The three DNA methyltransferase (DNMT)-inhibiting cytosine nucleoside analogues, azacitidine, decitabine and zebularine, which are currently studied as nonintensive therapy for myelodysplastic syndromes and acute myeloid leukemia (AML), differ in structure and metabolism, suggesting that they may have differential molecular activity. We investigated cellular and molecular effects of the three substances relative to cytarabine in Kasumi-1 AML blasts. Under in vitro conditions mimicking those used in clinical trials, the DNMT inhibitors inhibited proliferation and triggered apoptosis but did not induce myeloid differentiation. The DNMT inhibitors showed no interference with cell-cycle progression whereas cytarabine treatment resulted in an S-phase arrest. Quantitative methylation analysis of hypermethylated gene promoters and of genome-wide LINE1 fragments using bisulfite sequencing and MassARRAY suggested that the hypomethylating potency of decitabine was stronger than that of azacitidine; zebularine showed no hypomethylating activity. In a comparative gene expression analysis, we found that the effects of each DNMT inhibitor on gene transcription were surprisingly different, involving several genes relevant to leukemogenesis. In addition, the gene methylation and expression analyses suggested that the effects of DNMT-inhibiting cytosine nucleoside analogues on the cellular transcriptome may, in part, be unrelated to direct promoter DNA hypomethylation, as previously shown by others.

No abstract

Human chromosomal regions enriched in segmental duplications are subject to extensive genomic reorganization. Such regions are particularly informative for illuminating the evolutionary history of a given chromosome. We have analyzed 866 kb of Y-chromosomal non-palindromic segmental duplications delineating four euchromatin/heterochromatin transition regions (Yp11.2/Yp11.1, Yq11.1/Yq11.21, Yq11.23/Yq12, and Yq12/PAR2). Several computational methods were applied to decipher the segmental duplication architecture and identify the ancestral origin of the 41 different duplicons. Combining computational and comparative FISH analysis, we reconstruct the evolutionary history of these regions. Our analysis indicates a continuous process of transposition of duplicated sequences onto the evolving higher primate Y chromosome, providing unique insights into the development of species-specific Y-chromosomal and autosomal duplicons. Phylogenetic sequence comparisons show that duplicons of the human Yp11.2/Yp11.1 region were already present in the macaque-human ancestor as multiple paralogs located predominantly in subtelomeric regions. In contrast, duplicons from the Yq11.1/Yq11.21, Yq11.23/Yq12, and Yq12/PAR2 regions show no evidence of duplication in rhesus macaque, but map to the pericentromeric regions in chimpanzee and human. This suggests an evolutionary shift in the direction of duplicative transposition events from subtelomeric in Old World monkeys to pericentromeric in the human/ape lineage. Extensive chromosomal relocation of autosomal-duplicated sequences from euchromatin/heterochromatin transition regions to interstitial regions as demonstrated on the pygmy chimpanzee Y chromosome support a model in which substantial reorganization and amplification of duplicated sequences may contribute to speciation.

Summary A‐kinase anchor protein 12 (AKAP12) is a scaffold protein that participates in mitotic regulation and other signalling processes and probably exerts tumour suppressor function. We hypothesized that epigenetic repression of the AKAP12 gene might occur in malignant myeloid disorders. This study demonstrated that the 5′ CpG island of AKAP12 was unmethylated in normal haematopoietic progenitors and granulocytes but exhibited profound methylation in Kasumi‐1 and SKNO‐1 leukaemic myeloblasts. Correspondingly, AKAP12 was expressed in normal progenitors but transcriptionally silent in leukaemic blasts. Re‐expression of AKAP12 in Kasumi‐1 and SKNO‐1 cells was accomplished by treatment with MS275 alone or in combination with zebularine, indicating epigenetic mechanisms of gene repression. AKAP12 hypermethylation was found in one case of refractory anaemia with excess blasts (RAEB) and two cases of acute myeloid leukaemia (AML) in a panel of 21 blood or bone marrow samples from children with malignant myeloid disorders including refractory cytopenia, RAEB, juvenile myelomonocytic leukaemia and AML. While AKAP12 function has not been previously linked to leukaemogenesis, our results raise the possibility that epigenetic silencing of AKAP12 is involved in myeloid malignancies.

Cordeddu et al recently reported the discovery of a specific SHOC2 gene mutation underlying a variant of the neuro-cardio-facio-cutaneous (NCFC) syndrome family. 1 The common denominator of mutations associated with this group of disorders is their involvement in the dysregulation of the Ras-mitogen-activated protein kinase (MAPK) pathway. 2 Mutant SHOC2 undergoes aberrant N-myristoylation that results in constitutive membrane targeting. This in turn is thought to sustain RAF1-stimulated MAPK activation. 1 The Ras-MAPK pathway is central also to the pathophysiology of juvenile myelomonocytic leukemia (JMML) and related myeloproliferative neoplasms. 3 Leukemogenic perturbation of the Ras-MAPK pathway in nonsyndromic children results from somatic lesions of the same genes that cause NCFC syndromes when mutated in the germ line, as exemplified by PTPN11 4 and KRAS. 5 Moreover, some disorders of the NCFC spectrum (notably Noonan syndrome and neurofibromatosis type 1 [NF-1]) constitute a predisposition for the development of myeloproliferative neoplasms in childhood.Together, these findings provide a strong rationale to investigate the possible occurrence of somatic SHOC2 mutations in nonsyndromic JMML. Mutations affecting Ras pathway-related genes can be defined in approximately 80% of JMML cases (PTPN11 35%, KRAS/NRAS 25%, CBL 10%, NF1 11%) and are, with very few exceptions, mutually exclusive in the same patient. We performed SHOC2 mutation analyses in a cohort of 22 JMML cases preselected for the absence of mutations in PTPN11, KRAS/NRAS or CBL and without clinical NF-1 features. All children had been enrolled in the European Working Group of Myelodysplastic Syndromes in Childhood (EWOG-MDS) studies 98 or 2006, and samples had been taken after informed consent of patients' guardians. The entire SHOC2 coding sequence was analyzed by genomic sequencing in granulocyte DNA from bone marrow or peripheral blood of the 22 JMML patients. However, we discovered no pathologic sequence variations.In conclusion, we found no evidence of leukemogenic SHOC2 involvement in JMML. Although the genetic link between NCFC syndromes and JMML is well established for some Ras-MAPK pathway genes such as PTPN11 and KRAS, the absence of SHOC2 mutations in JMML underscores that this phenotypic duality is not a universal feature of all Ras-related genes. We have previously reported a similar observation for SOS1. 6 It is obvious that the leukemogenic potential of Ras-MAPK pathway mutations differs between individual genes.