Stability of gene expression and epigenetic profiles highlights the utility of patient-derived paediatric acute lymphoblastic leukaemia xenografts for investigating molecular mechanisms of drug resistance.

BACKGROUND: Patient-derived tumour xenografts are an attractive model for preclinical testing of anti-cancer drugs. Insights into tumour biology and biomarkers predictive of responses to chemotherapeutic drugs can also be gained from investigating xenograft models. As a first step towards examining the equivalence of epigenetic profiles between xenografts and primary tumours in paediatric leukaemia, we performed genome-scale DNA methylation and gene expression profiling on a panel of 10 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) tumours that were stratified by prednisolone response. RESULTS: We found high correlations in DNA methylation and gene expression profiles between matching primary and xenograft tumour samples with Pearson's correlation coefficients ranging between 0.85 and 0.98. In order to demonstrate the potential utility of epigenetic analyses in BCP-ALL xenografts, we identified DNA methylation biomarkers that correlated with prednisolone responsiveness of the original tumour samples. Differential methylation of CAPS2, ARHGAP21, ARX and HOXB6 were confirmed by locus specific analysis. We identified 20 genes showing an inverse relationship between DNA methylation and gene expression in association with prednisolone response. Pathway analysis of these genes implicated apoptosis, cell signalling and cell structure networks in prednisolone responsiveness. CONCLUSIONS: The findings of this study confirm the stability of epigenetic and gene expression profiles of paediatric BCP-ALL propagated in mouse xenograft models. Further, our preliminary investigation of prednisolone sensitivity highlights the utility of mouse xenograft models for preclinical development of novel drug regimens with parallel investigation of underlying gene expression and epigenetic responses associated with novel drug responses.

Hypermethylation and down-regulation of DLEU2 in paediatric acute myeloid leukaemia independent of embedded tumour suppressor miR-15a/16-1.

BACKGROUND: Acute Myeloid Leukaemia (AML) is a highly heterogeneous disease. Studies in adult AML have identified epigenetic changes, specifically DNA methylation, associated with leukaemia subtype, age of onset and patient survival which highlights this heterogeneity. However, only limited DNA methylation studies have elucidated any associations in paediatric AML. METHODS: We interrogated DNA methylation on a cohort of paediatric AML FAB subtype M5 patients using the Illumina HumanMethylation450 (HM450) BeadChip, identifying a number of target genes with p <0.01 and Δß >0.4 between leukaemic and matched remission (n = 20 primary leukaemic, n = 13 matched remission). Amongst those genes identified, we interrogate DLEU2 methylation using locus-specific SEQUENOM MassARRAY® EpiTYPER® and an increased validation cohort (n = 28 primary leukaemic, n = 14 matched remission, n = 17 additional non-leukaemic and cell lines). Following methylation analysis, expression studies were undertaken utilising the same patient samples for singleplex TaqMan gene and miRNA assays and relative expression comparisons. RESULTS: We identified differential DNA methylation at the DLEU2 locus, encompassing the tumour suppressor microRNA miR-15a/16-1 cluster. A number of HM450 probes spanning the DLEU2/Alt1 Transcriptional Start Site showed increased levels of methylation in leukaemia (average over all probes >60%) compared to disease-free haematopoietic cells and patient remission samples (<24%) (p < 0.001). Interestingly, DLEU2 mRNA down-regulation in leukaemic patients (p < 0.05) was independent of the embedded mature miR-15a/16-1 expression. To assess prognostic significance of DLEU2 DNA methylation, we stratified paediatric AML patients by their methylation status. A subset of patients recorded methylation values for DLEU2 akin to non-leukaemic specimens, specifically patients with sole trisomy 8 and/or chromosome 11 abnormalities. These patients also showed similar miR-15a/16-1 expression to non-leukaemic samples, and potential improved disease prognosis. CONCLUSIONS: The DLEU2 locus and embedded miRNA cluster miR-15a/16-1 is commonly deleted in adult cancers and shown to induce leukaemogenesis, however in paediatric AML we found the region to be transcriptionally repressed. In combination, our data highlights the utility of interrogating DNA methylation and microRNA in combination with underlying genetic status to provide novel insights into AML biology.

Gene-environment interaction in problematic substance use: interaction between DRD4 and insecure attachments.

To investigate the combined effect of an exon III variable number tandem repeat in the dopamine receptor gene (DRD4) and insecure attachment style on risk for tobacco, cannabis and alcohol use problems in young adulthood. It was hypothesized that (1) individuals with 5, 6, 7 or 8 repeats (labelled 7R+) would be at increased risk for problematic drug use, and (2) risk for drug use would be further increased in individuals with 7R+ repeats who also have a history of insecure parent-child attachment relations. Data were drawn from the Victorian Adolescent Health Cohort Study, an eight-wave longitudinal study of adolescent and young adult development. DRD4 genotypes were available for 839 participants. Risk attributable to the combined effects of 7R+ genotype and insecure attachments was evaluated within a sufficient causes framework under the assumptions of additive interaction using a two-by-four table format with a common reference group. 7R+ alleles were associated with higher tobacco, cannabis and alcohol use (binging). Insecure attachments were associated with higher tobacco and cannabis use but lower alcohol use. For tobacco, there was evidence of interaction for anxious but not avoidant attachments. For cannabis, there was evidence of interaction for both anxious and avoidant attachments, although the interaction for anxious attachments was more substantial. There is no evidence of interaction for binge drinking. Results are consistent with a generic reward deficit hypothesis of drug addiction for which the 7R+ disposition may play a role. Interaction between 7R+ alleles and attachment insecurity may intensify risk for problematic tobacco and cannabis use.

Paediatric leukaemia DNA methylation profiling using MBD enrichment and SOLiD sequencing on archival bone marrow smears.

BACKGROUND: Acute Lymphoblastic Leukaemia (ALL) is the most common cancer in children. Over the past four decades, research has advanced the treatment of this cancer from a less than 60% chance of survival to over 85% today. The causal molecular mechanisms remain unclear. Here, we performed sequencing-based genomic DNA methylation profiling of eight paediatric ALL patients using archived bone marrow smear microscope slides. FINDINGS: SOLiD™ sequencing data was collected from Methyl-Binding Domain (MBD) enriched fractions of genomic DNA. The primary tumour and remission bone marrow sample was analysed from eight patients. Four patients relapsed and the relapsed tumour was analysed. Input and MBD-enriched DNA from each sample was sequenced, aligned to the hg19 reference genome and analysed for enrichment peaks using MACS (Model-based Analysis for ChIP-Seq) and HOMER (Hypergeometric Optimization of Motif EnRichment). In total, 3.67 gigabases (Gb) were sequenced, 2.74 Gb were aligned to the reference genome (average 74.66% alignment efficiency). This dataset enables the interrogation of differential DNA methylation associated with paediatric ALL. Preliminary results reveal concordant regions of enrichment indicative of a DNA methylation signature. CONCLUSION: Our dataset represents one of the first SOLiD™MBD-Seq studies performed on paediatric ALL and is the first to utilise archival bone marrow smears. Differential DNA methylation between cancer and equivalent disease-free tissue can be identified and correlated with existing and published genomic studies. Given the rarity of paediatric haematopoietic malignancies, relative to adult counterparts, our demonstration of the utility of archived bone marrow smear samples to high-throughput methylation sequencing approaches offers tremendous potential to explore the role of DNA methylation in the aetiology of cancer.

Epigenetic deregulation in pediatric acute lymphoblastic leukemia.

Similar to most cancers, genome-wide DNA methylation profiles are commonly altered in pediatric acute lymphoblastic leukemia (ALL); however, recent observations highlight that a large portion of malignancy-associated DNA methylation alterations are not accompanied by related gene expression changes. By analyzing and integrating the methylome and transcriptome profiles of pediatric B-cell ALL cases and primary tissue controls, we report 325 genes hypermethylated and downregulated and 45 genes hypomethylated and upregulated in pediatric B-cell ALL, irrespective of subtype. Repressed cation channel subunits and cAMP signaling activators and transducers are overrepresented, potentially indicating a reduced cellular potential to receive and propagate apoptotic signals. Furthermore, we report specific DNA methylation alterations with concurrent gene expression changes within individual ALL subtypes. The ETV6-RUNX1 translocation was associated with downregulation of ASNS and upregulation of the EPO-receptor, while Hyperdiploid patients (> 50 chr) displayed upregulation of B-cell lymphoma (BCL) members and repression of PTPRG and FHIT. In combination, these data indicate genetically distinct B-cell ALL subtypes contain cooperative epimutations and genome-wide epigenetic deregulation is common across all B-cell ALL subtypes.

A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia.

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies.