Integrative Genomics Identifies the Molecular Basis of Resistance to Azacitidine Therapy in Myelodysplastic Syndromes

© 2017 The Author(s) Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only ∼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking individual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders

Transcriptional networks in acute myeloid leukemia

© 2019 Wiley Periodicals, Inc. Acute myeloid leukemia (AML) is a complex disease characterized by a diverse range of recurrent molecular aberrations that occur in many different combinations. Components of transcriptional networks are a common target of these aberrations, leading to network-wide changes and deployment of novel or developmentally inappropriate transcriptional programs. Genome-wide techniques are beginning to reveal the full complexity of normal hematopoietic stem cell transcriptional networks and the extent to which they are deregulated in AML, and new understandings of the mechanisms by which AML cells maintain self-renewal and block differentiation are starting to emerge. The hope is that increased understanding of the network architecture in AML will lead to identification of key oncogenic dependencies that are downstream of multiple network aberrations, and that this knowledge will be translated into new therapies that target these dependencies. Here, we review the current state of knowledge of network perturbation in AML with a focus on major mechanisms of transcription factor dysregulation, including mutation, translocation, and transcriptional dysregulation, and discuss how these perturbations propagate across transcriptional networks. We will also review emerging mechanisms of network disruption, and briefly discuss how increased knowledge of network disruption is already being used to develop new therapies

Functional Mutations Form at CTCF-Cohesin Binding Sites in Melanoma Due to Uneven Nucleotide Excision Repair across the Motif

© 2016 The Author(s) CTCF binding sites are frequently mutated in cancer, but how these mutations accumulate and whether they broadly perturb CTCF binding are not well understood. Here, we report that skin cancers exhibit a highly specific asymmetric mutation pattern within CTCF motifs attributable to ultraviolet irradiation and differential nucleotide excision repair (NER). CTCF binding site mutations form independently of replication timing and are enriched at sites of CTCF/cohesin complex binding, suggesting a role for cohesin in stabilizing CTCF-DNA binding and impairing NER. Performing CTCF ChIP-seq in a melanoma cell line, we show CTCF binding site mutations to be functional by demonstrating allele-specific reduction of CTCF binding to mutant alleles. While topologically associating domains with mutated CTCF anchors in melanoma contain differentially expressed cancer-associated genes, CTCF motif mutations appear generally under neutral selection. However, the frequency and potential functional impact of such mutations in melanoma highlights the need to consider their impact on cellular phenotype in individual genomes

Annotating function to differentially expressed LincRNAs in myelodysplastic syndrome using a network-based method

© The Author 2017. Published by Oxford University Press. All rights reserved. Motivation: Long non-coding RNAs (lncRNAs) have been implicated in the regulation of diverse biological functions. The number of newly identified lncRNAs has increased dramatically in recent years but their expression and function have not yet been described from most diseases. To elucidate lncRNA function in human disease, we have developed a novel network based method (NLCFA) integrating correlations between lncRNA, protein coding genes and noncoding miRNAs. We have also integrated target gene associations and protein-protein interactions and designed our model to provide information on the combined influence of mRNAs, lncRNAs and miRNAs on cellular signal transduction networks. Results: We have generated lncRNA expression profiles from the CD34þhaematopoietic stem and progenitor cells (HSPCs) from patients with Myelodysplastic syndromes (MDS) and healthy donors. We report, for the first time, aberrantly expressed lncRNAs in MDS and further prioritize biologically relevant lncRNAs using the NLCFA. Taken together, our data suggests that aberrant levels of specific lncRNAs are intimately involved in network modules that control multiple cancer-associated signalling pathways and cellular processes. Importantly, our method can be applied to prioritize aberrantly expressed lncRNAs for functional validation in other diseases and biological contexts. Availability and implementation: The method is implemented in R language and Matlab

Disruption of a 35 KB enhancer impairs CTCF binding and MLH1 expression in colorectal cells

© 2018 American Association for Cancer Research. Purpose: MLH1 is a major tumor suppressor gene involved in the pathogenesis of Lynch syndrome and various sporadic cancers. Despite their potential pathogenic importance, genomic regions capable of regulating MLH1 expression over long distances have yet to be identified. Experimental Design: Here, we use chromosome conformation capture (3C) to screen a 650-kb region flanking the MLH1 locus to identify interactions between the MLH1 promoter and distal regions in MLH1-expressing and nonexpressing cells. Putative enhancers were functionally validated using luciferase reporter assays, chromatin immunoprecipitation, and CRISPR-Cas9–mediated deletion of endogenous regions. To evaluate whether germline variants in the enhancer might contribute to impaired MLH1 expression in patients with suspected Lynch syndrome, we also screened germline DNA from a cohort of 74 patients with no known coding mutations or epimutations at the MLH1 promoter. Results: A 1.8-kb DNA fragment, 35 kb upstream of the MLH1 transcription start site enhances MLH1 gene expression in colorectal cells. The enhancer was bound by CTCF and CRISPR-Cas9–mediated deletion of a core binding region impairs endogenous MLH1 expression. A total of 5.4% of suspected Lynch syndrome patients have a rare single-nucleotide variant (G > A; rs143969848; 2.5% in gnomAD European, non-Finnish) within a highly conserved CTCF-binding motif, which disrupts enhancer activity in SW620 colorectal carcinoma cells. Conclusions: A CTCF-bound region within the MLH1-35 enhancer regulates MLH1 expression in colorectal cells and is worthy of scrutiny in future genetic screening strategies for suspected Lynch syndrome associated with loss of MLH1 expression

MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation

© 2016 Macmillan Publishers Limited. Aberrant ERG (v-ets avian erythroblastosis virus E26 oncogene homolog) expression drives leukemic transformation in mice and high expression is associated with poor patient outcomes in acute myeloid leukemia (AML) and T-acute lymphoblastic leukemia (T-ALL). Protein phosphorylation regulates the activity of many ETS factors but little is known about ERG in leukemic cells. To characterize ERG phosphorylation in leukemic cells, we applied liquid chromatography coupled tandem mass spectrometry and identified five phosphorylated serines on endogenous ERG in T-ALL and AML cells. S283 was distinct as it was abundantly phosphorylated in leukemic cells but not in healthy hematopoietic stem and progenitor cells (HSPCs). Overexpression of a phosphoactive mutant (S283D) increased expansion and clonogenicity of primary HSPCs over and above wild-type ERG. Using a custom antibody, we screened a panel of primary leukemic xenografts and showed that ERG S283 phosphorylation was mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling and in turn regulated expression of components of this pathway. S283 phosphorylation facilitates ERG enrichment and transactivation at the ERG +85 HSPC enhancer that is active in AML and T-ALL with poor prognosis. Taken together, we have identified a specific post-translational modification in leukemic cells that promotes progenitor proliferation and is a potential target to modulate ERG-driven transcriptional programs in leukemia

ERG promotes T-acute lymphoblastic leukemia and is transcriptionally regulated in leukemic cells by a stem cell enhancer

The Ets-related gene (ERG) is an Ets-transcription factor required for normal blood stem cell development. ERG expression is down-regulated during early T-lymphopoiesis but maintained in T-acute lymphoblastic leukemia (T-ALL), where it is recognized as an independent risk factor for adverse outcome. However, it is unclear whether ERG is directly involved in the pathogenesis of T-ALL and how its expression is regulated. Here we demonstrate that transgenic expression of ERG causes T-ALL in mice and that its knockdown reduces the proliferation of human MOLT4 T-ALL cells. We further demonstrate that ERG expression in primary human T-ALL cells is mediated by the binding of other T-cell oncogenes SCL/TAL1, LMO2, and LYL1 in concert with ERG, FLI1, and GATA3 to the ERG +85 enhancer. This enhancer is not active in normal T cells but in transgenic mice targets expression to fetal liver c-kit(+) cells, adult bone marrow stem/progenitors and early CD4(-)CD8(-) double-negative thymic progenitors. Taken together, these data illustrate that ERG promotes T-ALL and that failure to extinguish activity of stem cell enhancers associated with regulatory transcription factors such as ERG can contribute to the development of leukemia

Overexpression of ERG in cord blood progenitors promotes expansion and recapitulates molecular signatures of high ERG leukemias

© 2015 Macmillan Publishers Limited. High expression of the ETS family transcription factor ERG is associated with poor clinical outcome in acute myeloid leukemia (AML) and acute T-cell lymphoblastic leukemia (T-ALL). In murine models, high ERG expression induces both T-ALL and AML. However, no study to date has defined the effect of high ERG expression on primary human hematopoietic cells. In the present study, human CD34+ cells were transduced with retroviral vectors to elevate ERG gene expression to levels detected in high ERG AML. RNA sequencing was performed on purified populations of transduced cells to define the effects of high ERG on gene expression in human CD34+ cells. Integration of the genome-wide expression data with other data sets revealed that high ERG drives an expression signature that shares features of normal hematopoietic stem cells, high ERG AMLs, early T-cell precursor-ALLs and leukemic stem cell signatures associated with poor clinical outcome. Functional assays linked this gene expression profile to enhanced progenitor cell expansion. These results support a model whereby a stem cell gene expression network driven by high ERG in human cells enhances the expansion of the progenitor pool, providing opportunity for the acquisition and propagation of mutations and the development of leukemia