Project 4: Inhibiting Novel Autophagy Mediator ATG4B for Treating Glioblastoma

项目4：项目概要 通过抑制关键自噬介体ATG 4 B增强GBM细胞毒性治疗 胶质母细胞瘤（GBM）是最常见的恶性原发性脑肿瘤。尽管治疗包括 手术切除、放疗和化疗，大多数GBM患者在术后14至16个月内死亡。 诊断，强调迫切需要新的疗法来对抗这种致命的疾病。自噬是一 保守的分解代谢过程，通过调节细胞的能量平衡来维持体内平衡。癌 细胞利用自噬来清除受损的细胞器和聚集的蛋白质，并在高浓度下回收营养物质。 支持肿瘤生长。放射化疗（RT-TMZ）是治疗GBM的一线治疗方法，但 还激活肿瘤细胞中的自噬反应，从而保护细胞免于凋亡。 mTOR信号传导的抑制是癌症治疗中的常见靶标。然而，mTOR抑制也诱导 癌细胞的自噬因此，人们对抑制这种保护机制非常感兴趣， 治疗癌症非特异性自噬抑制剂如氯喹（CQ）和羟基-CQ（HCQ）正在被 在大量的临床试验中进行了研究。然而，这些化合物缺乏特异性， 与毒性相关，并可能降低其疗效。我们发现ATG 4 B，一种将LC 3 至LC 3-I/II，其是细胞中自噬过程所需的，在丝氨酸残基383处磷酸化（p-S383） 在患者来源的胶质瘤干细胞样细胞（胶质瘤起始细胞或GIC）中。我们还发现ATG 4 B S383 磷酸化增加GIC自噬活性，并且p-S383 ATG 4 B的肿瘤内表达 与GBM患者预后不良相关。相反，ATG 4 B的敲低，或非- 可磷酸化的ATG 4 B突变转基因（S383 A），抑制GSC自噬反应和致瘤性 移植到无胸腺小鼠的大脑中。此外，使用NCI的ATG 4 B的药理学抑制 抑制ATG 4 B酶活性和GBM致瘤性的化合物NSC 185058，显著增强RT 抑制GBM肿瘤生长并增加颅内移植GIC动物的存活率。 此外，NSC 185058还显著增强催化mTORC抑制剂AZD 2014的抑制作用 关于GIC基于这些强有力的数据和我们建立的不同遗传+的多个PDX GBM模型 表观遗传谱，在这个项目中，我们建议确定ATG 4 B抑制剂的抗GBM疗效 NSC 185058单药治疗和与RT-TMZ联合治疗;研究NSC 185058的治疗潜力 联合抑制ATG 4 B、mTOR和RT，并开发用于患者的NSC 185058， 抑制剂，作为单一药剂和与细胞毒性疗法组合，用于治疗患者的临床试验 复发性GBM该项目具有强大的分子机制基础和相关的治疗 假设，如果被证明是正确的，有可能对GBM的治疗产生积极影响， 其他恶性肿瘤。

PROJECT 4: PROJECT SUMMARY Enhancing GBM Cytotoxic Therapy Through Inhibition of Key Autophagy Mediator ATG4B Glioblastoma (GBM) is the most common and malignant primary brain tumor. Despite treatment consisting of surgical removal, radiation and chemotherapy, most patients with GBM die within 14 to 16 months after diagnosis, underscoring the urgent need for new therapies to combat this deadly disease. Autophagy is a conserved catabolic process that maintains homeostasis by regulating the energy balance of the cell. Cancer cells use autophagy to remove damaged organelles and aggregated proteins, and to recycle nutrients in high demand to support tumor growth. Radiochemotherapy (RT-TMZ) is the front-line treatments against GBM, but also activate the autophagic response in tumor cells, thus protecting the cells from undergoing apoptosis. Inhibition of mTOR signaling is a common target in cancer therapy. However, mTOR inhibition also induces autophagy in cancer cells. Consequently, there is immense interest in inhibiting this protective mechanism while treating cancer. Non-specific autophagy inhibitors like chloroquine (CQ) and hydroxy-CQ (HCQ) are being investigated in a large number of clinical trials. However, the lack of specificity of these compounds is , associated with toxicity and may diminish its efficacy. We discovered that ATG4B, an enzyme that converts LC3 to LC3-I/II, which is required for autophagy process in cells, is phosphorylated at serine residue 383 (p-S383) in patient-derived glioma stem-like cells (glioma initiating cells or GICs). We also found that ATG4B S383 phosphorylation increases GIC autophagic activity, and that intratumoral expression of p-S383 ATG4B correlates with poor prognosis in GBM patients. In contrast, knockdown of ATG4B, or expression of a non- phosphorylatable ATG4B mutant transgene (S383A), inhibits GSC autophagic response and tumorigenicity when engrafted in the brains of athymic mice. Furthermore, pharmacologic inhibition of ATG4B, using a NCI compound NSC185058 that inhibits ATG4B enzymatic activity and GBM tumorigenicity, markedly enhanced RT inhibition on GBM tumor growth and increases the survival of animals with intracranially engrafted GIC. Additionally, NSC185058 also markedly enhanced inhibitory effects by a catalytic mTORC inhibitor AZD2014 on GICs. Based on these strong data and our established multiple PDX GBM models of distinct genetic + epigenetic profiles, in this project, we propose to determine the anti-GBM efficacy of ATG4B inhibitor NSC185058, as monotherapy and in combination with RT-TMZ; investigate the therapeutic potential of combining inhibition of ATG4B, mTOR and RT and develop NSC185058 for use in patients, and test the ATG4B inhibitor, both as a single agent and in combination with cytotoxic therapy, in a clinical trial for treating patients with recurrent GBM. This project has a strong molecular mechanistic foundation and associated therapeutic hypothesis that, if proven correct, has the potential for positive impact on the treatment of GBM, and perhaps other malignancies.