Prolonged Mitogen-Activated Protein Kinase Kinase (MEK) Inhibition Induces Increase in Proteolysis and Compensatory Phosphorylation of MEK and Protein Kinase B (AKT) in Plexiform Neurofibroma Cells.

Plexiform neurofibromas associated with neurofibromatosis type I (pNF1s) are benign tumors caused by the complete loss of function of the NF1 gene, which encodes a negative regulator of the RAS/mitogen-activated protein kinase (MAPK) pathway. pNF1s carry a significant risk of progression to malignant peripheral nerve sheath tumors (MPNSTs), which are highly aggressive and largely incurable. FDA-approved mitogen-activated protein kinase kinase (MEK) inhibitors, selumetinib and mirdametinib, have shown ~30% tumor shrinkage in 70% and 42% pNF1 patients, respectively. However, not all pNF1s respond to MEK inhibition, and treatment is often associated with adverse effects such as dermatologic and gastrointestinal toxicities, underscoring the need for improved therapeutic strategies with minimal side effects. Here, we demonstrate that prolonged MEK inhibition increases proteolytic activity in 3D pNF1 tumor structures, consistent with enhanced extracellular matrix degradation. Prolonged treatment with four mechanistically and chemically distinct MEK inhibitors consistently reduced ERK phosphorylation, a downstream effector of the RAS/MAPK pathway, yet induced adaptive phosphorylation of MEK and AKT in pNF1 tumor cells. Phosphorylation of MEK is required for its catalytic activation and subsequent phosphorylation of ERK. Increased MEK phosphorylation in the presence of MEK inhibitors reflects upstream pathway reactivation but does not lead to ERK phosphorylation and activation because of the presence of the inhibitor. This response was also observed in MPNST cell lines treated with MEK inhibitors. These findings suggest that adaptive activation of upstream and parallel survival pathways may counteract the intended effects of MEK inhibition and support the rationale for combination strategies to improve therapeutic outcomes in NF1-associated tumors.