Published OnlineFirst April 24, 2018; DOI: 10.1158/1078-0432.CCR-18-0041 Cancer Therapy: Preclinical Clinical Cancer Research Tryptophan Metabolism Contributes to Radiation- Induced Immune Checkpoint Reactivation in Glioblastoma Pravin Kesarwani1, Antony Prabhu1, Shiva Kant1, Praveen Kumar2, Stewart F. Graham2, Katie L. Buelow1, George D. Wilson1, C. Ryan Miller3, and Prakash Chinnaiyan1,4 Abstract Purpose: Immune checkpoint inhibitors designed to revert tryptophan metabolism as a metabolic node specifictothe tumor-induced immunosuppression have emerged as potent mesenchymal and classical subtypes of glioblastoma. GDC- anticancer therapies. Tryptophan metabolism represents an 0919 demonstrated potent inhibition of this node and effec- immune checkpoint, and targeting this pathway's rate-limiting tively crossed the blood–brain barrier. Although GDC-0919 as enzyme IDO1 is actively being investigated clinically. Here, we a single agent did not demonstrate antitumor activity, it had a studied the intermediary metabolism of tryptophan metabolism strong potential for enhancing RT response in glioblastoma, in glioblastoma and evaluated the activity of the IDO1 inhibitor which was further augmented with a hypofractionated regimen. GDC-0919, both alone and in combination with radiation (RT). RT response in glioblastoma involves immune stimulation, Experimental Design: LC/GC-MS and expression profiling was reflected by increases in activated and cytotoxic T cells, which performed for metabolomic and genomic analyses of patient- was balanced by immune checkpoint reactivation, reflected by derived glioma. Immunocompetent mice were injected orthoto- an increase in IDO1 expression and regulatory T cells (Treg). pically with genetically engineered murine glioma cells and GDC-0919 mitigated RT-induced Tregs and enhanced T-cell treated with GDC-0919 alone or combined with RT. Flow cyto- activation. metry was performed on isolated tumors to determine immune Conclusions: Tryptophan metabolism represents a metabolic consequences of individual treatments. node in glioblastoma, and combining RT with IDO1 inhibition Results: Integrated cross-platform analyses coupling global enhances therapeutic response by mitigating RT-induced immu- metabolomic and gene expression profiling identified aberrant nosuppression. Clin Cancer Res; 1–12. Ó2018 AACR. Introduction failed. Therefore, developing innovative treatment strategies to improve outcome in glioblastoma is of clear importance (2). Glioblastoma is the most common adult primary brain tumor Recent clinical advancements using immune checkpoint inhi- (1). Despite continued advances in surgery and combined che- bitors designed to target tumor-mediated immune tolerance have moradiotherapy, clinical outcomes remain poor. An overwhelm- revolutionized our approach to cancer therapy and offers strong ing majority of tumors recur within a year of definitive therapy, promise in glioblastoma. Immune tolerance is important in and a very small percentage of patients survive beyond 3 years of normal physiology to prevent overreactivity of stimulated diagnosis. Unfortunately, concerted efforts to improve clinical immune responses to external pathogens and other stimuli. outcomes, including integration of molecularly targeted agents, Dysfunction of these immune response "brakes" may lead to a angiogenesis inhibitors, and vaccines to standard therapy, have all variety of disorders, including autoimmune diseases, type I dia- betes, inflammatory bowel disease, asthma, and allergies (3). Tumors have coopted multiple mechanisms to activate these "brakes" by inducing immunosuppressive signaling pathways, 1Department of Radiation Oncology, Beaumont Health, Royal Oak, Michigan. 2Metabolomics and Obstetrics/Gynecology, Beaumont Research Institute, creating a tolerogenic microenvironment allowing evasion of the Beaumont Health, Royal Oak, Michigan. 3Department of Pathology & Laboratory host immune response despite the presence of recognizable Medicine, Neurology, & Pharmacology, Lineberger Comprehensive Cancer Cen- antigens. Cytotoxic T lymphocyte–associated protein 4 (CTLA- ter and Neurosciences Center, University of North Carolina School of Medicine, 4) and programmed death-1 (PD-1), which negatively regulate 4 Chapel Hill, North Carolina. Oakland University William Beaumont School of T-cell activation, represent two specific immune checkpoints that Medicine, Royal Oak, Michigan. have received recent attention, with inhibitors targeting these Note: Supplementary data for this article are available at Clinical Cancer immune pathways demonstrating unprecedented clinical activity Research Online (http://clincancerres.aacrjournals.org/). in a variety of solid tumors (4, 5). Corresponding Author: Prakash Chinnaiyan, Beaumont Health System, 3811 The central nervous system (CNS) has been previously consid- West Thirteen Mile Road, Royal Oak, MI 48073. Phone: 248-551-4918; Fax: 248- ered an immune privileged site; however, growing data support a 551-1002; E-mail: [email protected] dynamic interaction between the CNS and the systemic immune doi: 10.1158/1078-0432.CCR-18-0041 system. Although the CNS lacks a traditional lymphatic system, Ó2018 American Association for Cancer Research. recent findings identified a rich lymphatic network in the dura www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst April 24, 2018; DOI: 10.1158/1078-0432.CCR-18-0041 Kesarwani et al. AHRs are also responsible for providing a tolerogenic pheno- Translational Relevance type in dendritic cells, resulting in increased production of Through integrative, cross-platform analyses coupling glob- Tregs and reduced type 1 Th1 cells (17, 18). Activation of AHRs al metabolomic profiling with gene expression arrays in over also results in the induction of various cytokines, such as TGFb, þ 100 patient-derived tumors, we identified aberrant tryptophan IL6, and IL1b, that are responsible for converting CD4 T cells metabolism as an important metabolic node and immune to inducible Tregs (iTreg) and maintaining the suppressive checkpoint in glioblastoma. We discovered that intermediar- ability of MDSCs (14, 19, 20). ies associated with tryptophan metabolism demonstrated Interestingly, a variety of tumors, including glioblastoma, have >90% accuracy in discriminating between low- and high-grade evolved mechanisms to coopt this potent mode of immune glioma, offering novel diagnostic implications. We then tolerance to evade the host immune system (21). This relationship extended these findings preclinically using a novel, immuno- between cancer and elevated tryptophan catabolism was first competent, genetically engineered mouse model of adult recognized in the 1950s with the identification of its metabolic astrocytoma with a potent, clinically relevant inhibitor of intermediaries in the urine of cancer patients (22, 23). The IDO1 tryptophan metabolism/IDO1. Through these studies, we pathway was later proposed to play a more direct role in tumor uncovered a potent synergy when combining an IDO1 inhib- immune evasion, demonstrating a more robust T-cell response itor with hypofractionated radiation and a novel mechanism and delayed growth in vivo following pathway inhibition (24). linking tryptophan metabolism with radiation-induced Several recent studies have demonstrated that tryptophan catab- immunosuppression involving immune checkpoint reactiva- olism, through IDO1 and/or TDO upregulation, is particularly tion. These encouraging findings support future clinical efforts relevant in glioblastoma. TDO-mediated pathway activation in a designed to combine IDO1 inhibition with hypofractionated panel of glioma cell lines resulted in inhibition of T-cell prolif- radiation in glioblastoma, offering the promise of harnessing a eration, and modulating this pathway influenced tumor growth patient's immune system to attack these otherwise recalcitrant (14). Wainwright and colleagues demonstrated significant IDO1 tumors. expression in glioblastoma that promoted an immunosuppres- sive environment through recruitment of Tregs (15) and went on to show that the therapeutic efficacy of IDO1 inhibition can be significantly enhanced when combined with other immune mater that is able to absorb and transport CSF into deep cervical checkpoint inhibitors (25). lymph nodes where CNS antigens have been reported (6, 7). The majority of studies evaluating the immune consequences Furthermore, glioblastomas produce an array of chemokines, of aberrant tryptophan metabolism have largely focused on such as IL8, CCL2, CXCL12, that are able to recruit immunosup- expression of its rate-limiting enzymes rather than the individ- pressive tumor-associated macrophages (TAM) and myeloid- ual metabolites involved in immunosuppression. In this study, fi derived suppressive cells (MDSC), furthering the tolerogenic using global metabolomic pro ling as a framework, we explore tumor microenvironment (8, 9). This paradigm shift has contrib- this immunomodulatory pathway from a metabolic perspec- uted toward a growing interest in the evaluation of immunother- tive, further validating its relevance in glioblastoma, and eval- apeutic approaches in glioblastoma, although early studies have uate the antitumor potential of the potent IDO1 inhibitor demonstrated limited clinical activity when used alone or in GDC-0919 in a novel, immunocompetent preclinical model combination with bevacizumab (10). of adult glioblastoma.