PROJECT SUMMARY Gliomas are the most common primary malignant brain tumors in adults and account for over 14,000 deaths annually in the United States. Glioblastoma (GBM, grade IV glioma) is the most common and deadly glioma subtype and is associated with a median overall survival of only ~15 months with aggressive therapy. Telomere maintenance mechanisms are a hallmark of cancer and are required to enable replicative immortality of malignant cells, including gliomas. To maintain telomeres, the majority of gliomas use the enzyme telomerase, which uses an RNA template and reverse transcription to extend telomeric repeats at the ends of chromosomes. In contrast, a subset of gliomas employs telomerase-independent mechanisms, termed Alternative Lengthening of Telomeres (ALT). ALT uses homologous recombination to maintain telomere length and is characterized by increased replicative stress in telomere regions and remodeling of telomeric chromatin architecture to an epigenetic state that is permissive to homologous recombination. ~10% of GBM cases and virtually all progressive grade II/III IDH-mutant astrocytomas harbor genetic alterations involved in the induction of ALT. In addition to ATRX mutations, we recently identified recurrent loss-of-function mutations in the SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1) gene as novel genetic mutations associated with the ALT phenotype in IDH-wildtype – TERT promoter wild-type GBM. SMARCAL1 encodes an annealing helicase that localizes to sites of DNA damage and replication stress and resolves stalled replication fork structures to facilitate fork progression within difficult-to-replicate DNA sequences, such as telomeres. Recent studies indicate that SMARCAL1 localizes to telomeric DNA in ALT- positive cancer cells with native ATRX-inactivating mutations and that SMARCAL1 helicase activity suppresses markers of replication stress and DNA damage at telomeres. Based on these observations, we hypothesize that SMARCAL1 activity is essential for resolving replication stress at telomeric regions and SMARCAL1 loss-of- function leads to an increase in unresolved replication fork collapse and DNA damage at telomeres in a manner that is permissive to homologous recombination and ALT. In Aim 1 we will use genetically engineered cell lines and cancer cell lines with native SMARCAL1 mutations to identify specific ALT pathway components required for telomere synthesis and cellular immortalization in the context of SMARCAL1 loss-of-function mutations. In Aim 2, we will use patient-derived glioma cell lines and xenografts to determine the extent to which SMARCAL1 helicase activity resolves replication stress at telomeres in ATRX-mutant GBM cell lines using ALT. Collectively, the proposed studies are expected to significantly advance our understanding of the role of SMARCAL1 in GBM telomere maintenance and are designed to establish proof of concept for therapeutic targe...