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.

儿童高级别胶质瘤（pHGG）是最具侵袭性的脑癌之一， 生存期（MS）约18个月1，2. pHGG的当前标准治疗包括肿瘤切除， 通过放疗和化疗，MS仅适度增加1-4。有限的主要原因 治疗结果是肿瘤复发，由pHGG的浸润性质引起， 免疫抑制性肿瘤微环境（TME）。半球型pHGG的主要亚型之一 编码组蛋白H3 F3 A 4中的G34 R/V取代，沿着ATRX和TP 53失活突变。 对pHGG发病机制中起作用的分子通路的研究需要准确的 重现pHGG显著特征并在大脑微环境中发育的小鼠模型 在一个有免疫能力的宿主体内我们的实验室创建了基因工程免疫活性pHGG小鼠模型 使用睡美人（SB）转座酶系统5，6。肿瘤携带遗传病变， pHGG的亚型，即，H3.3G34R与ATRX共表达，TP 53敲低。该pHGG模型中的宿主 显示出完整的免疫系统，从而能够对pHGG生物学的各个方面进行详细的机制研究 在体内，包括与TME免疫细胞的相互作用。我们对H3.3-G34 R与 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-G34 R pHGG.我们还观察到与免疫激活相关的BMP 2的上调， H3.3-G34 R中的“免疫应答的调节”和“I型干扰素的产生” pHGG模型。在此，我们提出确定H3.3-G34 R通过细胞和分子机制， 在体外调节小鼠和人H3.3G34R pHGG细胞对放疗和DDR抑制的反应 和体内。我们的目的是阐明H3.3-G34 R在DNA修复过程中的作用， 破坏剂和TME免疫细胞的重编程。我们建议测试H3.3G34R 重塑表观遗传景观，导致染色质状态和转录变化的改变。 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- H3.3G34R pHGG TME内免疫系统的通路介导的活化。我们将评估 染色质状态，并确定染色质可及性降低是否会损害DNA修复 在G34 R pHGG.我们还将评估G34 R pHGG中的基因组不稳定性是否介导免疫系统 通过cGAS-STING激活。我们还将在分子水平上定义表型多样性肿瘤， 使用scRNA-seq浸润H3.3-G34 R pHGG微环境中的免疫细胞簇;这将允许 让我们来揭示耐药性的机制。这些信息，将使我们能够揭开基因组成- H3.3-G34 R pHGG的定制治疗方式，如细胞周期检查点或DDR抑制剂 与放射治疗相结合。