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.

Pediatrich级神经胶质瘤（PHGGS）是脑癌最具侵略性的形式之一，中位数 生存（MS）约18个月1,2。 PHGG的当前护理标准，包括肿瘤切除 通过放射线和化学疗法，MS 1-4仅引起适度的增加。有限的主要原因 治疗结果是肿瘤复发，由PHGG的浸润性以及 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取代，以及ATRX和TP53灭活突变。 在PHGG发病机理中起作用的分子途径的投资需要准确 鼠标模型概括了PHGG的显着特征并在大脑的微环境中发展 在免疫能力的宿主中。我们的实验室创建了一般设计的免疫功能PHGG鼠标模型 采用睡美人（SB）转座酶系统5,6。肿瘤港遗传病变在 PHGG的亚型，即H3.3G34R与ATRX和TP53共表达。此PHGG模型中的主机 展示了完整的免疫系统，从而实现了有关PHGG生物学各个方面的详细机械研究 体内，包括与TME免疫细胞相互作用。我们关于H3.3-G34R的初步RNA-seq数据 H3.3-WT PHGG显示基因本体论的下调（GO）与DNA损伤反应有关（DDR） 在H3.3-G34R PHGG中。我们还观察到与免疫激活有关的GO的上调 H3.3-G34R中的反应，例如“免疫反应调节”和“ I型干扰素产生” PHGG模型。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细胞中对放疗和DDR抑制的反应 和体内。我们旨在阐明H3.3-G34R在DNA修复过程中所起的作用，响应DNA 破坏剂，以及TME免疫细胞的重编程。我们建议检验H3.3G34R的假设 重塑表观遗传景观，从而导致染色质状态和转录变化的变化。 这些改变会导致DDR障碍并诱导基因组不稳定性，从而导致CGAS-刺激 途径介导的H3.3G34R PHGG TME中免疫系统的激活。我们将评估 通过ATAC-SEQ染色质状态，并确定降低的染色质可及性是否会损害DNA修复 在G34R PHGG中。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 通过CGAS激活。我们还将在分子水平上定义表观上多样的肿瘤和 使用SCRNA-SEQ在H3.3-G34R PHGG微环境中浸润免疫细胞簇；这将允许 我们发现治疗性抗性的机制。这些信息将使遗传构成能够 针对H3.3-G34R PHGG的量身定制的治疗方法，例如细胞周期检查点或DDR抑制剂 与放射疗法结合。