Doctor of Philosophy, The Ohio State University, 2024, Biomedical Sciences

Pediatric cancer is the leading cause of death by disease in children, and approximately 43 children are expected to receive a cancer diagnosis each day. The paucity of approved treatments, many of which induce lasting negative effects on the health of survivors, and the dismal prognosis for patients who present with metastases at the time of diagnosis or recurrent/relapsed disease warrant more focused efforts on understanding the biology of pediatric cancers and developing novel treatments. Unlike adult cancers, which are usually characterized by genomic alterations and/or contributing lifestyle/environmental factors, pediatric cancers are more likely to be caused by epigenetic dysregulation or post-transcriptional processes such as alternative splicing. All premature mRNAs in a cell undergo splicing in order to remove introns and ligate exons together to produce a mature mRNA transcript that can then be translated into protein. Differential inclusion or exclusion of exons or introns leads to alternative splicing and the generation of mature mRNA transcripts that differ from the parent premature mRNA, which contributes to protein diversity in normal conditions and disease. Cancers hijack this process to selectively regulate the production of oncogenic isoforms, such as the IR-A isoform of the insulin receptor, and create a favorable environment for tumor progression and eventual metastasis. The objectives of the work presented in this dissertation are to 1) critically review the literature to ascertain what is known about alternative splicing in pediatric cancer, 2) study the role of alternative mRNA splicing in pediatric bone sarcomas, 3) test splice-switching oligonucleotides (SSOs) against splicing defects, and 4) utilize the U7 snRNA system to engineer an AAV that expresses SSO sequences as antisense RNAs. First, we reviewed the current literature on the role of alternative splicing in adult cancer, cancer predisposition syndromes, and pediatric cancer (open full item for complete abstract)

Committee: Dawn Chandler (Advisor); Emily Theisen (Committee Member); Steven Sizemore (Committee Member); Timothy Cripe (Advisor) Subjects: Biomedical Research; Molecular Biology; Oncology; Therapy

Doctor of Philosophy, The Ohio State University, 2024, Medical Science

Ewing sarcoma is a highly aggressive tumor of bone and muscle tissues, primar- ily affecting children, adolescent, and young adults. While the clinical outcome for patients with localized Ewing sarcoma have improved due to a combination of ap- proaches including surgery, radiation, and a high-dose chemotherapies, the same can not be said for patients with metastatic and relapsed diseases. The standard treatment regimen has seen little innovation over the past decades. This stagnation in therapeutic advancement reflects a critical gap in our knowledge, which, if addressed, could pave the way for more targeted interventions for Ewing sarcoma patients. Central to Ewing sarcoma pathogenesis is the EWS::FLI fusion oncoprotein. This fusion results from a translocation between chromosomes 11 and 22, merging the EWSR1 and FLI1 genes. EWS::FLI is an aberant transcription factor that dysregu- lates the gene expression profile of normal cells, leading to their malignant transfor- mation. The fusion of the EWS domain with the FLI domain endows the final protein a novel ability to bind at GGAA microsatellites, which are scattered across the hu- man genome in great numbers and with varied characteristics. EWS::FLI reprograms the epigenetic landscape by binding at GGAA repeats, opening nucleosome-wrapped DNA, recruiting chromatin modifiers, and altering chromatin architecture and the enhancer landscape. We investigated the role of the DBD-α4 helix in the FLI domain of the fusion protein in genome regulation. Employing a mutli-omics approach, we delineated the mechanisms by which this alpha-helix participates in the transcriptional activation capacity of EWS::FLI. Our studies unveiled that the DBD-α4 helix is required for col- lective binding at long and dense GGAA microsatellites, a process integral to various forms of genome regulation by EWS::FLI including formation of topologically asso- ciated domains (TADs), short-range loops, Ewing-specific enhancers, and pr (open full item for complete abstract)

Committee: Emily Theisen (Advisor) Subjects: Medicine; Oncology

Doctor of Philosophy, The Ohio State University, 2022, Molecular, Cellular and Developmental Biology

Ewing sarcoma is the second most common pediatric malignancy of bone and soft tissue. It is treated with highly aggressive therapy that involves surgery, radiation, and multiple chemo agents. However, the long-term survival rates are less than ideal (70% for localized disease, and less than 30%for metastatic disease) and the quality of life in survivors are diminished after intense therapy. A better understanding of the molecular underpinnings of this disease is therefore necessary to improve patient outcome. Genetically, Ewing sarcoma is characterized by a single chromosomal translocation event, t(11;22)(q24;q12) in ~85% of cases, that results in the expression of an oncogenic fusion transcription factor EWS/FLI. In fact, all cases of Ewing sarcoma express either EWS/FLI or a highly related FET/ETS fusion transcription factor as the sole recurring genomic aberration. Massively parallel sequencing have established Ewing sarcoma as a cancer with low mutational burden, and oncogenesis is thought to be driven by EWS/FLI mediated epigenetic reprogramming. The FLI portion of EWS/FLI contains an ETS type DNA binding domain (DBD) that allows EWS/FLI to bind consensus ETS sequences containing a single GGAA core motif (ACCGGAAGTG). The EWS portion is a low complexity domain (LCD), when fused to FLI allows EWS/FLI to bind DNA sequences containing repeats of GGAA motifs (GGAA-microsatellites) that otherwise remain inaccessible. EWS/FLI orchestrates widespread transcriptional dysregulation, including both activation and repression of thousands of genes, to promote oncogenic transformation in Ewing sarcoma. Upregulation of target genes correlate highly with EWS/FLI bound GGAA-microsatellite response elements that show length polymorphisms between cell lines and patient ethnicities. The EWS LCD has been shown to interact with and recruit histone modifying enzymes and chromatin remodelers to GGAA-microsatellites to induce epigenetic changes characteristic of enhancer elements. Thi (open full item for complete abstract)

Committee: Stephen Lessnick Dr. (Advisor) Subjects: Bioinformatics; Biology; Biomedical Research; Molecular Biology

MS, University of Cincinnati, 2022, Medicine: Clinical and Translational Research

Background: Maintaining dose-dense, interval-compressed chemotherapy improves survival in Ewing sarcoma patients but is limited by myelosuppression. Romiplostim is a thrombopoietin receptor agonist that may be useful in the treatment of chemotherapy-induced thrombocytopenia (CIT). Methods: Patients aged between 3 and 33 years with Ewing sarcoma from 2010-2020 were reviewed. CIT was defined as a failure to achieve 75,000 platelets per microliter by day 21 after the start of any chemotherapy cycle. Fisher's exact test was used for univariate analysis of categorical variables and Pearson's correlation coefficient was used to assess the association between continuous variables. Results: Twenty-seven out of 42 patients (64%) developed isolated CIT, delaying one to four chemotherapy cycles per patient. CIT occurred during consolidation therapy in 24/27(88.9%) and with ifosfamide/etoposide cycles in 24/27 (88.9%). Univariate analysis failed to identify risk factors for CIT. The use of radiation approached significance (p=0.06). Ten patients received romiplostim. The median starting dose was 3 (range 1-5) mcg/kg. Doses were escalated weekly by 1-2 mcg/kg to 4-10 mcg/kg and continued throughout chemotherapy. A higher romiplostim dose was associated with a higher change in average platelet counts from baseline r= 0.73 (p=0.04). No romiplostim-related adverse events were identified aside from mild headache. Conclusions: CIT is the primary reason for the inability to maintain treatment intensity in Ewing sarcoma. The concurrent use of romiplostim with chemotherapy is safe and feasible, and efficacy was associated with higher romiplostim doses.

Committee: Scott Langevin Ph.D. (Committee Member); Brian Turpin D.O. (Committee Member); Adam Lane (Committee Member) Subjects: Surgery

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Sciences

Ewing sarcoma is an aggressive bone and soft tissue-associated cancer affecting pediatric, adolescent, and young adult patients. Despite general improvement in pediatric cancer outcomes due to novel therapeutic options, Ewing sarcoma treatment, which consists of high-dose chemotherapy, radiation, and/or local surgical control, has remained largely unchanged for several decades and patients with metastatic disease continue to see poor outcomes. Although pediatric cancers often have far fewer mutational events than adult cancers, Ewing sarcoma is particularly interesting as the disease is often characterized by a sole chromosomal translocation event: These chromosomal translocations fuse one of the FET protein family members, a group of putative RNA-binding proteins, to a member of the ETS transcription factor family. As these FET/ETS fusion proteins have been determined to function as oncogenic transcription factors responsible for driving Ewing sarcomagenesis, it is critical that the biological mechanisms these fusions utilize to facilitate this process are elucidated. Despite discovery of several FET/ETS translocations, the majority of studies in the field focus on EWS/FLI, as it is the most common fusion observed in patients. Although these studies have provided a breadth of knowledge surrounding oncogenic function of the protein, there is a great deal of uncertainty how alternative FET/ETS fusions should be diagnosed and treated in the clinic. Herein, we characterize a novel FET/ETS fusion and perform the first comparative analysis on EWS/FLI and alternative, rarer FET/ETS fusion proteins. Our results reveal general similarities in DNA-binding and transcriptional regulation properties between the broad FET/ETS fusion group and provide the first tangible body of evidence to support that these fusions should indeed be classified as bona fide Ewing sarcoma tumors. Furthermore, we sought to characterize contributions of the FLI protein to overall EWS/FLI funct (open full item for complete abstract)

Committee: Stephen Lessnick MD/PhD (Advisor); Timothy Cripe MD/PhD (Committee Member); Lawrence Kirschner MD/PhD (Committee Member); Mark Parthun PhD (Committee Member) Subjects: Biomedical Research; Cellular Biology; Molecular Biology; Oncology

Doctor of Philosophy, The Ohio State University, 2018, Biomedical Sciences

Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is found predominantly in adolescents and young adults. The aggressive progression of Ewing sarcoma is due in part to the infiltration and activation of pro-tumor M2-like macrophages. Within the traditional classifications of inflammatory M1-like macrophages and immune suppressive M2-like macrophages is a broad panel of non-canonical macrophage subtypes whose actions in tumor progression and tumor resistance to immunotherapy are unknown. Oncolytic virotherapy that selectively replicates in and destroys cancer cells is a promising option for treating Ewing sarcoma through both direct infection of the cancer cells and activation of the host antitumor immune response. The effect of M1-like and M2-like tumor macrophages on oncolytic virus therapy, however, is variable among solid tumors and is unknown in Ewing sarcoma. We observed high activity of M2-like macrophage subtypes M(IL4) and M(IL10) in Ewing sarcoma xenograft models that experience a macrophage-dependent restriction of oncolytic virus antitumor efficacy. Macrophage depletion with Clodrosome or trabectedin greatly enhanced oncolytic virus antitumor efficacy of Ewing sarcoma xenografts by increasing tumor stroma inflammatory activity and decreasing angiogenic and immune suppressive signaling molecules. Our data suggest that combination of trabectedin and oncolytic virus therapy warrants testing in the clinical setting of pediatric sarcoma treatment. The presence of M(IL4) and M(IL10) tumor macrophages may be a cellular biomarker for determining antitumor efficacy of trabectedin and oncolytic virus combination therapy in a wider panel of solid tumor types.

Committee: Timothy Cripe (Advisor); Balveen Kaur (Committee Member); Kevin Cassady (Committee Member); Deborah Parris (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2018, Biomedical Sciences

Objective: Ewing Sarcoma is a pediatric bone malignancy initiated by a t(11;22) chromosomal translocation that produces the EWS/FLI oncoprotein. EWS/FLI transcriptionally activates and represses its target genes to mediate oncogenic reprogramming. Expression of its up-regulated targets correlates with EWS/FLI binding to associated GGAA-microsatellites, which show length polymorphisms. These microsatellite polymorphisms may critically affect EWS/FLI-responsiveness of key gene targets. For example, NR0B1 is necessary for EWS/FLI mediated oncogenic transformation, and we found a “sweet-spot” of 20-26 repeat length as optimal for EWS/FLI mediated transcriptional activity at NR0B1 through clinical observations and in vitro studies. The mechanism underlying this optimal length, however, is unknown. Methods: We explored the stoichiometry and binding affinity of EWS/FLI for different GGAA-repeat lengths through biochemical studies, including fluorescence polarization, ChIP-seq, and RNA-seq, combined with bioinformatics analysis. Additionally, use of EWS/FLI deletion constructs has been critical for elucidating the particular binding behavior of EWS/FLI at different microsatellite repeat lengths. Luciferase reporter assays, anchorage-independent growth and proliferation assays, as well as CRISPR technology have extended our findings to the in vivo setting. Finally, microscopy studies including use of confocal and transmission electron microscopy (TEM) have contributed visual characterization of the specific biochemical mechanisms we are investigating. Results: CRISPR-mediated deletion of the NR0B1 GGAA-microsatellite in Ewing sarcoma cells provided our field with the first in vivo evidence for the necessity of EWS/FLI binding at GGAA-microsatellites for anchorage dependent growth. Our biochemical studies, using recombinant ¿22 (a version of EWS/FLI containing only the FLI portion) demonstrate a stoichiometry of one monomer binding every two consecutive GGAA-repeats on (open full item for complete abstract)

Committee: Stephen Lessnick (Advisor); Michael Freitas (Committee Chair); Dennis Guttridge (Committee Member); Charles Bell (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2017, Medicine: Molecular and Developmental Biology

Hypoxia is a common feature in solid tumors which facilitates the development of therapeutically resistant and aggressive tumor phenotypes. As oxygen levels decrease, cellular metabolism and energy production becomes impaired and the potential for catastrophic DNA damage increases. To prevent cell death, tumor cells undergo several adaptations which prevent depletion of nutrients, minimize DNA damage, and re-acquire blood flow. The mechanisms behind tumor cell survival during hypoxia are poorly understood. Tumor neovascularization is a critical step in facilitating continued tumor growth. Accordingly anti- angiogenic therapy has shown significant success in preventing progression free survival however increased tumor aggression and alternative neovascularization methods result in decreased overall patient survival. Vasculogenic mimicry is one such proposed method of alternative neovascularization wherein tumor cells transdifferentiate and form de novo vascular structures. Although there are indications that this process is driven by hypoxia and is linked to aggressive tumor phenotypes, there is little known about the signaling pathways inducing such an adaptation. Evidence from in vitro studies suggest that activation of DNA damage repair proteins in proliferating hypoxic cells is critical for sustained proliferation and cell viability thus implicating repair proteins as potential therapeutic targets. In vivo studies demonstrating a correlation between hypoxia and the formation of γ-H2AX further support this potential. The evaluation of such therapeutic targets is limited by the inability of in vitro monolayer culture models to recreate therapeutically relevant aspects of a 3D in vivo tumor. Multicellular tumor spheroids (MCTS) represent a 3D in vitro culture model in which tumor cell adaptations to the hypoxic tumor microenvironment are recreated. These include the accumulation of metabolic waste, appearance of nutrient gradients, and development of prolif (open full item for complete abstract)

Committee: Rashmi Hegde Ph.D. (Committee Chair); Zelia Correa M.D. Ph.D. (Committee Member); Richard Lang Ph.D. (Committee Member); Qing Richard Lu Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. Ph.D. (Committee Member) Subjects: Cellular Biology