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...

). Finally, we demonstrate that RAS mutations, and not the lack of Homeobox gene A9 expression nor the expression of AF4-MLL are associated with poor outcome in t(4;11)-rearranged infants. We conclude that the presence of RAS mutations in Mixed Lineage Leukemiarearranged infant acute lymphoblastic leukemia is an independent predictor for a poor outcome. Therefore, future risk-stratification based on abnormal RAS-pathway activation and RAS-pathway inhibition could be beneficial in RAS-mutated infant acute lymphoblastic leukemia patients. Frequencies and prognostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemia in infants

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BackgroundTo investigate the etiology of MLH1 promoter methylation in mismatch repair (MMR) mutation-negative early onset MSI-H colon cancer. As this type of colon cancer is associated with high ages, young patients bearing this type of malignancy are rare and could provide additional insight into the etiology of sporadic MSI-H colon cancer.MethodsWe studied a set of 46 MSI-H colon tumors cases with MLH1 promoter methylation which was enriched for patients with an age of onset below 50 years (n = 13). Tumors were tested for CIMP marker methylation and mutations linked to methylation: BRAF, KRAS, GADD45A and the MLH1 -93G>A polymorphism. When available, normal colon and leukocyte DNA was tested for GADD45A mutations and germline MLH1 methylation. SNP array analysis was performed on a subset of tumors.ResultsWe identified two cases (33 and 60 years) with MLH1 germline promoter methylation. BRAF mutations were less frequent in colon cancer patients below 50 years relative to patients above 50 years (p-value: 0.044). CIMP-high was infrequent and related to BRAF mutations in patients below 50 years. In comparison with published controls the G>A polymorphism was associated with our cohort. Although similar distribution of the pathogenic A allele was observed in the patients with an age of onset above and below 50 years, the significance for the association was lost for the group under 50 years. GADD45A sequencing yielded an unclassified variant. Tumors from both age groups showed infrequent copy number changes and loss-of-heterozygosity.ConclusionSomatic or germline GADD45A mutations did not explain sporadic MSI-H colon cancer. Although germline MLH1 methylation was found in two individuals, locus-specific somatic MLH1 hypermethylation explained the majority of sporadic early onset MSI-H colon cancer cases. Our data do not suggest an intrinsic tendency for CpG island hypermethylation in these early onset MSI-H tumors other than through somatic mutation of BRAF.

Although B cells have been shown to be refractory to reprogramming into pluripotency, induced pluripotent stem cells (iPSCs) have been very recently generated, at very low efficiency, from human cord blood (CB)-and peripheral blood (PB)-derived CD191CD20 1 B cells using nonintegrative tetracistronic OSKM-expressing Sendai Virus (SeV). Here, we addressed whether cell ontogeny and hierarchy influence the reprogramming efficiency of the B-cell compartment. We demonstrate that human fetal liver (FL)-derived CD19 1 B cells are 110-fold easier to reprogram into iPSCs than those from CB/PB. Similarly, FL-derived CD341CD19 1 B progenitors are reprogrammed much easier than mature B cells (0.46% vs. 0.11%). All FL B-cell iPSCs carry complete VDJH rearrangements while 55% and 45% of the FL B-progenitor iPSCs carry incomplete and complete VDJH rearrangements, respectively, reflecting the reprogramming of developmentally different B progenitors (pro-B vs. pre-B) within a continuous differentiation process. Finally, our data suggest that successful B-cell reprogramming relies on active cell proliferation, and it is MYC-dependent as identical nonintegrative polycistronic SeV lacking MYC (OSKL or OSKLN) fail to reprogram B cells. The ability to efficiently reprogram human fetal primary B cells and B precursors offers an unprecedented opportunity for studying developmental B-lymphopoiesis and modeling B-cell malignances. STEM CELLS 2016;34:581-587 SIGNIFICANCE STATEMENTUsing tetracistronic OSKM-expressing nonintegrative Sendai vectors, we show that the developmental ontogeny and cellular hierarchy largely influences the reprogramming efficiency of human B cells into iPSCs. We also show that Ig/VDJH rearrangements do not constitute a reprogramming barrier and that B-cell reprogramming seems to rely on cell proliferation and it is cMYC-dependent. The ability to efficiently reprogram human fetal primary B cells and B precursors offers an unprecedented opportunity for studying developmental B-lymphopoiesis and modeling B-cell malignances.