Date of Award

2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

James M. Stafford

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a group of predominantly pediatric brain tumors with an average age of diagnosis of 6-7 years old, and a poor prognosis (median survival of ~1 year). Given the location of DIPG in the brainstem, surgical approaches are limited. Furthermore, the tumors have limited responsivity to traditional chemotherapy or radiotherapy; ergo new therapeutic options are needed. Recently, the drug ONC201 has emerged as a potential therapeutic option with outcomes sometimes surpassing progression-free, and expected survival outcomes. However, the selectivity of its effect, and mechanism in DIPG is still unclear. Here, we pursue a better understanding of ONC201, and its mechanism of action directly in DIPG patient-derived cell lines. First, we demonstrate that DIPG cell lines are among the most highly sensitive brain tumors, and significantly more sensitive than cells derived from normal brain tissue. Next, we directly show that activation of the mitochondrial protease, ClpP is the primary target of ONC201 in DIPG through ClpP mutagenesis, and pharmacology studies. Validating a role for ClpP as a vulnerability in DIPG is its high expression relative to normal brain tissue, and a significant positive correlation between ClpP protein levels, and ONC201 sensitivity in our cell line panel. We further demonstrated that ONC201 affects the spare respiratory capacity of the mitochondria with mitochondrial reactive oxygen species (ROS) production driving ONC201 toxicity in DIPG. Given the abnormal epigenome of DIPG, and its potential therapeutic targeting, we next probed how ONC201, and ClpP drive metabolic changes that may interact with the metabolome. A consistent hallmark of ClpP activation in DIPG was an elevated aKG/Succinate ratio, which indicates that ONC201 treatment affects the Krebs cycle. As the polycomb mark H3K27me3 was altered as a result of ONC201 treatment, we speculate these changes are linked to the TCA cycle disruption noted above. RNA-seq analysis further confirms the impact of ONC210 on DIPG by upregulating cell cycle, DNA replication, spliceosome, and RNA transport meanwhile downregulating ferroptosis, ribosome, oxidative phosphorylation, and mTOR signaling pathways. In conclusion, ONC201 hyperactivates ClpP resulting in impaired DIPG metabolism, mitochondrial damage, ROS production, and widespread epigenetic effects. Ultimately, we hope to elucidate whether ClpP targeting can be used to better diagnose, and improve therapeutic options in DIPG.

Language

en

Number of Pages

179 p.

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