Combinatorial pathway targeting approaches for BRAF fusions associated with pediatric low-grade gliomas.
INTRODUCTION: Activating BRAF-fusion mutations occur frequently in pediatric low-grade gliomas (PLGGs). We have previously shown targeting of KIAA1549-BRAF fusion by PLX4720, the research analog of vemurafenib, results in paradoxical activation of the mitogen activated protein kinase (MAPK) signal cascade. We hypothesized that BRAF-fusions could be targeted by downstream MEK inhibitors (MEKi), and that combinatorial targeting of BRAF-fusions could also prevent acquired resistance to single agent therapy. METHODS: BRAF-fusions were screened with a selection of clinically relevant MEKi and with PLX8394, a second-generation RAF inhibitor, in vitro in cell culture and soft agar. BRAF V600E and RAS mutant cells were also screened for comparison. MEKi/PLX8394 resistant BRAF-fusion cell lines were generated in vitro and interrogated by sequencing, immunoblotting, and RNA-seq analysis to identify potential co-targets. Combinatorial therapy was then tested in vitro and in vivo to confirm efficacy. RESULTS: BRAF-fusions in PLGGs can be targeted by MEKi and by the pan-RAF inhibitor PLX8394. AZD6244, MEK162, GDC0623, and GDC0973 all inhibited downstream ERK phosphorylation; however, GSK1120212 more robustly inhibited MAPK activation in the BRAF-fusion, BRAF V600E, and RAS mutant cells. Interestingly, the MEKi/PLX8394 resistant BRAF-fusion cells all showed activation of phosphatidylinositol 3-kinase (PI3K) pathway. BRAF-fusion parental cells were screened with RAD001, a targeted mTOR inhibitor, resulting in modest downstream inhibition. Co-targeting of the parental BRAF-fusions with GSK1120212 and RAD001 resulted in robust inhibition of down-stream signaling and in significant tumor inhibition, and prevented acquired resistance to targeted inhibition. CONCLUSION: Our work demonstrates that BRAF-fusion oncogenes can be effectively targeted with GSK1120212 as single agent but even more robustly with GSK1120212 and RAD001 co-targeting. This provides preclinical rationale for treating PLGGs harboring BRAF-fusion oncogenes with combinatorial MEK and mTOR inhibitors in clinical trial.