Background Helicobacter pylori (H. pylori) establishes lifelong colonization of the human gastric mucosa and promotes chronic inflammation associated with gastric cancer. Although macrophages play a central role in pathogen clearance, H. pylori persists despite sustained immune activation, suggesting perturbed macrophage responses. However, the mechanism behind this paradox remains unclear. Methodology: Here, we performed a comprehensive multi-omics analysis to define how H. pylori modulates macrophage polarization and transcriptional signatures controlling immune responses. We combined quantitative RNA-Seq, proteomics, and lipidomics of H. pylori infected THP-1 macrophages, complemented by confocal microscopy analysis, cytokine profiling and bacterial survival assays. Results H. pylori induced a rapid pro-inflammatory response characterized by increased cytokine and chemokine production and enhanced phagocytosis. Concurrently, macrophages underwent metabolic shift, including increased glycolysis, lipid remodeling, and lipid droplet accumulation. Time-resolved transcriptomics revealed a dynamic transition from early NF-κB dominated inflammatory responses to later STAT3 and HIF-1α associated regulatory and metabolic programs. Early induction of TNF-α and IL-10 was followed by sustained chemotactic signaling and partial attenuation of pro-inflammatory responses. Despite persisting induction of inflammatory cytokines such as IL-1β and IL-6, macrophages failed to fully clear internalized bacteria, suggesting the establishment of a non-resolving inflammatory state. Using isogenic H. pylori mutants, we found that CagA selectively enhanced HIF-1α stabilization, IL-6 secretion and increased bacterial survival, indicating that this virulence factor promotes bacterial persistence by modulating immuno-metabolic signaling. Conclusion Our findings demonstrate that H. pylori drives temporally structured macrophage responses, transitioning from early inflammatory activation to a hybrid state that supports bacterial persistence.

Abstract Background We investigated whether markers, genes or terms of the Human Phenotype Ontology associated with genetic or rare diseases (GARDs) that affect airway or lung function are associated with lung cancer. Methods Genes of interest were extracted from GARD (Genetic and Rare Diseases Information Center), OMIM (Online Mendelian Inheritance in Man®), ORPHANET and Monarch Initiative. Individual SNP, gene level and gene-set analyses were performed for 52,207 SNPs, 1677 genes or for 620 terms of the Human Phenotype Ontology. The analysis included 14,068 lung cancer cases and 12,390 cancer-free control subjects of European descent from the International Lung Cancer Consortium ILCCO. Results The marker rs56113850 (OR=0.893, 95%CI: 0.862-0.924) was associated with lung cancer (p=1.2x10-10). This marker is located in CYP2A6 as well as in an enhancer region of LTBP4, which is associated with cutis laxa. A suggestive significant association was observed for two markers associated with the DMD gene, which is linked to Duchenne muscular dystrophy. The gene sets "Abnormal circulating adrenocorticotropin concentration" and "Central nervous system neoplasm" were found to be significantly enriched with GARD genes, and can therefore be considered to be associated with lung cancer. Conclusions Genes associated with genetic and rare lung diseases do not generally appear to carry risk factors for lung cancer. However, genes associated with the hypothalamic-pituitary-adrenal axis show some, but rather weak or complex, associations with lung cancer. Tests at the gene level provide extremely inhomogeneous results, even when applied to the same data.

Background:Genome-wide association studies (GWAS) have identified numerous lung cancer susceptibility loci based on single nucleotide polymorphisms (SNPs), yet a substantial proportion of heritability remains unexplained. We therefore evaluated germline copy number variants (CNVs) as an underexplored source of genetic susceptibility and potential contributors to genomic instability in lung cancer. Methods:We conducted a genome-wide analysis of germline CNVs using 19,342 cases and 15,917 controls from the Transdisciplinary Research in Cancer of the Lung (TRICL) consortium, with replication in two independent cohorts. High-confidence CNVs were identified by integrating two CNV callers including PennCNV and modSaRa2. Association analyses were performed using both gene-based and CNV region-based approaches. Polygenic risk scores (PRS) were constructed from top loci, and functional validation was conducted using siRNA-mediated knockdown in lung fibroblast cells. Results:We identified CNVs in four genomic regions (1p36.22, 2q31.2, 6p21.32, and 19q13.32) significantly associated with lung cancer risk. Two loci (1p36.22 and 2q31.2) were consistently supported across both analytical strategies. A CNV-based PRS constructed from key genes (CLCN6, NFE2L2, OPA3, and PSMB8) was significantly associated with lung cancer risk and replicated across independent datasets. Functional assays demonstrated that knockdown of NFE2L2 and OPA3 increased endogenous DNA damage, supporting a role in genomic stability. Conclusions:Germline CNVs contribute to lung cancer susceptibility and may influence carcinogenesis through mechanisms related to genomic instability. Impact:These findings expand the genetic architecture of lung cancer and highlight CNVs as potential biomarkers for improving risk stratification and informing precision prevention strategies.

Abstract Background The global prevalence of metabolic diseases (MetDs) continues to rise and is associated with an increased cancer risk. Genome-wide association studies have identified numerous single nucleotide variants (SNVs) linked to MetDs, some within genes also implicated in tumorigenesis. G-quadruplexes (G4s) are non-canonical DNA secondary structures that regulate gene expression in diverse and context dependent ways. Variants affecting G4 structure may alter transcriptional efficiency. Notably, disease-associated variants are frequently located within or near regulatory elements and may overlap with cancer-associated alternative promoters. Results We systematically assess the overlap between MetD-associated SNVs and G4 motifs, and evaluate their effects on G4 stability, topology, and their potential to modulate the regulatory activity of G4s in alternative promoters. Approximately 0.9% to 1.6% of MetD-associated SNVs are located within G4 motifs (G4-SNVs), depending on the prediction tool. Effect alleles, those associated with risk or protection against MetDs, generally reduce G4 stability, regardless of their direction of association. Several G4-SNVs are mapped to cancer-associated alternative promoters, including the destabilizing MICB rs2855804 C/T variant and the stabilizing PLA2G6 rs2277844 G/A variants. In vivo G4 formation is confirmed by permanganate/S1 nuclease footprinting coupled with sequencing, while circular dichroism spectroscopy reveals allele-specific changes in G4 topology and stability. Integration of Hi-C data, histone modifications, transcription factor binding, and luciferase reporter assays further support their regulatory impact. Conclusions Although G4-SNVs are unlikely to be the sole disease drivers, they significantly influence transcriptional regulation, potentially contributing to allele-specific gene expression in MetD patients and their link to increased cancer risk.

Heterozygosity at human leukocyte antigen (HLA) loci may improve lung cancer immunosurveillance by increasing recognition of the tumor by the immune system. Previous studies utilizing data from population-level biobanks, such as the United Kingdom Biobank and FinnGen, have identified an association between germline HLA class II (HLA-II) heterozygosity and reduced lung cancer risk in smokers. In the present study, we evaluate the association between HLA heterozygosity and lung cancer in a large case-control study (15,302 cases and 14,580 controls) with imputed HLA allele-type information, comparing differences in HLA heterozygosity between smokers and non-smokers, among lung cancer subtypes, and at 2- and 4-digit HLA allele resolution. We identify a strong protective association of HLA-II heterozygosity in smokers compared to non-smokers, particularly at the HLA-DPB1 and HLA-DPA1 loci, and provide subtype-specific resolution. Finally, analysis of the additive effects of HLA allele heterozygosity in smokers identified significant associations with several 4-digit HLA alleles, including HLA-B∗08:01, HLA-A∗01:01, HLA-C∗07:01, HLA-DQA1∗05:01, HLA-DRB1∗03:01, and HLA-C∗03:04. Our study provides additional evidence, with added histologic subtype information, that germline HLA-II heterozygosity is inversely associated with lung cancer risk.

Abstract The histone methyltransferase PR domain containing protein 9 (PRDM9) is a key determinant of meiotic recombination in humans. It deposits activating histone marks thereby promoting recruitment of the meiotic recombination machinery. It recognizes DNA through a repetitive zinc-finger array that binds specific sequence motifs whose complementary G-rich strands can form DNA secondary structures, particularly G-quadruplexes (G4s). These may present an additional binding platform for PRDM9 and contribute to the formation of a chromatin environment permissive for meiotic recombination. We investigated the relationship between PRDM9 binding sites and G4 motifs using computational analyses of predicted and experimentally validated G4s and found that G4 motifs are among the most prevalent features at PRDM9 binding sites, with the strongest enrichment observed for highly stable G4s, largely independent of loop length. Using electrophoretic mobility shift assays, we further examined whether PRDM9 can bind short, single-stranded G4-forming oligonucleotides in addition to its canonical double-stranded DNA targets. PRDM9 directly bound folded G4 structures, and binding increased with G4 stability. This relationship was observed across different G4 motifs and following stabilization of the same G4 by increasing the potassium concentration or adding a G4-stabilizing ligand. PRDM9 also bound an artificial G4-forming sequence absent from the human genome, which was abolished when mutating the G4 motif to avoid structure formation. Together, these results support a model in which stable G4 structures facilitate PRDM9 recruitment by creating and discrete increased local chromatin accessibility, thereby contributing to the initiation of meiotic recombination. Graphical abstract

Premenstrual dysphoric disorder (PMDD) and its subclinical form categorized as premenstrual syndrome (PMS), are severe mood disorders characterized by cyclical depressive symptoms, anxiety, irritability, and other functional impairments, impacting a broad range of women during the late luteal phase. The estrogen receptor 1 (ESR1) gene encodes the estrogen receptor alpha (ERα) which plays a critical role in mediating estrogen signaling and regulates various physiological and psychological processes. In this study, we investigated the relationship between six single nucleotide polymorphisms (SNPs) in intron 4 of the ESR1 gene and premenstrual symptom severity, emphasizing symptom-genotype associations and SNP interactions. Results demonstrated that specific SNPs were linked to distinct symptom profiles, such as anxiety, difficulty concentrating, and sleep disturbances. Interactions between SNPs revealed both risk-enhancing and protective effects. These findings suggest that premenstrual symptoms may stem from a genotype-linked reduced sensitivity to ovarian hormones, providing a foundation for future research. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was in part funded by the Austrian Science Fund (P28261, W1233, P32276) and the European Research Council (ERC Starting Grant: 850953). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All studies were approved by the University of Salzburgs ethics committee (ethics vote numbers: GZ28/2018, GZ50/2020, GZ04/2018, GZ24/2022). All participants provided written informed consent to partake in the studies. All methods conform to the Code of Ethics of the World Medical Association (Declaration of Helsinki). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data will be made accessible upon reasonable request.

Despite lung cancer affecting all races and ethnicities, disparities are observed in incidence and mortality rates among different ethnic groups in the United States. Non-Hispanic African Americans had a high incidence rate of lung cancer at 55.8 per 100 000 people, as well as the highest death rate at 37.2 per 100 000 people from 2016 to 2020. While previous genome-wide association studies (GWAS) have identified over 45 susceptibility risk loci that influence lung cancer development, few GWAS have investigated the etiology of lung cancer in African Americans. To address this gap in knowledge, we conducted GWAS of lung cancer focused on studying African Americans, comprising 2267 lung cancer cases and 4264 controls. We identified three loci associated with lung cancer, one with lung adenocarcinoma, and four with lung squamous cell carcinoma in this population at the genomic-wide significance level. Among them, three novel loci were identified near VWF at 12p13.31 for overall lung cancer and GACAT3 at 2p24.3 and LMAN1L at 15q24.1 for lung squamous cell carcinoma. In addition, we confirmed previously reported risk loci with known or new lead variants near CHRNA5 at 15q25.1 and CYP2A6 at 19q13.2 associated with lung cancer and TRIP13 at 5p15.33 and ERC1 at 12p13.33 associated with lung squamous cell carcinoma. Further multi-step functional analyses shed light on biological mechanisms underlying these associations of lung cancer in this population. Our study highlights the importance of ancestry-specific studies for the potential alleviation of lung cancer burden in African Americans.

BACKGROUND:Lung adenocarcinoma (LUAD) in never-smokers is a major public health burden, especially among East Asian women. Polygenic risk scores (PRSs) are promising for risk stratification but are primarily developed in European-ancestry populations. We aimed to develop and validate single- and multi-ancestry PRSs for East Asian never-smokers to improve LUAD risk prediction. METHODS:PRSs were developed using genome-wide association study summary statistics from East Asian (8,002 cases; 20,782 controls) and European (2,058 cases; 5,575 controls) populations. Single-ancestry models included PRS-25, PRS-CT, and LDpred2; multi-ancestry models included LDpred2+PRS-EUR128, PRS-CSx, and CT-SLEB. Performance was evaluated in independent East Asian data from the Female Lung Cancer Consortium (FLCCA) and externally validated in the Nanjing Lung Cancer Cohort (NJLCC). We assessed predictive accuracy via AUC, with 10-year and (age 30-80) absolute risks estimates. RESULTS:The best multi-ancestry PRS, using East Asian and European data via CT-SLEB (clumping and thresholding, super learning, empirical Bayes), outperformed the best East Asian-only PRS (LDpred2; AUC = 0.629, 95% CI:0.618,0.641), achieving an AUC of 0.640 (95% CI : 0.629,0.653) and odds ratio of 1.71 (95% CI : 1.61,1.82) per SD increase. NJLCC Validation confirmed robust performance (AUC =0.649, 95% CI: 0.623, 0.676). The top 20% PRS group had a 3.92-fold higher LUAD risk than the bottom 20%. Further, the top 5% PRS group reached a 6.69% lifetime absolute risk. Notably, this group reached the average population 10-year LUAD risk at age 50 (0.42%) by age 41, nine years earlier. CONCLUSIONS:Multi-ancestry PRS approaches enhance LUAD risk stratification in East Asian never-smokers, with consistent external validation, suggesting future clinical utility.

BACKGROUND:Until now, no study has investigated the impact of allergen immunotherapy (AIT) on genome-wide DNA methylation in a longitudinal set-up. Herein, we investigated whether differences in DNA methylation occur in birch pollen allergic patients undergoing 6 months of birch pollen AIT, assessed alterations in methylation-based blood cell type composition, and correlated DNA methylation to serological AIT biomarkers. METHODS:We performed genome-wide DNA-methylation analysis on bisulfite-converted DNA derived from whole blood samples of 16 birch pollen-allergic patients (pre-/post-birch pollen AIT) and 15 placebo (pre-/post-placebo treatment). RESULTS:Our analysis identified cg22187251, located within a regulatory region upstream of the glucosaminyl (N-acetyl) transferase 2 (GCNT2) gene and cg22336863 upstream of the transcription start site of actin binding rho activating protein (ABRA), as hypermethylated. Functional assays revealed that these regions exhibit methylation-dependent promoter and enhancer activities. We identified differentially methylated positions within the HLA gene complex, and an AIT-specific increase of CD8+ T cell populations accompanied by a decrease in natural killer (NK) cell proportion. Strong to moderate correlations with clinical biomarkers (such as specific IgG4) were observed for 42% of the top 100 differentially methylated positions. CONCLUSION:GCNT2 and ABRA are implicated in Rho-signaling, a pathway involved in Th2 differentiation. GCNT2 modulates the SMAD-dependent TGF-β pathway, indicating a role in mediating AIT-induced immunotolerance. This is the first longitudinal study investigating DNA methylation changes induced by birch pollen AIT.

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.

DNA secondary structures are essential elements of the genomic landscape, playing a critical role in regulating various cellular processes. These structures refer to G-quadruplexes, cruciforms, Z-DNA or H-DNA structures, amongst others (collectively called 'non-B DNA'), which DNA molecules can adopt beyond the B conformation. DNA secondary structures have significant biological roles, and their landscape is dynamic and can rearrange due to various factors, including changes in cellular conditions, temperature, and DNA-binding proteins. Understanding this dynamic nature is crucial for unraveling their functions in cellular processes. Detecting DNA secondary structures remains a challenge. Conventional methods, such as gel electrophoresis and chemical probing, have limitations in terms of sensitivity and specificity. Emerging techniques, including next-generation sequencing and single-molecule approaches, offer promise but face challenges since these techniques are mostly limited to only one type of secondary structure. Here we describe an updated version of a technique permanganate/S1 nuclease footprinting, which uses potassium permanganate to trap single-stranded DNA regions as found in many non-B structures, in combination with S1 nuclease digest and adapter ligation to detect genome-wide non-B formation. To overcome technical hurdles, we combined this method with direct adapter ligation and sequencing (PDAL-Seq). Furthermore, we established a user-friendly pipeline available on Galaxy to standardize PDAL-Seq data analysis. This optimized method allows the analysis of many types of DNA secondary structures that form in a living cell and will advance our knowledge of their roles in health and disease.

Supplementary Table S7 shows the signals from interaction with smoking status for the identified variants in lung cancer.

Abstract Cigarette smoke, containing both nicotine and carcinogens, causes lung cancer. However, not all smokers develop lung cancer, highlighting the importance of the interaction between host susceptibility and environmental exposure in tumorigenesis. Here, we aimed to delineate the interaction between metabolizing ability of tobacco carcinogens and smoking intensity in mediating genetic susceptibility to smoking-related lung tumorigenesis. Single-variant and gene-based associations of 43 tobacco carcinogen–metabolizing genes with lung cancer were analyzed using summary statistics and individual-level genetic data, followed by causal inference of Mendelian randomization, mediation analysis, and structural equation modeling. Cigarette smoke–exposed cell models were used to detect gene expression patterns in relation to specific alleles. Data from the International Lung Cancer Consortium (29,266 cases and 56,450 controls) and UK Biobank (2,155 cases and 376,329 controls) indicated that the genetic variant rs56113850 C>T located in intron 4 of CYP2A6 was significantly associated with decreased lung cancer risk among smokers (OR = 0.88, 95% confidence interval = 0.85–0.91, P = 2.18 × 10−16), which might interact (Pinteraction = 0.028) with and partially be mediated (ORindirect = 0.987) by smoking status. Smoking intensity accounted for 82.3% of the effect of CYP2A6 activity on lung cancer risk but entirely mediated the genetic effect of rs56113850. Mechanistically, the rs56113850 T allele rescued the downregulation of CYP2A6 caused by cigarette smoke exposure, potentially through preferential recruitment of transcription factor helicase-like transcription factor. Together, this study provides additional insights into the interplay between host susceptibility and carcinogen exposure in smoking-related lung tumorigenesis. Significance: The causal pathway connecting CYP2A6 genetic variability and activity, cigarette consumption, and lung cancer susceptibility in smokers highlights the need for behavior modification interventions based on host susceptibility for cancer prevention.

Supplementary Methods and Figures

Supplementary Table S8 displays the sample size from independent Asian studies.

Supplementary Table S1 displays the number of ever- and never-smokers from each of the ten studies.

Supplementary Figure S4 shows the results of eQTL analysis of rs968516 in GTEx.

Supplementary Table S4 shows the imputation quality score of identified variants in different studies.