Abstract
Abstract Introduction: Pediatric histiocytic neoplasms are clonal hematopoietic disorders driven by mutations activating the mitogen-activated protein kinase (MAPK) pathway, such as BRAF- V600E. In non-BRAFV600E cases, we investigated alternative MAPK mutations and found two novel BRAF gene fusions. We have previously shown effective targeting of BRAF fusions (KIAA1549-BRAF) in pediatric low-grade gliomas (PLGGs) with paradox breaker RAF inhibitor (RAFi), PLX8394 and MEK inhibitors (MEKi). Here, we investigate the distinct responsiveness of novel BRAF fusions to RAFi therapies and explore the mechanistic basis of such differential responses compared to other BRAF fusions. Method: Two histiocytic patient tumors were analyzed using the CHOP Comprehensive Next- Gen Sequencing Solid Tumor Panel and a targeted RNA-seq panel for 106 fusion partner genes. Novel BRAF fusions identified were sub-cloned to create heterologous cell models. Immunoblotting of serum starved cells measured MAPK and PI3K pathway activation and soft agar assay tested for oncogenic phenotypes driven by BRAF fusions, along with response to PLX8394, LY3009120 (pan-RAF dimer inhibitor), and MEKi. Co-immunoprecipitation assays using Myc- and Flag-tagged BRAF fusion constructs assessed dimerization profiles, with or without inhibitors. Result: In the two M- and L-type histiocytic neoplasms assessed, we found novel and rare BRAF gene fusions, MTAP-BRAF and MS4A6A-BRAF, respectively. Both BRAF fusions activated the MAPK/ PI3K pathways and showed homo- and hetero-dimerization with BRAF and the respective N-terminal fusion partner. Compared to common BRAF fusions, MTAP-BRAF and MS4A6A- BRAF did not respond to PLX8394 due to no disruption of active fusion homo- and hetero-dimers, which was in turn was due to the untargeted, stable dimerization mediated by the N-terminal fusion partners. Conversely, we observed robust suppression with LY3009120 that bound fusion dimers and kept them in an inactivate confirmation. MEKi selumetinib and trametinib also suppressed fusion driven signaling and oncogenic phenotypes. Conclusion: We show that novel MTAP-BRAF and MS4A6A-BRAF fusions, found in histiocytic tumors, do not respond to RAFi PLX8394 but are targeted by LY3009120 or MEKi. Our finding that PLX8394 does not disrupt MTAP-BRAF or MS4A6A-BRAF dimerization due to contribution of N-terminal partners defines a novel paradigm for the distinct mechanisms sought by BRAF fusions in response to RAFi therapy. We show potent suppression of these mechanistically distinct BRAF fusions with MEKi or LY3009120 which function independent of vulnerability to fusion dimerization. Overall, this study highlights the unique and differential biology hijacked by BRAF fusions in response to RAFi and further warrants detailed mechanistic classification of BRAF fusions based on their responsiveness to targeted agents. Citation Format: Payal Jain, Lea F. Surrey, Joshua Straka, Pierre Russo, Richard Womer, Marilyn Li, Phillip B. Storm, Angela J. Waanders, Michael Hogarty, Jennifer Picarsic, Adam C. Resnick. Novel BRAF gene fusions in pediatric histiocytic neoplasms respond differentially to RAF targeted therapies based on dimerization profiles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 632.
Published Version
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