Enhancement of the Antileukemic Action of the Inhibitors of DNA and Histone Methylation: 5-Aza-2'-Deoxycytidine and 3-Deazaneplanocin-A by Vitamin C.

Epigenetic gene silencing by DNA methylation and histone methylation by EZH2 play an important role in the development of acute myeloid leukemia (AML). EZH2 catalyzes the trimethylation of histone H3-lysine 27-trimethylated (H3K27me3). These epigenetic alterations silence the expression of the genes that suppress leukemogenesis. Reversal of this gene silencing by 5-aza-2'-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, and by 3-deazaneplanocin-A (DZNep), an inhibitor of EZH2, results in synergistic gene reactivation and antileukemic interaction. The objective of this study is to determine if the addition of another epigenetic agent could further enhance the antileukemic action of these inhibitors of DNA and histone methylation. Vitamin C (Vit C) is reported to enhance the antineoplastic action of 5-Aza-CdR on AML cells. The mechanism responsible for this action of Vit C is due to its function as a cofactor of alpha-ketoglutarate-dependent dioxygenases (α-KGDD). The enhancement by Vit C of the catalytic activity of α-KGDD of the ten eleven translocation (TET) pathway, as well as of the Jumonji C histone demethylases (JHDMs), is shown to result in demethylation of DNA and histones, leading to reactivation of tumor suppressor genes and an antineoplastic effect. This action of Vit C has the potential to complement the antileukemic action of 5-Aza-CdR and DZNep. We observe that Vit C remarkably increases the antineoplastic activity of 5-Aza-CdR and DZNep against myeloid leukemic cells. An important step to bring this novel epigenetic therapy to clinical trial in patients with AML is the determination of its optimal dose schedule.

Epigenetic Modulation of Self-Renewal Capacity of Leukemic Stem Cells and Implications for Chemotherapy.

Most patients with acute myeloid leukemia (AML) have a poor prognosis. Curative therapy of AML requires the complete eradication of the leukemic stem cells (LSCs). One aspect of LSCs that is poorly understood is their low frequency in the total population of leukemic cells in AML patients. After each cell division of LSCs, most of the daughter cells lose their capacity for self-renewal. Investigations into the role of Isocitrate dehydrogenase (IDH) mutations in AML provide some insight on the regulation of the proliferation of LSCs. The primary role of IDH is to convert isocitrate to alpha-keto-glutarate (α-KG). When IDH is mutated, it converts α-KG to 2-hydroxyglutarate (2-HG), an inhibitor of the TET pathway and Jumonji-C histone demethylases (JHDMs). The demethylating action of these enzymes removes the epigenetic gene-silencing markers, DNA methylation, H3K27me3 and H3K9me2 and can lead to the differentiation of LSCs. This enzymatic action is blocked by 2-HG in mutated IDH (mut-IDH) AML patients, who can be induced into remission with antagonists of 2-HG. These observations suggest that there exists in cells a natural enzymatic mechanism that uses demethylation to reverse epigenetic gene-silencing, leading to a loss of the self-renewal capacity of LSCs. This mechanism limits the proliferative potential of LSCs. Epigenetic agents that inhibit DNA and histone methylation exhibit a synergistic antineoplastic action on AML cells. It is possible that the therapeutic potential of this epigenetic therapy may be enhanced by demethylation enzymes, resulting in a very effective treatment for AML.

Inhibition of DNA and Histone Methylation by 5-Aza-2'-Deoxycytidine (Decitabine) and 3-Deazaneplanocin-A on Antineoplastic Action and Gene Expression in Myeloid Leukemic Cells.

Epigenetic alterations play an important role in the development of acute myeloid leukemia (AML) by silencing of genes that suppress leukemogenesis and differentiation. One of the key epigenetic changes in AML is gene silencing by DNA methylation. The importance of this alteration is illustrated by the induction of remissions in AML by 5-aza-2'-deoxycytidine (5-AZA-CdR, decitabine), a potent inhibitor of DNA methylation. However, most patients induced into remission by 5-AZA-CdR will relapse, suggesting that a second agent should be sought to increase the efficacy of this epigenetic therapy. An interesting candidate for this purpose is 3-deazaneplanocin A (DZNep). This analog inhibits EZH2, a histone methyltransferase that trimethylates lysine 27 histone H3 (H3K27me3), a marker for gene silencing. This second epigenetic silencing mechanism also plays an important role in leukemogenesis as shown in preclinical studies where DZNep exhibits potent inhibition of colony formation by AML cells. We reported previously that 5-AZA-CdR in combination with DZNep exhibits a synergistic antineoplastic action against human HL-60 AML cells and the synergistic activation of several tumor suppressor genes. In this report, we showed that this combination also induced a synergistic activation of apoptosis in HL-60 cells. The synergistic antineoplastic action of 5-AZA-CdR plus DZNep was also observed on a second human myeloid leukemia cell line, AML-3. In addition, 5-AZA-CdR in combination with the specific inhibitors of EZH2, GSK-126, or GSK-343, also exhibited a synergistic antineoplastic action on both HL-60 and AML-3. The combined action of 5-AZA-CdR and DZNep on global gene expression in HL-60 cells was investigated in greater depth using RNA sequencing analysis. We observed that this combination of epigenetic agents exhibited a synergistic activation of hundreds of genes. The synergistic activation of so many genes that suppress malignancy by 5-AZA-CdR plus DZNep suggests that epigenetic gene silencing by DNA and histone methylation plays a major role in leukemogenesis. Targeting DNA and histone methylation is a promising approach that merits clinical investigation for the treatment of AML.

Three new cases of terminal deletion of the long arm of chromosome 7 and literature review to correlate genotype and phenotype manifestations.

Partial monosomy of the long arm of chromosome 7 has been characterized by wide phenotypic manifestations, but holoprosencephaly (HPE) and sacral agenesis have frequently been associated with this chromosomal deletion. A clear relationship between genotype and phenotype remains to be defined in the 7q deletion syndrome. Three patients (1, 2, and 3) were investigated with 7q terminal deletion and compared with similar deletion cases in the literature in order to stratify the phenotypes associated with 7q35 and 7q36 terminal deletion patients. Patients 1, 2, and 3 were carrying a de novo terminal deletion at bands 7q36.2, 7q35, and 7q36.1, respectively. In patient 3, a small Xq28 duplication was also identified by array-CGH. Our patients presented with heterogeneous phenotypic manifestations, which could imply the possible role of environmental factors (multifactorial inheritance), structural variations in the non-coding regions, penetrance, and/or polymorphism. The varying length of deletion was also taken into account. Growth retardation was the most frequent symptom found in both 7q35 and 7q36 patients we reviewed. The occurrence of HPE and sacral malformation together was seen in less than 10% of the reviewed cases in both kinds of deletion. HPE was associated mainly in cases with an unbalanced translocation.

Targeting of cancer stem cells by inhibitors of DNA and histone methylation.

INTRODUCTION: Curative chemotherapy should target cancer stem cells (CSCs). The key characteristics of CSCs are a block in differentiation and an epigenetic signature similar to embryonic stem cells (ESCs). Differentiation by ESCs and CSCs is suppressed by gene silencing through the polycomb repressive complex 2 (PRC2) and/or DNA methylation. PRC2 contains the EZH2 subunit, which catalyzes the trimethylation of histone 3 lysine 27, a gene silencing marker. It is possible to reverse this 'double lock' mechanism using a combination of inhibitors of EZH2 and DNA methylation (5-aza-2'-deoxycytidine), which exhibits remarkable synergistic antineoplastic activity in preclinical studies. AREAS COVERED: The authors discuss several specific EZH2 inhibitors that have been synthesized with antineoplastic activity. One such inhibitor, EPZ-6438 (E7438), has been shown to be effective against lymphoma in a Phase I study. The indirect EZH2 inhibitor, 3-deazaneplanocin-A (DZNep), also exhibits remarkable anticancer activity due to its inhibition of methionine metabolism. EXPERT OPINION: Agents that target EZH2 warrant Phase I trials. Due to its positive pharmacodynamics, DZNep merits a high priority for clinical investigation. Agents that show positive results in Phase I studies should be advanced to clinical trials for use in combination with 5-aza-2'-deoxycytidine due to the interesting potential of this epigenetic therapy to target CSCs.

Epigenetic therapy of acute myeloid leukemia using 5-aza-2'-deoxycytidine (decitabine) in combination with inhibitors of histone methylation and deacetylation.

BACKGROUND: The silencing of tumor suppressor genes (TSGs) by aberrant DNA methylation occurs frequently in acute myeloid leukemia (AML). This epigenetic alteration can be reversed by 5-aza-2'-deoxcytidine (decitabine, 5-AZA-CdR). Although 5-AZA-CdR can induce complete remissions in patients with AML, most patients relapse. The effectiveness of this therapy may be limited by the inability of 5-AZA-CdR to reactivate all TSGs due to their silencing by other epigenetic mechanisms such as histone methylation or chromatin compaction. EZH2, a subunit of the polycomb repressive complex 2, catalyzes the methylation of histone H3 lysine 27 (H3K27) to H3K27me3. 3-Deazaneplanocin-A (DZNep), an inhibitor of methionine metabolism, can reactivate genes silenced by H3K27me3 by its inhibition of EZH2. In a previous report, we observed that 5-AZA-CdR, in combination with DZNep, shows synergistic antineoplastic action against AML cells. Gene silencing due to chromatin compaction is attributable to the action of histone deacetylases (HDAC). This mechanism of epigenetic gene silencing can be reversed by HDAC inhibitors such as trichostatin-A (TSA). Silent TSGs that cannot be reactivated by 5-AZA-CdR or DZNep have the potential to be reactivated by TSA. This provides a rationale for the use of HDAC inhibitors in combination with 5-AZA-CdR and DZNep to treat AML. RESULTS: The triple combination of 5-AZA-CdR, DZNep, and TSA induced a remarkable synergistic antineoplastic effect against human AML cells as demonstrated by an in vitro colony assay. This triple combination also showed a potent synergistic activation of several key TSGs as determined by real-time PCR. The triple combination was more effective than the combination of two agents or a single agent. Microarray analysis showed that the triple combination generated remarkable changes in global gene expression. CONCLUSIONS: Our data suggest that it may be possible to design a very effective therapy for AML using agents that target the reversal of the following three epigenetic "lock" mechanisms that silence gene expression: DNA methylation, histone methylation, and histone deacetylation. This approach merits serious consideration for clinical investigation in patients with advanced AML.

Epigenetic action of decitabine (5-aza-2'-deoxycytidine) is more effective against acute myeloid leukemia than cytotoxic action of cytarabine (ARA-C).

Treatment of elderly patients with acute myeloid leukemia (AML) with standard cytarabine (ARA-C) chemotherapy can achieve some complete responses (CR), but the median overall survival is less than one year. New approaches should be investigated. The inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (decitabine, DAC), shows effectiveness in these patients, but was not approved by the US Federal Drug Administration. This decision was based on a clinical trial where DAC showed a median survival of 7.0 months as compared to standard ARA-C therapy or supportive care of 5.0 months. However, the difference was not statistically significant. Preclinical data indicate that DAC is much more effective against human AML than ARA-C. The key question is should these preclinical data also be used in the evaluation of new drugs for the clinical treatment of AML? The delayed epigenetic action of DAC is very different than the acute cytotoxic action of ARA-C and should be taken into account in the design clinical trials and evaluation of the response.

Outcomes during and after inpatient rehabilitation: comparison between adults and older adults.

OBJECTIVE: To compare changes on biopsychosocial variables between adults (< 65 years) and older adults (> or = 65 years) during and after an inpatient rehabilitation program. DESIGN: Comparative study. PARTICIPANTS: A total of 165 people 18 years and over admitted to an inpatient rehabilitation unit. METHODS: The participants were evaluated 3 times: at admission and discharge and 3 months later. Outcome measures were: Functional Autonomy Measurement System (SMAF), Modified Mini-Mental State Examination, Trail Making Test, Motor-Free Visual Perceptual Test, Visual Analogue Scale (VAS), Berg Balance Scale (BBS), Timed Up and Go (TUG), Jamar dynamometer, and General Well-being Schedule (GWBS). RESULTS: Both groups improved significantly between admission and discharge on the SMAF, BBS, TUG, VAS, Jamar dynamometer and GWBS. Although scores on many of the outcome measures differed at admission, the 2 groups improved similarly during rehabilitation. After 3 months, stability or a slight improvement was observed for both groups; however, younger participants improved more on mobility, balance, walking and grip strength. CONCLUSION: Although older participants had more disabilities at admission, they benefited as much as younger people from an intensive rehabilitation program with a comparable length of stay. However, younger participants continued to improve 3 months later.

Distribution of Salmonella in tissues following natural and experimental infection in pigs.

Clinical salmonellosis associated with Salmonella is increasingly reported in finishing swine. Since S. Typhimurium is often associated with these episodes and given that this serotype is among the most often reported in humans, we were interested to determine if various tissues and carcasses from animals coming from herds that were clinically affected were more likely to be contaminated by Salmonella compared to carcasses from animals raised in herds without any history of salmonellosis. Carcasses from animals from affected herds were significantly more contaminated by Salmonella while showing increased titers in antibodies directed against this bacterium. At the opposite, caecal contents and mesenteric lymph nodes from both groups of animals were similarly contaminated by Salmonella. In the second part of the study, we studied the persistence of the bacterium in various tissues after an experimental infection with S. Typhimurium. We found that, after the infection, Salmonella persisted for as many as 7 d in many extraintestinal tissues, while it was present in the feces of infected animals for all 14 d of the experiment. These findings indicated that carcasses from animals that experienced salmonellosis during their growth phase are more likely to be contaminated by this bacterium and that precautions must be taken in order to ensure that clinically affected animals should be kept on the farm for at least 7 d before being shipped for slaughter.

Expression of SMRTbeta promotes ligand-induced activation of mutated and wild-type retinoid receptors.

Nuclear receptors are ligand-modulated transcription factors regulated by interactions with corepressors and coactivators, whose functions are not fully understood. Acute promyelocytic leukemia (APL) is characterized by a translocation, t(15;17), that produces a PML/RARalpha fusion oncoprotein, whose abnormal transcriptional function is successfully targeted by pharmacologic levels of all-trans-retinoic acid (ATRA). Mutations in the ligand-binding domain of PML/RARalpha that confer resistance to ATRA have been studied by expression in nonhematopoietic cells, such as Cos-1. Here, we show that ATRA binding and transcriptional activation by the same PML/RARalpha mutant differ markedly between nonhematopoietic and leukemic cell lines. Differential expression of the corepressor isoform silencing mediator for retinoid and thyroid receptors beta (SMRTbeta) correlates with increased ligand binding and transcription by the mutant PML/RARalpha. Transient and stable overexpression of SMRTbeta in hematopoietic cells that only express SMRTalpha increased ATRA binding, ligand-induced transcription, and ATRA-induced cell differentiation. This effect may not be limited to abnormal nuclear receptors, because overexpression of SMRTbeta increased ATRA-induced binding and transcriptional activation of wild-type receptors PML/RARalpha and RARalpha. Our results suggest a novel role for the SMRTbeta isoform whereby its cell-specific expression may influence the binding and transcriptional capacities of nuclear receptors, thus providing new evidence of distinct functions of corepressor isoforms and adding complexity to transcriptional regulation.

Geriatric day hospital: who improves the most?

This study compared the changes in some bio-psychosocial variables (functional independence, nutritional risk, pain, balance and walking, grip strength, general well-being, psychiatric profile, perception of social support, leisure satisfaction, and caregivers' feeling of burden) in four categories of clients during their program at a geriatric day hospital (GDH). The study also evaluated whether or not improvements, if any, were maintained 3 months after discharge. One-hundred-and-fifty-one people, categorized by primary reason for admission, were assessed at the GDH with reliable and valid tools, at admission and at discharge. Three months after discharge, they were reassessed with the same tools. Overall, two categories of clients, stroke / neurological diseases and musculoskeletal disorders / amputations, improved the most. For the gait disorders and falls group, only the functional independence score improved, but not at a clinically significant level. Finally, clients in the cognitive function disorders / psychopathologies group improved the most on their well-being scores and caregivers' burden decreased the most. All gains were maintained up to 3 months after discharge, except for leisure satisfaction. With the exception of clients who attended the GDH because of gait disorders and falls, the improvements and maintenance achieved in each category occurred in the domains where improvement had been hoped for, because of the particular disabilities in question and because of the nature of the GDH services offered.

Response to histone deacetylase inhibition of novel PML/RARalpha mutants detected in retinoic acid-resistant APL cells.

Resistance to all-trans retinoic acid (ATRA) remains a clinical problem in the treatment of acute promyelocytic leukemia (APL) and provides a model for the development of novel therapies. Molecular alterations in the ligand-binding domain (LBD) of the PML/RARalpha fusion gene that characterizes APL constitute one mechanism of acquired resistance to ATRA. We identified missense mutations in PML/RARalpha from an additional ATRA-resistant patient at relapse and in a novel ATRA-resistant cell line, NB4-MRA1. These cause altered binding to ligand and transcriptional coregulators, leading to a dominant-negative block of transcription. These mutations are in regions of the LBD that appear to be mutational hot spots occurring repeatedly in ATRA-resistant APL patient cells. We evaluated whether histone deacetylase (HDAC) inhibition could overcome the effects of these mutations on ATRA-induced gene expression. Cotreatment with ATRA and TSA restored RARbeta gene expression in NB4-MRA1 cells, whose PML/RARalpha mutation is in helix 12 of the LBD, but not in an APL cell line harboring the patient-derived PML/RARalpha mutation, which was between helix 5 and 6. Furthermore, ATRA combined with TSA increases histone 4 acetylation on the RARbeta promoter only in NB4-MRA1 cells. Consistent with these results, the combined treatment induces differentiation of NB4-MRA1 only. Thus, the ability of an HDAC inhibitor to restore ATRA sensitivity in resistant cells may depend on their specific molecular defects. The variety of PML/RARalpha mutations arising in ATRA-resistant patients begins to explain how APL patients in relapse may differ in response to transcription therapy with HDAC inhibitors.