i Kinase Gene Fusions in Melanoma by Jacqueline Turner A thesis submitted to the Faculty of the Undergraduate School of the University of Colorado in partial fulfillment of graduating with Honors from the Department of Chemistry and Biochemistry 2017 i This thesis entitled: Recurrent Kinase Gene Fusions in Melanoma Written by Jacqueline Turner has been approved for the Department of Chemistry and Biochemistry _________________________________ _________________________________ Dr. Joseph Falke Dr. Robert Parson _________________________________ _________________________________ Dr. Jennifer Martin Dr. Natalie Ahn _________________________________ _________________________________ Dr. John Tentler Dr. William Robinson Date:_______________________ The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline ii Turner, Jacqueline (B.A., Department of Chemistry and Biochemistry) Kinase Fusions in Melanoma Thesis directed by Professor William Robinson Kinase gene fusions are a mechanism of alternative pathway activation and have been increasingly described in cancer, including malignant melanoma. The prevalence of kinase gene fusions across different subtypes in melanoma has not yet been reported. Additionally, few studies in melanoma have examined the responses of these kinase gene fusions to small molecule inhibitors. We used break-apart fluorescence in situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by immunohistochemistry (IHC) or sequencing. We identified a RET fusion in an acral lentiginous melanoma, an ARMC10-BRAF fusion in an unknown primary melanoma, and an AGK-BRAF fusion in a superficial spreading melanoma. This is the first report of a RET fusion in melanoma and the ARMC10-BRAF fusion has not been previously described in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. We went on to generate patient-derived xenograft (PDX) models of both BRAF fusions to show that both the ARMC10-BRAF and AGK-BRAF kinase fusions are sensitive to downstream mitogen-activated protein (MAP) kinase pathway inhibition. We characterized these responses and identified differential responses between the different BRAF gene fusions. Our data suggests gene fusions are more common than previously thought and are actionable therapeutic targets. Broader screening for kinase fusions in melanomas lacking known driver mutations should become part of routine clinical practice. iii Dedication This thesis is dedicated to Dr. William Robinson iv Acknowledgements I want to first thank Dr. William Robinson who has been a pivotal influence on my life. His patience, time, and support throughout my entire undergraduate career and throughout the course of this project been made me into the scientist I am today. When I first entered the lab, Dr. Robinson handed me a paper. It was titled “Kinase gene fusions are frequent in Spitz neoplasms and spitzoid melanomas”. He told me, this is what we want to do. From that point on I have been completely invested in studying kinase gene fusions and building this project from scratch. Dr. Robinson has always had faith in me. Even when I lost faith in myself, Dr. Robinson encouraged me to keep moving forward and working hard. He has provided me opportunities, guidance, and support without which I would not be where I am today. Dr. Robinson is a tremendous mentor and I am incredibly grateful for everything he has done for me. Thank you. Another outstanding mentor is Dr. Kasey Couts. Kasey is an amazing teacher and has put in so much time and energy to teaching me new techniques, developing my skills, and furthering my projects. Kasey is a wonderful role model who does her research diligently and with integrity. Kasey is an inspiration to me. She has taught me numerous protocols and procedures. Kasey has given me the tools I need to work independently at the bench. She also challenges me to think about the next step in the experiments, brings me into the creative process while designing projects, and has expanded so many more projects off of my initial data. Kasey and I have worked closely very closely and she even worked with me and taught me how to write an entire paper. I am extremely appreciative for all that she has done and I am very excited for working with her next year. v Without these incredible mentors providing support and guidance, this work would not be possible. The entire Robinson Lab including, Judson Bemis, Stacey Bagby, Carol Amato, Allison Applegate, Rita Gonzalez, Magdelena Glogowska, Steven Robinson, Dr. Jennifer Hintzsche, and Dr. Keith Wells have been such a big help in moving this project forward. I want to thank them for teaching me in the lab, answering all of my questions, and dealing with my boundless energy day-to-day. I also want to specifically acknowledge Stacey Bagby for her help with the patient- derived xenograft models. Additionally, I want to thank Maren Salzmann-Sullivan and Dr. Isabel Schlaepfer for teaching me how to run my first-ever western blot and always answering all of my questions. I have enjoyed all of our laughs in the lab and I look forward to working the next year together. A special thanks goes out to the donors of this research including the Heidi Horner Foundation, the Amy Davis Foundation, and the Moore Family Foundation. I hope that this work makes you proud. This research would not be where it is at without your generosity. Thank you to Dr. Marileila Varella-Garcia and her expertise on fluorescence in situ hybridization (FISH). Dr. Garcia allowed me to be extremely involved in the FISH studies and took the time and effort to train me throughout the process. I am so grateful to have worked with such a wonderful investigator. I wish to thank the entire melanoma Scientific Advisory Board including Dr. John Tentler, Dr. Yiqun Shellman, Dr. Aik-Choon Tan, Dr. Matthew Rioth, Dr. Theresa Medina, Dr. Joshua Wisell, and Dr. Neil Box for their guidance and support on this project. vi I want to thank my thesis committee including Dr. Joe Falke, Dr. Robert Parson, Dr. Natalie Ahn, Dr. Jennifer Martin, Dr. William Robinson, and Dr. Tentler. I am very excited to present you this research and am very grateful for your service on my committee. A special thanks to the patients who donated blood and tissue to the International Melanoma Biorepository and Research Laboratory. I wish to also acknowledge the entire melanoma tissue bank whose efforts make such a unique resource available for biomedical research. Lastly, I wish to thank my family and Ethan Cabral who have supported me throughout the entire process. I am so appreciative to have them in my life. vii Contents Chapter 1. Introduction……………………………………………………………………………1 1.1 The Mitogen-Activated Protein Kinase Pathway in Melanoma…………………………..1 1.2 The Etiology of Chromosomal Rearrangement…………………………………………...3 1.3 Kinase Gene Fusions in Melanoma……………………………………………………….5 1.4 Overview…………………………………………………………………………………..8 Chapter 2. Kinase Gene Fusions in Defined Subsets of Melanoma………………………………9 2.1 Introduction………………………………………………………………………………..9 2.1.1 Subsets of Melanoma……………………………………………………………..9 2.1.2 Gene Fusions as an Alternative Kinase Activation Mechanism in Melanoma…..10 2.1.3 Overview…………………………………………………………………………11 2.2 Materials and Methods………………………………………………………...................12 2.2.1 Melanoma Patient Samples…………………………………………………...….12 2.2.2 Break-apart Fluorescence In-situ Hybridization……………..………………….12 2.2.3 Immunohistochemistry………………………………………………………….13 2.2.4 Next-generation Whole Exome Sequencing…………………….………………13 2.2.5 Targeted RNA Sequencing……………………………………………………...14 2.2.6 RT-PCR, Sanger Sequencing……………………………………………………15 2.2.7 Quantitative Real-Time PCR……………………………………………………15 2.3 Results……………………………………………………………………………………17 2.3.1 Break-apart FISH Identifies Genomic Alterations in RET, ROS1, NTRK1, and BRAF……………………………………………………………………………………..17 viii 2.3.2 NTRK1, RET, and ROS1 Expression in Tumors with Rearrangements or Genomic Alterations……………………………………………………………………..21 2.3.3 Characterization of Patient BRAF Rearrangements……………………………..23 2.3.4 Classification of Genomic Alterations…………………………………………..26 2.3.5 Kinase Fusions Occur in Pan-negative Patient Samples………………………...29 2.4 Discussion………………………………………………………………………………..32 2.4.1 Gene Fusions and Rearrangements in Melanoma……………………………….32 2.4.2 Current Methods for Gene Fusion Detection……………………………………33 2.5 Overview…………………………………………………………………………………34 Chapter 3. BRAF Kinase Fusions Exhibit Differential Responses to Targeted Therapy In Vivo..35 3.1 Introduction………………………………………………………………………………35 3.1.1 Targeted Therapy in Melanoma…………………………………………………35 3.1.2 Success in Targeting BRAF Kinase Fusions in Melanoma……………………...36 3.1.3 Overview…………………………………………………………………………37 3.2 Materials and Methods…………………………………………………………………...38 3.2.1 Melanoma Patient Samples……………………………………………………...38 3.2.2 Patient-derived Xenograft (PDX) Treatment Models……………………………38 3.2.3 Immunoblotting………………………………………………………………….38 3.3 Results……………………………………………………………………………………40 3.3.1 BRAF Kinase Fusions are Effective Therapeutic Targets in Melanoma………..40 3.3.2 Differential