Targeting Epigenetic Changes Research Articles

Targeting Epigenetic Changes Mediated by Members of the SMYD Family of Lysine Methyltransferases

A comprehensive understanding of the mechanisms involved in epigenetic changes in gene expression is essential to the clinical management of diseases linked to the SMYD family of lysine methyltransferases. The five known SMYD enzymes catalyze the transfer of donor methyl groups from S-adenosylmethionine (SAM) to specific lysines on histones and non-histone substrates. SMYDs family members have distinct tissue distributions and tissue-specific functions, including regulation of development, cell differentiation, and embryogenesis. Diseases associated with SMYDs include the repressed transcription of SMYD1 genes needed for the formation of ion channels in the heart leading to heart failure, SMYD2 overexpression in esophageal squamous cell carcinoma (ESCC) or p53-related cancers, and poor prognosis associated with SMYD3 overexpression in more than 14 types of cancer including breast cancer, colon cancer, prostate cancer, lung cancer, and pancreatic cancer. Given the importance of epigenetics in various pathologies, the development of epigenetic inhibitors has attracted considerable attention from the pharmaceutical industry. The pharmacologic development of the inhibitors involves the identification of molecules regulating both functional SMYD SET (Suppressor of variegation, Enhancer of Zeste, Trithorax) and MYND (Myeloid-Nervy-DEAF1) domains, a process facilitated by available X-ray structures for SMYD1, SMYD2, and SMYD3. Important leads for potential pharmaceutical agents have been reported for SMYD2 and SMYD3 enzymes, and six epigenetic inhibitors have been developed for drugs used to treat myelodysplastic syndrome (Vidaza, Dacogen), cutaneous T-cell lymphoma (Zoinza, Isrodax), and peripheral T-cell lymphoma (Beleodag, Epidaza). The recently demonstrated reversal of SMYD histone methylation suggests that reversing the epigenetic effects of SMYDs in cancerous tissues may be a desirable target for pharmacological development.

Abstract LB-167: Novel LSD-1 inhibitor validation in new established clinically PFA patient-derived orthotopic xenograft (PDOX) models of ependymoma

Abstract Background: Ependymoma (EPN) is one of the most malignant tumors and surgical resection is the mainstay of treatment followed by re-irradiation and chemotherapy. However the benefit of this modality have not been clearly demonstrated. Posterior fossa A (PFA) EPN have the worst prognosis and driven by epigenetic changes, suggesting targeting epigenetic changes in PFA EPN can potentially be effective. The limited availability of a reliable translational platform has hampered efforts to understand tumor biology and the preclinical and clinical trials for EPN. In this study, we established a series of orthotopic EPN xenograft mouse models from fresh surgical specimens. We also examined the therapeutic efficacy of SYC-836, a novel LSD-1 inhibitor developed at Baylor College of Medicine, both in vitro and in vivo in these PDOX models. Material and Methods: Clinically and pathologically available PFA EPN samples were obtained from patients who had treatment in Texas Children's Hospital. Parts of the surgical samples were dissociated into single cells and implanted into the cerebellum of SCID mice (1x105 cells /2 uL FBS). Once tumorigenicity confirmed, xenograft tumor would be serially subtransplanted in mouse brains and be cultured in vitro to develop the permanent cell line. To test if xenograft tumors precisely reflect morphologic and pathologic characteristics of their parental tumors, we compared histology, and several key parameters in invasiveness, growth rate and microvessel density by IHC. To examine in vitro anti-tumor activities, primary cultured cells from an established PDOX model of PFA EPN (4423EPN) were subjected to SYC-836 at various concentrations (0-25uM). Cell viability was measured at 5 different time points over 14 days. To validate the drug's in vivo efficacy, two established PFA EPN PDOX models, 4423EPN and 2002EPN, were utilized. 40 mice per model were implanted and divided into 4 treatment groups: 1) control , 2) radiation/standard therapy (2 Gy XRT x 5 days), 3) SYC-836 only (15mg/kg IP x 28 days), and 4) combination (radiation + SYC-836 per regimen above). Animal survival times were analyzed using log rank analysis. Results: By now we have successfully developed 6 intracerebellum xenografts. It has since been serially subtransplanted in mouse for more than 3 generations and can be cryopreserved for long-term maintenance of tumorigenicity. We showed xenograft tumors precisely replicated morphologic and pathologic characteristics of their parental tumors. SYC-836 demonstrated effective cell killing in vitro in both time- and dose-dependent manner. IC50 was ~7.5uM. In vivo experiment was completed in 2 PFA EPN PDOX models. There were no survival benefit with either XRT only or SYC-836 only compared to control group; however, the combination treatment lead to significant improvement in animal survival. SYC-836 was well tolerated in mice. Conclusion: We have successfully established a series of clinically orthotopic EPN xenograft models and they well-matched with those of parental EPNs both preclinically and clinically. Our data showed that combining SYC-836 with radiation synergistically prolongs animal survival significantly, suggesting that SYC-836 may have a role in the clinical setting by either reducing radiation dosages, or be a potential adjuvant agent to other chemotherapy drugs in treatment for EPN. Citation Format: Huiyuan Zhang, Sibo Zhao, Yuchen Du, Lin Qi, Frank K. Braun, Mari Kogiso, Holly B. Lindsay, Sarah G. Injac, Laszlo Perlaky, Patricia Baxter, Jack M. Su, Xiao-Nan Li. Novel LSD-1 inhibitor validation in new established clinically PFA patient-derived orthotopic xenograft (PDOX) models of ependymoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-167.

Coumestrol Epigenetically Suppresses Cancer Cell Proliferation: Coumestrol Is a Natural Haspin Kinase Inhibitor.

Targeting epigenetic changes in gene expression in cancer cells may offer new strategies for the development of selective cancer therapies. In the present study, we investigated coumestrol, a natural compound exhibiting broad anti-cancer effects against skin melanoma, lung cancer and colon cancer cell growth. Haspin kinase was identified as a direct target protein of coumestrol using kinase profiling analysis. Histone H3 is a direct substrate of haspin kinase. We observed haspin kinase overexpression as well as greater phosphorylation of histone H3 at threonine 3 (Thr-3) in the cancer cells compared to normal cells. Computer modeling using the Schrödinger Suite program identified the binding interface within the ATP binding site. These findings suggest that the anti-cancer effect of coumestrol is due to the direct targeting of haspin kinase. Coumestrol has considerable potential for further development as a novel anti-cancer agent.

Role of methionine on epigenetic modification of DNA methylation and gene expression in animals

DNA methylation is one of the main epigenetic phenomena affecting gene expression. It is an important mechanism for the development of embryo, growth and health of animals. As a key nutritional factor limiting the synthesis of protein, methionine serves as the precursor of S-adenosylmethionine (SAM) in the hepatic one-carbon metabolism. The dietary fluctuation of methionine content can alter the levels of metabolic substrates in one-carbon metabolism, e.g., the SAM, S-adenosylhomocysteine (SAH), and change the expression of genes related to the growth and health of animals by DNA methylation reactions. The ratio of SAM to SAH is called ‘methylation index’ but it should be carefully explained because the complexity of methylation reaction. Alterations of methylation in a specific cytosine-guanine (CpG) site, rather than the whole promoter region, might be enough to change gene expression. Aberrant methionine cycle may provoke molecular changes of one-carbon metabolism that results in deregulation of cellular hemostasis and health problems. The importance of DNA methylation has been underscored but the mechanisms of methionine affecting DNA methylation are poorly understood. Nutritional epigenomics provides a promising insight into the targeting epigenetic changes in animals from a nutritional standpoint, which will deepen and expand our understanding of genes, molecules, tissues, and animals in which methionine alteration influences DNA methylation and gene expression.

Abstract 5058: Lysine specific demethylase-1 (LSD-1) Inhibitor SYC-836 in combination with radiation prolongs animal survival in patient-derived posterior fossa ependymoma xenograft mouse models

Abstract Background: Ependymoma (EPN) is the third most common malignant pediatric brain tumor. Current standard therapy include maximally safe surgical resection followed by radiation and lead to a 5-year overall survival of 50-71%. Recent molecular subgrouping of EPN has identified one group, posterior fossa A (PFA), which accounts for 45% of all EPN cases, to have one of the worst prognosis and it is driven by epigenetic changes, suggesting targeting epigenetic changes in PFA EPN can potentially be effective. In this study, we examined the therapeutic efficacy of SYC-836, a novel LSD-1 inhibitor compound developed at Baylor College of Medicine, both in vitro and in vivo in PDOX models of posterior fossa EPN. Methods: To examine in vitro anti-tumor activities, paired primary cultured cells (both as attached cells and neurospheres) from an established PDOX model of posterior fossa EPN (ICb-4423EPN) were subjected to SYC-836 at various concentrations (0-25uM). Cell viability and proliferation were measured using Cell Counting Kit-8 assay at 5 different time points over 14 days. To validate the drug’s in vivo efficacy, two established posterior fossa EPN PDOX models, ICb-4423EPN and ICb-2002EPN, were utilized. 40 eight weeks old SCID mice per model were implanted with tumor cells. They were divided into 4 treatment groups (10 mice/group) each: 1) control (DPBS, 10uL/kg IP daily x 28 days), 2) radiation/standard therapy (2 Gy focal XRT daily x 5 days), 3) SYC-836 only (15mg/kg IP daily x 28 days), and 4) combination (radiation + SYC-836 per regimen above). Animal survival times were analyzed using log rank analysis. Changes of histone lysine methylation were examined through western hybridization. Results: SYC-836 demonstrated effective cell killing in vitro against both attached and neurosphere cultured cells in both time- and dose-dependent manner. IC50 was ~7.5uM. In vivo experiment was completed in 1 of the 2 EPN PDOX models (ICb-2002EPN) with the second model ongoing. Median survival times for each group is as followed: control 136 days, radiation 148 days, SYC-836 only 136 days, combination 180 days. There were no survival benefit with either XRT only (P=0.205) or SYC-836 only (P=0.186) when compared to the control group; however, when used in combination, the treatment strategy lead to significant improvement in animal survival (P=0.004). SYC-836 was well tolerated in mice. Conclusion: Our data showed that combining SYC-836 with current standard therapy of radiation synergistically prolongs animal survival significantly, although as a single agent SYC-836 was not effective against posterior fossa ependymoma. Our data suggest that SYC-836 may have a role in the clinical setting by either reducing radiation dosages, or be a potential adjuvant agent to other chemotherapy drugs in our treatment approach for ependymoma. Citation Format: Sibo Zhao, Huiyuan Zhang, Lin Qi, Holly Lindsay, Yuchen Du, Mari Kogiso, Frank Braun, Sarah Injac, Laszlo Perlaky, Donald W. Parsons, Murali Chintagumpala, Adekunle Adesina, Yongcheng Song, Xiao-Nan Li. Lysine specific demethylase-1 (LSD-1) Inhibitor SYC-836 in combination with radiation prolongs animal survival in patient-derived posterior fossa ependymoma xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5058. doi:10.1158/1538-7445.AM2017-5058

Repurposing the anti-epileptic drug sodium valproate as an adjuvant treatment for diffuse intrinsic pontine glioma.

Targeting epigenetic changes in diffuse intrinsic pontine glioma (DIPG) may provide a novel treatment option for patients. This report demonstrates that sodium valproate, a histone deacetylase inhibitor (HDACi), can increase the cytotoxicity of carboplatin in an additive and synergistic manner in DIPG cells in vitro. Sodium valproate causes a dose-dependent decrease in DIPG cell viability in three independent ex vivo cell lines. Furthermore, sodium valproate caused an increase in acetylation of histone H3. Changes in cell viability were consistent with an induction of apoptosis in DIPG cells in vitro, determined by flow cytometric analysis of Annexin V staining and assessment of apoptotic markers by western blotting. Subsequently, immunofluorescent staining of neuronal and glial markers was used to determine toxicity in normal rat hippocampal cells. Pre-treatment of cells with sodium valproate enhanced the cytotoxic effects of carboplatin, in three DIPG cell lines tested. These results demonstrate that sodium valproate causes increased histone H3 acetylation indicative of HDAC inhibition, which is inversely correlated with a reduction in cell viability. Cell viability is reduced through an induction of apoptosis in DIPG cells. Sodium valproate potentiates carboplatin cytotoxicity and prompts further work to define the mechanism responsible for the synergy between these two drugs and determine in vivo efficacy. These findings support the use of sodium valproate as an adjuvant treatment for DIPG.

Icariin may benefit the mesenchymal stem cells of patients with steroid-associated osteonecrosis by ABCB1-promoter demethylation: a preliminary study.

In this study, we found out a previously undefined function of icariin which restored the dynamic balance between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) in patients with osteonecrosis of femoral head (ONFH) via ABCB1-promoter demethylation. These findings provided important information regarding potential implication of icariin targeting epigenetic changes for the treatment of steroid -associated ONFH. Here, we investigated whether icariin can also exert a beneficial role in the reactivation of MSCs in the patients with steroid-associated ONFH via ABCB1-promoter demethylation. Bone marrow was collected from the proximal femur in patients with steroid-associated ONFH (n = 20) and patients with new femoral neck fractures (n = 22), and then MSCs were isolated. We investigated cell viability, intracellular reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), P-glycoprotein (P-gp) activity, the transcript levels of ABCB1 and oxidative stress-related genes, methylation extent at CpG islands of ABCB1 promoter, and osteogenic and adipogenic differentiation ability of MSCs from the femoral neck fractures group and from the steroid-associated ONFH group treated with or without icariin. We observed that MSCs from the steroid-associated ONFH group showed reduced proliferation ability, elevated ROS level, depressed MMP, weakened osteogenesis, and enhanced adipogenesis while low P-gp activity, transcription level of ABCB1, and oxidative stress-related genes as well as aberrant CpG islands hypermethylation of ABCB1 were also noted in steroid-associated ONFH group. Treatment with icariin obviously induced de novo P-gp expression, decreased oxidative stress, and promoted osteogenesis. Icariin may be a potential drug targeting epigenetic changes for the treatment of steroid-associated ONFH.

Targeting Epigenetic Changes for the Reprogramming of Vascular Walls in Pulmonary Arterial Hypertension, the Role of Histone Deacetylases and their Inhibitors

Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vasculature as defined by an ele vated pulmonary vascular resistance (PVR) leading to a failure of the right heart and ultimately death [1]. Although the overall 5-year survival for PAH has significantly increased in the past years, none of the approved therapies have shown an ability to cure the disease. Current available treatments are limited, expensive, and often associated with significant, side effects. As such, furthering our understanding of the mechanisms underlying PAH development is important to improve patient management and outcomes. A number of evidence have shown that cellular and molecular changes occur in the pulmonary arteries in response to chronic hypoxic exposure [1,2], and primary lung diseases including chronic obstructive pulmonary disease, cystic fibrosis, etc. are associated with the presence of chronic hypoxia. In PAH, the pulmonary arteries (PAs) exhibit pathological proliferative vascular remodeling related to medial and adventitial thickening of pulmonary arterial walls. The excessive proliferation of smooth muscle cells as well as the accumulation of fibroblasts and myofibroblasts plays an important role in the medial and adventitial thickening respectively. Epigenetics describes that heritable changes in gene expression or cellular phenotype are caused by mechanisms other than changes in the underlying DNA sequence. Histone modification is one of the predominant epigenetic phenomena. It selectively modulates gene expressions that control many biological processes including cell proliferation. Histone deacetylases (HDACs) remove an acetyl group from lysine residues of target proteins to regulate a number of cellular processes. There are 18 HDAC enzymes which are identified and grouped into four classes. Class I, II, and IV share sequence similarity and are dependent on Zn2+ for enzymatic activity, whereas the class III sirtuins act through a distinct NAD+-dependent mechanism. Pulmonary vascular remodeling (PAR) is a characteristic pathological change of PAH. The development of PAR involves all layers of the vessel wall. It has been reported that adventitial fibroblasts in the pulmonary hypertensive vessel wall exhibit a hyper proliferative, inflammatory, and invasive phenotype. Hypoxia-induced PAR is characterized by the emergence of a distinct adventitial fibroblast population. This population exhibits a constitutively activated, pro-inflammatory phenotype that is capable of inducing recruitment, retension and pro-inflammatory activation of monocytes/microphages. Stenmark’s group has recently demonstrated that pulmonary adventitial fibroblasts from chronically hypoxic hypertensive calves (PH-Fibs) expressed a constitutive and persistent proinflammatory phenotype. The proinflammatory phenotype of PH-Fibs was associated with epigenetic alterations as demonstrated by increased activity of HDACs. Importantly, a reversal of this proinflammatory phenotype can be achieved with the use of class I HDAC inhibitors [3]. Another hallmark feature of PAR is excessive smooth muscle cell proliferation leading to medial thickening. In our high altitude longterm (LTH) sheep model, an obvious pulmonary arterial remodeling was observed. We found that the level of global histone acetylation was significantly decreased in LTH fetus PAs, and it was accompanied by the loss of the cyclin-dependent kinase inhibitor, p21, as opposed to the control. The treatment of the pulmonary arterial smooth cells (PASMC), isolated from LTH fetal lung, with a class I HDAC inhibitor markedly decreased their proliferation rate in part due to an increased expression of p21 at both the RNA and protein level. Interestingly, a class II HDAC inhibitor also showed an inhibitory effect on cell proliferation when used with a relative higher concentration. However, a class III inhibitor exhibited no effect on cell proliferation [4]. These findings suggested that epigenetic alterations caused by chronic hypoxia led to fetal PASMC proliferation and vessel remodeling as associated with vascular proliferative disease, and that this process is regulated by p21. This is consistent with a previous study which showed that a p53 gene deficiency decreased p21 expression levels that eventually resulted in hypoxia-induced PAH and vascular remodeling in mice [5]. The above studies correlate with in vivo data showing that small molecules (MGCD0103 and MS-275) that selectively block class I HDACs reduce hypoxia-induced PAH in a manner that associates with a blunted medial thickening of PAs and a reduced proliferation of PASMC in these vessels [6]. These findings support a unique role for HDACs as important targets for PAH, and highlight the potential of isoform-specific HDAC inhibitors for the treatment of cardiovascular diseases.

Enhanced Cytotoxic Effects of Combined Valproic Acid and the Aurora Kinase Inhibitor VE465 on Gynecologic Cancer Cells

Increasing evidence shows that targeting epigenetic changes including acetylation and deacetylation of core nucleosomal histones as well as Aurora kinases hold promise for improving the treatment of human cancers including ovarian cancer. We investigated whether the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), and the Aurora kinase inhibitor VE465 can have additive or synergistic effects on gynecologic cancer cells. We tested the in vitro antitumor activity of VPA and VE465, alone and in combination, in gynecologic cancer cells and assessed potential mechanisms of action. 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) analysis revealed that 72 h of treatment with VPA or VE465 alone induced dose-dependent cytotoxic effects in nine gynecologic cancer cell lines (ovarian: 2008/C13, OVCAR3, SKOV3, and A2780; cervical: ME180 and CaSki; endometrial: HEC-1B; and uterine sarcoma: MES-SA and MES-SA/D×5). Co-treatment with VPA and VE465 enhanced cytotoxic effects on five of these cell lines: ovarian: 2008/C13, A2780, and OVCAR3; endometrial: HEC-1B; and cervical: ME180. In ovarian 2008/C13 cells, co-treatment with VPA (2 mM) and VE465 (1 μM) induced more apoptosis than either VPA or VE465 alone. Western blot analysis showed that VPA alone increased the expression of cleaved PARP and p21 in a dose-dependent manner in 2008/C13 cells, while co-treatment with VPA and VE465 induced more cleaved PARP than treatment with VPA or VE465 alone did. The combined use of VPA and VE465 enhanced cytotoxic effects in some ovarian cancer cells, via enhanced induction of apoptosis. Targeting epigenetics with the HDAC inhibitor, in combination with Aurora kinase inhibitors, holds promise for more effective therapy of ovarian cancer.

Epigenetic Changes in Mitochondrial Superoxide Dismutase in the Retina and the Development of Diabetic Retinopathy

OBJECTIVETo investigate the role of epigenetic regulation of the manganese superoxide dismutase gene (sod2) in the development of diabetic retinopathy and the metabolic memory phenomenon associated with its continued progression after hyperglycemia is terminated.RESEARCH DESIGN AND METHODSStreptozotocin-induced diabetic rats were maintained in poor glycemic control (PC, GHb ∼12%) or in good glycemic control (GC, GHb ∼7.0%) for 4 months, or were allowed to maintain PC for 2 months, followed by GC for 2 additional months (PC-Rev). For experimental galactosemia, a group of normal rats were fed a 30% galactose diet for 4 months or for 2 months, followed by a normal diet for 2 additional months. Trimethyl histone H4 lysine 20 (H4K20me3), acetyl histone H3 lysine 9 (H3K9), and nuclear transcriptional factor NF-κB p65 and p50 at the retinal sod2 promoter and enhancer were examined by chromatin immunoprecipitation.RESULTSHyperglycemia (diabetes or galactosemia) increased H4K20me3, acetyl H3K9, and NF-κB p65 at the promoter and enhancer of retinal sod2, upregulated protein and gene expression of SUV420h2, and increased the interactions of acetyl H3K9 and NF-κB p65 to H4K20me3. Reversal of hyperglycemia failed to prevent increases in H4K20me3, acetyl H3K9, and NF-κB p65 at sod2, and sod2 and SUV420h2 continued to be abnormal. Silencing SUV420h2 by its small interfering RNA in retinal endothelial cells prevented a glucose-induced increase in H4K20me3 at the sod2 enhancer and a decrease in sod2 transcripts.CONCLUSIONSIncreased H4K20me3 at sod2 contributes to its downregulation and is important in the development of diabetic retinopathy and in the metabolic memory phenomenon. Targeting epigenetic changes may serve as potential therapeutic targets to retard the development and progression of diabetic retinopathy.

Valproate synergizes with purine nucleoside analogues to induce apoptosis of B‐chronic lymphocytic leukaemia cells

Resistance to chemotherapy and drug toxicity are two major concerns of chronic lymphocytic leukaemia (B-CLL) treatment by purine nucleoside analogues (PNA, i.e. fludarabine and cladribine). We hypothesized that targeting epigenetic changes might address these issues and evaluated the effect of the histone deacetylase inhibitor valproate (VPA) at a clinically relevant concentration. VPA acted in a highly synergistic/additive manner with fludarabine and cladribine to induce apoptosis of B-CLL cells. Importantly, VPA also restored sensitivity to fludarabine in B cells from poor prognosis CLL patients who became resistant to chemotherapy. Mechanism of apoptosis induced by VPA alone or combined with fludarabine or to cladribine was caspase-dependent and involved the extrinsic pathway. VPA, but neither fludarabine nor cladribine, enhanced the production of reactive oxygen species (ROS) and inhibition of ROS with N-acetylcysteine decreases apoptosis of CLL cells. VPA stimulates hyperphosphorylation of p42/p44 ERK, cytochrome c release and overexpression of Bax and Fas. Together, our data indicate that VPA may ameliorate the outcome of PNA-based therapeutic protocols and provide a potential alternative treatment in both the relapsed and front-line resistant patients and in patients with high risk features.

Role of RNA in the regulation of gene expression

The recent elucidation of the role of small RNAs in controlling gene expression, which was awarded the Nobel Prize in 2006, generated a significant paradigm shift in molecular biology. While the majority of work to date has centered on the role of small RNAs in modulating post-transcriptional silencing (i.e., the targeted degradation of mRNAs), there is a growing and relatively unrecognized field of study in which small RNAs have been shown to suppress gene expression at the transcriptional level (i.e., through targeting epigenetic changes to gene promoter loci).1 First discovered in plants and later defined in yeast, Candida elegans , and Drosophila , small RNAs have been shown to operate in the nucleus to direct stable long-term gene-silencing events.1 Recently, it has also become apparent that these same regulatory events can be exogenously controlled in human …

Valproate, a Histone Deacetylase Inhibitor, Enhances Purine Nucleoside Analogues Induced Apoptosis of B-Chronic Lymphocytic Leukemia Cells.

Resistance to chemotherapy and drug toxicity are two major concerns of Chronic Lymphocytic Leukemia (B-CLL) treatment by Purine Nucleoside Analogs (PNA, i.e. fludarabine and cladribine). We hypothesized that targeting epigenetic changes might address these issues and evaluated the effect of the histone deacetylase (HDAC) inhibitor valproate (VPA) at a clinically relevant concentration. We show that VPA acts in a highly synergistic/additive manner with 9-ß-D-arabinosyl-2-fluoroadenine-5′-monophosphate (F-ara-AMP, fludarabine, 1μM) and 2-chloro-2′-deoxyadenosine (CdA, cladribine, 1μM) to induce apoptosis of CLL cells. VPA, but neither fludarabine nor cladribine, enhances the production of Reactive Oxygen Species (ROS) and inhibition of ROS with N-acetylcysteine decreases apoptosis of CLL cells. VPA-induced apoptosis is caspase-dependent and involves the extrinsic initiation pathway. VPA stimulates hyperphosphorylation of p42/p44 Erk as well as overexpression of Bax, cytochrome c and Fas. These observations support a potential use of VPA alone or in combination with nucleosidic analogs in CLL therapeutic protocols allowing a reduction of their effective doses or improving their efficacy in PNA-resistant patients.