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Transcription Factor Activating Protein 4 Is Synthetic Lethal and a Master Regulator of MYCN Amplified Neuroblastoma

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Transcription Factor Activating Protein 4 Is Synthetic Lethal and a Master Regulator of MYCN Amplified Neuroblastoma Transcription Factor Activating Protein 4 is synthetic lethal and a master regulator of MYCN amplified neuroblastoma Shuobo Zhang Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2015 © 2015 Shuobo Zhang All rights reserved ABSTRACT Transcription Factor Activating Protein 4 is synthetic lethal and a master regulator of MYCN amplified neuroblastoma Shuobo Zhang Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, no drugs that target MYCN have yet been developed. Here, by combining a whole genome shRNA library screen and Master Regulator Inference Algorithm (MARINa) analysis, we identified Transcription Factor Activating Protein 4 (TFAP4) as a novel synthetic lethal interactor with MYCN amplification in neuroblastoma. Silencing TFAP4 selectively inhibits MYCN amplified neuroblastoma growth both in vitro and in xenograft mice models. TFAP4 expression is inversely correlated with patient survival in MYCN-high neuroblastoma. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as seen by increased neurite outgrowth, and up-regulation of neuronal markers. TFAP4 regulates a downstream signature similar to the signature of the oncogene anaplastic lymphoma kinase (ALK). Taken together, our results validate TFAP4 as an important master regulator in MYCN amplified neuroblastoma and a novel synthetic interactor with MYCN amplification. Thus, TFAP4 may be a novel drug target for neuroblastoma treatment. TABLE OF CONTENTS List of tables and figures .....................................................................................................ii Acknowledgements ..............................................................................................................v Chapter 1. Introduction .....................................................................................................1 Chapter 2. Materials and Methods .....................................................................................12 Chapter 3. TFAP4 is a master regulator and potential synthetic lethal candidate for MYCN amplified neuroblastoma ...................................................................21 Chapter 4. TFAP4 is synthetic lethal with MYCN amplification in neuroblastoma ......39 Chapter 5. TFAP4 inhibits differentiation of MYCN amplified neuroblastoma ............61 Chapter 6. Discussion .........................................................................................................80 References .............................................................................................................................87 i LIST OF TABLES AND FIGURES Chapter 1 Figure 1.1 Kaplan-Meier survival curve of infants <1 year old with metastatic disease (Stage IV). ................................................................................3 Figure 1.2 X-ray structure of MYC-MAX heterodimer binding on to DNA E-box ...........................................................................................4 Figure 1.3 Illustration of synthetic lethal agents selectively killing cells with gene A mutation. ...................................................................................7 Figure 1.4 Schematic illustration of MARINa. ......................................................10 Chapter 3 Figure 3.1 Neuroblastoma cell line and shRNA structure used in the synthetic lethal screen. ..................................................................................................27 Figure 3.2 Whole genome shRNA library screen identifies 218 synthetic lethal candidates with MYCN amplification. ..................................................28 Figure 3.3 MARINa analysis identifies top 25 master regulators in MYCN amplified neuroblastoma in NRC dataset. ................................29 Figure 3.4 MARINa analysis identifies top 25 master regulators in MYCN amplified neuroblastoma in NCI-TARGET dataset. .............................................30 Figure 3.5 Venn diagram of overlapping activated master regulators ...................31 Figure 3.6 Venn diagram of overlapping transcriptional regulators in the synthetic lethal screen and master regulators in MYCN amplified neuroblastoma .......................................................................................32 ii Figure 3.7 Rank of the overlapping master regulators in shRNA screen and in MARINa analysis. .................................................................................33 Figure 3.8 TFAP4 expression is inversely correlated with survival in MYCN-high neuroblastoma ...................................................................34 Chapter 4 Figure 4.1 MYCN directly upregulates TFAP4. ....................................................46 Figure 4.2 TFAP4 is upregulated in MYCN amplified neuroblastoma patients .....47 Figure 4.3 SHEP21N competition assay. ...............................................................48 Figure 4.4 c-MYC expression is significantly higher in stage 4 MYCN non- amplified neuroblastoma patients .........................................................49 Figure 4.5 MYC and MYCN expression in neuroblastoma cell lines. .....................50 Figure 4.6 Silencing TFAP4 by siRNA. ................................................................51 Figure 4.7 Silencing TFAP4 by siRNA only inhibits MYCN amplified neuroblastoma cell growth ....................................................................52 Figure 4.8 Knocking down of TFAP4 by two dox-inducible shRNAs in MYCN amplified cells .......................................................................................53 Figure 4.9 TFAP4 knocked down by dox-inducible shRNAs inhibits MYCN amplified neuroblastoma cell growth ....................................................54 Figure 4.10 Silencing TFAP4 inhibits colony formation of MYCN amplified neuroblastoma .......................................................................................55 Figure 4.11 Silencing of TFAP4 inhibits growth of MYCN amplified tumors ........56 Figure 4.12 Tumor weight of xenograft mice and TFAP4 protein level ..................57 Figure 4.13 Silencing TFAP4 inhibits growth of established tumors ......................58 iii Chapter 5 Figure 5.1 Family tree of helix-loop-helix transcription factors. ...........................62 Figure 5.2 Silencing TFAP4 induces neurite outgrowth in MYCN amplified neuroblastoma .......................................................................................68 Figure 5.3 Knocking down TFAP4 upregulates neuronal marker GAP43 expression.. ...........................................................................................69 Figure 5.4 Silencing TFAP4 slows G1/S progression. ...........................................70 Figure 5.5 Knocking down TFAP4 does not change CDKN1A expression.. .........71 Figure 5.6 Differentially expressed genes in MYCN amplified neuroblastoma after silencing TFAP4. ..........................................................................72 Figure 5.7 Silencing TFAP4 represses CCNE2 gene expression.. .........................73 Figure 5.8 Silencing TFAP4 represses ROCK2 and PAK4 gene expression. ........74 Figure 5.9 TFAP4 induces similar signature as anaplastic lymphoma kinase. ......75 Chapter 6 Figure 6.1 Schematic diagram of TFAP4 signaling in MYCN amplified neuroblastoma. ......................................................................................85 iv ACKNOWLEDGEMENTS First and foremost, I would like to thank my mentor, Dr. Darrell Yamashiro, for his guidance, mentorship and incredible support through the course of my graduate study. I am indebted to Darrell for giving me such a wonderful experience to learn, explore and grow as a scientist. Thank you also to my thesis committee members, Drs. Andrea Califano, Jan Kitajewski, Ken Olive, and Jose Silva, for all your advice on my project. I am grateful for the Pathology and Molecular Medicine graduate program at Columbia University for their support as well. I would then like to thank all the members of the Yamashiro lab, past and present, for supporting me through this wonderful journey. I could not have imagined working with such a diverse group of colleagues and friends. It has been a great joy to learn from every one of you. Thank you to Dr. Debarshi Banerjee, who always provided me great insights and suggestions. Thanks to Drs. Angela Kadenhe-Chiweshe and Alejandro Garcia, who taught me and performed the intrarenal xenograft surgeries in such a professional way. Thank you also to Ashish Jani, Jason Mitchell, John Andrews, Emily Sbiroli, Roderick Alfonso and Na Wei, for the assistance whenever I needed and for making lab so much fun. I wish you all every success in future. I am also extremely grateful for the support from Drs. Jessica Kandel and Angela Kadenhe-Chiweshe. Additionally, Briana Fitch and Stanley Cho, worked with me as summer students. They both made significant contributions in validating the synthetic lethal candidates. I extend a heartfelt thank you to the Califano lab, especially to my collaborator Dr. Gonzalo Lopez, who often intrigued me with a different perspective. It has been my pleasure to work with you and this work could not have been so great without your contribution. Thanks also to Jiyang v Yu, who introduced me to the bioinformatics world and donated his weekends to analyze the screen data for science and friendship.
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