Dominik Beck

Differential DNA repair underlies mutation hotspots at active promoters in cancer genomes

Promoters are DNA sequences that have an essential role in controlling gene expression. While recent whole cancer genome analyses have identified numerous hotspots of somatic point mutations within promoters, many have not yet been shown to perturb gene expression or drive cancer development. As such, positive selection alone may not adequately explain the frequency of promoter point mutations in cancer genomes. Here we show that increased mutation density at gene promoters can be linked to promoter activity and differential nucleotide excision repair (NER). By analysing 1,161 human cancer genomes across 14 cancer types, we find evidence for increased local density of somatic point mutations within the centres of DNase I-hypersensitive sites (DHSs) in gene promoters. Mutated DHSs were strongly associated with transcription initiation activity, in which active promoters but not enhancers of equal DNase I hypersensitivity were most mutated relative to their flanking regions. Notably, analysis of genome-wide maps of NER shows that NER is impaired within the DHS centre of active gene promoters, while XPC-deficient skin cancers do not show increased promoter mutation density, pinpointing differential NER as the underlying cause of these mutation hotspots. Consistent with this finding, we observe that melanomas with an ultraviolet-induced DNA damage mutation signature show greatest enrichment of promoter mutations, whereas cancers that are not highly dependent on NER, such as colon cancer, show no sign of such enrichment. Taken together, our analysis has uncovered the presence of a previously unknown mechanism linking transcription initiation and NER as a major contributor of somatic point mutation hotspots at active gene promoters in cancer genomes.

Genome-wide analysis of transcriptional regulators in human HSPCs reveals a densely interconnected network of coding and noncoding genes

Genome-wide combinatorial binding patterns for key transcription factors (TFs) have not been reported for primary human hematopoietic stem and progenitor cells (HSPCs), and have constrained analysis of the global architecture of molecular circuits controlling these cells. Here we provide high-resolution genome-wide binding maps for a heptad of key TFs (FLI1, ERG, GATA2, RUNX1, SCL, LYL1, and LMO2) in human CD34(+) HSPCs, together with quantitative RNA and microRNA expression profiles. We catalog binding of TFs at coding genes and microRNA promoters, and report that combinatorial binding of all 7 TFs is favored and associated with differential expression of genes and microRNA in HSPCs. We also uncover a previously unrecognized association between FLI1 and RUNX1 pairing in HSPCs, we establish a correlation between the density of histone modifications that mark active enhancers and the number of overlapping TFs at a peak, we demonstrate bivalent histone marks at promoters of heptad target genes in CD34(+) cells that are poised for later expression, and we identify complex relationships between specific microRNAs and coding genes regulated by the heptad. Taken together, these data reveal the power of integrating multifactor sequencing of chromatin immunoprecipitates with coding and noncoding gene expression to identify regulatory circuits controlling cell identity.

Activity of a heptad of transcription factors is associated with stem cell programs and clinical outcome in acute myeloid leukemia

Aberrant transcriptional programs in combination with abnormal proliferative signaling drive leukemic transformation. These programs operate in normal hematopoiesis where they are involved in hematopoietic stem cell (HSC) proliferation and maintenance. Ets Related Gene (ERG) is a component of normal and leukemic stem cell signatures and high ERG expression is a risk factor for poor prognosis in acute myeloid leukemia (AML). However, mechanisms that underlie ERG expression in AML and how its expression relates to leukemic stemness are unknown. We report that ERG expression in AML is associated with activity of the ERG promoters and +85 stem cell enhancer and a heptad of transcription factors that combinatorially regulate genes in HSCs. Gene expression signatures derived from ERG promoter-stem cell enhancer and heptad activity are associated with clinical outcome when ERG expression alone fails. We also show that the heptad signature is associated with AMLs that lack somatic mutations in NPM1 and confers an adverse prognosis when associated with FLT3 mutations. Taken together, these results suggest that transcriptional regulators cooperate to establish or maintain primitive stem cell-like signatures in leukemic cells and that the underlying pattern of somatic mutations contributes to the development of these signatures and modulate their influence on clinical outcome.

Opposing regulation of BIM and BCL2 controls glucocorticoid-induced apoptosis of pediatric acute lymphoblastic leukemia cells

Glucocorticoids are critical components of combination chemotherapy regimens in pediatric acute lymphoblastic leukemia (ALL). The proapoptotic BIM protein is an important mediator of glucocorticoid-induced apoptosis in normal and malignant lymphocytes, whereas the antiapoptotic BCL2 confers resistance. The signaling pathways regulating BIM and BCL2 expression in glucocorticoid-treated lymphoid cells remain unclear. In this study, pediatric ALL patient-derived xenografts (PDXs) inherently sensitive or resistant to glucocorticoids were exposed to dexamethasone in vivo. Microarray analysis showed that KLF13 and MYB gene expression changes were significantly greater in dexamethasone-sensitive than -resistant PDXs. Chromatin immunoprecipitation (ChIP) analysis detected glucocorticoid receptor (GR) binding at the KLF13 promoter to trigger KLF13 expression only in sensitive PDXs. Next, KLF13 bound to the MYB promoter, deactivating MYB expression only in sensitive PDXs. Sustained MYB expression in resistant PDXs resulted in maintenance of BCL2 expression and inhibition of apoptosis. ChIP sequencing analysis revealed a novel GR binding site in a BIM intronic region (IGR) that was engaged only in dexamethasone-sensitive PDXs. The absence of GR binding at the BIM IGR was associated with BIM silencing and dexamethasone resistance. This study has identified novel mechanisms of opposing BCL2 and BIM gene regulation that control glucocorticoid-induced apoptosis in pediatric ALL cells in vivo.

Genome-scale expression and transcription factor binding profiles reveal therapeutic targets in transgenic ERG myeloid leukemia

The ETS transcription factor ERG plays a central role in definitive hematopoiesis, and its overexpression in acute myeloid leukemia (AML) is associated with a stem cell signature and poor prognosis. Yet how ERG causes leukemia is unclear. Here we show that pan-hematopoietic ERG expression induces an early progenitor myeloid leukemia in transgenic mice. Integrated genome-scale analysis of gene expression and ERG binding profiles revealed that ERG activates a transcriptional program similar to human AML stem/progenitor cells and to human AML with high ERG expression. This transcriptional program was associated with activation of RAS that was required for leukemia cells growth in vitro and in vivo. We further show that ERG induces expression of the Pim1 kinase oncogene through a novel hematopoietic enhancer validated in transgenic mice and human CD34(+) normal and leukemic cells. Pim1 inhibition disrupts growth and induces apoptosis of ERG-expressing leukemic cells. The importance of the ERG/PIM1 axis is further underscored by the poorer prognosis of AML highly expressing ERG and PIM1. Thus, integrative genomic analysis demonstrates that ERG causes myeloid progenitor leukemia characterized by an induction of leukemia stem cell transcriptional programs. Pim1 and the RAS pathway are potential therapeutic targets of these high-risk leukemias.

Role of the erythropoietin receptor in ETV6/RUNX1-positive acute lymphoblastic leukemia.

Purpose: We explored the mechanisms leading to the distinct overexpression of EPOR as well as the effects of EPO signaling on ETV6/RUNX1-positive acute lymphoblastic leukemias. Experimental Design:ETV6/RUNX1-expressing model cell lines and leukemic cells were used for real-time PCR of EPOR expression. Proliferation, viability, and apoptosis were analyzed on cells exposed to EPO, prednisone, or inhibitors of EPOR pathways by [3H]thymidine incorporation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Annexin V/propidium iodide staining. Western blot analysis was done to detect activation of signaling proteins. Serum EPO levels and sequences of the EPOR (n = 53) as well as hemoglobin levels were taken from children with acute lymphoblastic leukemia enrolled in Austrian protocols. Results: We show here that ectopic expression of ETV6/RUNX1 induced EPOR up-regulation. Anemia, however, did not appear to influence EPOR expression on leukemic cells, although children with ETV6/RUNX1-positive leukemias had a lower median hemoglobin than controls. Exposure to EPO increased proliferation and survival of ETV6/RUNX1-positive leukemias in vitro, whereas blocking its binding site did not alter cell survival. The latter was not caused by activating mutations in the EPOR but might be triggered by constitutive activation of phosphatidylinositol 3-kinase/Akt, the major signaling pathway of EPOR in these cells. Moreover, prednisone-induced apoptosis was attenuated in the presence of EPO in this genetic subgroup. Conclusions: Our data suggest that ETV6/RUNX1 leads to EPOR up-regulation and that activation by EPO might be of relevance to the biology of this leukemia subtype. Further studies are, however, needed to assess the clinical implications of its apoptosis-modulating properties.

BloodChIP: a database of comparative genome-wide transcription factor binding profiles in human blood cells

The BloodChIP database (http://www.med.unsw.edu.au/CRCWeb.nsf/page/BloodChIP) supports exploration and visualization of combinatorial transcription factor (TF) binding at a particular locus in human CD34-positive and other normal and leukaemic cells or retrieval of target gene sets for user-defined combinations of TFs across one or more cell types. Increasing numbers of genome-wide TF binding profiles are being added to public repositories, and this trend is likely to continue. For the power of these data sets to be fully harnessed by experimental scientists, there is a need for these data to be placed in context and easily accessible for downstream applications. To this end, we have built a user-friendly database that has at its core the genome-wide binding profiles of seven key haematopoietic TFs in human stem/progenitor cells. These binding profiles are compared with binding profiles in normal differentiated and leukaemic cells. We have integrated these TF binding profiles with chromatin marks and expression data in normal and leukaemic cell fractions. All queries can be exported into external sites to construct TF–gene and protein–protein networks and to evaluate the association of genes with cellular processes and tissue expression.

An autonomous CEBPA enhancer specific for myeloid-lineage priming and neutrophilic differentiation

Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only.

Computational Prediction Models for Early Detection of Risk of Cardiovascular Events Using Mass Spectrometry Data

Early prediction of the risk of cardiovascular events in patients with chest pain is critical in order to provide appropriate medical care for those with positive diagnosis. This paper introduces a computational methodology for predicting such events in the context of robust computerized classification using mass spectrometry data of blood samples collected from patients in emergency departments. We applied the computational theories of statistical and geostatistical linear prediction models to extract effective features of the mass spectra and a simple decision logic to classify disease and control samples for the purpose of early detection. While the statistical and geostatistical techniques provide better results than those obtained from some other methods, the geostatistical approach yields superior results in terms of sensitivity and specificity in various designs of the data set for validation, training, and testing. The proposed computational strategies are very promising for predicting major adverse cardiac events within six months.

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Significance In this report we describe the generation of tissue-regenerative multipotent stem cells (iMS cells) by treating mature bone and fat cells transiently with a growth factor [platelet-derived growth factor–AB (PDGF-AB)] and 5-Azacytidine, a demethylating compound that is widely used in clinical practice. Unlike primary mesenchymal stem cells, which are used with little objective evidence in clinical practice to promote tissue repair, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner without forming tumors. This method can be applied to both mouse and human somatic cells to generate multipotent stem cells and has the potential to transform current approaches in regenerative medicine. Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.