Selective inhibition of BRAF and CRAF sensitizes NF1-deficient malignant peripheral nerve sheath tumors to MEK inhibitors

Background: Treatment for patients with malignant peripheral nerve sheath tumors (MPNST) is an unmet clinical need. Loss of NF1 in MPNST leads to hyperactivation of RAS oncoproteins, however little is known about relevant downstream oncogenic signaling and effective targeted therapies in MPNST are still lacking. Methods: Conditional gene expression, CRISPR-CAS9, and shRNA-mediated knockdown were used to perform gain/loss-of-function experiments to explore the effect of reconstituting the GTPase-activating protein-related domain of NF1 or knockdown of A/B/CRAF kinases on ERK signaling output and MPNST cell growth. Colony formation, cell proliferation and live cells imaging assays were performed to assess cell growth in response to genetic manipulations or drug treatments. Pathway enrichment analysis on RNA sequencing following drug perturbation, efficacy studies in cell-line-derived and patient-derived xenograft models, and immunoblotting/immunohistochemistry were conducted to assess tumor growth and ERK pathway activity in cells or in pharmacodynamic analyses of tumor xenografts. Results: NF1 loss activates RAS/ERK signaling through B/CRAF, and cell growth and ERK signaling of NF1-MPNST are dependent on B/CRAF, but not ARAF. Genetic or pharmacological inhibition of B/CRAF using paralog-selective RAF inhibitor (RAFi) significantly potentiates MEK inhibitor (MEKi) treatment through more effective suppression of ERK signaling and proliferation. This is shown in multiple traditional and patient-derived cell line and xenograft models, including those with acquired resistance to MEKi. Malignant Peripheral Nerve Sheath Tumor (MPNST) cell lines were treated with MEK inhibitor trametinib, BRAF/CRAF inhibitor LXH254, or the combination of both for 48hours. RNA sequencing was then performed and gene expression analysis was done from 3 cell lines.