The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown. Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations. A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P<0.001 for both comparisons). The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months vs. 41.1 months, P<0.001). DNMT3A mutations were associated with adverse outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 mutations, regardless of age, and were independently associated with a poor outcome in Cox proportional-hazards analysis. DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome. (Funded by the National Institutes of Health and others.)

Tetrahydrocannabinol (Δ⁹-THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UGT)-dependent glucuronidation. Many studies have shown that CYP2C9 and CYP3A4 are the primary enzymes responsible for these cytochrome P450-dependent oxidations, but little work has been done to characterize phase II metabolic pathways. In this study, we test the hypothesis that there are specific human UGTs responsible for classic cannabinoid metabolism. The activities of 12 human recombinant UGTs toward classic cannabinoids [cannabinol (CBN), cannabidiol (CBD), (–)-Δ⁸-THC, (–)-Δ⁹-THC, (±)-11-hydroxy-Δ⁹-THC (THC-OH), and (–)-11-nor-9-carboxy-Δ⁹-THC (THC-COOH)] were evaluated using high-performance liquid chromatography-tandem mass spectrometry and labeling assays. Despite activity by UGT1A1, 1A3, 1A8, 1A9, 1A10, and 2B7 toward CBN, CBD, THC-OH, and THC-COOH, only selected UGTs demonstrate sufficient activity for further characterization of steady-state kinetics. CBN was the most recognized substrate as evidenced by activities from hepatic UGT1A9 and extrahepatic UGT1A7, UGT1A8, and UGT1A10. These results may reflect the introduction of an aromatic ring to Δ⁹-THC, leading to favorable π stacking with phenylalanines in the UGT active site. Likewise, oxidation of Δ⁹-THC to THC-OH results in UGT1A9 and UGT1A10 activity toward the cannabinoid. Further oxidation to THC-COOH surprisingly leads to a loss in metabolism by UGT1A9 and UGT1A10, while creating a substrate recognized by UGT1A1 and UGT1A3. The resulting glucuronide of THC-COOH is the main metabolite found in urine, and thus these hepatic enzymes play a critical role in the metabolic clearance of cannabinoids. Taken together, glucuronidation of cannabinoids depends on upstream processing including enzymes such as CYP2C9 and CYP3A4.

Key Points: Decitabine treatment of in vitro expanded primary AML samples leads to global hypomethylation. Highly methylated CpGs are most affected by decitabine-induced hypomethylation, with little influence on transcriptional activity.

The Haemophilus influenzae HMW1 adhesin is a high-molecular weight protein that is secreted by the bacterial two-partner secretion pathway and mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. In recent work, we discovered that HMW1 is a glycoprotein and undergoes N-linked glycosylation at multiple asparagine residues with simple hexose units rather than N-acetylated hexose units, revealing an unusual N-glycosidic linkage and suggesting a new glycosyltransferase activity. Glycosylation protects HMW1 against premature degradation during the process of secretion and facilitates HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence. In the current study, we establish that the enzyme responsible for glycosylation of HMW1 is a protein called HMW1C, which is encoded by the hmw1 gene cluster and shares homology with a group of bacterial proteins that are generally associated with two-partner secretion systems. In addition, we demonstrate that HMW1C is capable of transferring glucose and galactose to HMW1 and is also able to generate hexose-hexose bonds. Our results define a new family of bacterial glycosyltransferases. Decoration of proteins with carbohydrates has an important impact on protein function throughout biology and has been recognized increasingly in pathogenic bacteria. Haemophilus influenzae is a common cause of both bacterial respiratory tract disease and bacterial invasive disease and initiates infection by colonizing the upper respiratory tract. The Haemophilus HMW1 adhesin is a large protein that resides on the bacterial surface and mediates bacterial attachment to respiratory epithelial cells, an essential step in the process of colonization. In recent work, we discovered that HMW1 is decorated at multiple sites with short carbohydrate units that serve to prevent degradation and to stabilize association with the bacterial surface. In the current study we identify the enzyme responsible for adding carbohydrate units at specific sites of HMW1. In addition, we demonstrate that this enzyme is capable of creating both carbohydrate-protein and carbohydrate-carbohydrate bonds. The amino acid sequence of this enzyme is similar to the sequences of proteins in several other bacteria, suggesting a new family of bacterial enzymes capable of creating carbohydrate-protein and carbohydrate-carbohydrate bonds.