Uncover the role of H3.3-G343R mutation in shaping the DNA damage response, anti-tumor immunity and mechanisms of resistance in glioma.

揭示 H3.3-G343R 突变在塑造神经胶质瘤 DNA 损伤反应、抗肿瘤免疫和耐药机制中的作用。

• 批准号: 10384185
• 负责人: Maria G Castro
• 金额: $ 52.79万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-15 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10384185
• 关键词: ATRX gene; Adaptive Immune System; Biological Assay; Biology; Blood; Brain; CD8-Positive T-Lymphocytes; Cause of Death; Cell Cycle Checkpoint; Cells; Cerebral hemisphere; Chemotherapy and/or radiation; Childhood; Childhood Brain Neoplasm; Childhood Glioma; Chromatin; Chromatin Structure; DNA Damage; DNA Repair; DNA Repair Disorder; Data; Defect; Development; Down-Regulation; Epigenetic Process; Excision; Exhibits; Frequencies; Gene set enrichment analysis; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Glioma; Goals; Histologic; Histones; Human; Immune; Immune response; Immune system; Immunity; Immunocompetent; Immunologic Memory; Impairment; In Vitro; Interferon Type I; Intracranial Neoplasms; Investigation; Ionizing radiation; Lead; Lesion; Link; Malignant Childhood Neoplasm; Malignant neoplasm of brain; Mediating; Modality; Modeling; Molecular; Mus; Mutation; Myeloid-derived suppressor cells; Nature; Nonhomologous DNA End Joining; Ontology; Pathogenesis; Pathway interactions; Pharmacology; Phenotype; Play; Preclinical Testing; Process; Production; Radiation therapy; Recurrence; Relaxation; Resistance; Role; Shapes; Sleeping Beauty; Stimulator of Interferon Genes; System; TP53 gene; Testing; Therapeutic; Transposase; Treatment Efficacy; Tumor Immunity; Tumor-infiltrating immune cells; Up-Regulation; Variant; adaptive immune response; brain parenchyma; chemotherapy; cytokine; experimental study; genetic makeup; homologous recombination; immune activation; immunoregulation; in vivo; inhibitor; knock-down; mouse model; mutant; nerve stem cell; novel therapeutic intervention; repaired; resistance mechanism; response; single-cell RNA sequencing; standard of care; therapy outcome; therapy resistant; transcriptome sequencing; treatment response; tumor; tumor microenvironment; tumor-immune system interactions; young adult

Pediatrichigh-grade gliomas (pHGGs) are one of the most aggressive forms of brain cancer, with a median survival (MS) of ~18 months 1,2. The current standard of care for pHGG, consisting of tumor resection followed by radiation and chemotherapy, elicits only a modest increase in MS 1-4. The main reasons for the limited therapeutic outcomes are tumor recurrence, caused by the infiltrative nature of pHGG, and the development of an immune-suppressive tumor microenvironment (TME). One of the main subtypes of hemispherical pHGG encodes for G34R/V substitutions in the histone H3F3A 4, along with ATRX and TP53 inactivating mutations. The investigation of the molecular pathways which play a role in the pathogenesis of pHGG requires accurate mouse models which recapitulate the salient features of pHGG and develop within the brain's microenvironment in an immune-competent host. Our lab created genetically engineered immune competent pHGG mouse models employing the Sleeping Beauty (SB) transposase system 5,6. Tumors harbor genetic lesions encountered in a subtype of pHGG, i.e., H3.3G34R co-expressed with ATRX and TP53 knock down. The host in this pHGG model exhibits an intact immune system, thus enabling detailed mechanistic studies on all aspects of pHGG biology in vivo, including interactions with the TME immune cells. Our preliminary RNA-Seq data on H3.3-G34R versus H3.3-Wt pHGG revealed downregulation of gene ontologies (GO) related to DNA Damage Response (DDR) in H3.3-G34R pHGG. We also observed upregulation of GOs related to the activation of the immune response, such as “Regulation of Immune Response” and “Type I Interferon Production”, in the H3.3-G34R pHGG model. Herein we propose to determine the cellular andmolecular mechanisms by which H3.3-G34R regulates the response to radiotherapy and DDR inhibition in mouse and human H3.3G34R pHGG cells in vitro and in vivo. We aim to elucidate the role played by H3.3-G34R in DNA repair processes, responseto DNA damaging agents, and in TME immune cells' reprogramming. We propose to test the hypothesis that H3.3G34R reshapes the epigenetic landscape, resulting in alterations in chromatin states and transcriptional changes. These alterations cause DDR impairment and induce genomic instability, which in turn leads to cGAS-STING- Pathway-mediated activation of the immune system within the H3.3G34R pHGG TME. We will assess chromatin states by ATAC-seq, and establish whether reduced chromatin accessibility impairs DNA repair in G34R pHGG. We will also evaluate whether genomic instability in G34R pHGG mediates the immune system activation via cGAS-STING. We will also define at the molecular level the phenotypically diverse tumor and infiltrating immune cell clusters within the H3.3-G34R pHGG microenvironment using scRNA-seq; this will allow us to uncover mechanisms of therapeutic resistance. This information, will enable uncovering genetic makeup- tailored therapeutic modalities for H3.3-G34R pHGG, such as cell cycle checkpoint or DDR inhibitors combined with radiotherapy.

儿童高级别神经胶质瘤 (pHGG) 是最具侵袭性的脑癌形式之一，中位数为 生存期 (MS) 约为 18 个月 1,2 当前 pHGG 的护理标准包括肿瘤切除术。 通过放疗和化疗，仅引起 MS 1-4 的适度增加，这是有限的主要原因。 治疗结果是由 pHGG 的浸润性质引起的肿瘤复发，以及 免疫抑制肿瘤微环境 (TME) 半球形 pHGG 的主要亚型之一。 编码组蛋白 H3F3A 4 中的 G34R/V 替换，以及 ATRX 和 TP53 失活突变。 对 pHGG 发病机制中起作用的分子途径的研究需要准确的 重现 pHGG 显着特征并在大脑微环境中发育的小鼠模型 我们的实验室在具有免疫能力的宿主中创建了具有免疫能力的基因工程 pHGG 小鼠模型。 使用睡美人 (SB) 转座酶系统 5,6，肿瘤具有遗传损伤。 pHGG 亚型，即与 ATRX 和 TP53 敲低共表达的 H3.3G34R 该 pHGG 模型中的宿主。 表现出完整的免疫系统，从而能够对 pHGG 生物学的各个方面进行详细的机制研究 体内，包括与 TME 免疫细胞的相互作用。 H3.3-Wt pHGG 揭示了与 DNA 损伤反应 (DDR) 相关的基因本体 (GO) 的下调 在 H3.3-G34R pHGG 中，我们还观察到与免疫激活相关的 GO 上调。 H3.3-G34R 中的反应，例如“免疫反应的调节”和“I 型干扰素的产生” pHGG 模型在此我们建议确定 H3.3-G34R 的细胞和分子机制。 调节小鼠和人 H3.3G34R pHGG 细胞对放疗和 DDR 抑制的体外反应 我们的目标是阐明 H3.3-G34R 在 DNA 修复过程和 DNA 反应中所发挥的作用。 我们建议检验 H3.3G34R 的假设。 重塑表观遗传景观，导致染色质状态的改变和转录的变化。 这些改变会导致 DDR 损伤并诱导基因组不稳定，进而导致 cGAS-STING- 我们将评估 H3.3G34R pHGG TME 内免疫系统的途径介导激活。 通过 ATAC-seq 检测染色质状态，并确定染色质可及性降低是否会损害 DNA 修复 我们还将评估 G34R pHGG 中的基因组不稳定性是否介导免疫系统。 我们还将在分子水平上定义表型多样化的肿瘤和 使用 scRNA-seq 渗透 H3.3-G34R pHGG 微环境中的免疫细胞簇； 我们揭示治疗耐药机制，这些信息将有助于揭示基因组成—— 针对 H3.3-G34R pHGG 的定制治疗方式，例如细胞周期检查点或 DDR 抑制剂 与放射治疗相结合。