/SMM patients (p 〈 0.02). CRM1 downregulation by shCRM1 lentiviruses significantly decreases MM cell viability regardless of drug sensitivity and p53 status. Importantly, SINE (KPT-185, KPT-251, KPT-276, and KPT-330) specifically blocked proliferation and decreased survival of MM cell lines (n=14) and patient MM cells (n=17) (LD50 〈 200 nM), cultured alone and with bone marrow stromal cells (BMSCs) or osteoclasts. Caspases 3, 8, and 9 were not induced by any SINE in BMSCs derived from MM patients, cultured either alone or with MM cells, under conditions inducing marked apoptosis of MM cells ( 〉 2-log differences). These agents potently enhanced nuclear accumulation of multiple CRM1 cargo tumor suppressor proteins p53, IκB, FOXO1A, FOXO3A, p27, and PP2A in MM cells. Transcripts of p53 and its downstream targets p21, PUMA, BAX were also induced by KPT-185, thereby inducing strong growth arrest and apoptosis. KPT-185 decreased MM oncogenes (c-myc, c-maf), anti-apoptosis molecules Mcl-1 and BCL-xL; increased pro-apoptotic protein BAX; as well as inhibited HSP70 and pIkBa. KPT-185 further blocked baseline and APRIL-induced NFkB p65 DNA-binding activity in MM cells. It triggered proteasome-dependent reduction of CRM1 protein; concurrently, KPT-185 and KPT-330 upregulated CRM1 mRNA. Furthermore, KPT-185 induced a number of tumor suppressing, regulatory, apoptotic and anti-inflammatory genes, i.e., p53, p21, PUMA, BAX, CHOP, C10orf10, MIC1, IκBα in MM1S cells in a dose-dependent manner, regardless of the presence of BMSCs. Cleavage of caspase 3 and PARP was markedly increased in MM1R cells treated with KPT-185 and bortezomib vs. either drug alone, validating that the combination of these agents triggered stronger cytotoxicity against MM cells. Combined treatment with dex and KPT-185 (or KPT-276) induced synergistic cytotoxicity against MM cells. Moreover, KPT-185 and KPT-330 impaired osteoclastogenesis and bone resorption via blockade of RANKL-induced NFκB activation in osteoclast precursor cells, without impacting osteoblasts and BMSCs (Abstract#48190). Importantly, SINEs (KPT-251 and KPT-276) suppressed MM cell growth (p 〈 0.01), diminished MM cell-induced osteolysis, and prolonged survival of SCID mice with diffuse human MM bone lesions (p=0.0004). Together, these results identify CRM1 as a promising novel target in MM, strongly supporting clinical development of SINE CRM1 antagonists to inhibit both MM cell growth and related bone disease. Disclosures: Landesman: Karyopharm Therapeutics Inc: Employment. Senapedis:Karyopharm Therapeutics Inc: Employment. Saint-Martin:Karyopharm Therapeutics Inc: Employment. Kashyap:Karyopharm Therapeutics Inc: Employment. Ying:Karyopharm Therapeutics Inc: Employment. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy. Richardson:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

Abstract: The 90-kDa heat shock protein (Hsp90) has become an important therapeutic target with ongoing evaluation in a number of malignancies. Although Hsp90 inhibitors have a high therapeutic index with limited effects on normal cells, they have been described to inhibit dendritic cell function. However, its effect on human immune effector cells may have significant clinical implications, but remains unexplored. In this study, we have evaluated the effects of Hsp90 inhibition on human T lymphocyte and NK cells, including their Ag expression, activation, proliferation, and functional activities. These studies demonstrate that Hsp90 inhibition irreversibly downregulates cell surface expression of critical Ags (CD3, CD4, CD8), the costimulatory molecule (CD28, CD40L), and αβ receptors on T lymphocytes, as well as activating receptors (CD2, CD11a, CD94, NKp30, NKp44, NKp46, KARp50.3) on NK cells. Hsp90 inhibition significantly reduced CD4 protein expression on T lymphocytes at both the cell surface and intracellular level, which was shown to be associated with aberrant regulation of Src-kinase p56Lck. Downregulation of the Ags triggered by Hsp90 inhibition on CD3+ T lymphocytes, both in CD4+ and CD8+ T cell subsets, was associated with a disruption in their cellular activation, proliferation, and/or IFN-γ production, when the inhibition occurred either in activated or inactivated cells. In addition, downregulation of key activating receptors on NK cells following Hsp90 inhibition resulted in decreased cytotoxicity against tumor cells. Therefore, these observations demonstrate the need to closely monitor immune function in patients being treated with a Hsp90 inhibitor and may provide a potential therapeutic application in autoimmune diseases.

Abstract: Abstract 3986 B cell-malignancies exhibit considerable immune dysfunction particularly in multiple myeloma (MM). We have previously demonstrated that in T cell-compartment, regulatory T helper cells are dysfunctional in multiple myeloma (MM) while Th17 cells are significantly elevated and IL-17 produced by them is associated with MM cell growth and survival as well as suppressed immune responses and bone disease. We have here investigated the B cell-subsets and their ability to re-program anti-tumor immunity in MM. We have first characterised four different B cell-subsets (B1a, B1b, B2 and regulatory B cells) using 10-color flow cytometric analysis in both peripheral blood and bone-marrow (BM) samples from MM patients compared with normal healthy donors. We observe that CD5+ B1a-B cells are significantly elevated in peripheral blood of MM patients (N=7) compared to healthy donor (N=15) (42±8% vs 13±3%, respectively, p 〈 0.05); while normal B cells (B2 cells) are significantly reduced in peripheral blood (29.8±6.5, p 〈 0.05) and in the BM samples (11±4.8, N=4, p 〈 0.05) of MM patients compared to healthy donors (59±3, and 60.2±2, N=10, respectively). We also observed that both B1b (47.9±18 vs. 22.8±4) and regulatory B cells (7.1±4.5 vs. 1.54±0.3) are elevated in BM samples of MM compared to healthy donors, however there were no differences in B1b and regulatory B cells in the peripheral blood of MM compared to healthy donor samples. Interestingly, in myeloma we observe higher levels of activated B cell subsets but lower levels of memory B cell subsets compared to healthy donors. These results, particularly very low levels of normal B cells in MM patients, may explain the decreased levels of uninvolved immunoglobulin in MM. As removal of B cell population has been shown to re-program T helper cell populations, we next investigated impact of B cell population on T cell activation. We activated normal PBMC via the anti-CD3 antibody, in the presence or absence of B or CD25+ cells and measured intra-cellular IFN-γ levels in CD69+ cells. We found that the absence of B cells significantly inhibited interferon-producing T cells compared to PBMC (by 43%; p 〈 0.05). Importantly, following removal of CD25+ cells, which consists of both Tregs and activated memory T cells, with or without B cells, we did not observe any difference in the inhibition of IFN-γ, indicating that B cells are influencing memory T cells rather than naïve T cells for the production of IFN-γ. This prompted us to identify the phenotypic signature of regulatory T cell populations when purified memory T cells are polarized with the regulatory T cell cocktail in presence or absence of B cells. We observed that B cells reduce FOXP3 expression by 18 %(N=5) and establish cognitive interactions with T cells. This occurred by increasing the expression of GITR (154%) and CTLA4 (54%); while reducing PD1 (−24%) and OX40 (−21%) expression on T cells without affecting HLA expression. We have also observed these improvements by B cell modulation on T cells in MM. Our results indicate that targeting these re-programmable capabilities of B cells to modulate T helper cell populations may enable us to improve T cell function in MM; and may improve immune function in MM and also allow us to enhance responses to vaccinations. Disclosures: Ghobrial: Millennium: Advisory Board Other; Novartis: Advisory Board, Advisory Board Other. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Treon:Onyx: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Cephalon: Consultancy; Avila: Consultancy. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

Abstract: Multiple myeloma (MM) is associated with significant immune dysfunction. The biological basis of this dysfunction remains ill defined. We have previously observed significantly decreased number of T regulatory (Treg) cells, as measured by Foxp3 expression, in both MGUS and MM compared to normal donors. Moreover, Treg cells in MM and MGUS are unable to suppress anti-CD3-mediated T cell proliferation. Here, we have further analyzed elements of bone marrow (BM) microenvironment that may be responsible for dysfunctional Treg cells in MM. Both cellular and soluble components of the BM microenvironment directly, by cell-cell interactions, as well as by production of soluble factors not only affect MM cell growth, survival, migration and drug resistance but also modulate function of immune components. Interactions between MM cells and bone marrow stromal cells trigger production of IL-6, as well as a number of other cytokines and chemokines including TNF-α , VGEF, IGF-1, SDF-1α , IL-1β , TGF-β , and MIP-1α /β with immunomodulatory activity. We and others have in fact demonstrated significantly elevated levels of serum IL-6 as well as soluble IL-6 (sIL-6) receptor in MM. Based on in vivo data that IL-6 can modulate Treg cell number and function in a murine model of asthma, we evaluated its role in Treg cell dysfunction in myeloma. We performed T cell proliferation assays with soluble anti-CD3 antibody with or without Treg cells from normal donors in presence or absence of IL-6/sIL-6R. We observed that the presence of IL-6 and/or sIL-6 receptor leads to loss of normal Treg cell suppressive activity on normal donor T cells (from inhibition of proliferation by 27% to increase in proliferation by 6% in presence of IL-6/sIL-6R, p=0.01). The Treg cell dysfunction observed here is similar to that observed in MM patients. Conversely, when Treg cells from MM patients were treated with IL-6 receptor super antagonist, Sant 7, prior to their addition to T cell proliferation assay, the suppressive activity of Treg cells was restored. Presence of untreated Treg cells was associated with 28% increase in T cell proliferation while Sant 7-treated Treg cells led to 35% inhibition of T cell proliferation (p=0.01). Additionally, we observed expansion of Foxp3+ Treg cell number in PBMC from MM patients following in vitro treatment with anti-IL-6 antibody. Furthermore, TGF-β , a known stimulator of Treg cell function was less effective in stimulating FOXP3+ Treg cells in PBMCs from MM patients (15.2%) compared to normal donor PBMCs (24%). Since Treg cells require interaction with antigen presenting cells (APC), we evaluated effects of IL-6 and TGF-β on the ability of APC to help suppressive activity of Treg cells. Prior exposure of mature dendritic cells to IL-6 and TGF-β abrogated Treg cell suppressive activity from 47% inhibition to 23% increase in T cell proliferation (p=0.01). In conclusion, we have observed that combination of IL6/sIL6R and TGF-β significantly affects Treg cell number and function. These cytokines are significantly expressed in the MM BM microenvironment and may be responsible for the observed Treg cell dysfunction. These cytokines thus may be targets to modulate immune responses in myeloma to enhance immune function and devise effective vaccination strategies in the future.

Abstract: We have previously demonstrated that Th17 cells, which produce IL-17A, are significantly elevated in peripheral blood and bone marrow (BM) of patients with Multiple Myeloma (MM) and IL-17A promotes MM cell growth and survival, both in vitro and in vivo via IL-17A receptor. We have recently evaluated and observed that anti-IL-17A monoclonal antibody (mAb) significantly inhibited MM cell growth in vitro, while IL-17A induced proliferation of MM cells compared to control. We have also observed significant down-regulation of IL-6 production by anti-IL-17A mAb in MM-BMSC co-culture. Importantly, the administration of anti-IL-17A mAb weekly for 4 weeks in the SCIDhu model of human myeloma, where MM cells grow within the human microenvironment in mice led to a significant inhibition of tumor growth compared to the control mice. This remarkable activity of anti-IL17 mAb raised the question of whether the myeloma cells themselves are a possible source of IL-17. In this study, we used transcriptome sequencing (RNA-Seq) data to evaluate the expression of IL-17A in primary CD138+ myeloma cells (N=17) compared to normal plasma cells (NPC) (N=5). Whereas none of the NPCs expressed IL-17A, it was significantly over-expressed in majority of MM cells. In addition, these data also showed that the expression of other IL-17 family members (IL-7B, C, D, E & F) and Th17-associated pro-inflammatory cytokines (IL-21, IL-22 & IL-23) were not significantly elevated in primary myeloma cells compared to normal donor plasma cells. We further validated these observations by IL-17 immunoblot showing IL17 expression in all MM cell lines and 10 out of 14 primary patient MM cells; confirmed IL-17 expression in MM cells by quantitative RT-PCR, and flow cytometry and by immuno-histochemistry and confocal microscopy. We observed that IL-17 knock down by IL-17-specific siRNA inhibited MM cell growth as well as their ability to induce IL-6 production in co-cultures with BMSC. Finally, expression profile data from 172 uniformly treated patients showed that patients with lower IL-17A expression had superior overall survival compared to those with higher expression. These data confirms that MM cells express IL-17 and targeting it with a mAb will abrogate the autocrine loop making it an attractive therapeutic target. Disclosures: No relevant conflicts of interest to declare.