Overcoming resistance to single-agent therapy for oncogenic BRAF gene fusions via combinatorial targeting of MAPK and PI3K/mTOR signaling pathways

Abstract

Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs.

Novel BRAF fusions in pediatric histiocytic neoplasms define distinct therapeutic responsiveness to RAF paradox breakers

“…A single report on BRAF-fusions in LCH 4 has shown unresponsiveness to BRAF-V600E specific inhibitors such as vemurafenib, termed first-generation RAF inhibitors (RAFi), but observed suppression by second-generation RAFi, PLX8394, and downstream MEK inhibition, similar to other pediatric glioma studies on BRAF-fusions 9,11 . Herein, we evaluated the responsiveness of novel MTAP-BRAF and MS4A6A-BRAF to such targeted inhibitors.…”

“…This is in contrast to PLX8394-mediated suppression of BRAF-fusion driven growth in the previously described LCH 4 and other cancers, such as the KIAA1549-BRAF fusion in pediatric glioma 9-11 . PLX8394 suppressed FAM131B-BRAF (a pediatric glioma derived fusion 12,13 ) and BRAF-V600E driven growth and signaling as well as actively disrupted FAM131B-BRAF dimers (Supplemental Figures 1B-D), highlighting therapeutic differences between MTAP-/MS4A6A-BRAF, BRAF-V600E and other BRAF-fusions.BRAF-fusions function as active homo-and heterodimers (with wild-type BRAF) to mediate cell signaling 9,11 . We found that MTAP-and MS4A6A-BRAF also mediate such protein-protein interactions in co-immunoprecipitation assays ( Figure 2C-D, DMSO lanes).…”

“…BRAF-fusions function as active homo-and heterodimers (with wild-type BRAF) to mediate cell signaling 9,11 . We found that MTAP-and MS4A6A-BRAF also mediate such protein-protein interactions in co-immunoprecipitation assays ( Figure 2C-D, DMSO lanes).…”

“…Despite the lack of cytologic atypia or increased mitotic rate by H&E stain, a Ki-67 proliferation index stain revealed increased staining in lesional cells up to 40%. Targeted RNA-seq identified a fusion of MS4A6A (NM_022349.3) exons 1-6 and BRAF (NM_004333.4) exons [11][12][13][14][15][16][17][18]. During staging, the patient was found to have PET-avid dissemination to lymph nodes and lung ( Figure 1N-P).…”

“…For molecular and therapeutic characterization, MTAP-BRAF and MS4A6A-BRAF were cloned and stably expressed in a heterologous cell model since patientderived cell lines were lacking. The NIH/3T3 cells model system was utilized due to its ability to reliably discern oncogenic fusion profiles [9][10][11] . In soft agar assays, both MTAP-BRAF and MS4A6A-BRAF expressing NIH3T3 showed a significant increase in colony count over control (p<0.0001, Figure 1R).…”