Mar 25, 2013 - Ñ1Ю. In our laboratory R2* measurements were performed using a custom-written software. The ROI included the entire liver profile in the slice, ...
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Methylation of KLF5 contributes to reduced expression in acute myeloid leukaemia and is associated with poor overall survival
Acute myeloid leukaemia (AML) is a genetically heterogeneous disease and a number of recurrent cytogenetic alterations are routinely used to stratify patients into risk groups and determine treatment options. Mutations in genes such as FLT3, NPM1, and CEBPA provide additional prognostic information, particularly within the intermediate cytogenetic risk group (Marcucci et al, 2011), however risk stratification remains imperfect and treatment strategies targeting the tumour biology associated with these lesions are still being investigated. Recently, recurrent mutations have been identified in epigenetic regulators, such as IDH1/2, TET2, and DNMT3A, and it is likely that epigenetically modified targets downstream of these mutations will provide biologically relevant markers for response to treatment with methyltransferase inhibitors, which have shown some efficacy to date in treatment of AML (Thomas, 2012). The zinc finger transcription factor Kr€ uppel-like factor 5 (KLF5) can act as a tumour suppressor which displays growth inhibitory effects in a number of solid tumours including prostate and oesophageal cancer (Dong & Chen, 2009). Accordingly, reduced expression of KLF5 has been associated with poor patient outcome in selected epithelial carcinomas (Kwak et al, 2008). In the haemopoietic system KLF5 plays a role in terminal myeloid differentiation, and we previously demonstrated hypermethylation of the KLF5 proximal promoter and intron 1 as functional mechanisms for down-regulation in AML (Humbert et al, 2011; Diakiw et al, 2012). In breast cancer, the methylation status of KLF5 intron 1 significantly correlated with overall patient survival (Kamalakaran et al, 2011), and hence we focused on this region for further analysis. KLF5 expression can also be reduced due to an A>G single nucleotide polymorphism (SNP rs3812852) in the promoter region, which is associated with increased susceptibility to schizophrenia and reduced risk of cardiac hypertension (Yanagi et al, 2008; Oishi et al, 2010). The aim of the current study was to investigate the effect of KLF5 intron 1 hypermethylation, and the genotype at SNP rs3812852, on KLF5 expression and clinical outcome in AML. Studies were performed on a retrospective cohort of 232 patients diagnosed with de novo or secondary AML at the Royal Adelaide and Queen Elizabeth Hospitals, South Australia (Table I). Bone marrow (BM) mononuclear cell samples were collected at diagnosis with approval from relevant
hospital ethics committees in accordance with the Declaration of Helsinki. 161 patients were treated using standard induction and consolidation chemotherapy protocols (Data S1, Table S1). Quantitative methylation analysis of KLF5 was performed using a matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry approach as previously described (Diakiw et al, 2012). Primer and amplicon sequences are given in Data S1. Samples with methylation of 25% were defined as hypermethylated ( 3 times the standard deviation above average methylation of normal mononuclear cell controls). The genotype at SNP rs3812852 in the KLF5 promoter (A or G allele) was determined using a MALDI-TOF mass spectrometry approach utilizing homogeneous MassExtend chemistry (Sequenom iPLEX GOLD; Australian Genome Research Facility, Brisbane, Qld, Australia). Figure 1A depicts the SNP and differentially-methylated region relative to the KLF5 genomic locus. Expression of KLF5 was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) as previously described (Diakiw et al, 2012). Assessment of genetic abnormalities and statistical analyses are outlined in Data S1. Consistent with our previous study utilizing a small test cohort (n = 27) (Diakiw et al, 2012), we found that hypermethylation of KLF5 intron 1 (MeHigh) was common in AML, with 116 of 232 patients (50%) demonstrating 25% methylation (Fig 1B). qRT-PCR performed on a subset of samples (RNA available for n = 85) demonstrated that mean KLF5 expression was significantly reduced in MeHigh samples by 36-fold relative to CD34+ controls, and by 17-fold relative to AML samples with low methylation (MeLow, Fig 1C). Methylation was inversely correlated with French-American-British subtype (M5), peripheral blood white cell count (WCC) at diagnosis, and with the most common DNMT3A mutation (R882 missense), which was 4-fold more prevalent in the MeLow group than in MeHigh patients (Table I). Of 232 AML samples, 28 patients (12%) were heterozygous for the less common G allele (G/A) at SNP rs3812852 in the KLF5 promoter, and one patient (
