The Interleukin 22 Pathway Interacts with Mutant KRAS to Promote Poor Prognosis in Colon Cancer Sarah Mccuaig1, David Barras2, Elizabeth H

Published OnlineFirst May 19, 2020; DOI: 10.1158/1078-0432.CCR-19-1086

CLINICAL CANCER RESEARCH | PRECISION MEDICINE AND IMAGING

The Interleukin 22 Pathway Interacts with Mutant KRAS to Promote Poor Prognosis in Colon Cancer Sarah McCuaig1, David Barras2, Elizabeth H. Mann1, Matthias Friedrich1, Samuel J. Bullers1, Alina Janney1, Lucy C. Garner1, Enric Domingo3, Viktor Hendrik Koelzer3,4,5, Mauro Delorenzi2,6,7, Sabine Tejpar8, Timothy S. Maughan9, Nathaniel R. West1, and Fiona Powrie1

ABSTRACT ◥ Purpose: The cytokine IL22 promotes tumor progression in Results: Transcriptomic analysis revealed a poor-prognosis murine models of colorectal cancer. However, the clinical subset of tumors characterized by high expression of IL22RA1, significance of IL22 in human colorectal cancer remains the alpha subunit of the heterodimeric IL22 receptor, and KRAS unclear. We sought to determine whether the IL22 pathway mutation [relapse-free survival (RFS): HR ¼ 2.93, P ¼ 0.0006; is associated with prognosis in human colorectal cancer, and to overallsurvival(OS):HR¼ 2.45, P ¼ 0.0023]. KRAS mutations identify mechanisms by which IL22 can influence disease showed a similar interaction with IL10RB and conferred the worst progression. prognosis in tumors with high expression of both IL22RA1 and Experimental Design: Transcriptomic data from stage II/III IL10RB (RFS: HR ¼ 3.81, P ¼ 0.0036; OS: HR ¼ 3.90, P ¼ 0.0050). colon cancers in independent discovery (GSE39582 population- Analysis of human colorectal cancer cell lines and primary tumor based cohort, N ¼ 566) and verification (PETACC3 clinical trial, organoids, including an isogenic cell line pair that differed only in N ¼ 752) datasets were used to investigate the association KRAS mutation status, showed that IL22 and mutant KRAS between IL22 receptor expression (encoded by the genes cooperatively enhance cancer cell proliferation, in part through IL22RA1 and IL10RB), tumor mutation status, and clinical augmentation of the Myc pathway. outcome using Cox proportional hazard models. Functional Conclusions: Interactions between KRAS and IL22 signaling interactions between IL22 and mutant KRAS were elucidated may underlie a previously unrecognized subset of clinically using human colorectal cancer cell lines and primary tumor aggressive colorectal cancer that could benefitfromtherapeutic organoids. modulation of the IL22 pathway.

Introduction inflammatory cytokines control several key features of malignancy, including cancer cell proliferation, survival, and dissemination (5). Colorectal cancer is a complex disease driven by the interplay Evidence from preclinical murine models of both sporadic and between tumor mutations, environmental factors, and aberrant immu- colitis-associated colorectal cancer has implicated IL23 and its down- nity (1, 2). Chronic intestinal inflammation (e.g., inflammatory bowel stream effector IL22 in the initiation and progression of colon disease) is a well-known risk factor for colorectal cancer, but colitis- tumorigenesis (6–9). Furthermore, IL22 has been associated with associated cancers account for only a small fraction of total tumor human gastric cancer progression (10) and has been described to incidence (3). However, colorectal cancers that are not associated with þ promote colorectal cancer stemness (11). IL23-responsive CD4 T colitis also elicit inflammatory responses, which are thought to be key cells and innate lymphoid cells secrete IL22 in the tumor microenvi- regulators of tumor progression and therapeutic resistance (4). Indeed, ronment (7, 8), where it signals through a heterodimeric receptor comprised of IL10 receptor beta (IL10RB) and IL22 receptor alpha 1 (IL22RA1; ref. 12). In the intestine, IL22RA1 expression is restricted to

1 the epithelium (13) and activates diverse signal transduction pathways Kennedy Institute of Rheumatology, University of Oxford, Oxford, United in response to IL22, including JAK/STAT3, MAPK, PI3K, and NF-kB, Kingdom. 2SIB Swiss Institute of Bioinformatics, Bioinformatics Core Facility, Lausanne, Switzerland. 3Department of Oncology, University of Oxford, Oxford, which collectively induce expression of genes involved in cell-cycle United Kingdom. 4Nuffield Department of Medicine, University of Oxford, progression and inhibition of apoptosis (14–16). Oxford, United Kingdom. 5Department of Pathology and Molecular Pathology, Oncogenic Ras isoforms modulate inflammatory pathways in University Hospital Zurich, Zurich, Switzerland. 6Ludwig Center for Cancer cancer by directly influencing inflammatory cytokine expression and 7 Research, University of Lausanne, Lausanne, Switzerland. Department of signal transduction (5). IL23, IL17, and IL22 are elevated in human Oncology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, 8 colorectal cancer, and their expression is modulated by the presence Switzerland. Molecular Digestive Oncology, KU Leuven, Leuven, Belgium. KRAS 9CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, of mutant Ras (17). Activating mutations in , a major Ras Oxford, United Kingdom. isoform, occur in 40% to 45% of colorectal cancers and drive constitutive activation of the Ras-Raf-MEK-ERK pathway (18). Note: Supplementary data for this article are available at Clinical Cancer KRAS Research Online (http://clincancerres.aacrjournals.org/). Although mutations are strongly associated with resistance to EGFR-targeted therapy, they do not correlate with resistance to Corresponding Author: Fiona Powrie, University of Oxford, Roosevelt Drive, chemotherapy (19–21). However, given the cytokine-modulating Headington, Oxford OX3 7YF, United Kingdom. Phone: 01865-612-600; E-mail: KRAS fi[email protected] capacity of the Ras family, mutations may be clinically significant in tumors with active cytokine signaling. Oncogenic KRAS Clin Cancer Res 2020;XX:XX–XX could influence virtually all signal transduction pathways downstream doi: 10.1158/1078-0432.CCR-19-1086 of IL22, but a specific interaction between IL22 signaling and KRAS 2020 American Association for Cancer Research. mutation has not been described.

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arrayexpress) on the A-AFFY-101 platform (customized Affymetrix Translational Relevance chip) and is a merge of E-MTAB-863 and E-MTAB-864 (26). Clinical IL22 promotes tumor progression in preclinical models of information on overall survival (OS) was available for 1,734 patients and colorectal cancer, but its importance in human colorectal cancer on relapse-free survival (RFS) for 1,499 patients. Gene expression remains unclear. Using a rigorous discovery/verification analysis of profiles of the patients in the combined dataset were merged at the tumor gene expression from over 1,000 patients, we have discov- gene level and then normalized and corrected for batch effects using the ered that among patients with colorectal cancer with high expres- Combat R package. For the individual analyses of the GSE39582 and sion of either or both subunits of the heterodimeric IL22 receptor, PETACC3 datasets, gene expression profiles from each of the datasets KRAS mutation confers poor prognosis. Functional studies were used independently (not normalized to the others). Tumors revealed that this association is due to an interaction between IL22 proximal to the splenic flexure were defined as proximal, and tumors signaling and oncogenic KRAS that enhances tumor cell prolifer- distal to the splenic flexure were classified as distal. ation through induction of the Myc pathway. These findings All stage II/III patients determined to have a KRAS-mutant tumor demonstrate that cell-intrinsic drivers of colorectal cancer can were included in the analysis. The KRAS mutation sites by codon interact with cell-extrinsic factors from the inflammatory micro- (where available) are detailed for each of the datasets in Supplementary environment to influence disease progression. Our data further Fig. S1B. justify the assessment of KRAS mutations in patients with colo- The interaction between mutant KRAS and all available cytokine/ rectal cancer, which has until now been clinically beneficial only for cytokine receptor genes was assessed by Cox proportional hazards tests prediction of cetuximab responsiveness, and suggest that for for interaction using the combined dataset. The top tertile was KRAS-mutant colorectal cancers with high IL22 receptor expres- considered high expression, and genes which yielded an uncorrected sion, closer monitoring and more aggressive or alternative ther- P value of less than 0.05 were considered for further analysis. apeutic strategies (e.g., antibody-based blockade of IL22) could be beneficial. Histotype analysis Tissue composition of the TCGA and an independent cohort, Stratification in COloRecTal cancer (S:CORT), was assessed by visual pathology review. The S:CORT cohort is composed of 385 In light of the strong evidence pointing toward a protumorigenic formalin-fixed, paraffin-embedded (FFPE) tumors from the FOCUS- role for IL22 in murine models of colorectal cancer, we sought to randomized clinical trial which assessed response to different chemo- determine the clinical importance of this cytokine in human disease therapy regimens in patients with advanced colorectal cancer (27). and to identify subtypes of colorectal cancer in which IL22 signaling Hematoxylin and eosin slides were scanned at high resolution with an may be particularly influential. Our results identify a previously Aperio scanner at a total magnification of 200. Tissue areas were unrecognized interaction between IL22 signaling and oncogenic KRAS annotated using the Indica Labs HALO digital pathology platform. that contributes to tumor cell proliferation and poor patient prognosis. Small image tiles representing tumor epithelium, desmoplastic stroma, inflamed stroma, mucin hypocellular stromal areas, necrosis, and glass background were generated from >1,500 pathologist-annotated tissue Materials and Methods areas. A deep neural network algorithm (Simoyan and Zisserman Prognostic study design VGG https://arxiv. org/abs/1409.1556) was trained to segment Four colorectal cancer transcriptomic datasets were analyzed in this and quantify individual tissue areas as described in ref. 28. Area study (Supplementary Fig. S1A). The French National Cartes measurements were normalized by total stromal content. Tumors d'Identite des Tumeurs (CIT) cohort (GSE39582; ref. 22) contains were stratified into molecular subgroups based on IL22RA1 mRNA 469 patients with stage II/III colorectal cancer who underwent surgery expression (as described above) and KRAS mutation status. between 1987 and 2007 at 7 different centers. Patients who received preoperative chemotherapy/radiotherapy were excluded from the Gene set enrichment analysis and consensus molecular subtype study. RNA from fresh-frozen primary tumors was assessed using analysis the HG-U133A Affymetrix platform, which contains one probe that Differential expression analysis between IL22RA1-high, KRAS MUT detects IL22RA1 (220056_at). and IL22RA1-high, KRAS-WT tumors in the combined dataset was The Pan-European Trials in Alimentary Tract Cancers (PETACC3) performed using the limma R package (version 3.24.4). Using the t cohort (NCT00026273) was used as a verification set (ArrayExpress:E- statistics from the differential expression analysis as a weighting factor, a MTAB-990). Data from stage II (n ¼ 108) and stage III (n ¼ 644) preranked gene set enrichment analysis (GSEA) using the GenePattern colorectal cancer were analyzed (23, 24). Gene expression data were interface from the Broad Institute (genepattern.broadinstitute.org/) was obtained using the ALMAC Colorectal Cancer DSA platform, which is performed. GSEA was run on MYC_TARGETS_V2 signature from the a customized Affymetrix chip that includes 61,528 probe sets mapping MSigDB portal (http://www.broadinstitute.org/gsea/msigdb). to 15,920 unique Entrez Gene IDs. The PETACC3 dataset contains Tumors from the combined dataset were classified according to the three different probes for IL22RA1. The probe used in our analysis was “consensus molecular subtypes” (CMS) of colorectal cancer defined by that which displayed the greatest variation in the dataset Guinney and colleagues (29). Tumors were then stratified according to (ADXCRAD_BX089163_s_at). Of note, the second probe was not IL22RA1 and KRAS mutation status (as described above). The pro- well mapped, and the third probe had a very narrow dynamic range. portion of tumors in each CMS in the molecular subtypes of interest is Finally, a merged dataset comprising patients with stage II/III displayed. colorectal cancer of GSE39582 (22), PETACC3 (23, 24), The Cancer Genome Atlas (TCGA; ref. 25), and ALMAC (26), representing 1,820 Colorectal cancer cell lines patients, was analyzed (referred to in tables as the “Combined” dataset). The DLD-1 isogenic pair was purchased from Horizon Discovery The ALMAC dataset was obtained from ArrayExpress (www.ebi.ac.uk/ Group [Parental Line (KRAS G13D): HD PAR-111 Passage 4;

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fi fi Wild-type reverted line (KRAS ): HD 105-040 Passage 4]. Experi- 37 C with 5% CO2. Cells were xed [Cyto x (BD)] and permeabilized ments with the isogenic pair were performed between passages 4–9. [Perm III Buffer (BD)] according to the manufacturer's protocol and KRAS mutation status in the isogenic pair was confirmed in-house stained for 1 hour at room temperature with fluorescently conjugated using the qBiomarker Somatic Mutation PCR Array Human KRAS antibodies to phosphorylated residues on intracellular signaling pro- Gene (Qiagen, Plate E Format, SMH-806ARE-12 337021). Colo205, teins of interest [anti-pSTAT3-Y705 (1:10; AF-647; BD; Clone: 4) and SW480, and DLD-1 cells were a generous gift from Dr. Simon anti-ERK1/2 (pT202/pY204; 1:10; AF-647; BD; Clone: 20A (RUO))]. Leedham (Wellcome Trust Centre for Human Genetics, Oxford, Flow cytometric analysis was performed on an LSR or Fortessa X-20 UK). Colo205 and DLD-1 cells were maintained in RPMI medium (BD). Data analysis was performed using FlowJo 10 software (Sigma) with 10% FBS and 1% penicillin/streptomycin (P/S; Sigma). (Tree Star). SW480 cells were cultured in DMEM (Sigma) with 10% FBS and 1% P/S. Cultures were maintained in 37 Cand5%CO2. All cell Proliferation assays lines were tested for mycoplasma, and determined to be mycoplas- Cells, 1 104 per well, were seeded in 48-well plates and allowed to ma free. adhere overnight. Cells were stimulated with 0 to 200 ng/mL IL22 or IL6 for 24, 48, and 72 hours. Cellular viability/proliferation was Cytokine stimulation experiments measured by adding 50 mg of MTT to each well 2 hours prior to the Cells, 3 104 per well in 48-well plates, were cultured overnight. end of the IL22 incubation. At the end of the incubation, supernatants Cytokines [recombinant human IL22 (R&D Systems) and recombi- were aspirated, formazan particles were solubilized with DMSO, and nant human IL6 (Peprotech)] were prepared in cell line–specific media samples transferred to a flat-bottom 96-well plate (Costar). Absor- at the concentrations indicated. All cytokine stimulation experiments bance was measured at 540 nm on a BMG SPECTROstar NANO in the DLD-1 isogenic pair were performed in serum-free conditions Microplate Reader (BMG Labtech). with 1X ITS (Insulin, Transferrin, Selenium; Sigma). Survival assays confirmed equal viability of both KRAS G13D and KRAS DLD-1 siRNA knockdown cells in serum-free media with 1X ITS. Cells, 3 104 per well in 48-well plates, were seeded and allowed to adhere overnight. siRNA was incubated with DharmaFect 2 (Dhar- RNA extraction, cDNA synthesis, and qPCR macon) in Opti-MEM (Thermo Fisher) for 20 minutes prior to RNA was extracted using the Qiagen RNeasy Mini Kit (Qiagen) transfection, and cells were transfected using 0.8 mL DharmaFect 2 according to the manufacturer's instructions. cDNA was synthesized and 40 nmol/L ON-TARGET Plus Human MYC SMARTpool (Dhar- using the High Capacity cDNA Kit (Applied Biosystems) according to macon) or ON-TARGET Plus control pool (Dharmacon). Media were the manufacturer's protocol using a C1000 Touch Thermal Cycler replaced with serum-free RPMI with 1X ITS (Sigma) after 48 hours. (Bio-Rad). Real-time qPCR analysis was performed using TaqMan Real-Time PCR assays (Life Technologies) and Precision Fast qPCR Primary colorectal cancer whole tissue qPCR analysis and Mastermix with ROX at a lower level, with inert blue dye (PrimerDe- organoid culture sign) and run on a ViiA7 Real-Time PCR System (Applied Biosys- Patients diagnosed with colorectal cancer scheduled for colectomy at DCT ¼ tems). Data were analyzed using the DCT method [2 where DCT the Churchill Hospital (Oxford, UK), were consented for research use – RPLPO CT(target gene) CT(endogenous control)]. was used as the endog- of their resected cancer and matched normal mucosa during a preop- enous reference gene for all qPCR analyses. erative appointment. Ethical approval for the study was provided by the National Research Ethics Committees of the UK National Health Immunoblotting Service (NHS) in accordance with the Declaration of Helsinki Cells were lysed using a solution of 50 mmol/L Tris, pH 6.8, (Reference numbers: 11/YH/0020, 16/YH/0247) under the HTA license 20 mmol/L EDTA, 5% SDS, 1 mmol/L DTT, and 10% glycerol. Equal 12217. The human samples used in this publication were ethically protein amounts (15–20 mg) were loaded into precast NuPAGE Novex sourced, and their research use was in agreement with the written 4%–12% Bis-Tris Gels (Life Technologies), separated by SDS-PAGE, informed consent. Tumor mutation status was obtained from the and transferred onto Immobilon-FL PVDF membranes (Millipore). Oxford Molecular Diagnostics Centre. Detailed protocols for human Membranes were blocked (Odyssey Blocking Buffer; Li-COR), incu- organoid culture were generously provided by Marc van de Wetering bated with primary antibody [total c-Myc (1:1,000; Cell Signaling (Hubrecht Institute, Netherlands). Fresh colorectal cancer biopsies (2 Technology; Clone: D84C12), b actin (1:4,000; Sigma; Clone: Ac-74)] mm3) were homogenized using Soft Tissue Homogenizing CK14 Kit overnight at 4C, and fluorescently conjugated secondary antibodies vials (Stretton Scientific) in a Precellys 24 (Bertin Instruments) homog- (goat anti-rabbit-800CW; 1:15,000; Li-COR; Clone: 925-32211) for enizer (30seconds at 3,500 RPM). RNA extraction, cDNA synthesis, and 1 hour at room temperature before detection using the Odyssey CLx qPCR analysis were performed as described above. detection system (Li-COR). Five to 7 mm fresh punch biopsies of resected tumor were used to establish organoid cultures as described (30). Genomic DNA was Flow cytometry extracted from organoids using the QiaAmp DNA MiniKit (Qiagen) For analysis of IL22RA1 expression, adherent cells were dissociated and used for KRAS mutation typing. KRAS mutation status was using TrypLE (Life Technologies), filtered through 70 mm filters, and determined using the qBiomarker Somatic Mutation PCR Array stained with anti-IL22RA1 (1:10; PE; R&D; Clone 305405) or matched Human KRAS Gene (Qiagen, Plate E Format, SMH-806ARE-12 isotype control (1:10; anti-mouse IgG1k; PE; R&D; Clone 11711), along 337021) assay according to the manufacturer's protocol. KRAS muta- with anti-EpCAM (1:100; CD326; FITC; Biolegend; Clone 9C4) and a tion status clinically determined from whole tissue sections was fixable viability dye (1:1,000; APC Cy7; eBioscience) for 30 minutes at compared with organoid KRAS mutation status to ensure concordance. room temperature before fixation in Fix/Lyse Solution (BD). For proliferation assays, organoids were dissociated to single cells For Phosflow experiments, 1 106 cells were stimulated in technical with TrypLE, resuspended in reduced growth factor basement mem- duplicates with 0 to 100 ng/mL IL22 (R&D Systems) for 30 minutes at brane extract 2 (BME 2; Cultrex, Amsbio), and seeded in 5 mL BME in

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IL22RA1 96-well plates. Cells were stimulated with 1 ng/mL IL22 in tumor performed to determine an optimal cutpoint based on log2 organoid culture media (30). Media were replaced after 48 hours. expression values in the discovery cohort (GSE39582). The presence or Ninety-one hours after initial seeding, the media were refreshed again absence of disease relapse at last follow-up was used as the binary with 1 ng/mL IL22 where appropriate and 10% Alamar Blue (BioRad) variable in this analysis. The IL22RA1 cutoff value associated with the and incubated for an additional 5 hours. Absorbance was measured at maximum Youden index (J ¼ sensitivity þ specificity 1) was found 570 and 600 nm as a reference on a BMG SPECTROstar NANO to be the 67th percentile. This value was used for all subsequent Microplate Reader (BMG Labtech). The percent difference in Alamar analyses of IL22RA1 in the discovery and verification datasets, and was Blue reduction for each condition compared with the no treatment also used for analyses of additional cytokine/cytokine receptor genes. condition was determined. Contingency analysis (Fisher exact test with Bonferroni multiple For confocal microscopy, organoids were seeded onto autoclaved comparisons correction) was used to assess association of clinicopath- 9-mm diameter glass coverslips (VWR) in 24-well plates. Note that 400 ologic features with IL22RA1 expression status. Univariate, multivar- mL of organoid media with 1 ng/mL IL22, as appropriate, were added, iate, and interaction analyses of RFS and OS were performed using and organoids were incubated for 30 minutes. Organoids were then Cox's proportional hazard regression models fitted with the survival R fixed on the slides with 4% paraformaldehyde and permeabilized with package. Interaction analyses were used to assess interactions between methanol at 20C for 10 minutes. Nonspecific antibody binding was KRAS mutation status and the expression of cytokine and cytokine blocked by incubation with 10% goat serum in TBS, and organoids receptor genes. HRs were estimated with model coefficients and 95% were stained with primary antibodies [Phospho-Stat3 (Tyr705) XP confidence intervals (CI), and P values were computed with Wald tests. Rabbit mAb (1:1,000; 9145; clone D3A7; Cell Signaling Technology) Time-to-event curves were prepared using Kaplan–Meier methods in and anti–E-cadherin (1:300; 610181; clone 36/E-cadherin; BD)] over- GraphPad Prism 7. night at 4C, followed by staining with secondary antibodies [goat anti- For the analysis of in vitro experiments in cell lines and ex vivo rabbit IgG Alexa Fluor 555 (1:400; Life Technologies; A32732) and experiments in primary organoids, all statistical tests were two-sided goat anti-mouse IgG Alexa Fluor 488 (1:400; Life Technologies; and are specified in figure legends with exact P values displayed. The A11001)] for 1 hour at room temperature. Nuclei were stained with number of independent experiments performed for each in vitro assay Hoechst 33258 (1:20,000; Life Technologies) for 10 minutes at room is indicated in figure legends. A data point for a given independent temperature. Coverslips were inverted and mounted onto Superfrost experiment is the average of technical replicates (for which 2–3 were Plus slides (VWR). Images were acquired on an Olympus FV1200 IX83 performed for each independent experiment). Differences were con- Confocal System. sidered significant when P < 0.05. All line graphs display mean SEM. For organoid experiments, single dots represent a value from an RNA-sequencing individual patient (often the average of technical triplicates). Paramet- Three independent experiments were performed with DLD-1 iso- ric tests were used when n < 8. These analyses were performed using genic cells at passages 6–8. RNA was extracted following the proce- GraphPad Prism 7. dures described above. Library preparation, RNA-sequencing, and quality control were performed by the High-Throughput Genomics Group (Wellcome Trust Centre for Human Genetics, Oxford, UK). Results Bioanalysis of RNA was performed, and all samples had RNA integrity KRAS mutation is prognostic only in colon cancers that express number (RIN) ¼ 10. Samples were normalized to 1 mg total RNA, and high amounts of the IL22 receptor gene the mRNA fraction was isolated by polyA selection. Libraries were To assess the role of the IL22 pathway in colorectal cancer, we first prepared using the TruSeq Stranded mRNA Kit (Illumina) and paired- examined expression of IL22RA1 in stage II/II colorectal cancer end sequenced using the Illumina HiSeq4000 platform (Illumina). specimens using transcriptomic data from a publicly available pop- “Strandedness” was tested as part of internal quality control, and the ulation-based cohort (GSE39582, N ¼ 566; ref. 22; Supplementary proportion of reads mapping to the correct strand was found to be Fig. S1A). Findings were validated using gene expression data from very high. Adapter trimming was performed using cutadapt, align- patients with stage II (N ¼ 108) and stage III (N ¼ 644) colorectal ment was performed using hisat2, indexing was performed with cancer enrolled in the PETACC3 (NCT00026273) phase III clinical SAMBAMBA, and mapped reads were quantified using featurecounts. trial (N ¼ 752; refs. 23, 24). Finally, we analyzed a merged dataset Principal component analysis (R package prcomp version 3.3.1) comprised of patients with stage II/III colorectal cancer from was performed on all samples. Pathway analyses were performed by GSE39582, PETACC3, and two additional independent cohorts, single-sample GSEA (ssGSEA) using the GenePattern web interface TCGA and ALMAC (referred to in tables as the “Combined” dataset; from the Broad Institute (genepattern.broadinstitute.org/). The Hall- Supplementary Fig. S1A). After excluding patients with missing data mark collection of well-annotated gene sets from the MSigDB portal or nonstage II/III tumors, 1,083 patients remained for analysis in the (http://www.broadinstitute.org/gsea/msigdb) was used. Enrichment combined dataset (see CONSORT diagram; Supplementary Fig. S1A). scores from ssGSEA were used for differential analysis using the IL22RA1 was expressed as a continuous variable with a roughly normal generalized linear model as implemented in the limma package distribution (Supplementary Fig. S1C). To determine a cutoff value for (version 3.30.0). Differentially expressed pathways with an adjusted classification of IL22RA1-high versus low status, we employed P value <0.01 were considered significant. Heat mapping was per- ROC statistics and derived the 67th percentile as the cutoff for all formed using the pheatmap package (version 1.0.8). Sequencing data subsequent analyses. High IL22RA1 expression was not significantly were deposited in NCBI's Gene Expression Omnibus and are available associated with patient gender, tumor–node–metastasis stage, through the GEO Series accession number GSE149262. KRAS mutation, or BRAF mutation, but was negatively associated with microsatellite instability (MSI) and proximal tumor location Statistical analyses (Supplementary Fig. S1D). For prognostic studies in tumor transcriptomic datasets, all analyses In the GSE39582 discovery cohort, IL22RA1 expression had no were performed using R software (version 3.03). ROC analysis was impact on RFS or OS (Fig. 1A; Supplementary Fig. S2A). Given that

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Figure 1. KRAS mutation confers poor prognosis in stage II/III colorectal cancers expressing high amounts of IL22RA1. A–F, Kaplan–Meier estimates of RFS in stage II/III patients in the GSE39582 cohort based on (A) IL22RA1 expression and (B) KRAS mutation status. RFS among (C) IL22RA1-high and (D) IL22RA1-low patients based on KRAS mutation status. RFS among IL22RA1-high patients with tumors (E) proximal or (F) distal to the splenic ﬂexure based on KRAS mutation status. IL22RA1-high and low is deﬁned by a cutpoint at the 67th percentile determined using ROC analysis. P values computed using Wald tests. HRs are univariate Cox proportional HRs with 95% CIs for the GSE39582 cohort.

Ras is a mediator of IL22 signaling, we next stratified patients based on Table S2). Previous studies have demonstrated poor prognosis and both IL22RA1 expression and KRAS mutation status for further response to palliative chemotherapy in advanced colorectal cancers survival analysis. Consistent with prior reports (21), activating KRAS with mucinous histology (31, 32). Given that mucinous adenocar- mutations were modestly associated with poor clinical outcome (HR ¼ cinoma is associated with proximal tumor location (33), we eval- 1.57; 95% CI, 1.11–2.23; P ¼ 0.0103; Fig. 1B; Supplementary Fig. S2B). uated differences in the mucinous composition of IL22RA1-high However, among cases with high IL22RA1 expression, KRAS muta- proximal versus distal tumors. A histologic analysis was performed tions were strongly associated with poor RFS (HR ¼ 2.93; 95% CI, on colorectal cancer samples from the TCGA and an independent 1.59–5.43; P ¼ 0.0006; Fig. 1C) and OS (HR ¼ 2.45; 95% CI, 1.38–4.36; cohort, S:CORT, composed of 385 FFPE tumors from the FOCUS- P ¼ 0.0023; Supplementary Fig. S2C). By contrast, KRAS mutations randomized clinical trial which assessed chemotherapy response in had no prognostic impact in patients with IL22RA1-low tumors (RFS patients with advanced colorectal cancer (27). There were no HR ¼ 1.16; 95% CI, 0.76–1.78; P ¼ 0.4840; OS HR ¼ 1.05; 95% CI, differences in the mucinous composition of IL22RA1-high distal 0.69–1.61; P ¼ 0.813; Fig. 1D; Supplementary Fig. S2D). These versus proximal tumors (Supplementary Fig. S3C). findings were validated in the PETACC3 clinical trial cohort (Table 1) Low immune cell infiltration is a poor prognostic factor for many and in the large combined cohort (RFS HR ¼ 2.05; 95% CI, 1.45–2.89; tumor types, including colorectal cancer (34). Because IL22RA1 is not P < 0.0001; OS HR ¼ 1.65; 95% CI, 1.09–2.50; P ¼ 0.018; Table 1). expressed on hematopoietic cells, high IL22RA1 expression in speci- Multivariate interaction analysis revealed that the IL22RA1–KRAS mens analyzed by bulk tumor transcriptomics could be a surrogate for interaction was independent of tumor site, MSI status, BRAF mutation tumors with a paucity of immune infiltrate. In the GSE39582 discovery status, and sex (Table 2). cohort, IL22RA1 expression was not correlated with markers of Notably, the prognostic effect of the IL22RA1–KRAS interaction antitumor immunity (GZMB, IFNG, and CD8a; Supplementary was most profound in proximal (right-sided) tumors (RFS HR ¼ 4.23; Fig. S3A). As a control, IFNG was compared with GZMB and CD8a 95% CI, 1.38–13.01; P ¼ 0.012; OS HR ¼ 9.41; 95% CI, 2.13–41.60; in the cohort. Both comparisons revealed significant correlations, by P ¼ 0.003; Fig. 1E and F; Supplementary Table S1). This obser- Pearson correlation analysis (Supplementary Fig. S3A). To better vation was independent of MSI and BRAF mutation, both of characterize the cellular composition of tumors according to IL22RA1 which are common features of proximal tumors (Supplementary and KRAS status, a histologic analysis was performed on colorectal

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Table 1. Univariate Coxph survival analysis of patients with stage II/III colorectal cancer in discovery (GSE39582), veriﬁcation (PETACC3), and combined cohorts based on IL22RA1 and KRAS mutation status.

RFS OS NPHR (95% CI) NPHR (95% CI)

GSE39582 IL22RA1high/IL22RA1low 149/288 0.1700 0.77 (0.54–1.12) 150/292 0.3330 0.84 (0.59–1.19) KRAS MUT/KRAS WT 163/274 0.0103 1.57 (1.11–2.23) 165/277 0.0533 1.40 (1.00–1.96) Within IL22RA1low: KRAS MUT/KRAS WT 115/173 0.4840 1.16 (0.76–1.78) 117/175 0.8130 1.05 (0.69–1.61) Within IL22RA1high: KRAS MUT/KRAS WT 48/101 0.0006 2.93 (1.59–5.43) 48/102 0.0023 2.45 (1.38–4.36) Within KRAS WT: IL22RA1high/IL22RA1low 101/173 0.0365 0.57 (0.34–0.97) 102/175 0.0650 0.64 (0.40–1.03) Within KRAS MUT: IL22RA1high/IL22RA1low 48/115 0.1860 1.43 (0.84–2.42) 48/115 0.1930 1.43 (0.84–2.43) PETACC3 IL22RA1high/IL22RA1low 217/429 0.4160 1.11 (0.86–1.43) 217/429 0.7300 0.95 (0.70–1.28) KRAS MUT/KRAS WT 254/392 0.0215 1.34 (1.04–1.72) 254/392 0.0051 1.51 (1.13–2.02) Within IL22RA1low: KRAS MUT/KRAS WT 175/254 0.2660 1.19 (0.87–1.63) 175/254 0.1260 1.32 (0.92–1.88) Within IL22RA1high: KRAS MUT/KRAS WT 79/138 0.0149 1.68 (1.11–2.56) 79/138 0.0070 2.00 (1.21–3.30) Within KRAS WT: IL22RA1high/IL22RA1low 138/254 0.8800 0.97 (0.68–1.39) 138/254 0.3890 0.83 (0.54–1.27) Within KRAS MUT: IL22RA1high/IL22RA1low 79/175 0.1040 1.38 (0.94–2.02) 79/175 0.3670 1.22 (0.79–1.89) Combined IL22RA1high/IL22RA1low 372/711 0.9200 0.99 (0.83–1.19) 439/879 0.4170 0.93 (0.77–1.12) KRAS MUT/KRAS WT 417/666 0.0006 1.43 (1.16–1.75) 481/837 0.0054 1.35 (1.09–1.66) Within IL22RA1low: KRAS MUT/KRAS WT 287/424 0.1800 1.19 (0.92–1.53) 338/541 0.5030 1.09 (0.84–1.42) Within IL22RA1high: KRAS MUT/KRAS WT 130/242 0.0000 2.05 (1.45–2.89) 143/296 0.0001 2.07 (1.44–2.96) Within KRAS WT: IL22RA1high/IL22RA1low 242/424 0.1390 0.80 (0.60–1.07) 296/541 0.0468 0.74 (0.55–1.00) Within KRAS MUT: IL22RA1high/IL22RA1low 130/287 0.0465 1.37 (1.00–1.87) 143/338 0.0660 1.36 (0.98–1.89)

Note: IL22RA1-high and -low is defined by a cutpoint at the 67th percentile determined using ROC analysis in the discovery cohort. This cutpoint was used to define IL22RA1-high and -low in all downstream analyses. Cox proportional HRs for RFS and OS are displayed with 95% CI. Significant results are bolded. Patient number (N) in each subgroup is indicated.

cancer samples from the TCGA and S:CORT cohorts. The tumor cell the highest expression tertile for each gene as “high”) and KRAS density, inflamed stromal cell density, and nonneoplastic cell count did mutation status in the combined cohort (Table 2). To determine not differ between the molecular subtypes of interest (Supplementary whether other cytokines and/or their cognate receptors interact with Fig. S3B). Taken together, these data suggest that IL22RA1-high KRAS mutation in terms of survival, a Cox proportional hazard tumors do not simply represent a subset of tumors with low immune interaction analysis was performed on all cytokine and cytokine infiltrate. receptor genes (classifying the highest expression tertile for each gene as “high”) and KRAS mutation status in the combined cohort. High expression of either or both subunits of the heterodimeric Although several cytokine/cytokine receptor genes interacted with IL22 receptor confers poor prognosis in KRAS-mutant KRAS mutation, IL22RA1 displayed the strongest poor-prognosis colorectal cancer interaction (Fig. 2A). Similarly, IL10RB, which encodes the second Clinical and preclinical studies have suggested that several cyto- subunit of the heterodimeric IL22 receptor, was also a strong interactor kines, including IL6 and IL17A, may influence colorectal cancer (Fig. 2A). Detailed survival analysis in the discovery cohort progression (6, 35–40). However, we detected no interaction between (GSE39582) revealed that like IL22RA1-high tumors, KRAS mutation IL6R (IL6 receptor) or IL17RA (IL17 receptor) expression (classifying was selectively associated with poor prognosis in IL10RB-high tumors

Table 2. Univariate and multivariate Coxph interaction analysis between mutant KRAS and cytokine receptor expression for stage II and III patients from combined cohort.

RFS OS Univariate P HR (95% CI) P HR (95% CI)

IL22RA1 KRAS MUT 0.0065 1.76 (1.17–2.63) 0.0179 1.65 (1.09–2.50) IL6R KRAS MUT 0.4763 1.16 (0.77–1.75) 0.2205 1.30 (0.85–1.98) IL17RA KRAS MUT 0.5082 1.15 (0.76–1.76) 0.4399 0.84 (0.55–1.30) Site KRAS MUT 0.9535 0.99 (0.66–1.48) 0.8421 0.96 (0.64–1.43) Multivariate (BRAF, MSI status, gender, tumor site) IL22RA1 KRAS MUT 0.0031 1.89 (1.24–2.89) 0.0640 1.52 (0.98–2.36) IL6R KRAS MUT 0.6282 1.11 (0.73–1.70) 0.3577 1.23 (0.79–1.93) IL17RA KRAS MUT 0.7419 1.08 (0.69–1.68) 0.6561 0.90 (0.56–1.43)

Note: The top tertile for each cytokine receptor gene was classiﬁed as “high”. Cox proportional HRs for RFS and OS are displayed with 95% CIs. Signiﬁcant interactions are bolded. Covariates included in the multivariate analysis were: BRAF mutation status, microsatellite stability/instability status, gender, and tumor site.

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Figure 2. High expression of either or both subunits of the heterodimeric IL22R confers poor prognosis in stage II/III KRAS-mutant colorectal cancer (CRC). A, Univariate Cox proportional hazard interaction analyses performed on all cytokines/cytokine receptor genes and KRAS mutation status in the combined cohort. The highest expression tertile for each gene was defined as “high.” Genes for whom the uncorrected P value was less than 0.05 are highlighted in blue (RFS HR < 1) and red (RFS HR > 1). B, RFS among stage II/III patients in the discovery cohort stratified based on both IL22RA1 and IL10RB expression and KRAS mutation status. IL22RA1-high/ low and IL10RB-high/low is defined by a cutpoint at the 67th percentile for each gene. Five-year (60-month) RFS proportions for each molecular subtype are displayed. C, Relative proportions of stage II/III patients in the discovery cohort categorized based on IL22RA1 expression, IL10RB expression, and KRAS mutation status. The 5-year survival of each molecular subtype is indicated.

(RFS HR ¼ 3.62; 95% CI, 1.95–6.70; P < 0.0001; OS HR ¼ 2.43; 95% CI, to IL22 in the KRAS wild-type Colo205 line (Fig. 3B). To determine 1.33–4.45; P ¼ 0.0039; Supplementary Fig. S4); this observation was whether proliferative differences were due specifically to the presence confirmed in the combined cohort (Supplementary Table S3). or absence of mutant KRAS, we employed a DLD-1 isogenic cell line To determine the effect of high expression of both IL22RA1 and system in which the parental line carries a heterozygous KRAS G13D IL10RB, patients were classified into six groups based on KRAS mutation (KRAS-MUT) that was removed to produce paired “wild- mutation status, IL22RA1, and IL10RB. KRAS-mutant tumors with type” cells (KRAS-WT). IL22RA1 expression at both the protein high expression of both cytokine receptor subunits displayed the (Fig. 3C) and mRNA (Fig. 3D) level was equivalent in the KRAS- lowest 5-year RFS rates (Fig. 2B). In the cohorts analyzed in this MUT and KRAS-WT isogenic cells, thus providing a pair of cell lines study, 18.9% of stage II/III colorectal cancers were KRAS mutant and with matched IL22RA1 expression, differing only in the presence or expressed high amounts of IL22RA1 and/or IL10RB compared with absence of mutant KRAS. As expected, phosphorylation of ERK1/2 18.8% of colorectal cancers that were KRAS mutant and expressed low (pERK1/2) was elevated at baseline in the KRAS-MUT cells compared amounts of both receptors (Fig. 2C). with their KRAS-WT counterparts based on flow cytometry analysis; but pERK1/2 was not induced by IL22 (Fig. 3E). In contrast, IL22 IL22-induced proliferation of colorectal cancer cells is induced activation of STAT3 (based on flow cytometry analysis of enhanced by KRAS mutation phosphorylation at Y705) in a dose-dependent fashion, but no differ- IL22 can promote epithelial proliferation in response to various ences were observed between KRAS-MUT and KRAS-WT cells stimuli (41–43). To investigate a possible functional interaction (Fig. 3F). In accordance with the equivalent STAT3 activation in between IL22 and mutant KRAS, we tested the proliferative response both lines, SOCS3 induction after IL22 stimulation was similar in the of a panel of KRAS-mutant and wild-type colorectal cancer cell lines to KRAS-MUT and KRAS-WT cells across a range of IL22 doses IL22. All three cell lines [Colo205 (KRAS-WT), DLD-1 (KRAS-MUT), (Fig. 3G). Next, we explored whether KRAS mutation alters the and SW480 (KRAS-MUT)] were functionally responsive to IL22, as proliferative response to IL22. Notably, although IL22 enhanced demonstrated by induction of SOCS3 (encoding suppressor of cyto- proliferation of both KRAS-MUT and KRAS-WT cells, this effect was kine signaling 3), a well-established direct transcriptional target of significantly greater in KRAS-MUT cells (Fig. 3H). Like IL22, IL6 is a STAT3 (ref. 44; Fig. 3A). However, there was no proliferative response well-known activator of STAT3. However, there was no difference in

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Figure 3. IL22 augments proliferation in KRAS-mutant cells. A, qPCR analysis of SOCS3 induction in Colo205 (KRAS-WT), DLD-1 (KRAS-MUT), and SW480 (KRAS-MUT) colorectal cancer cells after 24-hour IL22 stimulation (n ¼ 3). Statistical significance was determined using two-way ANOVA. B, Proliferation of Colo205, DLD-1, and SW480 cells following 24-, 48-, or 72-hour IL22 stimulation assessed by MTT assay. Raw 540-nm absorbances were blank corrected and normalized to the mean of the 0 ng/mL condition for each cell line and each independent experiment (n ¼ 3). Statistical significance was determined using two-way ANOVA. (C) Flow cytometric analysis [quantification and representative histogram (gray ¼ isotype control staining)] of IL22RA1 protein (n ¼ 5) and (D) qPCR analysis of IL22RA1 mRNA (n ¼ 3) at baseline in a DLD-1 isogenic cell line harboring an endogenous KRAS G13D mutation in the parental line (KRAS-MUT) that is deleted in the partner line (KRAS-WT). Statistical significance evaluated using unpaired t tests. E and F, Intracellular flow cytometric analysis of pERK1/2 or pSTAT3-Y705 in KRAS-MUT or WT DLD-1 isogenic cells stimulated for 30 minutes with IL22 (n ¼ 4). (Continued on the following page.)

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the proliferative response to IL6 between KRAS-MUT and WT cells by approximately half (Supplementary Fig. S5C). MYC knockdown (Fig. 3I). reduced IL22-induced cell proliferation in DLD-1 KRAS-MUT cells To validate our observations using cells derived from primary (Fig. 4G). tumors, we established organoid cultures from eight freshly resected Multiple cell types that are not responsive to IL22 in the tumor human colorectal cancer samples. Three of these tumors bore onco- microenvironment express MYC, complicating the interpretation of genic KRAS mutations. IL22 stimulation induced robust activation of MYC pathway analysis in whole tumor tissue. Nonetheless, to deter- STAT3 in these organoids (Fig. 3J). KRAS-mutant and wild-type mine whether the functional differences in MYC pathway induction in organoids were dissociated to single cells and seeded into organoid IL22RA1-high, KRAS-MUT versus WT cells are reflected in the culture conditions in the presence or absence of 1 ng/mL IL22 for colorectal cancer cohorts assessed in this study, we performed GSEA 96 hours. Consistent with data derived from cell lines, IL22 signifi- analysis on transcriptomic data from the combined cohort. The cantly increased the viable cell number in cultures of KRAS-MUT MYC_TARGETS_V2 pathway was not enriched in IL22RA1-high, organoids, but had no consistent effect on KRAS-WT organoids KRAS MUT tumors compared with IL22RA1-high, KRAS WT tumors (Fig. 3K). (Supplementary Fig. S6A). However, when tumors in the combined Together, these data indicate cooperativity between mutant KRAS cohort were classified into the CMS subtypes defined by Guinney and and IL22 signaling that promotes the proliferation and accumulation colleagues (29), IL22RA1 expression was associated with CMS2 status of cancer cells. regardless of KRAS mutation (Supplementary Fig. S6B). CMS2 is characterized by a molecular signature of epithelial MYC and WNT Myc is uniquely induced by IL22 in KRAS-mutant cells and activation. Moreover, qPCR analysis of IL22RA1 and MYC in pro- promotes cell proliferation spectively collected whole tumor biopsy tissue from patients under- To identify potential modes of interaction between IL22 and mutant going colectomies at the Churchill Hospital (Oxford, UK), revealed KRAS, we next performed unbiased transcriptomic analysis of DLD-1 that IL22RA1 positively correlates with MYC expression (Supplemen- isogenic cells following IL22 stimulation. RNA-sequencing was per- tary Fig. S6C). IL22RA1 is not a reported direct transcriptional target of formed on cells stimulated with 10 ng/mL IL22 for 2 hours to identify c-Myc. Indeed, MYC knockdown in DLD-1 cells did not alter IL22RA1 early transcriptional changes and after 24 hours to explore late changes expression compared with mock-transfected controls (Supplementary (Fig. 4A). Given that IL6R expression does not interact with mutant Fig. S6D). Therefore, elevated MYC expression in IL22RA1-high KRAS in a prognostically significant manner (Table 2), nor does it tumors could be a functional consequence of more active IL22 enhance proliferation in KRAS-mutant versus wild-type cells (Fig. 3I), signaling. we also stimulated cells with 10 ng/mL IL6 for 2 and 24 hours to identify pathways that are uniquely regulated by IL22 and not IL6 (Fig. 4A). Discussion Principal component analysis revealed that genotype (KRAS-MUT Inflammatory responses are observed in both colitis-associated and or WT), cytokine stimulation [no treatment (NT), IL22, IL6], and time sporadic colorectal cancer, but whether specific cytokines play ben- (2, 24 hours) were the major sources of transcriptomic variation in the eficial or deleterious roles in cancer is largely context dependent. dataset (Fig. 4B). GSEA analysis of 24-hour RNA-sequencing data Moreover, tumors develop adaptations to exploit the growth and revealed that among the Hallmark gene sets (MSigDB), only the Myc survival benefits conferred through cytokine signaling (5). Although pathway (“MYC_TARGETS_V2”) was uniquely induced by IL22 (and data from preclinical studies have suggested that IL22, or its upstream not IL6) in KRAS-MUT but not WT cells (Fig. 4C). MYC expression driver cytokine IL23, may be useful therapeutic targets for colorectal was significantly increased in KRAS-mutant versus wild-type cells after cancer, there has been a lack of compelling clinical evidence to support 2-hour IL22 stimulation (Fig. 4D). Although Myc target gene expres- this concept (6–9, 37, 39, 45, 46). Our study extends these previous sion was modestly increased by IL22 in KRAS-MUT cells after 2 hours efforts by demonstrating that IL22 signaling may be critical for a (Supplementary Fig. S5A), clear preferential induction of Myc targets specific subset of patients with colorectal cancer, defined by tumors in KRAS-MUT cells was observed after 24 hours (Fig. 4E). with high IL22 receptor expression and oncogenic KRAS mutations. Based on the results of our transcriptomic analyses, we predicted Furthermore, our data suggest that IL22 and oncogenic KRAS col- that c-Myc is preferentially induced in KRAS-MUT cells following laborate to promote cell proliferation via enhanced c-Myc activity. IL22 stimulation. Indeed, Western blot analyses demonstrated a These findings emphasize the important and often unpredictable marked time-dependent induction of c-Myc protein in KRAS-MUT effects that oncogenic mutations can exert on signals derived from cells following IL22 stimulation, whereas c-Myc did not accumulate in the tumor microenvironment. KRAS-WT cells (Fig. 4F; Supplementary Fig. S5B). To determine Prospective analysis of the PETACC3 cohort has previously whether the IL22-induced proliferation observed in KRAS-MUT cells demonstrated that KRAS mutations alone have no prognostic value was dependent on c-Myc, we used siRNA to suppress MYC expression for patients with stage II/III colorectal cancer who receive adjuvant in DLD-1 KRAS-MUT cells, which reduced c-Myc protein expression chemotherapy (21). Therefore, the negative prognostic effect of

(Continued.) Data are fold change in mean fluorescence intensity (MFI) over the MFI of the 0 ng/mL condition in the KRAS-WT line for each independent experiment. G, qPCR analysis of SOCS3 induction in DLD-1 isogenic cells after 24-hour IL22 stimulation (n ¼ 3). For dose–response experiments, statistical significance was determined using multiple unpaired t test comparing the KRAS-MUT with WT line at each IL22 dose (, P < 0.05). H and I, Proliferation of DLD-1 KRAS-MUT or WT isogenic cells following 24-, 48-, or 72-hour IL22 or IL6 stimulation assessed by MTT assay. Raw 540-nm absorbances were blank corrected and normalized to the mean of the 0 ng/mL condition for each cell line and each independent experiment [n ¼ 4 (IL22), n ¼ 3 (IL6)]. Statistical significance was determined using a two-way ANOVA and Sidak method to correct for multiple comparisons (, P < 0.0323; , P < 0.0021; , P < 0.0002; and , P < 0.0001). J, Representative confocal microscopic images of pSTAT3-Y705 induction in primary colorectal cancer organoids stimulated for 30 minutes with 1 ng/mL IL22. K, Proliferation of KRAS-MUT [N ¼ 3(N ¼ 2 KRAS G12D, N ¼ 1 KRAS G12V)] and WT (N ¼ 5) organoids treated with 1 ng/mL IL22 for 96 hours, assessed by Alamar Blue assay. Data are normalized to the NT condition for each line; each data point is the average of 3 technical replicates. Statistical significance was assessed using unpaired t tests. All data are mean SEM. iso, isotype control; MFI, mean fluorescence intensity; NT, no treatment; O.D., optical density.

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Figure 4. Mutant KRAS enhances IL22-induced expression of c-Myc and its downstream targets. A, Experimental workflow for RNA-sequencing analysis. DLD-1 KRAS-MUT or WT isogenic cells were stimulated for 2 or 24 hours with 10 ng/mL IL22 or 10 ng/mL IL6. RNA-sequencing was performed on the 2-hour and 24-hour samples. Three independent experiments were performed. B, Principal component analysis on all samples (n ¼ 36). C, Interaction analysis of ssGSEA scores to identify pathways uniquely regulated by IL22 in KRAS-MUT cells. Heatmap represents ssGSEA scores for each sample and displays only the significantly differentially regulated pathways (P adjusted < 0.01). D, MYC expression following 2-hour IL22 stimulation in DLD-1 isogenic cells. N ¼ 3 independent experiments. Statistical significance was assessed using unpaired t tests. E, Expression of genes in “MYC_TARGETS_V2” MSigDB Hallmark signature in 24-hour IL22-stimulated DLD-1 isogenic cells. Heatmap

represents row normalized log2(RPKMþ1) values. F, Total c-Myc expression in DLD-1 KRAS-MUT or WT isogenic cells stimulated with 10 ng/mL IL22 for 15 minutes (1500) to 24 hours (240) assessed by Western blot. Blot representative of three independent experiments. G, Proliferation of DLD-1 KRAS-MUT and WT cells in response to 48-hour 10 ng/mL IL22 treatment following 48-h MYC siRNA knockdown, assessed by MTT assay. Data are blank corrected 540-nm absorbances normalized to the MUT siscr NT condition for each independent experiment (n ¼ 4–9). Statistical signiﬁcance was assessed using unpaired t tests. NT, no treatment; siscr, cells transfected with nontargeting control siRNA; siMYC, cells transfected with MYC siRNA.

KRAS mutation in patients with IL22RA1-high tumors may be due to cytokine receptors that interact prognostically with mutant KRAS, a unique interaction between IL22 signaling and a constitutively IL22RA1 and IL10RB (which encode the IL22 receptor subunits) active KRAS pathway. Notably, in our screen for cytokines and were the only KRAS-interacting genes that showed a signiﬁcant

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negative prognostic association. Indeed, despite the well-described life (60, 61); both mechanisms could be at play in IL22-induced c-Myc tumor-promoting role of IL6 (35), which is similar to IL22 in its in KRAS-mutant cells. ability to activate STAT3, neither IL6 nor IL6R expression interacted Based on the evidence presented here, we propose a stratification prognostically with KRAS. Intriguingly, high interferon gamma strategy in which tumors from patients with colorectal cancer are receptor 1 (IFNGR1) expression interacted with KRAS and was subjected to standard KRAS mutation typing paired with quantifica- associated with improved prognosis, which is consistent with the tion of IL22 receptor expression. Although both IL22RA1 and IL10RB known antitumor properties of IFNg (47). contribute prognostic information, IL22RA1 expression is restricted to Much of the current understanding of the function and signaling epithelial cells in the colon and may thus be a more suitable biomarker. downstream of IL22 has come from studying its physiologic role in Colorectal cancers harboring KRAS mutations and high expression of microbial defense and tissue regeneration at barrier surfaces. Due to its IL22RA1 and/or IL10RB comprise approximately 19% of the total antiapoptotic and mitogenic effects, IL22 supports intestinal stem cell colorectal cancer population. Such patients are predicted to have lower integrity and tissue regeneration in models of colitis (48–50) and graft responsiveness to conventional chemotherapy, increased frequency of versus host disease (43). Recent evidence highlights its critical role in disease relapse, and reduced OS time. Beyond prognostic stratification, regulating DNA damage response pathways in intestinal epithelial our data suggest that pharmacologic blockade of IL22 signaling may be stem cells to protect from mutation acquisition and tumor develop- therapeutically beneficial for KRAS-mutant tumors. Notably, mono- ment (51). However, IL22 can exacerbate immune pathology when clonal antibodies targeting IL22 and its upstream driver IL23 have been produced chronically (52, 53). Based on our data, we propose that IL22 developed for inflammatory indications and were well tolerated in can also be pathogenic in colorectal cancers with adaptations that alter phase I and II studies (NCT00563524, NCT00883896, NCT01866007, the functional outcome of IL22 signaling. In agreement with previously NCT02203032, and NCT01225731). Furthermore, antibodies specific reported findings from Kryczek and colleagues (11), we found that to the p19 subunit of IL23 are now being investigated in phase II/III IL22 promotes expansion of DLD-1 cells, but only if they expressed trials in inflammatory bowel disease (NCT03926130, NCT03466411, constitutively active KRAS. This mutant KRAS-dependent prolifer- NCT03759288, and NCT03398135) and will provide gut-specific ative response to IL22 was similarly observed in primary colorectal safety and efficacy data. cancer organoids. Although we have revealed a novel interaction between KRAS and To date, the only evidence of a potential link between IL22 IL22 in colorectal cancer, several questions remain. For example, our signaling and oncogenic KRAS is derived from Kras-induced lung analyses focused on the direct effect of IL22 on cancer cell–intrinsic cancer models in which genetic ablation of Il22 (54) or Il22ra1 (55) pathways, but whether IL22 differentially affects the tumor microen- reduced tumor burden. Moreover, in a small cohort of patients with vironment based on KRAS mutation status is unknown. In addition, KRAS-mutant lung cancer (N ¼ 39), high IL22RA1 expression our cohorts were not sufficiently powered to study whether IL22 conferred poor recurrence-free survival (54). Intriguingly, evidence signaling interacts with constitutively active BRAF, an immediate from a preinvasive pancreatic neoplasia (PanIN) model revealed that downstream mediator of Ras signaling that is mutated in approxi- oncogenic KRAS and chronic pancreatitis synergistically induce IL22 mately 10% of colorectal cancers. Because IL22RA1 expression man- þ and IL17A production by CD4 and gd T cells and that oncogenic ifests as a continuous, nonbiphasic variable, further prospective studies KRAS augments IL17 receptor expression on PanIN epithelial assessing IL22RA1 expression are necessary (and ongoing in our cells (56). Based on contingency analyses in the tumor transcrip- laboratory) to establish a clinically relevant cutpoint for identifying tomic cohorts we studied, mutant KRAS and IL22RA1 expressions patients with high expression. appear to be independent variables in colon cancer. Consistent with Collectively, the evidence presented here suggests a method to both these findings, DLD-1 isogenic cells with or without mutant KRAS identify a subset of poor-prognosis colorectal cancer patients with had nearly identical IL22RA1 expression. This suggests that the KRAS-mutant tumors and to target these tumors using therapeutic interaction between IL22RA1 and mutant KRAS is at the level of blockade of the IL22 pathway. Interactions between cytokines and downstream signaling. oncogenic mutations are not limited to IL22 and KRAS. Exploration Previous studies of IL22-driven colorectal cancer attributed the and exploitation of cytokine–oncogene interactions more broadly may protumorigenic activity of IL22 to its STAT3-activating effect (7, 8, 11). be beneficial for precise patient stratification and the application of Interestingly, we observed no difference in the amount of IL22- immunomodulatory therapies in cancer. induced STAT3 activation between KRAS-mutant and wild-type DLD-1 cells. Moreover, the well-described STAT3-activator IL6 did Disclosure of Potential Conflicts of Interest not augment proliferation in KRAS-mutant versus wild-type cells, nor S. McCuaig, N.R. West, and F. Powrie are inventors on an unlicensed patent owned did expression of IL6 or IL6R interact with KRAS to impact prognosis. by Oxford University Innovation (Technology Transfer University of Oxford) which Therefore, the interaction between IL22 and KRAS is not due to describes a method for the treatment and prognosis of colorectal cancer, especially proximal colorectal cancer, and also relates to identifying patients with colorectal activation of STAT3 alone. Interestingly, the presence of mutant KRAS cancer who are likely to respond to therapy with an inhibitor of interleukin 22 enhanced IL22-induced expression of c-Myc and its downstream signaling. N.R. West is an employee/paid consultant for Genentech. F. Powrie is an target genes. c-Myc is one of the earliest described proto-oncogenes employee/paid consultant for GSK and Genentech, and reports receiving commercial and is a master transcriptional regulator of genes involved in cellular research grants from Roche and Janssen. No potential conflicts of interest were growth, proliferation, metabolism, and biosynthetic processes (57). disclosed by the other authors. Notably, the oncogenic activity of c-Myc is largely a consequence of ’ overexpression, and, in colorectal cancer, mutations within its Authors Contributions coding sequence are very rare (57). Myc expression and stability is Conception and design: S. McCuaig, N.R. West, F. Powrie Development of methodology: S. McCuaig, D. Barras, M. Friedrich, V.H. Koelzer, regulated at both the transcriptional and posttranslational level. MYC N.R. West STAT3 has been found to augment transcription (58, 59), Acquisition of data (provided animals, acquired and managed patients, provided whereas ERK1/2 and AKT activity downstream of constitutively active facilities, etc.): S. McCuaig, D. Barras, E.H. Mann, S.J. Bullers, A. Janney, L.C. Garner, Ras signaling enhances c-Myc protein stability and extends its half- E. Domingo, V.H. Koelzer, M. Delorenzi, S. Tejpar, T.S. Maughan, N.R. West

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Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Wetering of the Hubrecht Institute, Utrecht, for providing human organoid protocols. computational analysis): S. McCuaig, D. Barras, E.H. Mann, V.H. Koelzer, We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for M. Delorenzi, S. Tejpar, T.S. Maughan, N.R. West, F. Powrie Human Genetics (funded by Wellcome Trust grant reference 090532/Z/09/Z) for Writing, review, and/or revision of the manuscript: S. McCuaig, E.H. Mann, generation of the RNA-sequencing data and Nicholas Illott for assisting with NCBI E. Domingo, V.H. Koelzer, S. Tejpar, N.R. West, F. Powrie GEO data deposition. We thank the patients and their families for contributing Administrative, technical, or material support (i.e., reporting or organizing data, to this study. constructing databases): S. McCuaig, D. Barras, S.J. Bullers, V.H. Koelzer, This work was funded by an Oxford/CRUK Development Fund Grant (C25255/ M. Delorenzi A18085), an MRC Experimental Medicine Grant (MR/N02690X/1), and CRUK Study supervision: T.S. Maughan, N.R. West, F. Powrie OPTIMISTICC Grant (C10674/A27140). S. McCuaig was supported by the Rhodes Other (support with data acquisition via cell line experiments): A. Janney Trust. N.R. West was supported by an Irivington Institute Postdoctoral Fellowship (Cancer Research Institute). L.C. Garner is supported by a Wellcome Trust PhD Studentship (109028/Z/15/Z). E. Domingo is supported by the S:CORT Consortium Acknowledgments which is funded by a grant from the Medical Research Council and Cancer Research We thank the Oxford IBD Cohort Investigators (Carolina V. Arancibia-Carcamo; UK. This work was also supported by the NIHR Oxford Biomedical Research Centre. Adam Bailey; Ellie Barnes; Elizabeth Bird-Lieberman; Oliver Brain; Barbara Braden; The views expressed are those of the authors and not necessarily those of the NHS, the Jane Collier; James East; Alessandra Geremia; Lucy Howarth; Satish Keshav; Paul NIHR, or the Department of Health. Klenerman; Simon Leedham; Rebecca Palmer; Fiona Powrie; Astor Rodrigues; Jack Satsangi; Alison Simmons; Peter Sullivan; Simon Travis; Holm Uhlig) for helping to The costs of publication of this article were defrayed in part by the payment of page facilitate and coordinate GI specimen collection. Thank you to Cloe Vassart, James charges. This article must therefore be hereby marked advertisement in accordance Chivenga, Ngonidzashe Charumbira, and David Maldonado-Perez who consented with 18 U.S.C. Section 1734 solely to indicate this fact. patients and collected samples from the operating theatres for this study. We thank the team of pathologists at the JR Hospital for biopsying specimens and S. Page in the Oxford Molecular Diagnostics Centre for providing tumor mutation status data. We Received April 2, 2019; revised October 1, 2019; accepted May 14, 2020; thank Dr. Simon Leedham for generously providing cell lines and Dr. Marc Van de published ﬁrst May 19, 2020.

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IL22 Pathway and Mutant KRAS Promote Poor Prognosis in Colorectal Cancer

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AACRJournals.org Clin Cancer Res; 2020 OF13

The Interleukin 22 Pathway Interacts with Mutant KRAS to Promote Poor Prognosis in Colon Cancer

Sarah McCuaig, David Barras, Elizabeth H. Mann, et al.

Clin Cancer Res Published OnlineFirst May 19, 2020.

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