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There are approximately 44,000 cases of human papillomavirus–associated (HPV-associated) cancer each year in the United States, most commonly caused by HPV types 16 and 18. Prophylactic vaccines successfully prevent healthy people from acquiring HPV infections via HPV-specific antibodies. In order to treat established HPV-associated malignancies, however, new therapies are necessary. Multiple recombinant gorilla adenovirus HPV vaccine constructs were evaluated in NSG-β2m–/– peripheral blood mononuclear cell–humanized mice bearing SiHa, a human HPV16+ cervical tumor, and/or in the syngeneic HPV16+ TC-1 model. PRGN-2009 is a therapeutic gorilla adenovirus HPV vaccine containing multiple cytotoxic T cell epitopes of the viral oncoproteins HPV 16/18 E6 and E7, including T cell enhancer agonist epitopes. PRGN-2009 treatment reduced tumor volume and increased CD8+ and CD4+ T cells in the tumor microenvironment of humanized mice bearing the human cervical tumor SiHa. PRGN-2009 monotherapy in the syngeneic TC-1 model also reduced tumor volumes and weights, generated high levels of HPV16 E6–specific T cells, and increased multifunctional CD8+ and CD4+ T cells in the tumor microenvironment. These studies provide the first evaluation to our knowledge of a therapeutic gorilla adenovirus HPV vaccine, PRGN-2009, showing promising preclinical antitumor efficacy and induction of HPV-specific T cells, along with the rationale for its evaluation in clinical trials.

BackgroundWhile prophylactic human papillomavirus (HPV) vaccines will certainly reduce the incidence of HPV-associated cancers, these malignancies remain a major health issue. PDS0101 is a liposomal-based HPV therapeutic vaccine consisting of the immune activating cationic lipid R-DOTAP and HLA-unrestricted HPV16 peptides that has shown in vivo CD8+ T cell induction and safety in a phase I study. In this report, we have employed the PDS0101 vaccine with two immune modulators previously characterized in preclinical studies and which are currently in phase II clinical trials. Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domains of the transforming growth factor-β receptor type II (TGFβRII) fused to a human IgG1 monoclonal antibody blocking programmed cell death protein-1 ligand (PDL1), designed both as a checkpoint inhibitor and to bring the TGFβRII ‘trap’ to the tumor microenvironment (TME). NHS-interleukin-12 (NHS-IL12) is a tumor targeting immunocytokine designed to bring IL-12 to the TME and thus enhance the inflammatory Th1 response.MethodsWe employed TC-1 carcinoma (expressing HPV16 E6 and E7 and devoid of PDL1 expression) in a syngeneic mouse model in monotherapy and combination therapy studies to analyze antitumor effects and changes in immune cell types in the spleen and the TME.ResultsAs a monotherapy, the PDS0101 vaccine generated HPV-specific T cells and antitumor activity in mice bearing HPV-expressing mEER oropharyngeal and TC-1 lung carcinomas. When used as a monotherapy in the TC-1 model, NHS-IL12 elicited antitumor effects as well as an increase in CD8+ T cells in the TME. When used as a monotherapy, bintrafusp alfa did not elicit antitumor effects or any increase in T cells in the TME. When all three agents were used in combination, maximum antitumor effects were observed, which correlated with increases in T cells and T-cell clonality in the TME.ConclusionThese studies provide the rationale for the potential clinical use of combinations of agents that can (1) induce tumor-associated T-cell responses, (2) potentiate immune responses in the TME and (3) reduce immunosuppressive entities in the TME.

// Samuel T. Pellom Jr. 1, 2, 3 , Duafalia F. Dudimah 1 , Menaka C. Thounaojam 1 , Roman V. Uzhachenko 1 , Ashutosh Singhal 1 , Ann Richmond 4, 5, 6, 7, 8, 9 , Anil Shanker 1, 3, 7, 8, 9 1 Department of Biochemistry and Cancer Biology, School of Medicine, Meharry Medical College, Nashville, Tennessee, USA 2 Department of Microbiology and Immunology, School of Medicine, Meharry Medical College, Nashville, Tennessee, USA 3 School of Graduate Studies and Research, Meharry Medical College, Nashville, Tennessee, USA 4 Tennessee Valley Healthcare System, Nashville, Tennessee, USA 5 Department of Veterans Affairs, Nashville, Tennessee, USA 6 Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA 7 Host-Tumor Interactions Research Program, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University, Nashville, Tennessee, USA 8 Vanderbilt Center for Immunobiology, Vanderbilt University, Nashville, Tennessee, USA 9 Vanderbilt Center for Translational and Clinical Immunology, Vanderbilt University, Nashville, Tennessee, USA Correspondence to: Anil Shanker, email: ashanker@mmc.edu Keywords: proteasome inhibition, CD8 + T cells, immunosuppression, cancer immunotherapy, adoptive cell therapy Received: November 07, 2016 Accepted: December 07, 2016 Published: December 29, 2016 ABSTRACT Tumor-induced immune tolerance poses a major challenge for therapeutic interventions aimed to manage cancer. We explored approaches to overcome T-cell suppression in murine breast and kidney adenocarcinomas, and lung fibrosarcoma expressing immunogenic antigens. We observed that treatment with a reversible proteasome inhibitor bortezomib (1 mg/kg body weight) in tumor-bearing mice significantly enhanced the expression of lymphocyte-stimulatory cytokines IL-2, IL-12, and IL-15. Notably, bortezomib administration reduced pulmonary nodules of mammary adenocarcinoma 4T1.2 expressing hemagglutinin (HA) model antigen (4T1HA) in mice. Neutralization of IL-12 and IL-15 cytokines with a regimen of blocking antibodies pre- and post-adoptive transfer of low-avidity HA 518-526 -specific CD8 + T-cells following intravenous injection of 4T1HA cells increased the number of pulmonary tumor nodules. This neutralization effect was counteracted by the tumor metastasis-suppressing action of bortezomib treatments. In bortezomib-treated 4T1HA tumor-bearing mice, CD4 + T-cells showed increased IL-2 production, CD11c + dendritic cells showed increased IL-12 and IL-15 production, and HA-specific activated CD8 + T-cells showed enhanced expression of IFNγ, granzyme-B and transcription factor eomesodermin. We also noted a trend of increased expression of IL-2, IL-12 and IL-15 receptors as well as increased phosphorylation of STAT5 in tumor-infiltrating CD8 + T-cells following bortezomib treatment. Furthermore, bortezomib-treated CD8 + T-cells showed increased phosphorylation of mitogen-activated protein kinase p38, and Akt, which was abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitor. These data support the therapeutic potential of bortezomib in conjunction with other immunotherapies to augment the strength of convergent signals from CD8 + T-cell signaling molecules including TCR, cytokine receptors and downstream PI3K/Akt/STAT5 pathways to sustain CD8 + T-cell effector function in the tumor microenvironment.

Here we describe a novel bifunctional fusion protein, designated N-809. This molecule comprises the IL-15/IL15Rα superagonist complex containing the Fc-domain of IgG1 (N-803, formerly designated as ALT-803) fused to two single chain anti-PD-L1 domains. The fully human IgG1 portion of the N-809 molecule was designed to potentially mediate antibody dependent cellular cytotoxicity (ADCC). The studies reported here show that N-809 has the same ability to bind PD-L1 as an anti-PD-L1 monoclonal antibody. RNAseq studies show the ability of N-809 to alter the expression of an array of genes of both CD4+ and CD8+ human T cells, and to enhance their proliferation; CD8+ T cells exposed to N-809 also have enhanced ability to lyse human tumor cells. An array of genes was differentially expressed in human natural killer (NK) cells following N-809 treatment, and there was increased expression of several surface activating receptors; there was, however, no increase in the expression of inhibitory receptors known to be upregulated in exhausted NK cells. N-809 also increased the cytotoxic potential of NK cells, as shown by increased expression of granzyme B and perforin. The lysis of several tumor cell types was increased when either NK cells or tumor cells were exposed to N-809. Similarly, the highest level of ADCC was seen when both NK cells (from donors or cancer patients) and tumor cells were exposed to N-809. These studies thus demonstrate the multi-functionality of this novel agent.

Tributyltin (TBT), a toxic environmental contaminant, has been widely utilized for various industrial, agricultural and household purposes. Its usage has led to a global contamination and its bioaccumulation in aquatic organisms and terrestrial mammals. Previous studies suggest that TBT has debilitating effects on the overall immune function of animals, rendering them more vulnerable to diseases. TBT (at concentrations that have been detected in human blood) alters secretion of inflammatory cytokines from human lymphocytes ex vivo. Thus, it is important to determine if specified levels of TBT can alter levels of cytokines in an in vivo system. Mice were exposed to biologically relevant concentrations of TBT (200, 100 or 25 nM final concentrations). The quantitative determination of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL2, IL5, IL7, IL12βp40, IL13, IL15, keratinocyte chemoattractant (KC), macrophage inflammatory protein 1β (MIP), MIP2 and regulated on activation normal T-cell-expressed and secreted (RANTES) was performed in mouse sera by MAGPIX analysis and Western blot. Results indicated alterations (both decreases and increases) in several cytokines. The pro-inflammatory cytokines IFNγ, TNFα, IL-1β, IL-2, IL5, IL12βp40 and IL-15 were altered as were the chemokines MIP-1 and RANTES and the anti-inflammatory cytokine IL-13. Increases in IFNγ and TNFα were seen in the serum of mice exposed to TBT for less than 24 h. Levels of IL1β, IL-12 βp40, IL-5 and IL-15 were also modulated in mouse serum, depending on the specific experiment and exposure level. IL-2 was consistently decreased in mouse serum when animals were exposed to TBT. There were also TBT-induced increases in MIP-1β, RANTES and IL-13. These results from human and murine samples clearly suggest that TBT exposures modulate the secretion inflammatory cytokines.

Cancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well, such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Because T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T-cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c+CD8+ dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of costimulatory molecules, and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8+ T cells, stabilizing expression of T-cell receptor CD3ζ and IL2 receptor-α, while maintaining IFNγ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2−/− mice, bortezomib treatment after adoptive T-cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T-cell function in the context of cancer immunotherapy. Cancer Res; 75(24); 5260–72. ©2015 AACR.

Bortezomib is an inhibitor of the ubiquitin-proteasome proteolytic pathway responsible for intracellular protein turnover. Cellular proteins controlled by this pathway represent a diverse group of potential therapeutic targets, particularly in cancer cells, which exploit this proteasomal pathway to promote their growth and diminish apoptosis. Along with inhibiting the proteasome and thus sensitizing tumor cells to apoptosis, bortezomib may also have multiple effects on the host immune responses. This review summarizes the effects that bortezomib may play on immune cell subsets in various disease states in modifying lymphocyte receptors, ligands, the expression of various cytokines and chemokines and their downstream signaling. We also propose steps that can be taken to refine combinatorial strategies that include bortezomib to improve current immunotherapeutic approaches.

Butyrate is a histone deacetylase inhibitor implicated in many studies as a potential therapy for various forms of cancer. High concentrations of butyrate (>1.5 mM) have been shown to activate apoptosis in several cancer cell lines including prostate, breast, and leukemia. Butyrate is also known to influence multiple signaling pathways that are mediators of cytokine production. The purpose of this study was to evaluate the impact of high concentrations of butyrate on the cancer microenvironment vis-à-vis apoptosis, cellular migration, and capacity to modulate cytokine expression in cancer cells. The results indicate that high concentrations of butyrate induced a 2-fold activation of caspase-3 and reduced cell viability by 60% in U937 leukemia cells. Within 24 hours, butyrate significantly decreased the levels of chemokines CCL2 and CCL5 in HL-60 and U937 cells, and decreased CCL5 in THP-1 leukemia cells. Differential effects were observed in treatments with valproic acid for CCL2 and CCL5 indicating butyrate-specificity. Many of the biological effects examined in this study are linked to activation of the AKT and MAPK signaling pathways; therefore, we investigated whether butyrate alters the levels of phosphorylated forms of these signaling proteins and how it correlated with the expression of chemokines. The results show that butyrate may partially regulate CCL5 production via p38 MAPK. The decrease in p-ERK1/2 and p-AKT levels correlated with the decrease in CCL2 production. These data suggest that while promoting apoptosis, butyrate has the potential to influence the cancer microenvironment by inducing differential expression of cytokines.

The immunosuppressive tumor microenvironment disturbs host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8+ T lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8+ T cells. Moreover, bortezomib promoted CD8+ T cell nuclear factor-κB (NFκB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8+ T cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8+ T cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. We are also elucidating components of microRNA regulation affecting NICD-NFκB crosstalk. Our preliminary data suggest that bortezomib is also able to positively regulate miR-155 expression in CD8+ T cells from mice bearing tumor. As well as, miR-155 suppression downregulates bortezomib-induced increase in Notch target genes in T cells. We are currently investigating alternative proteasome inhibitors in order to understand whether bortezomib’s effect on miR-155 expression in CD8+ T cells is specific to bortezomib or primarily conducive to a proteasome inhibition effect. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death, but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on antitumor T cell functions. Citation Format: Ariana N. Renrick, Menaka C. Thounaojam, Portia Thomas, Samuel T. Pellom, Anil Shanker. Bortezomib enhances CD8+ T Lymphocyte antitumor effector function: Potential mechanism(s) via notch regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 647. doi:10.1158/1538-7445.AM2017-647

Meeting abstracts The immunosuppressive tumor microenvironment perturbs numerous immune regulatory networks and usurps host antitumor immunity. We discovered that tumor interferes with host hematopoietic Notch system in lung cancer patients [[1][1]]. The resultant decrease in immune Notch signaling

The effects of Tributyltin (TBT), a widespread environmental contaminant, on the induction of in vivo and ex vivo cytokine production and secretion were investigated. Human monocyte-depleted peripheral blood mononuclear cells and different groups of Balb/C mice were stimulated with desired concentrations of TBT (200-2.5 nM for human studies and 200-25 nM for mouse studies). The quantitative determination of IFNγ, TNFα and IL-1β was performed in mouse sera by MagPix analysis and western blot. The levels of these cytokines were measured at different time points: 6, 12, 24 and 48 h after injection. Analysis of IFNγ and TNFα in human cells were conducted by ELISA and western blot. MAPK and NF-κB pathways were inhibited to determine TBT-induced dysregulation of IFNγ and TNFα. Inhibition of the p38 and p44/42 pathways blocked the TBT-induced elevation of IFNγ, while the inhibition of the p38 pathway blocked the TBT-induced elevation of TNFα. Results showed that TBT increased the levels of IFNγ, TNFα and IL-1β in the sera of mice. IFNγ and TNFα secretion from human monocyte-depleted peripheral blood mononuclear cells also increased, showing striking agreement in the response to TBT between the mouse and human systems. These data suggest that TBT exposure can result in an inflammatory environment, which could predispose the host to various immunopathologies. Grant U54CA163066 from the National Institute of Health Citation Format: Shanieek Lawrence, Samuel T. Pellom, Anil Shanker, Margaret Whalen. Tributyltin-induced dysregulation of inflammatory cytokine levels in human and mouse immune cells. [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 934.

The immunosuppressive tumor microenvironment usurps host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8+T-lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Bortezomib also neutralized TGFβ-mediated suppression of IFNγ and granzyme B expression in activated CD8+T-cells. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8+T-cells. Moreover, bortezomib promoted CD8+T-cell nuclear factor-κB (NF-κB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8+T-cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8+T-cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. We are also elucidating components of microRNA regulation affecting NICD-NFκB crosstalk. Our preliminary data suggest that miR-155 plays a role in bortezomib-induced regulation of T cell function. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death, but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on anti-tumor T-cell functions. Citation Format: Ariana N. Renrick, Menaka C. Thounaojam, Duafalia F. Dudimah, Portia Thomas, Samuel T. Pellom, Roman V. Uzhachenko, Anil Shanker. Bortezomib enhances expression of effector molecules in antitumor CD8+ T lymphocytes by modulating Notch-NF-kB-miR-155 crosstalk. [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 4161.

Tributyltin (TBT) is an environmental contaminant that has been used for several industrial, agricultural and household purposes. Despite limited bans on the usage of Tributyltin, the stability and toxicity of the compound may prove to have negative effects on immune function in terrestrial mammals. Studies show that there is an increase in the incidences of tumors and decreased immune cell function in TBT-exposed mammals. TBT interferes with the ability of human natural killer (NK) cells to lyse target cells; it also alters the secretion of the pro-inflammatory cytokines Interferon gamma (IFNγ) and Tumor necrosis factor alpha (TNFα) from human immune cells ex vivo. Any dysregulation of the cytokine communication network may have detrimental effects on immune function, as cytokines play a key role in the regulation of immune responsiveness. There have been no in vivo studies on the effects of exposure to TBT on the cytokine communication network. The effects of 24 hour exposure to TBT on the production of IFNγ and TNFα from human immune cells were analyzed in an ex vivo system using western blotting. The serum of TBT-exposed mice was analyzed for changes in the levels of cytokine secretion and production using a Milliplex mouse cytokine/chemokine magnetic bead premixed 32 plex kit and western blotting; the effects of TBT-exposure was assessed using a time curve of 6h, 12h, 24h, 48h and control. TBT increased the levels of the cytokines IFNγ, TNFα, IL1β, IL5, IL7, IL12βp40, IL13, IL15, MIP1β, MIP2 and RANTES in the serum at a minimum of one time point. IFNγ and TNFα secretion and production from human cells also increased, showing striking agreement in the response to TBT between the human and mouse systems. Supported by: NIH grant 5U54CA163066-03 Citation Format: Shanieek T. Lawrence, Margaret Whalen, Samuel Pellom, Anil Shanker. Tributyltin alters the production and secretion of inflammatory cytokines from human and mouse immune cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2735. doi:10.1158/1538-7445.AM2015-2735

The immunosuppressive tumor microenvironment perturbs numerous immune regulatory networks and usurps host anti-tumor immunity. We discovered that tumor interferes with host hematopoietic Notch system in lung cancer patients. The resultant decrease in immune Notch signaling could be a major causative link in the inadequate induction of anti-tumor immunity. Interestingly, we found that tumor-induced decrease in immune Notch in various mouse solid tumor models could be restored therapeutically by the following two agents. Administration of a novel Delta-like ligand 1 (DLL1) multivalent cluster and the FDA-approved proteasome inhibitor drug bortezomib - which also sensitizes tumors to death signals - could activate Notch 1 signaling in lymphoid cells of tumor-bearing mice without increasing tumor cell proliferation or clonogenicity. Systemic activation of DLL1-Notch signaling could attenuate tumor vascularization as well as increase T cell infiltration in tumor, decrease proportion of regulatory T cells and enhance antitumor T cell function and memory in multiple mouse tumor models. New data also show that bortezomib affects the expression of notch receptors and ligands differentially in lymphocytes and in a wide range of solid tumor cells. Moreover, bortezomib administration increased the expression of Notch target genes Hes 1 and Hey 1 in thymus, lymph node, and spleen, as well as decreased the proportion of regulatory T cells and enhanced T cell production of IFN-γ in tumor-bearing mice. Results indicate that bortezomib-induced activation of Notch target genes Hes 1 and Hey 1 is through its inhibition of NFkB while its activation of another Notch target gene Deltex 1 is mediated via PI3K. The findings suggest a potential synergistic action of bortezomib and DLL1 activation of Notch signaling. The potential of modulating anti-tumor Notch signaling by the prototypic DLL1 cluster in combination with bortezomib presents exciting opportunities to uncover multi-pronged immune stimulatory regimens. Therapeutic restoration of immune Notch signaling could provide effective treatment and recurrence-free survival in cancer patients by breaking tumor resistance, enhancing immune surveillance, and sustaining robust anti-tumor immunity. Citation Format: Duafalia F. Dudimah, Samuel T. Pellom Jr., Roman V. Uzhachenko, David P. Carbone, Mikhail M. Dikov, Anil Shanker. Cancer therapy by resuscitating Notch immune surveillance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3642. doi:10.1158/1538-7445.AM2014-3642

Cytokines are important regulators of immune responsiveness. Alterations of cytokine secretion from immune cells would have the potential to disrupt both immune destruction of cancer cells and to alter the proliferation and invasiveness of tumor cells due to their capacity to act as growth factors, angiogenic factors, and inducers of epithelial-mesenchymal transition. Tributyltin (TBT) is a widespread environmental contaminant having been used in a variety of industrial applications and is found in human blood at levels ranging as high as 261 nM. Studies in human immune cells have shown that secretion of tumor necrosis factor alpha (TNFα) is altered by exposures to TBT. There have been no in vivo studies of the effects of exposure to TBT on the cytokine communication network. As it is not possible to examine alterations of this network in exposed humans, we examined the effects of TBT exposure in a mouse model using Milliplex mouse cytokine/chemokine magnetic bead premixed 32 plex kit. Serum and spleens of TBT-exposed mice were analyzed for changes in cytokine secretion/levels. TBT increased the levels of IL13 in the serum of non-tumor-bearing mice and increased IL1β, IL6, KC, and M-CSF in tumor-bearing mice. The spleens of both non-tumor-bearing and tumor-bearing mice showed increases in Eotaxin when exposed to TBT. Of significant interest is the fact that the presence of tumor causes increases in Eotaxin, IL12p40, M-CSF, and TNFα and exposure to TBT alone can cause increases in these same cytokines. Tumors are known to increase levels of cytokines that enhance their ability to survive. The results showing that TBT can amplify the levels of several cytokines that are increased by tumor suggest that TBT may have tumor-promoting abilities by modulating tumor growth and/or invasiveness. Experiments are under way to dissect cytokine signaling pathways that are affected by TBT. The findings will allow identification of molecular targets that are necessary to intercept the tumor promoting signaling following TBT exposure. Supported by: NIH grant 5U54CA163066-03 Citation Format: Shanieek Lawrence, Samuel Troy Pellom, Tasia Hurd-Brown, Anil Shanker, Margaret Whalen. Elevation of tumor-promoting cytokines in mice exposed to the environmental contaminant tributyltin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3191. doi:10.1158/1538-7445.AM2014-3191

Cancer growth interferes with the Notch signaling that plays diverse roles in lymphocyte commitment and differentiation, oncogenesis, and peripheral immune responses. Our studies show that the stimulation of hematopoietic Notch signaling following administration of a prototypic therapeutic agent clustered multivalent Delta-like Notch ligand (DLL1), in mouse lung tumor models can correct tumor-induced defects in lymphocyte differentiation and improve antitumor lymphocyte responses, without increasing tumor cell proliferation. Moreover, since the Notch signaling complex is inactivated via the proteasome, its inhibitor bortezomib, amongst its other potential effects, may sustain DLL1-mediated pharmacological activation of the Notch signaling. Our data demonstrate that the proteasome inhibitor bortezomib can selectively sensitize solid tumors to apoptotic signals by upregulating death receptors on tumor cells and amplifying caspase-8 activation in the extrinsic cell death pathway, without any adverse effect on the antigen-specific T cell proliferative or cytolytic functions in an in vivo setting. In the current study, we investigated the effects of bortezomib and DLL1 on Notch receptor/ligand system and its downstream signaling network with an aim to enhance cancer immunotherapy. Results show that bortezomib affects the expression of notch receptors and ligands differentially in lymphocytes and in a wide range of solid tumor cells: 4T1, PyMT, MDA-MB-231, C26, LLC, D459, and Renca-HA. Specifically, the expression of Notch 4 receptor and its ligand Jagged 1 was reduced in bortezomib-treated tumor cells. In addition, bortezomib treatment of tumor cells modulated Notch downstream targets Hes1, Hes3, Hey1, Deltex1 and Deltex3, which act as transcriptional regulators of tissue-specific differentiation. bortezomib also modulated the phosphorylation of Erk1/2 MAP kinases and AKT suggesting that bortezomib may mediate its effects through downstream phosphorylation of Erk targets, such as STATs and NFkB, or PI3K, which are key players in signaling cross-talk and intersect with Notch signaling. Noreover, bortezomib administration increased the expression of Notch target Hes1 in thymus, lymph node and spleen of tumor-bearing mice, suggesting a potential synergistic action of bortezomib and DLL1 activation of Notch signaling. These results suggest that tumor cell-death sensitizing bortezomib and pharmacological DLL1 cluster activating immunostimulatory Notch in tumor-bearing host follow common signal transduction pathways and can synergize in antitumor activity. These findings offer a novel three-pronged combinatorial therapeutic approach of immunostimulatory Notch ligand DLL1 and bortezomib to abrogate the immunosuppressive circuitries operating in the tumor microenvironment with a potential to reduce tumor burden and resuscitate tumor-specific immunity. Citation Format: Duafalia F. Dudimah, Roman V. Uzhachenko, Samuel T. Pellom, Asel K. Biktasova, Mikhail M. Dikov, David P. Carbone, Anil Shanker. Resuscitating immune surveillance in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3983. doi:10.1158/1538-7445.AM2013-3983

Notch signaling plays diverse roles in lymphocyte differentiation and tumor angiogenesis. Our recent findings implicate the role of the Notch 1 pathway in promoting antitumor immune effector functions. We explored the effects of Notch 1 stimulation on T cell immunity and its potential pharmacological activation using multivalent Delta-like Notch 1 ligand DLL1 cluster in combination with the proteasome inhibitor bortezomib, which could stabilize Notch 1 activation. Bortezomib was found to selectively sensitize solid tumor cells to apoptosis by upregulating death receptors on tumor cells and amplifying caspase-8 activation in the death-inducing signaling complex. This resulted in reduced pulmonary nodules of injected tumor cells in mice. Additionally, in an in vivo tumor setting, no adverse effects of bortezomib were observed on the antigen-specific T cell proliferative or cytolytic functions. But no long-term survival benefits could be obtained, possibly due to the immunosuppressive effects of the tumor. In our more recent studies, we observed that DLL1-specific immune Notch activation could enhance antitumor T cell immunity by overcoming tumor-associated immunosuppression in D459 and Lewis lung carcinoma models. The results offer a novel combinatorial approach to abrogate the immunosuppressive circuitries operating in the tumor microenvironment with a potential to reduce tumor burden and resuscitate tumor-specific immunity. This work has important translational implications for improving immunotherapy in cancer patients, and may provide insights into new strategies for cancer prevention. Citation Format: Duafalia F. Dudimah, Roman V. Uzhachenko, Samuel T. Pellom, Jr., Asel K. Biktasova, Mikhail M. Dikov, David P. Carbone, Anil Shanker. Resuscitating cancer immunosurveillance by combining Notch 1 and death receptor-activating therapy. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B93.

Kidney cancer is a malignant tumor that accounts for about two percent of all cancers in the United States. African American men and women account for a higher incidence rate of kidney cancer compared to other races. The higher rate of failure of current immunotherapeutic strategies is attributed to the immunosuppressive effects as well as modification of the immune-regulatory circuits brought about by cancers. With the aim of enhancing immune mechanisms against kidney cancer, we undertook a combinatorial approach to reveal the therapeutic potential of adoptive cell transfer regimens using mouse models in conjunction with molecular targeting drugs that sensitize kidney tumor cell-death. The proteasome inhibitor, bortezomib, selectively sensitizes kidney tumor cells to apoptosis by activating caspase-8 in the extrinsic apoptosis pathway. Moreover, our studies thus far have revealed that bortezomib can also enhance natural killer (NK) cell antitumor function. We observed that bortezomib upregulates Fas and NK cell activating receptor NKG2D ligand Rae1-on renal cancer Renca cells in vivo. Moreover, preliminary observations suggest that bortezomib can be combined with adoptive NK cell transfer to provide greater therapeutic benefits with little adverse effects in vivo. These new data predict the potential of combining adoptive NK cell transfer regimens with bortezomib-induced sensitization of kidney tumor cells to induce apoptosis. Such a combinatorial approach of NK cell transfer coupled with bortezomib treatment would have dual effects by sensitizing tumor cells to cell death and improve NK cell effector functions. This could prevent tumor metastasis by eliminating tumor escape variants and significantly benefit tumor immunotherapy efforts in kidney cancer. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B107.

Breast cancer mortality occurs primarily due to invasive metastasis and takes a disproportionately heavy toll in African American women. Clinical immunotherapy efforts prove largely ineffective, partly due to the numerous immune-regulatory circuits influenced by cancers, which alter the differentiation and function of immune cells, including T cells. The long-term objective of this study is to enhance immune mechanisms of cancer rejection to provide durable clinical benefits against breast cancer. Our recent work using mouse models has revealed the therapeutic potential of combinatorial adoptive cell transfer regimens along with immunostimulatory and tumor cell-death-sensitizing treatments. We found that the proteasome inhibitor bortezomib selectively sensitized mammary tumor cells to apoptosis by amplifying caspase-8 activation in the death-inducing signaling complex. This resulted in reduced lung metastases in mice. In addition, bortezomib could be combined with adoptive T cell transfer with no adverse effects on the antigen-specific T cell proliferative or cytolytic functions in vivo. However, no long-term survival benefits could be observed, likely due to immunosuppressive effects of tumor. More recently, we observed that Delta-like ligand (DLL1)-specific immune Notch activation could enhance antitumor T cell immunity by overcoming tumor-associated immunosuppression. These data predict the potential of combining immunostimulatory Notch activation by soluble clustered DLL1 ligand treatment and bortezomib-induced sensitization of breast tumor cells to apoptosis with T cell adoptive transfers. This could prevent tumor metastasis and significantly improve breast cancer therapy. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B108.