ALBERT

Beschreibung: Human outbreaks with avian influenza have been, so far, constrained by poor viral adaptation to non-avian hosts. This could be overcome via co-infection, whereby two strains share genetic material, allowing new hybrid strains to emerge. Identifying areas where co-infection is most likely can help target spaces for increased surveillance. Ecological niche modeling using remotely-sensed data can be used for this purpose. H5N1 and H9N2 influenza subtypes are endemic in Egyptian poultry. From 2006 to 2015, over 20,000 poultry and wild birds were tested at farms and live bird markets. Using ecological niche modeling we identified environmental, behavioral, and population characteristics of H5N1 and H9N2 niches within Egypt. Niches differed markedly by subtype. The subtype niches were combined to model co-infection potential with known occurrences used for validation. The distance to live bird markets was a strong predictor of co-infection. Using only single-subtype influenza outbreaks and publicly available ecological data, we identified areas of co-infection potential with high accuracy (area under the receiver operating characteristic (ROC) curve (AUC) 0.991).

Beschreibung: HOX genes encode a family of homeodomain transcription factors with important roles in hematopoiesis. Expression of HOX genes is also a common feature of acute myeloid leukemia (AML), and functional studies have suggested that HOX-dependent pathways may contribute to leukemogenesis. Although HOX expression is known to correlate with specific AML mutations, the patterns of expression of all 39 HOX genes in primary AML samples, and their relationships with recurrent AML mutations, are incompletely understood. In addition, little is known about the influence of AML mutations on DNA methylation at the HOX loci, and the relationship between HOX gene expression and methylation in AML. In this study, we carried out a combined analysis of gene expression data from microarray and RNA-sequencing platforms and genome-wide DNA array-based methylation from 189 primary AML samples that have been previously characterized by either whole-genome or whole exome sequencing. We also measured expression and methylation using the same platforms from normal bone marrow subsets, including CD34+ cells, promyelocytes, monocytes, neutrophils and lymphocytes, and obtained expression data from CD34+ hematopoietic precursors generated from in vitro differentiation of human embryonic stem cells. Our analysis confirmed previous work on the general patterns of HOX expression in AML. The HOXA and HOXB genes showed variation both within each cluster and across the AMLs, although high level expression was restricted to a subset of these genes, including HOXA3, HOXA5, HOXA7, HOXA9, HOXA10, HOXB2-HOXB4, and HOXB6, as well as HOX cofactor MEIS1; HOXC and HOXD genes were minimally expressed in all of the samples. These observations were orthogonally validated by RNA-seq, and with a targeted Nanostring expression platform. Consistent with previous studies, MLL-positive AML samples (n=11) expressed only HOXA genes and MEIS1. AML samples with CBFB-MYH11 rearrangements (n=12) showed expression of only MEIS1, and HOXB2-HOXB4 at moderate levels; RUNX1-RUNX1T1 (n=7) and PML-RARA (n=19) samples did not detectably express any HOX genes. In AMLs with a normal karyotype (n=85), we observed two distinct patterns; one pattern displayed little or no HOX gene expression (7/85; 8%), and another displayed canonical expression of a specific subset of the HOXA and HOXB genes and MEIS1 (78/85; 92%) with similar relative HOX gene expression levels in all cases. Comparison of this pattern with normal bone marrow revealed the same HOX expression pattern in normal CD34+ cells; additional analysis showed that this pattern was confined to hematopoietic stem/progenitor cells, but was not seen in more mature cells, including other CD34+ subsets, promyelocytes, monocytes and neutrophils. We also measured HOX gene expression in CD34+ hematopoietic precursors generated from in vitro differentiation of human embryonic stem cells, which revealed expression of only MEIS1 and the canonical HOXB genes, suggesting that activation of these genes may represent the earliest events in the HOX pathway of hematopoietic development. Correlation of HOX expression with recurrent AML mutations by gene set enrichment analysis demonstrated a significant association with NPM1 (P

Beschreibung: Mutations in DNMT3A (encoding one of two mammalian de novo DNA methyltransferases) are found in 〉30% of normal karyotype AML cases and correlate with poor clinical outcomes. Most DNMT3A mutations occur at position R882 within the catalytic domain (most commonly R882H) and are virtually always heterozygous. This over-representation suggests that mutations at R882 may result in gain-of-function or dominant-negative activity that contributes to leukemogenesis. However, how DNA methylation might be altered in DNMT3A-mutant cases of AML remains unclear, and no published study to date has addressed the effects of mixing wild-type (WT) and R882H DNMT3A. Importantly, mouse HSPCs deficient in Dnmt3a dramatically expand over time and have a concurrent defect in differentiation (Challen, GA et al. Nat Genet, 2011). Mice haploinsufficient for Dnmt3a, on the other hand, do not have a measurable defect in hematopoiesis. Collectively, these data suggest that the heterozygous R882 mutations probably cause more than a simple loss-of-function phenotype. We purified full-length, human WT and R882H DNMT3A using a mammalian tissue culture system to produce recombinant proteins for biochemical modeling of the de novo methylation potential of a DNMT3A-mutant AML cell. rhR882H DNMT3A exhibits roughly 10-20% of the de novo DNA methyltransferase activity of rhWT DNMT3A, similar to observations by other groups. We added increasing amounts of R882H DNMT3A to a fixed amount of WT DNMT3A and observed a linear increase in the net enzymatic activity, reflecting the summed activity of the two forms of DNMT3A in these 4-hour in vitro reactions. In contrast, 12-hour in vitro DNA methylation assays with mixed WT and R882H DNMT3A demonstrated net methylation less than the predicted summed activity of the two enzymes, suggesting that a dominant-negative effect of R882H DNMT3A may occur with a long equilibration time. To better simulate an AML cell with a heterozygous R882H mutation, we co-transfected HEK293T cells with equal amounts of poly-His-tagged WT and R882H DNMT3A expression vectors. Subsequently co-purified (i.e. in vivo-mixed) WT and R882H DNMT3A exhibited a striking reduction in methyltransferase activity, with total activity similar to R882H DNMT3A alone (Figure 1A). TSQ mass spectrometry allowed us to verify the presence and quantify the relative concentration of WT and R882H DNMT3A in our co-purified samples. We exploited a novel tryptic cleavage site in DNMT3A produced by the R882H mutation to generate standard concentration curves using recombinant peptides distinguishing the two protein forms. Our co-purified enzyme preparations had WT:R882H ratios ranging from 0.79 to 1.60; all demonstrated the dominant-negative effect of R882H. DNMT3A is a processive enzyme, catalyzing multiple methyl-group transfers before dissociating from target DNA. This is dependent on the ability of WT DNMT3A to form homo-oligomers (tetramers and larger), which was recently shown to be disrupted by the R882H mutation using the catalytic domain of DNMT3A produced in E.coli (Holz-Schietinger, C et al. JBC, 2012). We therefore postulated that the dominant-negative effect of R882H may be due to the disruption of WT DNMT3A oligomerization. Using a Superose 6 size exclusion column, we confirmed the tetramerization defect of R882H DNMT3A relative to WT DNMT3A. Notably, in vivo-mixed (co-purified) WT and R882H DNMT3A complexes exhibited a pattern of oligomerization identical to R882H DNMT3A alone. However, WT and R882H DNMT3A mixed in vitro exhibited a distribution of oligomers corresponding to the expected average of the WT and R882H curves (Figure 1B). These data demonstrate that production of equal amounts of WT and R882H DNMT3A within the same cell provides an environment where R882H DNMT3A can exert a potent dominant-negative effect on WT DNMT3A. Furthermore, our data suggest that this effect is associated with diminished formation of tetramers when WT and R882H DNMT3A are complexed together. Thus, the R882H mutation has two distinct consequences that affect DNMT3A activity in AML cells: 1) it severely reduces its own de novo methyltransferase activity, and 2) it disrupts the ability of WT DNMT3A to form functional tetramers. These two effects severely reduce total DNMT3A activity in AML cells, and may explain why this mutation is virtually always heterozygous in AML samples, since homozygosity would not further reduce DNMT3A activity. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 1329 De novo CpG methylation is catalyzed by two enzymes (DNMT3A and DNMT3B), while DNMT1 is responsible for maintenance methylation during cell replication. DNMT3L, a catalytically inactive protein, interacts with and influences DNMT3A and DNMT3B target preference and methylation kinetics. Recurrent mutations in DNMT3A have been found in over 20% of patients with acute myeloid leukemia (AML) and have been associated with poor clinical outcomes (Ley, TJ et al. NEJM, 2010). Greater than 50% of DNMT3A mutations are found at position R882 within the catalytic domain. Because R882H mutations in AML are nearly always heterozygous, because the mutant allele is expressed at the same level as the corresponding WT allele (Ley, TJ et al. NEJM, 2010), and because the mutant enzyme has reduced methyltransferase activity (Yamashita, Y et al. Oncogene, 2010; Holz-Schietinger, C et al. JBC, 2012), it has been suggested that the R882H mutation contributes to leukemogenesis by leading to haploinsufficiency for DNMT3A. However, mice haploinsufficient for Dnmt3a exhibit normal hematopoiesis, while HSPCs lacking Dnmt3a exhibit increased self-renewal and decreased differentiation after serial transplantation (Challen, GA et al. Nat Genet, 2011). To address this conundrum, we have studied the R882H mutation in a setting that mimics the intrinsic de novo methylation capacity of a typical AML cell. Using expression array and RNA-Seq data from 178 AML patients, we discovered that DNMT3L is not expressed in AML cells, and that DNMT3A is expressed on average 2.3-fold higher than DNMT3B. Interestingly, 92% of AML patients predominantly express inactive splice variants of DNMT3B, regardless of FAB or mutational profile (median ratio of inactive to active DNMT3B transcripts is 3.1:1). Given that the inactive splice variant DNMT3B3 is the most highly expressed isoform in most patients in our cohort, we explored the functional interactions between WT DNMT3A, R882H DNMT3A, and DNMT3B3 using recombinant enzymes made in eukaryotic cells. In vitro methylation of plasmid DNA (pcDNA3.1) with 3H-SAM using purified recombinant full-length human DNMT3A protein confirmed that the R882H mutation severely reduces the catalytic activity of DNMT3A, resulting in an enzyme with ∼10% of the activity of the WT enzyme. These results were verified by independent in vitro methylation experiments analyzed by bisulfite sequencing, which also revealed that the CpG-flanking sequence preferences of WT and R882H DNMT3A are identical and consistent with the expected “TNCGCY” motif previously described (Wienholz, BL et al. PLoS Genet, 2010). Mixing WT and R882H DNMT3A at equimolar ratios resulted in no significant changes in CpG-flanking sequence preference (compared to WT or R882H enzyme alone; Spearman correlation between WT DNMT3A and WT+R882H DNMT3A = 0.99). In contrast, mixing WT and R882H DNMT3A at equimolar ratios in a 12-hour methylation assay demonstrated that R882H DNMT3A exerts an inhibitory effect on the catalytic activity of WT DNMT3A in vitro. Instead of increasing net methylation activity by a predicted 10% (summing the activity of the two individual enzymes), R882H DNMT3A led to a 20% reduction in the measured methylation. Similarly, the addition of catalytically inactive DNMT3B3 to WT DNMT3A resulted in a mean decrease in methylation of 38%. Combining equimolar amounts of WT DNMT3A, R882H DNMT3A, and DNMT3B3 led to an additive inhibition of methylation compared to WT DNMT3A alone (62% decrease; p 〈 0.001; Figure 1). This scenario closely mimics the ratio of these enzymes in AML cells, and our data therefore suggest that the additive inhibitory effects of R882H DNMT3A and DNMT3B3 could severely reduce the total de novo methylation activity of DNMT3A in AML cells. The reduction of enzyme activity below haploinsufficient levels may be important for AML pathogenesis, and these findings provide a mechanism to achieve these levels. Figure 1: The de novo methyltransferase activity of WT DNMT3A is inhibited by R882H DNMT3A and DNMT3B3. Mixing equimolar amounts of WT DNMT3A, R882H DNMT3A, and DNMT3B3 leads to additive inhibition of methylation by 62% (p 〈 0.001). Figure 1:. The de novo methyltransferase activity of WT DNMT3A is inhibited by R882H DNMT3A and DNMT3B3. Mixing equimolar amounts of WT DNMT3A, R882H DNMT3A, and DNMT3B3 leads to additive inhibition of methylation by 62% (p 〈 0.001). Disclosures: Ley: Washington University: Patents & Royalties.