Samuel T. Pellom

Development of Proteasome Inhibitors as Therapeutic Drugs

The proteasome is a multi-unit enzyme complex found in the cytoplasm and nucleus of all eukaryotic cells and is responsible for degradation of unneeded or damaged intracellular proteins by proteolysis, a chemical reaction that breaks peptide bonds. Proteasome inhibition presents a promising approach to cancer therapy by targeting the proteasome function in tumor cells. Delineating the success of bortezomib in the treatment of multiple myeloma and mantle cell lymphoma, this review explores various proteasome inhibitors, currently in development, as molecular targeting agents in the fight against cancer. Proteasome inhibitors can be used alone or in combination with other conventional cancer therapies to sensitize tumor cells to cell death by various mechanisms and improve therapeutic benefits.

Modulatory effects of bortezomib on host immune cell functions.

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.

Characterization of the guinea pig animal model and subsequent comparison of the behavioral effects of selective dopaminergic drugs and methamphetamine.

Although not commonly used in behavior tests guinea pigs may offer subtle behavior repertoires that better mimic human activity and warrant study. To test this, 31 Hartley guinea pigs (male, 200–250 g) were evaluated in PhenoTyper cages using the video‐tracking EthoVision XT 7.0 software. Results showed that guinea pigs spent more time in the hidden zone (small box in corner of cage) than the food/water zone, or arena zone. Guinea pigs exhibited thigmotaxis (a wall following strategy) and were active throughout the light and dark phases. Eating and drinking occurred throughout the light and dark phases. An injection of 0.25 mg/kg SCH23390, the dopamine D1 receptors (D1R) antagonist, produced significant decreases in time spent in the hidden zone. There were insignificant changes in time spent in the hidden zone for guinea pigs treated with 7.5 mg SKF38393 (D1R agonist), 1.0 mg/kg sulpiride (D2R antagonist), and 1.0 or 10.0 mg/kg methamphetamine. Locomotor activity profiles were unchanged after injections of saline, SKF38393, SCH23390, and sulpiride. By contrast, a single injection or repeated administration for 7 days of low‐dose methamphetamine induced transient hyperactivity but this declined to baseline levels over the 22‐h observation period. Guinea pigs treated with high‐dose methamphetamine displayed sustained hyperactivity and travelled significantly greater distances over the circadian cycle. Subsequent 7‐day treatment with high‐dose methamphetamine induced motor sensitization and significant increases in total distances moved relative to single drug injections or saline controls. These results highlight the versatility and unique features of the guinea pig for studying brain–behavior interactions. Synapse 68:221–233, 2014.

Bortezomib improves adoptive T cell therapy by sensitizing cancer cells to FasL cytotoxicity

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.

Increased Cell Surface Free Thiols Identify Effector CD8+ T Cells Undergoing T Cell Receptor Stimulation

Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8+ T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8+ T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8+ T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8+ T cells. Finally, CSFT levels on effector CD8+ T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8+ T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.

Prospects of combining adoptive cell immunotherapy with bortezomib

Bortezomib, also known as PS-341 and VelcadeTM, is a front-line drug approved in the USA and EU for the treatment of multiple myeloma [1] and mantle cell lymphoma [2]. Its use has been extended lately to advanced stage non-small-cell lung cancer [3]. Bortezomib inhibits the ubiquitin-proteasome proteolytic pathway, which cancer cells rely heavily upon due to their high rate of aberrant protein generation and need of rapid protein turnover. Therefore, interference with the proteasome system became an attractive therapeutic strategy against cancer. Intracellular pathways requiring protein degradation for activation, such as the NFκB pathway, which maintain cancer cell resistance to apoptosis, are also one of the targets of this drug. The reversible nature of proteasome inhibition by bortezomib sets it apart from other commercially available proteasome inhibitor drugs. While bortezomib’s boronic acid moiety prevents it from being transported out of the cell by the multidrug resistance system, its transient half-life (9–15 h) makes it tolerable to patients when given at the doses of 1 mg/kg body weight, approximately 15–20 nM by blood volume. Bortezomib can sensitize solid tumor cells to apoptosis by amplifying tumor cell caspase-8 activation in the deathinducing signaling complex following death receptor ligation by death ligands such as TRAIL or its receptor agonist monoclonal antibodies [4,5]. Unfortunately, clinical trials in patients with solid tumors showed that bortezomib lacks therapeutic efficacy as a single agent. Moreover, there were concerns over the negative side effects reported on immune cells, particularly on CD4 T cells and dendritic cells, when using high doses of bortezomib [6–8]. New data show that administration of the proteasome inhibitor bortezomib (1 mg/kg body weight; ≤20 nM) has complementary immunostimulatory effects following adoptive transfer of CD8 T cells in mice bearing kidney or mammary adenocarcinomas. It was observed that lymphocyte effector functions were significantly improved following bortezomib treatment by mechanisms involving the upregulation of multiple Notch components and lymphocyte activation signaling molecules [9,10]. These results are in contrast with the effects of bortezomib observed in vitro where the doses used were high (>100 nM) – doses that cannot be extrapolated to the therapeutic situation in vivo. Only 15–20 nM concentrations of bortezomib are found to be tolerable in vivo in both mice and humans [5,11]. Growing evidence suggests that bortezomib given at 15–20 nM doses exhibits an intrinsic ability to increase the levels of immunostimulatory cytokines, and components of Notch and NFκB signaling pathways in lymphocytes, thereby amplifying their effector functions either directly or indirectly. Thus, bortezomib could be an attractive candidate for exploration in combination with antitumor T-cell and/or natural killer cell adoptive therapy [12,13]. Prospects of combining adoptive cell immunotherapy with bortezomib

Validation of research trajectory 1 of an Exposome framework: Exposure to benzo(a)pyrene confers enhanced susceptibility to bacterial infection.

The exposome provides a framework for understanding elucidation of an uncharacterized molecular mechanism conferring enhanced susceptibility of macrophage membranes to bacterial infection after exposure to the environmental contaminant benzo(a)pyrene, [B(a)P]. The fundamental requirement in activation of macrophage effector functions is the binding of immunoglobulins to Fc receptors. FcγRIIa (CD32a), a member of the Fc family of immunoreceptors with low affinity for immunoglobulin G, has been reported to bind preferentially to IgG within lipid rafts. Previous research suggested that exposure to B(a)P suppressed macrophage effector functions but the molecular mechanisms remain elusive. The goal of this study was to elucidate the mechanism(s) of B(a)P-exposure induced suppression of macrophage function by examining the resultant effects of exposure-induced insult on CD32-lipid raft interactions in the regulation of IgG binding to CD32. The results demonstrate that exposure of macrophages to B(a)P alters lipid raft integrity by decreasing membrane cholesterol 25% while increasing CD32 into non-lipid raft fractions. This robust diminution in membrane cholesterol and 30% exclusion of CD32 from lipid rafts causes a significant reduction in CD32-mediated IgG binding to suppress essential macrophage effector functions. Such exposures across the lifespan would have the potential to induce immunosuppressive endophenotypes in vulnerable populations.

Abstract 647: Bortezomib enhances CD8+T Lymphocyte antitumor effector function: Potential mechanism(s) via notch regulation

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

Abstract 4161: Bortezomib enhances expression of effector molecules in antitumor CD8+ T lymphocytes by modulating Notch-NF-kB-miR-155 crosstalk

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.

Abstract 934: Tributyltin-induced dysregulation of inflammatory cytokine levels in human and mouse immune cells

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA 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.