Integrome signatures of lentiviral gene therapy for SCID-X1 patients.

Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.

NetBID2 provides comprehensive hidden driver analysis.

Many signaling and other genes known as "hidden" drivers may not be genetically or epigenetically altered or differentially expressed at the mRNA or protein levels, but, rather, drive a phenotype such as tumorigenesis via post-translational modification or other mechanisms. However, conventional approaches based on genomics or differential expression are limited in exposing such hidden drivers. Here, we present a comprehensive algorithm and toolkit NetBID2 (data-driven network-based Bayesian inference of drivers, version 2), which reverse-engineers context-specific interactomes and integrates network activity inferred from large-scale multi-omics data, empowering the identification of hidden drivers that could not be detected by traditional analyses. NetBID2 has substantially re-engineered the previous prototype version by providing versatile data visualization and sophisticated statistical analyses, which strongly facilitate researchers for result interpretation through end-to-end multi-omics data analysis. We demonstrate the power of NetBID2 using three hidden driver examples. We deploy NetBID2 Viewer, Runner, and Cloud apps with 145 context-specific gene regulatory and signaling networks across normal tissues and paediatric and adult cancers to facilitate end-to-end analysis, real-time interactive visualization and cloud-based data sharing. NetBID2 is freely available at https://jyyulab.github.io/NetBID .

Single-cell analysis reveals the Comma-1D cell line as a unique model for mammary gland development and breast cancer.

The mammary gland epithelial tree contains two distinct cell populations, luminal and basal. The investigation of how this heterogeneity is developed and how it influences tumorigenesis has been hampered by the need to perform studies on these populations using animal models. Comma-1D is an immortalized mouse mammary epithelial cell line that has unique morphogenetic properties. By performing single-cell RNA-seq studies, we found that Comma-1D cultures consist of two main populations with luminal and basal features, and a smaller population with mixed lineage and bipotent characteristics. We demonstrated that multiple transcription factors associated with the differentiation of the mammary epithelium in vivo also modulate this process in Comma-1D cultures. Additionally, we found that only cells with luminal features were able to acquire transformed characteristics after an oncogenic HER2 (also known as ERBB2) mutant was introduced in their genomes. Overall, our studies characterize, at a single-cell level, the heterogeneity of the Comma-1D cell line and illustrate how Comma-1D cells can be used as an experimental model to study both the differentiation and the transformation processes in vitro.

miR-424/503 modulates Wnt/ß-catenin signaling in the mammary epithelium by targeting LRP6.

During the female lifetime, the expansion of the epithelium dictated by the ovarian cycles is supported by a transient increase in the mammary epithelial stem cell population (MaSCs). Notably, activation of Wnt/ß-catenin signaling is an important trigger for MaSC expansion. Here, we report that the miR-424/503 cluster is a modulator of canonical Wnt signaling in the mammary epithelium. We show that mammary tumors of miR-424(322)/503-depleted mice exhibit activated Wnt/ß-catenin signaling. Importantly, we show a strong association between miR-424/503 deletion and breast cancers with high levels of Wnt/ß-catenin signaling. Moreover, miR-424/503 cluster is required for Wnt-mediated MaSC expansion induced by the ovarian cycles. Lastly, we show that miR-424/503 exerts its function by targeting two binding sites at the 3'UTR of the LRP6 co-receptor and reducing its expression. These results unveil an unknown link between the miR-424/503, regulation of Wnt signaling, MaSC fate, and tumorigenesis.

SLFN11 is Widely Expressed in Pediatric Sarcoma and Induces Variable Sensitization to Replicative Stress Caused By DNA-Damaging Agents.

Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA-damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress and has been implicated as a potential biomarker to predict sensitivity to DNA-damaging agents (DDA). SLFN11 expression was quantified in 220 children with solid tumors using IHC. Sensitivity to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN) was assessed in sarcoma cell lines, including SLFN11 knock-out (KO) and overexpression models, and a patient-derived orthotopic xenograft model (PDOX). SLFN11 was expressed in 69% of pediatric sarcoma sampled, including 90% and 100% of Ewing sarcoma and desmoplastic small round-cell tumors, respectively, although the magnitude of expression varied widely. In sarcoma cell lines, protein expression strongly correlated with response to TAL and IRN, with SLFN11 KO resulting in significant loss of sensitivity in vitro and in vivo Surprisingly, retrospective analysis of children with sarcoma found no association between SLFN11 levels and favorable outcome. Subsequently, high SLFN11 expression was confirmed in a PDOX model derived from a patient with recurrent Ewing sarcoma who failed to respond to treatment with TAL + IRN. Selective inhibition of BCL-xL increased sensitivity to TAL + IRN in SLFN11-positive resistant tumor cells. Although SLFN11 appears to drive sensitivity to replicative stress in pediatric sarcomas, its potential to act as a biomarker may be limited to certain tumor backgrounds or contexts. Impaired apoptotic response may be one mechanism of resistance to DDA-induced replicative stress.

Cross-talk between GLI transcription factors and FOXC1 promotes T-cell acute lymphoblastic leukemia dissemination.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly malignant pediatric leukemia, where few therapeutic options are available for patients which relapse. We find that therapeutic targeting of GLI transcription factors by GANT-61 is particularly effective against NOTCH1 unmutated T-ALL cells. Investigation of the functional role of GLI1 disclosed that it contributes to T-ALL cell proliferation, survival, and dissemination through the modulation of AKT and CXCR4 signaling pathways. Decreased CXCR4 signaling following GLI1 inactivation was found to be prevalently due to post-transcriptional mechanisms including altered serine 339 CXCR4 phosphorylation and cortactin levels. We also identify a novel cross-talk between GLI transcription factors and FOXC1. Indeed, GLI factors can activate the expression of FOXC1 which is able to stabilize GLI1/2 protein levels through attenuation of their ubiquitination. Further, we find that prolonged GLI1 deficiency has a double-edged role in T-ALL progression favoring disease dissemination through the activation of a putative AKT/FOXC1/GLI2 axis. These findings have clinical significance as T-ALL patients with extensive central nervous system dissemination show low GLI1 transcript levels. Further, T-ALL patients having a GLI2-based Hedgehog activation signature are associated with poor survival. Together, these findings support a rationale for targeting the FOXC1/AKT axis to prevent GLI-dependent oncogenic Hedgehog signaling.

An integrative ENCODE resource for cancer genomics.

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

Network Approaches for Dissecting the Immune System.

The immune system is a complex biological network composed of hierarchically organized genes, proteins, and cellular components that combat external pathogens and monitor the onset of internal disease. To meet and ultimately defeat these challenges, the immune system orchestrates an exquisitely complex interplay of numerous cells, often with highly specialized functions, in a tissue-specific manner. One of the major methodologies of systems immunology is to measure quantitatively the components and interaction levels in the immunologic networks to construct a computational network and predict the response of the components to perturbations. The recent advances in high-throughput sequencing techniques have provided us with a powerful approach to dissecting the complexity of the immune system. Here we summarize the latest progress in integrating omics data and network approaches to construct networks and to infer the underlying signaling and transcriptional landscape, as well as cell-cell communication, in the immune system, with a focus on hematopoiesis, adaptive immunity, and tumor immunology. Understanding the network regulation of immune cells has provided new insights into immune homeostasis and disease, with important therapeutic implications for inflammation, cancer, and other immune-mediated disorders.

The corrected gene proximity map for analyzing the 3D genome organization using Hi-C data.

BACKGROUND: Genome-wide ligation-based assays such as Hi-C provide us with an unprecedented opportunity to investigate the spatial organization of the genome. Results of a typical Hi-C experiment are often summarized in a chromosomal contact map, a matrix whose elements reflect the co-location frequencies of genomic loci. To elucidate the complex structural and functional interactions between those genomic loci, networks offer a natural and powerful framework. RESULTS: We propose a novel graph-theoretical framework, the Corrected Gene Proximity (CGP) map to study the effect of the 3D spatial organization of genes in transcriptional regulation. The starting point of the CGP map is a weighted network, the gene proximity map, whose weights are based on the contact frequencies between genes extracted from genome-wide Hi-C data. We derive a null model for the network based on the signal contributed by the 1D genomic distance and use it to "correct" the gene proximity for cell type 3D specific arrangements. The CGP map, therefore, provides a network framework for the 3D structure of the genome on a global scale. On human cell lines, we show that the CGP map can detect and quantify gene co-regulation and co-localization more effectively than the map obtained by raw contact frequencies. Analyzing the expression pattern of metabolic pathways of two hematopoietic cell lines, we find that the relative positioning of the genes, as captured and quantified by the CGP, is highly correlated with their expression change. We further show that the CGP map can be used to form an inter-chromosomal proximity map that allows large-scale abnormalities, such as chromosomal translocations, to be identified. CONCLUSIONS: The Corrected Gene Proximity map is a map of the 3D structure of the genome on a global scale. It allows the simultaneous analysis of intra- and inter- chromosomal interactions and of gene co-regulation and co-localization more effectively than the map obtained by raw contact frequencies, thus revealing hidden associations between global spatial positioning and gene expression. The flexible graph-based formalism of the CGP map can be easily generalized to study any existing Hi-C datasets.

Measuring the reproducibility and quality of Hi-C data.

BACKGROUND: Hi-C is currently the most widely used assay to investigate the 3D organization of the genome and to study its role in gene regulation, DNA replication, and disease. However, Hi-C experiments are costly to perform and involve multiple complex experimental steps; thus, accurate methods for measuring the quality and reproducibility of Hi-C data are essential to determine whether the output should be used further in a study. RESULTS: Using real and simulated data, we profile the performance of several recently proposed methods for assessing reproducibility of population Hi-C data, including HiCRep, GenomeDISCO, HiC-Spector, and QuASAR-Rep. By explicitly controlling noise and sparsity through simulations, we demonstrate the deficiencies of performing simple correlation analysis on pairs of matrices, and we show that methods developed specifically for Hi-C data produce better measures of reproducibility. We also show how to use established measures, such as the ratio of intra- to interchromosomal interactions, and novel ones, such as QuASAR-QC, to identify low-quality experiments. CONCLUSIONS: In this work, we assess reproducibility and quality measures by varying sequencing depth, resolution and noise levels in Hi-C data from 13 cell lines, with two biological replicates each, as well as 176 simulated matrices. Through this extensive validation and benchmarking of Hi-C data, we describe best practices for reproducibility and quality assessment of Hi-C experiments. We make all software publicly available at http://github.com/kundajelab/3DChromatin_ReplicateQC to facilitate adoption in the community.

MrTADFinder: A network modularity based approach to identify topologically associating domains in multiple resolutions.

Genome-wide proximity ligation based assays such as Hi-C have revealed that eukaryotic genomes are organized into structural units called topologically associating domains (TADs). From a visual examination of the chromosomal contact map, however, it is clear that the organization of the domains is not simple or obvious. Instead, TADs exhibit various length scales and, in many cases, a nested arrangement. Here, by exploiting the resemblance between TADs in a chromosomal contact map and densely connected modules in a network, we formulate TAD identification as a network optimization problem and propose an algorithm, MrTADFinder, to identify TADs from intra-chromosomal contact maps. MrTADFinder is based on the network-science concept of modularity. A key component of it is deriving an appropriate background model for contacts in a random chain, by numerically solving a set of matrix equations. The background model preserves the observed coverage of each genomic bin as well as the distance dependence of the contact frequency for any pair of bins exhibited by the empirical map. Also, by introducing a tunable resolution parameter, MrTADFinder provides a self-consistent approach for identifying TADs at different length scales, hence the acronym "Mr" standing for Multiple Resolutions. We then apply MrTADFinder to various Hi-C datasets. The identified domain boundaries are marked by characteristic signatures in chromatin marks and transcription factors (TF) that are consistent with earlier work. Moreover, by calling TADs at different length scales, we observe that boundary signatures change with resolution, with different chromatin features having different characteristic length scales. Furthermore, we report an enrichment of HOT (high-occupancy target) regions near TAD boundaries and investigate the role of different TFs in determining boundaries at various resolutions. To further explore the interplay between TADs and epigenetic marks, as tumor mutational burden is known to be coupled to chromatin structure, we examine how somatic mutations are distributed across boundaries and find a clear stepwise pattern. Overall, MrTADFinder provides a novel computational framework to explore the multi-scale structures in Hi-C contact maps.

Loregic: a method to characterize the cooperative logic of regulatory factors.

The topology of the gene-regulatory network has been extensively analyzed. Now, given the large amount of available functional genomic data, it is possible to go beyond this and systematically study regulatory circuits in terms of logic elements. To this end, we present Loregic, a computational method integrating gene expression and regulatory network data, to characterize the cooperativity of regulatory factors. Loregic uses all 16 possible two-input-one-output logic gates (e.g. AND or XOR) to describe triplets of two factors regulating a common target. We attempt to find the gate that best matches each triplet's observed gene expression pattern across many conditions. We make Loregic available as a general-purpose tool (github.com/gersteinlab/loregic). We validate it with known yeast transcription-factor knockout experiments. Next, using human ENCODE ChIP-Seq and TCGA RNA-Seq data, we are able to demonstrate how Loregic characterizes complex circuits involving both proximally and distally regulating transcription factors (TFs) and also miRNAs. Furthermore, we show that MYC, a well-known oncogenic driving TF, can be modeled as acting independently from other TFs (e.g., using OR gates) but antagonistically with repressing miRNAs. Finally, we inter-relate Loregic's gate logic with other aspects of regulation, such as indirect binding via protein-protein interactions, feed-forward loop motifs and global regulatory hierarchy.