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

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.

这项重新提交的R03申请旨在确定剪接因子（SF）中常见的复发突变如何