ORCID ID
Graduation Date
Fall 12-19-2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Programs
Interdisciplinary Graduate Program in Biomedical Sciences
First Advisor
Gloria Borgstahl
Abstract
Mitoxantrone (MX) has broad applications as a chemotherapeutic across cancer types. Its abilities to inhibit topoisomerase 2 and intercalate DNA are well characterized. However, despite years in the clinic, researchers continue to identify non-canonical targets of MX, underlining our limited understanding of MX’s complex cellular interactions. More recently, MX was identified as an inhibitor of the RAD52:RPA protein-protein interaction, which is a critical complex in several DNA repair pathways and in homologous recombination (HR)-deficient cancer cells. Subsequent studies showed heightened sensitivity across several HR-deficient cell lines. However, the mechanisms underlying RAD52:RPA complex disruption and MX-sensitivity in HR-deficient cancer cells were unclear. This information is key to understanding MX’s therapeutic value and interpreting MX-sensitivity in HR-deficient cancer.
The studies presented in Chapter 2 looked to characterize the direct interaction between MX and RAD52. This was done using surface plasmon resonance, limited proteolysis, dynamic light scattering, and nano-differential scanning fluorimetry. Each of these investigations provided evidence of a direct interaction between MX and RAD52. The results suggested that MX interacts near or within the RPA-binding domain of RAD52, and it modulates RAD52 stability and architecture in a dose-dependent manner. These studies provide insight into how RAD52 dynamics are altered upon MX-interaction, which may contribute to disrupting RAD52’s interaction with RPA.
The studies outlined in Chapter 3 examine MX-sensitivity in HR-deficient UWB1.289 ovarian cancer cells and survey the proteome-wide binding profile of MX. First, an MX-biotin probe (MXP) and a negative control (MXP-NC) were used in a proof-of-concept study to demonstrate their utility as molecular probes of MX. These molecular tools were then used to survey the cellular targets of MX in HR-deficient UWB1.289 cells. In parallel, shotgun proteomics was used to assess the molecular consequences of MX-treatment. Proteomic analyses of MX-treated HR-deficient UWB1.289 cells showed unique downregulation of pathways necessary for genomic stability, including single-strand annealing, alongside significant upregulation of proteins related to ribosome biogenesis and RNA processing. Chemoproteomic analysis using MXP corroborated these results. This work helps to characterize the target landscape of MX and provides insight into off-target effects and MX action in HR-deficient cancer.
Recommended Citation
Wallin, Savanna A., "Deciphering Mechanisms of Mitoxantrone Action in Homologous Recombination Deficient Ovarian Cancer" (2025). Theses & Dissertations. 1011.
https://digitalcommons.unmc.edu/etd/1011
Comments
2025 Copyright, the authors