Targeting the Kynurenine Pathway for the Treatment of Cisplatin-Resistant Lung Cancer Dan J

Published OnlineFirst October 18, 2019; DOI: 10.1158/1541-7786.MCR-19-0239

MOLECULAR CANCER RESEARCH | METABOLISM

Targeting the Kynurenine Pathway for the Treatment of Cisplatin-Resistant Lung Cancer Dan J. M. Nguyen1, George Theodoropoulos1, Ying-Ying Li1, Chunjing Wu1, Wei Sha1, Lynn G. Feun2, Theodore J. Lampidis3, Niramol Savaraj1,2, and Medhi Wangpaichitr1,4

ABSTRACT ◥ Cisplatin resistance is a major barrier in the effective treat- cells. Exposing CR cells to antioxidant (TIRON) results in ment of lung cancer. Cisplatin-resistant (CR) lung cancer cells suppression of IDO1 activity and confers resistance to IDO1 do not primarily use glucose but rather consume amino acids inhibition, indicating an interrelationship between ROS and such as glutamine and tryptophan (Trp) for survival. CR cells IDO1. Because KYN plays a critical role in reprogramming activate the kynurenine (KYN) pathway (KP) to cope with na€ve T cells to the immune-suppressive regulatory T-cell excessive reactive oxygen species (ROS) and maintain homeo- (T-reg) phenotype, we observed higher expression of TGFb, þ þ stasis for growth and proliferation. Consequently, indoleamine FoxP3, and CD4 CD25 in mice bearing CR tumors compared 2,3-dioxygenase-1 (IDO1) becomes an essential enzyme for CR with tumors from cisplatin-sensitive counterparts. cells’ survival because it initiates and regulates the first step in the KP. Increased IDO1 activities and ROS levels are found in Implications: Findings suggest that the enzyme-inhibitory activ- CR cells versus cisplatin-sensitive lung cancer. Importantly, ity and antitumor efficacy of IDO1 inhibitors rely in part on ROS significantly greater KYN/Trp ratio (P ¼ 0.005) is detected in levels, arguing that IDO1 expression alone may be insufficient serum of patients who fail cisplatin when compared with na€ve to determine the clinical benefitsforthisclassofexperimental treatment. Knocking down IDO1 using shRNA or IDO1 inhi- cancer drugs. Importantly, IDO1 inhibitors may be more suitable bitors heightens ROS levels and results in a significant growth to treat patients with lung cancer who failed cisplatin therapy inhibitory effect only on CR cells and not on cisplatin-sensitive than na€ve treatment patients.

Introduction will develop drug resistance; thus, cisplatin resistance remains the major obstacle for the effective treatment of lung cancer. Surgery is the best treatment approach for early-stage lung Our research focus has been to elucidate molecular differences in cancer; nonetheless, most patients already have locally advanced metabolic heterogeneity observed among patients with sensitive and or metastatic disease at the time of diagnosis. Anti-PD1 (pembro- resistant tumors. We have previously reported that cisplatin-resistant lizumab) in combination with chemotherapy was recently approved (CR) lung cancer cells possessed increased numbers of mitochondria as first-line treatment of patients with non–small cell lung cancer and consumed higher rates of oxygen than nonresistant cancer cells, (NSCLC) with stage 3B who are not candidates for surgical resec- resulting in significantly higher (2-fold to 3-fold) basal levels of reactive tion. However, patients’ tumors must express PD-L1 and have no oxygen species (ROS; refs. 2, 3). In addition, we showed that CR tumors EGFR or anaplastic lymphoma kinase mutation in order to receive are no longer addicted to glucose for energy and do not utilize classic this therapy. Although these combination therapies offer a longer aerobic glycolysis [Warburg effect; (4, 5)]. By switching to oxidative duration of response than other second-line chemotherapy, the metabolism (OXMET), CR cells increase cellular energy or metabolic overall response rate in NSCLC is low (15%–20%; ref. 1). Subse- demand and outstrip the glutamine supply, making glutamine the quently, cisplatin-based chemotherapy alone and in combination conditionally essential amino acid for cell survival. Besides glutamine, with radiation remains the primary modality of treatment. Despite CR cells are also auxotrophic for arginine due to the lack of argini- early positive responses to cisplatin, all patients with lung cancer nosuccinate synthase 1 (ASS1), which is a key enzyme in the synthesis of arginine from citrulline (2, 6, 7). Lack of ASS1 expression therefore makes arginine an essential amino acid for CR tumors as well. Because 1Department of Veterans Affairs, Miami VA Healthcare System, Research Ser- of these phenomena, CR cells are collectively dependent more on vice, Miami, Florida. 2Department of Medicine, Hematology/Oncology, Miller OXMET for survival and are susceptible to amino acid deprivation. 3 School of Medicine, University of Miami, Miami, Florida. Department of Cell Here we show that these metabolic derangements have led us to the 4 Biology, Miller School of Medicine, University of Miami, Miami, Florida. Depart- discovery that L-tryptophan (Trp) catabolism is also upregulated in ment of Surgery, Cardiothoracic Surgery, Miller School of Medicine, University of Miami, Miami, Florida. CR cells. Although Trp is an essential amino acid, required for protein synthesis and as the precursor of serotonin and melatonin, the Note: Supplementary data for this article are available at Molecular Cancer catabolism of Trp can also generate kynurenine (KYN) via the Research Online (http://mcr.aacrjournals.org/). kynurenine pathway (KP). This pathway is responsible for the catab- Corresponding Authors: Medhi Wangpaichitr, Miami VA Healthcare System, olism of approximately 95% of ingested Trp not used for protein 1201 NW 16th Street, Research 151, Miami, FL 33125. Phone: 305-575- 7000x14496; Fax: 305-575-3375; Email: [email protected] and synthesis or the serotonin pathway (8, 9) and not limited to its originally derived role in the biogenesis of nicotinamide adenine Niramol Savaraj, [email protected] þ dinucleotide (NAD ; ref. 10). At the first step, Trp is oxidized through Mol Cancer Res 2019;XX:XX–XX indoleamine 2,3-dioxygenase-1 and 2 (IDO1 and IDO2) or Trp 2,3- doi: 10.1158/1541-7786.MCR-19-0239 dioxygenase-2 (TDO2) to form formylkynurenine, which is later 2019 American Association for Cancer Research. degraded to KYN (11). However, TDO2 is primarily present in the

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liver and is not expressed in lung tissue or lung tumor (12). IDO2 is Measurement of patients’ plasma expressed in human tumors (13), and recent genetic data have Patients undergoing cisplatin treatment were recruited in this study implicated it functionally in drug-resistant pancreatic ductal adeno- at the Miami VA Healthcare System. All procedures were approved by carcinoma resistant to DNA-damaging radiotherapy (14). But the the institution review board (ref.no.1190748-1). Participants prospec- significance of the relationship between cisplatin resistance and IDO2 tively provided informed consent for the use of their specimen in activity in lung cancer remains elusive and needs to be explored in research. future. Blood samples were drawn and centrifuged at 1,000 g for 10 IDO1 is the unique enzyme similar to superoxide dismutase-1 minutes to extract plasma and then stored at 80C. Plasma (SOD1) that exploits superoxide as a substrate (15, 16). We have samples (100 mL) were placed into a centrifugal tube and the previously reported that CR cells possessing elevated basal levels of same volume of 5% perchloric acid solution was added. Next, the ROS also expressed higher superoxide dismutase-1 (SOD1) when samples were placed in a vortex mixer for 30 seconds and then compared with parental cell counterparts (3). Increasing SOD1 levels maintained at room temperature for 15 minutes to allow plasma and IDO1 activities are crucial mechanisms for the capture of ROS/ protein precipitation. Finally, the samples were centrifuged at superoxide and maintenance of homeostasis in CR cells. Importantly, 12,000 g for 5 minutes and 20-mL samples of supernatant were increased IDO1 activity results in greater KYN production, which collected for analysis. Samples were analyzed by Agilent 1290 plays a key role in reprogramming na€ve T cells to the immune- Infinity high-performance liquid chromatography (HPLC) system suppressive regulatory T-cell (T-reg) phenotype (17, 18). Thus, it on Phenomenex Kinetex biphenyl column (2.1 mm 50 mm; 2.6 seems that adaptations in CR cells confer a survival advantage in an mm). HPLC conditions: buffer A: 0.1% trifluoroacetic acid in environment with elevated basal levels of ROS, which may otherwise water, buffer B: 0.1% trifluoroacetic acid, 70% acetonitrile in water, be lethal to normal cells (3). These findings are supported by recent UV detector: 225 nm/230 nm, linear gradient from 5%B to 50%B reports that IDO levels impact outcomes in patients with lung cancer in 5 minutes. and gliomas receiving radiotherapy that damages DNA as well as immunoradiotherapy (19, 20). Hence, links have been established Animal studies between metabolic alterations in oxidative processes and amino acid Procedures and mice protocol were approved by the Institutional catabolism and tumor responsiveness to treatment that may be utilized Animal Care and Use Committee of the Miami VA Healthcare System to affect outcomes. (Animal Welfare Assurance Number: A3739-01). Male or female Here, we show that IDO1 activity is dependent on ROS levels in C57BL6/N mice (ages 5 weeks, Jackson laboratory) were used to CR cells. Increased basal level of ROS heightens IDO1 activity and establish in vivo allograft using LLC versus LLC-CR. Mice were in turn hyperactivates the KP in CR cells in vitro, in vivo,andin inoculated subcutaneously with 2.5 106 cells on the dorsal lumbo- patients who failed cisplatin treatment. Inhibiting KP using IDO1 sacral region. Tumor growth was evaluated twice a week by measuring inhibitor further increases ROS in CR cells beyond their tolerance tumor volume according to the following formula: tumor volume ¼ limit, which ultimately leads to cell death. By investigating a new width2 length 0.5. Experiment was ended when either W or L paradigm between CR cells and their microenvironment and by reached the final set value of 10 mm. targeting their distinct metabolic features, we believe that our studies will identify a population of patients with lung cancer who Growth inhibition and cytotoxicity assay will benefit greatly from future chemoimmunotherapy and have Cells were seeded in 24-well dishes and treated with various improved outcomes. concentrations of IDO1 inhibitors (i.e., Epacadostat). The procedure was described previously (21, 25). Briefly, the culture media and the trypsinized cells were collected and this mixture was centrifuged at Materials and Methods 400 g for 5 minutes. The supernatant was discarded, resuspended in Cell lines and reagents 1 mL of Hank's buffer, and assayed for live cells and death cells using Two pairs of parental versus cisplatin-resistant human NSCLC cells trypan blue exclusion method. (A vs. ALC and FA vs. FC) and a pair of mouse Lewis lung cells (LLC vs. LLC-CR) were used. Cell line “A” was established with pleural fluid Western blot analysis from a patient with adenocarcinoma. FA was established from a poorly Cells were seeded at 1 105/mL onto 60-mm dishes, treated, differentiated squamous cell carcinoma. Cellular characteristics have collected, lysed, and immunoblotted with indicated antibody. been previously characterized (21–22, 24) and routinely tested for Detailed procedure was described in our previous publica- mycoplasma infection (MycoAlert; Lonza cat#LT07-710). These cell tions(21,25).Briefly, cell lysis was completed by sonication and lines were not derived from the patients reported here in this study. the total protein was separated on an SDS-PAGE, transferred onto LLC and LL24 were purchased (ATCC). We established their cisplatin- a polyvinylidene difluoride membrane (Millipore), and immuno- resistant variants using previously published methods (22, 23). Note: blotted with indicated primary antibody. Antibody to IDO1 ALC and FC exhibit 7-fold resistance to cisplatin. LLC-CR exhibits (cat#NBP1-87702) was purchased from Nuvous. Antibodies to: 3-fold resistance. LL24 is normal lung fibroblast. Supplementary Table hypoxia-inducible factor-1a (HIF1a; cat#610958) were purchased s1 shows the ID50 values for the sensitive and resistant cell lines. from BD Biosciences; Phospho-Aryl Hydrocarbon Receptor (AHR) Recombinant human IFNg was purchased from BioLegend (AHR; cat#GTX113124) and FoxP3 (GTX107737) were purchased (cat#570204). IDO1 inhibitors [Epacadostat (cat#T3545), PF- from GeneTex; AHR (cat# A1451) was purchased from ABclonal; 06840003 (cat#T4307), NLG-919 (cat#T1806), and indoximod (cat# Antibody to LAT1 (cat#5347) was purchased from Cell Signaling. T6543)] were purchased from TargetMol. Dimethoxyflavone (DMF; All antibody dilutions were at 1:1,000, except for Actin (Sigma; cat#D6571), CH-223191 (cat#C8124), TIRON (4,5-dihydroxy-1,3- cat#A5441) which was diluted at 1:10,000. Bands were measured þ benzenedisulfonic; cat#172553), and NAD were purchased from using a molecular imager ChemiDoc system with Quality One Sigma. software (Bio-Rad).

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Qualitative real-time PCR medium was removed, and cells were washed three times with fresh, qRT-PCR was carried out as previously described (2). Briefly, 1 mgof “cold” RPMI serum-free medium with Trp. Then cells were lysed with RNA was used for cDNA synthesis. The primers for qRT-PCR were 500 mL of 1 N NaOH, collected in 1.5-mL Eppendorf tubes and designed with PerlPrimer for SYBR Green fluorophore (see Supple- gently vortexed (2). Protein analysis was conducted on 250-mL mentary Methods). Forty-cycle amplification was used and the data samples using the Micro BCA Protein Assay Kit (Thermo Fisher were analyzed with CFX Manager software from Bio-Rad. To calculate Scientific; cat#23235). Radioactivity in the remaining sample was the relative mRNA level, we used the DDCt method. The level of mRNA measured as counts per minute, and normalized to protein, using a was corrected with that of GAPDH or actin. PerkinElmer Tri-Carb 2810TR liquid scintillation spectrometer with QuantaSmart (2). Knockdown experiment For stable knockdown (shRNA) of IDO1, ALC cells were trans- IDO1 activity assay fected with 1 mg of pGFP-C-shLenti plasmid (OriGene) expressing An IDO1 assay kit (BioVision; cat#K972-100) was used to measure shIDO1 (NM_002164) or scrambled control using lipofectamine IDO1 activity. Briefly, 5 106 cells were seeded onto 100-mm dishes, 2000 (Thermo Fisher Scientific) transfection reagent (see Supple- treated, collected, and homogenized with IDO1 assay buffer. IDO1 mentary Methods). After 24 hours, transfection medium Opti- activities were calculated by the amount of N-formylkynurenine MEM was exchanged to RPMI1640 containing 5 mg/mL of G418. produced/(Reaction time Amount of protein in well). Samples were GFP as a reporter was used to evaluate target gene knocked down measured at Ex/Em ¼ 402/488 nm. efficiency. For siRNA, cell lines A were transfected with 1 nmol/L of þ HIF1a SMARTpool siRNA (NM_005566) or siControl (Dharmacon) NAD concentration assay using INTERFERin transfection reagent (Polyplus) as previously A NAD/NADH assay kit (Cayman Chemical; cat#600480) was used þ described (25). to measure total cellular NAD concentration. Cells were seeded in 96- þ well at 1 104 and grow overnight. Total NAD was detected at Real-time assay of oxygen consumption 450 nm according to manufacturer's instruction. Simultaneous multiparameter metabolic analysis of cell populations in culture was performed in the Seahorse XFe24 extracellular flux Amino acid analyzer analyzer (Seahorse Bioscience) as described by Wu and colleagues (26). Physiologic fluids or culture medium were collected and the Bio- All cell lines were cultured in growth medium for 24 hours in plate chrom 30 amino acid analyzer (using ion exchange chromatography) before real-time metabolic analysis. At the start of the assay, growth was used to analyze free amino acids (30–40 in the complete panel) in medium was removed and replaced with assay medium. Basal oxygen samples (2). Values were reported compared with normal ranges in consumption rate (OCR) and extracellular acidification rate (ECAR) controls. Samples used in ion exchange chromatography are separated of the cells were measured. Oligomycin, Carbonyl cyanide 4-(trifluor- in an analytical fashion with an ion exchange column and buffers of omethoxy)phenylhydrazone (FCCP), and rotenone were used to increasing pH and ionic strength. After postcolumn derivatization inhibit ATP synthase, uncouple OXPHOS, and inhibit mitochondrial with ninhydrin, and colorimetric intensity recordings at 570 nm and complex 1, respectively. After XF assay, cells were harvested by trypsin- 440 nm, the amino acids separated by chromatography were ethylenediaminetetraacetic acid treatment and counted. The number detected (2). Amino acid concentration was calculated by comparing of cells per well were used to normalize OCR and ECAR. the peak area of a particular amino acid to the peak area of an internal standard of known concentration and then multiplying by its specific Assay of intracellular ROS/H2O2 response factor from calibration. As previously described (3), cells were collected and intracellular H2O2 was measured by incubating with 10 mmol/L of CM-H2DCFDA IHC staining (Life Technologies; cat#C2938) at 37C for 30 minutes in the dark. IHC staining was performed according to our routine methodology Then the cells were washed once with PBS and centrifuged to remove with some modification (2). Target retrieval solution (citric buffer; pH impermeable reagents. Cells were resuspended in 500 mL of PBS and 6.0) was used to enhance the staining. Samples were incubated analyzed either by fluorimeter [FLUOstar OPTIMA, BMG Labtech overnight with CD4 (GeneTex; cat#GTX50984), CD25 (Biorbyt; (excitation at 485 nm and emission at 520 nm)] or by flow cytometer cat#orb389314), and TGFb (Cell Signaling; cat#3709) primary anti- (CytoFLEX, Beckman). bodies at 1:100 in antibody dilution solution (Dako S3022). Anti-IDO- 1 Antibody (clone 4B7; Millipore cat# MABF850) which has been LAT1 localization validated in IDO / mouse was diluted at 1:50. Secondary antibody For cell surface LAT1 staining, fluorochrome-conjugated antibodies solution (LSAB2 Kits Dako, biotinylated antibody solution; were used to detect LAT1/CD98 (BioLegend; cat#315603). Cells were cat#K0675) was added for 30 minutes, washed, and streptavidin washed in PBS and resuspended in 250 mL of FACS buffer (PBS, 1% conjugated to peroxidase (HRP) solution was added for 30 minutes. fetal calf serum, 0.1% NaN3) with LAT antibody. Cells were incubated DAB Chromogen (Dako, cat#K3466) was used and then counter- for 30 minutes on ice, followed by three washes, and resuspended in stained with hematoxylin. 500 mL of PBS. Live cells were gated according to their forward scatter and side scatter. Data were acquired using CytoFLEX (Beckman) flow Immunofluorescent staining cytometer. Cells were treated with and without AHR antagonist, washed with PBS, and fixed with 4% paraformaldehyde for 30 minutes at room Trp uptake temperature. Cells were incubated with PBS containing 0.2%Triton Seeded cells were cultured in complete RPMI media for 24 hours, at X-100 (antibody diluent) for 15 minutes at room temperature and which point media was replaced with fresh Trp and serum-free RPMI subsequently blocked with PBS containing 5% BSA for 1 hour. Cells media with “hot” (5 mCi L-[5-3H(N)] Trp; ref. 2). Next, the hot were incubated with anti-AHR antibody (cat#GTX113124 at 1:1,000 in

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antibody diluent,) overnight at 4C. Secondary antibody (1:250; Alexa treatment baseline (Fig. 1H). Consistently, we did not find an increase Fluor 555) was added for 1 hour. DAPI (Vector Laboratories; in KYN/Trp ratio in patients (patients 4–6) who continued to respond cat#H1500) was used for nuclei staining. Cells were examined under to cisplatin treatment. a fluorescence microscope (Keyence BZ-X710) to determine localiza- Together, our data illustrate that CR cells consumed Trp at a higher þ tion of AHR. Our Keyence microscope also equipped with hybrid cell rate specifically for KYN production but not for production of NAD . count (an algorithm software that allows accurate quantification for Importantly patients who failed cisplatin therapy, in our small sam- phase contrast). pling, possessed significantly higher ratio of KYN/Trp, the indicator of IDO1 activity. These results suggest that increased IDO activity is Statistical analysis involved in disease progression in lung cancer, possibly through All statistical analyses were performed from three separate biolog- immunosuppressive effects. ical replicates that were isolated and analyzed in technical triplicates. Separate measurements using the two-tailed t test and the results were The KYN/AHR/ARNT axis is a crucial modulator of metabolism expressed as mean SD. A P value of less than 0.01 or 0.05 was in CR cells considered as statistically very significant and significant, respectively. Recent discoveries revealed that KYN can serve as an endogenous The medium or plasma concentrations of KYN or Trp, as well as KYN/ ligand for the Aryl Hydrocarbon Receptor (AHR) in cancer cells (28). Trp ratios approximated a parametric distribution; therefore, data KYN formed a complex with AHR and translocated into the nucleus to were presented as means SD. Because of the small sample size, the modulate their target genes as a ligand-activated transcription fac- associations between serum biomarker levels and time point variables tor (29). To further elucidate the unique role of AHR, immunofluo- were evaluated with a Mann–Whitney U test. rescence staining showed that AHR localized with high intensity to the nucleus of CR cells, whereas less accumulation was observed in parental counterparts, implicating that KP is specifically active in CR Results cells (Fig. 2A). To determine that AHR can be activated by KYN, we The KP contributes to higher KYN/Trp ratio in patients who fail added exogenous KYN (100 mmol/L) and assessed AHR localization. cisplatin As depicted, treatment with KYN increased the nuclear distribution of We have reported that CR lung cancer cells were less dependent on AHR in both parental and CR cells. We then determined that KYN was the glycolytic pathway but instead utilized mitochondria for biogen- specific to AHR activation in CR cells by inhibiting AHR translocation esis to catabolize glutamine as an alternative carbon skeleton with either 10 mmol/L of DMF (an AHR antagonist) or 1 mmol/L of source (2, 6). Here, we first compared oxygen consumption among CH-223191 (AHR inhibitor). AHR was significantly less accumulated resistant and sensitive cells using the Seahorse flux analyzer. In in the nucleus after treatments. Importantly, exposure with AHR response to adding oligomycin, FCCP, and rotenone (Supplementary inhibitors also resulted in suppression of IDO1 activities, whereas Fig. S1A), CR cells consumed significantly higher rates of oxygen addition of KYN increased IDO1 activity only in CR cells (Fig. 2B). (Fig. 1A) and thus had higher levels of ATP production than their Furthermore, we have previously demonstrated that CR cells pos- parental cells counterparts (Supplementary Fig. S1B). Adding to sess very low levels of HIF1a due to metabolic reprogramming these metabolic alterations and the reliance on amino acids, we (ref. 6; Fig. 2C). ARNT or HIF1b is a known binding partner of both further discovered that CR cells also uptake significantly higher HIF1a and AHR (29, 30). Thus, in the absence of HIF1a, it is possible amounts of Trp (Fig. 1B). It is noteworthy that even though Trp that ARNT is now available to bind and form a new partner with KYN/ þ can be used for de novo biosynthesis of NAD (essential coenzyme of AHR and initiate the transcription of genes, which favor the survival/ þ all organisms’ redox system), Trp is less efficient and a poor NAD proliferation of CR cells. Immunoblot showed that CR cells expressed þ precursor in vivo. Trp will be diverted only to NAD synthesis when higher basal level of AHR as well as its phosphorylated form (Fig. 2C). its supply exceeds enzymatic capacity (27). Thus, the main source of This is interesting because others have shown that AHR can be fully þ NAD is from salvage pathways, which require the uptake of other activated upon phosphorylation (31). To characterize the role of the þ NAD precursors such as nicotinic acid. Consistent with these KYN/AHR/ARNT axis, we assayed the expression of the AHR-target findings, we have found that although uptake of Trp is significantly gene LAT1 [L-type amino acid transporter-1; a known amino acid þ increased in CR cells as showed in Figure 1B, the NAD levels and transporter for large neutral amino acids such as arginine, leucine, and quinolate acid phosphoribosyltransferase (QPRT), a key enzyme in Trp (32, 33)] with and without adding KYN. As shown in Figure 2C, þ NAD biosynthesis, expressions were significantly decreased when LAT1 protein expressions were higher in CR cells; then we assayed for compared with parental cell counterparts (Fig. 1C and D). Hence, functional LAT1 that localized to the cell surface using the flow these findings demonstrate that CR cells actively utilized KP but did cytometer (Fig. 2D1). LAT1 was further augmented upon addition þ not engage in de novo synthesis of NAD . of KYN in CR cells (Fig. 2D2), whereas no increase in LAT1 was Next, we evaluated Trp levels in cell culture and found that Trp observed after exposing parental cells to KYN (Fig. 2D3), suggesting concentrations were significantly reduced in CR cells’ media (Fig. 1E). that HIF1a still maintained its complex with ARNT in parental cells. We then assayed for extracellular KYN levels (Fig. 1F), and used the To determine the role of HIF1a in parental cells, we knocked down KYN/Trp ratio as an indicator of IDO1 activity. A significantly higher HIF1a using siRNA and exposed AsiHIF1a to KYN. Increased LAT1 mean KYN/Trp ratio in CR cells’ media (as compared with parental expression was observed in AsiHIF1a after treatment (Fig. 2D4) and cell counterparts) implied that IDO1 activity was elevated in CR cells increased Trp uptake was found in CR cell after KYN treatment as (Fig. 1G). To gain insight into the physiologic role of KP, we assayed anticipated. But importantly, Trp uptake was increased in AsiHIF1a and the KYN/Trp ratio in serum of patients with cancer prior to and after exposure to KYN further increased Trp uptake (Fig. 2E), suggesting cisplatin/radiotherapy. All patients with cancer from our study pos- that downregulation of HIF1a levels will lead to an increase in AHR/ sessed higher basal levels of KYN/Trp ratio when compared with ARNT complex formation (Fig. 2G). We used four different unique healthy controls. Patients (patients 1–3) who failed cisplatin possessed sequences to silence IDO1 expression in order to determine efficacy of significantly higher KYN/Trp ratios when compared with the pre- silencing and consistency of effect (Fig. 2F). All knockdowns of IDO1

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A Oxygen consumption B TRP uptake 60,000 800 Parental Resistant Parental CR 700 50,000 600 40,000 500 400 30,000 300 20,000 200 CPM/Cell/Min

OCR (pmol/min) 100 10,000 0 0 A ALC FA FC LL24 A ALC FA FC

C NAD+ D QPRT mRNA level 120 Parental CR 1.2 Parental CR 100 1 80 0.8 60 0.6 nmol/L 40 0.4 20 0.2 0 0 A ALC FA FC A ALC FA FC mRNA relative expression

E Extracellular TRP level F Extracellular KYN level 25 14 Parental CR 12 Parental CR 20 10 15 8 6 10 4 m mol/L /cell m mol/L /cell 2 5 0 0 A ALC FA FC A ALC FA FC

G H KYN/TRP ratio in serum Fail cisplatin 0.4 KYN/TRP ratio in media Pretreat After cycle 3 0.35 0.025 Parental CR 0.3 0.02 0.25 0.015 0.2 Responsive 0.15 0.01

culture media 0.1 patient’s serum patient’s KYN/TRP ratio in KYN/TRP ratio in 0.005 0.05 0 0 A ALC FA FC Pt 1 Pt 2 Pt 3 Pt 4 Pt 5 Pt 6 Healthy ctrl Patients

Figure 1. CR lung cancer cells relied on OXMET and utilized KP, which contributed to the production of kynurenine (KYN) in CR tumors. A, Parent (A and FA) and CR (ALC and FC) cells were assayed for baseline oxygen consumption using Seahorse XFe24 extracellular flux analyzer. The rate of oxygen consumption was six times higher in CR than parental cells (, P ¼ 0.009 and , P ¼ 0.003). LL24 was used as control. B, Tryptophan (Trp) uptake was determined in parental versus CR cells using L-[5-3H (N)] Trp. The rate of Trp uptake was higher in CR cells (, P ¼ 0.02, , P ¼ 0.007). C, Total cellular NADþ concentrations were detected in parental versus CR cell lines. CR possessed lower basal levels of NADþ (, P ¼ 0.02; , P ¼ 0.006). D, Relative mRNA levels of QPRT. QPRT mRNA was significantly decreased in CR cells. GAPDH was used as internal control. The results shown in the graph were calculated with the DDCt method by setting the mRNA level of parental cells as 1 (, P ¼ 0.04, , P ¼ 0.01). E, Extracellular Trp concentration in culture medium was determined by amino acid analyzer. Lower levels of Trp were found in CR cells’ culture media (, P ¼ 0.04, , P ¼ 0.02). F, Extracellular KYN concentration in culture medium (amino acid analyzer). Higher levels of KYN were found in CR cells’ culture media (, P ¼ 0.01, , P ¼ 0.005). G, Extracellular KYN/Trp ratio in culture medium was determined by HPLC. CR cells possessed significantly greater KYN/Trp ratio (, P ¼ 0.003, , P ¼ 0.01) than control. H, KYN/Trp ratio in patients’ serum (HPLC). Patients 1 to 3 failed cisplatin after third cycle of cisplatin (, P ¼ 0.002, , P ¼ 0.004, , P ¼ 0.005). Patients 4 to 6 continued to respond to cisplatin after the third cycle of treatment. Pt, patient.

(shIDO1) suppressed LAT1 (Fig. 2F) and CYP1B1 (also a known CR tumors possess higher IDO1 activity and immunosuppressive AHR target gene; Supplementary Fig. S2) expressions in CR cells. phenotype in vivo Thus, our ﬁndings strongly suggest that the KYN/AHR/ARNT axis The tumor microenvironment with increased KYN levels can plays the unique modulator role in CR cells’ metabolism, and facilitate differentiation of na€ve T cells into T-reg cells (17, 18), increased Trp uptake can lead to further KYN secretion in CR cells. creating a highly immunosuppressive environment that favors

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Figure 2. Modulator role of KYN/AHR/ARNT axis in CR cells metabolism. A, Immunofluorescence (IF) staining of cells with 1:1,000 AHR antibody (red) and DAPI (blue nuclei). CR cells (ALC) possessed significantly higher intensity of AHR expression in the nucleus when compared with parental cells (, P ¼ 0.008). DMF or CH223191 significantly reduced AHR accumulation in CR cells (, P ¼ 0.007, , P ¼ 0.001; respectively) whereas exposure to KYN markedly increased AHR accumulation in the nucleus of both parental and CR cells. Bar graph indicated quantification of IF intensity (RFU/cell) using hybrid cell count. B, Addition of 100 mmol/L of KYN increased IDO1 activity in CR cells substantially (, P ¼ 0.03) but not significantly (NS) in parental cells. Treatment of 10 mmol/L of DMF or 1 mmol/L of CH223191 resulted in significant suppression of IDO1 activities (, P ¼ 0.006, , P ¼ 0.002, respectively). C, Immunoblot of lung cancer cell lines showed that resistant variants did not possess HIF1a but expressed higher levels of AHR and LAT1. Actin was used as a loading control. D, Flow cytometry analysis of surface LAT1 in lung cancer cell lines. D1: CR cells possessed higher surface LAT1 when compared with parental counterparts. D2: Treatment of KYN at 100 mmol/L for 48 hours further enhanced LAT1 expression in CR cells. D3: KYN treatment did not increase LAT1 expression in parental cells. D4: Knocking down HIF1a in parental cells resulted in increased LAT1 expression after KYN treatment. E, Knocking down HIF1a in parental cells increased Trp uptake and further increased Trp uptake upon exposure to KYN (100 mmol/L). Consistently, treatment of KYN further heightened Trp uptake in CR cells (48 hours; , P ¼ 0.02, , P ¼ 0.006). F, Knocking down IDO1 (shIDO1) in ALC suppressed LAT1 expression. Sh-A to D represent four unique shRNA sequences. G, Diagram illustrating the binding partners of ARNT (HIF1b). When HIF1a is downregulated, ARNT formed a new binding partner with AHR/KYN and initiated the transcription of genes that favored proliferation and increased Trp uptake, which can lead to further KYN secretion in CR cells.

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the growth of CR cells. To determine that our in vitro cell LLC and LLC-CR were then allografted into C57BL6/N mice. After model is indeed representative of in vivo settings, we created tumors developed, we then assayed for T-reg phenotypes. þ þ mouse CR cells (LLC-CR) from the Lewis lung mouse cell line CD4 CD25 and FoxP3 (forkhead family transcriptional regulator), (LLC). LLC-CR possessed 3- to 4-fold resistance to cisplatin which are the lineage-speciﬁc marker of T-reg cells, were higher in (Supplementary Fig. S3) and yielded 2-fold higher basal levels of LLC-CR when compared with parental counterpart, which further ROS (Fig. 3A). LAT1 and IDO1 activities were increased in LLC- indicated the involvement of KP in CR tumors (Fig. 3D). Furthermore, CR,thussuggestingstrongevidenceofKPutilization(Fig. 3B we also found higher expression of TGFb in LLC-CR tumors. TGFb and C). is a well-known immunosuppressive cytokine that is released by

Figure 3. Increased IDO1 activity and immune-suppressive phenotype were found in CR tumor. A, ROS analysis of mouse (LLC vs. LLC-CR) cells detected by CM-H2DCFDA probe. LLC-CR expressed 2-fold higher basal level of ROS. B, Immunoblot showed that LLC-CR also expressed higher levels of LAT1 protein. C, LLC-CR possessed higher IDO1 activity (, P ¼ 0.04). D, IHC staining of intratu- moral CD4þ, CD25þ (arrow), FoxP3þ (arrow), TGFb, and IDO1. Higher T-reg densities were found in mice bearing CR tumor than control. Box graph indicated quantiﬁcation of IHC (intensity/mm2) using hybrid cell count.

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T-reg (34) and has been shown to activate IDO1 biosynthesis (35, 36). activity. Consequently, these results may explain why many studies In fact IDO1 expression was elevated in mouse allografts of LLC-CR have shown insignificant correlation between IDO1 expressions and cells compared with allografts of LLC as seen through IHC with a clinic–pathologic parameters. mouse-validated antibody (Fig. 3D; IDO1). Together, our data suggest that increased IDO1 activity found in CR cells can create a highly CR cells were hypersensitive to IDO inhibitors immunosuppressive environment that favors the growth of tumors, To determine that IDO1 plays an important role in CR cell thus making CR tumors good candidates for IDO1 treatment therapy. proliferation and survival, we assayed for growth inhibitory effects using commercially available IDO1 inhibitors (Epacadostat, PF- Evaluation of IDO1 expression alone may be insufficient to 06840003, and NLG-919), as well as indoximod, which targets determine clinical benefits IDO2 (37). Only CR cells were hypersensitive to IDO1 inhibitors Depletion of Trp induces signaling events in T cells leading to with epacadostat showing to have the best efficacy for growth inhi- anergy and because Trp is a precursor for both serotonin and the KP bition at 12 mmol/L 0.5 (Fig. 5A). In addition, IFNg treatment (Fig. 4A), we assessed the expression of Trp hydroxylase (TPH), the further enhanced the cytotoxicity of epacadostat only in CR cells rate-limiting step in the serotonin pathway. TPH mRNA levels were (Fig. 5B) providing additional validation of CR cell's dependence on extremely low and downregulated in our NSCLC cell line models IDO. Henceforth, we selected the use of epacadostat to carry out the (Supplementary Fig. S4A). We then assayed for IDO1, IDO2, and subsequent experiments of IDO1 inhibition. We then determined the TDO2 (the rate-limiting step in KP) expressions. Even in the absence basal level of ROS in parental versus CR cells. ALC and FC cells of IFNg (cytokine inducer of IDO1), IDO1 mRNA expression was possessed 2.5- and 3.5-fold higher basal level of ROS (Fig. 5C, significantly higher in CR cells when compared with parental counter- respectively), and inhibiting IDO1 led to significantly higher ROS parts (Fig. 4B). However to our surprise, a surge in CR cells’ mRNA accumulation only in CR cells (Fig. 5D). It is noteworthy that expression did not significantly increase IDO1 protein levels (Fig. 4C). epacadostat did not affect IDO1 protein level as determined by IDO2 mRNA expressions were higher in CR cells but not as significant Western blot analysis (Fig. 5E), suggesting that epacadostat inhibits when compared with IDO1 (Supplementary Fig. S4B). As for TDO2, IDO1 enzymatic activity and not its expression in cells. this enzyme was not expressed in NSCLC cells but expressed only in brain cancer A172 cells utilized as positive controls (Supplementary Sensitivity to IDO1 inhibitor is correlated with higher Fig. S4C), thus indicating that TDO2 was not required in the formation ROS levels of KYN in lung cancer cells. We then assayed for IDO1 activity to It is also noteworthy that IFNg raised IDO1 protein expression determine whether increased Trp uptake in CR cells was being utilized slightly but not significantly in both parental and CR cell counterparts. by the KP. As depicted in Figure 4D, all CR cells possessed significantly We postulate here that the expression levels of IDO1 alone may not be higher IDO1 activity, which was further enhanced with IFNg treat- a sole indicator of IDO1 inhibitor sensitivity, and the efficacy of IDO1 ment but not in parental cells. inhibition may depend on ROS levels. In further support of this notion, Our data strongly support the concept that the level of IDO1 we found that a stepwise increase in cisplatin resistance (Fig. 6A)is expression alone may not be the crucial factor in determining its correlated with the progressive increase in ROS production (Fig. 6B)

Figure 4. IDO1 expression alone is not the only factor in determining its activity. A, Diagram illustrat- ed that tryptophan can be used to generate serotonin and KYN. B, Relative mRNA levels of IDO1. Total RNAs extracted from these cells were reverse-transcribed and subsequently used as template for real-time quan- titative PCR. GAPDH was used as internal control. The results shown in the graph were calculated with the DDCt method by setting the IDO1 mRNA level of LL24 cells as 1 (, P ¼ 0.005, , P ¼ 0.01). C, Immunoblot of lung cancer cell lines treated with and without IFNg (20 ng/mL) for 24 hours. No signiﬁcant differences in IDO1 protein expression levels were observed between parental and CR cells. D, Signiﬁcantly higher IDO1 activity was found in CR cells when compared with parental cell counterparts (, P ¼ 0.02, , P ¼ 0.03) and was further enhanced with IFNg (20 ng/mL) treatment for 24 hours (, P ¼ 0.03, , P ¼ 0.008).

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Figure 5. Antitumor activity of IDO inhibitors in CR cells. A, Growth-inhibitory effect of various IDO inhibitors (epacadostat: EPA, NLG-919: NLG, PF-06840003: PF, or indoximod: INDO) for 48 hours showed that CR cells were sensitive to IDO1 inhibitors with epacadostat yielding the best efficacy ( , P ¼ 0.03, , P ¼ 0.02). B, ID50 dosage of epacadostat alone and in combination with 20 ng/mL of IFNg. Combination treatment enhances epacadostat toxicity only in CR cells. (mean SD of three experiments, 48 hours). C, ROS analysis detected by CM-H2DCFDA probe indicated that CR cells expressed higher basal levels of ROS. D, ROS levels were heightened when treated with 15 mmol/L of epacadostat for 48 hours (, P ¼ 0.009, , P ¼ 0.005). Bar graph represents the relative fluorescent units/cell via fluorometer plate reader. E, Immunoblot of lung cancer cell lines treated with and without epacadostat (15 mmol/L/mL) for 48 hours. and escalating sensitivity to epacadostat (Fig. 6C). These findings found no further effect in the absence of IDO (ALCshIDOA&B), suggested that when cancer cells acquired greater resistance to cis- consistent with the specificmodulationofROSbyIDOinthese platin, ROS production also progressively increased. Consequently, cells. More importantly, silencing IDO suppressed CR cell growth, activation of KP is amplified leading to increasing sensitivity to IDO1 which further implicated the important role of KP in proliferation inhibitor. of CR tumors (Fig. 6E). Moreover, the chemical inhibition of IDO To establish the relationship between IDO1 and ROS, in our lung with epacadostat in already knocked down cells did not result in cancer and CR models, we inhibited IDO1 using shRNA as previ- further cell death indicating the specificity of the inhibitor for the ously shown (Fig. 2F). As we expected, knockdown (shIDO1) IDO1 enzyme. Exposing CR cells to 1 mmol/L of TIRON (mito- enhanced ROS production in ALCshIDOA&B due to the fact that chondria-localized antioxidant; ref. 38) resulted in significant sup- IDO metabolizes ROS as a substrate (Fig. 6D). The addition of pression of ROS production (Fig. 6F). Importantly, treatment of epacadostat to inhibit IDO only increased ROS in shCTRL and TIRON decreased IDO1 activity (Fig. 6G) and conferred resistance

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Figure 6. Sensitivity to IDO1 inhibitor is depen-

dent on higher ROS levels. A, ID50 dosage of cisplatin in parental cell line “A” and its cisplatin resistance variants. B, Intracellular ROS production measured by ﬂuorescence intensity

using CM-H2DCFDA probe. C, ID50 dosage of epacadostat (EPA) and its cisplatin resistance variants. CR2, CR4, and CR6 possessed 2-, 4-, and 6-fold resistance to cisplatin, respectively (mean SD of three experiments, 48 hours). D, Knocking down of IDO1 (shIDO-A and shIDO-B) further enhanced ROS level in CR cells (, P ¼ 0.02,, P ¼ 0.03); however, treatment with 15 mmol/L of epacadostat for 48 hours did not produce additional ROS accumulation in ALC- shIDO. E, Growth-inhibitory effect of epacadostat (48 hours) on ALC and ALCshIDO. Epacadostat signiﬁcantly suppressed ALC cells’ growth (, P ¼ 0.002), and no signiﬁcant growth inhibition was observed in the knocked down clones. F, Intracellular ROS production. Antioxidant (TIRON) suppressed ROS production in CR cells (, P ¼ 0.003, , P ¼ 0.006). G, Treatment of TIRON (48 hours) attenuated IDO1 activity (, P ¼ 0.03) in CR cells. H, TIRON treatment conferred resistance to epacadostat in CR cells (48 hours; , P ¼ 0.02). I, CR cells do not primarily utilize glucose but rather consume amino acids such as glutamine and tryptophan for survival. This metabolic switch is due to increased ROS production hyper- activating KP to balance oxidative stress and maintain cellular growth and proliferation. KYN is oxidized through indoleamine 2,3-dioxygenase (IDO) and plays a key role in reprogramming na€ve T cells to the immune-suppressive Treg phenotype. Further increase in ROS by inter- fering with tumor metabolism via IDO1 inhibition will selectively target these cisplatin-resistant lung cancer cells.

to epacadostat (Fig. 6H) in CR cells. Taken together, our results to treatment (2, 6). Among these metabolic changes is the use of indicated that the IDO1 activity, as well as sensitivity to IDO1 glutamine and fatty acids by CR cells as a carbon skeleton source in inhibitor, was dependent on ROS levels. metabolism instead of glucose (2, 6). Another important ﬁnding was the detection of signiﬁcantly higher basal levels of ROS accumulation in proliferating CR cells (3). Here, we have uncovered a unique Discussion metabolic difference wherein CR cells possess higher basal levels Over the past few years, we have discovered that metabolic repro- of ROS, which in turn activates the KP for Trp catabolism. Impor- gramming in CR cells contributes to various mechanisms of resistance tantly, KP generates KYN, which has been shown to reprogram na€ve

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T cells to the immune-suppressive regulatory T-cell (T-reg) phenotype often expressed in late-stage lung tumors with no study conducted in (Fig. 6I). CR tumors (Supplementary Fig. S5). In addition, many studies have Because ROS is a substrate for IDO1 in the oxidation reactions reported no significant correlation between IDO1 expression and yielding KYN, in this study we inhibited IDO1 [i.e., IDO inhibitor clinicopathologic parameters (52, 53), although the concept of poten- (epacadostat) or shIDO] leading to higher ROS (Fig. 6). This tial therapeutic efficacy for IDO1 inhibitors has been promoted (46). induction in ROS accumulation pushed CR cells beyond a threshold But the higher basal levels of ROS we have reported in CR cells will lead of ROS tolerance resulting in greater growth inhibition. Moreover, to a higher efficacy of the IDO1 inhibitor to decrease the level of KYN. epacadostat did not suppress IDO1 protein expression levels, Our studies have evaluated the metabolic background of these tumors, suggesting that epacadostat inhibits IDO1 enzymatic activity and adding another novel level of molecular effectors that can influence not its expression in cells. Nevertheless, these results clearly illus- IDO1 activity and any acquired resistance to the immune response. trate that survival of CR cells can be influenced by changes in ROS Thus, we believe that IDO1 expression level alone should not be used as levels. a sole factor in the determination of clinical benefit/tumor response to To further investigate the specific role of Trp in CR cells, we IDO1 inhibitor. þ investigated the production of KYN and NAD . As described previ- Activation of IDO1 enzymatic activity requires ROS as a cofactor; ously, Trp is oxidized through IDO1 to generate KYN. Here, we hence, activity along with expression should be evaluated in the reported that CR cells consumed Trp at a higher rate and generated clinic. In previous work, we reported that parental (or na€ve þ more KYN but not NAD . In fact, we have also previously reported treatment) cancer cells can upregulate both GSH and thiore- that NMNAT2 (a cytosolic enzyme that represents the final step in the doxin-1 (TRX1) antioxidant activities to keep ROS in check where þ biosynthesis of NAD in both de novo and salvage pathways) expres- CR cells cannot (3). Therefore, in the presence of active antiox- sion was significantly lower in CR cells (2). These findings strongly idants, our findings here may explain why less IDO1 activity was confirm that Trp is being used as a precursor for KYN production in found in parental tumors despite the expression of IDO1 protein. CR cells. The Trp consumption rate can also be utilized to stratify As for IDO2, a recent report showed that indoximod, which acts tumors on the basis of this metabolic attribute. downstream of IDO1/2 to suppress mTORC1 activity, exerted a Increased amino acid transport [via evaluation of LAT1 (CD98)] variety of benefits as an immunometabolic adjuvant (54). This is has also been reported in many types of cancers including lung interesting because we have previously found that certain CR cells cancer, providing some insight into molecular modifications in possessed higher mTORC1 activities, and inhibiting mTOR with tissue metabolism along tumor development (39, 40). In fact, all CCI-779 can restore cisplatin sensitivity (22) in these CR cells. In our CR cells possess higher LAT1 levels when compared with the future, we plan to investigate the role of IDO2 in CR tumors by parental cell counterparts, and treatment with the IDO1 inhibitor inhibiting both IDO2 and mTOR in our tumor models. suppressed LAT1 expression. Interestingly, we previously reported A recent phase III ECHO-301/KEYNOTE-252 (NCT02752074) that CR cells possessed higher expression of the glutamate/cystine/ clinical trial tested the efficacy of epacadostat in combination with xCT antitransporter pump (2), and LAT1 is a major component of pembrolizumab in patients with advanced melanoma previously this plasma membrane antiporter. LAT1 facilitates the cellular untreated with PD-1 or PD-L1 checkpoint inhibitors (55). The trial uptake of extracellular cystine in exchange for intracellular gluta- evaluated the potential enhancement of anti-PD-1 therapy via IDO1 mate and plays a key role in glutathione (GSH) synthesis. The inhibition by gathering data on progression-free survival, overall activity of CD98/xCT-mediated cystine uptake in cancer cells is survival, and adverse events but did not yield satisfactory results and known to be highly associated with tumor growth and chemore- was stopped early showing no indication that Epacadostat provided an sistance (41, 42). Thus, the important role of LAT1 in altered tumor increased benefit. Various limitations to the study parameters have metabolism is not limited to Trp uptake but also extends to the been proposed such as the degree of adequate IDO1 inhibition within homeostatic regulation of antioxidant and ROS. the tumor based on dosage and through sufficient drug exposure, use of Interestingly, we did not find an increase in LAT1 levels in combination therapies with DNA-damaging modalities, or the use of parental cells after KYN treatment, even though more AHR was IDO and TDO inhibition including dual inhibitors and AHR inhibi- translocated into nucleus. This may be due to the fact that parental tors (56). Perhaps future studies of such IDO and other inhibitors cells possessed significantly higher levels of HIF1a, which has a should include the evaluation of some metabolic factors such as much better binding affinity to HIF1b than AHR (30, 43). Thus, in elevated catabolism of Trp that affect the tumor microenvironment the presence of HIF1a,ARNT(HIF1b) is not readily available to and the suppression of immunity (57). bind and form a complex with KYN/AHR. In addition, we have Herein, we have gathered specific evidence to describe the effects of previously reported that CR cells secrete less lactate (2, 6) which IDO, ROS, the KYN/Trp ratio in lung-mediated modifications of the may very well impact the fate of T cells. Lactic acid can be taken up tumor microenvironment, and the potential suppression of antitumor by neighbor cells, including immune cells, to perpetuate the gly- immunity. We believe that higher ROS levels together with signifi- colytic pathway and upregulate HIF1a (44, 45). In fact, effector T cantly increased serum KYN/Trp ratios can be used to select patients cells (T-eff) rely on glycolysis to expand and this growth is greatly for future treatment using IDO1 inhibitors. Such a stratification or dependent on HIF1a (46). A diminished amount of lactate may patient grouping may increase efficacy in epacadostat trials. It is also prevent a robust immune response to the neighboring tumor. noteworthy that even though patients with cancer from our study Furthermore, mice deficient in HIF1a fail to mount a strong T- possessed higher basal levels of KYN/Trp ratio when compared with cell response and have increased T-reg populations (47, 48). All of healthy controls, these levels were negligible when compared with these findings support a concept wherein the metabolic modifica- patients who failed cisplatin and radiotherapy. Thus, the question tions in CR tumors provide a growth advantage, which in turn remains on how high is high enough to use the KYN/Trp ratio as a impacts immune cells in the tumor microenvironment. biomarker for predicting the efficacy of IDO inhibitors. Our work has Although high IDO expression is a suggested predictor for poor some limitations with only six patients, but we are currently recruiting prognosis and not associated with improved survival (49, 49, 51), it is more patients to our study who have failed cisplatin and radiotherapy.

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In the context of a secondary treatment compendium based on IDO Analysis and interpretation of data (e.g., statistical analysis, biostatistics, inhibition, the KYN/Trp ratio may simultaneously signal a need for a computational analysis): D.J.M. Nguyen, G. Theodoropoulos, Y.-Y. Li, C. Wu, shift away from cisplatin therapy for some patients in anticipation of W. Sha, T.J. Lampidis, N. Savaraj, M. Wangpaichitr Writing, review, and/or revision of the manuscript: D.J.M. Nguyen, the development of resistance. G. Theodoropoulos, Y.-Y. Li, L.G. Feun, T.J. Lampidis, N Savaraj, M. Wangpaichitr We firmly believe that IDO inhibitors may be more beneficial Administrative, technical, or material support (i.e., reporting or organizing data, in the treatment of patients with lung cancer who failed constructing databases): G. Theodoropoulos, M. Wangpaichitr cisplatin therapy than na€ve treatment patients. The data pre- Study supervision: M. Wangpaichitr sented herein provide a more comprehensive molecular inves- tigation of metabolic pathway components in lung tumor cells Acknowledgments that are resistant to cisplatin, characterizing variations that are The authors thank Drs. Mustafa Tekin and Jingyu Huang (Division of Clinical and Translational Genetics, University of Miami) for helping them proving to be valuable in survival mechanisms in resistant lung with amino acid levels analysis. This work is supported by BLR&D Merit cancer cells. Review Award (1BX003328; to N. Savaraj) and by BLR&D Career Development Award-2 (1K2BX001289) and Merit Review Award (1I01BX004371; to M. Disclosure of Potential Conflicts of Interest Wangpaichitr). No potential conflicts of interest were disclosed. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Authors’ Contributions advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Conception and design: Y.-Y. Li, T.J. Lampidis, N. Savaraj, M. Wangpaichitr this fact. Development of methodology: W. Sha, M. Wangpaichitr Acquisition of data (provided animals, acquired and managed patients, provided Received March 6, 2019; revised July 9, 2019; accepted October 15, 2019; facilities, etc.): D.J.M. Nguyen, C. Wu, W. Sha, M. Wangpaichitr published first October 18, 2019.

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AACRJournals.org Mol Cancer Res; 2019 OF13

Targeting the Kynurenine Pathway for the Treatment of Cisplatin-Resistant Lung Cancer

Dan J. M. Nguyen, George Theodoropoulos, Ying-Ying Li, et al.

Mol Cancer Res Published OnlineFirst October 18, 2019.

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