Supplementary Information includes: Supplementary Methods providing additional information on experimental techniques and data analysis; Supplementary Figures S1-S14 providing critical control experiments and raw data analyses for experiments presented in the main manuscript; and Supplementary Tables S1-S10 summarizing data presented in the main manuscript as well as listing reagents.

Intensity ratios of methylated to unmethylated DNA for TSG promoters in paired primary hLSCC (LC) and normal lung squamous cell (NL) samples from 18 individuals.

Intensity ratios of methylated to unmethylated DNA for TSG promoters in paired primary hLSCC (LC) and normal lung squamous cell (NL) samples from 18 individuals.

Key Points Tolerizing liposomes encapsulate a high-affinity islet CD4+ mimotope with calcitriol. Liposome administration s.c. at onset of hyperglycemia suppresses diabetes progression. Mimotope-specific T cells cross-regulate IGRP-specific CD8+ T cells in PLN. Visual Abstract Ag-specific tolerizing immunotherapy is considered the optimal strategy to control type 1 diabetes, a childhood disease involving autoimmunity toward multiple islet antigenic peptides. To understand whether tolerizing immunotherapy with a single peptide could control diabetes driven by multiple Ags, we coencapsulated the high-affinity CD4+ mimotope (BDC2.5mim) of islet autoantigen chromogranin A (ChgA) with or without calcitriol (1α,25-dihydroxyvitamin D3) into liposomes. After liposome administration, we followed the endogenous ChgA-specific immune response with specific tetramers. Liposome administration s.c., but not i.v., induced ChgA-specific Foxp3+ and Foxp3− PD1+ CD73+ ICOS+ IL-10+ peripheral regulatory T cells in prediabetic mice, and liposome administration at the onset of hyperglycemia significantly delayed diabetes progression. After BDC2.5mim/calcitriol liposome administration, adoptive transfer of CD4+ T cells suppressed the development of diabetes in NOD severe combined immunodeficiency mice receiving diabetogenic splenocytes. After BDC2.5mim/calcitriol liposome treatment and expansion of ChgA-specific peripheral regulatory T cells. IFN-γ production and expansion of islet-specific glucose-6-phosphatase catalytic subunit–related protein–specific CD8+ T cells were also suppressed in pancreatic draining lymph node, demonstrating bystander tolerance at the site of Ag presentation. Thus, liposomes encapsulating the single CD4+ peptide, BDC2.5mim, and calcitriol induce ChgA-specific CD4+ T cells that regulate CD4+ and CD8+ self-antigen specificities and autoimmune diabetes in NOD mice.

Background Dysbiosis of the gut microbiota has been implicated in the pathogenesis of many autoimmune conditions including type 1 diabetes (T1D). It is unknown whether changes in the gut microbiota observed in T1D are due to environmental drivers, genetic risk factors, or both. Here, we have performed an analysis of associations between the gut microbiota and T1D genetic risk using the non-obese diabetic (NOD) mouse model of T1D and the TwinsUK cohort. Results Through the analysis of five separate colonies of T1D susceptible NOD mice, we identified similarities in NOD microbiome that were independent of animal facility. Introduction of disease protective alleles at the Idd3 and Idd5 loci ( IL2 , Ctla4 , Slc11a1 , and Acadl ) resulted in significant alterations in the NOD microbiome. Disease-protected strains exhibited a restoration of immune regulatory pathways within the gut which could also be reestablished using IL-2 therapy. Increased T1D disease risk from IL-2 pathway loci in the TwinsUK cohort of human subjects resulted in some similar microbiota changes to those observed in the NOD mouse. Conclusions These findings demonstrate for the first time that type 1 diabetes-associated genetic variants that restore immune tolerance to islet antigens also result in functional changes in the gut immune system and resultant changes in the microbiota.

Malignant pleural mesothelioma (MPM) is a deadly cancer that is caused by asbestos exposure and that has limited treatment options. The current standard of MPM diagnosis requires the testing of multiple immunohistochemical (IHC) markers on formalin-fixed paraffin-embedded tissue to differentiate MPM from other lung malignancies. To date, no single biomarker exists for definitive diagnosis of MPM due to the lack of specificity and sensitivity; therefore, there is ongoing research and development in order to identify alternative biomarkers for this purpose. In this study, we utilized primary MPM cell lines and tested the expression of clinically used biomarker panels, including CK8/18, Calretinin, CK 5/6, CD141, HBME-1, WT-1, D2-40, EMA, CEA, TAG72, BG8, CD15, TTF-1, BAP1, and Ber-Ep4. The genomic alteration of CDNK2A and BAP1 is common in MPM and has potential diagnostic value. Changes in CDKN2A and BAP1 genomic expression were confirmed in MPM samples in the current study using Fluorescence In situ Hybridization (FISH) analysis or copy number variation (CNV) analysis with digital droplet PCR (ddPCR). To determine whether MPM tissue and cell lines were comparable in terms of molecular alterations, IHC marker expression was analyzed in both sample types. The percentage of MPM biomarker levels showed variation between original tissue and matched cells established in culture. Genomic deletions of BAP1 and CDKN2A, however, showed consistent levels between the two. The data from this study suggest that genomic deletion analysis may provide more accurate biomarker options for MPM diagnosis.

The identification of acquired CALR mutations in patients with essential thrombocythaemia (ET) or myelofibrosis (MF) has meant that disease-initiating mutations can now be detected in about 90% of all patients with a myeloproliferative neoplasm (MPN). Here, we show that only those CALR mutations that cause a +1 frameshift, thereby altering the carboxy-terminus of calreticulin, promote cytokine independence in vitro; in-frame deletions were not functional, and are unlikely to be the pathogenetic mutation underlying some MPN cases. Expression of the thrombopoietin receptor, MPL, was also necessary for factor-independence. Although the CALR mutations are considered to occur only in JAK2 V617F-negative cases and in a heterozygous state, progenitor genotyping revealed that this is not always true. Notably, CALR mutation-positive MPNs can be polyclonal: in one case, two distinct CALR mutation-positive subpopulations could be identified; in another, separate populations of JAK2 V617F-positive and CALR-mutated cells were present. Mitotic recombination involving chromosome 19 in a third instance resulted in the emergence of a CALR mutation-homozygous subclone. Collectively, our studies demonstrate that occasional patients with CALR mutation-positive ET or MF carry other MPN-initiating genetic mutations (including JAK2 V617F), acquire "secondary mutations" before or after the CALR mutation, or evolve over time to being CALR mutation-homozygous.

Enhancement of regulatory T‐cell (Treg) function is the goal of many immunotherapies aimed at treating type 1 diabetes (T1D). The use of interleukin (IL)‐2 is hindered by its effects on other populations such as effector T cells and NK cells. Combination therapies aimed at suppressing effector T cells while using IL‐2 to expand Tregs could be beneficial and have been trialed in T1D patients. We have investigated a combination therapy using IL‐2 and αCD11a blocking antibody to simultaneously expand Tregs and suppress the activation and migration of autoreactive T cells. When non‐obese diabetic mice were treated with low‐dose IL‐2/anti‐IL‐2 complexes (IL‐2c) and αCD11a, significant Treg expansion occurred in both the spleen and pancreas. Activation and IFNγ production by islet‐specific T cells was robustly suppressed in the periphery following IL‐2c/αCD11a treatment. Surprisingly, combination therapy accelerated diabetes onset compared with control treatments. Analysis of IL‐2 responsive populations found that combination therapy increased the activation of CD8+ T cells and natural killer (NK) cells specifically within the pancreas despite concomitant Treg expansion. Blocking effector T‐cell migration with the inhibitor FTY720 together with IL‐2c treatment also resulted in intra‐pancreatic expansion of effector cell populations. Thus, inhibiting effector T‐cell migration into the islets unleashes islet‐resident pathogenic effectors in the presence of low doses of exogenous IL‐2.

BACKGROUND:We aimed to identify prognostic blood biomarkers using proteomics-based approaches in malignant pleural mesothelioma (MPM).METHODS:Plasma samples from 12 MPM patients were used for exploratory mass spectrometry and ELISA analyses. The significance of secreted protein acidic and rich in cysteine (SPARC) was examined in sera from a Dutch series (n=97). To determine the source of the circulating SPARC, we investigated SPARC expression in MPM tumours and healthy controls, as well as the expression and secretion from cell lines and xenografts.RESULTS:Secreted protein acidic and rich in cysteine was identified as a putative prognostic marker in plasma. Validation in the Dutch series showed that the median survival was higher in patients with low SPARC compared with those with high SPARC (19.0 vs 8.8 months; P=0.01). In multivariate analyses, serum SPARC remained as an independent predictor (HR 1.55; P=0.05). In MPM tumour samples, SPARC was present in the tumour cells and stromal fibroblasts. Cellular SPARC expression was higher in 5 out of 7 cell lines compared with two immortalized mesothelial lines. Neither cell lines nor xenograft tumours secreted detectable SPARC.CONCLUSIONS:Low circulating SPARC was associated with favourable prognosis. Secreted protein acidic and rich in cysteine was present in both tumour cells and stromal fibroblasts; and our in vitro and in vivo experiments suggest that stromal fibroblasts are a potential source of circulating SPARC.

Background: Malignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets.Methods: Utilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy.Results: We identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p (P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the (normal) mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration.Conclusion: These functional data implicating KCa1.1 in MPM cell migration support our integrative approach using MPM gene expression datasets to identify novel and potentially druggable targets.

Top 20 Enriched microRNA Families Extracted from the Four Gene Expression Datasets. Table S2. Pathway Enrichment Analysis of Gene Targets of the miR-17 family members. Table S3. Primers and TaqMan Assay IDs for RT-qPCR and siRNA and Mimic Sequences. Table S4. Characteristics of Patients Analyzed in Fig. 2h and i. Table S5. Individual P values for Fig. 3a, f and g. Figure S1. TGFBR2 mRNA down regulated followed miR-17-5p transfection. Figure S2. MPM cell viability was not affected by transfection with miR-17-5p mimic or siRNA. Figure S3. Effect of KCNMA1 down-regulation on cell cycle in MPM cells. Figure S4. KCNMA1 down-regulation and miR-17-5p did not induce MPM cell apoptosis. Figure S5. Migration of MPM cell lines treated with miR-17-5p mimic or KCNMA1 siRNAs. Figure S6. Invasion of MPM cell lines treated with miR-17-5-5p mimic or KCNMA1 siRNAs. Figure S7. Paxilline did not sensitize MPM cells to cisplatin or gemcitabine. (DOCX 10063 kb)

Malignant pleural mesothelioma (MPM) is an asbestos-induced cancer with poor prognosis that displays characteristic alterations in microRNA expression. Recently it was reported that the expression of a subset of microRNAs can distinguish between MPM and adenocarcinoma of the lung. However, the functional importance of these changes has yet to be investigated. We compared expression of miR-192, miR-193a-3p and the miR-200 family in normal pleura and MPM tumor specimens and found a statistically significant reduction in the levels of miR-193a-3p (3.1-fold) and miR-192 (2.8-fold) in MPM. Transfection of MPM cells with a miR-193a-3p mimic resulted in inhibition of growth and an induction of apoptosis and necrosis in vitro. The growth inhibitory effects of miR-193a-3p were associated with a decrease in MCL1 expression and were recapitulated by RNAi-mediated MCL1 silencing. Targeted delivery of miR-193a-3p mimic using EDV minicells inhibited MPM xenograft tumour growth, and was associated with increased apoptosis. In conclusion, miR-193a-3p appears to have importance in the biology of MPM and may represent a target for therapeutic intervention.

In our study, we investigated the role of ZNF677 in non-small cell lung cancers (NSCLC). By comparing ZNF677 expression in primary tumor (TU) and in the majority of cases also of corresponding non-malignant lung tissue (NL) samples from > 1,000 NSCLC patients, we found tumor-specific downregulation of ZNF677 expression (adjusted p-values < 0.001). We identified methylation as main mechanism for ZNF677 downregulation in NSCLC cells and we observed tumor-specific ZNF677 methylation in NSCLC patients (p < 0.0001). In the majority of TUs, ZNF677 methylation was associated with loss of ZNF677 expression. Moreover, ZNF677 overexpression in NSCLC cells was associated with reduced cell proliferation and cell migration. ZNF677 was identified to regulate expression of many genes mainly involved in growth hormone regulation and interferon signalling. Finally, patients with ZNF677 methylated TUs had a shorter overall survival compared to patients with ZNF677 not methylated TUs (p = 0.013). Overall, our results demonstrate that ZNF677 is trancriptionally regulated by methylation in NSCLCs, suggest that ZNF677 has tumor cell growth suppressing properties in NSCLCs and that ZNF677 methylation might serve as prognostic parameter in these patients.

Long noncoding RNAs are an important class of RNAs that have been found to have diverse functions. Their discovery has stimulated a new area of cancer research and is providing new insights into chromatin regulation and transcriptional control. They have been implicated in a variety of cancers and have demonstrated their potential as diagnostic and prognostic markers in the circulation. Deciding how best to target these molecules, in terms of deciphering function and mechanism of action, is critical for determining their suitability as drug targets. Future research will need to focus on how they affect the intricate network of gene regulation that is only now starting to be uncovered.

SUMMARY Genomic technologies have revolutionized the way we study and understand cancer. The advent of next-generation sequencing technology in particular is now starting to change the clinical management of non-small-cell lung cancer. These technologies have helped us to refine prognostication and identify new driver mutations that can allow subselection of patients for therapeutic intervention. However, several limitations and challenges must be overcome before these technologies are widely accepted in diagnostic laboratories. It will be important for clinicians and diagnostic laboratories to consider sample type, analytical platform, cost, data security and ethics, and the bioinformatics challenges associated with 'big data', before widespread integration to the clinic. If these challenges can be overcome, then genomics has the potential to change clinical management of lung cancer.

7580 Background: Supervised proteomic analyses (discovery with isobaric tag for relative and absolute quantitation [iTRAQ] and validation with enzyme-linked immunosorbent assay [ELISA]) using 12 MPM patients from two phase II studies identified circulating SPARC as a promising prognostic marker, with high SPARC level associated with poor survival (p<0.01). We aimed to determine the independent prognostic significance of circulating SPARC in an independent cohort of MPM patients. Methods: Serum samples from MPM patients treated at the Netherlands Cancer Institute between 1995 and 2011 were included in this validation study. The samples were collected at the time of diagnosis and stored at -80°C, with clinical data collected prospectively. SPARC concentration was measured using the SEA791Hu ELISA kit (USCN Life Science Inc) according to the manufacturer’s instruction. The primary end point of the study was overall survival (OS) from the date of sample collection, determined by the Kaplan-Meier method. The independent prognostic value of SPARC (categorised into high vs. low according to median concentration) was examined using Cox regression analysis, incorporating known prognostic factors in a multivariate model, including age (<60 vs. ≥60 years), gender (male vs. female), stage (I-II vs. III-IV) and histological subtype (epithelial vs. non-epithelial). Results: A total of 97 patients were included: median age 60 years; 85% male; 50% stage I-II; and 75% epithelial subtype. Treatment received included: 67% chemotherapy; 22% radical surgery; and 18% supportive care alone. The median OS was 13.5 months (92 patients deceased). The median SPARC concentration was 1745µg/ml. In the multivariate analyses, male gender (median OS: 10.8 vs. 22.4 months; p=0.05), stage III-IV (median OS: 10.1 vs. 17 months; p=0.05) and high SPARC level (median OS: 9.3 vs.19 months; p=0.05) were independently associated with poor OS. Conclusions: This validation study is the first to demonstrate the prognostic role of circulating SPARC in MPM, with high SPARC level being an independent predictor of poor OS.

Abstract To discover new tumor-suppressor genes (TSG), we developed a functional genomics approach in which immortalized but nontumorigenic cells were stably transduced with large-scale shRNA pools and tested for tumor formation in mice. Identification of shRNAs in resulting tumors revealed candidate TSGs, which were validated experimentally and by analyzing expression in human tumor samples. Using this approach, we identified 24 TSGs that were significantly downregulated in human lung squamous cell carcinomas (hLSCC). Amplification of fibroblast growth factor receptor 1 (FGFR1), which aberrantly increases FGFR signaling, is a common genetic alteration in hLSCCs. Remarkably, we found that 17 of the TSGs encode repressors of FGFR signaling. Knockdown of 14 of these TSGs transformed immortalized human bronchial epithelial cells and, in most cases, rendered them sensitive to FGFR inhibitors. Our results indicate that increased FGFR signaling promotes tumorigenesis in many hLSCCs that lack FGFR1 amplification or activating mutations. Significance: A functional genomics approach identifies new lung TSGs whose loss aberrantly increases FGFR signaling to promote tumorigenesis. These TSGs are frequently downregulated in hLSCCs, indicating that increased FGFR signaling promotes tumorigenesis in many hLSCCs lacking FGFR1 amplification or activating mutations. Cancer Discov; 4(10); 1168–81. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1103