The role of SMARCAL1 in glioma telomere maintenance.

项目总结 胶质瘤是成人最常见的原发恶性脑肿瘤，死亡人数超过14,000人 每年在美国。胶质母细胞瘤(GBM，IV级)是最常见和最致命的胶质瘤。 亚型，与积极治疗的中位总生存期只有15个月有关。端粒 维持机制是癌症的一个标志，是实现复制永生所必需的 恶性细胞，包括胶质瘤。为了维持端粒，大多数胶质瘤使用端粒酶， 它使用RNA模板和逆转录来延长染色体末端的端粒重复序列。 相反，胶质瘤的一部分采用端粒酶不依赖的机制，称为选择性延长。 端粒(ALT)。ALT使用同源重组来维持端粒长度，其特征是 端粒区域复制应力增加和端粒染色质结构重塑 允许同源重组的表观遗传状态。约10%的GBM病例和几乎所有 进行性II/III级IDH突变星形细胞瘤存在与ALT诱发有关的基因改变。在……里面 除了ATRX突变外，我们最近还在SMARCAL1中发现了反复出现的功能丧失突变 (Swi/SNF相关、基质相关、肌动蛋白依赖的染色质调节子，A-like 1)基因为新基因 IDH-Wildtype-TERT启动子野生型GBM与ALT表型相关的基因突变。 SMARCAL1编码一种退火型解旋酶，定位于DNA损伤和复制应激部位，并 解决停滞不前的复制分叉结构，以促进难以复制的DNA中的分叉进展 序列，例如端粒。最近的研究表明，SMARCAL1定位于ALT-1中的端粒DNA。 带有天然ATRX失活突变和SMARCAL1解旋酶活性抑制的阳性癌细胞 复制应激和端粒DNA损伤的标志物。基于这些观察，我们假设 SMARCAL1活性是解决端粒区复制应激和SMARCAL1缺失的关键 功能导致未解决的复制叉缩和端粒DNA损伤的增加 这是允许同源重组和ALT的。在目标1中，我们将使用基因工程细胞系 和带有天然SMARCAL1突变的癌细胞株，以确定所需的特定ALT途径组件 在SMARCAL1功能丧失突变的背景下，用于端粒合成和细胞永生化。在……里面 目的2，我们将使用患者来源的胶质瘤细胞系和异种移植来确定SMARCAL1在多大程度上 解旋酶活性使用ALT解决ATRX突变的GBM细胞系中端粒的复制压力。总而言之， 预计拟议的研究将显著促进我们对SMARCAL1在GBM中的作用的理解 端粒维持，旨在为SMARCAL1的治疗靶向建立概念证据 携带ATRX突变的ALT阳性胶质瘤。

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 targeting of SMARCAL1 in ALT-positive gliomas harboring ATRX mutations.