PROJECT SUMMARY Histologically characterized by neoplastic stromal cells and abundant vasculature, hemangioblastomas (HBs) occur as sporadic lesions (~70%, predominantly in the cerebellum) and in familial forms associated with von Hippel-Lindau (VHL) disease, an inherited multisystem tumor disorder characterized by HBs and other benign and malignant visceral neoplasms (e.g., clear cell renal cell carcinoma (RCC)). Central nervous system (CNS) HBs, which are frequently multiple or recurrent in VHL disease, may not be resectable, and an effective drug treatment is not available. Although biallelic inactivation of the VHL tumor suppressor gene by pathogenic mutations, promoter hypermethylation and/or loss of VHL-bearing chromosome 3p25 have been identified in 47% to 64% of both familial (germline mutations) and sporadic (somatic mutations) CNS HBs, the underlying pathogenic mechanisms responsible for HBs remain incompletely understood. By whole exome sequencing of archived VHL disease-associated and sporadic cerebellar HBs and matched cerebellum and/or blood cells (n=23, age 24-63), we found 314 pathogenic and/or likely deleterious mutations (both germline and somatic). In a significant number of cases (14/23, 61%), we identified the germline fibroblast growth factor receptor 4 (FGFR4) p.G388R variant, 8 of which were VHL wild-type and 2 had multiple/recurrent cerebellar HBs; the other 6 cases had both FGFR4 p.G388R and VHL mutations, 1 of which had multiple/recurrent cerebellar HBs and RCC. FGFR4 p.G388R is a pathogenic activating mutation known to enhance basal signal transducer and activator of transcription 3 (STAT3) signaling, resulting in increased HIF-1α mRNA transcription, STAT3- and HIF-1 target gene expression, angiogenesis, and possibly increased tumor susceptibility. We hypothesize that in addition to VHL inactivation, a significant number of VHL disease-associated and sporadic cerebellar HBs harbor germline FGFR4 p.G388R variant that activates STAT3 signaling and target gene expression in these tumors. We also hypothesize that gene promoter hypermethylation (e.g. VHL) and/or chromosomal alterations (e.g., EGFR amplification) may coexist with FGFR4 p.G388R variant and could together contribute to VHL disease-associated and sporadic cerebellar HB pathogenesis. Based on our initial results, we propose (Aim 1a) to validate in a larger cohort of archived cerebellar HBs our novel finding that a significant number of both VHL disease-associated and sporadic HBs harbor germline FGFR4 p.G388R. We also propose (Aim 1b) to define whether FGFR4 p.G388R activates the JAK-STAT-HIF signaling pathway in these tumors and (Aim 2) to demonstrate whether gene promoter hypermethylation and/or chromosomal alterations may co-exist with FGFR4 p.G388R variant and could together contribute to VHL disease-related and sporadic cerebellar HB pathogenesis. We anticipate that our proposed work could have a significant impact on the genetic testing and counseling of p...