ORIGINAL ARTICLE MN1 overexpression is driven by loss of DNMT3B methylation activity in inv(16) pediatric AML NSD Larmonie 1 , TCJM Arentsen-Peters 1 , A Obulkasim 1 , D Valerio 1 , E Sonneveld 2 , AA Danen-van Oorschot 1 , V de Haas 2 , D Reinhardt 3 , M Zimmermann 3 , J Trka 4 , A Baruchel 5 , R Pieters 6 , MM van den Heuvel-Eibrink 6 , CM Zwaan 1 and M Fornerod 1 In acute myeloid leukemia (AML), specific genomic aberrations induce aberrant methylation, thus directly influencing the transcriptional programing of leukemic cells. Therefore, therapies targeting epigenetic processes are advocated as a promising therapeutic tool for AML treatment. However, to develop new therapies, a comprehensive understanding of the mechanism(s) driving the epigenetic changes as a result of acquired genetic abnormalities is necessary. This understanding is still lacking. In this study, we performed genome-wide CpG-island methylation profiling on pediatric AML samples. Six differentially methylated genomic regions within two genes, discriminating inv(16)(p13;q22) from non-inv(16) pediatric AML samples, were identified. All six regions had a hypomethylated phenotype in inv(16) AML samples, and this was most prominent at the regions encompassing the meningioma (disrupted in balanced translocation) 1 (MN1) oncogene. MN1 expression primarily correlated with the methylation level of the 3′ end of the MN1 exon-1 locus. Decitabine treatment of different cell lines showed that induced loss of methylation at the MN1 locus can result in an increase of MN1 expression, indicating that MN1 expression is coregulated by DNA methylation. To investigate this methylation-associated mechanism, we determined the expression of DNA methyltransferases in inv(16) AML. We found that DNMT3B expression was significantly lower in inv(16) samples. Furthermore, DNMT3B expression correlated negatively with MN1 expression in pediatric AML samples. Importantly, depletion of DNMT3B impaired remethylation efficiency of the MN1 exon-1 locus in AML cells after decitabine exposure. These findings identify DNMT3B as an important coregulator of MN1 methylation. Taken together, this study shows that the methylation level of the MN1 exon-1 locus regulates MN1 expression levels in inv(16) pediatric AML. This methylation level is dependent on DNMT3B, thus suggesting a role for DNMT3B in leukemogenesis in inv(16) AML, through MN1 methylation regulation. Oncogene advance online publication, 11 September 2017; doi:10.1038/onc.2017.293 INTRODUCTION Approximately 30% of pediatric acute myeloid leukemia (AML) patients achieve complete remission relapse after being treated with conventional chemotherapy. 1–4 Thus, better therapies are needed. AML-associated chromosomal aberrations lead to the expression of differentiation-inhibiting fusion proteins. For exam- ple, RUNX1-RUNX1T1 fusion proteins suppress the expression of RUNX1 target genes by recruiting histone deacetylase complexes to their promoters. 5 Histone methyltransferase KMT2A (MLL) fusion proteins bind to histone methyltransferase DOT1L complexes, causing the dysregulated transcription of DOT1L complex target genes. 6–8 CBFB-MYH11 oncogenic fusion proteins, resulting from inv(16)(p13;q22) chromosomal aberrations, occupy H3 acetylation- enriched regions. 9 These and similar AML-associated chromoso- mal aberrations cause transcriptional disruption of crucial genes involved in normal myeloid cell differentiation by directly affecting the function of epigenetic regulators. Epigenetic changes are stable, heritable and reversible chro- matin modifications. Therefore, the use of compounds inhibiting epigenetic processes, for example, DNA methyltransferase (DNMT) inhibitors, in combination with conventional chemotherapy is becoming an attractive therapeutic strategy in an attempt to reverse the effect that aberrant epigenetic processes have on normal cell differentiation. 2 Thus, epigenetic-targeted therapies might be a promising solution for more effective therapy in pediatric AML. To this end, more insight into the mechanism(s) behind changes in epigenetic processes as the result of AML-causing genetic abnormalities is required. Epigenetic changes caused by aberrant DNA methylation often result in the silencing of tumor suppressor genes through the CpG-island hypermethylation of mainly promoter regions. Hence, several studies used genome-wide DNA methylation profiling, primarily in adult AML, in an attempt to delineate the role of epigenetic changes on AML pathogenesis. 10–16 In this study, we performed genome-wide CpG-island methylation profiling on pediatric AML samples. We identified specific methylation patterns discriminating inv(16) from non-inv(16) AML. The myeloid oncogene meningioma (disrupted in balanced translocation) 1 (MN1), which is overexpressed in CBFB-MYH11-expressing cells, 17,18 was identified as most significantly differentially methylated. Despite previous efforts to delineate the cause of MN1 overexpression in inv(16) AML, the driving molecular mechanism had not yet been identified. Here we show that the hypomethylation of an MN1 exon-1 region determines MN1 1 Department of Pediatric Oncology/Hematology, Erasmus MC-Sophia Children’s Hospital, Rotterdam, The Netherlands; 2 Dutch Childhood Oncology Group (DCOG), The Hague, The Netherlands; 3 Department of Pediatric Oncology/Hematology, Medical High School, Hannover, Germany; 4 Pediatric Hematology/Oncology, 2nd Medical School, Charles University, Prague, Czech Republic; 5 CHU de Paris-Hôpital Robert Debré, Paris, France and 6 Princess Maxima Center for Pediatric Oncology, Utrecht, The Netherlands. Correspondence: Dr M Fornerod, Department of Pediatric Oncology and Hematology, Erasmus MC-Sophia Children’s Hospital, Wytemaweg 80, Rotterdam 3015 CN, The Netherlands. E-mail: m.fornerod@erasmusmc.nl Received 31 August 2016; revised 9 June 2017; accepted 17 July 2017 Oncogene (2017), 1 – 9 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0950-9232/17 www.nature.com/onc