Chromatin-modifying Agents Research Articles

The multifaceted functions of long non-coding RNA HOTAIR in neuropathologies and its potential as a prognostic marker and therapeutic biotarget.

Long non-coding RNAs (lncRNAs) are progressively being perceived as prominent molecular agents controlling multiple aspects of neuronal (patho)physiology. Amongst these is the HOX transcript antisense intergenic RNA, often abbreviated as HOTAIR. HOTAIR epigenetically regulates its target genes via its interaction with two different chromatin-modifying agents; histone methyltransferase polycomb-repressive complex 2 and histone demethylase lysine-specific demethylase 1. Parenthetically, HOTAIR elicits trans-acting sponging function against multiple micro-RNA species. Oncological research studies have confirmed the pathogenic functions of HOTAIR in multiple cancer types, such as gliomas and proposed it as a pro-oncological lncRNA. In fact, its expression has been suggested to be a predictor of the severity/grade of gliomas, and as a prognostic biomarker. Moreover, a propound influence of HOTAIR in other aspects of brain heath and disease states is just beginning to be unravelled. The objective of this review is to recapitulate all the relevant data pertaining to the regulatory roles of HOTAIR in neuronal (patho)physiology. To this end, we discuss the pathogenic mechanisms of HOTAIR in multiple neuronal diseases, such as neurodegeneration, traumatic brain injury and neuropsychiatric disorders. Finally, we also summarize the results from the studies incriminating HOTAIR in the pathogeneses of gliomas and other brain cancers. Implications of HOTAIR serving as a suitable therapeutic target in neuropathologies are also discussed.

Oral Decitabine/Cedazuridine Is a Tolerable and Effective Ambulatory Regimen for Patients with Advanced Myeloproliferative Neoplasms

Background: Treatment options are limited for patients (pts) with advanced myeloproliferative neoplasms (MPNs). Most pts become refractory to JAK2 inhibitor (JAKi) therapy by 3 years with subsequent survival of only 11-16 months. Sequential treatment with chromatin-modifying agents including decitabine (D) can upregulate CXCR4 expression, correct abnormal trafficking of myelofibrosis (MF) stem cells, and eliminate JAK2V617F+ MF stem and progenitor cells in preclinical models ( Wang et al, Cancer Res 2019, Blood 2010). Combination therapy with intravenous D and ruxolitinib (Rux) had a favorable effect on overall survival (OS) in a phase 2 study of MPN-accelerated/blast phase pts ( Mascarenhas et al, Blood Adv 2020) and prolonged survival in a cohort of DIPSS-plus high-risk MF pts ( Bader et al, Leuk Res 2015). Oral decitabine/cedazuridine (DC), therefore, represents an attractive ambulatory treatment option for pts with advanced MPNs. Methods: We retrospectively reviewed the electronic health records of pts with advanced high-risk MF (AHR-MF) or MPN- accelerated phase (MPN-AP) seen at our institution that were treated with DC with or without a JAKi from 1/2021-7/2023. MPN-AP was defined by the presence of 10-19% blasts in peripheral blood/ bone marrow. AHR-MF was defined by the presence of 4-9% circulating blasts or 5-9% blasts in the bone marrow, and/or refractoriness to a JAKi. Results: A total of 14 pts, 7 each with MPN-AP and AHR-MF, respectively received DC therapy (Table 1). Most pts (8/14 or 57%) were older than 75 years. All pts were classified as high risk/very high-risk (MIPPS70 plus v.2). Ten (71%) pts had one or more high molecular risk mutations ( ASXL1, EZH2, SRSF2, IDH1/2, U2AF1), with the most prevalent being ASXL1 mutations in 8/14 (57%) pts. Other somatic mutations included NF1, KRAS, NRAS, BRAF, GATA2, STAG2, DNMT3A and TET2 genes. Eight of the 14 pts (57%) had either an unfavorable or very high-risk karyotype (MIPSS70 plus v.2). Thirteen pts had previously failed JAKi therapy. The median number of cycles of DC therapy was 2 (range,1-6) and 4 (range,1-26) for MPN-AP and AHR-MF pts, respectively and 4/7 pts received a concurrent JAKi in each group. Two pts in AHR-MF cohort were successfully bridged to transplant. Two pts in the MPN-AP group discontinued treatment after cycle 1 due to neutropenic sepsis and progression to blast phase, respectively. Among MPN-AP pts who received ≥ 2 cycles of DC, circulating blasts were reduced to ≤ 5% in 4/5 pts, normalization of thrombocytopenia and improvement in hemoglobin by ≥ 1.5 g/dl each occurred in 3/5 pts (Fig.1). In the AHR-MF cohort, 6/7 pts received ≥ 2 cycles of DC with elimination of circulating blasts in one pt. WBC and platelet count were effectively reduced in 4/4 and 2/2 pts with baseline WBC of >50x 10 3/uL and platelet count >1000 x 10 3/uL, respectively. Spleen size reduction was achieved in two pts with combination of DC + Rux who were previously refractory to a JAKi. One pt achieved complete resolution of palpable splenomegaly after 2 cycles of combination therapy. The predominant treatment-related adverse event was neutropenic fever in three pts requiring hospitalization. DC cycle length was reduced from 5 to 3/28 days in 7/14 pts, which reduced the degree of therapy related neutropenia and anemia. Pts remained ambulatory during treatment except for hospitalizations related to neutropenic fever. With a median follow-up of 109 days (range, 28 days - 29 months), median OS has not been reached. Three pts in the MPN-AP cohort progressed to MPN-blast phase (BP), including two pts who only received one cycle of DC. Two pts with MPN-BP died after 28 and 68 days of DC therapy, respectively. None of the AHR-MF pts progressed to MPN-AP/BP. The most durable response was observed in a pt who has received a total of 26 cycles of DC + Rux with stable disease for >2 years and loss of BRAF and KRAS mutations after 9 months of treatment. Conclusions: DC therapy appears to be a well-tolerated and effective oral ambulatory regimen with clinically meaningful responses in elderly, transplant ineligible pts as well as a bridge to transplant in high-risk pts with advanced MPNs. The ease of outpatient administration significantly reduces pt treatment burdens and positively impacts their quality of life. These data provide a rationale for studying oral decitabine/cedazuridine +/- JAKi in a prospective manner, especially in pts with AHR-MF refractory to JAKi prior to evolution MPN-AP/BP.

Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells.

RationaleThe human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation.Methods and resultsHere, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2'-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network-like structures in the presence of VEGF.ConclusionChromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tube-forming cells in a 2D matrix in VEGF-dependent manner.

Abstract IA09: Non-mutational resistance to anti-cancer drugs

Abstract “Rationally-targeted” oncology drugs can deliver significant therapeutic benefit in genomically-defined subsets of cancer patients. However, the clinical benefit associated with these agents is substantially limited by the nearly invariable emergence of drug resistance over time. In many cases, specific mutational mechanisms of resistance have been elucidated, presumably reflecting the clonal selection of stochastically-generated mutations that were present prior to treatment; however, additional pre-clinical and clinical findings implicate non-mutational mechanisms as well. We have identified and characterized a small subpopulation of reversibly “drug-tolerant” cells. The drug-tolerant phenotype is associated with a distinct chromatin state, and is transiently acquired and relinquished at low frequency by individual cells within the population, implicating the dynamic regulation of phenotypic heterogeneity. The drug-tolerant subpopulation can be selectively ablated by treatment with certain chromatin-modifying agents, potentially yielding a therapeutic opportunity. Furthermore, this subpopulation appears to contain the precursors of drug-resistant clones that acquire mutations during, rather than prior to, drug treatment. Additional studies of non-mutational drug resistance mechanisms implicate the rewiring of growth factor-responsive cellular signaling pathways as a common survival mechanism within tumor cell populations. These findings suggest that cancer cell populations employ various epigenetically-determined survival strategies to establish sufficient phenotypic variation among cells in order to protect the population from eradication by potentially lethal exposures. Citation Format: Jeff Settleman. Non-mutational resistance to anti-cancer drugs. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA09.

Decreased expression of connective tissue growth factor in non-small cell lung cancer is associated with clinicopathological variables and can be restored by epigenetic modifiers.

PurposeRecent studies indicated undisputed contribution of connective tissue growth factor (CTGF) in the development of many cancers, including non-small cell lung cancer (NSCLC). However, the functional role and regulation of CTGF expression during tumorigenesis remain elusive. Our goal was to determine CTGF transcript and protein levels in tumoral and matched control tissues from 98 NSCLC patients, to correlate the results with clinicopathological features and to investigate whether the CTGF expression can be epigenetically regulated in NSCLC.MethodsWe used quantitative PCR, Western blotting and immunohistochemistry to evaluate CTGF expression in lung cancerous and histopathologically unchanged tissues. We tested the impact of 5-Aza-2′-deoxycytidine (5-dAzaC) and trichostatin A (TSA) on CTGF transcript and protein levels in NSCLC cells (A549, Calu-1). DNA methylation status of the CTGF regulatory region was evaluated by bisulfite sequencing. The influence of 5-dAzaC and TSA on NSCLC cells viability and proliferation was monitored by the trypan blue assay.ResultsWe found significantly decreased levels of CTGF mRNA and protein (both p < 0.0000001) in cancerous tissues of NSCLC patients. Down-regulation of CTGF occurred regardless of gender in all histological subtypes of NSCLC. Moreover, we showed that 5-dAzaC and TSA were able to restore CTGF mRNA and protein contents in NSCLC cells. However, no methylation within CTGF regulatory region was detected. Both compounds significantly reduced NSCLC cells proliferation.ConclusionsDecreased expression of CTGF is a common feature in NSCLC; however, it can be restored by the chromatin-modifying agents such as 5-dAzaC or TSA and consequently restrain cancer development.Electronic supplementary materialThe online version of this article (doi:10.1007/s00432-016-2195-3) contains supplementary material, which is available to authorized users.

Loss of DAB2IP expression in human urothelial carcinoma is associated with poorer recurrence-free survival.

The purpose of this study is to investigate the clinical relevance of deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP) expression in human urothelial carcinoma (UC). We studied DAB2IP protein expression by immunohistochemistry in 130 UCs (90 of the bladder and 40 of the upper urinary tract) and 79 adjacent normal tissues and assessed its prognostic value in terms of recurrence-free and progression-free survival in superficial bladder UC. Twelve human UC cell lines were examined for DAB2IP messenger RNA (mRNA) and protein expression using quantitative RT-PCR and western blotting. Selected cell lines were used to study the effect of treatment with chromatin-modifying agents (5-aza-2'-deoxycytidine, Trichostatin A, or both) on DAB2IP expression. Of 90 bladder tumors, 50 (56%) and, of 40 upper tract UC, 11 (28%) were positive for DAB2IP immunostaining (bladder cancer versus upper tract UC, p=0.003). In 65 superficial cases of bladder cancer loss of DAB2IP, expression was significantly associated with decreased recurrence-free survival (p=0.046), but not with progression-free survival. Most human urothelial cancer cell lines consistently express DAB2IP mRNA and protein, without any relation to S-phase kinase protein expression. After treatment with either 5-aza-2'-deoxycytidine or Trichostatin A or both, the low DAB2IP-expressing bladder cancer cell lines BFTC905 and BFTC909 showed increased DAB2IP mRNA expression. DAB2IP protein levels are higher in bladder cancer than in upper tract UC and in superficial bladder cancer. This is associated with longer recurrence-free survival. Epigenetic regulation of DAB2IP protein appears to play an important role in human urothelial carcinoma.

The pan-HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR-mutated and -wildtype non-small cell lung cancer cells.

BackgroundThe receptor tyrosine kinase (RTK) EGFR is overexpressed and mutated in NSCLC. These mutations can be targeted by RTK inhibitors (TKIs) such as erlotinib. Chromatin-modifying agents may offer a novel therapeutic approach by sensitizing tumor cells to TKIs.MethodsThe NSCLC cell lines HCC827 (EGFR mutant, adenocarcinoma), A549 (EGFR wt, adenocarcinoma) and NCI-H460 (EGFR wt, large cell carcinoma) were analyzed by SNP6.0 array. Changes in proliferation after panobinostat (LBH-589, PS) and erlotinib treatment were quantified by WST-1 assay and apoptosis by Annexin V/7-AAD flow cytometry. Abundance of target proteins and histone marks (acH3, H3K4me1/2/3) was determined by immunoblotting.ResultsAs expected, the EGFR wt cell lines A549 and NCI-H460 were quite insensitive to the growth-inhibitory effect of erlotinib (IC50 70-100 μM), compared to HCC827 (IC50 < 0.02 μM). All three cell lines were sensitive to PS treatment (IC50: HCC827 10 nM, A549 20 nM and NCI-H460 35 nM). The combination of both drugs further reduced proliferation in HCC827 and in A549, but not in NCI-H460. PS alone induced differentiation and expression of p21WAF1/CIP1 and p53 and decreased CHK1 in all three cell lines, with almost no further effect when combined with erlotinib. In contrast, combination treatment additively decreased pEGFR, pERK and pAKT in A549. Both drugs synergistically induced acH3 in the adenocarcinoma lines. Surprisingly, we also observed induction of H3K4 methylation marks after erlotinib treatment in HCC827 and in A549 that was further enhanced by combination with PS.ConclusionPS sensitized lung adenocarcinoma cells to the antiproliferative effects of erlotinib. In these cell lines, the drug combination also had a robust, not previously described effect on histone H3 acetylation and H3K4 methylation.

Role of epigenetic reprogramming in hematopoietic stem cell function.

Epigenetic regulatory networks determine the fate of dividing hematopoietic stem cells (HSCs). Prior attempts at the ex-vivo expansion of transplantable human HSCs have led to the depletion or at best maintenance of the numbers of HSCs because of the epigenetic events that silence the HSC gene-expression pattern. The purpose of this review is to outline the recent efforts to use small molecules to reprogram cultured CD34 cells so as to expand their numbers. Chromatin-modifying agents (CMAs) reactivate the gene-expression patterns of HSCs that have been silenced as they divide ex vivo. Increasing evidence indicates that CMAs act not only by promoting HSC symmetrical self-renewal divisions, but also by reprogramming progenitor cells, resulting in greater numbers of HSCs. The use of such CMAs for these purposes has not resulted in malignant transformation of the ex-vivo treated cell product. The silencing of the gene-expression program that determines HSC function after ex-vivo culture can be reversed by reprogramming the progeny of dividing HSCs with transient exposure to CMAs. The successful implementation of this approach provides a strategy which might lead to the development of a clinically relevant means of manufacturing increased numbers of HSCs.

Epigenetic priming of non-small cell lung cancer cell lines to the antiproliferative and differentiating effects of all-trans retinoic acid.

The retinoic acid signaling pathway, crucial for differentiation, is silenced by epigenetic mechanisms in many cancers. Epigenetically active, chromatin-modifying agents offer a novel treatment approach, by reactivating aberrantly silenced genes in tumor cells and by sensitizing them to subsequent treatments. We hypothesized that the treatment of non-small cell lung cancer (NSCLC) cells with a histone deacetylase (HDAC) inhibitor may prime them to the antiproliferative and differentiating activity of all-trans retinoic acid. The NSCLC cell lines A549, NCI-H460 and HCC827 were treated with ATRA (2 µM) and the pan-HDAC inhibitor panobinostat (LBH589; 10-35 nM). While treatment with ATRA alone showed only very modest effects, panobinostat reduced cellular proliferation by at least 50 %. Notably, the combination of panobinostat and ATRA had additive and synergistic effects, respectively, on growth inhibition and differentiation, with almost no cytotoxicity. Effects were strongest in A549, followed by the EGFR-mutant HCC827, and least pronounced in NCI-H460. Global histone H3 acetylation was strongly induced by panobinostat; interestingly, ATRA alone had also an effect on histone acetylation, which was synergistically enhanced when the HDAC inhibitor was added. The combination of the two drugs additively decreased expression of phospho-ERK and phospho-AKT, whereas p53 and p21(CIP1/WAF1) proteins were both induced. Panobinostat sensitized, to varying degrees, all three cell lines to the antiproliferative and differentiating effects of ATRA, with synergistic histone H3 acetylation. Combination therapy with an epigenetic drug and ATRA may offer an alternative to aggressive chemotherapy even in primary ATRA-insensitive tumors, such as adenocarcinomas of the lung.

Use of IL3 and chromatin‐modifying reagents valproic acid and 5‐aza‐2′‐deoxycytidine to affect mobilized peripheral blood CD34+ cell fate decisions

Culture of blood CD34(+) cells with chromatin-modifying agents can lead to an increase in marrow repopulating cells and in the case of valproic acid increased erythroid cell colony formation. We undertook research to help understand what effects these reagents have on mobilized peripheral blood (MPB) CD34(+) cells. Mobilized peripheral blood was obtained under informed consent and ethics committee approval from nine patients and allograft donors. Epigenetic modifiers valproic acid and 5-aza-2'-deoxycytidine were used singly or in combination with each other and with IL3 when culturing mobilized peripheral blood CD34(+) cells. Cultured cells were subsequently used in flow cytometry and colony-forming unit assay experiments. Addition of IL3 to the in vitro cell growth medium improved the expansion and maintained the functionality of CD34(+) cells. Valproic acid and IL3 also work synergistically to increase the numbers of CD34(+) /CD36(+) double-positive cells. We found that an apparent increase in red cell colony formation was a result of a decrease in white cell colonies, with no overall increase in red cell colonies when equivalent numbers of CD34(+) cells are plated. Mobilized peripheral blood CD34(+) stem and progenitor cells are affected by chromatin-modifying agents and IL3 giving higher numbers of CD34(+) /CD36(+) double-positive erythroid progenitors.

Differential Effects of Epigenetic Modifiers on the Expansion and Maintenance of Human Cord Blood Stem/Progenitor Cells

Epigenetic therapies, including DNA methyltransferase and histone deacetylase (HDAC) inhibitors, are increasingly being considered to treat hematological malignancies, but their effects on normal hematopoietic stem cells (HSCs) remain largely unexplored. We compared the effects of several HDAC inhibitors, including valproic acid (VPA) and trichostatin A (TSA), alone or in combination with 5-aza-2′-deoxycytidine (5azaD) on the expansion of HSCs. VPA induced the highest expansion of CD34+CD90+ cells and progenitor cells compared with other HDAC inhibitors or the sequential addition of 5azaD/TSA in culture. Xenotransplantation studies demonstrated that VPA prevents HSC loss, whereas 5azaD/TSA treatment leads to a net expansion of HSCs that retain serial transplantation ability. 5azaD/TSA-mediated HSC expansion was associated with increased histone acetylation and transient DNA demethylation, which corresponded with higher gene transcript levels. However, some genes with increased transcript levels lacked changes in methylation. Importantly, a global microarray analysis revealed a set of differentially expressed genes in 5azaD/TSA- and VPA-expanded CD34+ cells that might be involved in the expansion and maintenance of transplantable HSCs, respectively. In summary, our data indicate that treatment of HSCs with different chromatin-modifying agents results in either the expansion or maintenance of HSCs, an observation of potential therapeutic importance.

Transient Activation Of Inflammatory Molecules By Epigenetic Modifiers Likely Plays a Role In Expansion Of Transplantable Human Cord Blood Stem/Progenitor Cells

Recent studies indicate that inflammatory molecules, including interferon, possess stimulatory effects which trigger excessive proliferation leading to hematopoietic stem cell (HSC) exhaustion and bone marrow (BM) failure. We hypothesized that transient activation of inflammatory molecules by chromatin modifying agents (CMA) stimulates proliferation of transplantable HSC in culture. We previously demonstrated that sequential in vitro addition of a hypomethylating drug, 5-aza-2'-deoxycytidine (5azaD) followed by addition of a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) during human umbilical cord blood CD34+ cell culture results in expansion of serially transplantable long term HSC (Araki et al. Blood 2007, Exp Hematol 2009). However treatment with valproic acid (VPA), a HDAC inhibitor, only expands short term HSC, which are not serially transplantable (Araki et al. Abst. 827, Blood 2010). This failure of VPA to expand long-term repopulating HSC is not however due to poor BM homing (homing efficiency VPA-expanded cells 1.68 ± 0.35%, n = 10 NOD/SCID mice, 5azaD/TSA-expanded cells 0.39 ± 0.056%, n = 7, p = 0.008). Molecular studies indicate that expression of genes implicated in HSC self-renewal (HoxB4, Bmi1, Ezh2, and GATA2) are equally increased in 5azaD/TSA or VPA expanded CD34+ cells (n = 3, control vs. 5azaD/TSA or control vs. VPA p < 0.05). Similarly, increased HoxB4, Ezh2 and PU.1 protein levels are observed in VPA or 5azaD/TSA expanded cells compared to controls. Our global microarray data (Affymetrix chip HG U133 plus 2.0, n = 3) reveals a set of 113 differentially expressed genes preferentially detectable in 5azaD/TSA expanded cells associated with transplantable HSC expansion in culture and 278 HSC maintenance genes differentially expressed in CD34+ cells expanded in VPA or 5azaD/TSA, but not in control. Differential expression of genes representative of HSC expansion or maintenance gene sets were validated by real time qPCR. Intriguingly, functional pathway analyses of the 113 HSC expansion genes indicate the importance of inflammation signaling pathways (genes such as S100A8, Alox5). Furthermore, global genomic DNA methylation analysis of uncultured vs expanded, enriched CD34+ cells by LINE-1 methylation assay indicates transient hypomethylation of 5azaD/TSA-expanded cells at day 3 (55.4 ± 2.2 vs pre–treatment levels 77.3 ± 1.9) which later returned towards baseline (69.7 ± 2.1), corresponding with recovery of DNMT1 expression. Notably, control cultures or CD34+ cells expanded in VPA do not display changes in LINE-1 methylation (78.5 ± 2.2 and 77.2 ± 2 respectively, n = 2, mean % ± SE of 4 CpG sites). Interestingly, ELISA assays of conditioned medium from 5azaD/TSA-expanded cells reveals that levels of the inflammatory mediator leukotriene B4 (LTB4; synthesized from arachidonic acid by the Alox5 gene products, 5-lipooxygenase and LTA4 hydrolase) are 426.87 ± 101.7 pg/ml, significantly greater than that of control cultures (109.5 ± 11.6 pg/ml; n = 3, p = 0.03). VPA-treated cultures had slightly higher levels than controls (346.8 ± 138.8 pg/ml; p = 0.16). Similarly 5azaD/TSA IL-1β levels were significantly greater than controls (10.1 ± 0.8 pg/ml vs 6.0 ± 0.6 pg/ml, n= 2, triplicate wells, p = 0.05). IL-1β levels from VPA cultures were 10.1 ± 4.5 pg/ml (p = n.s.). Notably, expansion of CD34+CD90+ cells in the presence of 5azaD/TSA is reduced by 47.6% by addition of dexamethasone, an anti-inflammatory agent, without affecting total cell numbers. Furthermore, demethylation of CpG sites of the promoters of inflammatory/stress response genes correspond with higher expression of these genes in CMA-expanded cultures (e.g., S100A8 and Cyp11A1; pre treatment: 76.42% ± 4.91% and 80.73 % ± 4.16%, post 5azaD/TSA treatment 54.5% ± 3.84% and 54.29% ± 3.72% respectively; n = 2, mean ± SE of 3 to 11 CpG sites methylation). S100A8 is a known agonist of toll-like receptor 4, which plays a role in the maintenance and proliferation of endothelial progenitor cells, in keeping with the influence of systemic infection on HSC cycling and of interferon on HSC proliferation. Our studies, for the first time, identify inflammatory molecules regulated by epigenetic mechanisms that are potentially important in governing HSC fate choices. Further studies should determine the mechanisms by which CMAs influence HSC expansion or maintenance through inflammatory and other stress response pathway genes. Disclosures:No relevant conflicts of interest to declare.

Chromatin-Modifying Agents Reactivate Embryonic Renal Stem/Progenitor Genes in Human Adult Kidney Epithelial Cells but Abrogate Dedifferentiation and Stemness

Recent studies have suggested that epigenetic modulation with chromatin-modifying agents can induce stemness and dedifferentiation and increase developmental plasticity. For instance, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has been shown to promote self-renewal/expansion of hematopoietic stem cells and facilitate the generation of induced pluripotent stem cells (iPSCs). Previously, we observed that downregulation of embryonic renal stem/progenitor genes in the adult kidney was associated, at least in part, with epigenetic silencing. Therefore, we hypothesized that VPA may alter the expression of these genes and reprogram mature human adult kidney epithelial cells (hKEpCs) to a stem/progenitor-like state. Here, using quantitative RT-PCR and flow cytometry [fluorescence-activated cell sorting (FACS)] analysis, we show in VPA-treated primary cultures of human adult and fetal kidney significant reinduction of the renal stem/progenitor markers SIX2, OSR1, SALL1, NCAM, and PSA-NCAM. Robust SIX2 mRNA re-expression was confirmed at the protein level by western blot and was associated with epigenetic changes of the histones at multiple sites of the SIX2 promoter leading to gene activation, significantly increased acetylation of histones H4, and methylation of lysine 4 on H3. Furthermore, we could demonstrate synergistic effects of VPA and Wnt antagonists on SIX2 and also OSR1 reinduction. Nevertheless, VPA resulted in upregulation of E-CADHERIN and reduction in VIMENTIN, preventing the skewing of hKEpCs towards a more replicative mesenchymal state required for clonogenic expansion and acquisition of stem cell characters, altogether inducing cell senescence at early passages. These results demonstrating that chromatin-modifying agents prevent dedifferentiation of hKEpCs have important clinical implications as they may limit ex-vivo self-renewal/expansion and possibly the in vivo renal regenerative capacity initiated by dedifferentiation.

Histone Acetylation-Mediated Regulation of the Hippo Pathway

The Hippo pathway is a signaling cascade recently found to play a key role in tumorigenesis therefore understanding the mechanisms that regulate it should open new opportunities for cancer treatment. Available data indicate that this pathway is controlled by signals from cell-cell junctions however the potential role of nuclear regulation has not yet been described. Here we set out to verify this possibility and define putative mechanism(s) by which it might occur. By using a luciferase reporter of the Hippo pathway, we measured the effects of different nuclear targeting drugs and found that chromatin-modifying agents, and to a lesser extent certain DNA damaging drugs, strongly induced activity of the reporter. This effect was not mediated by upstream core components (i.e. Mst, Lats) of the Hippo pathway, but through enhanced levels of the Hippo transducer TAZ. Investigation of the underlying mechanism led to the finding that cancer cell exposure to histone deacetylase inhibitors induced secretion of growth factors and cytokines, which in turn activate Akt and inhibit the GSK3 beta associated protein degradation complex in drug-affected as well as in their neighboring cells. Consequently, expression of EMT genes, cell migration and resistance to therapy were induced. These processes were suppressed by using pyrvinium, a recently described small molecule activator of the GSK 3 beta associated degradation complex. Overall, these findings shed light on a previously unrecognized phenomenon by which certain anti-cancer agents may paradoxically promote tumor progression by facilitating stabilization of the Hippo transducer TAZ and inducing cancer cell migration and resistance to therapy. Pharmacological targeting of the GSK3 beta associated degradation complex may thus represent a unique approach to treat cancer.

The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

Quantifying the contribution of chromatin dynamics to stochastic gene expression reveals long, locus-dependent periods between transcriptional bursts

BackgroundA number of studies have established that stochasticity in gene expression may play an important role in many biological phenomena. This therefore calls for further investigations to identify the molecular mechanisms at stake, in order to understand and manipulate cell-to-cell variability. In this work, we explored the role played by chromatin dynamics in the regulation of stochastic gene expression in higher eukaryotic cells.ResultsFor this purpose, we generated isogenic chicken-cell populations expressing a fluorescent reporter integrated in one copy per clone. Although the clones differed only in the genetic locus at which the reporter was inserted, they showed markedly different fluorescence distributions, revealing different levels of stochastic gene expression. Use of chromatin-modifying agents showed that direct manipulation of chromatin dynamics had a marked effect on the extent of stochastic gene expression. To better understand the molecular mechanism involved in these phenomena, we fitted these data to a two-state model describing the opening/closing process of the chromatin. We found that the differences between clones seemed to be due mainly to the duration of the closed state, and that the agents we used mainly seem to act on the opening probability.ConclusionsIn this study, we report biological experiments combined with computational modeling, highlighting the importance of chromatin dynamics in stochastic gene expression. This work sheds a new light on the mechanisms of gene expression in higher eukaryotic cells, and argues in favor of relatively slow dynamics with long (hours to days) periods of quiet state.

Histone deacetylase inhibitors (HDACIs): multitargeted anticancer agents

Histone deacetylase (HDAC) inhibitors are an emerging class of therapeutics with potential as anticancer drugs. The rationale for developing HDAC inhibitors (and other chromatin-modifying agents) as anticancer therapies arose from the understanding that in addition to genetic mutations, epigenetic changes such as dysregulation of HDAC enzymes can alter phenotype and gene expression, disturb homeostasis, and contribute to neoplastic growth. The family of HDAC inhibitors is large and diverse. It includes a range of naturally occurring and synthetic compounds that differ in terms of structure, function, and specificity. HDAC inhibitors have multiple cell type-specific effects in vitro and in vivo, such as growth arrest, cell differentiation, and apoptosis in malignant cells. HDAC inhibitors have the potential to be used as monotherapies or in combination with other anticancer therapies. Currently, there are two HDAC inhibitors that have received approval from the US FDA for the treatment of cutaneous T-cell lymphoma: vorinostat (suberoylanilide hydroxamic acid, Zolinza) and depsipeptide (romidepsin, Istodax). More recently, depsipeptide has also gained FDA approval for the treatment of peripheral T-cell lymphoma. Many more clinical trials assessing the effects of various HDAC inhibitors on hematological and solid malignancies are currently being conducted. Despite the proven anticancer effects of particular HDAC inhibitors against certain cancers, many aspects of HDAC enzymes and HDAC inhibitors are still not fully understood. Increasing our understanding of the effects of HDAC inhibitors, their targets and mechanisms of action will be critical for the advancement of these drugs, especially to facilitate the rational design of HDAC inhibitors that are effective as antineoplastic agents. This review will discuss the use of HDAC inhibitors as multitargeted therapies for malignancy. Further, we outline the pharmacology and mechanisms of action of HDAC inhibitors while discussing the safety and efficacy of these compounds in clinical studies to date.

Evaluation of Genotoxicity of Chromatin Modifying Agents Expanded Hematopoietic Graft in a Non-Human Primate Model.

AbstractAbstract 2994▪▪This icon denotes a clinically relevant abstractThe suboptimal numbers of hematopoietic stem cells (HSC) present in human umbilical cord blood (CB) grafts results in a significant delay in blood cell reconstitution or graft failure, particularly in adult patients following transplantation. Using chromatin modifying agents (CMAs), including 5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA), we developed an ex-vivo expansion strategy that permits 7-fold expansion of transplantable HSC (Araki et al. Blood 2007, Exp Hematol 2009). Here we performed validation studies in non-human primate (NHP) baboons to examine the potential genotoxicity of CMA-expanded bone marrow (BM) graft. To our knowledge, there have been no reports suggesting 5azaD exacerbate chromosomal instability in human. We have demonstrated that the sequential addition of low-dose 5azaD (5 micromolar) and TSA (5 ng/ml) to human CB CD34+ cells does not result in a persistent depletion of Dnmt1 transcripts and protein levels. In this global methylation analysis using a LINE-1 assay indicates that DNA demethylation, which occurs with CMA treatment in culture, is transient: LINE-1 methylation of CD34+ cells in the absence or presence of CMA was 78.5% ± 2.2% and 55.4 % ± 2.2% respectively at day 3 while methylation levels of treated CD34+ cells returns to 69.7% ± 2.1% by day 9. In an attempt to evaluate the genotoxic potential of low-dose 5azaD/TSA, we transplanted baboons with CMA-expanded autologous BM grafts following a myeloablative dose of IV busulfan (4 mg/kg/day × 4 days). Although the initial pre-expansion CD34+ cell dose of CMA-expanded BM grafts was low (0.68, 2.64 and 5.18 × 106 CD34+ cells/kg respectively), all three baboons engrafted following transplantation. Following peripheral blood (PB) cell count recovery we examined BM CD34 and colony-forming cell (CFC) counts. Of note, the absolute number of CD34+ cells in the BM following hematopoietic reconstitution (926.0 ± 142.8 /microliter) was 3.5 fold higher than pre-transplant levels (257.9 ± 56.2 /microliter). However, the absolute number of CFC in the BM was 2.2 fold lower than pre-transplant levels. BM biopsies performed on the 3 baboons serially prior to, and 56, 120 and 400 days post-transplant respectively to examine hematopoietic recovery, myeloid-to-erythroid ratio and cellularity did not demonstrate myelodysplasia or malignancy. There was tri-lineage hematopoiesis evident in the BM in all 3 baboons with 50 to 70% cellularity. Further evaluation of the safety of 5azaD/TSA-expanded grafts was performed using three independent methods. Firstly, 150 NOD/SCID mice who received 5azaD/TSA expanded human CB grafts have not displayed any signs of tumor formation after primary or secondary transplantation. Secondly, karyotyping of CMA-expanded human CB graft or baboon CD34+ cells prior to and following transplant (day 266) reveals no detectable chromosomal anomalies. Thirdly, we used Flow-FISH, an in situ flow cytometry-based assay to measure relative telomere lengths (RTL) of purified CD11a+ (97.59% ± 0.38%) leukocytes from baboon BM prior to and following transplantation. The majority of CD11a+ leukocytes are short-lived which indirectly reflects the telomere lengths of the respective HSC from which they are derived. Our results show that the RTL of Jurkat cells were 10.6% ± 0.37%, while human CB cell RTLs were 19.7% ± 1.2%. The pre- and post-transplant (day 56) BM cells of one baboon (PA7619) displayed (average) RTL values of 13.48% ± 0.76 % and 13.96% ± 0.68% respectively. In another animal (PA7963), average RTL values were determined serially using Flow-FISH on BM CD11a+ enriched cells at multiple time-points post-transplant up to day 417 which displayed mean RTL of 17.66 % ± 0.52% while pre-transplant RTL was 15.39% ± 0.62%. In summary, the mean RTL values of baboon leukocytes were stable and post-transplant values were comparable to pre-transplant levels. Importantly, year-long monitoring shows no evidence of hematologic malignancies as determined by PB counts, BM examination (including cytogenetics) and measurements of leukocyte RTL values in a clinically relevant NHP model. These studies offer preliminary evidence that CMA-mediated expansion of CD34+ cells transferred into primates (and human CB CD34+ cells transferred into mice) is not genotoxic and does not induce hematopoietic malignancies, of relevance to use of human CMA-expanded CB grafts in clinical trials. Disclosures:No relevant conflicts of interest to declare.

Histone Deacetylase Inhibitors Promote the Ex Vivo Expansion of Cord Blood CD34+ Cells in Serum Free Cultures Accompanied by the Upregulation of Pluripotency Genes

AbstractAbstract 345Previously, we have reported that various chromatin modifying agents(CMA) can epigenetically alter HSC fate decisions resulting in increased numbers of cord blood(CB)- SCID repopulating cells(SRC) and/or erythroid progenitor cells depending upon the CMA utilized (Blood. 2011,117(17):4632–41). We have hypothesized that treatment of CB CD34+ cells with histone deacetyalse inhibitors(HDACIs) might expand the numbers of CB-SRC and that such cell products could be used as allogeneic stem cell grafts. We developed serum free(SF) and containing(SC) culture conditions and assessed the ability of several HDACIs including valproic acid(VPA), scriptaid(SCR) and CAY10433(C433) to expand CB-SRC Each of these HDACIs were shown to inhibit both class I and II HDACs. The addition of VPA to SF cultures led to a >6,000 fold expansion of CD34+ cells and >25,000-fold expansion of CD34+CD90+ cells as compared to primary cells(PC). By contrast, SC cultures resulted in only a 54-fold expansion of CD34+ cells and a 75-fold expansion of CD34+90+ cells as compared to PC in the presence of VPA.In order to examine the functional characteristics of HDACIs treated CB-CD34+ cells in SF cultures we evaluated aldehyde dehydrogenase activity(ALDH) in these expanded CD34+ cells. A far greater proportion of CD34+ cells were ALDH+ following treatment with VPA in SF cultures as compared to SC cultures (82.2±8.6%, versus 37.0±0.9%). By contrast incubation in the SC cultures induced the generation of greater proportion of ALDH-CD34+ cells as compared to SF cultures (27.0±5.0% versus 3.7±2.0%). These findings indicate that the inclusion of serum to such cultures favors HSC differentiation and eventual HSC depletion while SF conditions favor expansion of more primitive HPC. Purified ALDH+CD34+ cells from SF cultures supplemented with HDACIs contained far greater absolute numbers of BFU-E and CFU-Mix (8.4×107±6.7×107/CB collection) as compared to SC cultures (1.4×107±8.8×106/CB collection; One way ANOVA p=.02). These data indicate that based upon their degree of ALDH activity and functional potential different phenotypically and functionally defined subsets of CD34+ cells are generated in SF as compared to SC cultures.In order to further examine the effects of VPA on the pluripotency of expanded CD34+ cells we examined the expression of Sox2, Oct4 and Nanog in CD34+ cells re-isolated following 7 days of treatment with HDACIs in SF and SC cultures. RNA and protein expression levels of Sox2, Oct4 and Nanog genes were greater in cells generated in SF cultures supplemented with HDACIs than that observed in CD34+ cells cultured in SC. Sox2 and Oct4 bind to the promoter of Nanog and up-regulate Nanog expression. In the SF but not SC cultures VPA treatment resulted in the up-regulation of Nanog indicating the functionality of the Sox2/Oct4 interactions. We also documented using confocal microscopy the elevated expression of the proteins derived from these pluripotency genes both in the cytoplasm and nucleus of CD34+ cells re-isolated after a week of treatment with each of the HDACIs.The functional potential of the re-isolated CD34+ cells following a week of treatment with VPA in SF medium was evaluated by transplanting the expanded cells into NOD/SCID/gamma(c)null mice. SRC capable of multi-lineage hematopoietic differentiation were detected in the marrow of recipients of VPA treated grafts 13-weeks after transplantation (%CD45, 33.5±13.4% with VPA treated grafts versus 13.0±7.7% with grafts generated in the presence of CA). Bone marrow cells from primary recipients receiving HDACI treated CD34+ cells grafts were also capable of generating greater numbers of cells belonging to myeloid and lymphoid lineages(%CD45, 3.3±1.0%) as compared to grafts receiving CA(%CD45, 0.65±0.16%) 15 weeks after their transplantation into secondary recipients.These data indicate that inhibitors of class I and II HDACs under SF culture conditions provide an effective pharmacological approach which can be utilized to promote the ex vivo expansion of functional CB-SRC which are characterized by high ALDH activity and expression of a variety of pluripotency genes. These findings indicate the importance of epigenetic events in determining phenotype and function of dividing SRC in vitro. Disclosures:Hoffman:NYSTEM: Research Funding.

Rules of engagement for base excision repair in chromatin

Most of the DNA in eukaryotes is packaged in tandemly arrayed nucleosomes that, together with numerous DNA- and nucleosome-associated enzymes and regulatory factors, make up chromatin. Chromatin modifying and remodeling agents help regulate access to selected DNA segments in chromatin, thereby facilitating transcription and DNA replication and repair. Studies of nucleotide excision repair (NER), single strand break repair (SSBR), and the homology-directed repair (HDR), and non-homologous end-joining (NHEJ) double strand break repair pathways have led to an "access-repair-restore" paradigm, in which chromatin in the vicinity of damaged DNA is disrupted, thereby enabling efficient repair and the subsequent repackaging of DNA into nucleosomes. When damage is extensive, these repair processes are accompanied by cell cycle checkpoint activation, which provides cells with sufficient time to either complete the repair or initiate apoptosis. It is not clear, however, if base excision repair (BER) of the ~20,000 or more oxidative DNA damages that occur daily in each nucleated human cell can be viewed through this same lens. Until recently, we did not know if BER requires or is accompanied by nucleosome disruption, and it is not yet clear that anything short of overwhelming oxidative damage (resulting in the shunting of DNA substrates into other repair pathways) results in checkpoint activation. This review highlights studies of how oxidatively damaged DNA in nucleosomes is discovered and repaired, and offers a working model of events associated with BER in chromatin that we hope will have heuristic value.