D. Codner

Suppression of IFN-Induced Transcription Underlies IFN Defects Generated by Activated Ras/MEK in Human Cancer Cells

Certain oncolytic viruses exploit activated Ras signaling in order to replicate in cancer cells. Constitutive activation of the Ras/MEK pathway is known to suppress the effectiveness of the interferon (IFN) antiviral response, which may contribute to Ras-dependent viral oncolysis. Here, we identified 10 human cancer cell lines (out of 16) with increased sensitivity to the anti-viral effects of IFN-α after treatment with the MEK inhibitor U0126, suggesting that the Ras/MEK pathway underlies their reduced sensitivity to IFN. To determine how Ras/MEK suppresses the IFN response in these cells, we used DNA microarrays to compare IFN-induced transcription in IFN-sensitive SKOV3 cells, moderately resistant HT1080 cells, and HT1080 cells treated with U0126. We found that 267 genes were induced by IFN in SKOV3 cells, while only 98 genes were induced in HT1080 cells at the same time point. Furthermore, the expression of a distinct subset of IFN inducible genes, that included RIGI, GBP2, IFIT2, BTN3A3, MAP2, MMP7 and STAT2, was restored or increased in HT1080 cells when the cells were co-treated with U0126 and IFN. Bioinformatic analysis of the biological processes represented by these genes revealed increased representation of genes involved in the anti-viral response, regulation of apoptosis, cell differentiation and metabolism. Furthermore, introduction of constitutively active Ras into IFN sensitive SKOV3 cells reduced their IFN sensitivity and ability to activate IFN-induced transcription. This work demonstrates for the first time that activated Ras/MEK in human cancer cells induces downregulation of a specific subset of IFN-inducible genes.

HLA-DP Epitope Typing Using Monoclonal Antibodies

We have made a panel of murine anti-DP monoclonal antibodies for serological typing of HLA-DP polymorphisms; they can be used in microcytotoxicity (for 7 epitopes) and binding assays (for 8 epitopes). The antibodies detect polymorphic differences in both alpha and beta chains. As immunogens we sometimes used B-lymphoblastoid lines or purified DP molecules but mostly used mouse fibroblast transfectants expressing DP molecules. The DP beta genes were made from a cloned DPB1*0201 gene by replacing its major area of polymorphism with matching stretches of DNA amplified from other alleles; cloned DPA1*01 and DPA1*02 genes were used for transfection along with the beta chain genes. The monoclonal antibodies showed reaction patterns that correlated with the presence of particular amino-acid sequence motifs; thus none of the antibodies is allele-specific. They bind instead to epitopes which are found on a number of different HLA-DP types. We have constructed frequency tables so that the epitope (motif) data can be interpreted as the most likely genotype in each case. The basic assumption to justify this work is that HLA-DP matching or mismatching will likely influence transplant outcome, particularly in bone marrow transplantation. The present challenge is to define permissive and nonpermissive combinations of HLA-DP; it may be that matching for epitopes, rather than for full alleles, will help to resolve this issue.

Activation of ERα Signaling Differentially Modulates IFN-γ Induced HLA-Class II Expression in Breast Cancer Cells

The coordinate regulation of HLA class II (HLA-II) is controlled by the class II transactivator, CIITA, and is crucial for the development of anti-tumor immunity. HLA-II in breast carcinoma is associated with increased IFN-γ levels, reduced expression of the estrogen receptor (ER) and reduced age at diagnosis. Here, we tested the hypothesis that estradiol (E2) and ERα signaling contribute to the regulation of IFN-γ inducible HLA-II in breast cancer cells. Using a panel of established ER− and ER+ breast cancer cell lines, we showed that E2 attenuated HLA-DR in two ER+ lines (MCF-7 and BT-474), but not in T47D, while it augmented expression in ER− lines, SK-BR-3 and MDA-MB-231. To further study the mechanism(s), we used paired transfectants: ERα+ MC2 (MDA-MB-231 c10A transfected with the wild type ERα gene) and ERα− VC5 (MDA-MB-231 c10A transfected with the empty vector), treated or not with E2 and IFN-γ. HLA-II and CIITA were severely reduced in MC2 compared to VC5 and were further exacerbated by E2 treatment. Reduced expression occurred at the level of the IFN-γ inducible CIITA promoter IV. The anti-estrogen ICI 182,780 and gene silencing with ESR1 siRNA reversed the E2 inhibitory effects, signifying an antagonistic role for activated ERα on CIITA pIV activity. Moreover, STAT1 signaling, necessary for CIITA pIV activation, and selected STAT1 regulated genes were variably downregulated by E2 in transfected and endogenous ERα positive breast cancer cells, whereas STAT1 signaling was noticeably augmented in ERα− breast cancer cells. Collectively, these results imply immune escape mechanisms in ERα+ breast cancer may be facilitated through an ERα suppressive mechanism on IFN-γ signaling.

Integrated Genomics Identifies Convergence of Ankylosing Spondylitis with Global Immune Mediated Disease Pathways

Ankylosing spondylitis(AS), a highly heritable complex inflammatory arthritis. Although, a handful of non-HLA risk loci have been identified, capturing the unexplained genetic contribution to AS pathogenesis remains a challenge attributed to additive, pleiotropic and epistatic-interactions at the molecular level. Here, we developed multiple integrated genomic approaches to quantify molecular convergence of non-HLA loci with global immune mediated diseases. We show that non-HLA genes are significantly sensitive to deleterious mutation accumulation in the general population compared with tolerant genes. Human developmental proteomics (prenatal to adult) analysis revealed that proteins encoded by non-HLA AS risk loci are 2-fold more expressed in adult hematopoietic cells.Enrichment analysis revealed AS risk genes overlap with a significant number of immune related pathways (p < 0.0001 to 9.8 × 10-12). Protein-protein interaction analysis revealed non-shared AS risk genes are highly clustered seeds that significantly converge (empirical; p < 0.01 to 1.6 × 10-4) into networks of global immune mediated disease risk loci. We have also provided initial evidence for the involvement of STAT2/3 in AS pathogenesis. Collectively, these findings highlight molecular insight on non-HLA AS risk loci that are not exclusively connected with overlapping immune mediated diseases; rather a component of common pathophysiological pathways with other immune mediated diseases. This information will be pivotal to fully explain AS pathogenesis and identify new therapeutic targets.

Private rare deletions in SEC16A and MAMDC4 may represent novel pathogenic variants in familial axial spondyloarthritis.

Objective Axial spondyloarthritis (AxSpA) represents a group of inflammatory axial diseases that share common clinical and histopathological manifestations. Ankylosing spondylitis (AS) is the best characterised subset of AxSpA, and its genetic basis has been extensively investigated. Given that genome-wide association studies account for only 25% of AS heritability, the objective of this study was to discover rare, highly penetrant genetic variants in AxSpA pathogenesis using a well-characterised, multigenerational family. Methods HLA-B*27 genotyping and exome sequencing was performed on DNA collected from available family members. Variant frequency was assessed by mining publically available datasets and using fragment analysis of unrelated AxSpA cases and unaffected controls. Gene expression was performed by qPCR, and protein expression was assessed by western blot analysis and immunofluorescence microscopy using patient-derived B-cell lines. Circular dichroism spectroscopy was performed to assess the impact of discovered variants on secondary structure. Results This is the first report identifying two rare private familial variants in a multigenerational AxSpA family, an in-frame SEC16A deletion and an out-of-frame MAMDC4 deletion. Evidence suggests the causative mechanism for SEC16A appears to be a conformational change induced by deletion of three highly conserved amino acids from the intrinsically disordered Sec16A N-terminus and RNA-mediated decay for MAMDC4. Conclusions The results suggest that it is the presence of rare syntenic SEC16A and MAMDC4 deletions that increases susceptibility to AxSpA in family members who carry the HLA-B*27 allele.

Assessing Prognosis in Rheumatoid Arthritis Using Monoclonal Antibodies and Flow Cytometry

The HLA-DR4 association with rheumatoid arthritis (RA) was revealed in several pioneering observations by Stastny and others between 1974 and 1980 [1]. It is interesting that even in those early days there was an awareness that DR4 was not just associated with RA but was associated with severity of RA, as will be discussed later. As knowledge of the HLA system advanced and as further studies of RA were undertaken it became evident that the HLA association was by no means straightforward. For example, when it was found that DR4 was supertypic to a series of alleles - the subtypes of DR4 - it was noted that only a few of them were associated with RA; indeed one (DwlO or DRB1*0402) actually appeared to be protective. Later DR1 and DR14 were associated in some studies and DR10 was prominent in others. This confusing situation was considerably clarified when Gregersen et al. in 1987 [2] put forward the “shared epitope hypothesis”. These authors realised, from examination of the amino-acid sequence data that the alleles which were associated with RA all had a similar sequence in the beta chain, QKRAA or QRRAA at positions 70-74. This unifying hypothesis showed that the closest association between HLA and RA is with the shared sequence which lies on the edge of the MHC groove midway along one of its alpha helices. This is referred to as the “shared epitope”. The epitope concept leads into the purpose of this report which is to describe a series of monoclonal antibodies to DR4 and some of its subtypes as well as to the shared epitope. As will be discussed later, these antibodies may be useful in determining prognosis in early cases of RA.

OP0206 Interactions Between Smoking and Methylation Status is Highly Predictive of Radiographic Progression in Ankylosing Spondylitis

Background The variability in the rate of radiographic progression in ankylosing spondylitis (AS) and the role of smoking, as an independent risk factor for radiographic progression, has been well documented. We have recently identified multiple epigenetic variants associated with ankylosis in AS using a genome-wide epigenetic approach. In this study, we examined the recently identified epigenetic markers in an independent AS cohort to assess radiographic progression and determine if an interaction exists with smoking status. Methods Our replication cohort consisted of 76 AS patients (16 females; 32 smokers; 56 HLA-B27+) that had serial radiographs on average 3 years apart (range 1.2 to 7.5 yrs). Of the 76 patients, 35 patients exhibited radiographic progression (change in mSASSS score >0). The mean radiographic progression for the entire group was 0.99 mSASSS/yr. The DNA methylation experiments on 17 CpG sites were designed using EpiTyper and performed on a Sequenom MassArray 4 system. The association between methylation score and radiographic progression rate was examined by multiple linear regressions after controlling for age of onset and gender. An interaction between methylation score and smoking status was introduced into the model to examine the association in the smokers and non-smokers, respectively. The Akaike information criterion (AIC) was used to assess the goodness of fit. Methylation sites with more than 15% of data missing were excluded from the analysis. The total methylation score of 7 CpG sites had the best goodness of fit. Results In the univariate analysis, high total methylation score was significantly associated with less radiographic progression (β=-0.97; 95%CI=-1.91:-0.02; p=0.04). Smokers demonstrated worse radiographic progression than non-smokers, but it was not statistically significant (p=0.62). In the multivariate analysis, the interaction between methylation score and smoke status was statistically significant (p=0.008). Among smokers, high total methylation score was significantly associated with less radiographic progression (β=-2.01; 95%CI=-3.26:-0.75; p=0.0017). No such association was observed among non-smokers. Conclusions This is the first study to demonstrate how epigenetic factors can influence radiographic progression in AS. Specifically, the results from this study reveal a significant association between smoking, methylation status and radiographic progression in AS. Disclosure of Interest None declared

OP0200 Global DNA Methylation Patterns Differ Between Responders and Non-Responders in Psoriatic Arthritis Patients Treated with Tumor Necrosis Factor-α Inhibitors

Background Although tumor necrosis factor-α inhibitors TNFi are well established treatment for psoriatic arthritis (PsA), the efficacy can vary greatly between patients. In this study, we examined the genome-wide epigenetic changes among TNFi responders and TNFi secondary failures in PsA patients Methods Twenty-one PsA patients (15 males; 6 females) were considered TNFi responders of which 13 were treated with etanercept and 8 with adalumimab. The median follow up duration was 18 months. Twenty patients (5 males; 15 females) were secondary TNFi failures of which 15 were treated with etanercept and 5 with adalumimab. The median follow up duration was 36 months. Genome-wide DNA methylation profiling was performed using the Illumina HumanMethylation450k Beadchip, which measures ∼480,000 different CpG sites per sample and covers 96% of RefSeq genes. The methylation level at each CpG site was measured by β values varying from 0 (no methylation) to 1 (100% methylation). Analysis was performed using a t-test after a Bonferroni-Holm correction was applied. Regions of interest were selected based on β value (β diff>5%) and p-value (p<0.01). Results After quality control filtering, 384,599 and 368,863 CpG sites were evaluated for TNFi responders and TNFi failures, respectively. Analysis revealed 72 and 91 CpG sites of interest in the TNFi responder group and in the TNFi failure group, respectively. Top gene candidates for TNFi responders included TRAPPC9 (β diff=0.056; p=3.42x10-6 - functions as an activator of NFkB); CCR6 (β diff=0.053; p=0.0028 - regulates the migration and recruitment of dentritic and T cells); and PSORSC13 (β diff=0.035; p=0.003). Top candidate genes for TNFi secondary failures included CD70 (encoded protein is a ligand for TNFRSF27/CD27), and TNFRSF1B (a member of the TNF receptor superfamily, that mediates most of the metabolic effects of TNFα). Conclusions These preliminary results demonstrate that the global DNA methylation pattern differs between TNFi responders and secondary failures. They also serve to highlight the possible importance of epigenetic (methylation) changes with respect to the TNFα signaling pathway. High priority candidate regions and genes identified in this study warrant further validation. Disclosure of Interest None declared

Abstract 804: Class II transactivator (CIITA) transcription is reduced by the ER-E2 signaling pathway in breast cancer cells

The immune response is regulated by the interaction of CD4 + T-cells with human leukocyte antigen class II (HLA-II). HLA-II genes code for HLA-DR that display peptides from antigens, which have undergone proteolysis in the endocytic pathway. Peptide loading of HLA-DR molecules also requires the co-chaperones; Invariant chain (Ii) and HLA-DM. Regulation of HLA-II occurs mainly at the transcription level, through interaction of transcription factors with the class II transactivator (CIITA). We previously reported that discordant expression of HLA-II in breast cancer associated with decreased estrogen receptor (ER) levels and younger age at diagnosis, suggesting that the estrogen-ER signaling pathway is implicated in regulating HLA-II in breast carcinoma cells. Moreover, using an experimental model of ER + and ER − breast cancer cell lines, we found that estradiol (E 2 ) augmented expression in the ER − cell line but decreased expression in the ER + transfectant. To address whether ER and E 2 interfere with CIITA expression, we performed Western blotting and quantitative real-time PCR in the same experimental cell system. CIITA expression was markedly reduced in ER + , but not in ER − cells; furthermore, CIITA inversely correlated with ER levels. CIITA promoter activity was analysed using a luciferase reporter gene construct in cell lines, treated or not with E 2 and/or ICI 182,780 (ER antagonist). CIITA luciferase activity was reduced in ER + cells and was further inhibited by E 2 and was restored by ICI. This suggests that ER may be recruited to the CIITA promoter and indirectly interfere with transcription of HLA-II genes. We further analyzed HLA-II in a wild type ER + line, MCF-7, which was stimulated with IFN-γ or stably transfected with CIITA. HLA-DR surface expression, ascertained by flow cytometry, was upregulated in cells grown in E 2 -depleted medium. Western blotting confirmed that ER degradation by ICI in MCF-7 correlated with up regulation of CIITA and HLA-II. In aggregate, these results indicate that the E 2 -ER pathway interferes with CIITA and HLA-II expression. Investigations such as this should improve our understanding of how ER and estrogen modulate the antigen presentation capabilities and immune recognition of breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2011-804