Haploinsufficiency plays a crucial role in Neurofibromatosis (NF1), an autosomal dominant genetic disorder impacting over 120,000 individuals in the United States. Current therapeutic approaches focus on specific components of NF1 signaling, for example inhibiting MEK signaling pathways in tumors, thus failing to address the broad range of signaling and symptoms associated with NF1. Given that NF1 is characterized by both autosomal dominance and haploinsufficiency (lack of normal protein), upregulating protein expression of the remaining wild-type NF1 allele has the ability to compensate for loss of function from the mutant allele, thus alleviating a broad range of NF1 symptoms and overall disease progression. Infixion proposes to build a luciferase reporting assay to evaluate NF1 expression against known drugs, including 50+ compounds, across six drug classes, already identified by Infixion to correlate with increased NF1 expression. By confirming that these candidate drugs activate NF1 transcription, increase NF1 protein expression, and normalize Ras pathway signaling in NF1 +/- Schwann cells, and by further screening libraries of known drugs for increased NF1 expression, we propose a novel path of NF1 drug discovery that will impact a broad range of NF1 patients and symptoms, in a preventative manner, and without regard to the wide spectrum of NF1 genetic mutations. Research Background. Increasing NF1 expression via transfection reverses abnormal Ras activation resulting from NF1 loss (Wallis, et al. 2018). Transcriptional activation in other genetic conditions such as Willams-Beuren Syndrome, and Supravalvular Aortic Stenosis compensates for haploinsufficiency (Giordano, et al. 2012). Lastly, overcoming haploinsufficiency in another autosomal dominant condition (Sim1 induced obesity; mouse model) was recently shown using a Crispr/dCas9 transcriptional activator (Ahituv, et al. 2019).
Specific Aims. 1) Construct a luciferase reporter assay engineered into the endogenous NF1 gene of a well characterized, publically available, immortalized NF1 +/- Schwann cell line (Wallace et al. 2016; data published at Synapse.org). Validate assay using a viral transcription factor developed by Infixion using Crispr/dCas9 to upregulate NF1. 2) Deploy this NF1 luciferase reporter assay to screen 50+ known drugs shown by Infixion to correlate with increased NF1 expression across various cell/tissue types. Next, use this validated luciferase reporter to screen a 13,000+ compound repurposing library of known drugs available from Scripps Research Institute (known as ReFrame). 3) The top hits from Aim 2 screens will be evaluated, utilizing both immortalized and iPSC derived NF1+/- Schwann cells, for the following: a) ability to induce NF1 mRNA and protein expression, using qPCR and Westerns, b) the impact on Ras signaling (pERK, ELK-1, AKT, etc.) utilizing a targeted quantitative mass spec proteomics assay, c) their broader impact on gene expression in Schwann cells utilizing RNAseq, d) impact on cell proliferation, and e) their safety profile based on published data from previous trials. The goal is to prioritize not more than 3-5 candidates to take forward into a Phase 2 evaluation.

Public Health Relevance

Neurofibromatosis Type 1 (NF1) is an autosomal dominant genetic disorder that impacts an estimated 1 in 3000 people worldwide, and one that is characterized by a complex set of symptoms shown to be dependent on haploinsufficiency (i.e., the lack of normal protein). By upregulating NF1 protein expression from the normal allele by two-fold, and thus restoring normal levels of functional NF1 protein in tissues important in NF1, we target a range of NF1 symptoms by eliminating the haploinsufficient environment. We propose to develop and utilize NF1-specific luciferase reporter and proteomic assays, along with standard small molecule evaluation tools, to characterize a small molecule library of repurposed drugs for their ability to upregulate NF1 transcription, and to further evaluate the resulting impact of ?hits? on NF1 protein expression, cell proliferation, gene expression specificity, as well as downstream pathways (such as Ras, MEK, mTOR) known to be dysregulated by mutations in the NF1 gene.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1)
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Caporello, Emily Laura
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Infixion Bioscience, Inc.
United States
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