Specific promoter methylation identifies different subgroups of MLL-rearranged infant acute lymphoblastic leukemia, influences clinical outcome, and provides therapeutic options

Abstract

MLL-rearranged infant acute lymphoblastic leukemia (ALL) remains the most aggressive type of childhood leukemia, displaying a unique gene expression profile. Here we hypothesized that this characteristic gene expression signature may have been established by potentially reversible epigenetic modifications. To test this hypothesis, we used differential methylation hybridization to explore the DNA methylation patterns underlying MLLrearranged ALL in infants. The obtained results were correlated with gene expression data to confirm gene silencing as a result of promoter hypermethylation. Distinct promoter CpG island methylation patterns separated different genetic subtypes of MLL-rearranged ALL in infants. MLL translocations t(4;11) and t(11;19) characterized extensively hypermethylated leukemias, whereas t(9;11)-positive infant ALL and infant ALL carrying wildtype MLL genes epigenetically resembled normal bone marrow. Furthermore, the degree of promoter hypermethylation among infant ALL patients carrying t(4; 11) or t(11;19) appeared to influence relapse-free survival, with patients displaying accentuated methylation being at high relapse risk. Finally, we show that the demethylating agent zebularine reverses aberrant DNA methylation and effectively induces apoptosis in MLLrearranged ALL cells. Collectively these data suggest that aberrant DNA methylation occurs in the majority of MLLrearranged infant ALL cases and guides clinical outcome. Therefore, inhibition of aberrant DNA methylation may be an important novel therapeutic strategy for MLL-rearranged ALL in infants. (Blood. 2009;114:5490-5498) IntroductionAlthough long-term survival rates in childhood acute lymphoblastic leukemia (ALL) exceed 80%, 1 the survival chances of infants (Ͻ 1 year of age) still range between 20% and 50%. 2 Approximately 80% of infants with ALL carry chromosomal translocations involving the MLL (mixed lineage leukemia) gene, 3 fusing the N-terminal portion of the MLL gene to the C-terminal region of one of its translocation partner genes. The most frequent MLL translocations among infant ALL patients are t(4;11), t(11;19), and t(9;11), 2,4 giving rise to the fusion proteins MLL-AF4, MLL-ENL, and MLL-AF9. These chimeric MLL fusion proteins exhibit pronounced transforming capacities 5 and independently contribute to an unfavorable prognosis. 2,6 As a member of the trithorax gene family, MLL is involved in transcriptional regulation. 7 Therefore, structural alterations of this gene may be expected to affect its function, presumably leading to transcriptional deregulation. Not surprisingly, in recent gene expression profiling studies, 8,9 the authors characterized MLL-rearranged ALL as a unique type of leukemia that is genetically clearly separable from other ALL subtypes. Because epigenetic modifications affect gene expression patterns, 10 we hypothesized that the specific gene expression profiles associated with MLL-rearranged infant ALL may well be driven by epigenetic changes, which recently have been established to play important role...

MicroRNAs in acute leukemia: from biological players to clinical contributors

“…86 Moreover high levels of DNMT1 may be responsible for the genome-wide hypermethylation that characterizes t(4;11)/AF4-MLL-positive ALL and was linked to an increased risk of relapse. 72 None of the reactivated miRNAs was associated with DNA hypermethylation in adult or pediatric non-MLL precursor B-ALL. Their hypermethylation-related reduced expression levels are therefore specific for MLL-rearranged ALL.…”

“…The locus of the precursor of miR-126/miR-126* is embedded in a 287-bp CpG island and the high expression of 21 Similarly, high expression of miR-196b involved in leukemogenesis was associated with CpG island hypomethylation of the promoter region of the miR-196b/HOXA locus in MLL-rearranged ALL, which is on itself remarkable because MLL-rearranged ALL is characterized by genome-wide CpG island hypermethylation. 57,72 The MLL fusion product, however, may be directly involved in the mechanism underlying the transcriptional activation of the miR-196b/HOXA locus as it has been demonstrated that this fusion product recruits DOT1L histone methyltransferase leading to dimethylation of histone H3 lysine 79 residues (H3K79me2). This H3K79 dimethylation allows further chromatin remodelling and opens up the entire HOXA cluster including the embedded miR-196b gene.…”

Connectivity mapping identifies HDAC inhibitors for the treatment of t(4;11)-positive infant acute lymphoblastic leukemia

“…12 Detailed procedures are described in the Supplementary Methods. The genome-wide DNA methylation data has been deposited in the NCBI Gene Expression Omnibus 18 under GEO Series accession number GSE18400 as part of our recently published paper on DNA methylation patterns in MLL-rearranged infant ALL.…”

“…The genome-wide DNA methylation data has been deposited in the NCBI Gene Expression Omnibus 18 under GEO Series accession number GSE18400 as part of our recently published paper on DNA methylation patterns in MLL-rearranged infant ALL. 12 Gene expression profiling using affymetrix GeneChips Gene expression profiles (Affymetrix (Santa Clara, CA, USA), HGU133plus2.0 GeneChips) were generated for t(4;11)-positive infant ALL cases (n ¼ 15) and healthy pediatric bone marrow samples (n ¼ 7), using the same samples for which DNA methylation profiles were produced. The exact methodology is described elsewhere, 10 and briefly outlined in the Supplementary Methods.…”

“…11 In addition, we recently demonstrated that t(4;11)-positive infant ALL is further characterized by the presence of distinct genome-wide DNA methylation patterns, displaying hypermethylation at multiple gene promoters leading to transcriptional silencing of the associated genes. 12 In the present study, we revisited our DNA methylation profiles and found that, besides vast amounts of hypermethylated genes, a number of genes appeared to be inaccurately hypomethylated. As similar analyses in healthy bone marrow samples revealed that these genes normally remain methylated and suppressed during hematopoiesis, hypomethylation of these genes in t(4;11)-positive infant ALL seems to represent leukemia-specific deregulation of transcription.…”