Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis Type 1 (NF1) is a highly penetrant autosomal dominant genetic disorder where mutations in the tumor suppressor gene NF1 leads to decreased neurofibromin. The most debilitating manifestation is the presence of complex multilineage Schwann cell-derived plexiform neurofibromas (PN). Historically, little clinical success has been achieved targeting PN through surgery or chemotherapies. I performed an shRNA library screen of patient-derived Schwann cell lines to identify novel therapeutic targets to disrupt PN formation and progression. An shRNA library screen of human kinases and Rho-GTPases was performed in NF1-/- and paired NF1 competent immortalized Schwann cell lines. Following sequencing, candidates were identified. We previously developed a novel mouse model of NF1 wherein a neural crest specific Postncre targeted loxp-flanked Nf1 that replicated the PN found in patients. Additional cohorts of mice were generated with biallelic deletion of Rac1 (Nf1f/fRac1f/f Postn-Cre+; DKO ). Mice were aged for 9 months and peripheral nerves were harvested and fixed in formalin. Peripheral nerve size was measured and tumors were identified through blinded analysis of hematoxylin and eosin and Masson’s Trichrome (collagen) stained slides. Rho family members, including RAC1, were identified as candidates through an shRNA library screen. Genetic disruption of Rac1 in the Schwann cell lineage resulted in the prevention of tumor formation in DKO mice, as observed by peripheral nerve size and histological analysis. I observed an average of 14.8 +/- 2.65 tumors per mouse in the Nf1f/f Postnviii Cre+ cohort compared to 0 tumors in the DKO (p<0.0001). Following an shRNA library screen, RAC1 was identified as a candidate to modulate PN formation. Biallelic deletion of Rac1 in vivo prevented PN formation. I demonstrate that a candidate identified in an shRNA library screen can translate to an biological effect in a mouse model of PN.

BACKGROUND: Aberrant MET receptor tyrosine kinase (RTK) activation leads to invasive tumor growth in different types of cancer. Overexpression of MET and its ligand hepatocyte growth factor (HGF) occurs more frequently in glioblastoma (GBM) than in low-grade gliomas. Although we have shown previously that HGF-autocrine activation predicts sensitivity to MET tyrosine kinase inhibitors (TKIs) in GBM, whether it initiates tumorigenesis remains elusive. METHODS: Using a well-established Sleeping Beauty (SB) transposon strategy, we injected human and cDNA together with a short hairpin siRNA against (SB-hHgf.Met.ShP53) into the lateral ventricle of neonatal mice to induce spontaneous glioma initiation and characterized the tumors with H&E and immunohistochemistry analysis. Glioma sphere cells also were isolated for measuring the sensitivity to specific MET TKIs. RESULTS: Mixed injection of SB-hHgf.Met.ShP53 plasmids induced de novo glioma formation with invasive tumor growth accompanied by HGF and MET overexpression. While glioma stem cells (GSCs) are considered as the tumor-initiating cells in GBM, both SB-hHgf.Met.ShP53 tumor sections and glioma spheres harvested from these tumors expressed GSC markers nestin, GFAP, and Sox 2. Moreover, specific MET TKIs significantly inhibited tumor spheres' proliferation and MET/MAPK/AKT signaling. CONCLUSIONS: Overexpression of the HGF/MET axis along with p53 attenuation may transform neural stem cells into GSCs, resulting in GBM formation in mice. These tumors are primarily driven by the MET RTK pathway activation and are sensitive to MET TKIs. The SB-hHgf.Met.ShP53 spontaneous mouse glioma model provides a useful tool for studying GBM tumor biology and MET-targeting therapeutics.