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.

（请保持言语，不要PDF） 大约80％的MDS患者患有贫血，这是发病率的主要原因。贫血通常是大细胞，并且总是反映出无效的红细胞生成。但是，为什么在骨髓中交​​配时红细胞细胞死亡尚不确定，从而促使这项投资。尤其不清楚的是，为什么在50-75％的骨髓被肿瘤细胞代替，而许多正常（非塑性）细胞仍然存在时，贫血发生在MDS早期（即出现）。我们以前已经表明，当CFU-E/Prothrosblast会在细胞内血红素（合成的有毒樱桃）超过球蛋白（蛋白质）时死亡。这可能是由于血红素出口失败（FLVCR1缺失的小鼠）或核糖体蛋白质单倍氨酸弥倍不足，翻译受损以及球蛋白合成缓慢（MDS-5Q和Diamond Blackfan贫血（DBA）患者）。由于血红素合成正常启动，但球蛋白的翻译速度很慢，因此血红素超过了FLVCR的出口能力，并诱导高水平的ROS和细胞死亡。为了描述导致MDS-5Q和DBA细胞红细胞死亡的共享机制，我们研究了用Cite-Seq（细胞表面蛋白的抗体条形码）和RNA测序研究单骨髓细胞。我们将总转录组与Scvelo伪次分析一致，其中包括未完全处理的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，我们表明MDS-5Q患者骨髓中的所有红细胞细胞都具有转录变化，隐含的血红素毒性。几乎所有的骨髓红细胞生成都发生在红细胞岛（EBIS）内，该结构完成了中央巨噬细胞（“护士细胞”）和约10-50个成熟的红细胞。中央巨噬细胞的作用的前景是将血红素安全地从CFU-E/PRORERERERETHROMBLASTs循环到邻近的Avid-Avid后来的红细胞。有趣的是，5q+（非塑性）细胞也受到损害，无法扩展和重新构成红细胞生成。我们假设，在中央巨噬细胞中肿瘤（5q-）红系前体的大量血红素影响超过了可以代谢为铁，然后以铁蛋白或铁pi以铁蛋白导出的铁的影响。这损害了巨噬细胞支持共遵守正常（5q+）细胞成熟的能力，因此巨噬细胞的分化均分化。为了检验这一假设，我们将首先显示在这项R56资助的研究中使用EBI培养系统对人EBI进行建模的可行性和相关性。这将提供所需的数据，以证明对MDS-5Q患者以及其他低和低中间风险的MDS MDS患者对红细胞分化的影响及其对红细胞分化的影响是合理的。