Clinically tested therapies of mature B-cell malignancies based on DNA and histone methylation

ALK-positive anaplastic large cell lymphoma (ALK+ ALCL) is a T-cell non-Hodgkin lymphoma characterized by the presence of the fusion oncogene nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Tumor cells control the expression of key genes in different manners: mutations, translocations or epigenetically. Our group have shown that in this tumor a plethora of genes are silenced by NPM-ALK through DNA methylation. However, recent evidence showed that some of the genes downregulated by DNA methylation might have already been silenced prior to the appearance of DNA methylation. Herein, we investigate the possible role of histone methylation in the silencing of genes and its relationship to DNA methylation. To better understand the possible players in the silencing of genes in ALK+ ALCL we used T-cell transformed with NPM-ALK and recorded over time the changing in gene expression, DNA methylation and histone marks. Using RNAseq comparing transformed cells at different time points, we noticed that downregulated genes in ALK+ ALCL became gradually silenced also in our system. Next, we investigated the DNA methylation (meDNA) on their promoters, and we confirmed a progressive increase of meDNA but clearly slower compared to the silencing of the genes, or if compared to the meDNA status of established ALK+ ALCL cell lines. Finally, we explored the presence of silencing histone marks on the promoter of the silenced genes using qPCR-ChIP. Among the repressive histone marks evaluated, H3K27me3 stood up as possible candidate for the gene silencing. Therefore, we look at the different gene expression changes after the inhibition of either meDNA, using a DNMT1 inhibitor (GSK3685032), or histone methylation, by EZH2 inhibitor (EPZ6438). RNAseq data showed the re-expression of downregulated genes after EZH2 and/or DNMT1 inhibition providing clear insights on their epigenetic regulation. The co-presence of H3K27me3 and meDNA on the promoters of those genes points towards a crosstalk between DNA and histone methylation machineries. Despite it is clear how NPM-ALK regulates DNA methylation, nothing is known about NPM-ALK and EZH2. To investigate their relationship, we treated ALK+ cells with ALK inhibitor, and we observed a decrease of EZH2 expression at protein and mRNA level. STAT3 inhibition, through napabucasin or by STAT3 silencing using Crispr-cas9 technology, resulted, likewise, in the downregulation of EZH2. Moreover, ChIPseq showed STAT3 presence on EZH2 promoter, further confirming this mechanism. Altogether, those data showed that NPM-ALK regulates EZH2 expression through STAT3. In conclusion, we provide, not only novel information on the mechanism in the downregulation of genes in ALK+ ALCL, but also new insight on the understanding of the epigenetics machinery and the crosstalk between histone and DNA methylation, for long time considered two mutually exclusive mechanisms, or even antagonist. Citation Format: Cosimo Lobello, Shengchun Wang, Shilpa Rao, Jan Pawlicki, Nashwa Mansoor, James L. Riley, Reza Nejati, Wasik A. Mariusz. Crosstalk between histone and DNA methylation in the development and progression of ALK+ ALCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7015.

During mammalian development, chromatin is dynamically reprogrammed first in developing gametes and then after fertilization in the preimplantation embryo [1, 2]. In gametes, following an erasure phase that removes somatic DNA methylation, sex-specific DNA methylation is acquired. For female germ cells, this acquisition of methylation occurs in the growing oocyte, with an increasing progression of DNA methylation coincident with increasing oocyte size [1]. By the time the oocyte is fully grown, acquisition of DNA methylation is complete. During preimplantation development, DNA methylation is maintained primarily at imprinted genes and repetitive elements during global methylation erasure. This is followed by initiation of de novo DNA methylation late in preimplantation development [1]. During these chromatin remodeling periods, dynamic changes in histone methylation are also occurring. Importantly, these methylation patterns are critical to the overall health of gametes and embryos [1–4]. Both DNA and histone methylation are catalyzed by methyltransferases, which transfer a methyl group from the universal donor, S-adenosylmethionine (SAM), to CpG dinucleotides or histone tails [3–5]. Production of SAM is dependent on the 1-carbon folate pathway [3–5]. Thus, folates play a fundamental role in regulating DNA and histone methylation [3, 4]. In keeping with the critical reprogramming events described above, folate accumulation during oocyte and preimplantation development likely is essential to foster correct DNA and histone methylation patterns during gametogenesis and early development. Folates are essential nutrients that are acquired from our diet [3, 4]. Thus, a key question is how the oocyte and early embryo acquire and take up their folate pools. This question was address by Kooistra et al. [5] in their paper ‘‘Folate Transport in Mouse Cumulus-Oocyte Complexes and Preimplantation Embryos’’ published in this issue of Biology of Reproduction. Using gene expression studies, Kooistra et al. [5] show that mouse cumulus-oocyte complexes and oocytes harbor transcripts for the reduced folate carrier SLC19A1, an anion exchanger. In preimplantation embryos, Slc19a1 transcript abundance is very low due to the lack of embryonic gene activation. By comparison, folate receptor FOLR1 mRNA was present in preimplantation embryos beginning at the 2-cell stage but was lacking in oocytes and zygotes. This ying-yang expression pattern indicates that two distinct mechanisms may be operating in cumulus-oocyte complexes and preimplantation embryos [5]. To functionally characterize SLC19A1 and FOLR1 activity in cumulus-oocyte complexes, germinal vesicle oocytes, and mouse preimplantation embryos, transport experiments were employed to measure uptake of folates and the antifolate methotrexate, whereas biochemical inhibitors were used to distill the uptake mechanisms. The results indicate that folate transport occurs predominately through SLC19A1 in cumulusoocyte complexes but that FOLR1 is the principal uptake mechanism in embryos from the 2-cell through blastocyst stages. However, most interesting are the final experiments showing that SLC19A1 regulates cumulus cell folate uptake, with little to no uptake in fully grown oocytes. Thus, we are left with an intriguing question: What is the origin of endogenous folates in oocytes and zygotes? A careful look at the methods shows that fully grown oocytes were analyzed by Kooistra et al. Thus, it is reasonable to conclude that oocyte folate accumulation must occur during the early stages of folliculogenesis. Experiments now need to examine growing oocytes to determine when and through what mechanism folate uptake occurs. We are also left with the intriguing paradox that in vitro embryo development often occurs in non-folate supplemented culture medium. Does this mean that the early embryo relies on oocyte folate stores? And if so, why do early embryos have functional FOLR1 folate uptake? Future studies will also need to address whether variation in folate store accumulation is an important determinant of oocyte maturation and eventual embryonic developmental competence. We must also consider the implication of the Kooistra et al. study on DNA and histone methylation. With regard to DNA methylation in fully grown oocytes, acquisition would be complete. Thus, there may be no further need for folate uptake in fully grown oocytes. However, what would be the ramifications if oocytes failed to store sufficient levels of folates? One repercussion of reduced folate stores during the methylation acquisition phase in growing oocytes may be aberrant establishment of imprinted methylation. Interestingly, we have observed that initiation of methylation acquisition is impaired at the imprinted Peg1 gene in connexin 37-null oocytes [6]. We postulated that this may be due to reduced stores of a critical metabolite normally transported from granulosa cells to the oocyte via gap junctions. Another Correspondence: E-mail: mmann22@uwo.ca

Die Methylierung der genomischen DNA und Histonen im\n Kern eukaryotischer Zellen spielt eine wichtige Rolle\n in epigenetischen Prozessen wie z. B. Imprinting, Gene\n Dosage Compensation , und epigenetische Repression der\n Genexpression. Studien in Pilzen, Pflanzen und\n Vertebraten unterstützen ein Model, wonach die\n Methylierung von Histonen, insbesondere die\n Methylierung der Lysinreste 9 (H3K9) und/oder 27 im\n Histon 3, die de novo Methylierung von DNA auslöst. Im\n Gegensatz dazu, ist nur wenig über die Mechanismen\n bekannt, die für de novo Methylierung in\n Modelorganismen wie z. B. Drosophila melanogaster\n verantwortlich sind. Drosophila exprimiert\n Schlüsselfaktoren für die Methylierung von Histonen and\n DNA: a.) Histonmethyltransferasen (HMT), die H3K9\n methylieren und entscheidende Rollen für die\n Etablierung and Aufrechterhaltung von Heterochromatin\n und Genrepression spielen; b.) Eine\n DNA-Methyltransferase (dDNMT2), die überwiegend CpA und\n CpT-Motive im Drosophila Genom methyliert; c.)\n Methyl-CpG binding domain (MBD) Proteine, die\n methylierte DNA binden und DNA Methylierung in\n biologische Aktivität übersetzen. Ein Mitglied der\n Drosophila MBD-Proteinfamilie ist Medusa (MDU), welches\n sowohl ein MBD- als auch ein SET-Motiv enthält. Das\n SET-Motiv methyliert H3K9 in vitro und In Drosophila.\n Die Anwesenheit eines MBD und Set Motivs in MDU\n unterstützt die Hypothese, dass MDU an der Methylierung\n von H3K9 und DNA in Drosophila beteiligt ist. In dieser\n Arbeit habe ich die funktionale Beduetung von MDU\n bezüglich der Genexpression and DNA Methylierung\n untersucht. In vitro HMT-Experimente gekoppelt mit\n Western blot and Chromatin\n Immunpräzipitationsexperimenten ergaben, dass MDU H3K9\n in vitro und in vivo tri-methyliert. MDU-vermittellte\n Methylierung von H3K9 resultiert in Repression der\n Zielgentranskription in Drosophila Zellkultur, woraus\n abgeleitet werden kann, dass MDU als Repressor der\n Transkription wirkt. Das MBD-Motiv von MDU bindet\n methylierte CpA DNA Sequenzen in vitro, und besitzt\n eine intrinsische methylierte-DNA Binding saffinität.\n Die Ergebnisse molekularer and genetischer Studien\n zeigen, dass MDU die Transkription des Tumorsuppressor\n Gens retinoblastoma family protein (Rbf), einem\n Schlüsselregulator der Zellproliferation und\n differenzierung, reprimiert. Die Untersuchungen zur\n Funktion von MDU in der Regulation der Expression von\n Rbf unterstützen ein Model wonach tri-methylierung von\n H3K9 durch MDU die dDNMT2-abhängige de novo\n Methylierung in der cis-regulatorischen enhancer \n Region von Rbf auslöst. Sobald DNA Methylierung\n platziert ist, bindet das MBD-Motif von MDU an\n methylierte DNA und induziert eine selbstangetriebene \n DNA-Histone Mehtylierungskaskade, die zur Ausbreitung\n von DNA und H3K9 Methylierung auf dem Rbf Genlocus\n führt und letztendlich Repression der Rbf Transkription\n bewirkt. Die Ergebnisse dieser Arbeit entschlüsseln die\n Funktion von MDU in der Repression der Genexpression,\n ergeben einen Mechanismus für die Etablierung der de\n novo DNA Methylierung in Drosophila, und deuten auf\n eine wichtige Rolle der bifunktionalen MBD/SET Proteine\n für die Kontrolle der Proliferation und Differenzierung\n von Zellen hin.

Background:Osteoarthritis (OA) remains one of the most common osteopathy for centuries, which can be attributed to multiple risk factors including mechanical and biochemical ones. More and more studies verified that inflammatory cytokines play important roles in the progression of OA, such as tumor necrosis factor-alpha (TNF-α). In this study, we aimed to investigate the relationship between epigenetic manifestations of TNF-? and the pathogenesis of OA.Methods:Totally, 37 OA patients’ cartilage was collected through the knee joint and 13 samples of articular cartilage as healthy control was collected through traumatic amputation. Real-time PCR, Western blot and ELISA analysis were performed to observe the expression of target genes and proteins in collected samples.Results:Compared with the healthy control group, TNF-? was over-expressing in cartilage which was collected from OA patients. DNA hypomethylation, histone hyperacetylation and histone methylation were observed in the TNF-? promoter in OA compared with normal patients, and we also studied series of enzymes associated with epigenetics. The results showed that by increasing DNA methylation and decreasing histone acetylation in the TNF-? promoter, and TNF-? over-expression in OA cartilage was suppressed, histone methylation has no significant correlation with OA.Conclusion:In conclusion, the changes of epigenetic status regulate TNF-α expression in the cells, which are pivotal to the OA disease process. These results may give us a better understanding of OA and may provide new therapeutic options.

Alternative splicing (AS) increases transcript and proteomic diversity of intron containing genes and has emerged as a pervasive property of eukaryotic genes. DNA methylation is a regulator of gene expression which is present in many eukaryotes. Recently, chromatin structure and histone modifications have been shown to regulate AS in humans (1), but it is unknown if this also occurs in plants. Here, I investigated the regulatory role that DNA and histone methylation have on AS in Arabidopsis thaliana. I utilized the SR protein gene family as a model system to examine changes in isoform abundance in response to chemical inhibition of DNA and histone methylation using the histone deacetylase inhibitor Tricostatin A (TSA) and the DNA demethylating agent 5-Azadeoxycytidine (Azad). RT-PCR revealed that 12 SR genes had isoforms that had altered abundance in response to TSA and Azad-C. Antagonistic effects were found when both drugs were applied since changes in splicing were only seen for 10 of the SR genes. AS patterns of the SR genes are known to change according to organ and developmental stage (2) and DNA methylation is also known to change over the lifespan of the plant (3). I investigated if organ type and developmental patterns of AS are disrupted in a DNA hypermethylation mutant. Splicing patterns of the SR genes displayed tissue and developmental specific changes in the DNA hypermethylation mutant. Computational analysis of three different DNA methylation mutants was performed using a publically available Illumina dataset (4). Widespread changes in AS was detected in each of the mutants across all types of AS. Changes in methylcytosine content within the coding region of the gene did not account for a large proportion of the novel AS events detected. Splice site sequence analysis of introns uniquely retained in each of the mutant genotypes uncovered sequence changes around the functionally important sequence elements. The results of my thesis indicate for the first time that AS in plants is regulated in part by changes in DNA methylation and histone modifications.

Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.

Although there is growing interest in the possibility that alterations in histone methylation may play a role in carcinogenesis, it has not been explored adequately in humans. Similarly, there are no reports of associations between this and a similar epigenetic event, DNA methylation. Using immunohistochemical staining, we compared the methylation of DNA and histones in histopathologically normal oral epithelium, dysplastic oral lesions, and squamous cell cancers (SCCs) from subjects with squamous cell cancer (n = 48) with those of normal oral epithelium from subjects without oral cancer (n = 93) who were matched on age and race. Monoclonal antibodies specific for 5 methyl cytosine (5-mc), lysine 4 of histone H3 (H3-Lys4), and lysine 9 of histone H3 (H3-Lys9) were used in this study. The percentages of cells positive and a weighted average of the immunostaining intensity scores were calculated for each of these tissues, and Spearman correlation analyses were employed to study associations between DNA and histone methylation. Correlations between DNA and histone methylation, H3-Lys4 and H3-Lys9 were positive and statistically significant in all tissue types; they were strongest in normal oral epithelium from non-cancer subjects (n = 0.63, p < 0.001 and r = 0.62, p < 0.001 respectively). Similarly, the positive correlations betweenH3-Lys4 and H3-Lys9 were statistically significant in all tissue types and strongest in normal oral epithelium from non-cancer subjects (r = 0.77, p < 0.001). Patterns of DNA and histone methylation are similar in tissues across the spectrum of oral carcinogenesis, and there is a significant positive association between these two epigenetic mechanisms.

Oncogenesis is driven by the accumulation of genetic and epigenetic alterations that result in dysregulation of key oncogenes, tumor suppressor genes, and DNA repair/housekeeping genes. One of the major clinical needs is the discovery and clinical validation of new molecular biomarkers using non-or minimally invasive procedures to assist early diagnosis, prognosis and prediction of response to treatment. Histone methylation has profound effects on nuclear functions such as transcriptional regulation, maintenance of genome integrity and epigenetic inheritance. On the other hand, aberrant DNA methylation can be detected in several biological fluids of patients and could be served as a potential tumor biomarker. In the present chapter we describe latest developments on histone and DNA methylation based biomarkers in Lung cancer.

Gastric cancer (GC), one of the most common malignancies worldwide, is a heterogeneous disease developing from the accumulation of genetic and epigenetic changes. One of the most critical epigenetic alterations in GC is DNA and histone methylation, which affects multiple processes in the cell nucleus, including gene expression and DNA damage repair (DDR). Indeed, the aberrant expression of histone methyltransferases and demethylases influences chromatin accessibility to the DNA repair machinery; moreover, overexpression of DNA methyltransferases results in promoter hypermethylation, which can suppress the transcription of genes involved in DNA repair. Several DDR mechanisms have been recognized so far, with homologous recombination (HR) being the main pathway involved in the repair of double-strand breaks. An increasing number of defective HR genes are emerging in GC, resulting in the identification of important determinants of therapeutic response to DDR inhibitors. This review describes how both histone and DNA methylation affect DDR in the context of GC and discusses how alterations in DDR can help identify new molecular targets to devise more effective therapeutic strategies for GC, with a particular focus on HR-deficient tumors.

Choline is an essential nutrient that, via its metabolite betaine, serves as a donor of methyl groups used in fetal development to establish the epigenetic DNA and histone methylation patterns. Supplementation with choline during embryonic days (E) 11-17 in rats improves memory performance in adulthood and protects against age-related memory decline, whereas choline deficiency impairs certain cognitive functions. We previously reported that global and gene-specific DNA methylation increased in choline-deficient fetal brain and liver, and these changes in DNA methylation correlated with an apparently compensatory up-regulation of the expression of DNA methyltransferase Dnmt1. In the current study, pregnant rats were fed a diet containing varying amounts of choline (mmol/kg: 0 (deficient), 8 (control), or 36 (supplemented)) during E11-17, and indices of histone methylation were assessed in liver and frontal cortex on E17. The mRNA and protein expression of histone methyltransferases G9a and Suv39h1 were directly related to the availability of choline. DNA methylation of the G9a and Suv39h1 genes was up-regulated by choline deficiency, suggesting that the expression of these enzymes is under negative control by methylation of their genes. The levels of H3K9Me2 and H3K27Me3, tags of transcriptionally repressed chromatin, were up-regulated by choline supplementation, whereas the levels of H3K4Me2, associated with active promoters, were highest in choline-deficient rats. These data show that maternal choline supply during pregnancy modifies fetal histone and DNA methylation, suggesting that a concerted epigenomic mechanism contributes to the long term developmental effects of varied choline intake in utero.

The SRA Methyl-Cytosine-Binding Domain Links DNA and Histone Methylation

Decoding the Epigenetic Language of Neuronal Plasticity

Colorectal cancer (CRC) ranks among the most common malignant cancers of the digestive system, and its initiation and progression are closely related to both genetic and epigenetic mechanisms. Three major forms of modifications, viz. DNA methylation, RNA m6A methylation, and histone methylation, play important roles in regulating gene expression at various stages of transcription and translation. These methylation processes are dynamic and reversible, relying on the functions of methyltransferases, demethylases, and methylation-binding proteins. Extensive studies have shown that DNA, RNA m6A, and histone methylation significantly impact multiple pathological and physiological processes in CRC, including carcinogenesis, recurrence, metastasis, resistance to both radiotherapy and chemotherapy, as well as immune regulation. Advances in high-throughput sequencing and laboratory techniques have facilitated the identification of methylation regulation enzymes with aberrant expression at the DNA, RNA, and protein levels, revealing their clinical potential for early diagnosis and treatment of CRC. The upstream regulatory mechanisms controlling these methylation regulation enzymes are crucial for understanding alterations in methylation patterns. Current evidence identifies several key mechanisms, including posttranslational modifications, epigenetic regulation, and genetic alterations, which collectively influence the expression, activity, and stability of methyltransferases, demethylases, and binding proteins. These mechanisms thereby modulate the dynamic methylation landscape across various biological contexts. Furthermore, the complex crosstalk among DNA, RNA m6A, and histone methylation is increasingly being elucidated, highlighting a need for further investigation in CRC. In this review, we systematically summarize the molecular mechanisms, clinical applications, and crosstalk involving DNA methylation, RNA m6A methylation, and histone methylation, along with their related enzymes in the development of CRC. This review aims to provide new insights and directions that underscore the significant role of epigenetic methylation modifications and their associated enzymes in CRC.

BackgroundModifications of DNA and histones in various combinations are correlated with many cellular processes. In this study, we investigated the possible relationship between histone H4 tetraacetylation, DNA methylation and histone H3 dimethylation at lysine 9 during mitosis in maize root meristems.ResultsTreatment with trichostatin A, which inhibits histone deacetylases, resulted in increased histone H4 acetylation accompanied by the decondensation of interphase chromatin and a decrease in both global H3K9 dimethylation and DNA methylation during mitosis in maize root tip cells. These observations suggest that histone acetylation may affect DNA and histone methylation during mitosis. Treatment with 5-azacytidine, a cytosine analog that reduces DNA methylation, caused chromatin decondensation and mediated an increase in H4 acetylation, in addition to reduced DNA methylation and H3K9 dimethylation during interphase and mitosis. These results suggest that decreased DNA methylation causes a reduction in H3K9 dimethylation and an increase in H4 acetylation.ConclusionsThe interchangeable effects of 5-azacytidine and trichostatin A on H4 acetylation, DNA methylation and H3K9 dimethylation indicate a mutually reinforcing action between histone acetylation, DNA methylation and histone methylation with respect to chromatin modification. Treatment with trichostatin A and 5-azacytidine treatment caused a decrease in the mitotic index, suggesting that H4 deacetylation and DNA and H3K9 methylation may contain the necessary information for triggering mitosis in maize root tips.

Epigenetic small molecule modulators of histone and DNA methylation