Condensed Chromatin Structure Research Articles

Mechanisms of action and resistance in histone methylation-targeted therapy

Epigenomes enable the rectification of disordered cancer gene expression, thereby providing new targets for pharmacological interventions. The clinical utility of targeting histone H3 lysine trimethylation (H3K27me3) as an epigenetic hallmark has been demonstrated1–7. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumour cells remain unclear. Here we show the potency and mechanisms of action and resistance of the EZH1–EZH2 dual inhibitor valemetostat in clinical trials of patients with adult T cell leukaemia/lymphoma. Administration of valemetostat reduced tumour size and demonstrated durable clinical response in aggressive lymphomas with multiple genetic mutations. Integrative single-cell analyses showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, including tumour suppressor genes. Nevertheless, subsequent long-term treatment encounters the emergence of resistant clones with reconstructed aggregate chromatin that closely resemble the pre-dose state. Acquired mutations at the PRC2–compound interface result in the propagation of clones with increased H3K27me3 expression. In patients free of PRC2 mutations, TET2 mutation or elevated DNMT3A expression causes similar chromatin recondensation through de novo DNA methylation in the H3K27me3-associated regions. We identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Targeting epigenetic drivers and chromatin homeostasis may provide opportunities for further sustained epigenetic cancer therapies.

Spt5 C-terminal repeat domain phosphorylation and length negatively regulate heterochromatin through distinct mechanisms.

Heterochromatin is a condensed chromatin structure that represses transcription of repetitive DNA elements and developmental genes, and is required for genome stability. Paradoxically, transcription of heterochromatic sequences is required for establishment of heterochromatin in diverse eukaryotic species. As such, components of the transcriptional machinery can play important roles in establishing heterochromatin. How these factors coordinate with heterochromatin proteins at nascent heterochromatic transcripts remains poorly understood. In the model eukaryote Schizosaccharomyces pombe (S. pombe), heterochromatin nucleation can be coupled to processing of nascent transcripts by the RNA interference (RNAi) pathway, or to other post-transcriptional mechanisms that are RNAi-independent. Here we show that the RNA polymerase II processivity factor Spt5 negatively regulates heterochromatin in S. pombe through its C-terminal domain (CTD). The Spt5 CTD is analogous to the CTD of the RNA polymerase II large subunit, and is comprised of multiple repeats of an amino acid motif that is phosphorylated by Cdk9. We provide evidence that genetic ablation of Spt5 CTD phosphorylation results in aberrant RNAi-dependent nucleation of heterochromatin at an ectopic location, as well as inappropriate spread of heterochromatin proximal to centromeres. In contrast, truncation of Spt5 CTD repeat number enhanced RNAi-independent heterochromatin formation and bypassed the requirement for RNAi. We relate these phenotypes to the known Spt5 CTD-binding factor Prf1/Rtf1. This separation of function argues that Spt5 CTD phosphorylation and CTD length restrict heterochromatin through unique mechanisms. More broadly, our findings argue that length and phosphorylation of the Spt5 CTD repeat array have distinct regulatory effects on transcription.

Rhamnetin, a nutraceutical flavonoid arrests cell cycle progression of human ovarian cancer (SKOV3) cells by inhibiting the histone deacetylase 2 protein

Overexpression of HDAC 2 promotes cell proliferation in ovarian cancer. HDAC 2 is involved in chromatin remodeling, transcriptional repression, and the formation of condensed chromatin structures. Targeting HDAC 2 presents a promising therapeutic approach for correcting cancer-associated epigenetic abnormalities. Consequently, HDAC 2 inhibitors have evolved as an attractive class of anti-cancer agents. This work intended to investigate the anti-cancer abilities and underlying molecular mechanisms of Rhamnetin in human epithelial ovarian carcinoma cells (SKOV3), which remain largely unexplored. We employed various in vitro methods, including MTT, apoptosis study, cell cycle analysis, fluorescence microscopy imaging, and in vitro enzymatic HDAC 2 protein inhibition, to examine the chemotherapeutic sensitivity of Rhamnetin in SKOV3 cells. Additionally, we conducted in silico studies using molecular docking, MD simulation, MM-GBSA, DFT, and pharmacokinetic analysis to investigate the binding interaction mechanism within Rhamnetin and HDAC 2, alongside the compound’s prospective as a lead candidate. The in vitro assay confirmed the cytotoxic effects of Rhamnetin on SKOV3 cells, through its inhibition of HDAC 2 activity. Rhamnetin, a nutraceutical flavonoid, halted at the G1 phase of the cell cycle and triggered apoptosis in SKOV3 cells. Furthermore, computational studies provided additional evidence of its stable binding to the HDAC 2 protein’s binding site cavity. Based on our findings, we conclude that Rhamnetin effectively promotes apoptosis and mitigates the proliferation of SKOV3 cells through HDAC 2 inhibition. These results highlight Rhamnetin as a potential lead compound, opening a new therapeutic strategy for human epithelial ovarian cancer. Communicated by Ramaswamy H. Sarma

Roles of Specialized Chromatin and DNA Structures at Subtelomeres in Schizosaccharomyces pombe.

Eukaryotes have linear chromosomes with domains called telomeres at both ends. The telomere DNA consists of a simple tandem repeat sequence, and multiple telomere-binding proteins including the shelterin complex maintain chromosome-end structures and regulate various biological reactions, such as protection of chromosome ends and control of telomere DNA length. On the other hand, subtelomeres, which are located adjacent to telomeres, contain a complex mosaic of multiple common segmental sequences and a variety of gene sequences. This review focused on roles of the subtelomeric chromatin and DNA structures in the fission yeast Schizosaccharomyces pombe. The fission yeast subtelomeres form three distinct chromatin structures; one is the shelterin complex, which is localized not only at the telomeres but also at the telomere-proximal regions of subtelomeres to form transcriptionally repressive chromatin structures. The others are heterochromatin and knob, which have repressive effects in gene expression, but the subtelomeres are equipped with a mechanism that prevents these condensed chromatin structures from invading adjacent euchromatin regions. On the other hand, recombination reactions within or near subtelomeric sequences allow chromosomes to be circularized, enabling cells to survive in telomere shortening. Furthermore, DNA structures of the subtelomeres are more variable than other chromosomal regions, which may have contributed to biological diversity and evolution while changing gene expression and chromatin structures.

Changes in nascent chromatin structure regulate activation of the pro-fibrotic transcriptome and myofibroblast emergence in organ fibrosis

Cell reprogramming to a myofibroblast responsible for the pathological accumulation of extracellular matrix is fundamental to the onset of fibrosis. Here, we explored how condensed chromatin structure marked by H3K72me3 becomes modified to allow for activation of repressed genes to drive emergence of myofibroblasts. In the early stages of myofibroblast precursor cell differentiation, we discovered that H3K27me3 demethylase enzymes UTX/KDM6B creates a delay in the accumulation of H3K27me3 on nascent DNA revealing a period of decondensed chromatin structure. This period of decondensed nascent chromatin structure allows for binding of pro-fibrotic transcription factor, Myocardin-related transcription factor A (MRTF-A) to nascent DNA. Inhibition of UTX/KDM6B enzymatic activity condenses chromatin structure, prevents MRTF-A binding, blocks activation of the pro-fibrotic transcriptome, and results in an inhibition of fibrosis in lens and lung fibrosis models. Our work reveals UTX/KDM6B as central coordinators of fibrosis, highlighting the potential to target its demethylase activity to prevent organ fibrosis.

Durable Clinical Impacts and Mechanisms of Action and Resistance in EZH1/2-Targeting Epigenetic Therapy

Background: Epigenomes allow the rectification of disordered gene expressions, thereby providing new targets for pharmacological interventions in cancer therapy. The clinical utility of targeting histone H3 lysine tri-methylation (H3K27me3) as an epigenetic hallmark has been demonstrated. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumor cells remain unclear. Results and Discussion: We conducted intensive molecular monitoring of three patients enrolled in the valemetostat first-in-human phase 1 clinical trial (NCT02732275) for adult T-cell leukemia/lymphoma (ATL). Valemetostat was administered orally once daily (200 mg/day) until a sign of disease progression could be observed. The number of abnormal lymphocytes (Ably) in these three patients drastically decreased after one week of treatment. All patients could stay on valemetostat single agent for more than 2 years with acceptable safety and durable response. PBMC samples (n = 55) were collected from these three patients immediately before (Pre) and during treatment, as well as during the progressive disease (PD) stage. Flow cytometry-based histone methylation assay demonstrated that tumor H3K27me3 level was generally high in the Pre-samples, but the valemetostat treatment reduced it to a normal level in all patients. In addition, ChIP sequencing (ChIP-seq) could directly assess how valemetostat affects the tumor epigenome. The pre-treatment tumor cells showed an overall increasing trend in H3K27me3. For all H3K27me3 peaks (51,683 peaks), 85.9% of the peak signals decreased at the average fold-change of 0.742 after the valemetostat treatment. Focusing on the gene loci, an overall decrease could be observed around the TSS and across the gene body. The characteristic H3K27me3 clusters initially observed in the tumor cells were demethylated. We also conducted integrative multilayered single-cell analyses (scATAC-seq and scRNA-seq) and showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, such as tumor suppressor genes (TSGs), in all patients. These results demonstrated that valemetostat sufficiently restored the epigenome of the tumor cells close to the healthy state, leading to clinical improvements. Although all tested cases showed a durable response for more than 2 years, these responses were eventually interrupted due to the recurrence of ATL. We identified characteristic somatic mutations of EZH2 during clonal repopulation at PD. The identified amino acid substitutions were all located at the interface between EZH2 and valemetostat. Direct evaluation of the H3K27me3 level in the PD sample harboring the acquired EZH2 mutation showed that tumor cells in a hypermethylated state were repopulating. The scATAC-seq analysis also showed strong chromatin compaction of the same gene set occurred in the rapidly propagating resistant clone, indicating that the clonally selected mutations should be responsible for resistance. In patients free of the EZH2 resistant mutations, we detected TET2 mutation or elevated DNMT3A expression in the propagated resistant clones. Genome-wide DNA methylation analysis revealed increased DNA methylation near the TSS in the resistant clones. The genes characterized by the H3K27me3-related chromatin condensation were restored by valemetostat treatment but were again strongly re-condensed by mCpG gain at PD. Consequently, the gene regulation of several important TSGs restored by valemetostat was repressed again. Furthermore, in the single-cell platform, we successfully identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Conclusion: This study illustrates, for the first time, the molecular and cellular dynamics in patients in response to a molecular-targeting inhibitor designed for histone methyltransferases. Eliminating H3K27me3 leads to the reprogramming of the cancer epigenome, thereby exerting a sustained benefit on tumors. Targeting chromatin homeostasis might provide opportunities for further sustained epigenetic therapies.

Individual variation in buffalo somatic cell cloning efficiency is related to glycolytic metabolism.

Mammalian individuals differ in their somatic cell cloning efficiency, but the mechanisms leading to this variation is poorly understood. Here we found that high cloning efficiency buffalo fetal fibroblasts (BFFs) displayed robust energy metabolism, looser chromatin structure, high H3K9 acetylation and low heterochromatin protein 1α (HP1α) expression. High cloning efficiency BFFs had more H3K9ac regions near to the upstream of glycolysis genes by ChIP-seq, and involved more openness loci related to glycolysis genes through ATAC-seq. The expression of these glycolysis genes was also found to be higher in high cloning efficiency BFFs by qRT-PCR. Two key enzymes of glycolysis, PDKs and LDH, were confirmed to be associated with histone acetylation and chromatin openness of BFFs. Treatment of low cloning efficiency BFFs with PS48 (activator of PDK1) resulted in an increase in the intracellular lactate production and H3K9 acetylation, decrease in histone deacetylase activity and HP1α expression, less condensed chromatin structure and more cloning embryos developing to blastocysts. These results indicate that the cloning efficiency of buffalo somatic cells is associated with their glycolytic metabolism and chromatin structure, and can be improved by increasing glycolytic metabolism.

Mechanisms Contributing to the Dysregulation of miRNA-124 in Pulmonary Hypertension.

Chronic pulmonary hypertension (PH) is a fatal disease characterized by the persistent activation of pulmonary vascular cells that exhibit aberrant expression of genes including miRNAs. We and others reported that decreased levels of mature microRNA-124 (miR-124) plays an important role in modulating the activated phenotype of pulmonary vascular cells and HDAC inhibitors (HDACi) can restore the levels of mature miR-124 and reverse the persistently activated phenotype of PH vascular cells. In this study, we sought to determine the mechanisms contributing to reduced levels of miRNAs, as well as how HDACi restores the levels of reduced miRNA in PH vascular cells. We found that pulmonary artery fibroblasts isolated from IPAH patients (PH-Fibs) exhibit reduced levels of mature miR-124 and several other miRNAs including let-7i, miR-224, and miR-210, and that these reduced levels can be restored by HDACi. Using miR-124 expression in human PH-Fibs as a model, we determined that reduced miR-124 gene transcription, not decreased expression of miRNA processing genes, is responsible for reduced levels of mature miR-124 in human PH-Fibs. Using both DNase I Sensitivity and chromatin immunoprecipitation assays, we found that the miR-124-1 gene exhibits a more condensed chromatin structure in human PH-Fibs, compared to corresponding controls. HDACi relaxed miR-124-1 chromatin structure, evidenced by increased levels of the open chromatin mark H3K27Ac, but decreased levels of closed chromatin mark H3K27Me3. Most importantly, the delivery of histone acetyltransferase (HAT) via CRISPR-dCas9-HAT and guiding RNAs to the promoter of the miR-124-1 gene increased miR-124-1 gene transcription. Thus, our data indicate epigenetic events play important role in controlling miR-124 and likely other miRNA levels and epigenetic regulators such as HDACs appear to be promising therapeutic targets for chronic PH.

Epigenetically Upregulated T-Type Calcium Channels Contribute to Abnormal Proliferation of Embryonic Neural Progenitor Cells Exposed to Valproic Acid.

Valproic acid is a clinically used mood stabilizer and antiepileptic drug. Valproic acid has been suggested as a teratogen associated with the manifestation of neurodevelopmental disorders, such as fetal valproate syndrome and autism spectrum disorders, when taken during specific time window of pregnancy. Previous studies proposed that prenatal exposure to valproic acid induces abnormal proliferation and differentiation of neural progenitor cells, presumably by inhibiting histone deacetylase and releasing the condensed chromatin structure. Here, we found valproic acid up-regulates the transcription of T-type calcium channels by inhibiting histone deacetylase in neural progenitor cells. The pharmacological blockade of T-type calcium channels prevented the increased proliferation of neural progenitor cells induced by valproic acid. Differentiated neural cells from neural progenitor cells treated with valproic acid displayed increased levels of calcium influx in response to potassium chloride-induced depolarization. These results suggest that prenatal exposure to valproic acid up-regulates T-type calcium channels, which may contribute to increased proliferation of neural progenitor cells by inducing an abnormal calcium response and underlie the pathogenesis of neurodevelopmental disorders.

Rice homolog of Sin3-associated polypeptide 30, OsSFL1, mediates histone deacetylation to regulate flowering time during short days.

Rice homolog of Sin3-associated polypeptide 30, OsSFL1, mediates histone deacetylation to regulate flowering time during short days.

Discovery of Primarolides A and B from Marine Fungus Asteromyces cruciatus Using Osmotic Stress and Treatment with Suberoylanilide Hydroxamic Acid.

Advances in whole-genome sequencing of many fungal species has revealed the presence of numerous “silent” biosynthetic genes, highlighting their potential to produce a wide variety of natural products. These silent biosynthetic genes are regulated in part by their highly condensed chromatin structure, which can be modified to allow transcription in response to external stimuli. In this study, Asteromyces cruciatus was subjected to both epigenetic modification and osmotic stress to enhance the production of new natural products. This “cooperative induction” strategy led to the isolation and characterization of two new polyketides from a fermentation of A. cruciatus treated with suberoylanilide hydroxamic acid and sodium chloride. The metabolic profiles of the control and treated samples were assessed using ultra-high performance liquid chromatography high-resolution electrospray ionization mass spectrometry (UHPLC-HRESIMS) metabolomic analysis, highlighting the upregulation of two new polyketides, primarolides A and B. These compounds were purified using reversed-phase flash chromatography followed by high-performance liquid chromatography, and their planar structures were established using NMR spectroscopy.

Slowing Down Ageing: The Role of Nutrients and Microbiota in Modulation of the Epigenome.

The human population is getting ageing. Both ageing and age-related diseases are correlated with an increased number of senescent cells in the organism. Senescent cells do not divide but are metabolically active and influence their environment by secreting many proteins due to a phenomenon known as senescence associated secretory phenotype (SASP). Senescent cells differ from young cells by several features. They possess more damaged DNA, more impaired mitochondria and an increased level of free radicals that cause the oxidation of macromolecules. However, not only biochemical and structural changes are related to senescence. Senescent cells have an altered chromatin structure, and in consequence, altered gene expression. With age, the level of heterochromatin decreases, and less condensed chromatin is more prone to DNA damage. On the one hand, some gene promoters are easily available for the transcriptional machinery; on the other hand, some genes are more protected (locally increased level of heterochromatin). The structure of chromatin is precisely regulated by the epigenetic modification of DNA and posttranslational modification of histones. The methylation of DNA inhibits transcription, histone methylation mostly leads to a more condensed chromatin structure (with some exceptions) and acetylation plays an opposing role. The modification of both DNA and histones is regulated by factors present in the diet. This means that compounds contained in daily food can alter gene expression and protect cells from senescence, and therefore protect the organism from ageing. An opinion prevailed for some time that compounds from the diet do not act through direct regulation of the processes in the organism but through modification of the physiology of the microbiome. In this review we try to explain the role of some food compounds, which by acting on the epigenetic level might protect the organism from age-related diseases and slow down ageing. We also try to shed some light on the role of microbiome in this process.

DNA damage accumulation, defective chromatin organization and deficient DNA repair capacity in patients with rheumatoid arthritis

We investigated the DNA damage response and repair network in 18 patients with active rheumatoid arthritis and tested the hypothesis that treatment influences this network. A 3-fold increase of endogenous DNA damage (single- and double-strand breaks) was detected in patient-derived peripheral blood mononuclear cells than controls (alkaline comet assay; mean ± SD Olive Tail Moment of 11.8 ± 7.3 versus 4.3 ± 2.2, p < .001). Patients exhibited significantly higher formation of DNA damage (oxidative stress and abasic sites), deficient global genome repair and more condensed chromatin structure than controls. Twelve weeks following treatment, chromatin structure loosened, global genome repair capacity was restored, oxidative stress and abasic sites decreased and levels of endogenous DNA damage reached control values in all 8 patients examined. We conclude that deregulated chromatin organization, deficient DNA repair capacity and augmented formation of DNA damage, which are reversible after treatment, contribute to the accumulation of endogenous DNA damage in rheumatoid arthritis.

Change in Nucleosome Dynamics During Stress Responses in Plants.

The dynamic nature of chromatin is the basis for the regulation of various biological processes in eukaryotic organisms. Nucleosome, the basic unit of chromatin in eukaryotes, undergo various reversible posttranslational modifications (PTM) in response to both external and internal cues. This PTM is recognized by different reader molecules, which facilitates the recruitment of various chromatin remodeling proteins that modulate the chromatin structure. In plants, the promoters of active genes are associated with higher lysine acetylation of histones H3 and H4, and these modifications are recognized by Bromo-domain (BRM) containing chromatin remodelers. This leads to the remodeling of the nucleosome at promoter regions, thereby increasing accessibility of the transcription machinery. It also plays a role in transcriptional repression when enriched in repressed genes. Lysine methylation recruits methyl-binding domain-containing proteins such as LIKE HETEROCHROMATIN PROTEIN1 (LHP1), which facilitates a more condensed chromatin structure that further inhibits access by the transcriptional machinery. In this article, protocols to study the regulation of chromatin conformations and nucleosome dynamics in plants in response to different stress signals are provided.

Structural Basis of Heterochromatin Formation by Human HP1

Heterochromatin plays important roles in transcriptional silencing and genome maintenance by the formation of condensed chromatin structures, which determine the epigenetic status of eukaryotic cells.The trimethylation of histone H3 lysine 9 (H3K9me3), a target of heterochromatin protein 1 (HP1), is a hallmark of heterochromatin formation. However, the mechanism by which HP1 folds chromatin-containing H3K9me3 into a higher-order structure has not been elucidated. Here we report the three-dimensional structure of the H3K9me3-containing dinucleosomes complexed with human HP1α, HP1β, and HP1γ, determined by cryogenic electron microscopy with a Volta phase plate. In the structures, two H3K9me3 nucleosomes are bridged by a symmetric HP1 dimer. Surprisingly, the linker DNA between the nucleosomes does not directly interact with HP1, thus allowing nucleosome remodeling by the ATP-utilizing chromatin assembly and remodeling factor (ACF). The structure depicts the fundamental architecture of heterochromatin.

Chromatin Histone Modifying and DNA Repair Inhibition Enhances the Anti-Myeloma Activity of Melphalan

Repair of DNA and, thus, preservation of the genetic code are critical for normal cellular function. However, tumor cells use DNA repair pathways to develop resistance to chemotherapy. Therefore, inhibiting DNA repair may override this drug resistance. Herein, we further investigate the mechanistic basis of the link between DNA repair efficiency and response to anti-myeloma therapy and provide evidence that histone modifiers might prove efficacious when used in combination with DNA-damaging agents.We studied clonal bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of 26 unselected newly diagnosed multiple myeloma (MM) patients (12F/14M; median age 60 years, range 42-66) who responded (n=17) or did not respond (n=9) to subsequent melphalan therapy. PBMCs from healthy controls (n=25) were also included in the study. Cells were ex vivotreated with melphalan alone or in the presence of histone deacetylase inhibitor HDACi) vorinostat or the Non-Homologous End-Joining (NHEJ) inhibitor SCR7 and the extent of the DNA damage formation/repair (monoadducts by using Southern blot analysis; interstrand crosslinks by using quantitative-PCR; double-strand breaks repair using immunofluorescence quantification of γH2AX foci by confocal microscopy), the induction of the apoptotic pathway (using a photometric enzyme-immunoassay) and the local chromatin condensation were evaluated. Finally, the expression of a focused panel of 84 genes involved in DNA damage response pathways (ATM/ATR signaling, DNA repair pathways, cell cycle regulation, apoptosis) was also examined.Both BMPCs and PBMCs from responders to melphalan therapy showed more condensed chromatin structure and slower rates of DNA repair activity (P<0.0022) compared to non-responders. Moreover, apoptosis rates of both BMPCs and PBMCs were inversely correlated with individual DNA repair efficiency, being higher in responders’ cells compared to those of non-responders (P=0.0011). PBMCs from MM patients showed higher looseness of chromatin structure, increased DNA repair activity and higher apoptosis rates compared to healthy controls. Microarray analyses of untreated PBMCs consistently point to an altered expression of several DNA damage response-related genes in MM patients’ responders to subsequent therapy compared to non-responders. Particularly, responders’ PBMCs showed upregulation of ATR, BLM, DDB1, EXO1, FANCA, MRE11A, MSH2, PCNA, RAD50, RAD51, RPA1, XPA, XPC, XRCC1 and TP53 genes and downregulation of ATM, CDC25C, CDKN1A, CHEK2, ERCC1, MPG, PNKP and UNG genes compared to non-responders (in all cases, fold changes between groups were >2, P<0.001), suggesting that perturbation in the molecular components of DNA damage response pathways plays an important role in the therapeutic action of the genotoxic drugs. Interestingly, co-treatment of cells with melphalan and the NHEJ inhibitor SCR7 significantly reduced the rates of DNA damage repair and increased melphalan sensitivity of both BMPCs and PBMCs. Moreover, co-treatment of BMPCs with melphalan and the HDACi vorinostat resulted in hyperacetylation of histone H4, increased DNA damage burden and higher apoptosis rates of these cells. On the other hand, co-treatment of PBMCs with melphalan in the presence of vorinostat did not significantly alter the melphalan-induced DNA damage burden and the apoptosis rates of these cells. Interestingly, using Western blot analysis, we found that vorinostat decreased the levels of critical DNA repair proteins (Rad50, Mre11, Ku70, Ku86, DNA-PKcs) in malignant BMPCs, while it did not suppressed these DNA proteins in PBMCs, a finding that can explain, in part, the selectivity of vorinostat in causing cell death in malignant cells at concentrations that cause little or no normal cell death.Collectively, responders to melphalan therapy are characterized by slower rates of DNA repair mechanisms, resulting in higher accumulation of the cytotoxic DNA lesions, which in turn triggers the induction of the apoptotic pathway, a priority for successful clinical outcome. The enhancement of melphalan cytotoxicity by DNA repair modifiers offers a promising strategy toward treatment of MM and improvement of existing regimens. DisclosuresTerpos:Novartis: Honoraria; Celgene: Honoraria; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Genesis: Consultancy, Honoraria, Other: Travel expenses; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria.

DNA repair of myeloma plasma cells correlates with clinical outcome: the effect of the nonhomologous end-joining inhibitor SCR7

AbstractDNA repair activity of malignant cells seems to influence therapeutic outcome and patients' survival. Herein, we investigated the mechanistic basis for the link between DNA repair efficiency and response to antimyeloma therapy. Nucleotide excision repair (NER), interstrand cross-links repair (ICL/R), double-strand breaks repair (DSB/R), and chromatin structure were evaluated in multiple myeloma (MM) cell lines (melphalan-sensitive RPMI8226; melphalan-resistant LR5) and bone marrow plasma cells (BMPCs) from MM patients who responded (n = 17) or did not respond (n = 9) to subsequent melphalan therapy. The effect of DSB/R inhibition was also evaluated. Responders' BMPCs showed slower rates of NER and DSB/R (P < .0022), similar rates of ICL/R, and more condensed chromatin structure compared with nonresponders. Moreover, apoptosis rates of BMPCs were inversely correlated with individual DNA repair efficiency and were higher in responders' cells compared with those of nonresponders (P = .0011). Similarly, RPMI8226 cells showed slower rates of NER and DSB/R, comparable rates of ICL/R, more condensed chromatin structure, and higher sensitivity than LR5 cells. Interestingly, cotreatment of BMPCs or cell lines with DSB/R inhibitors significantly reduced the rates of DSB/R and increased melphalan sensitivity of the cells, with the nonhomologous end-joining inhibitor SCR7 showing the strongest effect. Together, responders' BMPCs are characterized by lower efficiencies of NER and DSB/R mechanisms, resulting in higher accumulation of the extremely cytotoxic ICLs and DSBs lesions, which in turn triggers the induction of the apoptotic pathway. Moreover, the enhancement of melphalan cytotoxicity by DSB/R inhibition offers a promising strategy toward improvement of existing antimyeloma regimens.

Stilbenoids remodel the DNA methylation patterns in breast cancer cells and inhibit oncogenic NOTCH signaling through epigenetic regulation of MAML2 transcriptional activity.

DNA hypomethylation was previously implicated in cancer progression and metastasis. The purpose of this study was to examine whether stilbenoids, resveratrol and pterostilbene thought to exert anticancer effects, target genes with oncogenic function for de novo methylation and silencing, leading to inactivation of related signaling pathways. Following Illumina 450K, genome-wide DNA methylation analysis reveals that stilbenoids alter DNA methylation patterns in breast cancer cells. On average, 75% of differentially methylated genes have increased methylation, and these genes are enriched for oncogenic functions, including NOTCH signaling pathway. MAML2, a coactivator of NOTCH targets, is methylated at the enhancer region and transcriptionally silenced in response to stilbenoids, possibly explaining the downregulation of NOTCH target genes. The increased DNA methylation at MAML2 enhancer coincides with increased occupancy of repressive histone marks and decrease in activating marks. This condensed chromatin structure is associated with binding of DNMT3B and decreased occupancy of OCT1 transcription factor at MAML2 enhancer, suggesting a role of DNMT3B in increasing methylation of MAML2 after stilbenoid treatment. Our results deliver a novel insight into epigenetic regulation of oncogenic signals in cancer and provide support for epigenetic-targeting strategies as an effective anticancer approach.

Abstract B17: Epigenetic regulation of NOTCH oncogenic signaling in breast cancer

Abstract Alterations in DNA methylation occur at different stages of cancer, including initiation, and may underlie up-regulation of genes with oncogenic functions. The NOTCH pathway is often overactive in breast cancer and plays roles in cancer development and progression. It is therefore a possible target for anti-cancer strategies. However, the mechanisms of NOTCH regulation in mammary carcinogenesis remain unclear which hinders the development of effective approaches to target this oncogenic pathway. Interestingly, certain dietary compounds such as polyphenols with a stilbenoid structure suppress the NOTCH signals in cancer and were shown in our study to modify epigenetic marks in genes positively regulating the pathway, including MAML2. In the present study, using two polyphenols, resveratrol from grapes and pterostilbene from blueberries, we investigate the implication of DNA methylation in regulation of NOTCH in breast cancer. Non-invasive MCF10CA1h and invasive MCF10CA1a human breast cancer cell lines were used as an experimental model. Following genome-wide DNA methylation analysis with Illumina 450K BeadChip array, pyrosequencing and QPCR were performed to assess methylation and expression of MAML2 and NOTCH target genes. Chromatin immunoprecipitation (ChIP) was applied to determine binding of epigenetic enzymes to MAML2 regulatory region. Depletion with siRNA was applied to establish functional link between MAML2 silencing and NOTCH activity. We found 4,293 CpG sites differentially methylated upon 9-day treatment of MCF10CA1h breast cancer cells with 15µM resveratrol as compared to untreated cells (0.05&amp;lt;differential methylation&amp;lt;-0.05, p&amp;lt;0.03, Wilcoxon rank-sum test). Among these changes, 3,508 CpGs corresponding to 1,707 genes were hypermethylated and functionally associated with oncogenic signaling pathways, including NOTCH. As the array data indicated, resveratrol led to re-methylation of enhancer region of MAML2 that is a coactivator of NOTCH target genes. The 15-20% increase in methylation of MAML2 was confirmed by pyrosequencing in breast cancer cells with both high and low invasive potential. Pterostilbene, an analog of resveratrol, exerted similar effects on MAML2 methylation state at 10µM concentration. Along with methylation of the MAML2 enhancer region, the compounds decreased MAML2 expression by 30-60%. Stronger MAML2 down-regulation was achieved in invasive cancer cells as compared to non-invasive cells after exposure to either resveratrol or pterostilbene. Epigenetic silencing of MAML2 was associated with downregulation of NOTCH target genes, including HES1 (30-50%), HEY1 (30-60%), and NOTCH1 (40-65%). Depletion of MAML2 with siRNA mimicked polyphenols' effects leading to suppression of NOTCH pathway. It supports a functional link between MAML2 and NOTCH signaling. The most profound effects were observed in invasive MCF10CA1a cells where further analyses revealed 2-fold increase in trimethylation of H3K27 (repressive histone mark) and 30% decrease in acetylation of H3K9 (activating mark) at MAML2 enhancer region upon exposure to resveratrol. The condensed chromatin structure was associated with binding of DNMT3B to the tested fragment of MAML2 upon polyphenol treatment whereas no binding was detected in untreated cells. It suggests the role of DNMT3B in increased methylation of MAML2, which is consistent with increased DNMT3B expression upon treatment with polyphenols. Our results establish a role for epigenetic modifications in regulation of NOTCH oncogenic signaling in breast cancer. It constitutes a novel insight into regulation of oncogenic signals in cancer and provides support for epigenetic-targeting strategies as an effective anti-cancer approach. This study was supported by the Purdue University Center for Cancer Research, Indiana CTSI (UL1TR001108), and Women's Global Health Institute granted to BS. Citation Format: Katarzyna Lubecka, Lucinda Kurzava, Kirsty Flower, Hannah Buvala, Dorothy Teegarden, Ignacio Camarillo, James M. Flanagan, Barbara Stefanska. Epigenetic regulation of NOTCH oncogenic signaling in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B17.

The State of Chromatin Condensation, the Expression of Genes Involved in DNA Damage Response and the DNA Repair Capacity Affect the Drug Sensitivity of PBMCs of Myeloma Patients Treated with Melphalan

Melphalan is an interstrand cross-link (ICL)-inducing agent and one of the most active chemotherapeuticdrugs in the treatment of multiple myeloma (MM). However, the molecular mechanisms contributing to differential response of MM patients to melphalan are poorly understood. Herein, we investigated the underlying mechanisms in processing and repair of melphalan-induced DNA lesions and their potent contribution to the outcome of anti-myeloma therapy.We studied peripheral blood mononuclear cells (PBMCs) from 15 newly diagnosed multiple myeloma (MM) patients (8M/7F; median age 61 years) responders (≥PR, n=9) and non-responders (< PR, n=6) to subsequent melphalan therapy. PBMCs from 25 healthy controls were also included in this study (HC; 14M/11F, median age 58 years). PBMCs were ex vivo treated with melphalan and the extent of the DNA damage formation/repair (monoadducts by using Southern blot; ICLs by using a quantitative PCR-based assay; double strand breaks (DSBs) using immunofluorescence quantification of γH2AX foci), the induction of the apoptotic pathway by using a photometric enzyme-immunoassay and the local chromatin condensation by using micrococcal nuclease digestion were examined. Finally, the expression of a focused panel of 84 genes involved in DNA damage response (DDR) pathways (ATM/ATR signaling, DNA repair pathways, cell cycle regulation, apoptosis) was also evaluated.Following ex vivo treatment of PBMCs with melphalan, in all individuals examined biphasic DNA repair kinetics were observed, including a fast first-phase of repair and a much slower second phase. Interestingly, the accumulation of monoadducts was inversely correlated with the first-phase repair capacity of the PBMCs, being significantly higher in HC than in responders and lowest in non-responders (all P<0.001). The second phase repair capacity showed no differences in all individuals analyzed. Also, although ICLs "unhooking" rates were similar in all individuals, accumulation of ICLs was significantly higher in HC compared to responders' PBMCs (P<0.01), due to higher levels of monoadducts that are precursors of ICLs left unrepaired in these cells, resulting in higher formation of ICLs. Minimal amounts of ICLs were observed in non-responders. Moreover, DSBs burden was significantly higher in HC than in responders, due to higher accumulation of ICL, which are precursors of DSBs and lower rates of DSB repair in these cells (P<0.01). Again, minimal amounts of DSBs were observed in non-responders.Interestingly, apoptosis rate of PBMCs was inversely correlated with the repair efficiency of both the first-phase monoadducts and the DSBs, with the apoptosis being significantly higher in HC compared to responders and lowest in non-responders (all P<0.05). In untreated PBMCs, we found an inverse correlation between the local chromatin condensation and the repair capacity. Therefore, we observed a progressive, significant increase in the looseness of the local chromatin structure, from HC to MM patients, with responders showing more condensed chromatin structure compared to non-responders (all P<0.05). Interestingly, by using a-amanitin, an inducer of chromatin condensation, PBMCs from non-responders showed the DNA repair capacity and the melphalan sensitivity similar to PBMCs from responders, suggesting that the state of the chromatin structure contributes to the response to melphalan therapy.Finally, microarray analyses of untreated PBMCs consistently point to an altered expression of several DNA damage response-related genes in MM patients compared to HC. Particularly, responders' PBMCs showed upregulation of 4 genes (ATR, CHEK2, XPA, XRCC1) and downregulation of 5 genes (ATM, MPG, UNG, CDKN1A, CDC25C) compared to non-responders (in all cases, fold changes between groups were >2, P<0.001), suggesting that changes in the molecular components of the DDR pathways correlate with the outcome of melphalan therapy.We conclude that the state of chromatin condensation, the expression of genes involved in DDR pathways and the repair capacity of monoadducts and DSBs affect the drug sensitivity of PBMCs and maybe used for the prediction of response of myeloma patients to melphalan therapy. DisclosuresTerpos:Celgene: Honoraria, Other: travel expenses; Novartis: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel expenses. Munshi:celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees. Dimopoulos:Janssen-Cilag: Honoraria; Novartis: Honoraria; Genesis: Honoraria; Amgen: Honoraria; Onyx: Honoraria; Celgene: Honoraria; Janssen: Honoraria.