METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer.

Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N7-methylguanosine (m7G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7G tRNA methylation in cancer cell translation control and tumour biology.

Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response.

DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor in children and adolescents. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming and the DNMT1 enzyme has been demonstrated to have an important role in both maintaining the epigenome and controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor proliferation through a mechanism that is independent on DNA methylation. Depletion of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 phase, and DNA damage, as revealed by the induction of γH2AX foci. These effects elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 mutated cells, persistent micronuclei and increased DNA instability was observed. Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 as an adjuvant agent in treating EWS.

Refinement of computational identification of somatic copy number alterations using DNA methylation microarrays illustrated in cancers of unknown primary.

High-throughput genomic technologies are increasingly used in personalized cancer medicine. However, computational tools to maximize the use of scarce tissues combining distinct molecular layers are needed. Here we present a refined strategy, based on the R-package 'conumee', to better predict somatic copy number alterations (SCNA) from deoxyribonucleic acid (DNA) methylation arrays. Our approach, termed hereafter as 'conumee-KCN', improves SCNA prediction by incorporating tumor purity and dynamic thresholding. We trained our algorithm using paired DNA methylation and SNP Array 6.0 data from The Cancer Genome Atlas samples and confirmed its performance in cancer cell lines. Most importantly, the application of our approach in cancers of unknown primary identified amplified potentially actionable targets that were experimentally validated by Fluorescence in situ hybridization and immunostaining, reaching 100% specificity and 93.3% sensitivity.

Experimental and Bioinformatic Approaches to Studying DNA Methylation in Cancer.

DNA methylation is an essential epigenetic mark. Alterations of normal DNA methylation are a defining feature of cancer. Here, we review experimental and bioinformatic approaches to showcase the breadth and depth of information that this epigenetic mark provides for cancer research. First, we describe classical approaches for interrogating bulk DNA from cell populations as well as more recently developed approaches for single cells and multi-Omics. Second, we focus on the computational analysis from primary data processing to the identification of unique methylation signatures. Additionally, we discuss challenges such as sparse data and cellular heterogeneity.

Chromatin-Based Classification of Genetically Heterogeneous AMLs into Two Distinct Subtypes with Diverse Stemness Phenotypes.

Global investigation of histone marks in acute myeloid leukemia (AML) remains limited. Analyses of 38 AML samples through integrated transcriptional and chromatin mark analysis exposes 2 major subtypes. One subtype is dominated by patients with NPM1 mutations or MLL-fusion genes, shows activation of the regulatory pathways involving HOX-family genes as targets, and displays high self-renewal capacity and stemness. The second subtype is enriched for RUNX1 or spliceosome mutations, suggesting potential interplay between the 2 aberrations, and mainly depends on IRF family regulators. Cellular consequences in prognosis predict a relatively worse outcome for the first subtype. Our integrated profiling establishes a rich resource to probe AML subtypes on the basis of expression and chromatin data.

Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils.

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils.

The reference epigenome and regulatory chromatin landscape of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease.

Decoding the DNA Methylome of Mantle Cell Lymphoma in the Light of the Entire B Cell Lineage.

We analyzed the in silico purified DNA methylation signatures of 82 mantle cell lymphomas (MCL) in comparison with cell subpopulations spanning the entire B cell lineage. We identified two MCL subgroups, respectively carrying epigenetic imprints of germinal-center-inexperienced and germinal-center-experienced B cells, and we found that DNA methylation profiles during lymphomagenesis are largely influenced by the methylation dynamics in normal B cells. An integrative epigenomic approach revealed 10,504 differentially methylated regions in regulatory elements marked by H3K27ac in MCL primary cases, including a distant enhancer showing de novo looping to the MCL oncogene SOX11. Finally, we observed that the magnitude of DNA methylation changes per case is highly variable and serves as an independent prognostic factor for MCL outcome.

Distinct Trends of DNA Methylation Patterning in the Innate and Adaptive Immune Systems.

DNA methylation and the localization and post-translational modification of nucleosomes are interdependent factors that contribute to the generation of distinct phenotypes from genetically identical cells. With 112 whole-genome bisulfite sequencing datasets from the BLUEPRINT Epigenome Project, we analyzed the global development of DNA methylation patterns during lineage commitment and maturation of a range of immune system effector cells and the cancers that arise from them. We show clear trends in methylation patterns that are distinct in the innate and adaptive arms of the human immune system, both globally and in relation to consistently positioned nucleosomes. Most notable are a progressive loss of methylation in developing lymphocytes and the consistent occurrence of non-CG methylation in specific cell types. Cancer samples from the two lineages are further polarized, suggesting the involvement of distinct lineage-specific epigenetic mechanisms. We anticipate broad utility for this resource as a basis for further comparative epigenetic analyses.

Whole-genome fingerprint of the DNA methylome during human B cell differentiation.

We analyzed the DNA methylome of ten subpopulations spanning the entire B cell differentiation program by whole-genome bisulfite sequencing and high-density microarrays. We observed that non-CpG methylation disappeared upon B cell commitment, whereas CpG methylation changed extensively during B cell maturation, showing an accumulative pattern and affecting around 30% of all measured CpG sites. Early differentiation stages mainly displayed enhancer demethylation, which was associated with upregulation of key B cell transcription factors and affected multiple genes involved in B cell biology. Late differentiation stages, in contrast, showed extensive demethylation of heterochromatin and methylation gain at Polycomb-repressed areas, and genes with apparent functional impact in B cells were not affected. This signature, which has previously been linked to aging and cancer, was particularly widespread in mature cells with an extended lifespan. Comparing B cell neoplasms with their normal counterparts, we determined that they frequently acquire methylation changes in regions already undergoing dynamic methylation during normal B cell differentiation.

Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers.

While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.