Fugen Li

Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia.

Familial platelet disorder with predisposition to acute myelogenous leukaemia (FPD/AML, MIM 601399) is an autosomal dominant disorder characterized by qualitative and quantitative platelet defects, and propensity to develop acute myelogenous leukaemia (AML). Informative recombination events in 6 FPD/AML pedigrees with evidence of linkage to markers on chromosome 21q identified an 880-kb interval containing the disease gene. Mutational analysis of regional candidate genes showed nonsense mutations or intragenic deletion of one allele of the haematopoietic transcription factor CBFA2 (formerly AML1) that co-segregated with the disease in four FPD/AML pedigrees. We identified heterozygous CBFA2 missense mutations that co-segregated with the disease in the remaining two FPD/AML pedigrees at phylogenetically conserved amino acids R166 and R201, respectively. Analysis of bone marrow or peripheral blood cells from affected FPD/AML individuals showed a decrement in megakaryocyte colony formation, demonstrating that CBFA2 dosage affects megakaryopoiesis. Our findings support a model for FPD/AML in which haploinsufficiency of CBFA2 causes an autosomal dominant congenital platelet defect and predisposes to the acquisition of additional mutations that cause leukaemia.

Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity

Cells differentiate when transcription factors bind accessible cis-regulatory elements to establish specific gene expression programs. In differentiating embryonic stem cells, chromatin at lineage-restricted genes becomes sequentially accessible, probably by means of ‘pioneer’ transcription factor activity, but tissues may use other strategies in vivo. Lateral inhibition is a pervasive process in which one cell forces a different identity on its neighbours, and it is unclear how chromatin in equipotent progenitors undergoing lateral inhibition quickly enables distinct, transiently reversible cell fates. Here we report the chromatin and transcriptional underpinnings of differentiation in mouse small intestine crypts, where notch signalling mediates lateral inhibition to assign progenitor cells into absorptive or secretory lineages. Transcript profiles in isolated LGR5+ intestinal stem cells and secretory and absorptive progenitors indicated that each cell population was distinct and the progenitors specified. Nevertheless, secretory and absorptive progenitors showed comparable levels of H3K4me2 and H3K27ac histone marks and DNase I hypersensitivity—signifying accessible, permissive chromatin—at most of the same cis-elements. Enhancers acting uniquely in progenitors were well demarcated in LGR5+ intestinal stem cells, revealing early priming of chromatin for divergent transcriptional programs, and retained active marks well after lineages were specified. On this chromatin background, ATOH1, a secretory-specific transcription factor, controls lateral inhibition through delta-like notch ligand genes and also drives the expression of numerous secretory lineage genes. Depletion of ATOH1 from specified secretory cells converted them into functional enterocytes, indicating prolonged responsiveness of marked enhancers to the presence or absence of a key transcription factor. Thus, lateral inhibition and intestinal crypt lineage plasticity involve interaction of a lineage-restricted transcription factor with broadly permissive chromatin established in multipotent stem cells.

The androgen receptor cistrome is extensively reprogrammed in human prostate tumorigenesis

Master transcription factors interact with DNA to establish cell type identity and to regulate gene expression in mammalian cells. The genome-wide map of these transcription factor binding sites has been termed the cistrome. Here we show that the androgen receptor (AR) cistrome undergoes extensive reprogramming during prostate epithelial transformation in man. Using human prostate tissue, we observed a core set of AR binding sites that are consistently reprogrammed in tumors. FOXA1 and HOXB13 colocalized at the reprogrammed AR binding sites in human tumor tissue. Introduction of FOXA1 and HOXB13 into an immortalized prostate cell line reprogrammed the AR cistrome to resemble that of a prostate tumor, functionally linking these specific factors to AR cistrome reprogramming. These findings offer mechanistic insights into a key set of events that drive normal prostate epithelium toward transformation and establish the centrality of epigenetic reprogramming in human prostate tumorigenesis.

Oncogenic deregulation of EZH2 as an opportunity for targeted therapy in lung cancer

UNLABELLED As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways, leading to epigenetic transformation independent of canonical growth factor pathway activation. As such, tumors feature a transcriptional program distinct from KRAS- and EGFR-mutant mouse lung cancers, but shared with human lung adenocarcinomas exhibiting high EZH2 expression. To target EZH2-dependent cancers, we developed a potent open-source EZH2 inhibitor, JQEZ5, that promoted the regression of EZH2-driven tumors in vivo, confirming oncogenic addiction to EZH2 in established tumors and providing the rationale for epigenetic therapy in a subset of lung cancer. SIGNIFICANCE EZH2 overexpression induces murine lung cancers that are similar to human NSCLC with high EZH2 expression and low levels of phosphorylated AKT and ERK, implicating biomarkers for EZH2 inhibitor sensitivity. Our EZH2 inhibitor, JQEZ5, promotes regression of these tumors, revealing a potential role for anti-EZH2 therapy in lung cancer. Cancer Discov; 6(9); 1006-21. ©2016 AACR.See related commentary by Frankel et al., p. 949This article is highlighted in the In This Issue feature, p. 932.

Integration of multiethnic fine-mapping and genomic annotation to prioritize candidate functional SNPs at prostate cancer susceptibility regions

Interpretation of biological mechanisms underlying genetic risk associations for prostate cancer is complicated by the relatively large number of risk variants (n = 100) and the thousands of surrogate SNPs in linkage disequilibrium. Here, we combined three distinct approaches: multiethnic fine-mapping, putative functional annotation (based upon epigenetic data and genome-encoded features), and expression quantitative trait loci (eQTL) analyses, in an attempt to reduce this complexity. We examined 67 risk regions using genotyping and imputation-based fine-mapping in populations of European (cases/controls: 8600/6946), African (cases/controls: 5327/5136), Japanese (cases/controls: 2563/4391) and Latino (cases/controls: 1034/1046) ancestry. Markers at 55 regions passed a region-specific significance threshold (P-value cutoff range: 3.9 × 10(-4)-5.6 × 10(-3)) and in 30 regions we identified markers that were more significantly associated with risk than the previously reported variants in the multiethnic sample. Novel secondary signals (P < 5.0 × 10(-6)) were also detected in two regions (rs13062436/3q21 and rs17181170/3p12). Among 666 variants in the 55 regions with P-values within one order of magnitude of the most-associated marker, 193 variants (29%) in 48 regions overlapped with epigenetic or other putative functional marks. In 11 of the 55 regions, cis-eQTLs were detected with nearby genes. For 12 of the 55 regions (22%), the most significant region-specific, prostate-cancer associated variant represented the strongest candidate functional variant based on our annotations; the number of regions increased to 20 (36%) and 27 (49%) when examining the 2 and 3 most significantly associated variants in each region, respectively. These results have prioritized subsets of candidate variants for downstream functional evaluation.

FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ER-positive breast cancer

Significance One of the mechanisms of endocrine resistance in estrogen receptor α (ER)-positive (+) breast cancer is the cross-talk between the ER and growth factor receptor pathways leading to altered ER activity and a reprogrammed ER-dependent transcriptome. However, key mediators of this ER-dependent transcriptional reprogramming remain elusive. Here we demonstrate that forkhead box protein A1 (FOXA1) up-regulation via gene amplification or overexpression contributes to endocrine resistance and increased invasiveness phenotypes by altering the ER-dependent transcriptome. We further show that IL-8, one of the top altered FOXA1/ER effectors, plays a key role in mediating these phenotypes and is a potential target to treat ER+/FOXA1-high breast cancer. Our findings provoke a new interplay of FOXA1 in the ER transcriptional program in endocrine-resistant breast cancer. Forkhead box protein A1 (FOXA1) is a pioneer factor of estrogen receptor α (ER)–chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we report preclinical evidence for a role of FOXA1 in Endo-R breast cancer as well as evidence for its clinical significance. FOXA1 is gene-amplified and/or overexpressed in Endo-R derivatives of several breast cancer cell line models. Induced FOXA1 triggers oncogenic gene signatures and proteomic profiles highly associated with endocrine resistance. Integrated omics data reveal IL8 as one of the most perturbed genes regulated by FOXA1 and ER transcriptional reprogramming in Endo-R cells. IL-8 knockdown inhibits tamoxifen-resistant cell growth and invasion and partially attenuates the effect of overexpressed FOXA1. Our study highlights a role of FOXA1 via IL-8 signaling as a potential therapeutic target in FOXA1-overexpressing ER-positive tumors.

Embryonic transcription factor SOX9 drives breast cancer endocrine resistance

Significance Resistance to endocrine treatment remains a significant clinical obstacle. ESR1 mutations were found to be the mechanism of endocrine resistance in a substantial number of patients with metastatic ER-positive breast. However, these mutations are primarily linked to aromatase inhibitor resistance and are not strongly associated with tamoxifen resistance. Herein, we show that tamoxifen treatment promotes a RUNX2–ER complex, which mediates an altered ER cistrome that facilitates the up-regulation of SOX9. We show that up-regulation of SOX9, an embryonic transcription factor with key roles in metastases, is a driver of endocrine resistance in the setting of tamoxifen treatment. Our data provide putative targets for the development of new strategies to treat tamoxifen-resistant breast cancer. The estrogen receptor (ER) drives the growth of most luminal breast cancers and is the primary target of endocrine therapy. Although ER blockade with drugs such as tamoxifen is very effective, a major clinical limitation is the development of endocrine resistance especially in the setting of metastatic disease. Preclinical and clinical observations suggest that even following the development of endocrine resistance, ER signaling continues to exert a pivotal role in tumor progression in the majority of cases. Through the analysis of the ER cistrome in tamoxifen-resistant breast cancer cells, we have uncovered a role for an RUNX2–ER complex that stimulates the transcription of a set of genes, including most notably the stem cell factor SOX9, that promote proliferation and a metastatic phenotype. We show that up-regulation of SOX9 is sufficient to cause relative endocrine resistance. The gain of SOX9 as an ER-regulated gene associated with tamoxifen resistance was validated in a unique set of clinical samples supporting the need for the development of improved ER antagonists.

Validation study of the LAMBDA model for predicting the BRCA1 or BRCA2 mutation carrier status of North American Ashkenazi Jewish women

lambda is a model that estimates the probability an Ashkenazi Jewish (AJ) woman carries an ancestral BRCA1 or BRCA2 mutation from her personal and family cancer history. lambda is relevant to clinical practice, and its implementation does not require a computer. It was developed principally from Australian and UK data. We conducted a validation study using 1286 North American AJ women tested for the mutations 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2. Most had a personal or family history of breast cancer. We observed 197 carriers. The area under the receiver operator characteristic (ROC) curve (a measure of ranking) was 0.79 [95% confidence interval (CI) = 0.77–0.81], similar to that for the model‐generating data (0.78; 95% CI = 0.75–0.82). lambda predicted 232 carriers (18% more than observed; p = 0.002) and was overdispersed (p = 0.009). The Bayesian computer program brcapro gave a similar area under the ROC curve (0.78; 95% CI = 0.76–0.80), but predicted 367 carriers (86% more than observed; p < 0.0001), and was substantially overdispersed (p < 0.0001). Therefore, lambda is comparable to brcapro for ranking AJ women according to their probability of being a BRCA1 or BRCA2 mutation carrier and is more accurate than brcapro which substantially overpredicts carriers in this population.

ChiLin: a comprehensive ChIP-seq and DNase-seq quality control and analysis pipeline

BackgroundTranscription factor binding, histone modification, and chromatin accessibility studies are important approaches to understanding the biology of gene regulation. ChIP-seq and DNase-seq have become the standard techniques for studying protein-DNA interactions and chromatin accessibility respectively, and comprehensive quality control (QC) and analysis tools are critical to extracting the most value from these assay types. Although many analysis and QC tools have been reported, few combine ChIP-seq and DNase-seq data analysis and quality control in a unified framework with a comprehensive and unbiased reference of data quality metrics.ResultsChiLin is a computational pipeline that automates the quality control and data analyses of ChIP-seq and DNase-seq data. It is developed using a flexible and modular software framework that can be easily extended and modified. ChiLin is ideal for batch processing of many datasets and is well suited for large collaborative projects involving ChIP-seq and DNase-seq from different designs. ChiLin generates comprehensive quality control reports that include comparisons with historical data derived from over 23,677 public ChIP-seq and DNase-seq samples (11,265 datasets) from eight literature-based classified categories. To the best of our knowledge, this atlas represents the most comprehensive ChIP-seq and DNase-seq related quality metric resource currently available. These historical metrics provide useful heuristic quality references for experiment across all commonly used assay types. Using representative datasets, we demonstrate the versatility of the pipeline by applying it to different assay types of ChIP-seq data. The pipeline software is available open source at https://github.com/cfce/chilin.ConclusionChiLin is a scalable and powerful tool to process large batches of ChIP-seq and DNase-seq datasets. The analysis output and quality metrics have been structured into user-friendly directories and reports. We have successfully compiled 23,677 profiles into a comprehensive quality atlas with fine classification for users.

Integrative analysis reveals the transcriptional collaboration between EZH2 and E2F1 in the regulation of cancer-related gene expression

Overexpression of EZH2 is frequently linked to the advanced and metastatic stage of cancers. The mechanisms of its oncogenic function can be context specific, and may vary depending on the protein complexes that EZH2 interacts with. To identify novel transcriptional collaborators of EZH2 in cancers, a computational approach was developed that integrates protein–DNA binding data, cell perturbation gene expression data, and compendiums of tumor expression profiles. This holistic approach identified E2F1, a known mediator of the Rb tumor suppressor, as a transcriptional collaborator of EZH2 in castration-resistant prostate cancer. Subsequent analysis and experimental validation found EZH2 and E2F1 cobind to a subset of chromatin sites lacking H3K27 trimethylation, and activate genes that are critical for prostate cancer progression. The collaboration of EZH2 and E2F1 in transcriptional regulation is also observed in diffuse large B-cell lymphoma cell lines, where activation of the transcriptional network is concordant with the cellular response to the EZH2 inhibitor. Implications: The direct collaboration between EZH2 and Rb/E2F1 pathway provides an innovative mechanism underlying the cascade of tumor progression, and lays the foundation for the development of new anticancer targets/strategies. Mol Cancer Res; 14(2); 163–72. ©2015 AACR.