Identifying ERK5 and its Associated Protein Interactome as Novel Druggable Targets in Diffuse Midline Glioma, H3K27-altered.

Introduction: Diffuse midline glioma (DMG) is an inoperable, aggressive, and lethal primary pediatric brain tumor with a median survival of 8-11 months. There is an unmet clinical need to develop novel therapies to improve patient outcomes.

Methods: The histone H3 (H3K27M) mutation, leading to a global reduction of H3K27 trimethylation, is a defining characteristic of DMG, H3K27-altered (DMG-H3K27a), and leads to activation of pro-tumorigenic signaling pathways. In preliminary studies, we found that H3K27M mutations deregulated receptor tyrosine kinases (RTKs) and activated RAS signaling. Using a siRNA screen, we identified ERK5, but not ERK1/2, as a novel effector of RAS pathway signaling in DMG-H3K27a cells. Collectively, we hypothesized that ERK5 is essential for DMG-H3K27a growth.

Results: Genetic and pharmacologic suppression of ERK5 inhibited DMG-H3K27a tumor growth, promoted apoptosis, and reduced proliferative capacity in vitro and in vivo. We performed quantitate protein phosphorylation profiling and in vitro validation and rescue experiments to determine that ERK5 functioned to stabilize MYC, a known proto-oncogene. Indeed, ERK5 inhibition reduced MYC levels and increased DMG-H3K27a cell doubling time. To determine ERK5’s protein-protein interactome and mechanistic regulators, we successfully developed a biotin-based BiolD2 system for ERK5. BiolD2 is a highly specific system that can detect a greater number of protein-protein interactions as compared to traditional methods. BiolD2 mass spectrometry analysis identified significant differences between ERK5 and ERK1/2’s interactome in DMG-H3K27a. Preliminary exploratory analysis of the ERK5 interactome using Gene Ontology and the STRING database showed significant associations between ERK5 and proteins involved in solute transport, trace-nutrient-related functions, and endosomal trafficking.

Conclusion : We have identified the H3K27M mutation as a driver of deregulated RTK activity and RAS pathway activation, and ERK5 as a novel therapeutic target in DMG-H3K27a. Utilization of the BiolD2 system may provide a further mechanistic understanding of ERK5’s downstream effector functions and generate additional druggable targets in H3K27M gliomas.