The studies proposed in this application make use of a novel mouse model of NF2 (Postn-Cre;Nf2flox/flox mice) that closely recapitulates important aspects of human NF2 disease, including a fully-penetrant schwannoma phenotype, and hearing and vestibular impairment resulting from the development of vestibular schwannomas. Utilizing this mouse model, we have taken a candidate approach to identify which proteins downstream of NF2 are key signaling intermediates required for schwannoma genesis. Previous studies have identified NF2 as an endogenous inhibitor of the kinase PAK1, and elevated levels of PAK1 kinase activity have been observed in primary human schwannoma samples. Importantly, the relevance of PAK1 kinase activity to NF2-deficient tumorigenesis has not been explored utilizing a spontaneous in-vivo schwannoma model. To address this question, we have intercrossed systemic PAK1 knockout mice (Pak1-/-) with Postn-Cre;Nf2flox/flox mice to generate Postn-Cre;Nf2flox/flox mice;Pak1-/- mice. In preliminary studies, we have observed that genetic ablation of Pak1 prevents tumor development and vestibular schwannoma-related hearing loss observed in Postn-Cre;Nf2flox/flox mice.
In Aim 1 of this proposal, we will comprehensively characterize the phenotype of Postn-Cre;Nf2flox/flox mice;Pak1-/- mice in terms of auditory function, tumor development, and survival. Functional assessment of hearing in mice will largely be determined through click and tone-evoked auditory brainstem response (ABR) testing. Given PAK1's role as a crucial node in a complex network of oncogenic signaling pathways, it remains unclear which pathways and substrates are affected downstream of hyperactive PAK1 in NF2-deficient tumors.
In Aim 2, we propose to elucidate the key molecular substrates that are activated by PAK1 in NF2-deficient tumor cells as a strategy to further characterize the basic pathophysiology of NF2- deficient cell types and identify additional therapeutic targets. Multiple studies from other labs have previously characterized NF2 as a novel regulator of mTORC signaling and protein translation. The mechanism by which NF2 regulates mTORC activation remains unknown. Given our preliminary data suggesting PAK1 is required for NF2-deficient schwannoma development in Postn-Cre;Nf2flox/flox mice, we hypothesized that PAK kinase activity could be the mediator of mTORC hyperactivity and deregulated translation in NF2-deficient cell types. Preliminary data in cell lines indicates this s indeed the case, with attenuation of PAK kinase activity decreasing biochemical markers of translational activity downstream of mTORC. The studies in Aim 2 are designed to further dissect this unexplored connection between PAK and deregulated mTORC activity in vitro using siRNA knockdown, lentiviral overexpression of PAK mutant constructs, and pharmacologic inhibition of PAKs. These results will then be validated in vivo through whole lysate and immunohistochemical studies.
Human NF2 disease is characterized by multiple schwannoma tumors, vestibular schwannoma-related hearing loss early in life, and premature mortality. Importantly, there are no medical alternatives to surgical resection for these tumors, and surgical complications invariably contribute to morbidity and mortality in these patients. Utilizing a genetically-engineered mouse model of NF2 that accurately recapitulates multiple aspects of human NF2 disease, we will genetically validate a potential therapeutic target for schwannomas, PAK1, while also performing studies to link the aberrant activation of PAK kinases in schwannomas to other biochemical pathways known to be dysregulated in these tumors to identify additional therapeutic opportunities for NF2 patients.
