Qijing Li, Ph.D. - US grants

One of the key features of a functioning immune system is its ability to distinguish antigens of foreign origin from those derived endogenously and to mount an immune response against the former. Antigen response and differentiation of CD4 T cells elicit and orchestrate the initial onset and progression of a wide range of autoimmune diseases. Understanding intrinsic regulations of CD4 T cell function has direct implications for the development of novel therapeutics to treat autoimmune diseases. While the protein-based signal transduction machinery downstream of T cell antigen recognition has been thoroughly studied, we have recently become aware of a novel and crucial element dictating T cell fate [unreadable] microRNA (miRNA). mir-17-92 is a gene cluster that encodes six different miRNAs, whose important roles in tumorigenesis have been well established, however, their impact on T cell regulation and associated molecular mechanisms are almost entirely unknown. Our preliminary studies indicated that this cluster dictates the T cell based autoimmune reaction through a complex mechanism. The objective of this project is to comprehensively dissect the function of mir-17-92 cluster during T cell antigen responses and differentiation. Timely and precise manipulation of these miRNAs should be an effective approach to suppress T cell responsiveness for immune tolerance. We aim to establish these miRNAs as targets for autoimmune therapy.

DESCRIPTION (provided by applicant): Regulatory mechanisms of miR-19b, a novel mediator of T cell autoimmunity: The inappropriate activation and differentiation of CD4 T cells elicits and orchestrates the onset and progression of a wide range of autoimmune diseases. Understanding CD4 T cells' intrinsic regulatory mechanisms has direct implications for the development of novel therapeutics to treat these diseases. While the protein-based signal transduction machinery downstream of T cell antigen recognition has been thoroughly studied, we have recently become aware of a novel and crucial element dictating T cell fate-microRNA (miRNA). mir-17-92 is a gene cluster encoding six different miRNAs, whose important tumor-cell-intrinsic roles in cancer have been well established. However, we have recently discovered that the mir-17-92 cluster also potentiates anti-tumor immunity in a T-cell-intrinsic manner. Furthermore, our preliminary studies indicate that mir-17-92 dictates the progression of CD4 T cell-mediated autoimmunity, principally through the activity of the cluster's mir-19b component. We aim to discover the molecular mechanism underpinning miR-19b's pro- autoimmune regulatory function, and thereby to establish miR-19b as a potential target for therapy of autoimmune diseases.

Summary of Work Malignant gliomas confer a dismal prognosis and have an inherent tendency to recur despite the most aggressive of therapies. Recently, significant progress has been made in both understanding the genetic foundation of these tumors as well as how to harness the power of the immune system to target these most devastating tumors. While there has been substantial evidence in support of using T cells to treat established tumors in mice and humans, the translational value of these treatments has been limited by the simplicity of glioma models used for their development: mostly relying on transplanted tumors that do not recapitulate the immune microenvironment present in human gliomas. To this end, this proposal seeks to develop a spontaneous and genetically faithful mouse glioma model by combining the initial IDH1R132H driver mutation with other most common mutations. These models will maintain the integrity of the immune microenvironment of the brain and best recapitulating human glioma. With these models, we will also assess the interplay between glioma development and the immune response as dictated by different genetic mutations. Our second aim is to identify IDH1R132H reactive T cell antigen receptors (TCRs). We aim to improve the current technology which employs single cell RNAseq to clone both the TCR? and TCR? chains. We will optimize the current cloning procedures to dramatically reduce the reagent expense and labor cost, which is essential for its clinical application. This aim will be extended into a translational direction, aim 3, through genetic engineering of T cells to redirect their antigen specificity against IDH1R132H. This aim is essentially a proof-of-principle study for TCR-T adoptive transfer immunotherapy in our immune-competent mouse glioma models. Combined, novel animal models and technologies developed in this study will help elucidate the complexities underlying glioma genetics and how these commonly mutated genes contribute to glioma development, progression, and glioma-specific immune suppression mechanisms. In addition, this will also facilitate the development and implementation a novel strategy for glioma immunotherapy.

Immune checkpoint blockade therapy has delivered unprecedented success in the treatment of melanoma and lung cancer. However, as exciting as this is, even with combined inhibition of PD-1 and CTLA4, only a portion of cancer patients were observed with objective responses and an even smaller percentage of them reached long term remission. Therefore, it is imperative to identify specific mechanisms that determine the efficacy of checkpoint targeting, and to develop novel therapeutic strategies synergizing with current treatments. Although the mechanism of checkpoint blockade resistance has not been precisely determined by changes in any novel biomarkers, a consensus has been reached that more favorable responses are observed in patients with T cell-inflamed ?hot? tumors. At the molecular level, the inflamed tumor microenvironment is characterized by activation of T and NK cells, effector molecules for cytolytic functions, chemokines for T and NK cell recruitment, type I interferons (IFN-I), and interferon-responsive genes. The anti-virus role of IFN-I was discovered decades ago, whereas its anti-tumor mechanism was more recently elucidated. The emerging scientific premise supports the hypothesis that there exists a plausible strategy to improve the immune ?readiness? of a tumor, and to overcome tumor resistance to checkpoint blockade therapy by elevating the level of intratumoral IFN-I. In this regard, our preliminary results show that inhibiting the expression of one epigenetic modifier, ubiquitin like with PHD and ring finger domains 1 (UHRF1), in lung cancer cells dramatically triggers IFN-I responses and ultimately intratumoral T cell accumulation. Surprisingly, UHRF1 deficient tumor cells also become resistant to IFN-I-induced PD-L1 surface expression. Furthermore, genetic deletion of UHRF1 impairs the proliferation and function of regulatory T cells (iTregs), a stromal cell population in the tumor mass that carries out an immunosuppressive function. Taken together, we hypothesize that targeting UHRF1 represents a comprehensive strategy to reverse immunosuppression in the tumor microenvironment. In this study, we will test this hypothesis by three specific aims. Aim 1 will determine molecular mechanisms through which tumoral UHRF1 remodels the tumor microenvironment. Aim 2 will determine the mechanism by which UHRF1 regulates T cell activation. Aim 3 will determine the pre-clinical efficacy of a therapeutic strategy combining UHRF1 suppression and PD-1 or CTLA-4 blockade against lung cancer. Since a small molecule UHRF1 inhibitor prototype has been developed, the success of this project will establish a novel and feasible target for tumor microenvironmental reprogramming, and lay a scientific foundation for combination therapy with checkpoint blockade against lung cancer.

Nerve infiltration has been implicated in the formation and progression of several solid tumor types including prostate, gastric, and pancreatic cancers. However, despite its neural origin, melanoma innervation has not been previously reported. In our preliminary studies, we discovered that melanoma tumor tissues from patient samples and mouse models are highly innervated. This innervation is dependent on tumor cell expression of nerve growth factor (NGF), as targeted NGF depletion eliminates intratumoral nerve fibers. Importantly, melanoma denervation via NGF knockdown or chemical sympathectomy dramatically reduces tumor burdens by remodeling the tumor microenvironment (TME). This TME reprogramming is associated with increased cytokine and chemokine expression, CD103+ DC activation, and CD8+ T cell recruitment, suggesting that NGF or nerve-derived neurotransmitters support tumor growth by suppressing antitumor immunity. Importantly, we confirmed this inverse correlation between tumor innervation and inflammation using clinical samples: melanomas expressing low levels of NGF are immunologically hot and associated with improved patient survival. These findings inspire our central hypothesis that NGF-mediated innervation of the tumor microenvironment can be exploited pharmacologically to reverse immunosuppression. In this study, we will test this hypothesis with the following three aims: Aim 1 will dissect molecular mechanisms of NGF-mediated remodeling of the intratumor immune microenvironment. Aim 2 will dissect molecular mechanisms by which NGF regulates T cell activation. Aim 3 will determine the pre-clinical efficacy of a therapeutic strategy combining NGF axis inhibition and immune checkpoint blockade against melanoma. Findings from the proposed studies will lay the foundation upon which potential combinational therapies can be developed to combat this disease.

Abstract Lung cancer is the most common cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases and is generally diagnosed at advanced stages, requiring multimodal therapy involving radiation, chemotherapy, and targeted therapies. Despite these medical interventions, the five- year survival rates of NSCLC patients are less than 5%, highlighting the need for innovative and more effective strategies to treat NSCLC. Dysregulation of cyclin-dependent kinases (CDKs), such as CDK4 and CDK6, occurs in 70% of NSCLC patients and results in aberrant cellular proliferation and tumorigenesis. Palbociclib (PD-03329, trade name Ibrance) is the first cyclin dependent kinase 4 and 6 inhibitor to be approved for breast cancer and is currently investigated as a monotherapy for other solid tumors, including NSCLC. While palbociclib has shown initial improvements in progression-free survival in a phase II clinical trial for recurrent or metastatic NSCLC patients, over half of patients either experience adverse effects or develop resistance and disease progression after eight weeks of treatment. Palbociclib achieves its therapeutic effect by arresting cells in G1 phase and promoting an irreversible cell cycle arrest known as cellular senescence. Senescence was initially thought to suppress tumorigenesis; however, growing evidence has suggested that senescent cells can paradoxically promote tumorigenesis and cancer relapse by altering the surrounding tumor microenvironment. The use of senolytic therapies to promote synthetic lethality may bypass the negative side effects of senescence and enhance the efficacy of palbociclib by either driving palbociclib-treated cells towards apoptosis rather than senescence. Through genetic screening, we identified thrombomodulin (THBD), a potent anticoagulant endothelial receptor, as a novel senolytic target for palbociclib-induced senescence. THBD-mediated signaling was upregulated during palbociclib-induced senescence in NSCLC cancer cell lines and served as a critical regulator of NSCLC cell fate and survival, as inhibition of THBD signaling in NSCLC cells attenuated senescence and promoted apoptosis. Importantly, inhibiting the activity of THBD downstream signaling by an FDA-approved drug caused senescent NSCLC cells to apoptose under treatment of palbociclib. Built on these findings, we propose two specific aims to fully investigate the mechanism by which THBD signaling mediates the senescent program induced by palbociclib and validate this pathway as a target to induce synthetic lethality in palbociclib- treated NSCLC cells both in vitro and in vivo for combinational therapy with the ultimate goal to develop preclinical and clinical trials to improve overall NSCLC patient outcome.

In brain tumors like glioblastoma (GBM), failures to develop an effective vaccine and achieve immune checkpoint inhibition have been attributed to the extraordinary antigenic intratumoral heterogeneity of this disease. To overcome this, successful immunotherapy for GBM will require antitumor T cells with increased magnitude and functionality (potency) and T cells targeting multiple antigens simultaneously (diversity). We have identified 3 strategies to accomplish these goals. First, we will confirm that conjoining neoantigen major histocompatibility complex class I (MHCI) epitope peptides with the universal tetanus P30 class II epitope markedly increases the potency of T cell responses and unveils T cells responses against MHC I antigens that are otherwise non-immunogenic, resulting in de novo immune responses capable of inducing antitumor efficacy. Second, we will administer P30 in the tumor microenvironment to stimulate P30-specific CD4+ T cell help. Help provided to CD8+ T cells at the tumor during the effector stage has been shown to improve the magnitude and persistence of CD8+ tumor infiltrating lymphocytes. Third, we will engage a novel, clinically-available checkpoint agonist ??CD27) and program cell death protein 1 (PD-1) blockade. Stimulating CD27 on antigen-engaged, CD4+ and CD8+ T cells increases the immunogenicity and memory of low-affinity CD8 epitopes, and improves the survival, effector function, and migratory capacity of activated T cells. However, as CD27 stimulation can cause expression of inhibitory PD-1 on T cells, we will also explore PD-1 blockade as a way of limiting this escape mechanism and further enhancing efficacy. We propose that multi-antigen P30-conjoined class I neoantigen vaccination with the novel checkpoint agonist ?CD27 and PD-1 blockade will increase the potency and diversity of neoantigen-specific CD8+ T cell responses, resulting in improved antitumor efficacy. Thus, despite a low mutational burden in GBM, our strategy should enable potent neoantigen-specific T cell responses against a breadth of targets to engender efficacy against heterogeneous tumor. Our Specific Aims are: 1. To determine if multi-antigen, conjoined neoantigen vaccination improves survival in mice with heterogeneous intracerebral glioma; 2. To determine if the addition of class II antigen at the tumor site improves efficacy in these tumors; 3. To determine if ?CD27, alone or in combination with PD-1 blockade, increases the potency and diversity of tumor-specific T cell responses and antitumor efficacy against heterogeneous tumors.