Project Summary Pediatric low-grade gliomas (pLGGs) are collectively the most common brain tumor of childhood and associated with devastating and life-long morbidity. pLGGs most often arise from single genetic driver events involving the BRAF oncoprotein. This finding has paved the way for precision medicine approaches for pLGG, leveraging MEK, BRAF and pan-RAF inhibitors. Research within our prior SPORE program identified the RAF inhibitor tovorafenib as a promising compound for BRAF-altered pLGGs and generated structural insights into its function. Our Phase 0/1 Clinical Trial of tovorafenib led to breakthrough drug designation by the FDA and set the stage for a company-sponsored Phase 2 trial (FIREFLY-1) of tovorafenib in recurrent pLGG. While early results are encouraging, response to tovorafenib is variable across patients for unclear reasons and there remain important unmet needs for children with pLGG. There is a need to better understand RAF alterations within pLGG at the cellular level and investigate if more specific RAF inhibitors, or combination therapies, could improve the therapeutic window and efficacy. Additionally, many children with pLGGs will be cured by standard of care (i.e. surgery and chemotherapy), and it is difficult to predict who needs targeted therapies and who can be spared additional, potentially toxic treatments. Even children who are ultimately cured by standard of care are followed with periodic MRIs that can be stressful for patients and parents alike. Non-invasive prognostic technologies are needed to better define risk of recurrence in pLGG. To address these needs, we have three specific aims: (1) to define RAF and MEK inhibitors with enhanced efficacy and tolerability for targeted inhibition for pLGG, (2) to conduct a genomic “audit” of WT RAF expression in pLGG, and (3) to develop non- invasive “child friendly” predictors of which children with pLGG are most likely to require and respond to RAF antagonists. For (1), we will determine if (and how) other RAF isoforms aside from BRAF play a role in pLGG tumor development and assess which RAF and MEK inhibitors, alone or in combination, most selectively target the relevant RAF isoforms. For (2), we will leverage novel single-cell genomic profiling and mass spectrometry technologies to probe the expression and activation state of RAF isoforms (A, B, and C) in surgically resected pLGGs. The aim 2 study plan may explain why certain BRAF-altered tumors do not respond as well to tovorafenib. For (3), we will utilize emerging, imaging-based artificial intelligence (AI) techniques to analyze longitudinal MRI scans from a large multi-institutional database and the FIREFLY-1 tovorafenib study. We will determine how volumetric trajectories change over time in pLGG patients on and off therapy, and if trajectories can predict clinical progression. We will determine if voxel-based deep learning analysis of longitudinal MRI can better predict recurrence and select patients who...