Systematic elucidation of transcriptional network necessary for initiation and maintenance of high-risk neuroblastoma

Neuroblastoma is a heterogeneous pediatric malignancy originating from the developing sympathetic nervous system, with poor long-term survival for high-risk patients (~40%). About half of advanced neuroblastomas harbor high-level amplification of the MYCN gene, and these tumors show few, if any, additional driver lesions. Despite significant increase in the body of knowledge of genetics in neuroblastoma, all the high-risk patients follow similar therapeutic procedures and little advancement has been made on molecular target based therapies. The major challenge is to dissect the complexity and heterogeneity of these tumors to find driver genes and activated pathways that are essential for the survival of these cancer cells. We used an integrated systems biology approach to define the core regulatory machinery responsible for maintenance of an aggressive neuroblastoma phenotypic state. In the first part of the thesis, I will discuss our computational approach to decipher the tumor heterogeneity by subtype classification, followed by identification of master regulator protein modules for three distinct molecular subtypes of high-risk neuroblastomas, which were validated in a large independent cohort of cases. We propose that such modules are responsible for integrating the effect of mutations in upstream pathways and for regulating the genetic programs and pathways necessary for tumor state implementation and maintenance. The second part of the thesis is focused on experimental validation of putative master regulators in the subtype of neuroblastomas associated with MYCN amplification. By using RNAi screening followed by experimental and computational analyses to elucidate the interdependencies between the top master regulators, we identified TEAD4-MYCN positive feedback loop as a major tumor maintenance mechanism in this subtype. While MYCN regulates TEAD4 transcriptionally, TEAD4 regulates MYCN through transcriptional and post-translational mechanisms. Jointly, MYCN and TEAD4 regulate 90% of inferred MR proteins and causally orchestrate 70% of the subtype-specific gene expression signature. TEAD4 gene expression was associated with neuroblastoma patient survival independently of age, tumor stage and MYCN status (P=2.1e-02). In cellular assays, MYCN promoted growth and repressed differentiation, while TEAD4 activated proliferation and DNA damage repair programs, the signature hallmarks of MYCN-amplified neuroblastoma cells. Specifically, TEAD4 was shown to induce MYCN-independent proliferation by transactivating key genes implicated in high-risk neuroblastoma pathogenesis, including cyclin-dependent kinases, cyclins, E2Fs, DNA replication factors, checkpoint kinases and ubiquitin ligases. The critical role of the core master regulator module in controlling tumor cell viability, both in vitro and in vivo, and its clinical relevance as a prognostic factor highlights TEAD4 as a novel and highly effective candidate target for therapeutic intervention. In this thesis, we demonstrate that interrogation of tumor specific regulatory networks with patient-derived gene expression signatures can effectively elucidate molecular subtypes as well as the core transcriptional machinery driving subtype specific hallmarks. This approach enables identification of oncogenic and non-oncogenic dependencies of high-risk neuroblastoma and is applicable to other tumor subtypes

A Systems Biology Approach to Transcription Factor Binding Site Prediction

The elucidation of mammalian transcriptional regulatory networks holds great promise for both basic and translational research and remains one the greatest challenges to systems biology. Recent reverse engineering methods deduce regulatory interactions from large-scale mRNA expression profiles and cross-species conserved regulatory regions in DNA. Technical challenges faced by these methods include distinguishing between direct and indirect interactions, associating transcription regulators with predicted transcription factor binding sites (TFBSs), identifying non-linearly conserved binding sites across species, and providing realistic accuracy estimates.We address these challenges by closely integrating proven methods for regulatory network reverse engineering from mRNA expression data, linearly and non-linearly conserved regulatory region discovery, and TFBS evaluation and discovery. Using an extensive test set of high-likelihood interactions, which we collected in order to provide realistic prediction-accuracy estimates, we show that a careful integration of these methods leads to significant improvements in prediction accuracy. To verify our methods, we biochemically validated TFBS predictions made for both transcription factors (TFs) and co-factors; we validated binding site predictions made using a known E2F1 DNA-binding motif on E2F1 predicted promoter targets, known E2F1 and JUND motifs on JUND predicted promoter targets, and a de novo discovered motif for BCL6 on BCL6 predicted promoter targets. Finally, to demonstrate accuracy of prediction using an external dataset, we showed that sites matching predicted motifs for ZNF263 are significantly enriched in recent ZNF263 ChIP-seq data.Using an integrative framework, we were able to address technical challenges faced by state of the art network reverse engineering methods, leading to significant improvement in direct-interaction detection and TFBS-discovery accuracy. We estimated the accuracy of our framework on a human B-cell specific test set, which may help guide future methodological development

Correlating measurements across samples improves accuracy of large-scale expression profile experiments

Gene expression profiling technologies suffer from poor reproducibility across replicate experiments. However, when analyzing large datasets, probe-level expression profile correlation can help identify flawed probes and lead to the construction of truer probe sets with improved reproducibility. We describe methods to eliminate uninformative and flawed probes, account for dependence between probes, and address variability due to transcript-isoform mixtures. We test and validate our approach on Affymetrix microarrays and outline their future adaptation to other technologies

A human B-cell interactome identifies MYB and FOXM1 as master regulators of proliferation in germinal centers

Assembly of a mixed interaction network specific to human B cells.Identification and validation of master regulators of germinal center reaction.MYB and FOXM1 are synergistic master regulators of proliferation in germinal center B cells and control a new protein complex involving replication and mitotic-related genes

Quality of labor and birth care in Sindh Province, Pakistan: Findings from direct observations at health facilities.

This study presents data from the first observation of labor, childbirth and immediate newborn care in a clinical setting in Sindh, the second most populous province of Pakistan. Trained midwives observed 310 births at 126 district level referral facilities and primary health care facilities in 10 districts of Sindh where the USAID-funded Maternal Child Health Integrated Program (MCHIP) was implemented. The facility participation rate was 78%. The findings show that monitoring vital signs during the initial examination was conducted for less than one-in-ten women. Infection prevention practices were only observed for one-in-four women. Modesty was preserved for less than half of women. In spite of an absence of monitoring during the first and second stages of labor, providers augmented labor with oxytocin in two-thirds of births. To prevent post-partum hemorrhage, oxytocin was administered within a minute of birth in 51% of cases. Immediate drying of the baby was nearly universal and eight out of ten babies were wrapped in a dry towel. Newborn vital signs and the baby's weight were taken in one-in-ten cases. Breastfeeding was initiated during the first hour of birth in 18% of cases. A support-person was present during labor and birth for 90% of women. While quality of care is poor across all facilities, the provision of care at district-level referral facilities was even lower quality than at primary health care facilities. This is because dais or assistants without formal training provided labor, birth, and newborn care for 40% of deliveries during night shifts at referral facilities. This study found many examples of suboptimal practice by skilled birth attendants across all levels of health facilities. There remains an urgent need to improve quality of service provision among skilled birth attendants in Pakistan

Cross-cohort analysis identifies a TEAD4-MYCN positive-feedback loop as the core regulatory element of high-risk neuroblastoma

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module—centered around a TEAD4–MYCN positive feedback loop—emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN -amplified (MYCNAmp) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo. Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas. SIGNIFICANCE: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4–MYCN positive feedback loop in MYCNAmpneuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention