Global hypomethylation in childhood asthma identified by genome-wide DNA-methylation sequencing preferentially affects enhancer regions.

BACKGROUND: Childhood asthma is a result of a complex interaction of genetic and environmental components causing epigenetic and immune dysregulation, airway inflammation and impaired lung function. Although different microarray based EWAS studies have been conducted, the impact of epigenetic regulation in asthma development is still widely unknown. We have therefore applied unbiased whole genome bisulfite sequencing (WGBS) to characterize global DNA-methylation profiles of asthmatic children compared to healthy controls. METHODS: Peripheral blood samples of 40 asthmatic and 42 control children aged 5-15 years from three birth cohorts were sequenced together with paired cord blood samples. Identified differentially methylated regions (DMRs) were categorized in genotype-associated, cell-type-dependent, or prenatally primed. Network analysis and subsequent natural language processing of DMR-associated genes was complemented by targeted analysis of functional translation of epigenetic regulation on the transcriptional and protein level. RESULTS: In total, 158 DMRs were identified in asthmatic children compared to controls of which 37% were related to the eosinophil content. A global hypomethylation was identified affecting predominantly enhancer regions and regulating key immune genes such as IL4, IL5RA, and EPX. These DMRs were confirmed in n = 267 samples and could be linked to aberrant gene expression. Out of the 158 DMRs identified in the established phenotype, 56 were perturbed already at birth and linked, at least in part, to prenatal influences such as tobacco smoke exposure or phthalate exposure. CONCLUSION: This is the first epigenetic study based on whole genome sequencing to identify marked dysregulation of enhancer regions as a hallmark of childhood asthma.

The whole-genome landscape of medulloblastoma subtypes.

Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.

Genome-wide DNA methylation sequencing identifies epigenetic perturbations in the upper airways under long-term exposure to moderate levels of ambient air pollution.

While the link between exposure to high levels of ambient particulate matter (PM) and increased incidences of respiratory and cardiovascular diseases is widely recognized, recent epidemiological studies have shown that low PM concentrations are equally associated with adverse health effects. As DNA methylation is one of the main mechanisms by which cells regulate and stabilize gene expression, changes in the methylome could constitute early indicators of dysregulated signaling pathways. So far, little is known about PM-associated DNA methylation changes in the upper airways, the first point of contact between airborne pollutants and the human body. Here, we focused on cells of the upper respiratory tract and assessed their genome-wide DNA methylation pattern to explore exposure-associated early regulatory changes. Using a mobile epidemiological laboratory, nasal lavage samples were collected from a cohort of 60 adults that lived in districts with records of low (Simmerath) or moderate (Stuttgart) PM10 levels in Germany. PM10 concentrations were verified by particle measurements on the days of the sample collection and genome-wide DNA methylation was determined by enzymatic methyl sequencing at single-base resolution. We identified 231 differentially methylated regions (DMRs) between moderately and lowly PM10 exposed individuals. A high proportion of DMRs overlapped with regulatory elements, and DMR target genes were involved in pathways regulating cellular redox homeostasis and immune response. In addition, we found distinct changes in DNA methylation of the HOXA gene cluster whose methylation levels have previously been linked to air pollution exposure but also to carcinogenesis in several instances. The findings of this study suggest that regulatory changes in upper airway cells occur at PM10 levels below current European thresholds, some of which may be involved in the development of air pollution-related diseases.

Gene Expression in Solitary Fibrous Tumors (SFTs) Correlates with Anatomic Localization and NAB2-STAT6 Gene Fusion Variants.

Solitary fibrous tumors (SFTs) harbor recurrent NAB2-STAT6 gene fusions, promoting constitutional up-regulation of oncogenic early growth response 1 (EGR1)-dependent gene expression. SFTs with the most common canonical NAB2 exon 4-STAT6 exon 2 fusion variant are often located in the thorax (pleuropulmonary) and are less cellular with abundant collagen. In contrast, SFTs with NAB2 exon 6-STAT6 exon 16/17 fusion variants typically display a cellular round to ovoid cell morphology and are often located in the deep soft tissue of the retroperitoneum and intra-abdominal pelvic region or in the meninges. Here, we employed next-generation sequencing-based gene expression profiling to identify significant differences in gene expression associated with anatomic localization and NAB2-STAT6 gene fusion variants. SFTs with the NAB2 exon 4-STAT6 exon 2 fusion variant showed a transcriptional signature enriched for genes involved in DNA binding, gene transcription, and nuclear localization, whereas SFTs with the NAB2 exon 6-STAT6 exon 16/17 fusion variants were enriched for genes involved in tyrosine kinase signaling, cell proliferation, and cytoplasmic localization. Specific transcription factor binding motifs were enriched among differentially expressed genes in SFTs with different fusion variants, implicating co-transcription factors in the modification of chimeric NGFI-A binding protein 2 (NAB2)-STAT6-dependent deregulation of EGR1-dependent gene expression. In summary, this study establishes a potential molecular biologic basis for clinicopathologic differences in SFTs with distinct NAB2-STAT6 gene fusion variants.

Lipomatous Solitary Fibrous Tumors Harbor Rare NAB2-STAT6 Fusion Variants and Show Up-Regulation of the Gene PPARG, Encoding for a Regulator of Adipocyte Differentiation.

Solitary fibrous tumors (SFTs) harbor activating NAB2-STAT6 gene fusions. Different variants of the NAB2-STAT6 gene fusion have been associated with distinct clinicopathologic features. Lipomatous SFTs are a morphologic variant of SFTs, characterized by a fat-forming tumor component. Our aim was to evaluate NAB2-STAT6 fusion variants and to further study the molecular genetic features in a cohort of lipomatous SFTs. A hybrid-capture-based next-generation sequencing panel was employed to detect NAB2-STAT6 gene fusions at the RNA level. In addition, the RNA expression levels of 507 genes were evaluated using this panel, and were compared with a control cohort of nonlipomatous SFTs. Notably, 5 of 11 (45%) of lipomatous SFTs in the current series harbored the uncommon NAB2 exon 4-STAT6 exon 4 gene fusion variant, which is observed in only 0.9% to 1.4% of nonlipomatous SFTs. Furthermore, lipomatous SFTs displayed significant differences in gene expression compared with their nonlipomatous counterparts, including up-regulation of the gene peroxisome proliferator activated receptor-γ (PPARG). Peroxisome proliferator activated receptor-γ is a nuclear receptor regulating adipocyte differentiation, providing a possible explanation for the fat-forming component in lipomatous SFTs. In summary, the current study provides a possible molecular genetic basis for the distinct morphologic features of lipomatous SFTs.

Unraveling most abundant mutational signatures in head and neck cancer.

Genomic alterations are a driving force in the multistep process of head and neck cancer (HNC) and result from the interaction of exogenous environmental exposures and endogenous cellular processes. Each of these processes leaves a characteristic pattern of mutations on the tumor genome providing the unique opportunity to decipher specific signatures of mutational processes operative during HNC pathogenesis and to address their prognostic value. Computational analysis of whole exome sequencing data of the HIPO-HNC (Heidelberg Center for Personalized Oncology-head and neck cancer) (n = 83) and TCGA-HNSC (The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma) (n = 506) cohorts revealed five common mutational signatures (Catalogue of Somatic Mutations in Cancer [COSMIC] Signatures 1, 2, 3, 13 and 16) and demonstrated their significant association with etiological risk factors (tobacco, alcohol and HPV16). Unsupervised hierarchical clustering identified four clusters (A, B, C1 and C2) of which Subcluster C2 was enriched for cases with a higher frequency of signature 16 mutations. Tumors of Subcluster C2 had significantly lower p16INK4A expression accompanied by homozygous CDKN2A deletion in almost one half of cases. Survival analysis revealed an unfavorable prognosis for patients with tumors characterized by a higher mutation burden attributed to signature 16 as well as cases in Subcluster C2. Finally, a LASSO-Cox regression model was applied to prioritize clinically relevant signatures and to establish a prognostic risk score for head and neck squamous cell carcinoma patients. In conclusion, our study provides a proof of concept that computational analysis of somatic mutational signatures is not only a powerful tool to decipher environmental and intrinsic processes in the pathogenesis of HNC, but could also pave the way to establish reliable prognostic patterns.

Somatic mutations and promotor methylation of the ryanodine receptor 2 is a common event in the pathogenesis of head and neck cancer.

Genomic sequencing projects unraveled the mutational landscape of head and neck squamous cell carcinoma (HNSCC) and provided a comprehensive catalog of somatic mutations. However, the limited number of significant cancer-related genes obtained so far only partially explains the biological complexity of HNSCC and hampers the development of novel diagnostic biomarkers and therapeutic targets. We pursued a multiscale omics approach based on whole-exome sequencing, global DNA methylation and gene expression profiling data derived from tumor samples of the HIPO-HNC cohort (n = 87), and confirmed new findings with datasets from The Cancer Genome Atlas (TCGA). Promoter methylation was confirmed by MassARRAY analysis and protein expression was assessed by immunohistochemistry and immunofluorescence staining. We discovered a set of cancer-related genes with frequent somatic mutations and high frequency of promoter methylation. This included the ryanodine receptor 2 (RYR2), which showed variable promoter methylation and expression in both tumor samples and cell lines. Immunohistochemical staining of tissue sections unraveled a gradual loss of RYR2 expression from normal mucosa via dysplastic lesion to invasive cancer and indicated that reduced RYR2 expression in adjacent tissue and precancerous lesions might serve as risk factor for unfavorable prognosis and upcoming malignant conversion. In summary, our data indicate that impaired RYR2 function by either somatic mutation or epigenetic silencing is a common event in HNSCC pathogenesis. Detection of RYR2 expression and/or promoter methylation might enable risk assessment for malignant conversion of dysplastic lesions.

Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children.

Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype-related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular "commuting" enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole-genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non-regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c-Jun N-terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood.

Paediatric and adult soft tissue sarcomas with NTRK1 gene fusions: a subset of spindle cell sarcomas unified by a prominent myopericytic/haemangiopericytic pattern.

Neoplasms with a myopericytomatous pattern represent a morphological spectrum of lesions encompassing myopericytoma of the skin and soft tissue, angioleiomyoma, myofibromatosis/infantile haemangiopericytoma and putative neoplasms reported as malignant myopericytoma. Lack of reproducible phenotypic and genetic features of malignant myopericytic neoplasms have prevented the establishment of myopericytic sarcoma as an acceptable diagnostic category. Following detection of a LMNA-NTRK1 gene fusion in an index case of paediatric haemangiopericytoma-like sarcoma by combined whole-genome and RNA sequencing, we identified three additional sarcomas harbouring NTRK1 gene fusions, termed 'spindle cell sarcoma, NOS with myo/haemangiopericytic growth pattern'. The patients were two children aged 11 months and 2 years and two adults aged 51 and 80 years. While the tumours of the adults were strikingly myopericytoma-like, but with clear-cut atypical features, the paediatric cases were more akin to infantile myofibromatosis/haemangiopericytoma. All cases contained numerous thick-walled dysplastic-like vessels with segmental or diffuse nodular myxohyaline myo-intimal proliferations of smooth muscle actin-positive cells, occasionally associated with thrombosis. Immunohistochemistry showed variable expression of smooth muscle actin and CD34, but other mesenchymal markers, including STAT6, were negative. This study showed a novel variant of myo/haemangiopericytic sarcoma with recurrent NTRK1 gene fusions. Given the recent introduction of a novel therapeutic approach targeting NTRK fusion-positive neoplasms, recognition of this rare but likely under-reported sarcoma variant is strongly encouraged.

Recurrent mutations within the amino-terminal region of ß-catenin are probable key molecular driver events in sinonasal hemangiopericytoma.

Sinonasal hemangiopericytoma (SN-HPC) is an uncommon, site-specific, low-grade mesenchymal neoplasm of probable perivascular myoid cell origin. In contrast to solitary fibrous tumors of soft tissue and sinonasal tract origin, SN-HPCs were recently shown to lack recurrent NAB2-STAT6 fusion variants. Other molecular alterations known to occur in some of soft tissue perivascular myoid cell neoplasms were also absent in SN-HPC; thus, the molecular pathogenesis of SN-HPCs remained unknown. Guided by whole-genome sequencing combined with RNA sequencing of an index case, we analyzed a total of six SN-HPCs for mutations within the amino-terminal region of the gene CTNNB1 (cadherin-associated protein), ß 1, 88 kDa, encoding ß-catenin. All six cases showed missense mutations, with amino acid substitutions clustering at positions 33 to 45, corresponding to the recognition site of the ß-catenin destruction complex. Similar CTNNB1 mutations have been described in a variety of epithelial and mesenchymal neoplasms. These mutations prevent ß-catenin phosphorylation and proteasomal degradation but promote its nuclear accumulation and subsequent increased transcription of Wingless-related integration site target genes. Consistent with these molecular findings, ß-catenin IHC showed consistent diffuse and strong nuclear staining of the tumor cells in all six SN-HPCs. Our results highlight, for the first time, CTNNB1 mutations as the likely initiating molecular events driving SN-HPC tumorigenesis, which places SN-HPC among the growing family of ß-catenin-driven mesenchymal neoplasms.

Solitary fibrous tumors/hemangiopericytomas with different variants of the NAB2-STAT6 gene fusion are characterized by specific histomorphology and distinct clinicopathological features.

Recurrent somatic fusions of the two genes, NGFI-A-binding protein 2 (NAB2) and STAT6, located at chromosomal region 12q13, have been recently identified to be presumable tumor-initiating events in solitary fibrous tumors (SFT). Herein, we evaluated a cohort of 52 SFTs/hemangiopericytomas (HPCs) by whole-exome sequencing (one case) and multiplex RT-PCR (all 52 cases), and identified 12 different NAB2-STAT6 fusion variants in 48 cases (92%). All 52 cases showed strong and diffuse nuclear positivity for STAT6 by IHC. We categorized the fusion variants according to their potential functional effects within the predicted fusion protein and found strong correlations with relevant clinicopathological features. Tumors with the most common fusion variant, NAB2ex4-STAT6ex2/3, corresponded to classic pleuropulmonary SFTs with diffuse fibrosis and mostly benign behavior and occurred in older patients (median age, 69 years). In contrast, tumors with the second most common fusion variant, NAB2ex6-STAT6ex16/17, were found in much younger patients (median age, 47 years) and represented typical HPCs from deep soft tissue with a more aggressive phenotype and clinical behavior. In summary, these molecular genetic findings support the concept that classic pleuropulmonary SFT and deep-seated HPC are separate entities that share common features but correlate to different clinical outcome.

Differentially methylated regions within lung cancer risk loci are enriched in deregulated enhancers.

Genome-wide association studies (GWAS) have identified SNPs linked with lung cancer risk. Our aim was to discover the genes, non-coding RNAs, and regulatory elements within GWAS-identified risk regions that are deregulated in non-small cell lung carcinoma (NSCLC) to identify novel, clinically targetable genes and mechanisms in carcinogenesis. A targeted bisulphite-sequencing approach was used to comprehensively investigate DNA methylation changes occurring within lung cancer risk regions in 17 NSCLC and adjacent normal tissue pairs. We report differences in differentially methylated regions between adenocarcinoma and squamous cell carcinoma. Among the minimal regions found to be differentially methylated in at least 50% of the patients, 7 candidates were replicated in 2 independent cohorts (n = 27 and n = 87) and the potential of 6 as methylation-dependent regulatory elements was confirmed by functional assays. This study contributes to understanding the pathways implicated in lung cancer initiation and progression, and provides new potential targets for cancer treatment.

Mutational mechanisms shaping the coding and noncoding genome of germinal center derived B-cell lymphomas.

B cells have the unique property to somatically alter their immunoglobulin (IG) genes by V(D)J recombination, somatic hypermutation (SHM) and class-switch recombination (CSR). Aberrant targeting of these mechanisms is implicated in lymphomagenesis, but the mutational processes are poorly understood. By performing whole genome and transcriptome sequencing of 181 germinal center derived B-cell lymphomas (gcBCL) we identified distinct mutational signatures linked to SHM and CSR. We show that not only SHM, but presumably also CSR causes off-target mutations in non-IG genes. Kataegis clusters with high mutational density mainly affected early replicating regions and were enriched for SHM- and CSR-mediated off-target mutations. Moreover, they often co-occurred in loci physically interacting in the nucleus, suggesting that mutation hotspots promote increased mutation targeting of spatially co-localized loci (termed hypermutation by proxy). Only around 1% of somatic small variants were in protein coding sequences, but in about half of the driver genes, a contribution of B-cell specific mutational processes to their mutations was found. The B-cell-specific mutational processes contribute to both lymphoma initiation and intratumoral heterogeneity. Overall, we demonstrate that mutational processes involved in the development of gcBCL are more complex than previously appreciated, and that B cell-specific mutational processes contribute via diverse mechanisms to lymphomagenesis.

Divergence of imprinted genes during mammalian evolution.

BACKGROUND: In contrast to the majority of mammalian genes, imprinted genes are monoallelically expressed with the choice of the active allele depending on its parental origin. Due to their special inheritance patterns, maternally and paternally expressed genes might be under different evolutionary pressure. Here, we aimed at assessing the evolutionary history of imprinted genes. RESULTS: In this study, we investigated the conservation of imprinted genes in vertebrate genomes and their exposition to natural selection. In a genome-wide comparison, orthologs of imprinted genes show a stronger divergence on cDNA and protein level in mammals. This pattern is most pronounced for maternally expressed genes in rodents in comparison to their non-rodent orthologs. The divergence is not attributable to increased mutation of CpG positions. It is contrasted by strong conservation of paternally expressed genes in mouse and rat. Interestingly, we found that the early divergence of imprinted genes was accompanied by an unusually strict conservation of their paralogs. CONCLUSIONS: The apparent degeneration of maternally expressed genes may reflect a relaxation of selective pressure due to counteracting effects on maternal and embryonic fitness. Functional redundancy provided by the presence of highly conserved (non-imprinted) paralogs may have facilitated the divergence. Moreover, intensification of imprinting in modern rodents seems to have shifted the evolutionary fate of imprinted genes towards strong purifying selection.

Imprinted genes show unique patterns of sequence conservation.

BACKGROUND: Genomic imprinting is an evolutionary conserved mechanism of epigenetic gene regulation in placental mammals that results in silencing of one of the parental alleles. In order to decipher interactions between allele-specific DNA methylation of imprinted genes and evolutionary conservation, we performed a genome-wide comparative investigation of genomic sequences and highly conserved elements of imprinted genes in human and mouse. RESULTS: Evolutionarily conserved elements in imprinted regions differ from those associated with autosomal genes in various ways. Whereas for maternally expressed genes strong divergence of protein-encoding sequences is most prominent, paternally expressed genes exhibit substantial conservation of coding and noncoding sequences. Conserved elements in imprinted regions are marked by enrichment of CpG dinucleotides and low (TpG+CpA)/(2·CpG) ratios indicate reduced CpG deamination. Interestingly, paternally and maternally expressed genes can be distinguished by differences in G+C and CpG contents that might be associated with unusual epigenetic features. Especially noncoding conserved elements of paternally expressed genes are exceptionally G+C and CpG rich. In addition, we confirmed a frequent occurrence of intronic CpG islands and observed a decelerated degeneration of ancient LINE-1 repeats. We also found a moderate enrichment of YY1 and CTCF binding sites in imprinted regions and identified several short sequence motifs in highly conserved elements that might act as additional regulatory elements. CONCLUSIONS: We discovered several novel conserved DNA features that might be related to allele-specific DNA methylation. Our results hint at reduced CpG deamination rates in imprinted regions, which affects mostly noncoding conserved elements of paternally expressed genes. Pronounced differences between maternally and paternally expressed genes imply specific modes of evolution as a result of differences in epigenetic features and a special response to selective pressure. In addition, our data support the potential role of intronic CpG islands as epigenetic key regulatory elements and suggest that evolutionary conserved LINE-1 elements fulfill regulatory functions in imprinted regions.

Integrative Analysis of Multi-omics Data Identified EGFR and PTGS2 as Key Nodes in a Gene Regulatory Network Related to Immune Phenotypes in Head and Neck Cancer.

PURPOSE: Malignant progression exhibits a tightly orchestrated balance between immune effector response and tolerance. However, underlying molecular principles that drive the establishment and maintenance of the tumor immune phenotype remain to be elucidated. EXPERIMENTAL DESIGN: We trained a novel molecular classifier based on immune cell subsets related to programmed death-ligand 1 (PD-L1) and interferon γ (IFNγ) expression, which revealed distinct subgroups with higher (cluster A) or lower (subcluster B3) cytotoxic immune phenotypes. Integrative analysis of multi-omics data was conducted to identify differences in genetic and epigenetic landscapes as well as their impact on differentially expressed genes (DEG) among immune phenotypes. A prognostic gene signature for immune checkpoint inhibition (ICI) was established by a least absolute shrinkage and selection operator (LASSO)-Cox regression model. RESULTS: Mutational landscape analyses unraveled a higher frequency of CASP8 somatic mutations in subcluster A1, while subcluster B3 exhibited a characteristic pattern of copy-number alterations affecting chemokine signaling and immune effector response. The integrative multi-omics approach identified EGFR and PTGS2 as key nodes in a gene regulatory network related to the immune phenotype, and several DEGs related to the immune phenotypes were affected by EGFR inhibition in tumor cell lines. Finally, we established a prognostic gene signature by a LASSO-Cox regression model based on DEGs between nonprogressive disease and progressive disease subgroups for ICI. CONCLUSIONS: Our data highlight a complex interplay between genetic and epigenetic events in the establishment of the tumor immune phenotype and provide compelling experimental evidence that a patient with squamous cell carcinoma of the head and neck at higher risk for ICI treatment failure might benefit from a combination with EGFR inhibition.

Whole-genome fingerprint of the DNA methylome during chemically induced differentiation of the human AML cell line HL-60/S4.

Epigenomic regulation plays a vital role in cell differentiation. The leukemic HL-60/S4 [human myeloid leukemic cell line HL-60/S4 (ATCC CRL-3306)] promyelocytic cell can be easily differentiated from its undifferentiated promyelocyte state into neutrophil- and macrophage-like cell states. In this study, we present the underlying genome and epigenome architecture of HL-60/S4 through its differentiation. We performed whole-genome bisulphite sequencing of HL-60/S4 cells and their differentiated counterparts. With the support of karyotyping, we show that HL-60/S4 maintains a stable genome throughout differentiation. Analysis of differential Cytosine-phosphate-Guanine dinucleotide methylation reveals that most methylation changes occur in the macrophage-like state. Differential methylation of promoters was associated with immune-related terms. Key immune genes, CEBPA, GFI1, MAFB and GATA1 showed differential expression and methylation. However, we observed the strongest enrichment of methylation changes in enhancers and CTCF binding sites, implying that methylation plays a major role in large-scale transcriptional reprogramming and chromatin reorganisation during differentiation. Correlation of differential expression and distal methylation with support from chromatin capture experiments allowed us to identify putative proximal and long-range enhancers for a number of immune cell differentiation genes, including CEBPA and CCNF Integrating expression data, we present a model of HL-60/S4 differentiation in relation to the wider scope of myeloid differentiation.

NOTCH target gene HES5 mediates oncogenic and tumor suppressive functions in hepatocarcinogenesis.

NOTCH receptor signaling plays a pivotal role in liver homeostasis and hepatocarcinogenesis. However, the role of NOTCH pathway mutations and the NOTCH target gene HES5 in liver tumorigenesis are poorly understood. Here we performed whole-exome sequencing of 54 human HCC specimens and compared the prevalence of NOTCH pathway component mutations with the TCGA-LIHC cohort (N = 364). In addition, we functionally characterized the NOTCH target HES5 and the patient-derived HES5-R31G mutation in vitro and in an orthotopic mouse model applying different oncogenic backgrounds, to dissect the role of HES5 in different tumor subgroups in vivo. We identified nonsynonymous mutations in 14 immediate NOTCH pathway genes affecting 24.1% and 16.8% of HCC patients in the two independent cohorts, respectively. Among these, the HES5-R31G mutation was predicted in silico to have high biological relevance. Functional analyses in cell culture showed that HES5 reduced cell migration and clonogenicity. Further analyses revealed that the patient-derived HES5-R31G mutant protein was non-functional due to loss of DNA binding and greatly reduced nuclear localization. Furthermore, HES5 exhibited a negative feedback loop by directly inhibiting the NOTCH target HES1 and downregulated the pro-proliferative MYC targets ODC1 and LDHA. Interestingly, HES5 inhibited MYC-dependent hepatocarcinogenesis, whereas it promoted AKT-dependent liver tumor formation and stem cell features in a murine model. Thus, NOTCH pathway component mutations are commonly observed in HCC. Furthermore, the NOTCH target gene HES5 has both pro- and anti-tumorigenic functions in liver cancer proposing a driver gene dependency and it promotes tumorigenesis with its interaction partner AKT.