Neurofibromatosis 2 (NF2) is characterized by vestibular schwannomas, and meningiomas that show loss of the NF2 tumor suppressor gene. Meningiomas arise from meningothelial arachnoid cap cells and are the most common primary intracranial tumors in adults. NF2 inactivation is frequently associated with sporadic meningiomas, particularly in primary atypical (WHO grade II) tumors. Meningiomas that progress despite surgery and radiation are responsible for significant morbidity and mortality. Therefore, effective systemic therapies are greatly needed. For meningioma modeling and preclinical drug screening, we employ human primary meningioma (MN) cell lines derived from surgically resected tumors and CRISPR-Cas9 genome-edited isogenic, human arachnoidal cell lines (ACs) that either express or lack NF2. Employing these models, we established that NF2-deficient meningiomas reveal aberrant activation of mTORC1/mTORC2 signaling, which led to clinical trials for NF2 and meningiomas. Recently, we undertook large-scale kinome, transcriptome and drug screening studies in our AC and MN cell models to identify other potential targets. Kinome and transcriptome data revealed increased activation and expression of several EPH receptor family tyrosine kinases (EPH-RTKs), Src family kinase members (SFKs) and c-KIT, which are all targets of dasatinib, which is recently published. Our ‘omics approach also identified other interesting candidates, including ligands NRG1, HBEGF and apelin, and AMPK-related kinases, particularly NUAK2, to be consistently upregulated in the kinome and transcriptome of NF2-null AC/MN cells. Our recent results suggest that NRG1 expression and ERBB3 signaling is regulated by mTORC1 signaling. We plan to examine whether NRG1 alone or factors such as HBEGF and APLN are also regulated by mTOR or play a role in downstream signaling in NF2-null MN cells. We propose to understand the mechanism and biological significance of elevated expression and activation of NUAK2 in meningioma. Further, our large-scale drug screening efforts, in collaboration with NIH-NCATS, revealed a set of proteasome pathway related drugs exhibiting cytotoxic effects in NF2-null cells. Here we propose to examine three different drugs targeting the proteasome (provided by Millennium-Takeda Pharmaceuticals), alone and combined with TAK-228 (mTOR inhibitor) in 5 grade I, 5 grade II and 5 grade III MN lines with NF2 loss. More importantly, we propose to undertake single cell RNAseq along with array CGH in NF2-deficient meningiomas and their corresponding primary cell lines to define tumor heterogeneity and correlate with drug response. Anti-tumor efficacy of proteasome drugs will also be evaluated in orthotopic NF2- deficient benign and malignant meningioma models. Our overall approach of (i) leveraging the ‘omics and drug screening results, (ii) characterizing tumor heterogeneity and correlating with drug response, and (iii) testing potential drugs in orthotopic NF2-deficient i...