Project 4: Inhibiting Novel Autophagy Mediator ATG4B for Treating Glioblastoma

项目4：项目总结 通过抑制关键的自噬调节因子ATG4B加强GBM细胞毒治疗 胶质母细胞瘤(GBM)是最常见和最恶性的原发脑肿瘤。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 它强调迫切需要新的治疗方法来抗击这一致命疾病。自噬是一种 保守的分解代谢过程，通过调节细胞的能量平衡来维持动态平衡。癌 细胞使用自噬来移除受损的细胞器和聚集的蛋白质，并在高密度下循环营养。 支持肿瘤生长的需求。放化疗(RT-TMZ)是治疗GBM的一线治疗方法，但 还可以激活肿瘤细胞中的自噬反应，从而保护细胞免受凋亡。 抑制mTOR信号转导是肿瘤治疗中的一个常见靶点。然而，mTOR抑制也会导致 癌细胞中的自噬。因此，人们对抑制这一保护机制非常感兴趣，同时 治疗癌症。像氯喹(CQ)和羟基-CQ(HCQ)这样的非特异性自噬抑制剂正在 在大量临床试验中进行了调查。然而，这些化合物缺乏特异性的是， 与毒性有关，并可能降低其疗效。我们发现ATG4B，一种转化Lc3的酶 Lc3-I/II，这是细胞自噬过程所必需的，在丝氨酸残基383位被磷酸化(p-S383) 在患者来源的胶质瘤干细胞(胶质瘤启动细胞或GICs)中。我们还发现ATG4B S383 磷酸化增加GIC自噬活性，p-S383ATG4B在肿瘤内的表达 与GBM患者预后不良有关。相反，ATG4B的敲除，或非 可磷酸化ATG4B突变体转基因(S383A)抑制GSC自噬反应和致瘤性 植入无瘤小鼠的大脑。此外，使用NCI对ATG4B的药理抑制 抑制ATG4B酶活性和基底膜致瘤性的化合物NSC185058显著增强RT 抑制GBM肿瘤生长并提高GIC脑内植入动物的存活率。 此外，NSC185058还显著增强了催化mTORC抑制剂AZD2014的抑制作用 在GIC上。基于这些强有力的数据和我们建立的多个不同基因的PDX GBM模型 表观遗传学特征，在本项目中，我们建议确定ATG4B抑制剂的抗GBM效果 NSC185058，作为单一疗法并联合RT-TMZ；研究其治疗潜力 联合抑制ATG4B、mTOR和RT并开发用于患者的NSC185058，并检测ATG4B 作为单一药物和与细胞毒治疗联合使用的抑制剂，用于治疗患者的临床试验 伴有复发的基底膜。这个项目有强大的分子机制基础和相关的治疗 假说，如果被证明是正确的，有可能对GBM的治疗产生积极影响，也许 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.