NF1 and SPRED1/2 Cooperate in RAS-Independent Functions, Modulating MAPK-AKT and RRAS/RRAS2 Signaling

The neurofibromatosis type 1 (NF1) gene product, neurofibromin, is a critical negative regulator of the RAS-MAPK pathway primarily through its GTPase-activating protein (GAP) activity on RAS. While its direct interaction with SPRED is known to enhance this regulation, a significant portion of neurofibromin's structural domains and their mechanistic functions remain poorly understood, especially beyond canonical RAS-MAPK signaling. This gap limits our comprehensive understanding of NF1 pathology and the development of diverse therapeutic strategies for conditions like plexiform neurofibromas.

Researchers investigated RAS-independent functions of NF1 and SPRED1/2 using CRISPR-Cas9 methods to ablate NF1 or SPRED1/2 in isogenic "RASless" mouse embryonic fibroblast (MEF) cell lines. These MEF lines expressed either wild-type KRAS4b or an oncogenic KRAS mutation. They assessed MAPK-AKT signaling, RRAS and RRAS2 GTPase activity, and performed a transcriptome microarray analysis on knockout MEF cells. Findings were corroborated in Schwann cell models derived from Neurofibromatosis type I patients, specifically plexiform neurofibroma cells or unaffected nerve cells, with abrogated NF1 or neurofibromin RAS-GAP activity.

Loss of SPRED1/2 phenocopied NF1 loss, demonstrating their cooperative requirement to modulate MAPK-AKT signaling. Crucially, this cooperation was found to regulate biochemical and signaling functions independently of RAS. Loss of NF1 or SPRED1/2 also led to a potent suppression of the RAS family GTPases, RRAS and RRAS2, a phenomenon occurring independently of RAS or AKT pathway activation. A transcriptome microarray analysis revealed a specific subset of RAS-independent, NF1-SPRED1/2-dependent gene signatures. These genes were also directly regulated by the RAS-GTPase function of neurofibromin, highlighting a broader regulatory role. The modulation of these NF1-SPRED1/2-dependent downstream signaling effectors was further corroborated in patient-derived Schwann cell models.

The study provides compelling evidence for RAS-independent functions that are dependent on the cooperation of NF1 and SPRED1/2, uncoupled from canonical MAPK signaling.