Therapeutic targeting of the SWI/SNF chromatin remodeler to regulate GBM chemosensitivity

胶质母细胞瘤（GBM）是成人脑中最常见的原发性恶性肿瘤，并且是最常见的恶性肿瘤之一。 毁灭性的癌症，因为它总是致命的。胶质母细胞瘤的血管周围龛和坏死区 富含对治疗具有高度抗性的干细胞样肿瘤细胞，称为GBM干细胞（GSC）。 在GBM肿瘤中，也存在固有抗性或获得抗性的分化细胞 治疗因此，存在鉴定和靶向分子途径的关键未满足的需求， 促进GBM癌症的干性，这将增强GBM对目前批准的疗法的敏感性。这 我们的主要发现是：1）SWI/SNF的BRG 1催化亚基 染色质重塑复合物促进GBM GSC表型和DNA烷基化抗性 用于治疗GBM患者的药物如替莫唑胺（TMZ）; 2）PFI-3，一种小分子BRG 1 溴结构域（BRD）抑制剂（BRI）通过降低GSC对DNA烷化剂的敏感性来改善GSC对DNA烷化剂的敏感性。 3）开发了克服TMZ抗性GBM抗性的下一代BRI 细胞系，包括有效的BRG 1特异性BRI（IV-255）;和4）BRI增加亚类 干扰素（IFN）反应基因的预测，以提高抗肿瘤宿主免疫反应。 我们的中心假设是靶向BRG 1的BRD选择性地破坏GBM GSC的维持， 其增强GBM对批准的疗法的敏感性并增加GBM免疫反应性。目标1 我们将恢复BRG 1-KD（敲低）GSC和TMZ抗性GBM细胞中的BRG 1表达， 阻止染色质结合的野生型BRG或突变型BRG 1 BRD。我们将研究 BRG 1 BRD在体外和体内维持GBM恶性表型和治疗敏感性的作用 vivo.在目标2中，我们将改进BRI结构并开发更多的小分子BRG 1 BRI。我们将 定义BRI在体外和体内对癌症干性和对GBM的治疗敏感性的活性，以及 评估BRD结合的选择性、药物样性质并优化BBB通透性。抗癌 BRI对颅内GBM肿瘤的疗效将在免疫功能低下和 免疫活性GBM小鼠模型，以确定其对肿瘤相关免疫细胞的影响。在Aim中 我们将定义BRD靶向BRG 1如何重编程转录以促进GBM免疫反应性 并通过进行全局转录组分析（RNA-seq）来抑制肿瘤发生， 与BRG 1和RNA聚合酶II特异性ChIP。这些BRD调控的基因将定义BRG 1- 依赖转录组，它也将确定候选分子途径， BRG 1抑制剂靶向肿瘤细胞，以获得癌细胞的合成杀伤力。我们的总体 我们的目标是用BRI结合目前采用的疗法治疗性靶向BRG 1， 迫切需要治疗这种毁灭性的脑癌的方法。

Glioblastoma (GBM) is the most common primary malignancy of the adult brain and is among the most devastating cancers as it is invariably lethal. The perivascular niche and necrotic regions of GBM tumors is enriched for stem-like tumor cells called GBM stem cells (GSCs) that are highly resistant to therapy. Within GBM tumors there are also differentiated cells that are intrinsically resistant, or acquire resistance to, therapy. Thus, there is a critical unmet need to identify and target the molecular pathways that promote GBM cancer stemness, which will enhance GBM sensitivity to currently approved therapies. This proposal is premised on several of our key findings: 1) the BRG1 catalytic subunit of the SWI/SNF chromatin remodeling complex promotes the GBM GSC phenotype and resistance to DNA alkylating agents such as temozolomide (TMZ) used to treat GBM patients; 2) PFI-3, a small molecule BRG1 bromodomain (BRD) inhibitor (BRI), improves GSC sensitivity to DNA alkylating agents by reducing GSC stemness; 3) next generation BRIs were developed that overcome the resistance of TMZ-resistant GBM cell lines, including a potent BRG1-specific BRI (IV-255); and 4) BRIs increase expression of a subclass of interferon (IFN) response genes that are predicted to enhance the anti-tumoral host immune response. Our central hypothesis is that targeting the BRD of BRG1 selectively disrupts GBM GSCs maintenance, which enhances GBM sensitivity to approved therapies and increases GBM immunoreactivity. In Aim 1 we will restore BRG1 expression in BRG1-KD (knockdown) GSCs and in TMZ resistant GBM cells with either wild-type BRG or mutant BRG1 BRD that prevents chromatin binding. We will examine the role of the BRG1 BRD in maintaining the GBM malignant phenotype and therapeutic sensitivity in vitro and in vivo. In Aim 2 we will refine the BRI structure and develop additional small molecule BRG1 BRIs. We will define BRI activity on cancer stemness and therapeutic sensitivity on GBM in vitro and in vivo, and assess selectivity for BRD binding, drug-like properties and optimize BBB penetrance. The anticancer efficacy of BRIs on intracranial GBM tumors will be tested in both immunocompromised and immunocompetent GBM mouse models to define their impact on tumor-associated immune cells. In Aim 3 we will define how BRD targeting of BRG1 reprograms transcription to promote GBM immunoreactivity and inhibit tumorigenesis by performing global transcriptome analysis (RNA-seq), which will be coupled with BRG1 and RNA polymerase II specific ChIP. These BRD-regulated genes will define the BRG1- dependent transcriptome in GSCs, and it also will identify candidate molecular pathways that could be pharmacologically targeted with BRG1 inhibition to get cancer cell synthetic lethality. Our overarching goal is to target BRG1 therapeutically with BRIs combined with presently employed therapies as a novel and urgently required therapeutic approach for this devastating form of brain cancer.