Targeting EGFR/FOXG1-mediated resistance to ONC201 in H3K27M-mutant diffuse midline glioma

项目总结/摘要 背景和长期目标：患有弥漫性中线胶质瘤（DMG）的儿童和年轻人 携带H3 K27 M突变的人很少存活超过两年，并且没有经过证实的治疗方法 辐射多巴胺受体DRD 2拮抗剂ONC 201诱导TNF相关凋亡的转录- 诱导配体（TRAIL），导致多种肿瘤细胞类型的凋亡。ONC 201另外结合并 激活线粒体蛋白酶ClpP，导致乳腺癌细胞中的细胞死亡 活性氧化磷酸化（Oxygenic phosphorylation，OXPHOS）。ONC 201在H3 K27 M-DMG中处于早期使用阶段，但其 其机制和疗效尚不清楚。在我们的初步数据中，ONC 201在鼠H3 K27 M-1中是有效的。 突变型神经胶质瘤在我们未发表的临床数据（n=50例H3 K27 M患者）中，ONC 201使PFS和总体PFS加倍。 在一些具有多种持续反应的临床亚组（例如丘脑H3 K27 M）中的生存（OS）。尽管 尽管ONC 201在H3 K27 M-DMG中的这种令人印象深刻的功效，但我们不知道ONC 201在H3 K27 M-DMG肿瘤细胞中如何起作用， 如何改善耐药肿瘤的反应。 在肿瘤测序分析中，ONC 201耐药与EGFR的高表达相关， 脑发育转录因子FOXG 1。FOXG 1表达也是最强的阴性预测因子， 人神经胶质瘤细胞系中的ONC 201敏感性。为了优先考虑哪些患者应该接受治疗 ONC 201和产生合理的联合治疗，迫切需要阐明其机制 在H3 K27 M细胞中的ONC 201敏感性。我们的中心假设是ONC 201在H3 K27 M-DMG中有效 通过靶向ClpP和EGFR信号传导介导ONC 201抗性的具有活性OXPHOS的肿瘤 通过FOXG 1使OXPHOS失活。这是基于我们在K27 M-DMG细胞中的初步数据，其显示 在促进OXPHOS的培养基中培养的细胞对ONC 201具有改善的应答，和（iii）EGFR 敲低降低FOXG 1基因组结合并改善ONC 201应答。 方法：在具体目标1中，我们将确定以下因素的影响：（i）ClpP线粒体靶向相对于DRD 2 拮抗和（ii）糖酵解相对于OXPHOS，在H3 K27 M-DMG细胞对ONC 201的敏感性。具体目标 2、确定FOXG 1在H3 K27 M-DMG生物能量学中的作用。在具体目标3中，我们将确定 EGFR抑制影响H3 K27 M-DMG中ONC 201诱导的线粒体应激的能力和基线 EGFR/FOXG 1肿瘤染色以预测ONC 201反应。我们希望能够确定EGFR 和FOXG 1影响H3 K27 M-DMG肿瘤中的线粒体生物能量学和ONC 201敏感性。 意义：这一贡献预计将是重大的，因为它将允许临床医生优先考虑 H3 K27 M-DMG患者应接受ONC 201治疗，并为合理组合治疗提供平台。 治疗以改善ONC 201抗性。我们的翻译团队将很快开设一个多站点的国际 平台试验可以立即为H3 K27 M-DMG患者实施该提案的结果。

PROJECT SUMMARY / ABSTRACT Background and long-term objectives: Children and young adults with diffuse midline glioma (DMG) harboring H3K27M mutation rarely survive longer than two years and have no proven therapies beyond radiation. The dopamine receptor DRD2 antagonist ONC201 induces transcription of TNF-related apoptosis- inducing ligand (TRAIL), leading to apoptosis in multiple tumor cell types. ONC201 additionally binds and activates the mitochondrial protease ClpP, resulting in mitochondrial-mediated cell death in breast cancer cells with active oxidative phosphorylation (OXPHOS). ONC201 is in early phase use in H3K27M-DMG, but its mechanism and efficacy remains unknown. In our preliminary data, ONC201 is effective in murine H3 K27M- mutant gliomas. In our unpublished clinical data (n=50 H3K27M patients), ONC201 doubles PFS and overall survival (OS) in some clinical sub-groups (e.g. thalamic H3K27M) with multiple sustained responses. Despite this impressive efficacy in H3K27M-DMG, we do not know how ONC201 works in H3K27M-DMG tumor cells or how to improve responses in resistant tumors. In analysis of tumor sequencing, ONC201 resistance correlates with high expression of EGFR and the brain developmental transcription factor FOXG1. FOXG1 expression is also the strongest negative predictor of ONC201 sensitivity in human glioma cell lines. In order to prioritize which patients should be treated with ONC201 and to generate rationale combination therapies, there is a critical need to elucidate the mechanism of ONC201 sensitivity in H3K27M cells. Our central hypothesis is that ONC201 is effective in H3K27M-DMG tumors with active OXPHOS through targeting of ClpP and that EGFR signaling mediates ONC201 resistance by inactivating OXPHOS via FOXG1. This is based on our preliminary data in K27M-DMG cells, which shows that cells cultured in media that promotes OXPHOS have an improved response to ONC201, and (iii) EGFR knockdown reduces FOXG1 genomic binding and improves ONC201 response. Approach: In Specific Aim 1, we will determine the impact of: (i) ClpP mitochondrial targeting vs. DRD2 antagonism and (ii) glycolysis vs OXPHOS, in the sensitivity of H3K27M-DMG cells to ONC201. In Specific Aim 2, we will determine the role of FOXG1 in H3K27M-DMG bioenergetics. In Specific Aim 3, we will determine the ability of EGFR inhibition to impact ONC201-induced mitochondrial stress in H3K27M-DMG and for baseline EGFR/FOXG1 tumor staining to predict ONC201 response. We expect to define the mechanism by which EGFR and FOXG1 impact mitochondrial bioenergetics and ONC201 sensitivity in H3K27M-DMG tumors. Significance: This contribution is expected to be significant because it will allow clinicians to prioritize which H3K27M-DMG patients should be treated with ONC201 and provide a platform for rationale combinatorial treatments to improve ONC201 resistance. Our translational team will soon open a multi-site international platform trial that can implement the results of this proposal immediately for patients with H3K27M-DMG.