Abnormal epigenetic regulation has been implicated in oncogenesis. We report here the identification of somatic mutations by exome sequencing in acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5). We discovered mutations in DNMT3A (encoding DNA methyltransferase 3A) in 23 of 112 (20.5%) cases. The DNMT3A mutants showed reduced enzymatic activity or aberrant affinity to histone H3 in vitro. Notably, there were alterations of DNA methylation patterns and/or gene expression profiles (such as HOXB genes) in samples with DNMT3A mutations as compared with those without such changes. Leukemias with DNMT3A mutations constituted a group of poor prognosis with elderly disease onset and of promonocytic as well as monocytic predominance among AML-M5 individuals. Screening other leukemia subtypes showed Arg882 alterations in 13.6% of acute myelomonocytic leukemia (AML-M4) cases. Our work suggests a contribution of aberrant DNA methyltransferase activity to the pathogenesis of acute monocytic leukemia and provides a useful new biomarker for relevant cases.

Older patients with newly diagnosed acute myeloid leukemia (AML) in the phase 3 AZA-AML-001 study were evaluated at entry for cytogenetic abnormalities, and a subgroup of patients was assessed for gene mutations. Patients received azacitidine 75 mg/m2/day x7 days (n = 240) or conventional care regimens (CCR; n = 245): intensive chemotherapy, low-dose cytarabine, or best supportive care only. Overall survival (OS) was assessed for patients with common (occurring in ≥10% of patients) cytogenetic abnormalities and karyotypes, and for patients with recurring gene mutations. There was a significant OS improvement with azacitidine vs CCR for patients with European LeukemiaNet-defined Adverse karyotype (HR 0.71 [95%CI 0.51–0.99]; P = 0.046). Azacitidine-treated patients with -5/5q-, -7/7q-, or 17p abnormalities, or with monosomal or complex karyotypes, had a 31–46% reduced risk of death vs CCR. The most frequent gene mutations were DNMT3A (27%), TET2 (25%), IDH2 (23% [R140, 15%; R172, 8%]), and TP53 (21%). Compared with wild-type, OS was significantly reduced among CCR-treated patients with TP53 or NRAS mutations and azacitidine-treated patients with FLT3 or TET2 mutations. Azacitidine may be a preferred treatment for older patients with AML with Adverse-risk cytogenetics, particularly those with chromosome 5, 7, and/or 17 abnormalities and complex or monosomal karyotypes. The influence of gene mutations in azacitidine-treated patients warrants further study.

The DNA methylation program of the fruit fly Drosophila melanogaster is carried out by the single DNA methyltransferase gene dDnmt2, the function of which is unknown before. We present evidence that intactness of the gene is required for maintenance of the normal life span of the fruit flies. In contrast, overexpression of dDnmt2 could extend Drosophila life span. The study links the Drosophila DNA methylation program with the small heatshock proteins and longevity/aging and has interesting implication on the eukaryotic DNA methylation programs in general.DNA methylation at cytosines of the vertebrate genomes participates in the control of several interlinked biological processes, e.g. gene expression, cell growth, genomic imprinting, X chromosome inactivation, and embryogenesis, which in turn are mediated through chromatin remodeling (1, 2). The vertebrate DNA methyltransferases, including Dnmt1, Dnmt2, Dnmt3a, Dnmt3b, and Dnmt3L, are a family of proteins with highly conserved motifs in their carboxyl regions. Of these, Dnmt1 is the maintenance enzyme. Dnmt3a and Dnmt3b carry out the de novo methylation reaction. Unlike the above three enzymes, Dnmt2 lacks the N-terminal regulatory region (2). Although recent studies have demonstrated residual DNA methylation activities of the mammalian Dnmt2 proteins and the Drosophila ortholog dDnmt2 (3, 4), the latter of which is the single DNA methyltransferase responsible for genome methylation of the fruit flies (5-10), the functional role of the Dnmt2 proteins has remained unclear. We present evidence below that dDnmt2 is a regulator of the life span of Drosophila. EXPERIMENTAL PROCEDURES Drosophila Strains and Culture Conditions-dDnmt2GS12412 stock was a kind gift from Dr. T. Aigaki at Tokyo Metropolitan University. yw strain, the recipient stock of Gene Search (GS) insertion, was used as an internal control in the longevity experiment. UAS-dDnmt2 transgene was inserted in pUAST vector as described (10). The reference background for transgenic lines was w 1118 . UAS-dDnmt2 females were crossed with the da-GAL4 males or w 1118 males, and the life span of progeny was assessed. Flies were reared at 25°C and maintained in vials containing standard cornmeal agar medium. To monitor the life span, flies eclosed within 24 h were collected in single-sex groups of 20. They were then transferred to fresh medium and scored for survivorship once every 3 days until all of the flies died.Stress Resistance-For thermal stress test, 4-day-old flies were transferred to new vials, maintained, and scored for survivorship at 36°C. To examine the effect of starvation, flies were transferred to vials containing two 2.4-cm glass-fiber filter circles (Whatman) moisturized with 350 l of distilled water. The survivals were followed at 25°C. Distilled water was added to keep the filters moist during the test. Paraquat resistance was performed as follows. Whatman glass-fiber filter circles soaked with 350 l of 20 mM paraquat (Sigma) in 5% sucrose solution were placed in clean empty vials. To minimi...

Background Further advances of targeted cancer therapy require comprehensive in-depth profiling of somatic mutations that are present in subpopulations of tumor cells in a clinical tumor sample. However, it is unclear to what extent such intra-tumor heterogeneity is present and whether it may affect clinical decision making. To unravel this challenge, we established a deep targeted sequencing platform to identify potentially actionable DNA alterations in tumor samples. Methods We assayed 515 FFPE tumor samples and matched germline (475 patients) from 11 disease sites by capturing and sequencing all the exons in 201 cancer related genes. Mutations, indels and copy number data were reported. Results We obtained a 1000-fold average sequencing depth and identified 4794 non-synonymous mutations in the samples analyzed, which 15.2% were present at less than 10% allele frequency. Most of these low level mutations occurred at known oncogenic hotspots and are likely functional. Identifying low level mutations improved identification of mutations in actionable genes in 118 (24.84%) patients, among which 47 (9.8%) would otherwise be unactionable. In addition, acquiring ultra-high depth also ensured a low false discovery rate (less than 2.2%) from FFPE samples. Conclusion Our results were as accurate as a commercially available CLIA-compliant hotspot panel, but allowed the detection of a higher number of mutations in actionable genes. Our study revealed the critical importance of acquiring and utilizing high depth in profiling clinical tumor samples and presented a very useful platform for implementing routine sequencing in a cancer care institution.