Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Durbin, Adam D. et al. “Selective gene dependencies in MYCN- amplified neuroblastoma include the core transcriptional regulatory circuitry.” Nature genetics 50 (2018): 1240-1246 © 2018 The Author(s) As Published 10.1038/s41588-018-0191-z Publisher Springer Science and Business Media LLC Version Author's final manuscript Citable link https://hdl.handle.net/1721.1/124922 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Published as: Nat Genet. 2018 September ; 50(9): 1240–1246. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript HHMI Author Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry Adam D. Durbin*,1,2, Mark W. Zimmerman*,1, Neekesh V. Dharia*,1,2,3, Brian J. Abraham4, Amanda Balboni Iniguez1,3, Nina Weichert-Leahey1, Shuning He1, John M. Krill-Burger3, David E. Root3, Francisca Vazquez3, Aviad Tsherniak3, William C. Hahn3,5, Todd R. Golub1,3, Richard A. Young4,6,†, A. Thomas Look1,2,†,‡, and Kimberly Stegmaier1,2,3,†,‡ 1Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215 2Division of Pediatric Hematology/Oncology, Boston Children’s Hospital, Boston, MA, 02115 3The Broad Institute, Cambridge, MA, 02142 4Whitehead Institute for Biomedical Research, Cambridge, MA, 02142 5Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215 6Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142 Abstract Childhood high-risk neuroblastomas with MYCN gene amplification are difficult to treat effectively1. This has focused attention on tumor-specific gene dependencies that underlie tumorigenesis and thus provide valuable targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR-Cas9 approaches to detect genes involved in tumor cell growth and survival2–6, we identified 147 candidate gene dependencies selective for MYCN-amplified neuroblastoma cell lines, compared to over 300 other human cancer cell lines. We then used genome-wide ChIP-seq analysis to demonstrate that a small number of essential transcription factors: MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2, are members of the transcriptional core regulatory circuitry (CRC) that maintains cell state in MYCN-amplified neuroblastoma. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which act synergistically, in vitro and in vivo, with rapid downregulation of CRC transcription factor gene Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms †Corresponding Authors: Kimberly Stegmaier [email protected]; A. Thomas Look [email protected]; Richard A. Young [email protected]. *These authors contributed equally to the study. ‡These authors jointly supervised this work and represent co-principal investigators. Author Contributions: ADD, MWZ, NVD, KS, BJA, RAY and ATL conceived the study and designed the experiments. ADD and MWZ performed genomic meta-analyses, ChIP-seq, ATAC-seq, siRNA, low-throughput CRISPR-Cas9, pharmacologic and gene expression assays. NW and SH performed ChIP-seq and ATAC-seq experiments. ABI, ADD, NVD and KS performed and interpreted the drug activity and synergy studies. NVD, KS, JMK, FV, DER, AT, WCH and TRG performed and analyzed the genome-scale CRISPR-Cas9 screening experiments. NVD, BJA, and RAY performed computational analysis. ADD, MWZ, NVD, KS and ATL wrote the manuscript with input from all authors. Competing Interests: RAY is a founder and shareholder of Syros Pharmaceuticals, which is discovering and developing therapeutics directed at transcriptional pathways in cancer. KS and WCH consult for Novartis Pharmaceuticals as part of the Dana-Farber Cancer Institute/Novartis Drug Discovery Program. BJA is a shareholder of Syros Pharmaceuticals. No other potential conflicts of interest are declared. Durbin et al. Page 2 expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma that are essential for cell state and survival in this tumor. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author We Manuscript HHMI Author conducted systematic genome-scale CRISPR-Cas9 screens in 341 human cancer cell lines, including nine neuroblastoma lines with MYCN-amplification7. Our cell line panel also included an additional two neuroblastoma cell lines, SKNFI and SKNAS, which do not harbor MYCN-amplification but do express high levels of MYCN or c-MYC, respectively8. We identified 147 candidate genes that were selectively essential to the growth and survival of MYCN-amplified neuroblastoma cell lines (Supplementary Table 1). The guide RNAs targeting these genes showed greater depletion in the nine MYCN-amplified neuroblastoma cell lines than in the other human cancer cell lines representing 26 tumor types. Classification of the 147 dependencies by gene ontology molecular function revealed a diverse set of genes encoding proteins in numerous functional categories (Fig. 1a)9,10, with overrepresentation of ontologies for nucleic acid binding (12%, p=8.48×10−3) and transcription factor activity or binding (20%, p=6.78×10−3). Many dependency genes have not been previously associated with the sympathoadrenal cell lineage. Others have been reported to be involved in neural crest cell specification and neuroblastoma pathogenesis1,11–20. This set of transcriptional regulatory genes is highly connected by putative protein-protein interactions in the STRING database21 (Fig. 1b). Representative dependency scores in MYCN-amplified neuroblastoma compared to other tumor cells are shown in Fig. 1c, d for the genes HAND2 and ISL1, and for a subset of other genes with nucleic acid binding and/or transcription factor activities in Supplementary Fig. 1. Our results show that many transcription factor genes with selective dependency in MYCN- amplified neuroblastoma cells also displayed dependency in the two MYCN non-amplified cell lines studied so far, although additional MYCN non-amplified lines will need to be studied before firm conclusions can be drawn. We next performed transient small-interfering RNA (siRNA) knockdown targeting mRNAs that encode five transcription factors dependencies (HAND2, ISL1, PHOX2B, GATA3 and TBX2) to independently verify that these genes represent dependencies in MYCN-amplified neuroblastoma cell lines. Depletion of expression of these genes resulted in suppression of colony formation and induction of apoptosis (Fig. 1e, f and Supplementary Figs. 2, 3). Thus, MYCN-amplified neuroblastoma cells rely on a discrete set of genes that are either unique to, or only minimally shared with, other tumor types. The gene dependencies we identified suggest that diverse sets of complex pathways are activated to support the viability of MYCN-amplified neuroblastoma. Given the overrepresentation of transcription factor and nucleic acid binding activities among the 147 gene dependencies (Fig. 1a), we hypothesized that these genes were specifically required for transcriptional regulation in neuroblastoma cells. Previous studies have demonstrated that lineage-specific differences in cell state during development are determined by tissue- specific core regulatory circuits (CRCs) that may be subverted to drive malignant transformation22,23. Core regulatory circuitry is formed by transcription factors assembled into positive, feed-forward autoregulatory loops that establish and maintain cell lineage and identity through their extended regulatory networks24,25. CRC transcription factor genes are typically marked by extensive stretches of acetylation at histone H3-lysine 27 (H3K27ac), a Nat Genet. Author manuscript; available in PMC 2019 February 22. Durbin et al. Page 3 genomic feature characteristic of high levels of gene expression, termed super-enhancers (SEs)26,27 (Fig. 2a). To investigate the relevance of a CRC framework to the dependency genes identified earlier, we performed chromatin-immunoprecipitation coupled to high- HHMI Author ManuscriptHHMI Author Manuscript HHMI Author throughput Manuscript HHMI Author sequencing (ChIP-seq) for H3K27ac in five MYCN-amplified neuroblastoma cell lines, revealing a shared set of 77 genes associated with SEs across all five cell lines (Fig. 2b and Supplementary Table 2). As observed in the selective dependencies, the SE- associated genes in MYCN-amplified neuroblastoma cells were overrepresented among ontologies9,10 including nucleic acid binding (21%, p=3.93×10−3) and transcription factor activity or binding (22%, p=4.29×10−8). Examples of the H3K27ac signals of highly ranked transcription factor genes are shown in Fig. 2c and Supplementary Fig. 4. When these 77 SE-associated genes were compared with the 147 selective dependencies in MYCN-amplified neuroblastoma identified by the CRISPR-Cas9 screen, 11 of the SE- associated genes were selective dependencies and 10 of 11 (91%) of these genes encoded transcription factors (Fig. 2d). This represents 11 of 69 SE-associated genes targeted by specific guide RNAs (15.9%, p=1.11×10−10