Abstract 4384: Selective cross-cohort discovery of transcriptional mechanisms presiding over high-risk neuroblastoma subtype state maintenance
Authors: Rajbhandari Presha , Lopez Gonzalo , Yu Jiyang , Rodriguez-Barrueco Ruth , Alvarez Mariano , Martinez Daniel , Yarmarkovich Mark , Vandesompele Jo , Mestdagh Pieter , Silva Jose M. , Lasorella Anna , Iavarone Antonio , Maris John M. , Califano Andrea 
Published: July 2016

Abstract
BACKGROUND: Neuroblastoma (NBL) is the most common extracranial solid tumor in children. High-risk NBLs progress to metastatic disease and have 5-year survival of only ∼40%, despite intensive multimodal therapy. This malignancy is characterized by significant heterogeneity, both clinical and molecular, which is still poorly understood.
Rather than focusing on its initiating genetic events, which are highly idiosyncratic, we focused on the core regulatory machinery responsible for implementation and maintenance of tumor state. This approach led to elucidating three molecularly distinct subtypes of high-risk NBLs, as well as the core regulatory machinery responsible for their implementation and stability, including canalization and integration of mutational events and regulation of the genetic programs that represent the hallmarks of this disease.
METHODS: We dissected large-scale gene expression profiling data available from TARGET and NRC Consortium by clustering algorithm and established three subtypes of high-risk NBL, followed by identification of master regulators (MR)s of each subtype by Master Regulator Inference algorithm (Lefebvre, C. et al, 2010). We performed extensive experimental validation of MRs by both in-vitro and in-vivo RNAi mediated screening, using cell viability as readout. We then used a variety of experimental assays to elucidate the modular logic controlling disease state and to identify novel NBL subtype specific dependencies.
RESULTS: We identified unique MR protein modules for three distinct molecular subtypes of high-risk NBL, which were conserved across independent cohorts. Experimental MR validation identified a TEAD4-MYCN positive feedback loop as the key NBL state maintenance mechanisms in the MYCN amplification associated subtype. Jointly, MYCN and TEAD4 regulate 90% of inferred MR proteins and causally implement 70% of the subtype gene expression signature. Biologically, MYCN repressed differentiation and TEAD4 activated proliferation, two hallmarks of MYCN-amplified NBL. Specifically, TEAD4 was shown to induce MYCN-independent proliferation by transactivating key genes implicated in high-risk NBL pathogenesis, including cyclin-dependent kinases, cyclins, E2Fs, DNA replication factors, checkpoint kinases and ubiquitin ligases. Consistently, TEAD4 inhibition induced loss of NBL cell viability, thus providing novel therapeutic targets. TEAD4 activity was an outstanding predictor of survival, independent of outcome-related variables.
CONCLUSION: Our results show that the inference of transcriptional regulators driving distinct molecular subgroups when combined with functional analyses is valuable to uncover the regulatory modules required for sustaining the tumor subtypes. This approach can be used to successfully identify the functional bottlenecks of other cancer subtypes.
Citation Format: Presha Rajbhandari, Gonzalo Lopez, Jiyang Yu, Ruth Rodriguez-Barrueco, Mariano Alvarez, Daniel Martinez, Mark Yarmarkovich, Jo Vandesompele, Pieter Mestdagh, Jose M. Silva, Anna Lasorella, Antonio Iavarone, John M. Maris, Andrea Califano. Selective cross-cohort discovery of transcriptional mechanisms presiding over high-risk neuroblastoma subtype state maintenance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4384.