High-resolution Transcriptomic and Epigenetic Profiling Identifies Novel Regulators of COPD

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high-resolution multi-omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients

High-resolution transcriptomic and epigenetic profiling identifies novel regulators of COPD

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high-resolution multi-omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients

Sex differences in oncogenic mutational processes.

Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Quantitative Proteomics Identifies TCF1 as a Negative Regulator of Foxp3 Expression in Conventional T Cells

Regulatory T cells are important regulators of the immune system and have versatile functions for the homeostasis and repair of tissues. They express the forkhead box transcription factor Foxp3 as a lineage-defining protein. Negative regulators of Foxp3 expression are not well understood. Here, we generated double-stranded DNA probes complementary to the Foxp3 promoter sequence and performed a pull-down with nuclear protein in vitro, followed by elution of bound proteins and quantitative mass spectrometry. Of the Foxp3-promoter-binding transcription factors identified with this approach, one was T cell factor 1 (TCF1). Using viral over-expression, we identified TCF1 as a repressor of Foxp3 expression. In TCF1-deficient animals, increased levels of Foxp3(intermediate)CD25(negative) T cells were identified. CRISPR-Cas9 knockout studies in primary human and mouse conventional CD4 T (T-conv) cells revealed that TCF1 protects T-conv cells from inadvertent Foxp3 expression. Our data implicate a role of TCF1 in suppressing Foxp3 expression in activated T cells

Recurrent Mutations in EGR2 Direct Specific Epigenetic Reconfiguration in Chronic Lymphocytic Leukemia

Abstract Epigenetic alterations are universal in cancer and are important in establishing the malignant phenotype. Dissection of the factors that shape the tumor-specific epigenome may reveal insight into key aspects of tumorigenesis and therapeutic resistance. In chronic lymphocytic leukemia (CLL), we have previously found that broad changes in epigenetic patterns co-occur with the evolution of genetic alterations. We have also uncovered that aberrant patterning of DNA methylation in CLL involves excessive activity of a defined group of transcription factors (TFs), including the early growth response (EGR) TF family. Recent work has further revealed that recurrent mutations in EGR2 are associated with exceptionally poor clinical outcomes in CLL. The basis for the adverse association of EGR2 mutations in CLL is unclear. To explore the role of EGR2 mutations in CLL, we initially performed genome-wide DNA methylation analysis using Illumina arrays on CLL patients harboring EGR2 mutations (n=27) compared to EGR wild-type cases (n=265). We found that the three most common recurrent mutations, occurring at amino acid positions E356K, H384N and D411H within the DNA binding domain, are each associated with an exclusive subset of tumor-specific hypomethylated CpG sites. A search for TF sequence motifs at these loci revealed a strong enrichment of novel derivative EGR2 motifs that differ only marginally (usually by a single nucleotide) from the canonical EGR2 recognition sequence. Each recurrent mutation led to specific enrichment of a different derivative EGR2 motif. Furthermore, the canonical (wild-type) recognition sequence was not enriched, suggesting that mutations re-localize binding activity to derivate sequence motifs rather than simply altering binding affinity. Luciferase enhancer, proximity ligation and electrophoretic mobility shift assays confirmed that each EGR2 mutant protein specifically binds and enhances transcriptional activity only when the matched EGR2 derivative recognition motif is present. These results establish that derivative motif sequences may function as novel cryptic enhancers in the presence of the cognate EGR2 mutant TF. We performed multiomics profiling (DNA methylome, ATAC-seq, ChIP-seq and RNA-seq) to examine the nature of the epigenetic reconfiguration and the phenotypic impact of individual EGR2 mutations. Whole genome bisulfite sequencing of E356K- and H384N-mutated CLL samples (n=4 each) was used to reveal the full complement of recurrent differentially methylated regions (DMRs) across the genome, and recapitulated the mutually-exclusive pattern of DMRs between mutations. Overlaying DMRs with data from ChIP-seq and ATAC-seq experiments in the same samples revealed the nature of EGR2 mutation-specific chromatin reconfiguration to be remarkably mutation-specific. For E356K, hypomethylated DMRs are often associated with foci of accessible chromatin, EGR2 binding, and flanked by gains of H3K4me1 and H3K27ac, indicative of the acquisition of active enhancer function. Conversely, H384N mutations generated fewer DMRs and mainly directed the deposition of H3K4me1 only, indicative of gain of poised enhancers at these loci. RNA-sequencing analyses revealed that a subset of epigenetically reconfigured regions was associated with mutation-specific altered gene expression, and differences were virtually always associated with proximal gene activation. E356K and H384N mutations displayed highly differential gene expression patterns, with E356K exhibiting a greater impact on gene expression. Integrated analyses indicated that E356K mutations may specifically involve activated Notch signaling, revealed by the aberrant activation of Notch target genes and the mutual exclusivity of NOTCH1 mutations, further highlighted by enriched co-mutation of NOTCH1 in H384N-mutated CLL. Together these findings provide an exceptional example of the precise role that a singular TF may play in programming the epigenetic landscape. As there are no known TFs that naturally bind derivative EGR2 motifs, these mutant proteins provide insight into aberrant enhancer generation and the phenotypic impact of (re)directed TF binding in a human disease setting. Although these recurrent mutations are presently only known in CLL, these findings provide insight into the mechanisms that may surround other gain-of-function TF activity in various malignancies. Disclosures Kipps: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees. </jats:sec

DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia

Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program

Epigenetic Drug Treatment Globally Induces Cryptic Transcription Start Sites Encoded in Long Terminal Repeats

Abstract Epigenetic drugs are currently used for the treatment of several hematologic malignancies, but their mechanism of action remains poorly understood. By using a previously described reporter cell line for epigenetic reactivation of the DAPK1 locus, we have shown that epigenetic treatment causes transcription from uncharacterized intronic transcription start sites (TSSs), thereby generating DAPK1 mRNA with novel first exons. Based on these findings, we analyzed whether inhibition of DNA-Methyltransferases (DNMTs), Histone deacetylases (HDACs), or both resulted in the genome-wide induction of non-canonical TSSs. While epigenetic treatment altered expression of known promoter sites, we observed that both HDAC- and DNMT-inhibitors predominantly induced de novo transcription from cryptic promoters encoded in long-terminal repeat (LTR) retrotransposons. These LTR-associated 'treatment induced, not-annotated TSS' (TINATs) are currently not annotated and normally silenced in almost all cell types with the exception of testicular und thymic tissue. In the majority of cases, these TINATs arose most commonly from LTR12 elements, particularly LTR12C (which apparently provides 50% of all TINATs). TINAT activation after DNMT-inhibitors (DNMTi) coincided with DNA hypomethylation and gain in H3K4me3, H3K9ac, and H3K27ac histone marks. In contrast, HDAC-inhibitors (HDACi) induced only canonical TSSs in association with histone acetylation, but TINATs via a yet unknown mechanism. Nevertheless, both inhibitors convergently induced unidirectional transcription from identical TINAT sites. Moreover, we found a consensus GATA2 binding motif which strongly distinguished LTR12Cs with TINATs from LTR12Cs without TINATs, supporting that GATA2 is likely the upstream transcription factor responsible for TINAT activation. TINATs originating from non-canonical TSSs located within introns of protein-coding genes frequently spliced into downstream exons thereby creating LTR/non-LTR fusion transcripts that harbor novel in place of canonical exon sequence at their 5' end. The resulting transcripts encode truncated or chimeric open reading frames which translated into currently uncharacterized protein isoforms with predicted abnormal functions or immunogenic potential, the last one based on their foreign sequence and capability of being presented on MHC-class I molecules. In summary, we could show that DNMTi and/or HDACi do not predominantly alter the expression of canonical genes, but induce de novo transcription of LTRs especially of the LTR12 family, resulting in numerous fusion transcripts that encode novel protein isoforms which might have the potential to influence cell proliferation or might be an elegant explanation for the priming effect of epigenetic therapy. Ongoing experiments are investigating the functional mechanisms of TINAT reactivation upon epigenetic drug treatment and future proteomic approaches combined with T-cell cytotoxicity assays will further shed light on the interaction between epigenetic and immune therapy and the role of ERV-derived antigen presentation. Disclosures Lübbert: Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding. </jats:sec