Deciphering the molecular mechanism of ineffective erythropoiesis in MDS-5q

破译MDS-5q无效红细胞生成的分子机制

• 批准号: 10773217
• 负责人: Janis L Abkowitz
• 金额: $ 10万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-21 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10773217
• 关键词: 3-Dimensional; Age; Anemia; Antibodies; Apoptotic; Atherosclerosis; Bar Codes; Biopsy; Blood Cell Count; Bone Marrow; CD14 gene; CD34 gene; CFU-E; Cell Death; Cell Differentiation process; Cell Surface Proteins; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Cessation of life; Chromosome Deletion; Chromosomes; Data; Diagnosis; Diamond-Blackfan anemia; Dysmyelopoietic Syndromes; Elderly; Enzymes; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Ferritin; Funding; Genetic Transcription; Geography; Germ-Line Mutation; Globin; Goals; Health; Heart; Hematopoietic stem cells; Heme; Heme Iron; Human; Impairment; In Vitro; Investigation; Iron; Island; Link; Lung; Macrocytic Anemia; Macrophage; Marrow; Messenger RNA; Methods; Modeling; Molecular; Morbidity - disease rate; Mus; Mutation; Neurologic; Normal Cell; Nurses; Patients; Persons; Phenotype; Process; Pronormoblasts; Protein Biosynthesis; Proteins; Rattus; Reactive Oxygen Species; Recycling; Residual state; Ribosomal Proteins; Risk; Role; Sampling; Site; Somatic Mutation; Structure; System; Testing; Toxic effect; Translations; Umbilical Cord Blood; chelation; chromosome 5q loss; erythroid differentiation; human model; in vitro Model; in vivo; metal transporting protein 1; monocyte; mosaic; neoplastic; neoplastic cell; neurocognitive disorder; older patient; prevent; protein expression; reconstitution; single-cell RNA sequencing; therapeutic evaluation; trafficking; transcriptome; transcriptome sequencing

(PLEASE KEEP IN WORD, DO NOT PDF) Approximately 80% of MDS patients present with anemia, which is the major cause of morbidity. The anemia is generally macrocytic and always reflects ineffective erythropoiesis. However, why erythroid cells die while maturing in the bone marrow is uncertain, prompting this investigation. Especially unclear is why anemia occurs early in MDS (i.e. at presentation) when often 50-75% of the marrow is replaced by neoplastic cells and many normal (non-neoplastic) cells remain. We previously have shown that CFU-E/proerythroblasts die when their intracellular heme (a toxic chelate synthesized enzymatically) exceeds globin (a protein). This can result from failed heme export (Flvcr1-deleted mice) or from ribosomal protein haploinsufficiency, impaired translation, and slowed globin synthesis (MDS-5q and Diamond Blackfan anemia (DBA) patients). Since heme synthesis initiates normally, but globin translation is slowed, heme exceeds the export capacity of FLVCR and induces high levels of ROS and cell death. To delineate the shared mechanisms that lead to the death of maturing MDS-5q and DBA cells erythroid cells, we studied single marrow cells with CITE-seq (antibody barcoding of cell surface proteins) and RNA sequencing. We aligned total transcriptomes with SCVelo pseudotime analysis, which includes incompletely processed mRNAs, and then linked a cell’s surface protein expression to its unique transcriptome. Using this approach in preliminary studies, we showed that all erythroid cells in MDS-5q patient marrow have transcriptional changes implicating heme toxicity. Nearly all marrow erythropoiesis takes place within erythroblastic islands (EBIs), a structure comprised of a central macrophage (“nurse cell”) and ~10-50 maturing red cells. We suspect that the role of the central macrophage is to safely and efficiently recycle heme from CFU-E/proerythroblasts to neighboring iron-avid later erythroid cells. Interestingly, the 5q+ (non-neoplastic) cells are also compromised and fail to expand and reconstitute erythropoiesis. We hypothesize, that the large quantities of heme influx from neoplastic (5q-) erythroid precursors into the central macrophage exceeds that which can be metabolized to iron then stored as ferritin or exported via ferroportin. This compromises the macrophage’s ability to support the maturation of co-adherent normal (5q+) cells, and thus the differentiation of both normal and neoplastic erythroid precursors fail. In order to test this hypothesis we will first show the feasibility and the relevance of using an EBI culture system to model human EBI in this R56-funded study. This would provide the data needed to justify studies of heme-iron trafficking and its impact on red cell differentiation in MDS-5q patients, and perhaps other low and low-intermediate risk MDS patients with disabling anemia.

（请以 WORD 形式保存，请勿以 PDF 形式保存） 大约 80% 的 MDS 患者出现贫血，这是发病的主要原因。贫血通常是大红细胞性贫血，并且总是反映红细胞生成不足。然而，红细胞在骨髓中成熟的过程中死亡的原因尚不清楚，因此这一研究尤其不清楚。这就是为什么 MDS 早期（即就诊时）出现贫血的原因，此时通常 50-75% 的骨髓被肿瘤细胞和许多正常细胞取代我们之前已经证明，当细胞内血红素（一种酶促合成的有毒螯合物）超过球蛋白（一种蛋白质）时，CFU-E/原红细胞就会死亡，这可能是由于血红素输出失败（Flvcr1 缺失的小鼠）造成的。或来自核糖体蛋白单倍体不足、翻译受损和球蛋白合成减慢（MDS-5q 和 Diamond Blackfan 贫血 (DBA)）由于血红素合成正常启动，但球蛋白翻译减慢，血红素超过了 FLVCR 的输出能力，并诱导高水平的 ROS 和细胞死亡，以描述导致成熟 MDS-5q 和 DBA 细胞死亡的共同机制。红系细胞中，我们使用 CITE-seq（细胞表面蛋白抗体条形码）和 RNA 测序研究了单个骨髓细胞，我们使用 SCVelo 伪时间分析对总转录组进行了比对，其中包括不完全分析。处理 mRNA，然后将细胞的表面蛋白表达与其独特的转录组联系起来，在初步研究中使用这种方法，我们发现 MDS-5q 患者骨髓中的所有红细胞都有涉及血红素毒性的转录变化，几乎所有骨髓红细胞生成都发生在成红细胞内。岛（EBI），一种由中央巨噬细胞（“护士细胞”）和约 10-50 个成熟红细胞组成的结构，我们怀疑中央的作用。巨噬细胞的作用是安全有效地将血红素从 CFU-E/原红细胞回收到邻近的嗜铁后来红系细胞，从肿瘤性 (5q-) 红系前体流入中央巨噬细胞的血红素数量超过了可以代谢为铁然后储存为铁的量。这会损害巨噬细胞支持共粘附正常 (5q+) 细胞成熟的能力，因此正常和肿瘤性红系前体细胞的分化都会失败。为了检验这一假设，我们将首先证明其可行性。以及在这项 R56 资助的研究中使用 EBI 培养系统模拟人类 EBI 的相关性，这将为证明血红素铁贩运及其对影响的研究的合理性提供所需的数据。 MDS-5q 患者以及其他患有致残性贫血的低风险和中低风险 MDS 患者的红细胞分化。