Independent Prognostic Significance of Monosomy 17 and Impact of Karyotype Complexity in Monosomal Karyotype/Complex Karyotype Acute Myeloid Leukemia: Results from Four ECOG-ACRIN Prospective Therapeutic Trials

The presence of a monosomal karyotype (MK+) and/or a complex karyotype (CK+) identifies subcategories of AML with poor prognosis. The prognostic significance of the most common monosomies (monosomy 5, monosomy 7, and monosomy 17) within MK+/CK+ AML is not well defined. We analyzed data from 1,592 AML patients age 17–93 years enrolled on ECOG-ACRIN therapeutic trials. The majority of MK+ patients (182/195; 93%) were MK+/CK+ with 87% (158/182) having ≥5 clonal abnormalities (CK≥ 5). MK+ patients with karyotype complexity ≤4 had a median overall survival (OS) of 0.4y compared to 1.0y for MK- with complexity ≤4 (p < 0.001), whereas no OS difference was seen in MK+ vs. MK- patients with CK≥ 5 (p = 0.82). Monosomy 5 (93%; 50/54) typically occurred within a highly complex karyotype and had no impact on OS (0.4y; p = 0.95). Monosomy 7 demonstrated no impact on OS in patients with CK≥ 5 (p = 0.39) or CK ≤ 4 (p = 0.44). Monosomy 17 appeared in 43% (68/158) of CK≥ 5 patients and demonstrated statistically significant worse OS (0.4y) compared to CK≥ 5 patients without monosomy 17 (0.5y; p = 0.012). Our data suggest that the prognostic impact of MK+ is limited to those with less complex karyotypes and that monosomy 17 may independently predict for worse survival in patients with AML

Reverse engineering of TLX oncogenic transcriptional networks identifies RUNX1 as tumor suppressor in T-ALL

The TLX1 and TLX3 transcription factor oncogenes have a key role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL)(1,2). Here we used reverse engineering of global transcriptional networks to decipher the oncogenic regulatory circuit controlled by TLX1 and TLX3. This systems biology analysis defined T cell leukemia homeobox 1 (TLX1) and TLX3 as master regulators of an oncogenic transcriptional circuit governing T-ALL. Notably, a network structure analysis of this hierarchical network identified RUNX1 as a key mediator of the T-ALL induced by TLX1 and TLX3 and predicted a tumor-suppressor role for RUNX1 in T cell transformation. Consistent with these results, we identified recurrent somatic loss-of-function mutations in RUNX1 in human T-ALL. Overall, these results place TLX1 and TLX3 at the top of an oncogenic transcriptional network controlling leukemia development, show the power of network analyses to identify key elements in the regulatory circuits governing human cancer and identify RUNX1 as a tumor-suppressor gene in T-ALL

ETV6 mutations in early immature human T cell leukemias

A substantial proportion of adult T-ALL samples display gene expression and mutation characteristics of both T cell and acute myeloid leukemias; mutations in ETV6 are found exclusively within this new molecular subgroup of adult T-ALL patients

An Integrative Genomic and Epigenomic Approach for the Study of Transcriptional Regulation

The molecular heterogeneity of acute leukemias and other tumors constitutes a major obstacle towards understanding disease pathogenesis and developing new targeted-therapies. Aberrant gene regulation is a hallmark of cancer and plays a central role in determining tumor phenotype. We predicted that integration of different genome-wide epigenetic regulatory marks along with gene expression levels would provide greater power in capturing biological differences between leukemia subtypes. Gene expression, cytosine methylation and histone H3 lysine 9 (H3K9) acetylation were measured using high-density oligonucleotide microarrays in primary human acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) specimens. We found that DNA methylation and H3K9 acetylation distinguished these leukemias of distinct cell lineage, as expected, but that an integrative analysis combining the information from each platform revealed hundreds of additional differentially expressed genes that were missed by gene expression arrays alone. This integrated analysis also enhanced the detection and statistical significance of biological pathways dysregulated in AML and ALL. Integrative epigenomic studies are thus feasible using clinical samples and provide superior detection of aberrant transcriptional programming than single-platform microarray studies

Genomic analyses identify recurrent MEF2D fusions in acute lymphoblastic leukemia

Chromosomal rearrangements are initiating events in acute lymphoblastic leukaemia (ALL). Here using RNA sequencing of 560 ALL cases, we identify rearrangements between MEF2D (myocyte enhancer factor 2D) and five genes (BCL9, CSF1R, DAZAP1, HNRNPUL1 and SS18) in 22 B progenitor ALL (B-ALL) cases with a distinct gene expression profile, the most common of which is MEF2DBCL9. Examination of an extended cohort of 1,164 B-ALL cases identified 30 cases with MEF2D rearrangements, which include an additional fusion partner, FOXJ2; thus, MEF2D-rearranged cases comprise 5.3% of cases lacking recurring alterations. MEF2D-rearranged ALL is characterized by a distinct immunophenotype, DNA copy number alterations at the rearrangement sites, older diagnosis age and poor outcome. The rearrangements result in enhanced MEF2D transcriptional activity, lymphoid transformation, activation of HDAC9 expression and sensitive to histone deacetylase inhibitor treatment. Thus, MEF2D-rearranged ALL represents a distinct form of high-risk leukaemia, for which new therapeutic approaches should be considered.This work was supported in part by the American Lebanese Syrian Associated Charities of St. Jude Children’s Research Hospital; by a Stand Up to Cancer Innovative Research Grant and St. Baldrick’s Foundation Scholar Award (to C.G.M.); by a St. Baldrick’s Consortium Award (S.P.H.), by a Leukemia and Lymphoma Society Specialized Center of Research grant (S.P.H. and C.G.M.), by a Lady Tata Memorial Trust Award (I.I.), by a Leukemia and Lymphoma Society Special Fellow Award and Alex’s Lemonade Stand Foundation Young Investigator Awards (K.R.), by an Alex’s Lemonade Stand Foundation Award (M.L.) and by National Cancer Institute Grants CA21765 (St Jude Cancer Center Support Grant), U01 CA157937 (C.L.W. and S.P.H.), U24 CA114737 (to Dr Gastier-Foster), NCI Contract HHSN261200800001E (to Dr Gastier-Foster), U10 CA180820 (ECOG-ACRIN Operations) and CA180827 (E.P.); U10 CA180861 (C.D.B. and G.M.); U24 CA196171 (The Alliance NCTN Biorepository and Biospecimen Resource); CA145707 (C.L.W. and C.G.M.); and grants to the COG: U10 CA98543 (Chair’s grant and supplement to support the COG ALL TARGET project), U10 CA98413 (Statistical Center) and U24 CA114766 (Specimen Banking). This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract Number HHSN261200800001E

Base-Pair Resolution DNA Methylation Sequencing Reveals Profoundly Divergent Epigenetic Landscapes in Acute Myeloid Leukemia

We have developed an enhanced form of reduced representation bisulfite sequencing with extended genomic coverage, which resulted in greater capture of DNA methylation information of regions lying outside of traditional CpG islands. Applying this method to primary human bone marrow specimens from patients with Acute Myelogeneous Leukemia (AML), we demonstrated that genetically distinct AML subtypes display diametrically opposed DNA methylation patterns. As compared to normal controls, we observed widespread hypermethylation in IDH mutant AMLs, preferentially targeting promoter regions and CpG islands neighboring the transcription start sites of genes. In contrast, AMLs harboring translocations affecting the MLL gene displayed extensive loss of methylation of an almost mutually exclusive set of CpGs, which instead affected introns and distal intergenic CpG islands and shores. When analyzed in conjunction with gene expression profiles, it became apparent that these specific patterns of DNA methylation result in differing roles in gene expression regulation. However, despite this subtype-specific DNA methylation patterning, a much smaller set of CpG sites are consistently affected in both AML subtypes. Most CpG sites in this common core of aberrantly methylated CpGs were hypermethylated in both AML subtypes. Therefore, aberrant DNA methylation patterns in AML do not occur in a stereotypical manner but rather are highly specific and associated with specific driving genetic lesions

Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, ‘epigenetic’ drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.National Institutes of Health (U.S.) (Grant R37-HD04502

Deregulation of DUX4 and ERG in acute lymphoblastic leukemia

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL).1,2 Here, we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG are hallmarks of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases, and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt utilizes a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivating domains of ERG, but inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia, in which DUX4 deregulation results in loss-of-function of ERG, either by deletion or induction of expression of an isoform that is a dominant negative inhibitor of wild type ERG function

An Integrated Epigenomic Analysis Approach To Determine the Molecular Basis of Acute Leukemias

Abstract Acute leukemias are classified based on their immunologic, cytogenetic and morphologic characteristics. However, in most instances, response to treatment and survival probability cannot be accurately predicted, suggesting that the disease is even more complex and heterogeneous than can be shown with current techniques. Although prognostic value has been shown for certain gene expression profiles, expression profile studies are limited by the fact that only a snapshot of mRNA content is obtained in a basal state, failing to represent how genes will respond to different stresses, also failing to detect the roles of genes expressed at lower levels for which major changes in expression levels are often lost in the “noise” of expression arrays. In order to overcome these issues and to provide a more accurate molecular phenotype of acute leukemias, we established an integrated epigenomic and genomic high-throughput platform using novel techniques and custom high-density oligonucleotide arrays. We combine studies of i) genome-wide cytosine methylation using a novel technique we developed that provides accurate quantitative determination of DNA methylation levels, using genome-wide custom oligo arrays, ii) chromatin structure by ChIP on chip for histone code settings associated with active or repressed genes using 24 K promoter tiling arrays, iii) gene copy number by array-based comparative genomic hybridization (array CGH) at 6 kb resolution genome-wide and iv) 36 K gene expression arrays. Results are validated by single locus quantitative PCR techniques. Cross platform integration is facilitated by use of NimbleGen oligo arrays for all studies and analysis using novel bioinformatics and statistical models. We used this integrative analysis platform to perform an in-depth analysis of the epigenomic basis of AML and ALL using primary patient samples and cell lines. The data allowed us to generate a “gene activity index” which identified the ability of genes to be expressed to be characterized genome-wide in AML and ALL cells. This data also allowed a much more comprehensive analysis of pathways active in these cells to be identified in comparison to expression arrays alone. Our current studies apply integrative platform and gene activity indexing to large series of patients enrolled in multicenter clinical trials in order to provide high-resolution analysis of the molecular basis of acute leukemia