Deconvoluting Mechanisms of Acquired Resistance to RAF Inhibitors in BRAF-Mutant Human Glioma.

Purpose: Selective RAF-targeted therapy is effective in some patients with BRAF-mutated glioma, though emergent and adaptive resistance occurs through ill-defined mechanisms.

Experimental Design: Paired pre-/post- RAF inhibitor (RAFi)-treated glioma samples ( = 15) were obtained and queried for treatment-emergent genomic alterations using DNA and RNA sequencing (RNA-seq). Functional validation of putative resistance mechanisms was performed using established and patient-derived BRAF-mutant glioma cell lines.

Results: Analysis of 15 tissue sample pairs identified 13 alterations conferring putative resistance were identified among nine paired samples (including mutations involving , and ). We performed functional validation of mechanisms of resistance, including loss of , or , in BRAF-mutant glioma lines, and demonstrate they are capable of conferring resistance . Knockdown of CBL resulted in increased 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. RNA-seq analysis identified two emergent expression patterns in resistant samples, consistent with expression patterns of known glioma subtypes.

Conclusions: Resistance mechanisms to BRAFi in glioma are varied and may predict effective precision combinations of targeted therapy, highlighting the importance of a personalized approach.