CRAF gene fusions in pediatric low-grade gliomas define a distinct drug response based on dimerization profiles

Abstract

Pediatric low-grade gliomas (PLGGs) are commonly associated with BRAF gene fusions that aberrantly activate the mitogen-activated protein kinase (MAPK) signaling pathway. This has led to PLGG clinical trials utilizing RAF- and MAPK pathway-targeted therapeutics. Whole-genome profiling of PLGGs has also identified rare gene fusions involving another RAF isoform, CRAF/RAF1, in PLGGs and cancers occuring in adults. Whereas BRAF fusions primarily dysregulate MAPK signaling, the CRAF fusions QKI-RAF1 and SRGAP3-RAF1 aberrantly activate both the MAPK and phosphoinositide-3 kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathways. Although ATP-competitive, first-generation RAF inhibitors (vemurafenib/PLX4720, RAFi) cause paradoxical activation of the MAPK pathway in BRAF-fusion tumors, inhibition can be achieved with ‘paradox breaker’ RAFi, such as PLX8394. Here we report that, unlike BRAF fusions, CRAF fusions are unresponsive to both generations of RAFi, vemurafenib and PLX8394, highlighting a distinct responsiveness of CRAF fusions to clinically relevant RAFi. Whereas PLX8394 decreased BRAF-fusion dimerization, CRAF-fusion dimerization is unaffected primarily because of robust protein–protein interactions mediated by the N-terminal non-kinase fusion partner, such as QKI. The pan-RAF dimer inhibitor, LY3009120, could suppress CRAF-fusion oncogenicity by inhibiting dimer-mediated signaling. In addition, as CRAF fusions activate both the MAPK and PI3K/mTOR signaling pathways, we identify combinatorial inhibition of the MAPK/mTOR pathway as a potential therapeutic strategy for CRAF-fusion-driven tumors. Overall, we define a mechanistic distinction between PLGG-associated BRAF- and CRAF/RAF1 fusions in response to RAFi, highlighting the importance of molecularly classifying PLGG patients for targeted therapy. Furthermore, our study uncovers an important contribution of the non-kinase fusion partner to oncogenesis and potential therapeutic strategies against PLGG-associated CRAF fusions and possibly pan-cancer CRAF fusions.

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

“…Interestingly, second-generation RAFi PLX8394 also showed no suppression in MTAP-or MS4A6A-BRAF driven soft agar growth despite targeting MAPK/PI3K signaling (Figure 2A-B). 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][10][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.…”

“…We found that this difference arises due to the contribution of N-terminal partners, MTAP (exons 1-7) and MS4A6A (exons 1-6), to respective fusion dimerization that is unaffected by PLX8394 ( Figure 2C-D, lanes 3,7). Similar role of N-terminal partner accounts for differential response of CRAF-fusions to PLX8394 10 . Furthermore, we observed that Trunc-MTAP (exons 1-7) competitively substituted MTAP-BRAF homo-dimerization in a dose-dependent manner, suggesting preferential and potent protein interactions mediated by N-terminal partner in these histiocytic-specific BRAF-fusions ( Figure 2E).…”

“…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).…”

Targeting Alterations in the RAF–MEK Pathway

“…RAF1 fusions are observed in some cancers, typically resulting (much like BRAF fusions) in an aberrant gene product fusing the C-terminal CRAF kinase domain to an N-terminal fusion partner with a dimerization domain that leads to RAS-independent dimerization and activation of CRAF kinase activity. These fusions are found in low-grade pediatric gliomas, prostate cancer, melanoma, and pancreatic cancer (26,(31)(32)(33).…”

“…BRAF and CRAF (RAF1) fusions predominantly lead to C-terminal RAF kinase domains fused to an N-terminal partner with a dimerization domain, leading to constitutive RAS-independent dimerization and activation of RAF kinase activity. Accordingly, RAF monomer inhibitors are not effective against BRAF or CRAF fusions (31,70). In fact, RAF monomer inhibitors can induce paradoxical activation of BRAF and CRAF fusion constructs, likely through transactivation of the dimer partner, similar to what is observed with wild-type RAF dimers in the presence of activated RAS. "…”