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Forkhead Box F1 (FOXF1) Is an Essential Effector of the PAX3

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Forkhead Box F1 (FOXF1) Is an Essential Effector of the PAX3 Forkhead Box F1 (FOXF1) is an essential effector of the PAX3/FOXO1 oncogene in human alveolar rhabdomyosarcoma A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Program of Molecular and Developmental Biology in the College of Medicine on April 9, 2019 by David E. Milewski B.A. Miami University (2010) Dissertation Committee Dr. Tanya Kalin, MD PhD (Chair) Dr. Vladimir Kalinichenko, MD PhD Dr. Joseph Pressey, MD Dr. Doug Millay, PhD Dr. Lionel Chow, MD PhD P a g e | 2 Abstract Rhabdomyosarcoma (RMS) is an aggressive soft tissue neoplasm that displays characteristic of incomplete skeletal muscle differentiation. RMS can be subcategorized based on the presence of specific genetic alterations which can be associated with adverse patient outcome. The recurrent t(2;13) translocation defines the alveolar subtype of rhabdomyosarcoma (aRMS) and gives rise to a PAX3/FOXO1 fusion protein. RMS patients with this translocation have the poorest outcomes, despite the absence of cooperating mutations. Multiple studies have defined a gene signature associated with fusion-positive aRMS. While it is presumed that these genes may contribute to the malignant phenotype of aRMS, little mechanistic evidence exists for their role in aRMS. In this work, we study the biological and molecular contributions of Forkhead Box F1 (FOXF1), one of the most consistently and uniquely upregulated genes in fusion-positive aRMS. Using a combination of cell lines and newly established patient derived xenograft (PDX) models, we uncover a central role for FOXF1 in the pathogenesis of aRMS. We found that FOXF1 is uniquely and specifically induced in aRMS due to direct transcriptional regulation by PAX3/FOXO1 at distal enhancer elements. Knockdown or knockout of FOXF1 in aRMS revealed an important role for promoting the proliferation and survival of aRMS cells. Moreover, loss of FOXF1 was associated with widespread induction of spontaneous myogenic differentiation despite the presence of the PAX3/FOXO1 oncogene. In a primary human myoblast model, expression of FOXF1 alone was sufficient to block terminal myogenic differentiation and bypass myoblast senescence. Mechanistically, we found that FOXF1 establishes a unique epigenetic landscape by co-regulating enhancers with PAX3/FOXO1, MYOD1 and MYOG. In conclusion, we have identified the transcriptional activation of FOXF1 by PAX3/FOXO1 is a critical event for tumorigenesis and endows unique molecular mechanisms to fusion positive RMS. P a g e | 3 P a g e | 4 Acknowledgements I have had fantastic support from those inside and outside the laboratory. I would first like to thank my mentor, Dr. Tanya Kalin, for her strong support over the years. She has given me the freedom to pursue my research interests, even though some of them were outside the scope of the lab. She has always given great feedback on data and helped keep me focused on the goals for my projects. My committee has also helped steer me in the right direction and were always willing to meet to discuss ideas about the project. I would also like to thank the lab members of the Kalin and Kalinichenko labs. They have made a great lab environment where we can learn from each other and have a fun while we do our work. I would also like to thank Dr. Joseph Pressey, Dr. Sara Szabo, Dr. Brian Turpin for their trust and support with establishing the sarcoma PDX program at CCHMC. It has been such a unique, impactful experience for my career development and has really inspired me. Most importantly, I would like to thank my parents, Dr. Allan and Mary Kay Milewski, and my wonderful wife Maribeth Milewski for their support of my research. They have always been understanding of the long hours I spent in lab, gave me good advice, and made sure I relaxed and had fun when I was out of lab. They have helped make this such a fun experience. P a g e | 5 Table of Contents Title Page ....................................................................................................................................... 1 Abstract .......................................................................................................................................... 2 Acknowledgements ....................................................................................................................... 4 Table of Contents .......................................................................................................................... 5 List of Figures ................................................................................................................................ 7 List of Tables ............................................................................................................................... 10 Chapter 1: Introduction ............................................................................................................. 12 I. Overview of Rhabdomyosarcoma.......................................................................................... 12 a) Histological subtypes and clinical correlates .................................................................... 12 b) Genomic Landscape of Rhabdomyosarcoma .................................................................... 15 II. Identification of a fusion-positive aRMS gene signature ..................................................... 18 III. Forkhead Box F1 (FOXF1) ................................................................................................. 20 a) Features of the FOXF1 gene locus. ................................................................................... 20 b) Structural anatomy of the FOXF1 protein. ........................................................................ 22 d) Phenotypes of FOXF1+/- humans and FOXF1-/- mice. ...................................................... 23 e) Expression of FOXF1 in human cancer. ........................................................................... 24 Chapter 2: Tumor modeling using patient-derived xenografts (PDX): CCHMC Sarcoma PDX Initiative .............................................................................................................................. 26 I. Abstract .................................................................................................................................. 26 II. Introduction .......................................................................................................................... 26 III. Materials and Methods ........................................................................................................ 31 IV. Results ................................................................................................................................. 37 V. Discussion ........................................................................................................................... 51 Chapter 3: Transcriptional regulation of the FOXF1 gene locus in aRMS. ........................ 53 P a g e | 6 I. Abstract .................................................................................................................................. 53 II. Introduction .......................................................................................................................... 53 III. Materials and Methods ........................................................................................................ 54 IV. Results ................................................................................................................................. 57 V. Discussion ........................................................................................................................... 69 Chapter 4: Identification of FOXF1 as positive regulator of cell proliferation and a potent repressor of terminal myogenic differentiation ....................................................................... 71 I. Abstract ................................................................................................................................. 71 II. Introduction .......................................................................................................................... 71 III. Materials and Methods ....................................................................................................... 73 IV. Results ................................................................................................................................ 74 V. Discussion ............................................................................................................................ 92 Chapter 5: Genome-wide binding of FOXF1 in aRMS uncovers cooperative enhancer activation with PAX3/FOXO1 and myogenic regulatory factors ........................................... 94 I. Abstract .................................................................................................................................. 94 II. Introduction .......................................................................................................................... 94 III. Materials and Methods ........................................................................................................ 96 IV. Results ................................................................................................................................. 98 V. Discussion ........................................................................................................................
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