Modifications in chromatin, including DNA methylation and histone modification, are known to be important epigenetic determinants of gene transcription. DNA methylation levels fluctuate markedly in early mouse development. In preimplantation development, the mouse embryo undergoes active and passive genomic demethylation (3,15). This is restored at the time of implantation by the combined action of de novo and maintenance DNA methyltransferases (Dnmts). Studies of DNA methyltransferase 1-deficient (Dnmt1 Ϫ/Ϫ ) and Dnmt3a/ Dnmt3b-deficient (Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ]) mouse embryos have demonstrated that restoring DNA methylation is essential for development (13,19). Dnmt1 Ϫ/Ϫ and Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] embryos exhibit an early-lethal phenotype. At day 9.5 postcoitus, the embryos appear to have gastrulated but exhibit marked growth delay, having failed to turn or develop somites. In the presence of Dnmt1 deficiency, development is thought to fail because of cell death. Dnmt1-deficient embryoid bodies (EBs) aberrantly express Xist, down-regulate X-linked genes, and apoptose when induced to differentiate (20). Late-passage hypomethylated Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] embryonic stem (ES) cells are unable to form teratomas in vivo, but the cause of their differentiation failure has not been studied (4).Early embryonic development is characterized by high levels of Dnmt3a and Dnmt3b expression. These enzymes clearly have roles in initiating remethylation of the genome following preimplantation demethylation, but it is not known whether continued de novo methyltransferase activity is required for development once global remethylation has taken place. This was our reason for studying the differentiation of Dnmt[3a Ϫ/Ϫ , 3b Ϫ/Ϫ ] ES cells in vitro. These mutant ES cells were derived from fully methylated wild-type ES cells and would have been predicted to have retained most of their methylation because of the continued presence of the maintenance methyltransferase Dnmt1. In fact, while early-passage Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells are well methylated, DNA methylation levels fall progressively in culture (4). However, the rate of loss and the precise levels of methylation remaining have not been quantified. We have used a quantitative assay of DNA methylation to examine the effects of progressively decreasing genomic methylation levels on differentiation in vitro. Our studies reveal a clear but unexpected difference between the behaviors of hypomethylated Dnmt1 Ϫ/Ϫ and Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells in in vitro assays of differentiation. At very low levels of DNA methylation, Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells demonstrate an inability to initiate differentiation upon leukemia inhibitory factor (LIF) withdrawal, remaining viable and retaining markers characteristic of undifferentiated ES cells. MATERIALS AND METHODSES cell culture. ES cells were maintained on gelatin in a Glasgow modification of Eagle medium (Invitrogen) supplemented with 10% fetal calf serum, 100 M 2-mercaptoethanol, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 ...

Background:Multiple myeloma is a plasma cell disorder that is characterised by clonal proliferation of malignant plasma cells in the bone marrow, monoclonal paraprotein in the blood or urine and associated organ dysfunction. It accounts for approximately 1% of cancers and 13% of haematological cancers. Myeloma arises from an asymptomatic proliferation of monoclonal plasma cells termed monoclonal gammopathy of undetermined significance (MGUS).Methods:MicroRNA expression profiling of serum samples was performed on three patient groups as well as normal controls. Validation of the nine microRNAs detected as promising biomarkers was carried out using TaqMan quantitative reverse transcription PCR. MicroRNA levels in serum were normalised using standard curves to determine the numbers of microRNAs per μl of serum.Results:Three serum microRNAs, miR-720, miR-1308 and miR-1246, were found to have potential as diagnostic biomarkers in myeloma. Use of miR-720 and miR-1308 together provides a powerful diagnostic tool for distinguishing normal healthy controls, as well as patients with unrelated illnesses, from pre-cancerous myeloma and myeloma patients. In addition, the combination of miR-1246 and miR-1308 can distinguish MGUS from myeloma patients.Conclusion:We have developed a biomarker signature using microRNAs extracted from serum, which has potential as a diagnostic and prognostic tool for multiple myeloma.

• Data from Dnmt3a2/2 mice implicate Dot1l as a critical mediator of the malignant gene expression program of Dnmt3a-mediated leukemia.• Pharmacologic inhibition of DOT1L exerts potent antileukemic activity in DNMT3A-mutant human acute myeloid leukemia in vitro and in vivo.Mutations in DNA methyltransferase 3A (DNMT3A) are common in acute myeloid leukemia and portend a poor prognosis; thus, new therapeutic strategies are needed. The likely mechanism by which DNMT3A loss contributes to leukemogenesis is altered DNA methylation and the attendant gene expression changes; however, our current understanding is incomplete. We observed that murine hematopoietic stem cells (HSCs) in which Dnmt3a had been conditionally deleted markedly overexpress the histone 3 lysine 79 (H3K79) methyltransferase, Dot1l. We demonstrate that Dnmt3a 2/2 HSCs have increased H3K79 methylation relative to wild-type (WT) HSCs, with the greatest increases noted at DNA methylation canyons, which are regions highly enriched for genes dysregulated in leukemia and prone to DNA methylation loss with Dnmt3a deletion. These findings led us to explore DOT1L as a therapeutic target for the treatment of DNMT3A-mutant AML. We show that pharmacologic inhibition of DOT1L resulted in decreased expression of oncogenic canyon-associated genes and led to dose-and time-dependent inhibition of proliferation, induction of apoptosis, cell-cycle arrest, and terminal differentiation in DNMT3A-mutant cell lines in vitro. We show in vivo efficacy of the DOT1L inhibitor EPZ5676 in a nude rat xenograft model of DNMT3A-mutant AML. DOT1L inhibition was also effective against primary patient DNMT3A-mutant AML samples, reducing colony-forming capacity (CFC) and inducing terminal differentiation in vitro. These studies suggest that DOT1L may play a critical role in DNMT3A-mutant leukemia. With pharmacologic inhibitors of DOT1L already in clinical trials, DOT1L could be an immediately actionable therapeutic target for the treatment of this poor prognosis disease. (Blood. 2016;128(7):971-981)