Pharmacological targeting of Hedgehog (HH)/GLI has proven effective for certain blood, brain and skin cancers including basal cell carcinoma (BCC). However, limited response rates and the development of drug resistance call for improved anti-HH therapies that take synergistic crosstalk mechanisms and immune evasion strategies into account. In previous work, we demonstrated that cooperation of HH/GLI and Interleukin 6 (IL6)/STAT3 signaling drives BCC growth. Whether synergistic HH-IL6 signaling promotes BCC via the activation of immune evasion mechanisms remained unclear. HH-IL6 regulated immunosuppressive genes such as indoleamine 2,3-dioxygenase 1 (IDO1) were identified by gene expression profiling. IDO1 expression was evaluated in human BCC and melanoma models by qPCR and Western blot analyses. The cis-regulatory region of IDO1 was interrogated for HH-IL6-regulated GLI and STAT transcription factor binding and epigenetic modifications by targeted chromatin-immunoprecipitation and bisulfite pyrosequencing. Functional analyses of the immunosuppressive effects of IDO1 involved HPLC-MS measurements of its metabolites and the assessment of T cell proliferation via flow cytometry. Bioinformatic analyses of GLI-STAT cooperation were conducted on published bulk and single-cell RNA-seq data of human BCC and melanoma patients. We identified IDO1 as a target gene of cooperative GLI-STAT activity in BCC and melanoma. GLI1 and STAT3 transcription factors synergistically enhanced IDO1 expression by jointly binding to the cis-regulatory region of IDO1 and by increasing active chromatin marks at the histone level. In human melanoma cells, inhibition of GLI1 expression prevented the induction of IDO1 expression in response to IL6/STAT3 and IFNγ/STAT1 signaling. Pharmacological targeting of HH/GLI signaling reduced IDO1 expression, resulting in decreased production of the immunosuppressive metabolite kynurenine. Further, inhibition of GLI1 enhanced the efficacy of the selective IDO1 inhibitor epacadostat and rescued T cell proliferation by attenuating IDO1/kynurenine-mediated immunosuppression. Elevated expression of IDO1 correlated with active HH/GLI and JAK/STAT signaling in skin cancer patients supporting the clinical relevance of the mechanistic data presented. These results identify the immunosuppressive IDO1-kynurenine pathway as a novel pro-tumorigenic target of oncogenic GLI and STAT1/STAT3 cooperation. Our data suggest simultaneous pharmacological targeting of these signaling axes as rational combination therapy in melanoma and non-melanoma skin cancers.

Cystic fibrosis (CF) is a monogenic disease, characterized by massive chronic lung inflammation. The observed variability in clinical phenotypes in monozygotic CF twins is likely associated with the extent of inflammation. This study sought to investigate inflammation-related aberrant DNA methylation in CF twins and to determine to what extent acquired methylation changes may be associated with lung cancer. Blood-based genome-wide DNA methylation analysis was performed to compare the DNA methylomes of monozygotic twins, from the European CF Twin and Sibling Study with various degrees of disease severity. Putatively inflammation-related and differentially methylated positions were selected from a large lung cancer case-control study and investigated in blood by targeted bisulphite next-generation-sequencing. An inflammation-related locus located in the Plakophilin-3 (PKP3) gene was functionally analysed regarding promoter and enhancer activity in presence and absence of methylation using luciferase reporter assays. We confirmed in a unique cohort that monozygotic twins, even if clinically discordant, have only minor differences in global DNA methylation patterns and blood cell composition. Further, we determined the most differentially methylated positions, a high proportion of which are blood cell-type-specific, whereas others may be acquired and thus have potential relevance in the context of inflammation as lung cancer risk factors. We identified a sequence in the gene body of PKP3 which is hypermethylated in blood from CF twins with severe phenotype and highly variably methylated in lung cancer patients and controls, independent of known clinical parameters, and showed that this region exhibits methylation-dependent promoter activity in lung epithelial cells.

Pharmacological targeting of Hedgehog (HH)/GLI has proven effective for certain blood, brain and skin cancers including basal cell carcinoma (BCC). However, limited response rates and the development of drug resistance call for improved anti-HH therapies that take into account synergistic crosstalk mechanisms and immune evasion strategies.In previous work, we demonstrated that crosstalk of HH/GLI with pro-inflammatory Interleukin-6 (IL6) signaling drives BCC by promoting tumor cell proliferation [[1][1]]. In the present study, we screened for possible mechanisms of cancer immune evasion regulated by synergistic HH-IL6 signaling and identified the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) as a novel transcriptional target co-regulated by HH-IL6 signaling. Analysis of the cis -regulatory region of IDO1 by chromatin-immunoprecipitation revealed co-occupancy of this region by HH- IL6 induced GLI1 and STAT3 transcription factors along with active chromatin marks at the histone level. Elevated expression of IDO1 in human BCC with high-level HH and IL6 signatures supports the clinical relevance of our mechanistic data. Genetic inhibition of GLI1 expression prevented the induction of IDO1 expression in response to IL6/STAT3 and IFNγ/STAT1 signaling in human melanoma cells. Pharmacological targeting of HH signaling at the level of GLI proteins interfered with IDO1 expression and consequently prevented the production of the immunosuppressive metabolite kynurenine generated by active IDO1 from tryptophan. Further, inhibition of GLI1 enhanced the efficacy of the selective IDO1 inhibitor epacadostat. Of note, inhibition of HH/GLI signaling in melanoma cells not only reduced IDO1 expression but also interfered with the repression of T cell activation by attenuating IDO1/kynurenine-mediated immunosuppression. These data identify the immunosuppressive IDO1-kynurenine pathway as a novel pro-tumorigenic effector of oncogenic HH-IL6 and GLI-STAT cooperation. Our data suggest simultaneous pharmacological targeting of the HH/GLI, JAK/STAT and IDO1- kynurenine axis as rational combination therapy in skin cancers.### Competing Interest StatementThe authors have declared no competing interest.* BCC : basal cell carcinoma cpm : counts per million dox : doxycycline ENCODE : ENCyclopedia Of DNA Elements GLI : glioma associated oncogene homolog HH : Hedgehog ICB : immune checkpoint blockade ICBs : immune checkpoint blockers IDO1 : indoleamine 2,3-dioxygenase 1 IFNγ : Interferon-gamma IL6 : Interleukin-6 ISTD : internal standard JAK : Janus Kinase MDSCs : myeloid-derived suppressor cells PBMCs : peripheral blood mononuclear cells PTCH : Patched RNAi : RNA interference shRNA : short hairpin RNA SMO : Smoothened STAT : signal transducer and activator of transcription Treg : regulatory T cell [1]: #ref-1

Background and aims: Wnt signaling is involved in atherosclerotic plaque formation directly and indirectly by modulating cardiovascular risk factors. We investigated whether circulating concentrations of Wnt inhibitors are associated with cardiovascular events in subjects with intermediate cardiovascular risk. Methods: 904 non-diabetic subjects participating in the SAPHIR study were assessed. In the SAPHIR study, middle-aged women without overt atherosclerotic disease at study entry were followed up for 10 years. 88 patients of our study cohort developed cardiovascular disease at follow-up (CVD group). Subjects of the CVD group were 1: 2 case-control matched for age, sex, BMI and smoking behavior with subjects without overt cardiovascular disease after a 10 year-follow-up (control group). 18 patients of the CVD group and 19 subjects of the control group were retrospectively excluded due to fulfilling exclusion criteria. Baseline circulating sclerostin, dickkopf (DKK)-1, secreted frizzled-related protein (SFRP)-1 and Wnt inhibitory factor (WIF)-1 levels were assessed by ELISA. Results: Baseline systemic SFRP-1 and WIF-1 levels were significantly higher in patients with cardiovascular events (n = 70) when compared to healthy controls (n = 157) while DKK-1 and sclerostin levels were similar in both groups. Logistic regression analysis revealed WIF-1 as a significant predictor of future cardiovascular events. Conclusions: Our data suggest that increased SFRP-1 and WIF-1 levels precede the development of symptomatic atherosclerotic disease. Assessment of systemic WIF-1 levels, which turned out to be independently associated with CVD, might help to early identify patients at intermediate cardiovascular risk. (C) 2018 Elsevier B.V. All rights reserved.