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Triple-negative breast cancer (TNBC) afflicts women at a younger age than other breast cancers and is associated with a worse clinical outcome. This poor clinical outcome is attributed to a lack of defined targets and patient-to-patient heterogeneity in target antigens and immune responses. To address such heterogeneity, we tested the efficacy of a personalized vaccination approach for the treatment of TNBC using the 4T1 murine TNBC model. We isolated tumor membrane vesicles (TMVs) from homogenized 4T1 tumor tissue and incorporated glycosyl phosphatidylinositol (GPI)-anchored forms of the immunostimulatory B7-1 (CD80) and IL-12 molecules onto these TMVs to make a TMV vaccine. Tumor-bearing mice were then administered with the TMV vaccine either alone or in combination with immune checkpoint inhibitors. We show that TMV-based vaccine immunotherapy in combination with anti-CTLA-4 mAb treatment upregulated immunomodulatory cytokines in the plasma, significantly improved survival, and reduced pulmonary metastasis in mice compared to either therapy alone. The depletion of CD8(+) T cells, but not CD4(+) T cells, resulted in the loss of efficacy. This suggests that the vaccine acts via tumor-specific CD8(+) T cell immunity. These results suggest TMV vaccine immunotherapy as a potential enhancer of immune checkpoint inhibitor therapies for metastatic triple-negative breast cancer.

Immune checkpoint inhibitor (ICI) immunotherapy improved the survival of head and neck squamous cell carcinoma (HNSCC) patients. However, more than 80% of the patients are still resistant to this therapy. To test whether the efficacy of ICI therapy can be improved by vaccine-induced immunity, we investigated the efficacy of a tumor membrane-based vaccine immunotherapy in murine models of HNSCC. The tumors, grown subcutaneously, are used to prepare tumor membrane vesicles (TMVs). TMVs are then incorporated with glycolipid-anchored immunostimulatory molecules GPI-B7-1 and GPI-IL-12 by protein transfer to generate the TMV vaccine. This TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. The tumor-free mice survived for several months, remained tumor-free, and were protected following a secondary tumor cell challenge, suggesting that the TMV vaccine induced an anti-tumor immune memory response. However, no synergy with anti-PD1 mAb was observed in this model. In contrast, the TMV vaccine was effective in inhibiting MOC1 and MOC2 murine oral cancer models and synergized with anti-PD1 mAb in extending the survival of tumor-bearing mice. These observations suggest that tumor tissue based TMV vaccines can be harnessed to develop an effective personalized immunotherapy for HNSCC that can enhance the efficacy of immune checkpoint inhibitors.

BackgroundPD-L1 is one of the major immune checkpoints which limits the effectiveness of antitumor immunity. Blockade of PD-L1/PD-1 has been a major improvement in the treatment of certain cancers, however, the response rate to checkpoint blockade remains low suggesting a need for new therapies. Metformin has emerged as a potential new drug for the treatment of cancer due to its effects on PD-L1 expression, T cell responses, and the immunosuppressive environment within tumors. While the benefits of metformin in combination with checkpoint blockade have been reported in animal models, little remains known about its effect on other types of immunotherapy.MethodsVaccine immunotherapy and metformin were administered to mice inoculated with tumors to investigate the effect of metformin and TMV vaccine on tumor growth, metastasis, PD-L1 expression, immune cell infiltration, and CD8 T cell phenotype. The effect of metformin on IFN-γ induced PD-L1 expression in tumor cells was assessed by flow cytometry, western blot, and RT-qPCR.ResultsWe observed that tumors that respond to metformin and vaccine immunotherapy combination show a reduction in surface PD-L1 expression compared with tumor models that do not respond to metformin. In vitro assays showed that the effect of metformin on tumor cell PD-L1 expression was mediated in part by AMP-activated protein kinase signaling. Vaccination results in increased T cell infiltration in all tumor models, and this was not further enhanced by metformin. However, we observed an increased number of CD8 T cells expressing PD-1, Ki-67, Tim-3, and CD62L as well as increased effector cytokine production after treatment with metformin and tumor membrane vesicle vaccine.ConclusionsOur data suggest that metformin can synergize with vaccine immunotherapy to augment the antitumor response through tumor-intrinsic mechanisms and also alter the phenotype and function of CD8 T cells within the tumor, which could provide insights for its use in the clinic.

Dendritic cells (DCs) are the most effective antigen presenting cells for the development of T cell responses. The only FDA approved DC-based immunotherapy to date is Sipuleucel-T, which utilizes a fusion protein to stimulate DCs ex vivo with GM-CSF and simultaneously deliver the antigen PAP for prostate cancer. This approach is restricted by the breadth of immunity elicited to a single antigen, and to cancers that have a defined tumor associated antigen. Other multi-antigen approaches have been restricted by poor efficacy of vaccine adjuvants. We have developed a vaccine platform that consists of autologous DCs pulsed with cytokine-adjuvanted tumor membrane vesicles (TMVs) made from tumor tissue, that encapsulate the antigenic landscape of individual tumors. Here we test the efficacy of DCs pulsed with TMVs incorporated with glycolipid-anchored immunostimulatory molecules (GPI-ISMs) in HER2-positive and triple negative breast cancer murine models. Pulsing of DCs with TMVs containing GPI-ISMs results in superior uptake of vesicles, DC activation and cytokine production. Adaptive transfer of TMV-pulsed DCs to tumor bearing mice results in the inhibition of tumor growth, reduction in lung metastasis, and an increase in immune cell infiltration into the tumors. These observations suggest that DCs pulsed with TMVs containing GPI-GM-CSF and GPI-IL-12 can be further developed to be used as a personalized immunotherapy platform for cancer treatment.

AlumCauses cell death releases uratecrystals among DNA components into blood stream (rupture of endo-lysosome and release of uric acid which acts as a DAMP (Damage Associated Molecular Pattern)Induces TH2 response associated with Asthma, Allergies, and Eczema (IL4 àIgE, IL5àIgA)Immune response can form against these DNA proteins, antibodies against our own DNA potential risk

Breast cancer is the leading cause of death among female cancer patients. Our lab has developed a therapeutic cancer vaccine approach for triple negative breast cancer (TNBC). In this study, we investigated whether vaccine developed from a human TNBC cell line could effectively induce antitumor immunity, which control the growth of tumors in humanized mice implanted with the human TNBC cell line. Human CD34+ HSC implanted NSG-SGM3 from JAX labs and NOG-EXL mice from Taconic Biosciences used for the studies. Humanized NSG-SGM3 mice were vaccinated with 100 mg of the vaccine once every 2 weeks (3 doses). Control mice were injected with vehicle (PBS). One week after the last vaccination, mice were challenged with MDA-MB-231 TNBC cells subcutaneously in the hind flank. Body weight and tumor growth were monitored. All the mice in both groups have developed tumors, splenomegaly and metastatic lesions in the liver, lungs and spleen. However, all the mice also developed GvHD and need to be euthanized before completion of the study. We have also observed an allo-response (CD69+ T cells in control mice with MDA-MB-231 tumors) to the HLA-matched tumor cells but the tumors eventually developed into metastatic disease affecting multiple organs (liver, lungs and spleen) suggesting that the allo-response is not effective against the tumor. Since the tumor-bearing mice developed GvHD and becoming anemic even in a specific pathogen-free barrier facility, future studies will need to be tested in humanized mice which do not develop GvHD and live longer for carrying out vaccine immunotherapy studies. Funding: NIH/NCI R01 CA202763 (PS & CP).

Immunotherapy has emerged as a promising treatment of breast cancer; however, response rates to therapy remain far from ideal. We have developed a new platform for personalized vaccine immunotherapy that utilizes tumor tissue to generate tumor membrane vesicles (TMVs). These TMVs can be incorporated with immunostimulatory molecules such as IL-12 and B7-1 and used for immunization. In this study, we investigated the combinatorial effect of TMV-based vaccine immunotherapy, to induce an antitumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert antitumor effects by promoting CD8 T-cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while the combination with metformin resulted in greater inhibition of primary growth. TMV vaccination reduced the metastatic burden in the lungs while the combination with metformin further abrogated metastatic spread. Interestingly, TMV vaccination alone induced a significant increase in T-cell infiltration to the tumor, and combination with metformin caused a decrease in the accumulation of T cells, despite having better control of tumor growth. Further, we show that metformin can reduce PD-L1 expression within the tumor microenvironment and result in more IFN-γ-producing CD8 T cells within the tumor, providing a mechanistic insight for the improvement of TMV-based vaccine immunotherapy. Citation Format: Luis E. Munoz, Ramireddy Bommireddy, Haley L. Huang, Christopher D. Pack, Sampath Ramachandiran, Periasamy Selvaraj. Metformin reduces PD-L1 expression in the tumor and enhances the efficacy of vaccine-generated CD8 T cells in a murine model of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B32.

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in the United States. DLBCL comprises biologically distinct subtypes including germinal center-like (GCB) and activated-B-cell-like DLBCL (ABC). The most aggressive type, ABC-DLBCL, displays dysregulation of both canonical and noncanonical NF-κB pathway as well as genomic instability. Although, much is known about the tumorigenic roles of the canonical NF-kB pathway, the precise role of the noncanonical NF-kB pathway remains unknown. Here we show that activation of the noncanonical NF-κB pathway regulates chromosome stability, DNA damage response and centrosome duplication in DLBCL. Analysis of 92 DLBCL samples revealed that activation of the noncanonical NF-κB pathway is associated with low levels of DNA damage and centrosome amplification. Inhibiting the noncanonical pathway in lymphoma cells uncovered baseline DNA damage and prevented doxorubicin-induced DNA damage repair. In addition, it triggered centrosome amplification and chromosome instability, indicated by anaphase bridges, multipolar spindles and chromosome missegregation. We determined that the noncanonical NF-κB pathway execute these functions through the regulation of GADD45α and REDD1 in a p53-independent manner, while it collaborates with p53 to regulate cyclin G2 expression. Furthermore, this pathway regulates GADD45α, REDD1 and cyclin G2 through direct binding of NF-κB sites to their promoter region. Overall, these results indicate that the noncanonical NF-κB pathway plays a central role in maintaining genome integrity in DLBCL. Our data suggests that inhibition of the noncanonical NF-kB pathway should be considered as an important component in DLBCL therapeutic approach.

Introduction: Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% of all cancer related mortalities in the US. Currently, there is no cure for the advanced squamous cell carcinoma of the head and neck (SCCHN) thus development of efficacious therapies is urgently needed. To test whether vaccine-induced immunity inhibits tumor growth, we investigated efficacy of a tumor membrane-based vaccine immunotherapy in a murine SCCHN (SCC VII) model. Materials and Methods: The SCC VII tumors grown subcutaneously in C3H/HeJ mice were harvested to generate tumor membrane vesicles (TMVs). TMVs were then protein transferred with glycolipid-anchored immunostimulatory molecules mouse GPI-B7.1 and mouse GPI-IL-12 to generate the TMV vaccine. Mice were vaccinated with TMV vaccine either before (prophylactic) or after SCC VII tumor cell challenge (therapeutic) and tumor growth was monitored every 3 days. Survival was then assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Results: SCCVII cells express MHC I, CD44, CD47 and respond to IFN-γ in vitro. The therapeutic TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. Tumor-free mice remained protective from rechallenge with SCCVII cells. Conclusions: These observations suggest that tumor tissue-based vaccines can be harnessed to develop an effective immunotherapy for SCCHN. Funding: Supported by Head and Neck SPORE pilot funding from Emory Winship Cancer Institute. Citation Format: Ramireddy Bommireddy, Luis Munoz, Chrstopher D. Pack, Sampath Ramachandiran, Shaker JC Reddy, Janet Kim, Georgia Chen, Nabil F. Saba, Dong M. Shin, Periasamy Selvaraj. TMV vaccine inhibits growth of squamous cell carcinoma of head and neck in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4097.

Diffuse large B-cell lymphoma (DLBCL) has been categorized into two molecular subtypes that have prognostic significance, namely germinal center B-cell like (GCB) and activated B-cell like (ABC). Although ABC-DLBCL has been associated with NF-κB activation, the relationships between activation of specific NF-κB signals and DLBCL phenotype remain unclear. Application of novel gene expression classifiers identified two new DLBCL categories characterized by selective p100 (NF-κB2) and p105 (NF-κB1) signaling. Interestingly, our molecular studies showed that p105 signaling is predominantly associated with GCB subtype and histone mutations. Conversely, most tumors with p100 signaling displayed ABC phenotype and harbored ABC-associated mutations in genes such as MYD88 and PIM1. In vitro, MYD88 L265P mutation promoted p100 signaling through TAK1/IKKα and GSK3/Fbxw7a pathways, suggesting a novel role for this protein as an upstream regulator of p100. p100 signaling was engaged during activation of normal B cells, suggesting p100's role in ABC phenotype development. Additionally, silencing p100 in ABC-DLBCL cells resulted in a GCB-like phenotype, with suppression of Blimp, IRF4 and XBP1 and upregulation of BCL6, whereas introduction of p52 or p100 into GC cells resulted in differentiation toward an ABC-like phenotype. Together, these findings identify specific roles for p100 and p105 signaling in defining DLBCL molecular subtypes and posit MYD88/p100 signaling as a regulator for B-cell activation.

The inhibitors of apoptosis (IAP) are important regulators of apoptosis. However, little is known about the capacity of Smac mimetics (IAP inhibitor) to overcome virally associated-lymphoma's (VAL) resistance to apoptosis. Here, we explored the pro-apoptotic effect of a novel Smac mimetic, RMT5265.2HCL (RMT) in VAL cells. RMT improved the sensitivity to apoptosis in EBV- and to some extend in HTLV-1- but not in HHV-8-VAL. Furthermore, we identified that RMT promotes caspase 3 and 9 cleavage by inhibiting XIAP and inducing the mitochondrial efflux of Smac and cytochrome C. This investigation further support exploring the use of Smac inhibitors in VAL.

The process of apoptosis is essential for maintaining the physiologic balance between cell death and cell growth. This complex process is executed by two major pathways that participate in activating an executioner mechanism leading to chromatin disintegration and nuclear fragmentation. Dysregulation of these pathways often contributes to cancer development and resistance to cancer therapy. Here, we review the most recent discoveries in apoptosis regulation and possible mechanisms for resensitizing tumor cells to therapy.

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in the United States. DLBCL comprises biologically distinct subtypes including germinal center-like (GCB) and activated-B-cell-like DLBCL (ABC). The most aggressive type, ABC-DLBCL, displays dysregulation of both canonical and noncanonical NF-κB pathway as well as genomic instability. Although, much is known about the tumorigenic roles of the canonical NF-kB pathway, the precise role of the noncanonical NF-kB pathway remains unknown. Here we show that activation of the noncanonical NF-κB pathway regulates chromosome stability, DNA damage response and centrosome duplication in DLBCL. Analysis of 92 DLBCL samples revealed that activation of the noncanonical NF-κB pathway is associated with low levels of DNA damage and centrosome amplification. Inhibiting the noncanonical pathway in lymphoma cells uncovered baseline DNA damage and prevented doxorubicin-induced DNA damage repair. In addition, it triggered centrosome amplification and chromosome instability, indicated by anaphase bridges, multipolar spindles and chromosome missegregation. We determined that the noncanonical NF-κB pathway execute these functions through the regulation of GADD45α and REDD1 in a p53-independent manner, while it collaborates with p53 to regulate cyclin G2 expression. Furthermore, this pathway regulates GADD45α, REDD1 and cyclin G2 through direct binding of NF-κB sites to their promoter region. Overall, these results indicate that the noncanonical NF-κB pathway plays a central role in maintaining genome integrity in DLBCL. Our data suggests that inhibition of the noncanonical NF-kB pathway should be considered as an important component in DLBCL therapeutic approach.

Paraquat, N-methyl-4-phenyl-1,2,3,6 tetrahydropyridine, and rotenone have been shown to reproduce several features of Parkinson's disease in animal and cell culture models. Although these chemicals are known to perturb dopamine homeostasis and induce dopaminergic cell death, their molecular mechanisms of action are not well defined. We have previously shown that paraquat does not require functional dopamine transporter and does not inhibit mitochondrial complex I in order to mediate its toxic action (Richardson et al., 2005). In this study, we show that paraquat specifically oxidized the cytosolic form of thioredoxin and activated Jun N-terminal kinase (JNK), followed by caspase-3 activation. Conversely, 1-methyl-4-phenylpyridinium (MPP(+)) and rotenone oxidized the mitochondrial form of thioredoxin but did not activate JNK-mitogen-activated protein kinase and caspase-3. Loading cells with exogenous dopamine did not exacerbate the toxicity of any of these compounds. These data suggest that oxidative modification of cytosolic proteins is critical to paraquat toxicity, while oxidation of mitochondrial proteins is important for MPP(+) and rotenone toxicity. In addition, intracellular dopamine does not seem to exacerbate the toxicity of these dopaminergic neurotoxicants in this model.

Background: Multiple myeloma is a malignant plasma cell cancer that is increasing in frequency in today’s aging population. While proteasome inhibitors such as bortezomib have improved the outcome of treatment, many patients fail to achieve a complete remission and ultimately the disease progresses. With these new treatments come higher associated toxicities and significant healthcare costs for the drugs. Several new cancer therapies with companion diagnostics have been approved in parallel with the drug to guide physicians in selecting those patients who would specifically benefit from the drug. No such test exists for the use of bortezomib in multiple myeloma. We have previously demonstrated that the 3’ end of the nuclear factor kappa-B 2 (NFKB2/p100) gene, a member of the NFKB/Rel gene family, functions as a mediator of bortezomib apoptotic effect through the induction of caspase 8 activation. Methodology: We have investigated the loss of NF-kB2 3’end loss by quantitative PCR (QPCR) in 56 MM patients and by break-apart Fluorescent In-Situ Hybridization in the whole bone marrow of 31 patients as well as in sorted cells of 24 MM patients treated with bortezomib containing regimens. Quality of response was assessed after 4 cycles of treatment initiation. QPCR was measured in peripheral blood lymphocyte and CD138 positive cells from the bone marrow to generate a QPCR ratio. Results: 37 of the 56 newly diagnosed patient with MM achieved >partial response (PR), while 16 remained stable or progress to treatment. The mean QPCR ratio in responders was 1.69 ± 0.13, while in non-responder was 0.43 ± 0.1 (p Conclusion: loss of NF-kB2 3’ end is a potential companion diagnostic test for bortezomib. QPCR is more sensitive to detect NFKB2 3’end abnormalities. Disclosures Kelkar: Empire Genomics: Employment. Kaplan:Empire Genomics: Employment. Lonial:Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

2,3,5-Tris-(glutathion- S-yl)hydroquinone (TGHQ), a reactive metabolite of the nephrotoxicant hydroquinone, induces the ROS-dependent activation of MAPKs, followed by histone H3 phosphorylation and oncotic cell death in renal proximal tubule epithelial cells (LLC-PK1). Cell death and histone H3 phosphorylation are attenuated by pharmacological inhibition of p38 MAPK or ERK1/2 pathways. Because TGHQ, but not epidermal growth factor (EGF), induces histone H3 phosphorylation and cell death in LLC-PK1cells, we hypothesized that there are differences in the mechanisms by which TGHQ and EGF induce activation of the EGF receptor (EGFR). We therefore compared the relative ability of TGHQ, H2O2, and EGF to activate EGFR and MAPKs and found that p38 MAPK activation is EGFR independent, whereas ERK1/2 activation occurs mainly through EGFR activation. TGHQ, H2O2, and EGF induce different EGFR tyrosine phosphorylation profiles that likely influence the subsequent differential kinetics of MAPK activation. We next transfected LLC-PK1cells with a dominant negative p38 MAPK-expressing plasmid (pcDNA3-DNp38). TGHQ failed to induce phosphorylation of p38 MAPK and its substrate, MK-2, in pcDNA3-DNp38-transfected cells, indicating loss of function of p38 MAPK. In untransfected, pcDNA3 or pcDNA3-p38 (native)-transfected LLC-PK1cells, Hsp27 was intensively phosphorylated after TGHQ treatment, whereas in pcDNA3-DNp38-transfected cells, TGHQ failed to induce Hsp27 phosphorylation. Thus EGFR-independent p38 MAPK and EGFR-dependent ERK1/2 activation by TGHQ lead to the activation of two downstream signaling factors, i.e., histone H3 and Hsp27 phosphorylation, which have in common the potential ability to remodel chromatin.

The tuberous sclerosis-2 ( Tsc-2) gene is a suppressor of renal tumorigenesis and an early target of reactive oxygen species-induced renal cancer. Tuberin, the protein product of the Tsc-2 gene, participates in the regulation of cell proliferation, although the mechanism by which it suppresses proliferation is unknown. Quinol-thioether-transformed rat renal epithelial (QT-RRE) cell lines, derived from quinol-thioether-transformed primary renal epithelial cells from Eker rats, lack tuberin expression due to loss of heterozygosity of the Tsc-2 gene. These cell lines were used to examine the mechanism by which tuberin exerts its antiproliferative action. Loss of tuberin function correlates with high ERK activity ( 39 ), which could contribute to the formation of renal tumors. In this study, we sought to identify possible downstream effectors regulated by tuberin, using QT-RRE cells transfected with Tsc-2 cDNA to restore tuberin expression. Constitutively high ERK, B-Raf, and Raf-1 activities were observed in QT-RRE cells. However, restoration of tuberin expression in QT-RRE cells by transient transfection with Tsc-2 cDNA substantially decreased both ERK and B-Raf activity, with only modest changes in Raf-1 activity, suggesting tuberin functions as an upstream negative regulator of the ERK pathway. High ERK activity was not mediated through EGF receptor activation, but treatment with genistein demonstrated that protein kinases are involved in ERK cascade activation. The data indicate that loss of tuberin results in the upregulation of the ERK signaling pathway with subsequent increases in new DNA synthesis, and ultimately, tumor formation.

Extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK) were all rapidly activated in a ROS-dependent manner during 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-mediated oxidative stress and oncotic cell death in renal proximal tubule epithelial cells (LLC-PK1). TGHQ-induced phosphorylation of ERK1/2 and JNK MAPKs required epidermal growth factor receptor (EGFR) activation, whereas p38 MAPK activation was EGFR independent. In contrast to their established roles in cell survival, TGHQ-activated ERK1/2 and p38 MAPK (but not JNK) appear to contribute to cell death, since inhibition of ERK1/2 or p38 MAPKs with PD098059 or SB202190 respectively, attenuated TGHQ-mediated cell death. TGHQ increased AP-1 and NFkappaB DNA-binding activity, but whereas pharmacological inhibition of ERK1/2 or p38 MAPKs attenuated AP-1 DNA binding activity, it potentiated TGHQ-mediated NFkappaB activation. Consistent with a role for NFkappaB activation in the cytoprotective response to ROS in renal epithelial cells, an anti-NFkappaB peptide SN50 suppressed the protective effects of ERK inhibition (PD098059 treatment). The data provide evidence that the activation of MAPKs by ROS in renal epithelial cells plays an important role in oncotic cell death, and NF-kB is involved in the cytoprotective effects of PD098059.

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.

Abstract 2891 The proteasome inhibitor, bortezomib, is a powerful agent for the treatment of Multiple Myeloma (MM). However, many patients fail to achieve an optimal response. To identify biomarkers that could predict an optimal response to bortezomib, we assessed the role of the noncanonical NF-kB pathway in the response to bortezomib. Our in vitro studies identified that NF-kB2/p100 protein levels correlated directly with bortezomib sensitivity. Based on this finding, we measured at diagnosis the mRNA levels of different NF-kB2 regions in 64 patients schedule to receive bortezomib-based regimens. While no difference in expression of the 5'end regions was found between the CD138 (+) and CD138 (-) cells in these patients, half of them demonstrated low or absent 3'end expression levels in the CD138 (+) cells. Among forty-seven patients treated with four cycles of Bortezomib and dexamethasone, we found that low plasma cell NF-kB2 3'end levels were associated with a lower overall response rate (low: 47.8% vs. high: 96%, see table). In contrast, low plasma cell NF-kB2 3'end levels failed predict bortezomib response when lenalidomide or thalidome was added to the therapeutic regimen. To further understand the cause for NF-kB2 truncation, we are currently analyzing RNA sequencing in 3 non-responder patients and whole genome sequencing of 38 MM patients to determine the potential causative mechanisms. Preliminary results suggest that NF-kB2 truncation result from multiple genetic alterations, including: premature stop codons produced by ALU insertions, inversions and splicing variants. Overall, this study substantiates the basis of a more precise therapeutic algorithm according to NF-kB2 3'end plasma cell levels, which include the use of bortezomib and dexamethasone in patients with high NF-kB2 3'end plasma cell levels and immunomodulators or DNA damaging agents in patients with low NF-kB2 3'end plasma cell levels.BD+BTD or BRD++NF-kB2 3' end mRNA (CD 138(+)/CD138(−)LowHighLowHigh> Very Good Partial Response5 (21.7)9 (37.5)1 (11)4 (50)Partial Response6 (26.1)14 (58.3)7 (78)4 (50)Stable Disease4 (17.4)1 (4.2)0 (0)0 (0)Progressive disease8 (34.8)0 (0)1 (12)0 (0)Overall Response11 (47.8)23 (95.8)8 (88)8 (100)*Bortezomib + Dexamethasone**Bortezomib+Lenalidomide/Thalidomide+Dexamethasone Disclosures: Kaufman: Millenium, Onxy, Novartis, Keryx: Consultancy; Merck, Celgene: Research Funding.

Abstract 3240 Poster Board III-177 Lymphomas are heterogeneous diseases comprising multiple clinical and biological subgroups. Several studies have shown that genomic instability and constitutive activation of the NF-kB pathway are key features for lymphoma development (Shen M,Hematologica 2007). However, it remains unclear whether changes in activation of the canonical and non-canonical NF-kB pathways (rel-A/p50 and rel-B/p52, respectively) reflect a response to genomic instability and therefore promotes lymphomagenesis. To answer this question, we first demonstrated in two lymphoma cell lines (Daudi and OCI-Ly3) that DNA damage induced by Doxorubicin (2 mcg/mL) resulted in nuclear localization of rel-A and Rel-B. Then, to determine the role of each NF-kB pathway in DNA repair and centrosome duplication we compared the number of cells positive for phospo-H2aX (pH2aX) and centrosomes numbers (measured by gamma-tubulin) in p105-siRNA (canonical) and P100-siRNA (non-canonical) with luciferase-siRNA (control) expressing cells. Our results showed that the expression of p105 and p100 siRNAs increase the number of pH2aX (+) cells compared to control. Subsequently, a time course measuring pH2aX (+) cells was performed after treating p105, P100 and luciferase siRNA OCI-ly3 expressing cell lines with Doxorubicin (2 mcg/mL). In cells expressing luciferase siRNA, pH2aX (+) cells peak (60%) at 60 minutes (min) and return to normal at 120 min, in p105 siRNA –cells pH2aX peak at 90 min (90% + cells) and then decrease similarly to luciferase siRNA cells. P100 siRNA cells demonstrate a continuous increased in pH2aX (+) cells up to 80%. In addition, p100 siRNA expression was associated with centrosome amplification (>2 centrosomes in 20-30% of the cells vs. < 8% in p105 or Luciferase siRNA expressing cells). Also, the expression of NF-kB siRNAs delayed doxorubicin-induced phosphorylation of p53 (serine 15 – target of ATM) and CHK2. To evaluate whether the genomic instability caused by both NF-kB siRNAs affects tumor development, we performed xenograft experiments. Our results demonstrated that NF-kB siRNAs not only slow down tumor initiation but prevented tumor development (p105 siRNA= 8 days delayed and 22% were tumor free and p100 siRNA= 13.5 days delayed and 55% were tumor free compared to luciferase siRNA). To investigate these findings in primary tissues we measured the number of phospo-H2aX (+) cells and the levels of rel-A and rel-B nuclear localization in 40 primary lymphoma tumor samples. Our results demonstrated that phospo-H2aX levels inversely correlated with rel-B nuclear localization (r=-0.58, p Disclosures No relevant conflicts of interest to declare.