GENETICS OF PAEDIATRIC GLIOMAS

Pediatric low-grade gliomas are the most common type of brain tumor diagnosed in children, and represent a heterogeneous group of tumors, which are poorly classified based on histology and location, according to Payal Jain, a graduate student at the University of Pennsylvania, and lead author of a preclinical study investigating pediatric low-grade gliomas that are driven by CRAF fusion proteins rather than BRAF fusion proteins. The study found that CRAF fusions do not respond to RAF inhibitors and only show partial response to single-agent MEK inhibitors. They do, however, respond robustly to the combinatorial targeting of both the mitogen-activated protein kinase (MAPK) and PI3K signaling pathways.

The findings suggest the importance of molecularly stratifying patients with pediatric low-grade gliomas to identify appropriate treatment. The study (abstract A138) was presented at the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, November 5 to 9 in Boston.

Study Methodology

CRAF fusions were stably expressed in heterologous cell model systems (NIH 3T3 cells) and tested for differential activation of downstream signaling pathways via immunoblotting. BRAF fusion and vector control cell lines were used for comparison. Oncogenic properties of CRAF fusions were characterized via soft agar assays and flank xenografts in immuno-compromised mice.

First- and second-generation RAF inhibitors, PLX4720 (the research version of vemurafenib [Zelboraf]) and PLX8394 respectively, and MEKi were screened for suppression of CRAF fusion–mediated oncogenicity using immunoblotting and soft agar assays. The drugs that showed efficacy in these in vitro assays were further tested for efficacy in mice flank xenografts. Combinatorial drug therapy was also tested in vitro and in vivo.

Study Findings

CRAF fusions were found to activate the MAPK and PI3K pathways. As compared to BRAF fusions, CRAF fusions were not found to be responsive to any RAF inhibitors tested, including the paradox breaker PLX8394 (a second-generation RAF inhibitor).

AZD6244 and GSK1120212/trametinib (Mekinist), both clinically relevant MEK inhibitors, abrogated downstream phosphorylated ERK signaling and prevented growth of the stably expressing CRAF fusion NIH 3T3 cell lines in soft agar. In mice flank xenografts, GSK1120212 partially inhibited tumor growth driven by CRAF fusions. Since the CRAF fusions also activate the PI3K pathway, combinatorial targeting using a MEKi and mTOR inhibitor—GSK1120212 and RAD001/everolimus (Afinitor), respectively—was found to show robust tumor inhibition in mice flank xenografts.

“This study demonstrates that CRAF fusions do not respond to RAF inhibitors, show partial response to single-agent MEKi, but respond robustly to combinatorial targeting of both MAPK and PI3K pathways via GSK1120212 and RAD001. Therefore, CRAF fusions are distinct from BRAF fusions in terms of responsiveness to targeted therapies. These findings suggest molecular classification of PLGGs prior to treatment and also provide preclinical rationale for combination therapy for CRAF fusion expressing PLGGs,” concluded the study authors.

“Our studies emphasize the need for molecular subclassification of these low-grade [gliomas], which can guide personalized medicine approaches not only in low-grade [gliomas], but also in other cancers where the CRAF fusions have been coming up in large sequencing studies,” said Ms. Jain during a press briefing at the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

Ms. Jain and senior author Adam C. Resnick, PhD, declared no potential conflicts of interest.

Funding for this study was provided by A Kids’ Brain Tumor Cure Foundation, the National Institutes of Health, and the Damon Runyon Dale F. Frey Scientist Award.