Author ORCID Identifier

0000-0003-4483-5053

Date of Graduation

5-2021

Document Type

Dissertation (PhD)

Program Affiliation

Cell and Regulatory Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Dung-Fang Lee

Committee Member

Jeffrey T. Chang

Committee Member

Jeffrey A. Frost

Committee Member

John F. Hancock

Committee Member

Guang Peng

Committee Member

Lisa L. Wang

Abstract

Osteosarcoma is the most common bone malignancy in children and adolescents worldwide. Patients with Type II Rothmund-Thomson Syndrome (RTS) are highly predisposed to develop osteosarcoma, with 30% of patients in the largest cohort study developing osteosarcoma. Patients with Type II RTS have biallelic mutations in the DNA helicase RECQL4, which has been shown to have mitochondrial functions that include DNA replication and repair. We describe the generation of 12 induced pluripotent stem cell lines (iPSCs) from two paired RTS patient/ parental control (Family) sets. These iPSCs were validated, then differentiated to the bone precursor, mesenchymal stem cells (MSCs). After verification of the MSCs, we differentiated the MSCs to osteoblasts, which are thought to be the cells that form osteosarcoma. We hypothesized that transcriptional changes that occur during differentiation may be important during osteosarcomagenesis. During differentiation, increased oxidative phosphorylation related genes, specifically transcription of genes which encode complex I of the electron transport chain, were upregulated. We next demonstrated that the observed transcriptional changes led to phenotypic changes in enzyme activity. We then explored the nature of oxidative phosphorylation and glycolysis in RTS osteoblasts and found that maximal respiration and ATP production increased compared to Family osteoblasts. We then sought to explore inhibitors of oxidative phosphorylation that might reverse the phenotype of ATP production preferentially through the electron transport chain. xii We utilized the specific inhibitor of complex I, IACS-010759, and examined the nature of oxidative phosphorylation after treatment. We found significantly decreased maximal respiration and cell proliferation after treatment at physiological doses. Transcriptional changes that occurred after treatment demonstrated a decrease in cell cycling and tumor promoting pathways and a restoration of normal cellular processes, including upregulation in genes that may prevent tumor formation. This study helps shed light on the long understudied molecular pathogenesis of osteosarcoma and provides new insights for a more specific and clinically relevant therapeutic.

Keywords

osteosarcoma, oxidative phosphorylation, RECQL4, Rothmund-Thomson Syndrome, complex I, iPSC, osteoblasts, osteosarcomagenesis

Available for download on Friday, February 18, 2022

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