Abstract A24: Targeted synthetic lethal screens to identify acquired vulnerabilities in a novel class of ASCL1-dependent neuroendocrine non-small cell lung cancers
Authors: Augustyn Alexander , Borromeo Mark , Wang Tao , Shao Chunli , Dospoy Patrick , Huffman Kenneth , Carstens Ryan , Girard Luc , Behrens Carmen , Wistuba Ignacio , Xie Yang , Johnson Jane , Gazdar Adi , Minna John 
Published: August 2014

Abstract
Introduction: Gene expression signatures from large cohorts of lung tumors suggest that distinct neuroendocrine (NE) cancers with poor prognosis appear in ~10% of otherwise pathologically unremarkable NSCLCs. However, a complete molecular characterization is lacking because a similar subtype has yet to be identified in a significant number of lung cancer cell lines. Our goal is to develop a pre-clinical model for NSCLC-NE and use it to rationale targeted therapy for this important subtype of lung cancer. We are developing targeted siRNA-based synthetic lethal screens to identify acquired vulnerabilities in lung cancers by using “mini-libraries” of putative cancer stem cell genes (CSC, N = 40), nuclear receptors and their co-regulators (NR/CoReg, N = 120), chromatin remodelers (CR, N = 102), and ASCL1-regulated druggable genes (N = 40). The siRNA libraries were derived from multiple studies while the ASCL1-targeted library was developed from our own ChIP-Seq data.
Identifying NSCLC-NE Cell Lines: Using mRNA expression signatures from 207 lung cell lines (NSCLC/SCLC/HBEC) we identified a cluster of NSCLC cell lines (9% of NSCLC) that express genes indicative of a neuroendocrine phenotype (NSCLC-NE). A highly expressed gene in this class of cell lines is the lineage-specific transcription factor ASCL1, which is required for the development of pulmonary neuroendocrine cells. ASCL1 loss-of-function studies demonstrate dramatic cell death compared to controls, suggesting these cells are addicted to ASCL1 for survival.
ASCL1 ChIP-Seq Analysis: To better understand the molecular pathogenesis of ASCL1-dependent NSCLC-NEs and identify druggable therapeutic downstream targets, we performed ChIP-Seq analysis on six ASCL1+ and two ASCL1- cell lines. A 125-member “ASCL1-signature” was generated and tested on multiple resected NSCLC datasets (N &gt; 800 patients) for prognostic utility and tumors that exhibited the “ASCL1 signature” had significantly worse prognosis. A target of ASCL1 as determined by ChIP-Seq analysis is the anti-apoptotic regulator BCL2. BCL2 knockdown using siRNA as well as inhibition of BCL2 using a small molecule (ABT-263) results in cell death that is specific to ASCL1+/BCL2+ cells.
ASCL1 Upstream Regulation: A separate therapeutic avenue is to determine upstream regulators of ASCL1 and we have demonstrated that ASCL1 participates in a double-negative feedback loop with the MEK/ERK pathway. Pharmacological activation of the MEK/ERK pathway results in loss of ASCL1 mRNA and protein, and induces apoptosis in ASCL1-dependent NSCLC-NE lines. Additionally, siRNA-mediated knockdown of ASCL1 results in reciprocal activation of the MEK/ERK pathway even in the presence of an siRNA targeting MEK1. Combining MEK/ERK activators with ABT-263 results in dramatic induction of apoptosis in NSCLC-NE while sparing normal immortalized HBEC cells suggesting the possibility of a synthetic lethal combination therapy for this subset of tumors.
Synthetic Lethal Screens: We have completed synthetic lethal siRNA screens for the CSC, NR/CoReg, and CR mini-libraries using a panel of lung cancer lines. Optimal transfection conditions were established for each line and siRNA pools were used with multiple (n = 9) replicates providing great reproducibility (r &gt; 0.9). From these screens, preliminary data demonstrates that the NSCLC-NE tumor lines have response phenotypes distinct from all other classes of lung cancer.
Conclusions: We show that NE gene expression in NSCLC is of clinical relevance, and ASCL1 appears to be a lineage-dependent oncogene for NSCLC-NE. ASCL1 ChIP-Seq and siRNA screen data provide a roadmap for systematically searching for therapeutic targets such as BCL2 while work focused on targeting pathways upstream of ASCL1 also demonstrates clinical potential for NSCLC with neuroendocrine features. Our results indicate that lung cancers driven by ASCL1 have distinct acquired vulnerabilities. (Supported by NCI SPORE P50CA70907, CPRIT)
Citation Format: Alexander Augustyn, Mark Borromeo, Tao Wang, Chunli Shao, Patrick Dospoy, Kenneth Huffman, Ryan Carstens, Luc Girard, Carmen Behrens, Ignacio Wistuba, Yang Xie, Jane Johnson, Adi Gazdar, John Minna. Targeted synthetic lethal screens to identify acquired vulnerabilities in a novel class of ASCL1-dependent neuroendocrine non-small cell lung cancers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A24.