Abstract 3414: The pan-RAF inhibitor tovorafenib suppresses NF1-mutant glioma through upregulation of FOXO1 and triggering of oncogene-induced senescence
Authors: Alaali Lujain A. , Yuan Ming , Eberhart Chales , Raabe Eric 
Published: April 2023

Abstract
Pediatric low-grade glioma (pLGG) is the most common childhood brain tumor. Patients with pLGG often have significant lifelong disabilities due to the location of the tumor and tumor treatments, including surgery, chemotherapy, and radiation. The most common mutations in pLGG are in the mitogen-activated protein kinase MAPK pathway, including the neurofibromin, and the BRAF oncgene. Tovorafenib (pan-RAF inhibitor) has demonstrated promising pre-clinical results in multiple cancers. Tovorafenib targets monomeric and dimeric forms of wild-type and BRAF mutants. Our objective is to investigate the effects of tovorafenib in pLGG, comparing the response of cells with BRAFV600E compared to NF1-mutant cells with wild-type RAF. JHH-NF1-PA1 is a neurofibromin (NF1) mutant line derived from a patient with germline type 1 neurofibromatosis, and JHH-PXA-1 has the BRAFV600E mutation. In addition to tovorafenib, we also tested TAK632, a tool compound that targets RAF and has a different structure . We measured readouts such as pERK and pMEK, pS6 and pAKT473 by Western blot. We treated our cells with 100 nM tovorafenib for 2 hours and 4 hours, and we found an increase of pERK (P=0.0037) and pAKT473 in JHH-NF1-PA1 but a significant decrease pERK in JHH-PXA-1 “BRAF mutant”. We also, tested TAK632 at 100 nM for 4 hrs, and found a decrease of pERK and pAKT protein level in both cell lines. Tovorafenib caused an increase of growth in JHH-NF1-PA1 cells at 100 nM and 200 nM by eighteen percent (P&lt;0.05) and twenty-five percent (P&lt;0.05), respectively. We detect the IC50 value at 600 nM for NF1 mutant cell line. The JHH_PXA1 cell line did not show cell growth increase in any of the lower concentrations and the IC50 was approximately 400 nM. In JHH-NF1-PA1, we observe activation in AKT and ERK in Western blot at concentrations of 500nM, 1000nM and1500nM, but downregulation at 2000nM. We further investigated the relationship between AKT and ERK phosphorylation, and the cell growth inhibition in NF1 mutant cells. FOXO1 protein induces cell cycle arrest at G1 by modulating the expression of the CDK. FOXO1 status can be regulated by AKT and ERK phosphorylation. We observed increases of total FOXO1 as well asFOXO1 phosphorylation and acetylation with increasing tovorafenib concentrations in NF1-mutant glioma. Phosphorylation and acetylation are associated with increased activity and DNA binding of FOXO1. We also detected an increase of p21CIP1 with tovorafenib treatment, suggesting the induction of senescence, which is a known effect of activated FOXO1. Additional studies investigating the induction of senescence downstream of tovorafenib are underway. We are currently planning to test tovorafenib against orthotopic xenografts of NF1 mutant glioma compared to BRAFV600E mutant glioma. Our data suggest that tovorafenib may act differently in NF1 mutant cells compared to BRAFV600E mutant cells.
Citation Format: Lujain A. Alaali, Ming Yuan, Chales Eberhart, Eric Raabe. The pan-RAF inhibitor tovorafenib suppresses NF1-mutant glioma through upregulation of FOXO1 and triggering of oncogene-induced senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3414.