Title: Deconvoluting mechanisms of acquired resistance to RAF inhibitors in BRAF V600E mutant human glioma

Purpose : Selective RAF-targeted therapy is effective in some patients with BRAF V600E mutated glioma, though emergent and adaptive resistance occur through ill-defined mechanisms. Experimental Design: Paired pre-/ post- RAF inhibitor (RAFi)-treated glioma samples (N=15) were obtained and queried for treatment-emergent genomic alterations using DNA and RNA sequencing. Functional validation of putative resistance mechanisms was performed using established and patient-derived BRAF V600E -mutant glioma cell lines. Results: Analysis of 15 tissue sample pairs identified thirteen alterations conferring putative resistance were identified among nine paired samples (including mutations involving ERRFI1, BAP1, ANKHD1 , and MAP2K1 ). We performed functional validation of mechanisms of resistance, including loss of NF1, PTEN, or CBL , in BRAF V600E mutant glioma lines, and demonstrate they are capable of conferring resistance in vitro . Knockdown of CBL resulted in increased EGFR expression and phosphorylation, a possible mechanism for maintaining ERK signaling within the cell. Combination therapy with a MEKi or EGFR inhibitor was able to overcome resistance to BRAFi, in NF1 knockdown and CBL knockdown, respectively. Restoration of wild-type PTEN in B76 cells (PTEN-/-) restored sensitivity to BRAFi. We identified and validated CRAF upregulation as a mechanism of resistance in one resistant sample. RNAseq analysis identified two emergent expression patterns in resistant samples, consistent with expression patterns of known glioma subtypes. Conclusions: Resistance varied predict highlighting a personalized

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