Inhibition of NF1 Protein Degradation as a Treatment for NF1 Haploinsufficiency

PROJECT ABSTRACT. Haploinsufficiency plays a crucial role in Neurofibromatosis (NF1), an autosomal dominant genetic disorder impacting over 120,000 Americans. Current therapeutic approaches target downstream components of NF1 signaling, for example MEK inhibition in tumors, thus failing to address the broad range of signaling and symptoms associated with NF1 mutations. Given that NF1 is characterized by both autosomal dominance and haploinsufficiency (lack of normal protein), inhibiting NF1 protein degradation, causing a net increase in NF1 protein, has the potential to alleviate a broad range of NF1 symptoms and halt overall disease progression. Infixion proposes to identify and validate genes involved in NF1 protein ubiquitination and degradation, and to build a protein-tagged reporter assay to screen the impact of known drugs on NF1 protein levels, with a focus on modulators of NF1 protein degradation. By identifying candidate drugs inhibiting NF1 ubiquitination, we target an increase in overall NF1 protein, and thus a normalizing of Ras (and other) pathway signaling in individuals with NF1. We propose this as a novel path of NF1 drug discovery, with potential impact on a broad range of NF1 patients and symptoms, in a preventative manner, and applicable to the very wide spectrum of unique NF1 genetic mutations. Research Background. Increasing NF1 expression via transfection reverses abnormal Ras activation resulting from NF1 loss (Wallis, 2018; Mellert, 2018). Increased protein expression in other genetic conditions such as Willams-Beuren Syndrome, and Supravalvular Aortic Stenosis compensates for haploinsufficiency (Giordano, et al. 2012). Lastly, overcoming haploinsufficiency in other autosomal dominant conditions (Sim1; Pax6 genes) have shown an ability in vivo to correct symptoms. (Matharu, et. al. 2019; Rabiee, et. al. 2020). Specific Aims. 1) Identify regulators of NF1 protein stability using siRNA libraries as a genetic (knock-down) screen in NF1-relevant cell types. Identifying regulators of NF1 ubiquitination and subsequent degradation will allow for rational selection of additional libraries to screen for compounds to increase NF1 protein. 2) Construct an assay, engineering the endogenous NF1 gene to tag the NF1 protein, in a well characterized, publicly available (ATCC), immortalized NF1 +/- Schwann cell line. Validate assay using compounds already verified by Infixion to increase NF1 protein levels. 3) Deploy NF1 protein stabilization assay to screen a 13,000+ compound repurposing library of known drugs available from Scripps Research Institute (known as ReFrame), and other targeted libraries. The top hits from these screens will be evaluated utilizing immortalized Schwann, primary fibroblast and iPSC derived NF1+/- cells, for the following: a) ability to induce NF1 protein expression using Westerns/ELISA, b) impact on Ras signaling (pERK, ELK-1, AKT, etc.) utilizing a targeted quantitative mass spec proteomics assay, c) impact on cell proliferation, and d) safety profile based on published data. Program goal is to prioritize 3-5 candidate compounds for an SBIR Phase 2 pre-IND evaluation.