title: Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions characterizing pediatric astrocytomas.
abstract: Astrocytomas are the most common type of brain tumors in children. Activated BRAF protein kinase mutations are characteristic of pediatric astrocytomas with KIAA1549-BRAF fusion genes typifying low-grade astrocytomas and (V600E)BRAF alterations characterizing distinct or higher-grade tumors. Recently, BRAF-targeted therapies, such as vemurafenib, have shown great promise in treating V600E-dependent melanomas. Like (V600E)BRAF, BRAF fusion kinases activate MAPK signaling and are sufficient for malignant transformation; however, here we characterized the distinct mechanisms of action of KIAA1549-BRAF and its differential responsiveness to PLX4720, a first-generation BRAF inhibitor and research analog of vemurafenib. We found that in cells expressing KIAA1549-BRAF, the fusion kinase functions as a homodimer that is resistant to PLX4720 and accordingly is associated with CRAF-independent paradoxical activation of MAPK signaling. Mutagenesis studies demonstrated that KIAA1549-BRAF fusion-mediated signaling is diminished with disruption of the BRAF kinase dimer interface. In addition, the KIAA1549-BRAF fusion displays increased binding affinity to kinase suppressor of RAS (KSR), an RAF relative recently demonstrated to facilitate MEK phosphorylation by BRAF. Despite its resistance to PLX4720, the KIAA1549-BRAF fusion is responsive to a second-generation selective BRAF inhibitor that, unlike vemurafenib, does not induce activation of wild-type BRAF. Our data support the development of targeted treatment paradigms for BRAF-altered pediatric astrocytomas and also demonstrate that therapies must be tailored to the specific mutational context and distinct mechanisms of action of the mutant kinase.
authors.affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
authors.forename: Angela J
authors.initials: AJ
authors.lastname: Sievert
authors.forename: Shih-Shan
authors.initials: SS
authors.lastname: Lang
authors.forename: Katie L
authors.initials: KL
authors.lastname: Boucher
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Madsen
authors.forename: Erin
authors.initials: E
authors.lastname: Slaunwhite
authors.forename: Namrata
authors.initials: N
authors.lastname: Choudhari
authors.forename: Meghan
authors.initials: M
authors.lastname: Kellet
authors.forename: Phillip B
authors.initials: PB
authors.lastname: Storm
authors.forename: Adam C
authors.initials: AC
authors.lastname: Resnick
mesh_terms: D000818:Animals; D001254:Astrocytoma / Q000378:metabolism*; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D002648:Child; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D041681:NIH 3T3 Cells; D009368:Neoplasm Transplantation; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D010641:Phenotype; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D000077484:Vemurafenib
chemical_list: C568616:BRAF-KIAA1549 fusion protein, human; D004791:Enzyme Inhibitors; D007211:Indoles; D015514:Oncogene Proteins, Fusion; C528407:PLX 4720; D013449:Sulfonamides; D000077484:Vemurafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf