Biology of terminal R-loops in splicing factor mutant cancers

剪接因子突变癌症中末端 R 环的生物学

• 批准号: 10652900
• 负责人: Manoj M. Pillai
• 金额: $ 8.38万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10652900
• 关键词: ATAC-seq; Acute Myelocytic Leukemia; Alternative Splicing; Architecture; Automobile Driving; Biochemical; Biochemical Process; Biochemistry; Biological Assay; Biological Models; Biology; CRISPR/Cas technology; Cancer Etiology; Catalysis; Cell Line; Cells; Chemistry; Chromatin; Chronic Lymphocytic Leukemia; Clonal Evolution; Cytoplasm; Cytosine; DNA; DNA replication fork; Data; Data Set; Defect; Dependovirus; Development; Disease; Disease model; Disparate; Epigenetic Process; Event; Exons; Family; Gene Expression; Genetic Transcription; Genomic Instability; Growth; HDAC2 gene; Histone Deacetylase; Image; Investigation; Label; Lead; Ligation; Link; Location; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Messenger RNA; Metabolic; Molecular; Mutate; Mutation; Nature; Oncogenes; Oncogenic; Pathogenicity; Pathologic; Pathway interactions; Patients; Physiology; Play; Polymerase; Positioning Attribute; Production; Proteins; RNA; RNA Polymerase II; RNA Processing; RNA Splicing; RNA library; Recurrence; Regulation; Resolution; Role; S Phase Arrest; S phase; SRSF2 gene; Sampling; Site; Spliceosomes; Structure; Tissues; Transcript; Transcriptional Regulation; Translations; Tumor Suppressor Proteins; Uracil; Uveal Melanoma; cancer therapy; cancer type; epigenetic regulation; genome integrity; genome-wide; in vivo Model; mRNA Export; mRNA Stability; melanoma; mutant; nonsynonymous mutation; novel; novel therapeutics; nucleic acid structure; overexpression; response; small hairpin RNA; small molecule inhibitor; targeted treatment; transcriptome sequencing; tumor; tumorigenesis; ATAC-seq; Acute Myelocytic Leukemia; Alternative Splicing; Architecture; Automobile Driving; Biochemical; Biochemical Process; Biochemistry; Biological Assay; Biological Models; Biology; CRISPR/Cas technology; Cancer Etiology; Catalysis; Cell Line; Cells; Chemistry; Chromatin; Chronic Lymphocytic Leukemia; Clonal Evolution; Cytoplasm; Cytosine; DNA; DNA replication fork; Data; Data Set; Defect; Dependovirus; Development; Disease; Disease model; Disparate; Epigenetic Process; Event; Exons; Family; Gene Expression; Genetic Transcription; Genomic Instability; Growth; HDAC2 gene; Histone Deacetylase; Image; Investigation; Label; Lead; Ligation; Link; Location; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Messenger RNA; Metabolic; Molecular; Mutate; Mutation; Nature; Oncogenes; Oncogenic; Pathogenicity; Pathologic; Pathway interactions; Patients; Physiology; Play; Polymerase; Positioning Attribute; Production; Proteins; RNA; RNA Polymerase II; RNA Processing; RNA Splicing; RNA library; Recurrence; Regulation; Resolution; Role; S Phase Arrest; S phase; SRSF2 gene; Sampling; Site; Spliceosomes; Structure; Tissues; Transcript; Transcriptional Regulation; Translations; Tumor Suppressor Proteins; Uracil; Uveal Melanoma; cancer therapy; cancer type; epigenetic regulation; genome integrity; genome-wide; in vivo Model; mRNA Export; mRNA Stability; melanoma; mutant; nonsynonymous mutation; novel; novel therapeutics; nucleic acid structure; overexpression; response; small hairpin RNA; small molecule inhibitor; targeted treatment; transcriptome sequencing; tumor; tumorigenesis

This resubmitted R03 application seeks to determine how recurrent mutations common in splicing factors (SF) lead to changes in chromatin accessibility and epigenetic landscape, thereby contributing to oncogenesis. Recurrent mutations in SF are prevalent across multiple unrelated cancer types including acute myeloid leukemia, chronic lymphocytic leukemia, lung cancer and melanoma. Among the hundreds of splicing factors, only 4 are commonly mutated: SF3B1, SRSF2, U2AF1 and ZRSR2. SF mutations are mutually exclusive and non-synonymous, suggesting their role as tumor drivers with neomorphic function. Their well-established roles in splicing catalysis have led to the presumption that alternate splicing of tumor suppressors or oncogenes is the primary mechanism driving clonal evolution of mutant cells. While alternate splicing events are demonstrable in RNA-seq datasets, such changes are quite modest and restricted to specific mutational subtypes. This makes a direct role of alternative splicing in oncogenesis questionable. Recent studies have looked at alternate molecular mechanisms of oncogenesis including excess R-loops that arise in response to mutant SF expression. R-loops are 3 stranded structures of two DNA and one RNA molecules formed during transcription. R-loops are critical regulators of chromatin states, and when unchecked can lead to genome instability. These R-loops also cause S-phase arrest and can be rescued by the over-expression of RNAseH. Emerging evidence points to the close coordination between spliceosomal machinery and messenger RNA processing, including its 3' end cleavage and termination. We hence hypothesize that terminal R-loops arise from defective mRNA processing. In the first aim of this proposal, we will determine dynamics of transcription in SF3B1 and U2AF1 mutations through metabolic labeling of nascent RNA (transient transcript sequencing with Timelapse chemistry or TT-TL-seq). Location of RNA Polymerase II in relation to R-loops and stalled DNA replication forks will be determined through proximity ligation assay. The second aim will explore how epigenetic modifiers regulate the formation and resolution of such pathologic R-loops. Our preliminary results using a short hairpin RNA library reveals the role of histone deacetylase pathway in this regulation; hence we will explore the relation between HDAC and R-loops in this aim. Finally, we will determine chromatin accessibility in splicing factor mutant acute myeloid leukemia patient samples, since R-loops and open chromatin are highly correlated. Given the limited scope of the R03 mechanism, the proposal is primarily focused on biochemical studies linking aberrant RNA processing and R-loop formation. If successful, it will form the basis of comprehensive investigations into the mechanisms of oncogenesis conferred by SF mutations utilizing appropriate in vivo models.

该重新提交的R03应用程序旨在确定剪接因子（SF）中常见的突变如何发生 导致染色质的可及性和表观遗传景观的变化，从而导致肿瘤发生。 SF中的复发突变在多种无关的癌症类型中普遍存在，包括急性髓样 白血病，慢性淋巴细胞性白血病，肺癌和黑色素瘤。在数百个剪接因素中， 通常只有4个突变：SF3B1，SRSF2，U2AF1和ZRSR2。 SF突变是相互排斥的，并且 非同义词，表明它们是具有新形态功能的肿瘤驱动因素的作用。他们成熟的角色 在剪接催化中，导致推定肿瘤抑制剂或癌基因的交替剪接是 驱动突变细胞克隆进化的主要机制。而替代拼接事件是 在RNA-seq数据集中证明，此类更改非常适度，并且仅限于特定的突变 亚型。这使替代剪接在肿瘤发生中的直接作用值得怀疑。最近的研究 查看肿瘤发生的替代分子机制，包括响应于 突变的SF表达。 R环是两个DNA的3条结构，在 转录。 R环是染色质状态的关键调节剂，未经检查的何时会导致基因组 不稳定。这些R环也会引起S期停滞，可以通过RNASEH的过表达来挽救。 新兴证据表明，剪​​接体机械与Messenger RNA之间的密切协调 处理，包括其3'端的裂解和终止。因此，我们假设出现终端R环 来自缺陷的mRNA处理。在该提案的第一个目的中，我们将确定转录动力学 在SF3B1和U2AF1突变中，通过新生RNA的代谢标记（瞬态转录测序 及时化学或TT-TL-seq）。 RNA聚合酶II的位置与R环相关并停滞不前 DNA复制叉将通过接近连接测定确定。第二个目标将探索如何 表观遗传修饰符调节这种病理性R环的形成和分辨率。我们的初步结果 使用短发夹RNA库揭示了组蛋白脱乙酰基酶途径在此调节中的作用。因此，我们 将在此目标中探索HDAC与R-loops之间的关系。最后，我们将确定染色质 剪接因子突变体急性髓样白血病患者样品中的可及性，因为R环和开放 染色质高度相关。鉴于R03机制的范围有限，该提案主要是 专注于将异常RNA处理和R环形成联系起来的生化研究。如果成功，它将 构成了对SF赋予的肿瘤发生机制的全面研究的基础 利用合适的体内模型的突变。