Charting the differentiation topology of SF3B1 mutated clonal hematopoiesis (CH) and myelodysplastic syndromes (MDS) via a multi-omics single-cell toolkit

通过多组学单细胞工具包绘制 SF3B1 突变克隆造血 (CH) 和骨髓增生异常综合征 (MDS) 的分化拓扑图

• 批准号: 10366517
• 负责人: Omar Abdel-Wahab
• 金额: $ 69.8万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-15 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10366517
• 关键词: Address; Admixture; Adopted; Affect; Automobile Driving; Bar Codes; Blood Cells; Bone Marrow; Cell physiology; Cell surface; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Characteristics; Chromatin; Clonal Expansion; Complementary DNA; DNA; Data; Development; Disease; Dysmyelopoietic Syndromes; Epigenetic Process; Erythroid; Frequencies; Gene Expression; Genetic; Genetic Transcription; Genomics; Genotype; Growth; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic stem cells; Human; Individual; Knowledge; Lead; Length; Lesion; Link; Mediating; Methods; Minority; Mutate; Mutation; Nature; Patients; Pattern; Phenotype; Play; Population; Protein Isoforms; Proteins; RNA; RNA Splicing; RNA analysis; RNA-Binding Proteins; Recurrence; Resolution; Risk; Role; Sampling; Signal Transduction; Somatic Mutation; Spliceosomes; System; Techniques; Technology; Testing; Trees; Validation; Variant; base; bone cell; cell type; chromatin modification; cohort; driver mutation; hematopoietic differentiation; insight; interest; methylome; mouse model; multiple omics; mutant; mutational status; progenitor; programs; protein expression; self-renewal; single cell technology; single-cell RNA sequencing; transcriptome

SUMMARY Genomic analyses of thousands of MDS patients has established that mutations in RNA splicing factors are the most common class of genetic alterations in patients with MDS. In parallel, functional studies have revealed that several of these genetic lesions appear to drive aberrant hematopoietic self-renewal and differentiation that is characteristic of MDS. Specifically, mutations in splicing factor 3b subunit 1(SF3B1), a core spliceosome component, are among the most common in patients with MDS and lead to incorrect intronic branch point recognition. Despite these advances, our knowledge of the effects of this genetic alteration on downstream gene expression programs within actual disease initiating cells has been hampered by the coexistence of normal wildtype hematopoiesis together with the aberrant clone harboring somatic driver mutations. While some of these limitations are now beginning to be addressed with single cell genomics, performing a layered genomic analysis to simultaneously capture somatic mutations, gene expression, RNA splicing, and chromatin state in single cells has never been performed. Expression of RNA-binding proteins is in turn cell type dependent, necessitating the simultaneously profiling of gene expression, full-length cDNA and SF3B1 mutational status at the single cell level, allowing splicing to be examined in the proper cellular context. To address this challenge, we developed an array of multi-omic single-cell technologies that are capable of capturing multiple layers of information (e.g., genotypes, transcriptomes, methylomes, protein expression) from the same single cells. Moreover, we addressed the specific challenge of genotyping in scRNA-seq in single cells at high throughput by developing Genotyping of Transcriptomes (GoT). Importantly, GoT turns the admixture of mutant and wildtype hematopoiesis from a limitation to an advantage, enabling the direct comparison of mutant and wildtype cells within the same individual. Capitalizing on a unique cohort of bone marrow samples from individuals with MDS and CH, we now aim to apply and extend the multi-omics single-cell toolkit to test define how SF3B1 somatic mutations lead to clonal growth advantage. First, we will perform GoT across MDS and CH samples with canonical SF3B1 driver mutations. We will integrate GoT with Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE- seq) (GoT-CITE), to add the critical layer of cell surface markers to single-cell whole transcriptomes. Second, mutations in splicing factors are specifically associated with greater risk of transformation in CH. Therefore, we will develop and implement GoT-Splice, where long-read sequencing will be used to define splicing variation as a function of cell identity. Third, given the high importance of epigenetic patterning to hematopoietic stem cell identity, we will develop and apply targeted single-cell genotyping in the context of chromatin accessibility (GoT-ChA). This will allow us to unravel the regulatory underpinnings of SF3B1-driven CH and MDS.

概括 成千上万MDS患者的基因组分析已经确定RNA剪接因子中的突变是 MDS患者中最常见的遗传改变。同时，功能研究具有 揭示了这些遗传病变中的几个似乎促进了异常的造血自我更新和 MD的特征的分化。具体而言，剪接因子3B亚基1（SF3B1）中的突变，A 核心剪接体成分，是MDS患者最常见的，导致内含子不正确 分支点识别。尽管取得了这些进步，但我们对这种遗传改变的影响的了解 实际疾病中启动细胞中的下游基因表达程序受到了阻碍 正常野生型造血的共存以及带有躯体驱动器的异常克隆 突变。虽然这些局限性中的一些现在开始使用单细胞基因组学来解决 进行分层的基因组分析以同时捕获体细胞突变，基因表达，RNA 单细胞中的剪接和染色质状态从未进行过。 RNA结合蛋白的表达是 反过来，细胞类型依赖于基因表达，全长cDNA和 SF3B1在单细胞水平上的突变状态，可以在适当的细胞环境中检查剪接。 为了应对这一挑战，我们开发了一系列能够 从中捕获多层信息（例如，基因型，转录组，甲基组，蛋白质表达） 相同的单个单元格。此外，我们解决了单个SCRNA-SEQ中基因分型的具体挑战 通过发展转录组的基因分型（GOT），处于高吞吐量的细胞。重要的是，转弯 从限制到优势的突变体和野生型造血的混合，使直接 在同一个体内的突变体和野生型细胞的比较。 利用来自MDS和CH的个体的独特的骨髓样品，我们现在的目标是 应用并扩展多摩变单细胞工具包，以测试定义SF3B1体细胞突变如何导致克隆 增长优势。首先，我们将使用Canonical SF3B1驱动程序进行MDS和CH样品 突变。我们将通过测序与转录组和表位的细胞索引进行整合（cite- seq）（got-cite），将细胞表面标记的临界层添加到单细胞整个转录组中。第二， 剪接因子中的突变与CH中更大的转化风险特别相关。因此，我们 将开发和实现splice，其中将使用长阅读测序来定义剪接变化 作为细胞身份的函数。第三，鉴于表观遗传模式对造血茎的重要性很高 细胞身份，我们将在染色质可及性的背景下开发并应用目标的单细胞基因分型 （got-cha）。这将使我们能够揭开SF3B1驱动的CH和MD的调节基础。