DNA Methylation Signatures Identify Biologically Distinct Subtypes in Acute Myeloid Leukemia

Abstract: We hypothesized that DNA methylation distributes into specific patterns in cancer cells, which reflect critical biological differences. We therefore examined the methylation profiles of 344 patients with acute myeloid leukemia (AML). Clustering of these patients by methylation data segregated patients into 16 groups. Five of these groups defined new AML subtypes that shared no other known feature. In addition, DNA methylation profiles segregated patients with CEBPA aberrations from other subtypes of leukemia, defined four epigenetically distinct forms of AML with NPM1 mutations, and showed that established AML1-ETO, CBFb-MYH11, and PML-RARA leukemia entities are associated with specific methylation profiles. We report a 15 gene methylation classifier predictive of overall survival in an independent patient cohort (p < 0.001, adjusted for known covariates)

Characterization of the mouse adeno-associated virus AAVS1 ortholog

The nonpathogenic human adeno-associated virus (AAV) has developed a mechanism to integrate its genome into human chromosome 19 at 19q13.4 (termed AAVS1), thereby establishing latency. Here, we provide evidence that the chromosomal signals required for site-specific integration are conserved in the mouse genome proximal to the recently identified Mbs85 gene. These sequence motifs can be specifically nicked by the viral Rep protein required for the initiation of site-specific AAV DNA integration. Furthermore, these signals can serve as a minimal origin for Rep-dependent DNA replication. In addition, we isolated the mouse Mbs85 proximal promoter and show transcriptional activity in three mouse cell lines

Transepithelial projections from basal cells are luminal sensors in pseudostratified epithelia

Basal cells are by definition located on the basolateral side of several epithelia, and they have never been observed reaching the lumen. Using high-resolution 3D confocal imaging, we report that basal cells extend long and slender cytoplasmic projections that not only reach toward the lumen but can cross the tight junction barrier in some epithelia of the male reproductive and respiratory tracts. In this way, the basal cell plasma membrane is exposed to the luminal environment. In the epididymis, in which luminal acidification is crucial for sperm maturation and storage, these projections contain the angiotensin II type 2 receptor (AGTR2). Activation of AGTR2 by luminal angiotensin II, increases proton secretion by adjacent clear cells, which are devoid of AGTR2. We propose a paradigm in which basal cells scan and sense the luminal environment of pseudostratified epithelia and modulate epithelial function by a mechanism involving crosstalk with other epithelial cells

DNA Methylation Profiling Predicts Clinical Outcomes and Reveals Unique Insights Into the Molecular Complexity of Acute Myeloid Leukemia

Abstract Abstract 707 Epigenetic deregulation of genes through aberrant DNA methylation has been widely reported in cancer. We hypothesized that in AML this aberrant DNA methylation does not occur randomly, but rather occurs in specific and distinct patterns. Therefore, large-scale genome-wide analysis of the DNA methylome could help explain and define the complexity underlying leukemia biology and reveal the existence of epigenetically defined variants of AML. Using the HELP microarray assay, which measures DNA methylation at 50,000 CpG sites annotated to ∼14,000 promoters, we obtained DNA methylation profiles for 344 AML patients seen at Erasmus University Medical Center. Median follow-up based on survivors was 18.2 months (7-215); median age: 48 years (15-77). Unsupervised analysis (hierarchical clustering, correlation distance with Ward's clustering method) demonstrated that based on their methylation profiles AML patients distributed into 16 cohorts. 11 of these groups were also defined by the presence of specific molecular lesions: inv(16) [cluster 1], t(8;21) [cluster 3], t(15;17) [cluster 6], CEBPA-mutant [clusters 4 and 9], CEBPA-silenced [cluster 10] NPM1-mutant [clusters 12, 13, 14 and 16] and 11q23 abnormalities [cluster 11]. Enrichment for cases harboring a specific molecular lesion within a given cluster was determined using Fisher's exact test (p30%. The DNA methylation signatures of each cluster featured involvement of distinct gene networks and DNA regulatory elements, and displayed distinct degrees of hyper or hypomethylation with respect to normal CD34+ bone marrow cells. Of note, in spite of the variation in methylation across the 16 clusters, we identified a set of 45 genes that were almost universally aberrantly methylated (in >70% cases and present in at least 10/16 cluster signatures). This common epigenetic signature included the tumor suppressor PDZD2, the nuclear import proteins IPO8 and TNPO3, PIAS2, a regulator of MAP kinase signaling, CDK8, and CSDA, a regulator of CSF2. Gene expression profiling of the same patients indicated that at least 50% of these genes were also aberrantly silenced compared to normal CD34+ cells. Finally, we randomly divided the 344-patient cohort into a training group of 200 patients, a test group (n=95) and an independent validation group (n=49), and using the Supervised Principal Components algorithm identified a 15-gene methylation classifier that was predictive of OS (p<0.009) and event free survival (p<0.013). Furthermore, after adjustment for age, cytogenetic risk, NPM1, FLT3 and CEBPA status in a multivariate analysis, this classifier remained an independent risk factor for OS (Hazard ratio 1.29, 95% CI: 1.11-1.49; p<0.001). In summary, we have i) demonstrated that unique and distinct DNA methylation patterns characterize distinct forms of AML; ii) identified novel, epigenetically defined subgroups of AML with distinct clinical behavior; iii) revealed the presence of a consistently aberrantly methylated signature across AML subtypes, with confirmed silencing of the genes involved; and iv) report a 15-gene methylation classifier predictive of OS, and confirmed as an independent risk factor when adjusted for known AML covariates. Disclosures: No relevant conflicts of interest to declare

Genome-wide epigenetic analysis delineates a biologically distinct immature acute leukemia with myeloid/T-lymphoid features

Acute myeloid leukemia is a heterogeneous disease from the molecular and biologic standpoints, and even patients with a specific gene expression profile may present clinical and molecular heterogeneity. We studied the epigenetic profiles of a cohort of patients who shared a common gene expression profile but differed in that only half of them harbored mutations of the CEBPA locus, whereas the rest presented with silencing of this gene and coexpression of certain T-cell markers. DNA methylation studies revealed that these 2 groups of patients could be readily segregated in an unsupervised fashion based on their DNA methylation profiles alone. Furthermore, CEBPA silencing was associated with the presence of an aberrant DNA hypermethylation signature, which was not present in the CEBPA mutant group. This aberrant hypermethylation occurred more frequently at sites within CpG islands. CEBPA-silenced leukemias also displayed marked hypermethylation compared with normal CD34+ hematopoietic cells, whereas CEBPA mutant cases showed only mild changes in DNA methylation compared with these normal progenitors. Biologically, CEBPA-silenced leukemias presented with a decreased response to myeloid growth factors in vitro

Aberrant DNA hypermethylation signature in acute myeloid leukemia directed by EVI1

DNA methylation patterns are frequently dysregulated in cancer, although little is known of the mechanisms through which specific gene sets become aberrantly methylated. The ecotropic viral integration site 1 (EVI1) locus encodes a DNA binding zinc-finger transcription factor that is aberrantly expressed in a subset of acute myeloid leukemia (AML) patients with poor outcome. We find that the promoter DNA methylation signature of EVI1 AML blast cells differs from those of normal CD34+ bone marrow cells and other AMLs. This signature contained 294 differentially methylated genes, of which 238 (81%) were coordinately hypermethylated. An unbiased motif analysis revealed an overrepresentation of EVI1 binding sites among these aberrantly hypermethylated loci. EVI1 was capable of binding to these promoters in 2 different EVI1-expressing cell lines, whereas no binding was observed in an EVI1-negative cell line. Furthermore, EVI1 was observed to interact with DNA methyl transferases 3A and 3B. Among the EVI1 AML cases, 2 subgroups were recognized, of which 1 contained AMLs with many more methylated genes, which was associated with significantly higher levels of EVI1 than in the cases of the other subgroup. Our data point to a role for EVI1 in directing aberrant promoter DNA methylation patterning in EVI1 AMLs

Growth arrest-specific 1 binds to and controls the maturation and processing of the amyloid-β precursor protein

Alzheimer's disease (AD), the most common neurodegenerative disorder, is characterized by cerebral deposition of amyloid-β (Aβ), a series of peptides derived from the processing of the amyloid-β precursor protein (APP). To identify new candidate genes for AD, we recently performed a transcriptome analysis to screen for genes preferentially expressed in the hippocampus and located in AD linkage regions. This strategy identified CALHM1 (calcium homeostasis modulator 1), a gene modulating AD age at onset and Aβ metabolism. Here, we focused our attention on another candidate identified using this screen, growth arrest-specific 1 (Gas1), a gene involved in the central nervous system development. We found that Gas1 formed a complex with APP and controlled APP maturation and processing. Gas1 expression inhibited APP full glycosylation and routing to the cell surface by leading to a trafficking blockade of APP between the endoplasmic reticulum and the Golgi. Gas1 expression also resulted in a robust inhibition of APP transport into multivesicular bodies, further demonstrating that Gas1 negatively regulated APP intracellular trafficking. Consequently, Gas1 overexpression led to a reduction in Aβ production, and conversely, Gas1 silencing in cells expressing endogenously Gas1 increased Aβ levels. These results suggest that Gas1 is a novel APP-interacting protein involved in the control of APP maturation and processing