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.

(请保存在Word中，不要保存为PDF) 大约80%的MDS患者患有贫血，这是发病率的主要原因。贫血通常是巨核细胞增多，总是反映无效的红细胞生成。然而，红系细胞在骨髓中成熟时死亡的原因尚不清楚，这促使了这项研究。尤其不清楚的是，为什么在MDS早期(即发病时)会出现贫血，通常50-75%的骨髓被肿瘤细胞取代，而许多正常(非肿瘤)细胞仍然存在。我们之前已经证明，当CFU-E/前红细胞的细胞内血红素(一种酶合成的有毒螯合物)超过珠蛋白(一种蛋白质)时，它们就会死亡。这可能是由于血红素输出失败(Flvcr1缺失的小鼠)或核糖体蛋白单倍体不足、翻译受损和珠蛋白合成减慢(MDS-5q和钻石黑扇贫血(DBA)患者)。由于血红素合成正常，但珠蛋白翻译速度减慢，导致血红素超出FLVCR的输出能力，导致高水平的ROS和细胞死亡。为了阐明导致成熟的MDS-5q细胞和DBA细胞红系细胞死亡的共同机制，我们用CITE-SEQ(细胞表面蛋白抗体条码)和RNA测序对单个骨髓细胞进行了研究。我们将总转录本与SCVelo伪时间分析进行了比对，其中包括未完全处理的mRNAs，然后将细胞表面蛋白的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系细胞都有转录变化，这与血红素毒性有关。几乎所有的骨髓红细胞生成都发生在红母细胞岛(EBI)内，这是一种由中央巨噬细胞(“哺乳细胞”)和~10-50个成熟红细胞组成的结构。我们怀疑中央巨噬细胞的作用是安全和有效地将来自CFU-E/前红细胞的血红素循环到邻近的嗜铁的后来的红系细胞。有趣的是，5q+(非肿瘤)细胞也受到损害，无法扩大和重建红细胞生成。我们假设，从肿瘤(5q-)红系前体细胞流入中央巨噬细胞的大量血红素超过了可以代谢为铁然后以铁蛋白的形式储存或通过铁蛋白输出的量。这损害了巨噬细胞支持共贴附的正常(5q+)细胞成熟的能力，因此正常和肿瘤红系前体细胞的分化都失败。为了验证这一假设，我们将首先在这项由R56资助的研究中展示使用EBI培养系统来模拟人类EBI的可行性和相关性。这将提供必要的数据，以证明在MDS-5Q患者以及其他患有致残性贫血的低风险和低中风险MDS患者中，关于血红素-铁运输及其对红细胞分化的影响的研究是合理的。