IFNL4-ΔG Allele Is Associated with an Interferon Signature in Tumors And

Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/1078-0432.CCR-18-1060 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

IFNL4-G allele is associated with an interferon signature in tumors and survival of African-American men with prostate cancer

Wei Tang1, Tiffany A. Wallace1, Ming Yi2, Cristina Magi-Galluzzi3, Tiffany H. Dorsey1, Olusegun O. Onabajo4, Adeola Obajemu4, Symone V. Jordan1, Christopher A. Loffredo5, Robert M. Stephens2, Robert H. Silverman6, George R. Stark6, Eric A. Klein7, Ludmila Prokunina-Olsson4, and Stefan Ambs1

1 Laboratory of Human Carcinogenesis, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD, USA 2 Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA 3 Department of Pathology, Cleveland Clinic, Cleveland, OH, USA 4 Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, NCI, NIH, Bethesda, MD, USA 5 Cancer Prevention and Control Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA 6 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA 7 Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, OH, USA

Running title: Interferon signature and African-American prostate cancer patients

Key words: Prostate cancer, IFNL4, interferon signature, African-American, gene expression, disease recurrence, immune therapy, IDO1

Abbreviations: AA, African-American; EA, European-American; IFNL4, interferon-4; IRDS, interferon-related DNA damage resistance signature; IRG, interferon response genes; ISG, interferon-stimulated gene; IDO1, indoleamine-2,3-dioxygenase-1

Address all correspondence to:

Stefan Ambs, PhD, Laboratory of Human Carcinogenesis, NCI, NIH, Bldg.37/Room 3050B, Bethesda, MD 20892-4258. Phone 240-760-6836; Email: [email protected]

Conflict of interest: The authors declare no potential conflicts of interest.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/1078-0432.CCR-18-1060 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

TRANSLATIONAL RELEVANCE

Tumor interferon signaling has recently been shown to modulate response and resistance to

immune checkpoint blockade. Here, we describe a distinct and biologically relevant interferon

signature in prostate tumors that has a high prevalence in African-American patients. This

signature, known as “Interferon-related DNA Damage Resistance Signature” (IRDS), predicts

decreased disease-free survival. Moreover, we link its occurrence to a germline variant allele,

rs368234815-G, within the interferon λ4 (IFNL4) gene. This IFNL4 allele controls production of a type-III interferon, IFN-λ4, and is a known predictor of decreased viral clearance. Together, these observations indicate that IRDS and IFNL4 rs368234815-ΔG may have a function in the

tumor biology and survival of African-American patients, and influence immune therapy

outcomes, which should be examined in future studies.

ABSTRACT

Purpose: Men of African ancestry experience an excessive prostate cancer mortality that could

be related to an aggressive tumor biology. We previously described an immune-inflammation

signature in prostate tumors of African-American patients. Here, we further deconstructed this

signature and investigated its relationships with tumor biology, survival, and a common

germline variant in the interferon λ4 (IFNL4) gene.

Experimental Design: We analyzed gene expression in prostate tissue datasets and performed

genotype and survival analyses. We also overexpressed IFNL4 in human prostate cancer cells.

Results: We found that a distinct interferon signature that is analogous to the previously

described “Interferon-related DNA Damage Resistance Signature” (IRDS) occurs in prostate tumors. Evaluation of two independent patient cohorts revealed that IRDS is detected about

twice as often in prostate tumors of African-American than European-American men.

Furthermore, analysis in The Cancer Genome Atlas (TCGA) showed an association of increased

IRDS in prostate tumors with decreased disease-free survival. To explain these observations, we

assessed whether IRDS is associated with an IFNL4 germline variant (rs368234815-ΔG) that

controls production of IFN-λ4, a type-III interferon, and is most common in individuals of

African ancestry. We show that the IFNL4 rs368234815-ΔG allele was significantly associated

with IRDS in prostate tumors and overall survival of African-American patients. Moreover, IFNL4

overexpression induced IRDS in three human prostate cancer cell lines.

Conclusions: Our study links a germline variant that controls production of IFN-λ4 to the

occurrence of a clinically relevant interferon signature in prostate tumors that may

predominantly affect men of African ancestry.

INTRODUCTION

Prostate cancer incidence and mortality rates are highest among men of African ancestry (1-3).

Environmental exposures and ancestry-specific factors may influence prostate cancer biology

and cause a more aggressive disease in these men (4-11). We and others described an immune signature that is prevalent in prostate tumors of African-American (AA) patients and hypothesized that this signature affects tumor biology (5, 12-15). Here, we further investigated

this immune signature and discovered an interferon signature in tumors of AA patients that is

analogous to the previously described interferon-related DNA damage resistance signature,

also termed IRDS (16). IRDS includes 49 interferon-stimulated genes (ISGs) that are induced

through activation of the JAK-STAT pathway (17). IRDS was initially identified because of its

effects leading to resistance to ionizing radiation. It can be induced by persistent activation of

the JAK-STAT pathway by various exogenous and endogenous stimuli that generate an

interferon response (18, 19).

All interferons (type-I, type-II, type-III) induce sets of ISGs that are overlapping but also distinct. However, among the human interferons, the expression of the recently discovered IFN-

λ4, a type-III interferon, is uniquely controlled by genetics. Only carriers of the G allele for the

germline variant rs368234815-ΔG/TT in the IFNL4 gene can produce this interferon (20). This

allele is most common in individual of African ancestry (up to 80% allele frequency), while it is

less common in Europeans (~30%) and Asians (less than 10%) (20). Moreover, carriers of IFNL4 rs368234815-ΔG have an impaired ability to clear certain viral infections, such as hepatitis C

virus (HCV), spontaneously or after treatment (20). One of the mechanisms by which IFN-λ4 renders cells refractory to an antiviral response is the induction of a persistent gene signature

that resembles IRDS (20, 21). Thus, we hypothesized that because IFN-λ4 is produced more

commonly in men of African ancestry, it might explain the increased occurrence of IRDS in their

tumors, and be of clinical importance. Accordingly, we examined whether IFNL4 rs368234815-

ΔG is associated with the development of IRDS in prostate tumors and disease outcomes.

Consistent with our hypothesis, we found that IFNL4 rs368234815-ΔG is significantly associated

with both the presence of IRDS and an increased all-cause mortality among AA prostatectomy

patients.

MATERIALS AND METHODS

Study design, patient data, and cell lines. To compare gene expression profiles from prostate

tumors of AA and EA patients, we analyzed gene expression data from two patient cohorts that

were previously described in detail by us (12) and others (22). These two datasets are publicly

available (GSE6956, GSE21032) and consist of microarray data for primary prostate tumors from 33 AA and 36 EA men (12) and 24 AA (non-Hispanic) and 98 EA men (22). These men were

previously untreated prostatectomy patients with exception of 16 patients in the Taylor et al.

cohort who received neoadjuvant hormone therapy or chemotherapy. We examined the

prevalence of two previously reported interferon-related gene signatures in the tumors,

Interferon-related DNA Damage Resistance Signature (IRDS) (23) and Interferon Regulated

Genes (IRG) (24). As described in the publications, IRDS and IRG include 49 and 42 ISGs, respectively. To assess the association of IRDS with disease-free survival, we evaluated the publicly available TCGA prostate cancer data accessible through the Cancer Genomics Data

Server (CGDS, at http://www.cbioportal.org/public-portal) and hosted by the Computational

Biology Center at Memorial-Sloan-Kettering Cancer Center through C-Bioportal for Cancer

Genomics. In this large dataset, recurrence-free survival was available for 491 patients who are

mainly EA men (self-reported race/ethnicity: 270 white, 43 black, 6 Asian, others unknown). To

examine the association between IFNL4 rs368234815-ΔG allele and IRDS in prostate tumors, we

genotyped rs368234815-ΔG in available genomic DNA from 44 frozen tumors in the Wallace et

al. cohort (12). We also assessed genome-wide gene expression using a linear regression model in relation to 0, 1 and 2 copies of the ΔG allele and applying an additive model. The Wallace et al. study has been approved by institutional review boards, as described (12). To assess the

association of IFNL4 rs368234815-ΔG with disease recurrence and overall mortality, we isolated

genomic DNA from tumor-adjacent, non-cancerous prostate tissues that were obtained from

197 AA prostate cancer patients after a prostatectomy at the Cleveland Clinic (1987 to 2012).

According to follow-up through 2014, 92 patients had a PSA-defined prostate cancer

recurrence, and 29 died of various causes (Supplementary Table 1). Clinical information

including age at diagnosis, tumor stage, grade (Gleason score), vital status follow-up, and cause

of death, was available for these subjects. Collection of tissues and patient information was

reviewed and approved by the Cleveland Clinic IRB under protocol CCF IRB 313-773. Our

research followed the ethical guidelines set by the Declaration of Helsinki and informed consent

was obtained from all patients in the study.

Human prostate cancer cell lines 22Rv1 and PC-3 (from European-American donors) and

MDA-PCa-2b (from an African-American donor) were obtained from the American Type Culture

Collection (Manassas, VA) and have been regularly authenticated using a short tandem repeat

analysis with GenePrint10 and tested for Mycoplasma contamination.

IRDS in prostate tumors and associations with IFNL4-G and disease recurrence. The mRNA expression data for Wallace et al. (12) was available in-house while the data for Taylor et al.

(22) was downloaded as normalized log2 data from the cBio Cancer Genomics Portal

(http://cbio.mskcc.org/cancergenomics/prostate/data/). Normalized expression data were

subjected to a pathway-level comparative analysis using the Sample-Level Enrichment-Based

Pathway Ranking (SLEPR) method (25). SLEPR can be used to quantitatively evaluate gene set-

level expression patterns at the sample level (e.g., relative expression of a gene signature in a

tumor). Pathway-level enrichment assessment using SLEPR was applied to Gene Ontology

annotations (GO, www.geneontology) and to the two overlapping expression signatures, IRDS

and IRG. The IRDS and IRG gene lists were obtained from the two publications that described

these signatures (16, 24). Supplementary Table 2 shows the Affymetrix probeset composition of IRDS for the GeneChip HG-U133A 2.0 arrays. Evaluation of sample-level up-regulated genes

in a pathway or pre-defined gene signature was performed using the one-sided MADe method

(see SLEPR). Computation of pathway-level enrichment scores for each sample-level differentiated gene was performed based on 2x2 contingency tables using the Fisher’s exact

test. To determine the statistical significance of enrichment scores at the pathway/gene

signature level for class comparison (e.g., AA versus EA patients), a P value was calculated from

1000 permutations. False discovery rates (FDR) q-values were computed from permutated

data. Heatmaps to visualize enrichment scores for up-regulated genes in a pathway/gene

signature at the sample level (e.g., in an AA tumor) were generated using gradients of red color

which indicate the enrichment level [-log (permutated P value)]. All procedures for SLEPR and

visualization of findings in heatmaps are part of the WPS software (25). For the analysis of the

association between tumor IRDS and IFNL4 rs368234815-ΔG, enrichment scores for IRDS were dichotomized and tumors with an enrichment score of zero (no significant enrichment above background) were defined as IRDS-negative (n = 25), while the other tumors were defined as

IRDS-positive (n = 19).

The relationship between IRDS in prostate tumors with disease recurrence was

evaluated in RNA-seq data for the TCGA prostate cancer cohort of 491 patients. We extracted

the IRDS expression profile from the TCGA RNA-set data, leading to a 45-gene signature with all

genes being measurable expressed, and applied the consensus clustering method to identify

distinct tumor clusters with differential IRDS expression in the dataset. This was done using the

Bioconductor ConsensusClusterPlus package

(https://bioconductor.org/packages/release/bioc/html/ConsensusClusterPlus.html). This

method provides a quantitative assessment for determining the number of possible clusters

within the dataset. Initially, we tested 2 to 8 cluster groups. Final subgroups were defined

according to their cumulative distribution functions (CDF) and the Delta area under the CDF

curve, yielding 3 distinct clusters that represented the most robust clustering in our data. Then,

we calculated the IRDS score based on IRDS expression values and assigned those to the three

clusters, yielding robust differences between the clusters (low, medium, high). We then

accessed the association of these three IRDS expression groups with disease recurrence using

Cox regression modeling and visualized findings with a Kaplan-Meier plot.

IFNL4 rs368234815-ΔG genotyping. Genomic DNA was isolated from frozen tissues using the

DNeasy blood and tissue kit, (QIAGEN, Valencia, CA). To isolate DNA from formalin-fixed,

paraffin-embedded (FFPE) tissues, five-micron sections were deparaffinized and DNA was

extracted using the BiOstic FFPE Tissue DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA).

DNA quantity and quality were determined by NanoDrop 1000 (Thermo Scientific, Waltham,

MA) and 10ng of DNA was used for genotyping. A previously described TaqMan assay for

rs368234815 (20) was purchased from Life Technologies (Grand Island, NY) and used on the ABI

7900 (Applied Biosystems, Grand Island, NY) according to standard protocols. Genotyping success rates were 100% for the frozen tissues (44 tumors) and 98.5% for FFPE tissues

(194/197), with 100% concordance among duplicates.

Expression analysis of indoleamine-2,3-dioxygenase (IDO1) in prostate tumors. IDO1

expression was measured by quantitative RT-PCR using a Life Technologies TaqMan expression

assay (IDO1, Hs00984148_m1) and total RNA from prostate tumors of 21 AA and 22 EA men.

Characteristics of these patients have previously been described (12). In this cohort, AA and EA

patients are matched on Gleason score and pathological stage. Relative normalized expression

values were calculated as described (26), using the group mean Ct for the target and the

endogenous control 18s RNA. Fold differences were calculated as 2– ΔΔCt. Graphs were prepared

using ΔCt = Ct18S - Cttarget and plotted using GraphPad Prism 7.

Measurement of tryptophan in plasma samples. Levels of tryptophan, which is metabolized by

IDO1, were measured in plasma samples from randomly selected 50 AA and 50 EA population-

based controls (mean age: 63.4 and 63.7, respectively) and 50 AA and 50 EA prostate cancer patients (mean age: 61 and 62.4, respectively). These subjects were previously recruited into the NCI-Maryland Prostate Cancer Case-Control study (27). Tryptophan metabolite

concentrations were measured at SAIC/Leidos-NCI Frederick, Frederick, MD, using a described

High Performance Liquid Chromatography method (28). Tryptophan measurements were

obtained for 197 out of the 200 plasma samples (98.5%).

In vitro induction of an interferon signature with by overexpression of IFNL4 in human

prostate cancer cell lines. To examine IFN-λ4-induced gene expression, the human prostate

cancer cell lines, 22Rv1, PC-3, and MDA-PCa-2b, were transfected with a previously described

IFNL4 expression construct that generates IFN-λ4 protein with C-terminal Halo-tag (20). A GFP

reporter gene was used to monitor transfection efficiency. Transfection of the IFNL4-Halo

construct and empty Halo-control vector was performed in triplicates using Lipofectamine/LTX

and yielded robust overexpression of IFNL4 in the IFNL4-Halo-transfected cell lines without affecting cell viability (Supplementary Figure 1). The IFNL4 qRT-PCR was performed as described (20). In the experiments with 22Rv1 cells, we also added antibodies that block the activity of IFN-λ4 - 20 µg/ml of goat anti-α-IL10R2 antibody (R&D Systems, Minneapolis, MN) that blocks one receptor of IFN-λ4, or 20 µg/ml of a rabbit monoclonal anti-IFN-λ4 antibody

(Abcam, ab196984), that blocks IFN-λ4 directly. Cells were lysed after 48 hours and total RNA was extracted with the RNeasy kit with DNase I treatment (QIAGEN). One to 1.5 µg of total RNA was used to generate cDNA using the RT2 first strand kit (QIAGEN). The Human Type I Interferon

Response PCR array (QIAGEN) was used to evaluate expression of a panel of other relevant genes. This array includes 96 SYBR Green expression assays for type-I interferons (IFNA and

IFNB) and their receptors, ISGs and molecules involved in response and resistance to signaling, as well as positive and negative controls. Differences in expression between cells transfected with the IFNL4 construct and empty vector were evaluated with a multiple comparisons- adjusted two-tailed t-test. We also examined expression of two other type-III IFNs - IFNL1

(Hs00601677_g1) and IFNL3 (Hs04193050), and endogenous control 18s RNA (4319413E) with

Life Technologies TaqMan expression assays. For all TaqMan assays, gene expression was measured in Ct values and normalized by endogenous control 18s RNA. Analysis of the PCR

Arrays was performed using the GeneGlobe Data Analysis Software, in relation to a panel of positive and negative controls included on the array.

BrdU assay. Cells were pre-seeded in 96-well cell culture plates (Corning Life Sciences,

Tewksbury, MA) overnight and then cultured in either the complete culture medium or the medium without FBS, or transfected with either the IFNL4-Halo expression vector or the vector

control construct. After a 48-hour incubation, cells were assessed for cell proliferation using the

BrdU colorimetric ELISA assay (Roche) according to the provided protocol. Eight replicates were

analyzed per condition, and proliferation was calculated as relative ratio by normalization BrdU

incorporation to the control.

Statistical analysis. Analyses were conducted using SAS 9.2 (SAS Institute, Cary, NC) and R (R

Foundation for Statistical Computing; http://www.r-project.org/). All statistical tests were two-

sided. P < 0.05 was considered statistically significant. The non-parametric Mann-Whitney test

was used for group comparisons with continuous data and the Fisher’s exact test for

categorized data. Associations between a genotype and the occurrence of IRDS were estimated

with linear regression models. Survival analysis was conducted with a multivariate Cox

Proportional-Hazards Regression model under a log-additive hazards assumption. Disease-free

survival was defined from the date of prostatectomy to the date of recurrence (PSA-defined).

Overall survival was defined from the date of the prostate cancer diagnosis to the date of death

(Cleveland Clinic cohort). Survival analyses (disease-free, overall) were controlled for age (<65

or ≥65 years), stage (I, II or III, IV) and Gleason score (<7 or ≥7). Statistical significance was

determined using the Wald test.

RESULTS

Interferon-related DNA damage resistance signature (IRDS) is prevalent in prostate tumors of

AA men. We previously described an immune-inflammation signature in tumors of AA patients that showed up-regulation of genes involved in interferon signaling (12). Here, we examined

whether two interferon signatures, IRDS and IRG, are detectable in prostate tumors and more

prevalent in AA than EA patients. IRDS and IRG have been described independently but show

considerable similarity and could be functionally equivalent, and are both associated with

decreased breast cancer survival (23, 24). Our analysis found that both signatures are detected

about twice as often in tumors from AA than EA men in two independent datasets, Wallace et

al. and Taylor et al. (Figure 1A and B). For example, IRDS was detected at a frequency of 67%

and 42% in AA men in these two datasets, which is significantly higher than the observed

frequency in EA men (33% and 18%, respectively). We also investigated IRDS and IRG in cultured prostate cancer epithelial cells from 14 AA and 13 EA men. The expression data were previously published by Timofeeva et al. (29). This analysis showed that the two signatures can

be detected in 5 out of 14 (36%) cell cultures from AA men and 2 out of 13 (15%) for EA men

(Supplementary Figure 2).

Of the two signatures, IRDS and IRG, only IRDS has been characterized (17, 23, 30). We

therefore focused on IRDS in our follow-up analyses. While IRDS captures the expression of

many ISGs, others are not represented by this signature. Because indoleamine-2,3-dioxygenase

(IDO1) is an immune-suppressive ISG with a key function in cancer biology, but not an IRDS

gene (Supplementary Table 2), we examined its expression in prostate tumors from 21 AA and

22 EA patients from the Wallace et al. dataset using a qRT-PCR approach. The expression

analysis showed an approximately 2-fold increased tumor expression of IDO1 in AA men when

compared to EA men (Figure 1C), consistent with the two-fold higher prevalence of IRDS in AA

men. Since IDO1 uses tryptophan as a substrate for its enzymatic activity, we examined blood

tryptophan levels in AA and EA prostate cancer patients to assess if increased tumor IDO1 in AA

patients may affect these levels (Figure 1D). We found very similar mean blood tryptophan

levels in AA men (31.9 ± 8.1 µM) and EA men (32.1 ± 5.9 µM) without prostate cancer, and in EA

prostate cancer patients (31.9 ± 4.5 µM), but a modest decrease in blood tryptophan (by 8-9%)

among the AA cases (29.3 ± 7.6 µM; P = 0.02 when compared with EA cases; P = 0.08 when

compared to AA controls).

IRDS is associated with early disease recurrence. To examine whether IRDS is clinically relevant for prostate cancer patients, we analyzed available gene expression and cancer recurrence data for 491 prostate cancer patients in TCGA. We defined the occurrence of IRDS based on 45 IRDS

genes with measurable expression and used the RNA-sequencing data to categorize patients

into 3 groups with low, medium, and high IRDS expression in their tumors (Figure 2). The

Kaplan-Meier survival plot shows that an increased IRDS expression is associated with

decreased disease-free survival (Figure 2C). Using a Cox regression model, we estimate that

patients with high IRDS expression in their tumors have a two-fold increased hazard of an

earlier disease recurrence [hazard ratio (HR) of 2.09 (95% confidence interval (CI): 1.07-4.10 after controlling for age, disease stage, and Gleason score], when compared to patients with

low IRDS expression.

IFNL4 rs368234815-ΔG is associated with IRDS and a distinct expression profile in prostate

tumors. IFN-λ4 is a recently discovered type-III interferon that induces a similar set of ISGs that

constitute IRDS, and impairs an antiviral response, as shown for HCV (20). Production of IFN-λ4

is genetically controlled by a genetic variant, IFNL4 rs368234815-ΔG, that is most common

among subjects of African ancestry (20). Thus, we examined whether IFNL4 rs368234815-ΔG is

associated with the occurrence of IRDS in 44 prostate tumors that had available information on

IRDS and the IFNL4 rs368234815-ΔG/TT genotype. As shown in Table 1, homozygote carriers of

the ΔG allele (ΔG/ΔG) were significantly more likely to have IRDS-positive tumors than carriers

of the combined TT/TT and TT/ΔG genotypes. When we restricted the analysis to the 23

African-American patients in this cohort, we again found a significant association between the

ΔG/ΔG genotype and the occurrence of IRDS in prostate tumors (odds ratio: 8.2; 95% CI: 1.1 to

60.4). Of note, only AA men were carriers of the ΔG/ΔG genotype among the 44 men.

Further examination of the gene expression data revealed that the IFNL4 rs368234815-

ΔG is associated with increased expression of immune-, host defense-, and inflammation-

related genes in prostate tumors (Figure 3). Gene expression differences based on the ΔG genotype separated tumors into three distinct clusters. Cluster 1 was significantly enriched for tumors from AA men and carriers of the ΔG/ΔG genotype and showed ΔG allele dosage increase of expression of genes such as PTPRC, TNF, RSAD2, IFI44, NLRP3, CCL5, STAT1, CCL4,

IFI35, IRF9, ISG15, IFNG, and MX1, among others. Many of these are IRDS genes (e.g., IFI35,

IFI44, MX1, STAT1). This observation was further explored by a pathway analysis using Gene

Ontology (GO) biological processes, molecular function, and cellular component annotations.

The approach identified GO terms such as immune response, defense response and response to

virus, as being associated with IFNL4 rs368234815-ΔG (FDR < 5%). This observation was

corroborated by transient overexpression of IFNL4 in 22Rv1, PC-3, and MDA-PCa-2b human

prostate cancer cells. Expression of IFN-λ4 induced an interferon signature in these cells,

consistent with IRDS, under these experimental conditions (Figure 4A-C) while not affecting cell

growth (Supplementary Figure 1). The IFNL4-transfected cells showed induction of known ISGs

that are common to both IRDS and IRG, such as IFIT1-3, IFIH1, IFITM1, OAS1, OAS2, MX1, IRF7

and IRF9, when compared with vector control-transfected cells. Furthermore, when we

inhibited IFN-λ4 signaling with blocking antibodies targeting either the IFN-λ4 receptor, IL10R2,

or IFN-λ4, this signature was attenuated (Figure 4A).

IFNL4 rs368234815-ΔG is associated with overall survival of AA prostate cancer patients. To test whether IFNL4 rs368234815-ΔG may influence either disease-free or overall survival of prostate

cancer patients, we genotyped 194 AA prostatectomy patients from the Cleveland Clinic, as

described in Methods. Twenty-five (12.8%) of the patients carried the TT/TT genotype, 92

(47.4%) the TT/ΔG genotype, and 77 (39.7%) were homozygote for ΔG, which corresponds to a

63.4% ΔG allele frequency. These genotypes did not associate with the Gleason score of tumors, nor did we find an association with disease recurrence (P = 0.88) in this cohort.

However, the ΔG/ΔG genotype was associated with a significantly decreased overall survival of

these patients (Figure 4D). The Cox regression model showed that each copy of the ΔG allele

increased the risk of a prostate cancer death by about two-fold (HR = 2.07; 95% CI: 1.14 to 3.75

adjusted for age, disease stage, Gleason score; Ptrend = 0.01) compared with carriers of the

TT/TT genotype (Table 2).

DISCUSSION

In this study we show that a subset of prostate tumors manifests a distinct interferon signature,

IRDS, that has previously been linked to acquired resistance to radiation and chemotherapy,

increased metastases, and poor survival of breast cancer and glioblastoma patients (17-19, 23,

30, 31). We show that in prostate cancer patients, IRDS predicts decreased disease-free survival

and is significantly more prevalent in tumors of AA than EA patients. Furthermore, we

discovered that a germline variant in the IFNL4 gene was significantly associated with the

increased prevalence of IRDS and all-cause mortality in AA prostatectomy patients, suggesting

genetic predisposition to these clinical outcomes.

Up-regulated interferon signaling through the JAK-STAT signaling is part of IRDS and the

antiviral response (19), but is also triggered by DNA methyltransferase inhibitors (32) and occurs in various types of cancers (23, 31, 33, 34). In human triple-negative breast cancer, co- inactivation of p53 and ARF coincides with an oncogenic IFN-β-STAT1-ISG15 signaling signature

that is reminiscent of IRDS (34). However, in primary prostate tumors, the inactivation of p53

and ARF is rather uncommon (35, 36), indicating that the common occurrence of IRDS in

primary tumors of AA patients is likely caused by another mechanism. Investigations using

mouse models demonstrated that interferon signaling through STAT1 is pro-tumorigenic in

leukemia and colon cancer development through chronic inflammation-mediated

carcinogenesis (37, 38). These findings are consistent with our previous reports that tumors of

AA patients demonstrate a prominent immune-inflammation signature that may increase

tumor aggressiveness and can be targeted with anti-inflammatory drugs (12, 27). In a study by

Hardiman et al. (15), the effect of vitamin D supplementation on the prostate cancer

transcriptome was investigated in 10 AA and 17 EA patients. In line with our findings, the authors found an immune-inflammation signature in tumors of AA patients but also showed that this signature is diminished by vitamin D treatment.

Besides increasing inflammation and disease progression, the immune-inflammation signature in AA prostate tumors may cause resistance to radiation and chemotherapy, and T-

cell exhaustion. IRDS co-occurs as part of this immune-inflammation signature and has been

shown to increase resistance to radiation (30) while inhibition of the JAK-STAT pathway was

found to result in re-sensitization of docetaxel-resistant DU145 prostate cancer cells (39).

Others found that the JAK-STAT signaling increases cross-resistance in myeloma cell lines (40).

More recently, several investigations linked IRDS-like signatures to the clinical response to anti-

CTLA4 therapy and PD-1 blockade (41, 42). Accordingly, these signatures cause immune

suppression in melanomas and resistance to anti-CTLA4 therapy but increase the response rate

to PD-1 blockade. PD-L1 is an ISG and an important mediator of resistance to anti-CTLA4

therapy (41). PD-L1 expression may detrimentally affect AA prostate cancer patients because of

IRDS. Indeed, it was recently shown that expression of PD-L1 is increased in prostate tumors of

AA men, when compared with EA men, using immunohistochemistry (43). We did not find that

PD-L1 was significantly upregulated at the transcript level in AA prostate tumors in the Wallace et al. and TCGA datasets, suggesting that IRDS may regulate protein stability rather than

expression of PD-L1 in these tumors. However, we found that IDO1, another ISG, is up-

regulated on the mRNA level in AA prostate tumors, consistent with an immune-suppressive

environment in these tumors. Additional measurements of plasma tryptophan levels in our

study suggest that AA prostate cancer patients may have a higher turnover of this IDO1 substrate although the reduction of plasma tryptophan was rather modest.

Currently, there is no firm evidence that a viral infection is a cause of prostate cancer

(44), although a meta-analysis of 26 tissue-based case-control studies reported an association

between human papillomavirus positivity and the disease (45). Additionally, RNASEL, a gene

that protects against viral pathogens, may have an important tumor suppressor function in

prostate cancer (46, 47). Besides viral infections, spontaneous reactivation of endogenous

retroviruses may also lead to occurrence of IRDS. We reported the up-regulation of the human

endogenous retrovirus type K envelope protein in prostate tumors of AA patients (48).

Moreover, it was recently shown that treatment with DNA methyltransferase inhibitors can

lead to reactivation of endogenous retroviruses and an IRDS-like signature in melanoma

patients that increases the sensitivity to immune checkpoint therapy (32). Western diet may

also up-regulate interferon signaling (49).

With our current study, we provide the first evidence that IFNL4 rs368234815-ΔG could

be a predisposition factor for IRDS in prostate cancer patients. Although this effect is not

exclusive to AA men, but based on a much higher ΔG allele frequency in AA compared to EA patients (e.g., 62% in AA versus 34% in EA in the NCI-Maryland Prostate Cancer Case-Control

Study), this genetic predisposition would be most important for men of African ancestry. This stark difference in the ΔG allele frequency between AA and EA patients could account for some

of the known health disparity in outcomes of prostate cancer. IFN-λ4 protein is only produced

in individuals with IFNL4 rs368234815-ΔG and attenuates antiviral responses through negative

regulation of interferon signaling (21). We showed that IFN-λ4 induces an interferon signature

in three human prostate cancer cell lines but could not detect IFNL4 expression in human prostate tumors based on TCGA RNA-seq data. IFNL4 expression is known to be low and transient, but IFN-λ4 is potent even at very low levels (21, 50). Lastly, IFNL4 rs368234815-ΔG

was associated with overall survival of AA prostatectomy patients, indicating a broader

biological effect of this variant. We also found similar association with overall survival of AA prostatectomy patients for an intronic IFNL4 variant, rs12979860-T, that is in high linkage

disequilibrium with rs368234815-ΔG (Supplementary Figure 3). However, in contrast to IRDS,

we did not find an association of IFNL4 rs368234815-ΔG with decreased disease-free survival.

Perhaps, our analysis of 194 genotyped AA prostatectomy patients was underpowered to

detect this association, or the impact of IFNL4 rs368234815-ΔG on disease recurrence is weaker

than the effect of IRDS. Alternatively, IFNL4 rs368234815-ΔG may mainly affect outcomes of

late stage therapies such as chemotherapy and immunotherapy, or may have a detrimental impact on morbidities arising from long-term androgen ablation therapy, explaining the observed association of this genotype with overall survival.

Thus, future research is needed to define the role of IFN-λ4 in IRDS among prostate

cancer patients. Yet, based our findings, IFNL4 rs368234815-ΔG may have potential clinical use

for identifying prostate cancer patients with increased sensitivity to immune checkpoint

blockade therapy and thereby predicting disease outcomes.

ACKNOWLEDGEMENTS

This research was supported by the Intramural Research Program of the Center for Cancer

Research (ZIA BC010499 and ZIA BC010624, to S.A.) and Division of Cancer Epidemiology and

Genetics (ZIA CP010201, to L. P.-O.), NCI, NIH, the Prostate Cancer Foundation 2013 Ben

Franklin-PCF Special Challenge Award (G.R.S., S.A., and E.A.K), the Cleveland Clinic Prostate

Cancer Center of Excellence Award (to R.H.S., G.R.S., C.M.-G., and E.A.K.), and the National

Institute of Allergy and Infectious Diseases (NIAID), NIH grant R01AI135922 (to R.H.S.).

We thank the Cooperative Prostate Cancer Tissue Resource (CPCTR) for providing tissue

specimens and supporting data. We would also like to thank personnel at the University of

Maryland and the Baltimore Veterans Administration Hospital for their contributions with the

recruitment of subjects.

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Figure legends

Figure 1. Increased frequency of two interferon signatures, IRDS and IRG, in prostate tumors from African-American (AA) men. A. Analysis of 33 and 36 tumors from AA and European-

American (EA) men, respectively, in the Wallace et al. dataset (GSE6956). Prevalence of IRG and

IRDS: 55% and 67% in AA and 19% and 33% in EA. Permutated P value and false discovery rate

(FDR) for difference in IRDS frequency between AA and EA tumors: P = 1.6x 10-4; FDR = 3.7%.

Red, up-regulated expression. B. Analysis of 24 and 98 tumors from AA and EA men, respectively, in the Taylor et al. dataset (GSE21032). Prevalence of IRG and IRDS: 46% and 42% in AA and 17% and 18% in EA. Permutated P value and FDR for difference in IRDS frequency between AA and EA tumors: P = 0.006; FDR = 21%. Gene expression data were analyzed with the SLEPR method, as described under Methods. C. Increased mRNA expression of IDO1 in

prostate tumors from AA men. IDO1 TaqMan assay was used to compare expression in AA vs.

EA men. Lines = mean ΔCt values. Fold expression difference is derived from 2– ΔΔCt values. D.

Decreased abundance of tryptophan in plasma of AA prostate cancer patients. P values from two-sided Mann Whitney test. Shown are means ± S.D.

Figure 2. IRDS is associated with early disease recurrence in the TCGA prostate cancer cohort.

A. Expression of 45 IRDS genes identifies prostate tumors with low (group 1: n = 159), medium

(group 2: n=270), and high (group 3: n = 62) expression of IRDS (sum Z score). Hierarchical clustering based on gene expression of IRDS in 491 TCGA prostate tumors. Red, up-regulated expression. B. Boxplots representing the relationship between IRDS expression and the three

groups. Grouping based on IRDS sum Z score statistics. C. High IRDS expression in prostate

tumors is associated with decreased disease-free survival. Kaplan-Meier survival analysis. Log-

rank test (P < 0.05) and unadjusted hazard ratios (HR) from a Cox regression analysis with Ptrend.

Figure 3. IFNL4 rs368234815-ΔG is associated with a distinct expression profile in prostate

tumors (n = 44). Hierarchical clustering based on expression of 1,194 transcripts yields 3

distinct clusters and shows a significant association between IFNL4 rs368234815-ΔG and the pattern of gene expression (red, up-regulated genes). IFNL4 rs368234815 genotypes are shown above the heatmap in red for ΔG/ΔG, green for TT/ΔG, and blue for TT/TT. Cluster 1 is

significantly enriched for tumors from African-American men and carriers of the ΔG/ΔG genotype. Frame marks 511 probesets, representing 447 unique genes, with signal enrichment in cluster 1. Gene Ontology (GO) relationships for these genes are shown to the right and include immune response, defense response and response to virus (FDR < 5% for all associations). The 1,194 transcripts were selected because their expression was associated with

ΔG allele (P < 0.05) using a linear regression model where relationships with ΔG were analyzed

under an additive model (coded as 0, 1 and 2 ΔG alleles).

Figure 4. IFNL4 overexpression induces an interferon signature in vitro and IFNL4

rs368234815-ΔG associates with overall survival. IFN-λ4 induces an interferon signature in the

22Rv1 (A), PC-3 (B), and MDA-PCa-2b (C) human prostate cancer cells. Shown are heatmaps of

genes related to the human type I interferon response after transfection of cells with an IFNL4

expression construct. Red, up-regulated expression. In A. blocking antibodies were added to the

culture medium targeting the IL10R2 receptor and IFN-λ4. *Significantly different gene expression induced by IFNL4 overexpression vs. control. D. Association of IFNL4 rs368234815-

ΔG with overall survival among African-American prostate cancer patients (n=194). Carriers of

the ΔG/ΔG genotype experienced the worst survival. Kaplan-Meier survival curve by IFNL4

genotype. Log-rank test: P < 0.05.

Tables

Table 1. IFNL4 rs368234815-ΔG allele is associated with occurrence of IRDS in prostate tumors

Fisher’s Odds ratio (OR) IFNL4 Genotype, N (%) exact test All tumors, n=44 TT/TT or TT/G G/G P value Adj. OR (95% CI)† IRDS-negative 23 (92%) 2 (8%) < 0.001 15.7 (2.7 to 90.6) IRDS-positive 8 (42%) 11 (58%)

Only tumors from AA men, n=23 IRDS-negative 6 (75%) 2 (25%) 0.04 8.2 (1.1 to 60.4) IRDS-positive 4 (27%) 11 (73%) CI = confidence interval; AA = African-American †Adjusted for age at diagnosis and pathological stage

Table 2. IFNL4 rs368234815-ΔG allele is associated with decreased overall survival of AA prostate cancer patients

Risk allele RAF, % Genotype N (%) Crude HR (95% CI) Adjusted HR (95% CI)† rs368234815-ΔG 63.4 TT/TT 25 (12.9) 1.00 (reference) 1.00 (reference) TT/ΔG 92 (47.4) 1.47 (0.32-6.75) 2.07(0.44-9.66) ΔG/ΔG 77 (39.7) 3.26 (0.75-14.15) 4.28 (0.97-18.90) Total 194

per allele - 2.00 (1.09-3.67) 2.07 (1.14-3.75) Ptrend - 0.02 0.01

ΔG/ΔG vs TT/TT+TT/ΔG - 2.39 (1.14-5.01) 2.21 (1.05-4.69) P - 0.02 0.02 RAF = risk allele frequency in dataset (n=194), HR = hazard ratio; CI = confidence interval † Multivariable Cox regression model adjusted for age, disease stage (TNM) and Gleason score; Ptrend: ΔG/ΔG vs. TT/ΔG vs. TT/TT

IFNL4-∆G allele is associated with an interferon signature in tumors and survival of African-American men with prostate cancer

Wei Tang, Tiffany A. Wallace, Ming Yi, et al.

Clin Cancer Res Published OnlineFirst July 16, 2018.

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