Epi-miRNAs: Regulators of the Histone Modification Machinery in Human Cancer

Cancer is a leading cause of death and disability worldwide. Epigenetic deregulation is one of the most critical mechanisms in carcinogenesis and can be classified into effects on DNA methylation and histone modification. MicroRNAs are small noncoding RNAs involved in fine-tuning their target genes after transcription. Various microRNAs control the expression of histone modifiers and are involved in a variety of cancers. Therefore, overexpression or downregulation of microRNAs can alter cell fate and cause malignancies. In this review, we discuss the role of microRNAs in regulating the histone modification machinery in various cancers, with a focus on the histone-modifying enzymes such as acetylases, deacetylases, methyltransferases, demethylases, kinases, phosphatases, desumoylases, ubiquitinases, and deubiquitinases. Understanding of microRNA-related aberrations underlying histone modifiers in pathogenesis of different cancers can help identify novel therapeutic targets or early detection approaches that allow better management of patients or monitoring of treatment response.

Alterations of Chromatin Regulators in the Pathogenesis of Urinary Bladder Urothelial Carcinoma

Urothelial carcinoma (UC) is the most frequent histological type of cancer in the urinary bladder. Genomic changes in UC activate MAPK and PI3K/AKT signal transduction pathways, which increase cell proliferation and survival, interfere with cell cycle and checkpoint control, and prevent senescence. A more recently discovered additional category of genetic changes in UC affects chromatin regulators, including histone-modifying enzymes (KMT2C, KMT2D, KDM6A, EZH2), transcription cofactors (CREBBP, EP300), and components of the chromatin remodeling complex SWI/SNF (ARID1A, SMARCA4). It is not yet well understood how these changes contribute to the development and progression of UC. Therefore, we review here the emerging knowledge on genomic and gene expression alterations of chromatin regulators and their consequences for cell differentiation, cellular plasticity, and clonal expansion during UC pathogenesis. Our analysis identifies additional relevant chromatin regulators and suggests a model for urothelial carcinogenesis as a basis for further mechanistic studies and targeted therapy development.

Aberrant Histone Landscape in Juvenile Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric cancer characterized by excessive accumulation of monocytes/macrophages with myelodysplastic features and splenomegaly. Hematopoietic stem cell transplantation (HSCT) is the only curative option, but relapse occurs in ~50% of the patients. Mutations in the RAS pathway are the major drivers and DNA hypermethylation is associated with severe clinical phenotype. DNA methylation is linked to histone modifications in normal cell development and cancer pathogenesis. Gene expression levels are programmed by histone modifications. However, a comprehensive characterization of histone modifications in JMML is yet to be elucidated. It was reported that the CD34+ hematopoietic stem and progenitors cells (HSPCs) possess leukemia initiation potential (Caye et al., 2015; Krombholz et al., 2016; Louka et al., 2021; Yoshimi et al., 2017). We hypothesized that dysregulated histone modifications contribute to leukemogenesis in JMML. To this goal, we investigated epigenetic landscape at the single-cell level in JMML HSPCs using cytometry by time-of-flight (EpiTOF). We isolated HSPCs from the spleens of JMML patients with PTPN11 mutations which were collected at diagnosis, before treatment (provided by the European Working Groups of Myelodysplastic Syndromes). The CD34+ cells from JMML Spleens (n=5) and Bone Marrow (BM n=1) alongside healthy controls (cord blood CB n=5; BM n=2) were stained with antibodies against 15 immunophenotypic and 46 key histone markers and subsequently analyzed on a mass cytometer (Fluidigm, Helios). We found that a group of key histone markers were significantly downregulated in JMML in comparison to healthy CBs, including H4K16ac, H3K23ac, H3K56ac, H3K36me1, H3K9me1 and H3K27me2 (Fig.1). Dimensionality reduction analyses showed a global reduction in histone acetylation in all JMML spleen samples (~70%) when compared to CBs (average >95%). Our study revealed significant dysregulation of key histone markers and their respective modifiers (histone modifying enzymes) within the leukemogenic CD34+ compartment (Fig.2). Our findings demonstrated an aberrant epigenetic regulation in JMML patient samples with concurrent loss of histone acetylation and some histone methylation markers, which have been reported as hallmarks of other cancers. Further characterization of the epigenetic landscape in JMML was performed using transposase-accessible chromatin with high-throughput sequencing (bulk ATAC-seq). Notably, ATAC-seq data revealed significantly lower expression of key histone acetyltranferases (HAT), such as Kat6b and Kat8 (Fig.2) , which specifically regulate acetylation of H3K23 and H4K16 respectively. Additionally, an increased activity of histone deacetylase, HDAC9, was also observed in these samples (Fig.2). Collectively, our data suggests aberrant activities of histone modifying enzymes (HMEs) in JMML HSPCs (Fig.2), with both increased HDAC and decreased HAT activities observed in our ATAC-data. This finding is concurrent with our EpiTOF findings of global loss of histone acetylation in JMML (Fig.1A). It is known that histone modifications are easier to reverse when compared to DNA methylation, and we believe targeted inhibition of specific HDACs that we have found to be upregulated in JMML (such as HDAC9) might hold great therapeutic potential. Additionally, investigation into the impact of altered chromatin structure of JMML HSPCs on gene expression is currently ongoing using single-cell RNA-seq. In conclusion, our study identified a novel mechanism of epigenetic dysregulation in putative JMML leukemia initiating cells related to imbalanced activities of histone modifying enzymes which could potentially represent future therapeutic targets. Figure 1 Figure 1. Disclosures Bertaina: AdicetBio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees.

How the naked mole-rat resists senescence: a constraints-based theory

Here, I present a theory describing how the stabilization of constraints imposed on chromatin dynamics by the naked mole-rat's histone H1.0 protein—which in terminally differentiated cells constrains the accessibility of the nucleosome core particle for histone-modifying enzymes and chromatin remodeling factors—explains its resistance to both senescence and cancer. Further, this theory predicts that a mutant house mouse displaying such stabilization will be similarly resistant to both senescence and cancer. A proof-of-concept computational analysis is presented and two predictions for the direct testing of the theory are provided. These experiments comprise, as test subjects, mutant naked mole-rats synthesizing a house mouse (Mus musculus)-like histone H1.0, and mutant house mice synthesizing a naked mole-rat-like histone H1.0. The predictions are that the constraints on chromatin dynamics embodied by the respective mutant histone H1.0 proteins will negate the otherwise significant resistance to both senescence and cancer of the naked mole-rats and, conversely, confer such resistance to the house mice. A verification of these predictions will imply that constraints on chromatin dynamics embodied by naked mole-rat-like histone H1.0 proteins may confer significant resistance to both senescence and age-related cancer to otherwise senescence-prone and/or cancer-susceptible multicellular species, including humans.

Histone Modifying Enzymes as Targets for Therapeutic Intervention in Oesophageal Adenocarcinoma

Oesophageal adenocarcinoma (OAC) has a dismal prognosis, where curable disease occurs in less than 40% of patients, and many of those with incurable disease survive for less than a year from diagnosis. Despite the widespread use of systematic chemotherapy in OAC treatment, many patients receive no benefit. New treatments are urgently needed for OAC patients. There is an emerging interest in epigenetic regulators in cancer pathogenesis, which are now translating into novel cancer therapeutic strategies. Histone-modifying enzymes (HMEs) are key epigenetic regulators responsible for dynamic covalent histone modifications that play roles in both normal and dysregulated cellular processes including tumorigenesis. Several HME inhibitors are in clinical use for haematological malignancies and sarcomas, with numerous on-going clinical trials for their use in solid tumours. This review discusses the current literature surrounding HMEs in OAC pathogenesis and their potential use in targeted therapies for this disease.

CoREST has a conserved role in facilitating SPR-5/LSD1 maternal reprogramming of histone methylation

Maternal reprogramming of histone methylation is critical for reestablishing totipotency in the zygote, but how histone modifying enzymes are regulated during maternal reprogramming is not well characterized. To address this gap, we asked whether maternal reprogramming by the H3K4me1/2 demethylase SPR-5/LSD1/KDM1A, is regulated by the co-repressor protein, SPR-1/CoREST in C. elegans and mice. In C. elegans, SPR-5 functions as part of a reprogramming switch together with the H3K9 methyltransferase MET-2. By examining germline development, fertility and gene expression in double mutants between spr-1 and met-2, we find that spr-1 mutants are partially compromised for spr-5; met-2 reprogramming. In mice, we generated a separation of function Lsd1 M448V point mutation that compromises CoREST binding, but only slightly affects LSD1 demethylase activity. When maternal LSD1 in the oocyte is derived exclusively from this allele, the progeny phenocopy the increased perinatal lethality that we previously observed when LSD1 was reduced maternally. Together, these data are consistent with CoREST having a conserved function in facilitating maternal LSD1 epigenetic reprogramming.

In Vivo Silencing of Genes Coding for dTip60 Chromatin Remodeling Complex Subunits Affects Polytene Chromosome Organization and Proper Development in Drosophila melanogaster

Chromatin organization is developmentally regulated by epigenetic changes mediated by histone-modifying enzymes and chromatin remodeling complexes. In Drosophila melanogaster, the Tip60 chromatin remodeling complex (dTip60) play roles in chromatin regulation, which are shared by evolutionarily-related complexes identified in animal and plants. Recently, it was found that most subunits previously assigned to the dTip60 complex are shared by two related complexes, DOM-A.C and DOM-B.C, defined by DOM-A and DOM-B isoforms, respectively. In this work, we combined classical genetics, cell biology, and reverse genetics approaches to further investigate the biological roles played during Drosophila melanogaster development by a number of subunits originally assigned to the dTip60 complex.

Histone Modifying Enzymes in Gynaecological Cancers

Genetic and epigenetic factors contribute to the development of cancer. Epigenetic dysregulation is common in gynaecological cancers and includes altered methylation at CpG islands in gene promoter regions, global demethylation that leads to genome instability and histone modifications. Histones are a major determinant of chromosomal conformation and stability, and unlike DNA methylation, which is generally associated with gene silencing, are amenable to post-translational modifications that induce facultative chromatin regions, or condensed transcriptionally silent regions that decondense resulting in global alteration of gene expression. In comparison, other components, crucial to the manipulation of chromatin dynamics, such as histone modifying enzymes, are not as well-studied. Inhibitors targeting DNA modifying enzymes, particularly histone modifying enzymes represent a potential cancer treatment. Due to the ability of epigenetic therapies to target multiple pathways simultaneously, tumours with complex mutational landscapes affected by multiple driver mutations may be most amenable to this type of inhibitor. Interrogation of the actionable landscape of different gynaecological cancer types has revealed that some patients have biomarkers which indicate potential sensitivity to epigenetic inhibitors. In this review we describe the role of epigenetics in gynaecological cancers and highlight how it may exploited for treatment.

Running Against the Wnt: How Wnt/β-Catenin Suppresses Adipogenesis

Mesenchymal stem cells (MSCs) give rise to adipocytes, osteocytes, and chondrocytes and reside in various tissues, including bone marrow and adipose tissue. The differentiation choices of MSCs are controlled by several signaling pathways, including the Wnt/β-catenin signaling. When MSCs undergo adipogenesis, they first differentiate into preadipocytes, a proliferative adipocyte precursor cell, after which they undergo terminal differentiation into mature adipocytes. These two steps are controlled by the Wnt/β-catenin pathway, in such a way that when signaling is abrogated, the next step in adipocyte differentiation can start. This sequence suggests that the main role of Wnt/β-catenin signaling is to suppress differentiation while increasing MSC and preadipocytes cell mass. During later steps of MSC differentiation, however, active Wnt signaling can promote osteogenesis instead of keeping the MSCs undifferentiated and proliferative. The exact mechanisms behind the various functions of Wnt signaling remain elusive, although recent research has revealed that during lineage commitment of MSCs into preadipocytes, Wnt signaling is inactivated by endogenous Wnt inhibitors. In part, this process is regulated by histone-modifying enzymes, which can lead to increased or decreased Wnt gene expression. The role of Wnt in adipogenesis, as well as in osteogenesis, has implications for metabolic diseases since Wnt signaling may serve as a therapeutic target.

Epigenetic regulation of adipogenesis by histone-modifying enzymes

Many studies investigating the transcriptional control of adipogenesis have been published so far; recently the research is focusing on the role of epigenetic mechanisms in regulating the process of adipocyte development. Histone-modifying enzymes and the histone tails post-transcriptional modifications catalyzed by them, are fundamentally involved in the epigenetic regulation of adipogenesis. In our review, we will discuss recent advances in epigenomic regulation of adipogenesis with a focus on histone-modifying enzymes implicated in the various phases of adipocytes differentiation process from mesenchymal stem cells to mature adipocytes. Understanding adipogenesis, may provide new ways to treat obesity and related metabolic diseases.