Demethylating Agent Research Articles

MicroRNA-429 inhibits the migration and invasion of colon cancer cells by targeting PAK6/cofilin signaling.

MicroRNAs (miRs), a class of non-coding RNAs 18-25 nucleotides in length, can lead to mRNA degradation or inhibit protein translation by directly binding to the 3'-untranslational region (UTR) of their target mRNAs. The deregulation of miR-429 has been suggested to be involved in the development and progression of colon cancer. However, the detailed molecular mechanism involved remains to be determined. The aim of the present study was to investigate the role of miR-429 in the regulation of migration and invasion of colon cancer cells using RT-qPCR and western blotting. The results showed that the expression of miR-429 was reduced in colon cancer cell lines, when compared to a normal colon epithelial cell line. Treatment with DNA demethylation agent 5-aza-2'-deoxycytidine and histone deacetylase inhibitor phenylbutyrate (PBA), or transfection with the pre-miR-429 lentivirus plasmid led to the upregulation of miR-429 expression, as well as inhibition of migration and invasion in colon cancer cells. Investigation of the molecular mechanism showed that PAK6 was a novel target of miR-429, and the expression of PAK6 was upregulated in colon cancer tissues and cell lines, and was negatively regulated by miR-429 in colon cancer cells. Moreover, the cofilin signaling acted as a downstream effector of miR-429 in colon cancer cells. In conclusion, the results of the present study suggested that miR-429 inhibits the migration and invasion of colon cancer cells, partly at least, by mediating the expression of PAK6, as well as the activity of cofilin signaling. Therefore, miR-429 is as a potential molecular target for the treatment of colon cancer.

Targeting p53 in CML Chronic Phase Leukemia Stem Cells

Chronic myelogenous leukemia (CML) is a hematological disease resulting from generation of BCR/ABL oncogene. Resveratrol is an important phytoalexin which is contained in many plants and it has cytotoxic effects in various cancer types. Gene promoter methylation causes loss of tumor suppressor gene function in human cancer. CHD4 (Chromodomain/Helicase/DNAbinding domain) gene which contains a CpG island is a member of the cadherin family. In this study we aimed to evaluate the role of epigenetic modification of resveratrol in cadherin family genes in K562 human CML cell line. K562 cells were treated with 100 mM (IC50 dose) resveratrol depend on time and dosage during 72 hours and cytotoxicity was evaluated by using WST-1 assay. The RT-qPCR is used for gene expression analysis. Gene expression levels were evaluated by using RT Profiler PCR Array. RT-qPCR results showed that, CHD4 gene expression increased 3.45 fold, according to the control cells that untreated with resveratrol. Significant upregulation was found in CHD4 gene expression among the cadherin family genes in resveratrol treated K562 cells. CHD4 is affected by methylation and frequently is silenced in CML. Downregulation of CHD4 gene expression could trigger activation of the Wnt-pathway and cause aberant cell-to-cell interactions. After treatment with the resveratrol in K562 cells, CHD4 gene expression was upregulated. Resveratrol could increase the cadherin family gene expressions and activation of the Wntpathway is blocked in CML. Also, resveratrol could demethylate CpG island in CHD4 promoter region and increase CHD4 gene expression. Our results indicate that resveratrol may act as a demethylation agent on the promoter of tumor supresor genes such as CHD4 and increase the expression of these genes. Therefore, resveratrol could be used as a pioneering anti-tumorigenesis drug in CML. 411 Targeting p53 in CML Chronic Phase Leukemia Stem Cells Luke F. Peterson, Miao-Chia Lo, Yihong Lui, Diane Gianolla, Emilija Mitrikeska, Nicholas J. Donato, Craig N. Johnson, Shaomeng Wang, Jessica Mercer, Moshe Talpaz Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, USA; Division of Hematology and Oncology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA; Moores UCSD Cancer Center, University of California San Diego, La Jolla, CA, USA; DNA Sequencing and Microarray Core, University of Michigan, Ann Arbor, MI, USA; Department of Pharmacology and Medicinal Chemistry, University of Michigan, Ann Arbor, MI, USA

Resveratrol Increases CHD4 Gene Expression and Regulates Wnt Signaling in Chronic Myeloid Leukemia

Chronic myelogenous leukemia (CML) is a hematological disease resulting from generation of BCR/ABL oncogene. Resveratrol is an important phytoalexin which is contained in many plants and it has cytotoxic effects in various cancer types. Gene promoter methylation causes loss of tumor suppressor gene function in human cancer. CHD4 (Chromodomain/Helicase/DNA- binding domain) gene which contains a CpG island is a member of the cadherin family. In this study we aimed to evaluate the role of epigenetic modification of resveratrol in cadherin family genes in K562 human CML cell line. K562 cells were treated with 100 mM (IC50 dose) resveratrol depend on time and dosage during 72 hours and cytotoxicity was evaluated by using WST-1 assay. The RT-qPCR is used for gene expression analysis. Gene expression levels were evaluated by using RT 2 Profiler PCR Array. RT-qPCR results showed that, CHD4 gene expression increased 3.45 fold, according to the control cells that untreated with resveratrol. Significant upregulation was found in CHD4 gene expression among the cadherin family genes in resveratrol treated K562 cells. CHD4 is affected by methylation and frequently is silenced in CML. Downregulation of CHD4 gene expression could trigger activation of the Wnt-pathway and cause aberant cell-to-cell interactions. After treatment with the resveratrol in K562 cells, CHD4 gene expression was upregulated. Resveratrol could increase the cadherin family gene expressions and activation of the Wntpathway is blocked in CML. Also, resveratrol could demethylate CpG island in CHD4 promoter region and increase CHD4 gene expression. Our results indicate that resveratrol may act as a demethylation agent on the promoter of tumor supresor genes such as CHD4 and increase the expression of these genes. Therefore, resveratrol could be used as a pioneering anti-tumorigenesis drug in CML.

Activation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor gene expression following DNA demethylation in placental choriocarcinoma and transformed cell lines.

We characterised DNA methylation and gene expression of four tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4, DR5, DcR1 and DcR2 in three choriocarcinoma (JAR, JEG-3, BeWo) and two transformed (HTR-8/SVneo and HPT-8) cell lines. DR4 mRNA was detected in JAR, JEG-3, BeWo and HTR-8/SVneo cells, whereas DR5 was present in all detected cells. DcR1 transcripts were expressed only in JAR, JEG-3 and BeWo cells, whereas DcR2 transcripts were detected only in HTR-8/SVneo and HPT-8 cells. Hypermethylated DR4 promoter was observed in JAR, JEG-3, BeWo and HTR-8/SVneo cells, hypermethylated DcR1 promoter in HTR-8/SVneo and HPT-8 cells and hypermethylated DcR2 promoter in JAR, JEG-3 and BeWo cells. Restoration of DR4, DcR1 and DcR2 expression with decreased DNA methylation of these genes was induced by the DNA demethylation agent 5-aza-2'-deoxycytidine (5-aza-CdR) in trophoblast cells, whereas DR5 expression did not exhibit any change. Significant negative correlation between the expression and DNA methylation of these genes was also observed. In all tested cell lines, only HPT-8 demonstrated sensitivity to TRAIL-induced apoptosis. Combined treatment with 5-aza-CdR and TRAIL resulted in apoptosis in JAR, JEG-3, BeWo and HTR-8/SVneo cells but not in HPT-8 cells. The results indicate that DNA methylation is associated with TRAIL receptor expression and might be involved in trophoblast apoptosis.

Dietary compound isoliquiritigenin prevents mammary carcinogenesis by inhibiting breast cancer stem cells through WIF1 demethylation.

Breast cancer stem cells (CSCs) are considered as the root of mammary tumorigenesis. Previous studies have demonstrated that ISL efficiently limited the activities of breast CSCs. However, the cancer prevention activities of ISL and its precise molecular mechanisms remain largely unknown. Here, we report a novel function of ISL as a natural demethylation agent targeting WIF1 to prevent breast cancer. ISL administration suppressed in vivo breast cancer initiation and progression, accompanied by reduced CSC-like populations. A global gene expression profile assay further identified WIF1 as the main response gene of ISL treatment, accompanied by the simultaneous downregulation of β-catenin signaling and G0/G1 phase arrest in breast CSCs. In addition, WIF1 inhibition significantly relieved the CSC-limiting effects of ISL and methylation analysis further revealed that ISL enhanced WIF1 gene expression via promoting the demethylation of its promoter, which was closely correlated with the inhibition of DNMT1 methyltransferase. Molecular docking analysis finally revealed that ISL could stably dock into the catalytic domain of DNMT1. Taken together, our findings not only provide preclinical evidence to demonstrate the use of ISL as a dietary supplement to inhibit mammary carcinogenesis but also shed novel light on WIF1 as an epigenetic target for breast cancer prevention.

Recent advances in primary cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of skin-homing T-cell neoplasms, which represent approximately 75% of all primary cutaneous lymphomas. Currently available drug therapies, when effective, simply control disease and the only option for curing CTCL is stem cell transplant. In the last year, there has been an incredible effort made to improve the understanding and treatment of CTCL. Recent findings indicate that epigenetic aberrations are integral to active disease. Furthermore, multiple tumor-derived immunological factors have also been shown to inhibit viability, proliferation, and cytokine production of nonmalignant T cells. Several novel targeted therapies show great potential, most promising being antibody drug conjugates targeting surface markers such as CD30 in some CTCL subtypes. Additional attractive targets involve the global modulation of epigenetic markers such as demethylation agents or HDAC inhibitors, either as single agents or in combination therapies. This is a concise review of recent advances in the field of CTCL with special focus on research articles over the preceding year.

HLA-matched allogeneic stem cell transplantation improves outcome of higher risk myelodysplastic syndrome A prospective study on behalf of SFGM-TC and GFM.

Allogeneic hematopoietic stem cell transplantation (HSCT) is considered the only a curative treatment in patients with higher risk myelodysplastic syndrome (MDS), although demethylating agents (DMA) have been reported to improve survival. The advantage of HSCT over other treatment comes from retrospective studies and the aim of the current study was to prospectively test this hypothesis, analyzing in particular patients from the pre-transplant period to avoid the selection bias of performing transplantation. This study was conducted to compare overall survival in MDS patients candidates to transplantation according to donor availability. The majority of patients (76%) received a treatment with DMA after registration, 69% had a human leukocyte antigen (HLA)-identical donor, 70% of whom were transplanted. Baseline patient and disease characteristics were similar according to donor availability. Four-year overall survival was significantly better in patients with an HLA matched donor (37%) compared to patients without donor (15%). There was also evidence that this overall survival advantage was because of transplantation. Mortality risk was decreased after transplantation but it became significant only after the second year post transplant, because of early transplant-related mortality. Our results appear to justify, in higher risk MDS, a transplantation approach in all potential candidates who have an HLA identical donor.

Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny.

Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between de novo DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (POLGA)) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of POLGA in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of POLGA as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of POLGA leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein (GFAP). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of POLGA is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of POLGA and mtDNA copy number in the presence of a DNA demethylation agent that inhibits de novo methyltransferase 1 activity.

Age dependent changes in the LPS induced transcriptome of bovine dermal fibroblasts occurs without major changes in the methylome.

BackgroundBy comparing fibroblasts collected from animals at 5-months or 16-months of age we have previously found that the cultures from older animals produce much more IL-8 in response to lipopolysaccharide (LPS) stimulation. We now expand this finding by examining whole transcriptome differences in the LPS response between cultures from the same animals at different ages, and also investigate the contribution of DNA methylation to the epigenetic basis for the age-dependent increases in responsiveness.ResultsAge-dependent differences in IL-8 production by fibroblasts in response to LPS exposure for 24 h were abolished by pretreatment of cultures with a DNA demethylation agent, 5-aza-2′deoxycytidine (AZA). RNA-Seq analysis of fibroblasts collected from the same individuals at either 5 or 16 months of age and exposed in parallel to LPS for 0, 2, and 8 h revealed a robust response to LPS that was much greater in the cultures from older animals. Pro-inflammatory genes including IL-8, IL-6, TNF-α, and CCL20 (among many other immune associated genes), were more highly expressed (FDR < 0.05) in the 16-month old cultures following LPS exposure. Methylated CpG island recovery assay sequencing (MIRA-Seq) revealed numerous methylation peaks spread across the genome, combined with an overall hypomethylation of gene promoter regions, and a remarkable similarity, except for 20 regions along the genome, between the fibroblasts collected at the two ages from the same animals.ConclusionsThe fibroblast pro-inflammatory response to LPS increases dramatically from 5 to 16 months of age within individual animals. A better understanding of the mechanisms underlying this process could illuminate the physiological processes by which the innate immune response develops and possibly individual variation in innate immune response arises. In addition, although relatively unchanged by age, our data presents a general overview of the bovine fibroblast methylome as a guide for future studies in cattle epigenetics utilizing this cell type.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1223-z) contains supplementary material, which is available to authorized users.

Epigenetic inactivation and tumor-suppressor behavior of NGFR in human colorectal cancer.

The nerve growth factor receptor (NGFR/p75) is a potential tumor suppressor, but its role in colorectal cancer is unknown. Here, the hypermethylation status, biologic function, and clinical relevance were determined for p75NGFR in colorectal cancer. The methylation status and expression of p75NGFR were assessed in colorectal cancer cell lines and clinical tissues by bisulfite genomic sequencing (BGS), qRT-PCR, and immunoblot assay. Methylation of p75NGFR was frequently found in colorectal cancer, leading to its silencing or downregulation, and it was effectively restored by a demethylation agent. The overexpression of p75NGFR in multiple colorectal cancer cell model systems significantly inhibited cell proliferation (concomitant with G1-phase arrest), invasion, and colony formation and induced cell apoptosis. In contrast, p75NGFR knockdown significantly promoted proliferative and invasive phenotypes. Importantly, p75NGFR methylation was observed in the majority of primary colorectal cancer specimens and was associated with histologic grade and preoperative serum CA19-9 levels. Multivariate analysis indicated that patients who lack p75NGFR have reduced overall survival (64% vs. 75%, P = 0.028) and disease-free survival (61% vs. 72%, P = 0.034) compared with p75NGFR-positive patients. In conclusion, p75NGFR is predominantly silenced or downregulated in colorectal cancer, and its biologic activities are consistent with it being a relevant tumor suppressor. p75NGFR is a candidate tumor suppressor and has independent prognostic potential in colorectal cancer.

Sonic hedgehog produced by bone marrow-derived mesenchymal stromal cells supports cell survival in myelodysplastic syndrome.

The role of marrow microenvironment in the pathogenesis of myelodysplastic syndrome (MDS) remains controversial. Therefore, we studied the influence of bone marrow-derived mesenchymal stromal cells (BMSCs) from patients with different risk types of MDS on the survival of the MDS cell lines SKM-1 and MUTZ-1. We first demonstrated that the expression of Sonic hedgehog (Shh), smoothened (Smo), and glioma-associated oncogene homolog 1 (Gli1) was increased in MDS patients (n = 23); the increase in expression was positively correlated with the presence of high-risk factors. The Shh signaling inhibitor, cyclopamine, inhibited high-risk MDS BMSC-induced survival of SKM-1 and MUTZ-1 cells, suggesting a role for Shh signaling in MDS cell survival. Furthermore, cyclopamine-mediated inhibition of Shh signaling in SKM-1 and MUTZ-1 cells resulted in decreased DNMT1 expression and cell survival; however, exogenous Shh peptide had the opposite effect, suggesting that Shh signaling could regulate the expression of DNMT1, thereby modulating cell survival in MDS. In addition, the apoptosis of SKM-1 and MUTZ-1 cell increased significantly when cultured with cyclopamine and a demethylation agent, 5-Aza-2′-deoxycytidine. These findings suggest that Shh signaling from BMSCs is important in the pathogenesis of MDS and could play a role in disease progression by modulating methylation.

Demethylation of microRNA-142 induced by demethylation agents plays a suppressive role in osteosarcoma cells

Osteosarcoma (OS), the most common malignant bone tumor, occurs mainly in adolescents and young adults, with a morbidity of ∼5 cases per million. The expression levels of microRNAs (miRNAs) as tumor suppressors were recently found to be downregulated in OS. Certain alterations of miRNAs and the possible mechanisms through which miRNAs affect cell proliferation and migration in OS were recently found to be correlated with methylation epigenetic mechanisms. In this study, it was demonstrated that, due to hypermethylation, the expression level of miRNA-142 (miR-142) was significantly downregulated in OS tissues and cells compared with that in control samples. The present study demonstrated an increased expression of miR-142 in Saos-2 and MG63 cells treated with demethylation agents, suggesting that the effect of such agents on cell growth, inhibition of invasion and cell cycle retardation may be mediated by miR-142 in OS.

MiR-34c suppresses tumor growth and metastasis in nasopharyngeal carcinoma by targeting MET

Our previous microarray analysis indicated that miR-34c was downregulated in nasopharyngeal carcinoma (NPC). However, little is known about the function and molecular mechanism of miR-34c in NPC. In this study, miR-34c was found to be significantly downregulated in NPC cell lines and clinical tissues. Ectopic expression of miR-34c suppressed NPC cell viability, colony formation, anchorage-independent growth, cell migration and invasion in vitro, and inhibited xenograft tumor growth and lung metastasis in vivo. MET proto-oncogene (MET) was identified as a direct target of miR-34c using luciferase reporter assays, quantitative RT-PCR, western blotting and immunofluorescent staining. Overexpression of miR-34c markedly reduced MET expression at both the mRNA and protein levels. Knockdown of MET suppressed NPC cell proliferation, migration and invasion, whereas the restoration of MET rescued the suppressive effects of miR-34c. The demethylation agent 5-aza-2′-deoxycytidine (DAC) restored the expression of miR-34c in NPC cell lines. The promoter region of miR-34c was hypermethylated in NPC cells. In conclusion, miR-34c suppresses tumor growth and metastasis in NPC by targeting MET. The newly identified miR-34c/MET pathway provides further insights into the development and progression of NPC, and may represent a novel therapeutic target for NPC treatment.

Downregulated miR329 and miR410 promote the proliferation and invasion of oral squamous cell carcinoma by targeting Wnt-7b.

microRNA (miRNA) dysregulation contributes widely to human cancer but has not been fully assessed in oral cancers. In this study, we conducted a global microarray analysis of miRNA expression in 40 pairs of betel quid-associated oral squamous cell carcinoma (OSCC) specimens and their matched nontumorous epithelial counterparts. Eighty-four miRNAs were differentially expressed in the OSCC specimens compared with the matched tissue. Among these downregulated miRNAs, 19 miRNAs were found and mapped to the chromosome 14q32.2 miRNA cluster region, which resides within a parentally imprinted region designated as Dlk-Dio3 and known to be important in development and growth. Bioinformatic analysis predicted two miRNAs from the cluster region, miR329 and miR410, which could potentially target Wnt-7b, an activator of the Wnt-β-catenin pathway, thereby attenuating the Wnt-β-catenin signaling pathway in OSCC. Stable ectopic expression of Wnt-7b in OSCC cells overexpressing miR329 or miR410 restored proliferation and invasion capabilities abolished by these miRNA. Combining a demethylation agent and a histone deacetylase inhibitor was sufficient to reexpress miR329, miR410, and Meg3, consistent with epigenetic regulation of these miRNA in human OSCC. Specifically, arecoline, a major betel nut alkaloid, reduced miR329, miR410, and Meg3 gene expression. Overall, our results provide novel molecular insights into how betel quid contributes to oral carcinogenesis through epigenetic silencing of tumor-suppressor miRNA that targets Wnt-β-catenin signaling.

The high mobility group A2 protein epigenetically silences the Cdh1 gene during epithelial-to-mesenchymal transition.

The loss of the tumour suppressor E-cadherin (Cdh1) is a key event during tumourigenesis and epithelial–mesenchymal transition (EMT). Transforming growth factor-β (TGFβ) triggers EMT by inducing the expression of non-histone chromatin protein High Mobility Group A2 (HMGA2). We have previously shown that HMGA2, together with Smads, regulate a network of EMT-transcription factors (EMT-TFs) like Snail1, Snail2, ZEB1, ZEB2 and Twist1, most of which are well-known repressors of the Cdh1 gene. In this study, we show that the Cdh1 promoter is hypermethylated and epigenetically silenced in our constitutive EMT cell model, whereby HMGA2 is ectopically expressed in mammary epithelial NMuMG cells and these cells are highly motile and invasive. Furthermore, HMGA2 remodels the chromatin to favour binding of de novo DNA methyltransferase 3A (DNMT3A) to the Cdh1 promoter. E-cadherin expression could be restored after treatment with the DNA de-methylating agent 5-aza-2′-deoxycytidine. Here, we describe a new epigenetic role for HMGA2, which follows the actions that HMGA2 initiates via the EMT-TFs, thus achieving sustained silencing of E-cadherin expression and promoting tumour cell invasion.

Epidemiology of Adults AML in Nord-Pas De Calais and Picardy

IntroductionAML is a heterogeneous group of neoplasm according to leukemogenic mechanisms and has variable response to treatment. In addition to age and WBC count at diagnosis, cytogenetic and molecular abnormalities are key factors to assess prognosis. In this work, we evaluated the number of adult AML cases diagnosed between 2009 and 2012 in the French Nord Pas de Calais and Picardie regions (North of France). The aim of the study was the collection of epidemiology data, treatments proposed and their results in real life.Matérials and MethodsThe Nord Pas de Calais and Picardie observatory contains data of all adult patients (>18 years) with AML (>20% marrow blasts) diagnosed in the oncology and/or hematology departments of the following participating Hospitals: Amiens (A), Arras (Ar), Boulogne (B), Dunkerque (D), Lens (L), Lille (CHRU et CH St Vincent (Vi)), Roubaix (R) et Valenciennes (V), whose overall catchment population is close to 6 million inhabitants , allowing a population based registry. A disadvantage of registry data is that investigators cannot control data collection and quality. In our observatory, data were obtained from the declaration of all the participants centralized with a web access reporting data base. The declaration of patients in the database was made both by clinicians, cytogeneticists and molecular biologists. The following data were entered:, disease course, WBC counts, bone marrow blast %, cytogenetics, immunophenotype, molecular biology, treatment received, outcome. To optimize data quality, each kind of data was entered respectively by clinicians, cytogeneticists and molecular biologists. Data were cross validated and the follow up data were updated every years during follow-up of the patients. Data was collected as of October 2013ResultsBetween January 2009 and December 2012, 899 patients were enrolled in the observatory, respectively 185 (A), 33 (Ar), 43 (B), 66 (D), 85 (L), 197 (CHRU), 57 (Vi), 98 (R) and 135 (V). Median age was 67 years (range 17 to 97), 54% of patients were older than 65 years, M/F was 1.12 (425 female (47%) et 474 male (53%)). Cytogenetic studies were performed in 87% of the patients, immunophenotype in 57%, molecular biology screening for FLT3 and NPM1 mutation in 61%.Intent of treatment was conventional chemotherapy (CTi) 50%, demethylating agent (DMT) 18% and best supportive care (BSC) 32% + Low dose cytarabine (LDAC). The overall median survival was 269 days (624d < 65 y and 115d for > 65 y) (fig1). 15% of the patients (132pts) were included in a clinical trial (90 % <65 y and 10% >65 y). Median overall survival of the patients included in a clinical trial and aged <65y was 1121d vs 563d for patients not included in a clinical trial and aged <65y (p:ns).Of the 482 patients aged >65 y, MRC cytogenetic classification was unfavourable (139 pts; 29%), intermediate (209 pts; 43%), favourable (18 pts; 4%) and not done 116 pts (24%). Overall, in pts aged > 65, treatment was CTI 93 pts (19%), DMT 130 pts (27%), BSC 210 pts (44%), LDAC) 38 pts (8%), others 11 pts (2%). Among pts with unfavourable karyotype treatment was: Cti (14%), DMT (45%), LDAC (2%), BSC (38%) and others (1%). overall median survival in this unfavourable group was 222d with DMT vs 104d with LDAC vs 227d with CTI. In intermediate cytogenetics cases, treatment was Cti (24%), DMT (19%), LDAC (14%), BSC (42%) and other (1%), and median OS was 270d with DMT vs 189d with ARAC-LD vs 378d with Cti. Demethylating agent treatment results were different between MRC groups. In the intermediate MRC group of pts >65y, OS was better with CTI vs DMT (p:0.03), whereas in the unfavorable pts group aged >65y/o, DMT treatment seemed to be as effective than Cti (fig 2 and 3).ConclusionThis registry study gives a real life idea of the therapeutic intent in AML patients, stressing in particular the small proportion of patients who can be included in clinical trials due to comorbidity and also the small number of available clinical trials in elderly patients, due to the current lack of promising drug apart from IC (among pts>65y/o only 13 pts have been included). Our observatory shows that in pts >65 y/o the intent of treatment should take account MRC classification, CTI being more effective that DMT or LDAC only in intermediate cases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Randomised Assessment of Vosaroxin Monotherapy and Vosaroxin Combined with Low Dose Ara-C Versus Low Dose Ara-C in Older Patients with Acute Myeloid Leukaemia

The older patient with AML represents an important subgroup who are often not considered fit for traditional “3+7” type chemotherapy. Although demethylation agents and low dose Ara-c (LDAC) are superior to best supportive care, other options have so far failed to improve survival which is typically 3-4 months. Much of the problem could be attributed to chemoresistance of the leukaemic clone.Vosaroxin is a first in class anti-cancer quinolone which by intercalating into DNA has topoisomerase II inhibitory properties and can by-pass resistance mediated by P-glycoprotein or P53. This results in less free radical production, which has been associated with anthracycline toxicity. In preliminary studies in relapsed/refractory patients >70 yrs, remission rates of up to 30% were observed, with a low 30 day mortality of <10%. In preclinical testing we documented synergy with Ara-C. It was therefore considered to be a candidate treatment in our “Pick a Winner” trial design either alone or combined with LDAC.Two parallel studies. Study 1 (02/2012-11/2013) 104 untreated patients with AML or high risk MDS (>10% marrow blasts) equally randomised between Vosaroxin and LDAC. Study 2 (06/2012-08/2013) 104 patients equally randomised between Vosaroxin + LDAC vs LDAC. Treatment plan was for 4 courses of allocated therapy with the option to extend for patient benefit.Study 1: Median age 75 (60-89); 8/104 had MDS, 31/104 had secondary AML; the risk categories were 3 favourable, 37 intermediate and 64 poor. Study 2 Median age 75(60-91); 12/104 had MDS, 27/104 had secondary AML; 1 fav, 41 int and 62 poor. There were no significant differences between randomised arms. There was no cardiac/renal co-morbidity restriction to entry in either study. The median number of courses delivered (investigational vs LDAC) Study 1(1.0 vs 2.5); Study 2(1.0 vs 2.0). Vosaroxin was 75mg/m2 IV infusion on days 1 and 4. LDAC as 20mg twice a day SC days 1-10. Toxicities NCICTC v.3.RESULTS:Study 1: 28% had a complete marrow response (CR 15%; CRi 13%), with no difference between LDAC (CR 16%; CRi 14%) and Vosaroxin (CR 15%; Cri 11%). The 60 day mortality was greater in the Vosaroxin arm (38% vs 20%; HR 2.16 (1.05-4.43) p=0.04), with increased oral and GI toxicity, more resource use, antibiotics days and hospitalisation. Of those who did not respond, survival on the Vosaroxin arm was worse (1.9 vs 4.9mo: HR 1.68(1.04-2.71), p=0.03). In the responders the OS from response was better on LDAC (17.5 vs 6.9 mo, p=0.04). There was a trend for a better RFS in the LDAC arm (30% vs 8%). Because of the increased early mortality and inferior RFS the OS at 12 mo on vosaroxin was worse than LDAC (12% vs 31%; HR 1.94(1.26-3.00), p=0.003) with a median OS of 3.2 vs 9.0 mo respectively. Cause of death (Vosa vs LDAC: resistant 21 vs 23; infection 10 vs 8; recurrent disease 6 vs 5; cardiac 3 vs 0; pulmonary 1 vs 1; haemorrhage 1 vs 0; renal 0 vs 1; multiple 4 vs 3; other/unknown 2 vs 1).Study 2: 36% response (CR 22%; CRi 14%), no significant difference between LDAC (CR 20%; CRi 14%) and LDAC+ Vosaroxin (CR 25%; CRi 13%). The day 60 mortality was greater on the combo arm (36% vs 18% HR 2.12 (1.01-4.45), p=0.05). Of the non-responders those on the combo arm had worse survival (1.5 vs 4.1 mo; HR 1.92 (1.1-3.31), p=0.02). Survival of responders was not significantly different (median not reached vs 10.1 mo). No relapsed patient survived beyond 9 mo, although survival was better in the LDAC compared to the combo arm (8.8 vs 2.7 mo, p=0.05). The inferior outcome for patients on the combo arm who did not achieve CR or relapsed resulted in non-significantly worse OS (9.6 vs 3.1 mo HR 1.30 (0.81-2.07), p=0.3). Cause of death (Vosa+LDAC vs LDAC: resistant 16 vs 19; infection 9 vs 10; infection+haemorrhage 1 vs 0; recurrent disease 4 vs 4; cardiac 2 vs 0 other cancer 1 vs 0; renal 1 vs 0; other/unk 3 vs 1). In exploratory subgroup analysis we found no evidence of any subgroup that showed a survival benefit. According to the rules of Pick a Winner there had to be evidence of a superior remission rate; while this was passed the increased 60-day mortality and non-significantly worse survival (p=0.10 at time of DMEC meeting) led to a recommendation for closure.Conclusion: In spite of the attributes of Vosaroxin, we failed to find benefit in unselected patients classified as unfit for intensive chemotherapy, either as monotherapy, or in combination with LDAC.Acknowledgments: We are grateful to Sunesis for supporting this Investigator Initiated Study. DisclosuresNo relevant conflicts of interest to declare.

SHH Inhibitor PF-0449913 in Vitro Remodels Leukemic Niche into HSC Supportive Niche

BackgroundAcute myelogenous leukemia (AML) is a highly lethal disease with only 20% of 5 years survival. Most AML patients experience relapse usually leading to death. Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Growing evidence indicates that in the development of myeloid malignancies deregulation of stem cells activity is as important as deregulation of the BM microenvironment. Sonic hedgehog (SHH) pathway molecules in general and SMO in particular are well known to maintain the growth of cancer cells. However, it’s role in leukemia niche-stem cell interaction is not well defined. Therefore, here we investigate: 1/ how the small molecule, SMO antagonist, PF-0449913 impacts the AML BM microenvironment and 2/ how in turn, changes in the activity of AML BM niche cells contribute to its remodeling into HSC supportive niche.MethodsCD34- cell from bone marrow patients undergoing hip replacement surgery (normal BM) as well as AML bone marrow were utilized for the development of the primary human stromal monolayers. We investigated stromal cultures of primary normal BM (NBM, n=6) and AML (n=6). As a control human normal bone marrow stromal cell line HS-5 was used. Then human CD34+ cells were selected from AML primary samples (n=6). As a normal control, CD34+ cells from cord blood (CB, n=5), or NBM (n=5) were utilized for the co-culture experiments for up to 9 weeks and then plated in survival and self-renewal assays. PF-0449913 was added to stromal co-cultures or, as a pre-treatment, directly to stromal monolayers.ResultsIn order to examine the role of SMO regulation in LSC generation and maintenance, primary AML (n=6) or cord blood (n=3) CD34+ cells were co-cultures with normal BM (n=3) or AML (n=3) stroma in the presence of novel small molecule inhibitor (PF-0449913) for 2 weeks. It significantly reduced LSC survival and self-renewal, while spared HSC. These effects were not observed in the absence of stroma. Importantly, pre-treatment of the AML stroma with PF-0449913 for 1 week prior- to co-culture also resulted in LSC’s inhibition of survival and self-renewal.AML- and normal BM-derived stroma differ in their ability to support HSC and LSC: LSC (n=6) were capable to self-renew after 9 weeks of co-culture with both normal and AML stroma, while cord blood (n=4) as well as normal BM (n=5) HSC lost their self-renewal potential after only 2 weeks of co-culture with the AML stroma. PF-0449913 induced changes in AML stroma. which was previously unsupportive to HSC survival, it became compliant : self-renewal of HSC increased more than 3 times. It reversed also the inhibitory effect of AML stroma-derived conditioned media (CM).Pre-treatment of the HS-5 cells with CM from AML stroma for 4 weeks prior to co-culture experiments led to significant inhibition of the cord blood (n=3) and NBM (n=3) HSC survival and self-renewal. However, in CM obtained from AML stroma treated with PF-0449913 that effect was reversed.Combined treatment of AML cells (n=6) with PF-0449913 and de-methylating agent Vidaza (5-azacytodine) or CXCR4 antagonist AMD3100 resulted in even greater inhibition of LSC survival and self-renewal. However, Vidaza did not mediate changes in AML stroma.ConclusionsTogether these data indicate that, while both conditioned media and co-culture with AML stroma impaired HSC survival and self-renewal, co-culture conditions resulted in a greater reduction in survival and self-renewal capacity, implicating that cell-cell contact or unstable secreted factors exacerbate the effects. That suggests that microenvironmental cues play a key role in regulating normal HSC versus LSC survival and maintenance, and leukemic stroma exibit severely compromised ability to maintain normal HSCs, but effectively support LSCs. PF-0449913treatment of AML induces not only eradication of LSC, but also mediate changes in AML stroma, supporting HSC generation and maintenance.Simultaneous targeting of both AML niche and LSC could represent a novel avenue for treatment of AML patients. DisclosuresNo relevant conflicts of interest to declare.

Promoter methylation-mediated silencing of β-catenin enhances invasiveness of non-small cell lung cancer and predicts adverse prognosis.

β-Catenin plays dual role in adhesion complex formation and the Wnt signaling pathway. Although β-catenin expression appears to be upregulated and Wnt signaling pathway is activated in the majority of cancers, its expression level seems to be lost in non-small cell lung cancer (NSCLC). We previously reported that the promoter of β-catenin was hypermethylated in two NSCLC cell lines. In the current study, we expanded our analysis for the methylation status of β-catenin promoter region and its protein expression in seven NSCLC cell lines and a series of 143 cases of primary human lung cancer with adjacent non-neoplastic tissues. Quantitative methylation specific PCR (qMSP) analysis showed methylation of β-catenin promoter region in five NSCLC cell lines, with increased β-catenin protein levels upon 5′-Aza-2′-deoxycytidine (5-aza-dC) treatment. The methylation status in SPC (methylated) and A549 (unmethylated) was confirmed by bisulfite sequencing PCR. 5-Aza-dC treatment inhibited invasiveness of SPC but not A549. Immunofluorescence analysis showed membranous β-catenin expression was lost in SPC and could be re-established by 5-aza-dC, while Wnt3a treatment led to nuclear translocation of β-catenin in both SPC and A549. Dual-luciferase assays indicated that 5-aza-dC treatment caused no significant increase in Wnt signaling activity compared with Wnt3a treatment. The effect of demethylation agent in SPC can be reversed by β-catenin depletion but not E-cadherin depletion which indicated that the methylation mediated β-catenin silencing might enhance NSCLC invasion and metastasis in an E-cadherin independent manner. Subsequent immunohistochemistry results further confirmed that β-catenin promoter hypermethylation correlated with loss of immunoreactive protein expression, positive lymph node metastasis, high TNM stage and poor prognosis. The present study implicates β-catenin promoter hypermethylation in the mechanism of epigenetic changes underlying NSCLC metastasis and progression, thus indicating the potential of β-catenin as a novel epigenetic target for the treatment of NSCLC patients.

Epigenetic therapy for colorectal cancer.

Aberrations in epigenome that include alterations in DNA methylation, histone acetylation, and miRNA (microRNA) expression may govern the progression of colorectal cancer (CRC). These epigenetic changes affect every phase of tumor development from initiation to metastasis. Since epigenetic alterations can be reversed by DNA demethylating and histone acetylating agents, current status of the implication of epigenetic therapy in CRC is discussed in this article. Interestingly, DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) have shown promising results in controlling cancer progression. The information provided here might be useful in developing personalized medicine approaches.