TryptophanMetabolismContributestoRadiation- Induced …aberrant tryptophan metabolism as a metabolic...

Translational Cancer Mechanisms and Therapy

TryptophanMetabolismContributes to Radiation-Induced Immune Checkpoint Reactivation inGlioblastomaPravin Kesarwani1, Antony Prabhu1, Shiva Kant1, Praveen Kumar2,Stewart F. Graham2, Katie L. Buelow1, George D.Wilson1, C. Ryan Miller3, andPrakash Chinnaiyan1,4

Abstract

Purpose: Immune checkpoint inhibitors designed to reverttumor-induced immunosuppression have emerged as potentanticancer therapies. Tryptophan metabolism represents animmune checkpoint, and targeting this pathway's rate-limitingenzyme IDO1 is actively being investigated clinically. Here, westudied the intermediary metabolism of tryptophan metabo-lism in glioblastoma and evaluated the activity of the IDO1inhibitor GDC-0919, both alone and in combination withradiation (RT).

Experimental Design: LC/GC-MS and expression profilingwas performed for metabolomic and genomic analyses ofpatient-derived glioma. Immunocompetent mice wereinjected orthotopically with genetically engineered murineglioma cells and treated with GDC-0919 alone or combinedwith RT. Flow cytometry was performed on isolated tumors todetermine immune consequences of individual treatments.

Results: Integrated cross-platform analyses coupling glob-al metabolomic and gene expression profiling identified

aberrant tryptophan metabolism as a metabolic node spe-cific to the mesenchymal and classical subtypes of glioblas-toma. GDC-0919 demonstrated potent inhibition of thisnode and effectively crossed the blood–brain barrier.Although GDC-0919 as a single agent did not demonstrateantitumor activity, it had a strong potential for enhancingRT response in glioblastoma, which was further augmentedwith a hypofractionated regimen. RT response in glioblas-toma involves immune stimulation, reflected by increases inactivated and cytotoxic T cells, which was balanced byimmune checkpoint reactivation, reflected by an increasein IDO1 expression and regulatory T cells (Treg). GDC-0919mitigated RT-induced Tregs and enhanced T-cell activation.

Conclusions: Tryptophan metabolism represents a meta-bolic node in glioblastoma, and combining RT with IDO1inhibition enhances therapeutic response by mitigating RT-induced immunosuppression. Clin Cancer Res; 24(15); 3632–43.�2018 AACR.

IntroductionGlioblastoma is the most common adult primary brain tumor

(1). Despite continued advances in surgery and combined che-moradiotherapy, clinical outcomes remain poor. An overwhelm-ing majority of tumors recur within a year of definitive therapy,and a very small percentage of patients survive beyond 3 years ofdiagnosis. Unfortunately, concerted efforts to improve clinicaloutcomes, including integration of molecularly targeted agents,angiogenesis inhibitors, and vaccines to standard therapy, have all

failed. Therefore, developing innovative treatment strategies toimprove outcome in glioblastoma is of clear importance (2).

Recent clinical advancements using immune checkpoint inhi-bitors designed to target tumor-mediated immune tolerance haverevolutionized our approach to cancer therapy and offers strongpromise in glioblastoma. Immune tolerance is important innormal physiology to prevent overreactivity of stimulatedimmune responses to external pathogens and other stimuli.Dysfunction of these immune response "brakes" may lead to avariety 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,creating a tolerogenic microenvironment allowing evasion of thehost immune response despite the presence of recognizableantigens. Cytotoxic T lymphocyte–associated protein 4 (CTLA-4) and programmed death-1 (PD-1), which negatively regulateT-cell activation, represent two specific immune checkpoints thathave received recent attention, with inhibitors targeting theseimmune pathways demonstrating unprecedented clinical activityin a variety of solid tumors (4, 5).

The central nervous system (CNS) has been previously consid-ered an immune privileged site; however, growing data support adynamic interaction between the CNS and the systemic immunesystem. Although the CNS lacks a traditional lymphatic system,recent findings identified a rich lymphatic network in the dura

1Department of Radiation Oncology, Beaumont Health, Royal Oak, Michigan.2Metabolomics and Obstetrics/Gynecology, Beaumont Research Institute,Beaumont Health, Royal Oak, Michigan. 3Department of Pathology & LaboratoryMedicine, Neurology, & Pharmacology, Lineberger Comprehensive Cancer Cen-ter and Neurosciences Center, University of North Carolina School of Medicine,Chapel Hill, North Carolina. 4Oakland University William Beaumont School ofMedicine, Royal Oak, Michigan.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Prakash Chinnaiyan, Beaumont Health System, 3811West Thirteen Mile Road, Royal Oak, MI 48073. Phone: 248-551-4918; Fax: 248-551-1002; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0041

�2018 American Association for Cancer Research.

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mater that is able to absorb and transport CSF into deep cervicallymph nodes where CNS antigens have been reported (6, 7).Furthermore, glioblastomas produce an array of chemokines,such as IL8, CCL2, CXCL12, that are able to recruit immunosup-pressive tumor-associated macrophages (TAM) and myeloid-derived suppressive cells (MDSC), furthering the tolerogenictumor microenvironment (8, 9). This paradigm shift has contrib-uted toward a growing interest in the evaluation of immunother-apeutic approaches in glioblastoma, although early studies havedemonstrated limited clinical activity when used alone or incombination with bevacizumab (10).

Emerging studies have identified multifaceted strategies bywhich alterations in tumor metabolism may also contribute toa potent tolerogenic immune environment, thereby representinga line of next-generation immune checkpoints (11, 12). One ofthemost clinically advanced with particular relevance to glioblas-toma is tryptophanmetabolism, whosemost notable physiologicrole has been attributed to peripheral immune tolerance and fetalprotection frommaternal immune rejection in the placenta (13).Tryptophan is metabolized to kynurenine by the rate-limitingenzymes indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan2,3-dioxygenase (TDO; refs. 14, 15) contributing toward animmune tolerant environment at many levels. Increased metab-olism of tryptophan results in depletion of this critical metabolitein the microenvironment, resulting in cell-cycle arrest and/oranergy in effector T cells, while simultaneously fostering matu-ration and activation of regulatory T cells (Treg; ref. 16). Inaddition to this passive consequence of tryptophan metabolism,the resulting kynurenine is exported from the cell into the micro-environment, where it has the capacity to balance regulatory andeffector responses of the immune system. This includes bindingand activation of aryl hydrocarbon receptors (AHR), a cyto-plasmic transcription factor, resulting in reduced proliferationand infiltration of effector T cells (14). Kynurenine-activated

AHRs are also responsible for providing a tolerogenic pheno-type in dendritic cells, resulting in increased production ofTregs and reduced type 1 Th1 cells (17, 18). Activation of AHRsalso results in the induction of various cytokines, such as TGFb,IL6, and IL1b, that are responsible for converting CD4þ T cellsto inducible Tregs (iTreg) and maintaining the suppressiveability of MDSCs (14, 19, 20).

Interestingly, a variety of tumors, including glioblastoma, haveevolved mechanisms to coopt this potent mode of immunetolerance to evade thehost immune system(21). This relationshipbetween cancer and elevated tryptophan catabolism was firstrecognized in the 1950s with the identification of its metabolicintermediaries in the urine of cancer patients (22, 23). The IDO1pathway was later proposed to play a more direct role in tumorimmune evasion, demonstrating a more robust T-cell responseand delayed growth in vivo following pathway inhibition (24).Several recent studies have demonstrated that tryptophan catab-olism, through IDO1 and/or TDO upregulation, is particularlyrelevant in glioblastoma. TDO-mediated pathway activation in apanel of glioma cell lines resulted in inhibition of T-cell prolif-eration, and modulating this pathway influenced tumor growth(14). Wainwright and colleagues demonstrated significant IDO1expression in glioblastoma that promoted an immunosuppres-sive environment through recruitment of Tregs (15) and went onto show that the therapeutic efficacy of IDO1 inhibition can besignificantly enhanced when combined with other immunecheckpoint inhibitors (25).

The majority of studies evaluating the immune consequencesof aberrant tryptophan metabolism have largely focused onexpression of its rate-limiting enzymes rather than the individ-ual metabolites involved in immunosuppression. In this study,using global metabolomic profiling as a framework, we explorethis immunomodulatory pathway from a metabolic perspec-tive, further validating its relevance in glioblastoma, and eval-uate the antitumor potential of the potent IDO1 inhibitorGDC-0919 in a novel, immunocompetent preclinical modelof adult glioblastoma.

Materials and MethodsStudy approval

Allmice were housed in the AAALAC-accredited Animal facilitylocated at William Beaumont Research Institute (Royal Oak, MI).All animal studies were carried out under protocols approved bythe Institutional Animal Care and Use Committee at WilliamBeaumont Research Institute. Patient-derived tumors wereobtained from the H. Lee Moffitt Cancer Center (Tampa, FL),following Institutional Review Board and Human Subjectsapproval received from the H. Lee Moffitt Cancer Center TissueCore Facility as described previously (26).

Human tumor samples and metabolomic profilingAll tumors used in this analysis were newly diagnosed. Tumors

were fresh-frozen and their integrity and histology confirmed by astaff pathologist before aliquoting samples (26). Metabolomicstudies were conducted at Metabolon Inc using methodsdescribed previously (26). For box plots, raw area count wasnormalized and rescaled to set themedian equal to one, followedby inputtingminimum tomissing values. Rescaled datawere usedto calculate fold change by comparing GBM with low-gradeglioma (LGG).

Translational Relevance

Through integrative, cross-platform analyses coupling glob-al metabolomic profiling with gene expression arrays in over100patient-derived tumors,we identified aberrant tryptophanmetabolism as an important metabolic node and immunecheckpoint in glioblastoma. We discovered that intermediar-ies associated with tryptophan metabolism demonstrated>90%accuracy in discriminating between low- andhigh-gradeglioma, offering novel diagnostic implications. We thenextended these findings preclinically using a novel, immuno-competent, genetically engineered mouse model of adultastrocytoma with a potent, clinically relevant inhibitor oftryptophan metabolism/IDO1. Through these studies, weuncovered a potent synergy when combining an IDO1 inhib-itor with hypofractionated radiation and a novel mechanismlinking tryptophan metabolism with radiation-inducedimmunosuppression involving immune checkpoint reactiva-tion. These encouraging findings support future clinical effortsdesigned to combine IDO1 inhibition with hypofractionatedradiation in glioblastoma, offering the promise of harnessing apatient's immune system to attack these otherwise recalcitranttumors.

Radiation and Tryptophan Metabolism in Glioblastoma

www.aacrjournals.org Clin Cancer Res; 24(15) August 1, 2018 3633




Microarray and database analysisFor details, see Supplementary Methods.

Cell culture and reagentsHuman glioblastoma cell lines U87, T98G, (obtained from

ATCC), and U251 (obtained from CLS Cell Lines ServiceGmbH) were cultured in Minimum Essential Media (MEM).Human glioblastoma neural stem cells (GNS) MES326 andPN19 were generated and cultured in DMEM-F12 media withGlutaMAX, heparin, and B27 supplement as described previ-ously (27). The genetically engineered murine GBM cell lineTRP was cultured in MEM (28–30). Primary cell culture ofmouse splenocyte (T cells) and murine GBM tumor linesGL261 (obtained from NCI DTP, DCTD tumor repository)was performed in RPMI1640. All cell culture reagents werepurchased from GIBCO/Thermo Fisher Scientific and CorningLife Sciences. Tryptophan, kynurenine, p-dimethylaminoben-zaldehyde, glacial acetic acid, TCA, and IDO1 inhibitors 1-L-methyl tryptophan (1-L-MT) were purchased from SigmaAldrich. GDC-0919 was provided by Genentech.

Western blot analysis, flow cytometry, magnetic sorting, andELISA

For details, see Supplementary Methods.

Metabolite extraction and targeted liquid chromatographymass spectrometry

For details, see Supplementary Methods.

Immunocompetent tumor modelsC57BL/6 (H-2b, CD45.2)micewere exposed to 4Gy total body

irradiation one day prior to implantation to help tumor engraft-ment. Mice were injected with 2� 105 TRP (murine GBM) tumorcells in the brain for intracranial tumor model or 2 � 106 TRPtumor cells in the right flank for subcutaneous tumormodel. MRIwas performed on 6 to 8 days after tumor implant. After verifi-cation of tumors,mice were randomized into described treatmentarms. Detailed procedure is provided in Supplementary Materialsand Methods.

T-cell suppression assayFor details, see Supplementary Methods.

Kynurenine estimationFor details, see Supplementary Methods.

Statistical analysisComparisons across two different groups were performed

on original data using two-tailed Student t test. A log-rank testwas used for survival analyses. Comparisons between tumorvolumes in the subcutaneous tumor model were performedusing two-way ANOVA in combination with post hoc opera-tions to generate P values between RT and RT þ GDC-0919.ROC curves were constructed using metabolomics data forkynurenine and kynurenine/kynurenine acid ratios. Highestconcentrations of kynurenine and kynurenine/kynurenineacid ratios in LGG were used as thresholds for predictingglioblastoma from LGG. All statistical analyses were per-formed using Origin Pro 2016 software (Origin LabCorporation).

ResultsMetabolomic profiling identifies aberrant tryptophanmetabolism in glioblastoma

Metabolomic profiling was performed in glioma using a robustlibrary of standards enriched with intermediates of tryptophanmetabolism, comparing patient-derived glioblastoma (n¼ 80) toLGG (n ¼ 28). Tryptophan levels were modestly elevated inglioblastoma (1.44-fold, P � 0.011; Fig. 1A). Tryptophan hasseveral metabolic fates independent of the IDO1/TDO2 pathway,including serving as amediator for the formation of serotonin andindoles (Supplementary Fig. S1A). Global metabolomic profilingidentified several indoles, which may serve as both catabolicbyproducts and anabolic precursors of tryptophan, elevated inglioblastoma (Supplementary Fig. S1B). Of the identified meta-bolites associatedwith tryptophanmetabolism, kynurenine dem-onstrated the highest accumulation in glioblastoma when com-pared with LGG (6.07-fold, P � 0.0002; Fig. 1A). Interestingly,kynurenate, a metabolite immediately downstream of kynure-nine, was significantly lower in glioblastoma when comparedwith LGG (0.23-fold, P � 0.0004), and the resulting kynurenine/kynurenate ratio demonstrated the highest degree of sensitivity(91.1%) and specificity (96.5%) in discriminating between glio-blastoma and LGG in comparison with using kynurenine alone(sensitivity 60.8%; specificity 91.6%; P < 0.00001; Fig. 1A; Sup-plementary Fig. S1C). Using the TCGA database, we then soughtto determine whether aberrant expression of enzymes involvedwith tryptophan metabolism recapitulated these metabolic find-ings in glioma. Both IDO1 and TDO2 were elevated in glioblas-tomawhen compared with LGG and consistent with the observedaccumulation of kynurenate in LGG, we identified increasedexpression of its biosynthetic enzyme kynurenine aminotransfer-ase I (KYAT1) in these tumors when compared with glioblastoma(Fig. 1B).

Tryptophan metabolism is linked to molecular subtypes ofglioblastoma

We next sought to determine whether aberrant tryptophanmetabolism was unique to a specific glioblastoma molecularsubtype. Transcriptional profiling has revealed considerable inter-tumoral heterogeneity in glioblastoma (31). These include theproneural and mesenchymal subtypes, which have been identi-fied most consistently in glioblastoma. Their transcriptional pro-files are mutually exclusive and can be applied to approximatelyone half of all tumors. The proneural subtype is characterized bymutations in isocitrate dehydrogenase 1 (IDH1), frequent altera-tions in expression of p53 and platelet-derived growth factorreceptor, alpha polypeptide (PDGFR-a), and a transcriptionalsignature typically present in LGG. In contrast, mesenchymalglioblastoma is characterized by mesenchymal gene expression,including CD44 and chitinase 3-like 1 (YKL40) and attributed tomore aggressive disease. Other subtypes include classical andneural, which are characterized by EGFR and the expression ofneuronalmarkers, respectively.Of the 80 glioblastoma specimensmetabolomically profiled, 56 had paired tissue available fortranscriptional profiling, allowing for cross-platform analysis.Following molecular subtyping using methods described previ-ously (31) we demonstrated that tryptophan and kynurenineaccumulationwas specific to classical andmesenchymal subtypes,whereas kynurenate accumulation, the metabolite elevated inLGG, was only evident in the proneural subtype. The

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kynurenine/kynurenate ratio was able to discriminate betweenindividual molecular subtypes, with highest ratios present in theclassical and mesenchymal subtypes when compared with neuraland proneural tumors (Fig. 1C). The expression levels of enzymesinvolved in tryptophanmetabolism corroborated thesemetabolicfindings, with higher levels of IDO1 and TDO2 in classical andmesenchymal subtypes, whereas neural and proneural subtypesdemonstrated elevated expression of KYAT1 (Fig. 1D). Using aunique data repository generated by the IvyGAP,which used lasercapture microdissection to isolate RNA from different structuralregions of individual tumors that was subsequently molecularlyprofiled using RNA-seq, we recently uncovered a relationshipbetween subtype heterogeneity in glioblastoma and its uniquetumor microenvironment. In this study, we demonstrated thatcells obtained from the infiltrating, leading edge of a tumor nearlyexclusively harbored a proneural signature, whereas the mesen-chymal subtype was observed in perinecrotic regions within anindividual tumor (32). On the basis of these findings, we soughtto determine whether aberrant tryptophan metabolism wasunique in specific regions within an individual tumor. Consistentwith our previousfindings, increased IDO1/TDO2 expressionwaspresent in the necrotic and central core of tumor when comparedwith the periphery, which corresponds to the mesenchymal/classical and proneural molecular subtypes, respectively (Fig. 1E).

The IDO1 pathway is activated in glioblastoma cell linesAs cross-platform analysis coupling metabolomics with tran-

scriptional profiling identified aberrant tryptophan metabolism

as a metabolic phenotype in glioblastoma, we extended investi-gations to preclinical models. IDO1, which is typically undetect-able in cells at baseline, is rapidly induced by proinflammatorystimuli. Because of the multiple IFN-stimulated response ele-ments in the IDO1 promoter region, in vitro treatment of culturedcells with the T effector cells and proinflammatory cytokine IFNgleads to robust expression of this enzyme (33). Accordingly,baseline expression of IDO1 was low/undetectable in establishedhuman glioblastoma cell lines and robustly expressed followingtreatment with IFNg (Fig. 2A). Similar findings were observedwhen extended to novel, molecular subtype-specific GNSs, withmore robust expression in the mesenchymal GNSs when com-pared with proneural, which is consistent with our findings frompatient-derived tumors. To determine whether IFNg-inducedexpressionof IDO1 corresponded to increased tryptophanmetab-olism, kynurenine levels were evaluated. Consistent withincreased IDO1 expression, IFNg induced accumulation of kynur-enine in the media of glioblastoma cell lines grown in culture,confirming pathway activation (Fig. 2B). TDO2, on the otherhand, was inconsistently expressed in cell lines and interestinglyappeared to be downregulated by IFNg in several of the linestested (Supplementary Fig. S2A).

To begin to explore the immune consequence of aberranttryptophan metabolism in glioblastoma, we extended investiga-tions to an adult astrocytic, genetically engineered mouse (GEM)cell line to allow for in vivo studies using an immunocompetentmodel. Specifically, primary astrocyte lines were generated from aseries of conditional GEM in which core glioblastoma pathways

Figure 1.

Aberrant tryptophan metabolism represents a metabolic node in glioblastoma. A, Metabolomic profiling performed on patient-derived LGG (n ¼ 28) andglioblastoma (n ¼ 80) demonstrates differential accumulation of tryptophan (TRP), kynurenine (KYN), kynurenate (KYNA), and KYN/KYNA ratio. B, Expressionanalysis of enzymes involved in tryptophan metabolism in LGG and glioblastoma was performed using The Cancer Genome Atlas (TCGA). C and D, Cross-platformanalysis was performed coupling metabolomic profiles with gene expression arrays in glioblastoma (n ¼ 56). The relative accumulation of intermediaries oftryptophan metabolism (C) and enzymes driving tryptophan metabolism (D) were evaluated in the context of the tumors' molecular subtype, classified as classical(CL),mesenchymal (M), neural (N), or proneural (PN). E, Expression of enzymes involved in tryptophanmetabolism in the context of their anatomic structure derivedfrom the Ivy GAP database. Regions were classified as periphery (leading edge and infiltrating tumor), cellular tumor, and necrotic core (perinecrotic andpseudopalisading necrosis). �� , P < 0.00005; �� , P < 0.0005; �� , P < 0.005; � , P < 0.05.






were genetically targeted (30). Briefly, after Cre-mediated recom-bination in vitro, lines express (i) a truncation mutant of SV40large T antigen (T) from the humanGfappromoter that inactivatesall 3 Rb family proteins; (ii) a constitutively active KrasG12Dmutant (R); and/or (iii) a homozygous Pten deletion (P). As aninitial investigation, we performedmetabolomic profiling of TRPcells grown orthotopically, which harbor all 3 of these mutationsanddisplay themost aggressive phenotype (30),which confirmedpathway activation with high levels of kynurenine when com-pared with normal brain (Fig. 2C). Although we were unable toinduce IDO1 expression and kynurenine production in the TRPline and the mouse glioblastoma cell line GL261 in vitro using avariety of methods, including both human and mouse IFNg(Supplementary Fig. S2B), expression was clearly evident intumors grown in vivo (Fig. 2D).

GDC-0919 demonstrates potent inhibition of tryptophanmetabolism in glioblastoma and achieves biologically relevantconcentrations in the brain

As we established aberrant tryptophan metabolism in patient-derived glioblastoma, which was recapitulated in preclinicalmodels, wenext sought to determine the potential formolecularlytargeting this immunomodulatory pathway using the potent and

direct small-molecule IDO1 inhibitor GDC-0919 (navoximod),which is actively being investigated in clinical trials (34). As aninitial investigation, we evaluated levels of kynurenine followinggraded concentrations of GDC-0919 in our human glioblastomacell lines in vitro. GDC-0919 demonstrated potent inhibition ofkynurenine production, with median effective dose (ED50) con-centrations ranging from 7 nmol/L to 1.77 mmol/L and nearcomplete inhibition at 5 mmol/L (Fig. 3A). As we were unableto induce IDO1 expression in vitro in our mouse glioblastomamodels, we implemented an ex vivo approach to evaluate thepotential of GDC-0919 to inhibit kynurenine production in theTRP line. In these studies, TRP tumors grown subcutaneously inC57BL/6 mice were excised and disaggregated in cell culturemedia using DNase I and collagenase IV. Dissociated tumorswere then passed through a 70-mm cell strainer to obtain auniform cell suspension. The cell suspension was treated withIFNg , and supernatant was collected after 24 hours for kynurenineestimation. Using this approach, we demonstrated that TRP cellsendogenously produced kynurenine ex vivo, which was signifi-cantly increased with IFNg . Kynurenine production wascompletely inhibited in TRP ex vivo by GDC-0919 (5 mmol/L).Interestingly, in a parallel study where mice were treated withGDC-0919 (200 mg/kg, twice a day for 3 days) in vivo prior to

Figure 2.

The IDO1 pathway is activated in glioblastoma cell lines. A, Human glioblastoma cell lines (T98G, U87, U251, MES326, and PN19) were cultured in the presence orabsence of 50 ng/mL of human IFNg for 3 days and evaluated by Western blot analysis. B, Human cell lines were cultured in 250 mmol/L tryptophan andpresence or absence of 50 ng/mL of IFNg . Media supernatant was collected after 3 days for kynurenine estimation using Ehrlich method. Data, mean � SD. C,Orthotopic TRP tumors grown in C57BL/6micewere analyzed for kynurenine using LC/MS and comparedwith normalmurine brain.D, IDO1 expressionwas analyzedin TRP tumors isolated from C57BL/6 mice using Western blot analysis. Results are representative of at least three independent experiments. � , P < 0.05.

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tumor excision, TRP cells demonstrated diminished productionof both baseline and IFNg-induced kynurenine (Fig. 3B).

Next, we performed pharmacokinetic/pharmacodynamic stud-ies to determine the potential of GDC-0919 to cross the blood–brain barrier (BBB) and achieve biologically relevant concentra-tions in the brain. Using LC-MS, we quantified GDC-0919 levelsin normal brain (with no tumor implanted), intracranial tumors,subcutaneous tumors, and plasma of mice following treatmentwith GDC-0919 (200 mg/kg twice a day) for 3 days, with tissuebeing extracted 2 hours after the last dose. Importantly, GDC-0919 achieved biologically relevant concentrations in intracranialtumors (�15 mmol/L), which was comparable with levelsachieved in subcutaneous tumors. Furthermore, GDC-0919achieved biologically relevant concentrations in the normal brain(�5 mmol/L), which is particularly important in glioblastoma,based on the infiltrative nature of this malignancy (Fig. 3C). Inaddition, tryptophan and kynurenine levels were quantified in

both intracranial tumors and plasma from mice treated with theabove-described schedule, confirmingdrug activitywith increasedlevels of tryptophan and decreased levels of kynurenine followingGDC-0919 treatment (Fig. 3D).

GDC-0919 enhances radiation response in glioblastomaNext, we determined the antitumor potential of IDO1 inhibi-

tion using GDC-0919 in glioblastoma. As an initial investigation,we evaluated for growth delay in TRP cells grown in a subcuta-neous xenograft model. As a single agent, GDC-0919 did notdemonstrate antitumor activity (Supplementary Fig. S3A). Thesefindings were recapitulated using a first-generation IDO1 inhib-itor 1-L-MT (Supplementary Fig. S3B). As radiotherapy (RT) is astandard treatment in glioblastoma and emerging data haveidentified a potential for synergy when combining RT withimmune checkpoint blockade, we went on to evaluate the poten-tial of GDC-0919 to enhance RT response. Continuing with the

Figure 3.

GDC-0919 demonstrates potent inhibition of tryptophan metabolism in glioblastoma and achieves biologically relevant concentrations in the brain. A, Human GBMcell lines (T98G, U87, U251, and MES326) were cultured with human IFNg (50 ng/mL) and of tryptophan (250 mmol/L) and graded concentrations of theIDO1 inhibitor GDC-0919. The supernatant was collected after 3 days and evaluated for kynurenine. Datawere plotted and used to calculate ED50 values for individualcell lines. B, Murine TRP tumors (in vitro and ex vivo tumors) isolated from C57BL/6 mice and C57BL/6 mice treated with GDC-0919 (200 mg/kg twice a dayfor 3 days given orally) were cultured in the presence and absence of mouse IFNg (100 ng/mL) and tryptophan (250 mmol/L). The supernatant was collected after 3days and analyzed for kynurenine. C and D, C57BL/6 mice with subcutaneous or intracranial TRP tumors were treated with GDC-0919 (200 mg/kg twice aday for 3 days given orally). Tissue and plasmawere isolated after 3 days of treatment and analyzed for GDC-0919 (C) and tryptophan and kynurenine (in intracranialtumors) using LC/MS (D). Results obtained in mg/kg were converted to mmol/L by assuming mg/mL ratio to be 1. Data, mean � SD. � , P < 0.05. A minimumof 4 tissue samples were used for each experiment.






subcutaneous TRP model, GDC-0919 alone again demonstratedno antitumor activity, however, when combined with RT (6 Gy�1), demonstrated a significant growth delay when compared withRT alone (P < 0.005; Fig. 4A). Next, to allow for translating thesefindings clinically, we extended studies to an orthotopic, intra-cranial TRP tumor model. MRI was performed using our small-animal imaging platform prior to initiating therapy to confirmboth presence of intracranial tumors and equal volumes betweentreatment arms (Supplementary Fig. S4A and S4B). Similar toresults in the subcutaneous model, IDO1 inhibition using GDC-0919 alone did not demonstrate antitumor activity; however, atrend toward improved survival when combined with RT (6 Gy�1; P ¼ 0.06; Fig. 4B) was evident. As fractionation has beendescribed to further potentiate the synergy between RT andimmune checkpoint agents, we extended studies using a hypo-fractionated regimen (6 Gy � 3). As demonstrated in Fig. 4C,

IDO1 inhibition demonstrated potent antitumor activity whencombined with fractionated RT, with approximately one third ofmice demonstrating complete regression of tumors confirmed byMRI (Supplementary Fig. S4C) and increased survival whencompared with fractionated RT alone (P < 0.005).

Tryptophan metabolism contributes to radiation-inducedimmune checkpoint reactivation in glioblastoma

We went on to evaluate immunologic factors underlying theinterface between tryptophan metabolism and radiationresponse. As an initial investigation, correlative studies wereperformed using the experimental design detailed in Fig. 4B,consisting of mice with intracranial tumors treated with vehiclecontrol, GDC-0919, single-fraction RT, or the combination. Micewere sacrificed 5 days following initiation of therapy, and flowcytometry was performed on macrodissected tumors to evaluate

Figure 4.

GDC-0919 enhances radiation response in glioblastoma. TRP tumors were grown subcutaneously (A) or intracranially (IC; B and C) in immunocompetent C57BL/6mice. MRI was obtained to confirm the establishment of intracranial tumors prior to the initiation of therapy. Following establishment of tumors, mice wererandomized to treatment as described in schema. GDC-0919 was given orally at a concentration of 200 mg/kg twice a day (6 days a week) for 2 to 3 weeks. Eachtreatment arm consisted of a minimum of 6 mice. �� , P < 0.005.

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for a panel of immunologic markers. Similar to above efficacystudies, GDC-0919 alone did not appear to modulate immuneresponse in orthotopic TRP tumors, demonstrating no changesin percentage of Tregs (CD4þFoxP3þCD25þ), MDSCs(CD11bþGr1þ), or TAMs (CD11bþF4/80þ; Fig. 5A). As GDC-0919 alone did not appear to influence the number of Tregsquantitatively in ourmodel,we extendedour studies to determinewhether IDO1/kynurenine upregulation could contribute towardTreg function using a CD8þ T-cell proliferation/suppressionassay. Briefly, na€�ve CD8þ T cells and Tregs (CD4þCD25þ T cells)were isolated from splenocytes, and CD8þ T cells were thenstained with cell proliferation dye (CFSE). These cells were thenactivated (using anti-CD3 and anti-CD28 antibodies) and cocul-tured in the presence or absence of Tregs, kynurenine, or thecombination, for 3 days. Interestingly, neither kynurenine norTregs alone appeared to suppress the proliferation ofCD8þT cells.However, kynurenine appeared to impart suppressiveness toTregs, as the combination led to diminished proliferation ofCD8þ T cells (Fig. 5B).

Interestingly, we found that RT alone appeared to contributetoward an immunosuppressive environment in glioblastoma,including over a 50% increase in Tregs (from 14.7% to 23.6%of CD45þCD4þ cells). A trend in increase of MDSCs and TAMs

was alsoobserved.When combinedwithRT,GDC-0919 appearedtomitigate this immunosuppressive response, normalizing Tregs,MDSCs, and TAMs back to baseline levels (Fig. 5A). Next, weextended this line of investigation to studies using a fractionatedRT regimen, a combination that led to a significant survivaladvantage in our model (Fig. 4C). Similar to a single fractionschedule, fractionated RT also led to an increase in Tregs, whichwas attenuated by GDC-0919 (Fig. 5C). In addition to immuno-suppression, we sought to determine whether GDC-0919, RT, orthe combination influenced immune activation. No changes inthe percentage of T effector cells (CD8þ T cells) or helper T cells(CD4þ T cells) were observed in either treatment arm (Supple-mentary Fig. S5A and S5B). As radiation induces formation ofvarious neoantigens, thereby enhancing activation of cytotoxic Tcells (35), we extended our studies to determine whether changesin activated or cytotoxic T cells could be observed with the RT andGDC-0919 combination. Interestingly, fractionated RT alone ledto robust increases in both activated T cells (CD8þCD69þ) andcytotoxic T cells (CD8þGzmBþ) in the tumor (Fig. 6A). This RT-induced immune activation was also reflected by increased IFNglevels in the plasma of mice receiving fractionated RT in compar-isonwith controls (Fig. 6B).When combinedwith RT, GDC-0919led to further increases in both activated and cytotoxic T cells,

Figure 5.

Tryptophan metabolism contributes to radiation-induced immunosuppression in glioblastoma. A, Intracranial tumors were macrodissected from mice 5 daysfollowing the GDC-0919 and/or RT treatment schedule described in Fig. 4B and analyzed for immune correlates by flow cytometry, including presence of Tregs(CD4þFoxP3þCD25þ), MDSCs (CD45þCD11bþGr1þ), and tumor-associated macrophages (CD45þCD11bþF4/80þ). Scatter plot represents data from 5 to6 tumors from each group. B, To analyze the suppressive ability of Tregs, splenocytes from C57BL/6 mice were used for isolating CD8þ T cells and Tregs(CD4þCD25þ) usingmagnetic sorting. CFSE-labeledCD8þ T cellswere activated using plate-bound anti-CD3/CD28 antibody for 3 days in the presence or absence ofTregs (CD8:Tregs¼ 1:3) and kynurenine (50 mmol/L) and represented as flow histogram plots as CFSE dilution (proliferation) or no proliferation (suppression). Thebar graph represents cumulative data from three experiments. C, Intracranial tumors were macrodissected from mice 7 days following the GDC-0919 and/orfractionated RT treatment schedule described in Fig. 4C and analyzed for immune correlates by flow cytometry, including presence of Tregs (CD4þFoxP3þCD25þ).Scatter plot represents data as mean � SEM from 4 tumors in each group. � , P < 0.05.






which supports the observed enhanced antitumor response bythis combination (Fig. 6A). We went on to demonstrate increasedexpression of PD-1 in resected tumors following RT, suggesting anincrease in tumor-specific tumor infiltrating lymphocytes (Sup-plementary Fig. S5C; ref. 36).

We went on to explore potential mechanism(s) underlying thepotentiation of RT response with IDO1 inhibition. As both theantitumor and immune response following IDO1 inhibitionappeared RT-specific in our glioblastoma model, as an initialinvestigation, we sought to determinewhether RT could reactivateor further activate this immune checkpoint. As demonstratedin Fig. 6C, Western blot analysis performed on intracranial TRPtumors following fractionated RT demonstrated increased IDO1expressionwhen comparedwith controls, confirming RT-inducedpathway activation. As we have shown that RT stimulates both T-cell activation and cytotoxicity, we next sought to link thisobserved T-cell activation with increased IDO1 expression bydetermining whether activated T cells could in turn induce tryp-tophan metabolism, thereby contributing toward RT-inducedimmunosuppression. To test this possibility, we cocultured irra-diated and control U251 cells with HLA-A2þ na€�ve human

peripheral bloodmononuclear cells (PBMC). As expected, neithercontrol nor irradiated U251 cells demonstrated IDO1 pathwayactivation (Fig. 6D). Interestingly, coculturing U251 cells withna€�ve PBMCs appeared to stimulate T-cell activation, leading toIDO1 pathway activation (Fig. 6E) determined by increasedkynurenine production. Pathway activation was significantlypotentiated when PBMCs were cocultured with irradiated U251cells, providing further support linking RT response with trypto-phan metabolism in glioblastoma.

DiscussionThere have been several recent reports implicating aberrant

tryptophan metabolism with glioblastoma immune tolerance(14, 23, 37). A majority of these studies have indirectly evaluatedthis pathway through expression of its rate-limiting enzymesIDO1 and TDO. Results from this line of investigation weresomewhat inconsistent, reporting IDO1 expression in 8% to61.5% of glioblastoma using IHC staining in 60 and 39 tumors,respectively (38, 39). Furthermore, thedeterminationof increasedIDO1 expression in glioblastoma is also mixed, ranging from

Figure 6.

Radiation induces antitumor immune reactivation. A, Intracranial TRP tumors described in Fig. 4C were macrodissected from mice 7 days following the GDC-0919and/or fractionated RT treatment schedule and analyzed for activated (CD8þCD69þ) and cytotoxic (GzmBþCD8þ) T cells. Scatter plot represents data asmean� SEM from 4 tumors in each group. B, Plasma from mice harboring intracranial TRP tumors described in Fig. 4C were analyzed for mouse IFNg using ELISA.Scatter plot represents data from 4 mice from each group. C, IDO1 expression was analyzed in orthotopic TRP tumors isolated from C57BL/6 control miceand mice treated with f-RT and used to performWestern blot analysis for IDO1. Tubulin was used as loading control. D, Human PBMCs (HLA-A2þ) were coculturedwith U251 (HLA-A2þ) and 250 mmol/L of tryptophan for 3 days. Cell culture supernatant was analyzed for kynurenine and cells were used for Western blottingfor IDO1 (E). Results are representative of at least three independent experiments. �� , P < 0.005; � , P < 0.05.

Kesarwani et al.





medium to strong enzyme expression observed in 61.5% of thesetumors when using IHC to 29%when measured by mRNA levels.Our study is among thefirst to delineate this pathway at the level ofthe individual metabolites contributing to immunomodulation.In addition to further validating the relevance of this pathway ingliomagenesis, evaluating levels of individualmetabolites providea far more striking distinction between glioblastoma and LGGthan when determined through enzyme expression. For example,when quantifying relative values of kynurenine and using thehighest level of kynurenine detected in LGG as a threshold todefine pathway activation, approximately 60% of glioblastomademonstrated aberrant tryptophan metabolism. Furthermore,through globalmetabolomic profiling, we uncovered a previouslyundescribed, inverse relationship between kynurenine levels andits downstream metabolic intermediate kynurenate in glioma. Aratio generated by coupling these two metabolites led to a highlysignificant distinction between LGG and glioblastoma, with over90%of glioblastoma having a kynurenine/kynurenate ratio abovethe defined threshold (Supplementary Fig. S1C). These findingshave unique clinical applications. For example, the differentialaccumulation of tryptophan-related metabolites in LGG andglioblastoma can potentially be imaged throughMR spectroscopyand therefore has diagnostic implications. In addition, such imag-ing correlates may be extended into clinical trials, allowing for theenrichment in patients with tumors likely to respond to IDO1pathway inhibition or by supporting phase 0 investigationsdesigned to confirm target engagement of a specific compounddesigned to inhibit this pathway.

In addition to evaluating the IDO1 pathway from a metabolicperspective, integrated analyses coupling these global metabolo-mic signatures with gene expression profiles performed onmatched tumors provided further insight into the biologic impli-cations of this pathway. These cross-platform studies demonstrat-ed that tryptophan metabolism is specific to the mesenchymaland classical molecular subtypes of glioblastoma, which is con-sistent with recent work identifying immune heterogeneitybetween these individual subtypes (40). Our group has recentlyreported that rather than representing intertumoral heterogeneity,molecular subtypes in glioblastoma were reflective of regionalintratumoral heterogeneity, with the proneural subtype compris-ing the infiltrative edge of an individual tumor, whereas themesenchymal subtype was specific to the perinecrotic core(31). Consistent with these findings, evaluating regional expres-sion of IDO1 using the Ivy GAP Atlas demonstrated highestexpression of this enzyme in the perinecrotic core and lowest inthe infiltrative edge of individual tumors. As increased expressionof IDO1 has been recently described as a prognostic factor inglioblastoma (23), the observed differential expression of thisenzyme may contribute toward the therapeutic resistance typi-cally attributed to this region. In addition, these findings mayimply that aberrant tryptophan metabolism is a late event ingliomagenesis and rational combinatorial strategies designed toalso utilize agents specifically targeting the unique immune and/or signaling associated with tumor cell populations expanding inthe infiltrative edge may lead to more durable control in theseotherwise resistant tumors.

Aberrant tryptophan metabolism identified in patient-derivedglioblastoma was recapitulated in our preclinical models, withrobust pathway activation observed in all glioblastoma cell linestested. The IDO1 inhibitor GDC-0919 demonstrated potentinhibition of tryptophanmetabolism in these models, which was

measured by inhibition of kynurenine production, and impor-tantly, pharmacokinetic/pharmacodynamic studies demonstrat-ed the strong potential for this agent to cross the BBB and achievebiologically relevant concentrations in the brain, supportingfurther preclinical investigations with this compound in glio-blastoma. To determine the potential of GDC-0919 to serve as anovel immune checkpoint inhibitor in glioblastoma, we extend-ed studies in vivo using an adult nongermline GEM astrocyte cellline that recapitulated both the genotype and aggressive pheno-type of glioblastoma (29, 30) and displayed aberrant tryptophanmetabolism. Using this model, IDO1 inhibition with GDC-0919did not demonstrate antitumor activity as a single agent insubcutaneous and orthotopic tumors or modulate the tumors'immune profile at baseline (41, 42). Although molecular knock-down of IDO1 expression has been shown to influence glio-blastoma growth (43), our results are consistent with otherrecent publications demonstrating minimal activity in glioblas-toma when inhibiting IDO1 as a single targeted agent (44).Although we did not observe any changes in the absolutenumber of Tregs following IDO1 inhibition, we did uncover animportant role by which kynurenine modulates their activity,serving as a critical metabolite required for Treg-mediated sup-pression of CD8þ T-cell proliferation. Further work is needed toprecisely define the role kynurenine and its downstream meta-bolic intermediates play in modulating Treg function and deter-mine its mechanistic underpinnings.

AlthoughGDC-0919 did not demonstrate antitumor activity asa single agent in our glioblastoma model, we identified a uniquesynergy when combined with radiation, which was most notablewhen using a hypofractionated regimen. As recent strategiesdesigned to incorporate this agent in cancer treatment have beenlargely negative (45), these findings, which are consistent withrecent studies that have generated considerable interest in thepotential for immune checkpoint agents to be used in concert withradiation (46, 47), may provide a novel direction for continuedclinical trial development. Our results provide a novel insight intothe complex mechanisms that may be underlying these interac-tions, suggesting radiation may perturb cancer immunoediting atseveral levels. For example, in our working model, RT-inducedtumor cell death results in the release of tumor-associated antigen/peptides, ensued by antigen processing and presentation to T cells(35, 48). This, in turn, helps stimulate the immune system andclonal expansion as demonstrated by a robust increase in activatedand cytotoxic T cells, contributing toward an immunologic shiftfrom a state of "equilibrium" or "escape" back to an immunologicstate of "elimination" of tumor cells (49). However, this potentialfor an immunologic shift that would result in enhanced antitumoractivity appears to be dampened by parallel increases in RT-induced immunosuppression, most notably, a consistent increasein Tregs. Our data, along with another recent publication (50),suggests that theobserved increase in activatedandcytotoxic T cellsfollowing RT may in turn stimulate tryptophan metabolism,contributing to the observed increase in Tregs. Inhibiting trypto-phan metabolism mitigates the observed accumulation of RT-induced Tregs and attenuates their suppressiveness. This results ina further increase in T-cell activation and cytotoxicity, therebyenhancing antitumor activity by tipping the immunologic balancetoward a state of "elimination" (37), thereby providing a mech-anistic link between IDO1 inhibition and enhanced radiationresponse. Focused studies involving depletion of Tregs, and otherpotential targets of IDO1 inhibition, including MDSCs, are still






required to more definitively establish mechanisms governingpotentiation of RT response following IDO1 inhibition.

In summary, aberrant tryptophan metabolism represents animportant metabolic node in glioblastoma. Although its specificrole in gliomagenesis remains unclear, this pathway appears toplay a significant role in RT-induced immune checkpoint reacti-vation. GDC-0919 is a potent IDO1 inhibitor with the capacity ofcrossing the BBB and enhancing RT response by mitigating RT-induced immunosuppression, thereby shifting the immunologicbalance toward a state of "elimination," which was particularlystriking when using a hypofractionated approach. These encour-aging findings support clinical efforts designed to combine IDO1inhibition with hypofractionated RT in glioblastoma, offering thepromise of effectively harnessing a patient's own immune systemto attack these otherwise recalcitrant tumors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: P. Kesarwani, P. ChinnaiyanDevelopment of methodology: P. Kesarwani, P. Chinnaiyan

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Kesarwani, A. Prabhu, S. Kant, P. Kumar,S.F. Graham, K.L. Buelow, C.R. Miller, P. ChinnaiyanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Kesarwani, A. Prabhu, S. Kant, P. ChinnaiyanWriting, review, and/or revision of the manuscript: P. Kesarwani, P. Kumar,S.F. Graham, K.L. Buelow, G.D. Wilson, C.R. Miller, P. ChinnaiyanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P. Kesarwani, S. KantStudy supervision: P. Chinnaiyan

AcknowledgmentsThis work was supported by the NIH/NINDS (R21NS090087), ACS (RSG-

11-029-01), Bankhead-Coley Cancer Research Program, and Cancer ResearchSeed Grant Awards from Beaumont Heath (P. Chinnaiyan). C.R. Miller issupported by NIH/NCI (R01CA204136).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 5, 2018; revised March 6, 2018; accepted April 20, 2018;published first April 24, 2018.

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2018;24:3632-3643. Published OnlineFirst April 24, 2018.Clin Cancer Res Pravin Kesarwani, Antony Prabhu, Shiva Kant, et al. Checkpoint Reactivation in GlioblastomaTryptophan Metabolism Contributes to Radiation-Induced Immune

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