Methylation of <i><scp>KLF</scp>5</i> contributes to reduced expression in acute myeloid leukaemia and is associated with poor overall survival (2013)

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 Krüppel-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 1). 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 3·6-fold relative to CD34+ controls, and by 1·7-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 1). 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 (<1%) homozygous (G/G). The different alleles did not significantly associate with any clinical characteristics (Table SII). Mean KLF5 expression was significantly reduced (4·1-fold) in AML samples heterozygous for the SNP genotype (SNPG/A) compared to CD34+ controls (Fig 1D), but not relative to samples with the common A/A genotype (SNPA/A, P = 0·109). Whilst this may reflect limited sample size (SNPG/A expression n = 12), it is also possible that multiple additional regulatory mechanisms contribute to reduced KLF5 expression in AML. As our data does not support a direct correlation between SNP genotype and expression, the data on SNP genotype, in relation to clinical outcome, is not presented further. Univariate analysis revealed that MeHigh was associated with significantly reduced median overall survival (OS) relative to MeLow patients (10·4 vs. 20·7 months, Fig 1E). Methylation of KLF5 had a significant effect on survival within the intermediate cytogenetic risk group (median of 12·1 vs. 31·3 months for MeHigh and MeLow respectively), but did not segregate outcome in patients with favourable or adverse cytogenetics (Fig 1F). Multivariate analysis (Table SIII) confirmed that these effects were independent of other prognostic variables including age (≥60 years), WCC (≥50 × 109/l), FLT3-internal tandem duplication (ITD)/NPM1 risk status (low risk = FLT3-ITD−/NPM1+, high risk = all other combinations), and DNMT3A mutation in AML overall (P = 0·002, hazard ratio 2·376) and in intermediate risk AML (P = 0·003, hazard ratio 3·146). This study demonstrates that hypermethylation of KLF5 intron 1 contributes to reduced KLF5 expression in AML. The differentially-methylated region is highly conserved across multiple vertebrate species (Diakiw et al, 2012), which is consistent with the presence of an uncharacterized regulatory element. Methylation was inversely associated with DNMT3A R882 mutations, raising the possibility that KLF5 intron 1 is a methylation target downstream of the de novo methyltransferase DNMT3A. While the findings in this study will need to be repeated in independent and prospective cohorts to confirm their significance, our survival analyses suggest that methylation of KLF5 intron 1 may act as a functional biomarker which could potentially be used to identify AML patients with poor prognosis, particularly in the intermediate risk patient group. Given that demethylating agents have shown some success in treatment of poor risk and relapsed AML (Thomas, 2012), it will now be of interest to assess whether KLF5 methylation may be useful in predicting response to treatment with these agents. This work was supported by grants from the National Health and Medical Research Council (NHMRC #626946), Cancer Council of South Australia, and Medvet Sciences. The authors would like to thank Silke Danner (Department of Haematology, Royal Adelaide Hospital, Australia) for her assistance in compiling clinical data for the AML cohort. RJD was the principal investigator and takes primary responsibility for the paper. SMD designed and performed research, analysed data, and wrote the paper. ALB designed and performed research, analysed data, and wrote the paper. GAE contributed to patient sample processing, and NC performed mutation analysis of the AML patients. CHK, MP, LBT, AHW, and IDL analysed data and critically reviewed the paper. AHW receives clinical trial funding from Celgene. Table SI. Distribution of clinical characteristics and genetic lesions according to KLF5 intron 1 methylation status or promoter SNP rs3812852 genotype (treated patient cohort). Table SII. Distribution of clinical characteristics and genetic lesions according to KLF5 promoter SNP rs3812852 genotype. Table SIII. Statistical analysis of overall survival according to KLF5 intron 1 methylation status and promoter SNP rs3812852 genotype. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.