Pediatric Low-grade Gliomas with CRAF Gene Fusions are Therapeutically Distinct from Braf-Fusions Based on Dimerization Mediated by N-Terminal Fusion Partner
Pediatric low-grade gliomas (PLGGs) are the most common brain tumors in children mainly defined by activating gene fusions that dysregulate the mitogen-associated protein kinase (MAPK) pathway. Our lab has characterized the molecular mechanism of PLGG-associated KIAA1549-BRAF gene fusion and response to targeted MAPK pathway inhibitors. We have since gone on to explore non-BRAF gene fusions, specifically CRAF (or RAF1) fusions, QKI-RAF1 and SRGAP3-RAF1, as potential PLGG driver mutations. As CRAF and BRAF are shared targets of MAPK therapeutics, we sought to investigate the mechanistic and response of CRAF-fusions to clinically relevant RAF inhibitors (RAFi) and downstream pathway inhibitors. We focused on the role of N-terminal fusion partners and RAF dimerization for successful targeting. Using heterologous model systems, we found that CRAF-fusions do not respond to RAFi, show partial response to single-agent MEKi, but robustly respond to combinatorial targeting of both MAPK and PI3K pathways and novel RAF dimer inhibitors. Upon comparing the homo- and hetero-dimerization profiles of QKI-RAF1and BRAF-fusions in the presence of RAFi, we found that QKI-RAF1 retains robust homo- and hetero-dimerization that, in contrast, are disrupted in BRAF fusions that respond to RAFi. Interestingly, we found that CRAF-fusions’ dimerization is unaffected primarily due to robust protein-protein interactions mediated by the non-kinase N-terminal fusion partner, such as QKI. This suggests that dimerization is essential for MAPK pathway activation and determines responsiveness to RAFi. Furthermore, we found that the novel RAF dimer inhibitor, LY3009120, stabilized CRAF-fusions in an inactive dimer conformation and suppressed oncogenic potential. Overall, CRAF-fusions are distinct from BRAF-fusions in responsiveness to targeted therapies, especially due to effect of N-terminal fusion partner in mediating stable dimerization. Our study suggests that molecular classification of PLGGs should inform therapeutic intervention of RAF-altered PLGGs even within RAF-mutant subtypes.