Jonathan Powell - US grants

DESCRIPTION (adapted from the applicant s abstract): Whether a particular antigen will induce a tolerogenic or immunogenic response is determined by the context in which the antigen is encountered rather than by the antigen itself. T cell clones that engage their T cell receptor (TCR) in the absence of co- stimulation not only fail to produce IL-2 and proliferate, but are hyporesponsive upon subsequent full re-challenge; they are anergic. Current data suggests that it is not co-stimulation that prevents the induction of anergy, but rather TCR engagement in the absence of IL-2 induced cell cycle progression. Rapamycin, which inhibits cell cycle progression in G1, induces anergy in the presence of TCR engagement and co-stimulation, whereas hydroxyurea, which inhibits the cell cycle in S phase does not induce anergy under similar conditions. It is hypothesized that TCR engagement leads to the up-regulation of the negative regulatory factors that mediate anergy, and that IL-2 induced cell cycle progression from G1 to S phase serves to inactivate/degrade these factors. By examining the cyclin-dependent cascade, this application seeks to define the biochemical events that take place between G1 and S phase that are responsible for determining the anergic/non- anergic fate of an activated T cell. Once identified, candidate genes will be over-expressed in T cell clones in order to determine their effect on T cell activation. Although the precise mediators of anergy have yet to be elucidated, one mechanism for the maintenance of anergy appears to be the formation of a transcriptional inhibitory complex at the IL-2 promoter. This complex, which binds to the 180 site of the IL-2 promoter, contains a cAMP- responsive element binding protein (CREB)/cAMP-responsive element modulator (CREM) heterodimer and appears to be facilitated by the binding of the high mobility group protein HMG (I) Y. It is hypothesized that the CREB/CREM complex mediates the inhibition of IL-2 by recruiting co-repressors to the IL- 2 promoter. Initial experiments will determine if the over-expression of p300, a co-activator of IL-2 transcription, has the ability to overcome this transcriptional repression. Second, experiments will be performed to try to identify these co-repressors using DNA affinity precipitation. In addition, the potential role of histone acetylation as well as histone deacetylase activity in mediating this repression will be explored. Ultimately, understanding the pathways that mediate T cell inhibition might help in the generation of more specific immunosupressive agents for preventing allograft rejection and treating autoimmunity, as well as designing agents that inhibit these pathways in order to promote tumor immunity.

The ultimate outcome of T cell receptor engagement (TCR) is dictated not by the antigen but rather by the context in which the antigen is encountered. T cells that recognize antigen presented by activated APCs in the context of costimulation are activated, while T cells that recognize antigen presented by resting APCs are rendered tolerant. Our lab is interested in dissecting the pathways involved in TCR-induced tolerance vs activation. This proposal will focus on the role of the transcription factors Egr-2 and Egr-3 in inducing T cell anergy as well as the co-repressor/coactivator molecule NAB2 in facilitating TCR induced activation. Using T cell clones we will examine the regulation of Egr-2 and Egr-3 in anergic and non-anergic cells. We will inducibly overexpress Egr-2 and Egr-3 in order to demonstrate that these factors are the cyclosporin sensitive component of T cell anergy. In addition, we will utilize T cells from Egr-2 and Egr-3 knockout mice in order to determine the affect of these factors on T cell activation and tolerance induction. In order to determine the effect of these factors in vivo we will adoptively transfer TCR transgenic T cells from Egr- knockout mice into animals which have a tumor expressing cognate antigen. Using this system we will assess the abilities of such cells to eradicate tumor as well as their susceptibility to tumor induced tolerance. NAB2, was originally discovered as a protein which binds to and can either co-repress or co-activate Egr mediated transcription. Indeed, we have generated preliminary data that NAB2 is upregulated by TCR engagement and that the overexpression of NAB2 leads to an increase in IL-2 promoter mediated transcription. In this proposal we will characterize the regulation of NAB2 in T cells as well as its effects on T cell activation and the induction of tolerance. Furthermore, we will assess the ability of T cells which overexpress NAB2 to eradicate tumor in vivo. Understanding the discreet pathways that lead to activation and tolerance should provide insight into devising specific clinical interventions. In the case of cancer immunotherapy, the goal is to inhibit tumor induced tolerance while at the same time enhance tumor specific T cell activation. On the other hand, in transplantation and autoimmunity, the objective is to inhibit T cell activation while at the same time promote TCR induced tolerance.

[unreadable] DESCRIPTION (provided by applicant): The adenosine A2a receptor has recently been shown to play a critical role in negatively regulating immune responses in vivo. Our laboratory has been interested in dissecting the signals that promote TCR-induced activation versus tolerance. During the course of our studies we have found that the adenosine A2a receptor is highly upregulated during the induction of T cell anergy. Based upon preliminary data we hypothesize that A2a receptor engagement on T cells promotes the induction of T cell tolerance. Using T cell clones in vitro we will define the ability of A2a receptor agonists to promote T cell tolerance even in the setting of costimulation. Using A2a knockout mice we will define the specificity of the A2a receptor in promoting tolerance and the role of endogenous adenosine in promoting tolerance. Further we will define the role of the A2a receptor on antigen presenting cells (APCs) and target tissues. Using an in vivo model of T cell mediated autoimmunity we will demonstrate the ability of A2a receptor engagement to prevent T cell mediated death by promoting anergy and antigen specific Lag-3+ T regulatory cells. Interestingly, the tumor microenvironment contains high levels of adenosine. As such, we propose that tumor-derived adenosine facilitates the induction of tumor-specific T cell tolerance. We will test this hypothesis using A2a specific antagonists and T cells from the A2a knockout mice in a well defined murine model of prostate cancer. We predict that by inhibiting A2a receptor engagement, we will be able to prevent/overcome tumor-induced tolerance and thus enhance the efficacy of tumor vaccines. We will also define the mechanism of A2a-induced tolerance. Previously, we have shown that the binding of CREB and CREM to the -180 site of the IL-2 promoter plays an important role in repressing IL-2 transcription in anergic T cells. In as much as A2a engagement leads to the generation of cAMP, we will test the hypothesis that A2a engagement mediates its inhibitory effect in part by enhancing the binding of the CREB/CREM at this site. Furthermore, based on preliminary data we will test the hypothesis that the novel cAMP activated target EPAC is a critical downstream effector. Understanding the role and mechanism by which the A2a receptor promotes T cell tolerance should provide insight in terms of devising specific clinical targets. [unreadable] [unreadable] [unreadable]

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? DESCRIPTION (provided by applicant): During the first 5 years of this proposal, we have been able to define a central role for mTOR in regulating T cell activation, differentiation and function. Our studies have led to the development of a novel paradigm whereby mTOR integrates signals from the immune microenvironment to regulate the outcome of TCR recognition. Selective deletion of mTOR in T cells prevents the generation of Th1, Th2, Th17 effector T cells under normally activating conditions. In contrast, our studies reveal that the default outcome for antigen recognition in the absence of mTOR activity is to that of Foxp3+ regulatory T cells. mTOR signals via two complexes: mTOR Complex I (mTORC1) which is activated by the small GTPase Rheb and contains the adaptor protein Raptor and mTORC2 which contains the adaptor Rictor. We selectively deleted mTORC1 activity by creating Rheb-/- T cells. Such cells failed to differentiate into Th1 and Th17 cells but readily become Th2 cells. Alternatively, Rictor-/- T cells readily differentiate into Th1 and Th17 cells but fail to become T2 cells. Additionally, we have been able to show that the AGC kinase Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a downstream target of mTORC2 that reciprocally enhances Th2 differentiation will inhibiting Th1 differentiation. In parallel, our studies have revealed that mTORC1 is necessary for CD8+ effector T cell generation and function, while the inhibition of mTORC2 selectively enhances the generation of CD8+ memory T cells. In this proposal we seek to further understand the mechanisms by which mTOR regulates the outcome of TCR engagement: In Aim 1 We will define the mechanisms by which mTORC1 and mTORC2 coordinate metabolism and T cell differentiation and function. Specifically we will define distinct metabolic programs from Th1 and Th2 cells. In doing so we will reveal novel metabolic functions for GATA-3 as well as a novel role for regulating T cell function for the ribonucleoprotein YB-1. In Aim 2 we will demonstrate a role for SGK1 in regulating CD8+ memory T cell development through the selective phosphorylation of Foxo1. In Aim 3 We will test the hypothesis that TCR-induced influx of leucine leads to the recruitment of mTOR to the endosome by the Ragulator complex. Furthermore, we will test the hypothesis that the asymmetric partitioning of these signaling components can promote the generation of 2 daughter cells with distinct phenotypic and metabolic properties. Overall, by studying the molecules downstream of mTOR signaling we hope to identify novel targets for treating autoimmune disease, preventing transplantation rejection and enhance vaccines and immunity to infectious diseases and tumors.

In an effort to identify factors responsible for the maintenance of the anergic phenotype we employed a novel statistical algorithm, HAM (Hypothesis based Analysis of Microarrays) to interrogate a data base consisting of T cell stimulated under conditions that either promote full T cell activation or anergy. Among the genes identified by this approach was Sprouty 1 a member of an evolutionarily conserved family of inducible inhibitors that has been mostly studied in the field of Development. In particular Sprouty 1 has been implicated in negative feedback loops that impart spatial temporal constraints to intracellular signals. In this project we will dissect the mechanism by which Sprouty 1 inhibits T cell activation and maintains the anergic state. In Aim I we will determine the mechanisms governing the expression of Sprouty 1 and the precise biochemical mechanisms by which Sprouty 1 inhibits TCR-induced signaling. In Aim II, in collaboration with Project 2, we will determine how the structure and intracellular trafficking of Sprouty 1 dictates its inhibitory function. In Aim III using conditional Sprouty 1 null mice we will determine the role of Sprouty 1 in regulating viral, "self and tumor immune responses in vivo. Overall dissecting the mechanisms by which Sprouty 1 inhibits T cell function should provide important insight in terms of identifying novel pharmacologic targets. For example in autoimmune diseases and transplantation, the goal would be to inhibit T cell activation but leave Sprouty 1 signaling intact. Alternatively, for tumor immunity, developing strategies to inhibit Sprouty 1 function and thus enhance the anti-tumor response.

DESCRIPTION (provided by applicant): Nonmyeloablative bone marrow transplant offers a safe, potentially curative treatment for non-malignant hematological diseases such as sickle cell anemia. Unfortunately, successful nonmyeloablative transplant to treat sickle cell anemia has been limited due to immune-mediated graft rejection. Our research has demonstrated that rapamycin can promote regulatory T cell (Treg) differentiation of naive T cells and anergy of Th1 cells following T cell activation. We used these observations to develop a novel preparative regimen to inhibit rejection and graft versus host disease (GVHD) by promoting T cell tolerance. Our strategy reduces the frequency of alloreactive T cells with alemtuzumab, creates space for engraftment with low dose total body irradiation, and allows lymphocyte recovery under extended rapamycin treatment. In the matched sibling setting this approach has had great success, resulting in stable mixed chimerism that corrects their hematological phenotype of sickle cell anemia and reverses pulmonary hypertension. Based on this success, a second clinical trial was developed to expand the potential donor pool to include haploidentical related donors, greatly increasing potential availability of this therapy to patients. The new trial employs the same fundamental principles in the choice of preparative regimen with the addition of dose escalation of post transplant cyclophosphamide to further reduce alloreactive T cells that could contribute to rejection or GVHD. Peripheral blood samples from patients and donors on this trial will provide a unique opportunity to systematically investigate the role of T cell tolerance in promoting stable chimerism. We propose to do this by examination of mixed lymphocyte reactions (MLRs) and intracellular cytokine staining (ICS) from samples obtained pretransplant, posttransplant on rapamycin, and posttransplant after completion of rapamycin therapy. We will determine whether the continued presence of rapamycin is necessary to suppress allogeneic responses in vitro and whether the tolerance measured in the MLR is dependent on the presence of Tregs. We will test whether addition of rapamycin or Tregs is able to suppress the MLR from a patient who develops graft rejection or GVHD while receiving rapamycin. We will determine if clinical resistance to rapamycin in the form of rejection or GVHD corresponds to biochemical resistance to rapamycin at the level of mTOR target phosphorylation and whether a novel mTOR kinase inhibitor can overcome such biochemical resistance in vitro. A final aim is to determine whether prolonged mTOR inhibition interferes with antigen specific T cell cytokine production or leads to generation of antigen specific Tregs to clinically relevant CMV or influenza A. We believe that a better understanding of the immunologic consequences of mTOR inhibition will result in safer and more successful bone marrow transplantation, allowing expansion of this potentially curative therapy to a wider number of patients with chronic hematologic illnesses. PUBLIC HEALTH RELEVANCE (provided by applicant): Sickle cell anemia and other chronic anemias impose a huge burden of pain and suffering for patients and large health care costs related to hospitalizations and chronic transfusion/chelation therapy. In this proposal we seek to define the operative cellular and biochemical processes that promote T cell tolerance in a novel protocol to cure sickle cell disease with non-myeloablative bone marrow transplantation. It is hoped that the results of this study will allow safer application of this curative therapy to a greater number of patients and aid in improving tolerogenic therapy for autoimmunity and solid organ transplantation as well.

Every 3 minutes approximately one person in the United States is diagnosed with a blood cell cancer with an estimated 171,550 new cases in 2016. A projected 1,237,824 people are either living with the disease, or are in remission. In spite of the fact there are a number of approved therapies, the American Cancer Society estimates there will be almost 58,000 deaths this year alone. While a number of hematologic malignancies are cured using cytotoxic chemotherapy in younger patients (e.g. Hodgkins Disease, Acute Lymphocytic Leukemia, Diffuse Large Cell Lymphoma, Burkitt's lymphoma), the more intense side effects in older patients results in less sanguine outcomes. Thus an alternative approach is not only needed in general, but particularly desirable in older patients. We and others have shown that the transcription factor proto-oncogene c-Myc drives certain cancers to change their energy metabolic requirements, and become ?glutamine addicted? for their growth and survival. Lymphoma is a clear example of such a cancer. The glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) broadly blocks glutamine utilizing reactions critical for the synthesis of nucleic acids, proteins and the generation of alpha-ketoglutarate for energy metabolism. DON has shown robust efficacy in both lymphoma animal models and exploratory clinical studies, but its development was halted due to marked dose-limiting toxicities, many of which were gastrointestinal (GI)- related, as the GI system is highly dependent on glutamine utilization. We hypothesized that a novel cell- directed prodrug of DON which could deliver the drug selectively to the lymphoid cells would permit significant dose reduction, greatly alleviating the adverse events. The feasibility of this approach is supported by the recent success of Gilead's lymphoid cell-targeted prodrug of the antiviral agent tenofovir, called tenofovir alafenamide (TAF), which in Ph 3 clinical trials provided similar efficacy with a 30-fold dose reduction and less toxicity. By exploiting a similar concept yet taking a unique molecular design strategy, we have identified an initial lead DON prodrug, JHU-083, which preferentially delivers 30-fold more DON to peripheral blood mononuclear cells (PBMCs) versus human plasma, and exhibits similar efficacy to DON with substantially reduced toxicity in murine lymphoma models. Findings from a tissue distribution/tolerability study in swine confirms the PBMC targeting of the prodrug. In head-to-head comparison versus equimolar DON, the DON prodrug showed enhanced DON delivery to PBMCs and reduced delivery to GI tissues resulting in less GI pathology and fewer clinical symptoms. Although promising, JHU-083 is not ideal for translation as it exhibits high clearance. Thus, our main drug discovery focus will be to create novel DON prodrugs which remain intact in plasma and microsomes, such that their lymphocyte delivery of DON can be sustained. In this grant, two PIs with complimentary expertise will design novel DON prodrugs with optimized pharmacokinetic parameters and characterize their efficacy/ toxicity profiles in lymphoma mouse models. At the completion of these studies we will have developed a novel, robust and safe inhibitor of glutamine metabolism. Our studies will lay the ground work for the rapid introduction of such compounds into clinical trials.

In order to sustain their prodigious anabolic needs tumors employ a specialized metabolism that differs from untransformed somatic cells. This metabolism leads to a tumor microenvironment (TME) that is acidic, hypoxic and depleted of critical nutrients required by immune cells. In this context tumor metabolism itself is a checkpoint that inhibits immune mediated tumor destruction. It stands to reason that targeting tumor metabolism might represent a potent means of ?Conditioning? tumors for killing by immunotherapy. Glutamine is essential for the aggressive growth characteristics of tumors. In collaboration with Dr. Barbara Slusher, head of the Johns Hopkins Drug Discovery Program, we have developed a novel agent to inhibit glutamine metabolism. The drug, JHU-083, is a pro-drug of the glutamine antagonist 6-diazo-5-oxo-l- norleucine (DON). DON blocks glutamine-utilizing reactions critical for the synthesis of nucleic acids, proteins and the generation of alpha-ketoglutarate. As a prodrug of DON, JHU-083 itself is inactive and is then converted in plasma by plasma esterases and intracellularly by cathepsin which are enriched in tumors compared to normal cells. While DON can cause gut and bone marrow toxicity, JHU-083 at similar doses avoids these side effects because its conversion to DON is enriched in the tumor. In this proposal, using JHU-083 as a tool to inhibit tumor metabolism, we will test the hypothesis that inhibiting tumor glutamine metabolism will not only inhibit tumor growth but will render tumors more susceptible to killing by immunotherapy. Our preliminary data demonstrate that treatment of tumors with JHU-083 changes the TME such that it is less acidic, less hypoxic and more replete with nutrients leading to enhanced endogenous anti-tumor responses and markedly enhancing the efficacy of immunotherapy in the form of checkpoint blockade, ACT and A2aR blockade. Furthermore, our preliminary studies demonstrate at the doses employed to inhibit tumor growth and change the TME, JHU-083 also acts on TILs to inhibit exhaustion and oxidative stress and enhance productive activation and memory generation. Based on these preliminary studies we will pursue the following specific Aims: 1. We will test the hypothesis that targeting tumor glutamine metabolism will change the tumor microenvironment (TME) such that the tumor will be more susceptible to immune mediated destruction. 2. We will test the hypothesize that treating mice with JHU-083 will enhance the efficacy of immunotherapy in the form of checkpoint blockade and ACT. 3. We will test the hypothesis that inhibition of tumor glutamine metabolism with JHU-083 will enhance the efficacy of immunotherapy in the form of A2Ar antagonism. At the completion of these studies we will have defined a novel approach to enhance anti-tumor immunotherapy. Furthermore, our studies will provide important insight into the role of tumor metabolism as a hurdle to the efficacy of immunotherapy. Finally, our findings provide the preclinical rationale for the development of DON prodrugs as a novel means of treating cancer.

The ultimate goal of immune-engineering is to harness the exquisite specificity of the immune system to prevent and treat disease. However, it is becoming increasingly clear that cellular metabolic programming is not merely a consequence of immune cell activation but rather an integral component of promoting differentiation and function. The generation of cells with robust effector function is intimately dependent upon the generation of nucleic acid, lipid and protein substrates, and energy. Likewise, persistence or the induction of immunologic ?memory? is dependent upon metabolic reprogramming that fuels long term survival. In this context successful engineering of immune cells will entail modulation, programming and even reprogramming of energy and substrate-generating metabolic programs. Initially based on our studies dissecting the role of the mTOR pathway in regulating immune responses our lab has defined specific nodes/targets to regulate metabolic programming necessary to fuel effector function and long term persistence. TR&D 3 seeks to exploit these findings to target these critical nodes in order to engineer metabolic reprogramming of cells, thus maximizing function and persistence. To this end we will pursue the following Aims: Aim 1. Employing synthetic biology, genetically egineer cells with enhanced mTORC1 activity by knocking down/out/mutating TSC2, leading to enhanced effector function characterized by more robust glycolytic reprogramming. Deliverable: The creation of a reprogrammed ?stock? effector cell that can then be modified for an array of cellular therapies. Aim 2. By regulating glutamine metabolism, formulate growth and differentiation conditions that promote the generation of cells epigenetically programmed to persist when adoptively transferred in vivo and to maximally respond upon rechallenge. Deliverable: Metabolically optimized media for producing robust and persistent effector cells. Aim 3. Based on novel findings regarding the ability of SGK1 to promote both a memory and effector T cell phenotype, develop small molecule inhibitors of SGK1 that can metabolically reprogram T cells both ex vivo and in vivo. Deliverable: Small molecule inhibitors to enhance efficacy of Adoptive Cellular Therapy.

PROJECT SUMMARY mTOR plays a critical role in integrating signals from the immune microenvironment to regulate T cell activation, differentiation and function. We have been able to demonstrate that the Tuberous Sclerosis Complex 2 (TSC2) protein plays an important role in regulating mTORC1 activation in T cells. TSC2 is a RasGap protein that inhibits the activity of Rheb GTPase that in turn activates mTORC1. We have shown that genetic deletion of TSC2 in T cells leads to enhanced mTORC1 activity and a marked increase in CD8+ T cell effector function. However, while TSC2-/- T cells respond robustly to viruses and tumors, their persistent mTORC1 activity leads to a decrease in memory CD8+ T cell generation. Recently, the Kass lab has identified a novel phosphorylation site on TSC2 that regulates mTORC1 activity in cardiac myocytes. Phosphorylation of this site (S1365) leads to the inhibition of mTORC1 signaling. Mutating this site(S?A) leads to an increase mTORC1 activity and the development of worse heart disease and mortality from pressure-overload (PO) stress. Alternatively, creating a phosphomimetic (S?E) at this site mitigates mTORC1 activity and imparts protection from heart failure upon pressure overload. We hypothesized that the TSC2 (S1365) site might play an important role in regulating mTORC1 activity in T cells. Our preliminary studies demonstrate that upon TCR engagement this site is indeed phosphorylated. T cells harboring the SA mutation have unaltered mTORC1 activity in the non-stimulated condition (unlike TSC2-/- T cells), but show markedly increased activity upon TCR engagement. T cells with the SE mutation exhibit the opposite. Furthermore, phosphorylation of TSC2 (S1365) is markedly induced by hypoxia, low pH and reactive oxygen species suggesting that this pathway plays a critical role in integrating stress signals in order to regulate T cell differentiation and function. In this project we seek to define and understand a novel and selective mechanism of mTORC1 regulation in T cells. The overall goal of this proposal is to dissect the mechanisms by which phosphorylation of TSC2 at S1365 regulates mTORC1 activation in T cells, and consequently selectively regulates T cell activation, differentiation and function. Upon the completion of this proposal our findings will help elucidate novel and critical mTORC1 regulatory signaling mechanisms in T cells, and have implications for developing vaccines and engineering more robust T cells for Adoptive Cellular Therapy. This may in turn result in improved treatment strategies for preventing and treating infections as well as cancer.