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Determining the Function and Effects of a Novel FOXO1 Fusion Gene

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Determining the Function and Effects of a Novel FOXO1 Fusion Gene Determining the Function and Effects of a Novel FOXO1 Fusion Gene Tanner Wherley A thesis presented to the Faculty at Cincinnati Children’s Medical Center in partial fulfillment of the requirements for the degree of Master of Science in the Graduate School of Arts and Sciences Committee Chair: Rashmi Hedge, PhD Biomedical Research Technologies Program College of Medicine University of Cincinnati May 15th, 2020 Abstract: Rhabdomyosarcoma (RMS) is a heterogenous group of soft-tissue tumors with many underlying causes varying patient to patient. The two most encountered histological classifications, Alveolar and Embryonal, are driven by molecularly distinct mechanisms with an unknown fusion gene status in the later. Recent sequencing identified a novel fusion gene between FOXO1 and the Long Intergenic Non-coding RNA 00598 in an Embryonal RMS patient. Here we use C2C12 mouse myoblasts to investigate the resulting FOXO1-LINC fusion protein (FOXO1- LINC) and its role in the development of Embryonal RMS. We show that the FOXO1-LINC protein inhibits terminal differentiation of mouse myoblasts and disrupts the fusion of nuclei. The FOXO1-LINC protein acts as a dominant negative on FOXO1 function, blocking the activation of FOXO1 responsive luciferase production and target gene activation. Although expression of the FOXO1-LINC was not fully transformative, MyoG expression was inhibited during differentiation and the genotype/phenotype was mimicked using a constitutively active transcriptional co-activator with PDZ-binding motif (Taz) construct, suggesting potential roles for the fusion gene in the development of ERMS. ii iii Table of Contents: Abstract…………………………………….........................................................................….....….…..ii Table of Contents……………………………......................................................................……....…...iv Introduction……………………………….................................................................................….........v Experimental Design and Protocols………........................................................................................vi Results…............................................................................................................................................vii Conclusion..........................................................................................................................................x Statement of Work .............................................................................................................................xi Bibliography .......................................................................................................................................xii Supplemental Figures ........................................................................................................................xv iv Introduction: RMS is the most common soft-tissue sarcoma of children and young adults (1). Histologically, the two most common subtypes, Alveolar and Embryonal (ARMS and ERMS), display a variable degree of skeletal muscle differentiation (2). Morphologically, ARMS tumors are characterized by ‘primitive’, compact, small round blue cancer cells adhered to the outsides of alveoli like structures. Harder to distinguish pathologically, ERMS often resembles the developing embryonic muscle with variable cell density and expanded eosinophilic cytoplasm (3,4). In low to intermediate risk patients, surgical removal and chemotherapy regimens, such as vincristine and actinomycin-D, have contributed to a slow climb in overall survival, but the 5-year survival for patients with metastatic or recurrent tumors remains grim (5,6). Determining the oncogenic drivers and aberrant biological cellular pathways hijacked in these tumors is vital to develop targeted therapies for patients present and future, reducing chemotherapy toxicity and providing hope for patients with recurrent or metastatic growth (7). With the need of more accurate classifications reflecting patient prognosis and therapeutic strategies, classification parameters are shifting to also reflect the underlying tumor genomic landscape as Fusion Positive (FP) or Fusion Negative (FN) (2,8). Distinct to FP ARMS is a t(2;13) or t(1;13) PAX3/7-FOXO1 chimeric fusion protein, with few other PAX fusion artifacts noted (9,10). This chromosomal translocation fuses the DNA binding domain (DBD) of PAX with the potent transactivating domain (TAD) of FOXO1. Expression of a PAX3/7 fusion protein in combination with other genetic alterations drive oncogenicity in FP ARMS (11-13). A small subset of FN ARMS cases also exist, histologically diagnosed as ARMS, but lacking a PAX fusion gene (9,14). However, clinical analysis and microarray data reveal similar expression profiles between FN ARMS and FN ERMS (15,16). FN ERMS is known to be caused by many genetic alterations such as the loss of heterozygosity at 11.p15.5 and various mutations affecting receptor tyrosine kinase (RTK)/RAS/PIK3CA pathways (17,18). Despite these classification efforts, studying FP ERMS remains important as it is still poorly understood due to the small number of fusion genes identified in ERMS with different expressional profiles (19-22). For this report, ARMS and ERMS will refer to the histological patterning and diagnosis and FP/FN status will reflect the underlying genomic landscape affecting the tumor. The expression of muscle specific markers indicate myogenic progenitors are the cells of origin for RMS, however other mesenchymal origins are reported (23-26). Myogenesis is the process of generating muscle during development or in response to injury and is often recapitulated in-vitro using an established primary mouse myoblast line, C2C12 (27,28). These satellite-cell derived myoblasts rapidly proliferate in a nutrient rich environment, however differentiate into mature myotubules when serum starved (28,29). Directing this process, MYOD1 acts as the master regulator of skeletal muscle differentiation, activating the expression of muscle specific genes such as MYOG to induce cell cycle arrest and confirm terminal differentiation (30). In the development of RMS, while MYOD1 is still able to bind DNA, complete activation of its downstream targets is disrupted, suggesting the inhibition of important MYOD1 cofactors (31-33). For this reason, MYOD1 and its target genes are useful markers to confirm the muscular cell lineage and gage the degree of myogenesis in RMS (34,35). Initially discovered with the PAX3/7-FOXO1 fusion protein (11), the FOXO1 gene encodes a transcription factor acting downstream of many signaling pathways in response to starvation and stress (36). Mechanistically, this protein binds DNA as a monomer until phosphorylated at AKT phosphorylation sites (T24, S256, S319), where it then binds 14-3-3 proteins for nuclear removal (37,38). Early in-vivo studies revealed that FOXO1 is involved in muscle fiber type specification during embryogenesis, however its exact role was found to be conflicting (39,40). Muscle specific triple knockout of three Forkhead box O proteins 1, 3 and 4 confirm that FOXO factors play essential roles in regulation of autophagy and the ubiquitin-proteasome system in mature muscle (41). Nonetheless, FOXO1 may play a role in myogenic growth and differentiation with unidentified roles. Here we present preliminary and supportive in-vitro data for a novel fusion gene acting as a driver of ERMS. An 80kB deletion of Chromosome 13 results in the fusion of FOXO1 Exon 1 (E1) with Exon 5 (E5) of the Long Intergenic Non-coding RNA 00598. Determining the function and effects of the FOXO1-LINC fusion gene will help establish correct therapeutic strategies as preliminary data indicates this fusion gene is conserved in other soft-tissue sarcomas with an unidentified role. v Experimental Design and Protocols: Cell Culture- Both Human Embryonic Kidney (HEK) 293 and mouse C2C12 myoblast cells were maintained in Delbecco’s Modified Eagles Medium (DMEM, Gilbco) supplemented with 10% Fetal Bovine Serum (FBS) and 1:100 antibiotic-antimycotic (10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, 5 µg/mL of Gibco Amphotericin B). Cells were passaged at 90-95% confluency in a 10-cm dish (~7.5x106 cells total, depending on the cell line used), and maintained under a passage number of 18. Differentiating C2C12 were cultured in DMEM with 2% Horse Serum (2%HS) and anti-anti for the indicated number of days. Cells were incubated at 37˚C in 5% CO2. Media was changed after the first day of 2%HS treatment and then every two days. Plasmids- PiggyBac (PB) (System Biosciences) constructs were under a CAG promoter with a cytomegalovirus (CMV) enhancer, an internal ribosomal entry sight (IRES) and enhanced green fluorescent protein (eGFP). The PB-CAG-eGFP-IRES was used as the control (Ctl) and inserts were cloned into the PB-CAG-eGFP-IRES backbone using blunt end ligation. The PB-Flag-FOXO1-LINC-eGFP contains an N-terminal Flag Tag (DYKDDDDK) used for tracking the localization and immunoblotting (IB) for expression levels. The PB-Flag- caFOXO1 was created by subcloning the insert from a pcDNA3-Flag-FKHR-AAA mutant (Addgene plasmid #13508). The 3x Insulin Response Sequence (3xIRS) luciferase plasmid (3xIRS-luc) was in a pGL-2 promoter vector with a simian virus 40 (SV40) promoter (Addgene plasmid #13511). The PB-Flag-Taz-S4A-eGFP construct was given by Xiaohua Hu from Richard Lu’s laboratory. Stable Cell Line Generation- Transfection of C2C12 was difficult, often resulting in less than 20% eGFP+,
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