PI3 Kinase Inactivation in Myelodysplastic Syndrome

骨髓增生异常综合征中的 PI3 激酶失活

基本信息

批准号：
10476196
负责人：
Kira Gritsman
金额：
$ 10万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-17 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10476196
关键词：
1-Phosphatidylinositol 3-Kinase Abnormal Cell Address Alternative Splicing Anemia Animal Model Arginine Autophagocytosis Blood Blood Cells Bone Marrow Bone Marrow Transplantation CD34 gene Categories Cell Compartmentation Cell Line Cell Maturation Cells Chloroquine Clinical Cytokine Signaling Data Data Set Defect Development Dysmyelopoietic Syndromes Dysplasia Engineering Erythroid Erythropoiesis Event Excision Exons Flow Cytometry Gene Expression Profile Genes Genetic Genomic Instability Growth Factor Hematological Disease Hematopoiesis Hematopoietic Hematopoietic stem cells Histology Human IL3 Gene Impairment Knock-out Knockout Mice Lead Link Lipids Measures Mediating Messenger RNA Metformin Modeling Molecular Abnormality Morbidity - disease rate Mus Mutation Myelogenous NUP98 gene Nuclear PIK3CA gene PTEN gene Pancytopenia Pathogenesis Pathway interactions Patients Pattern Pharmaceutical Preparations Pharmacology Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiological Play Process Production Protein Isoforms Protein Kinase Proteins Proto-Oncogene Proteins c-akt Quality Control RNA Splicing RNA-Binding Proteins Regulation Reporting Reverse Transcriptase Polymerase Chain Reaction Role SRSF2 gene Sampling Serine Signal Transduction Spleen Spliceosomes TFRC gene Tamoxifen Testing Transplantation biological adaptation to stress cytokine cytopenia drug candidate erythroid differentiation exon skipping experimental study hematopoietic stem cell differentiation high risk improved inhibitor/antagonist mouse model mutant novel novel therapeutic intervention prevent progenitor stem cell biology stem cell function stem cell genes stem cell therapy stem cells therapeutic target

项目摘要

Regulation of Autophagy by PI3 Kinase in Myelodysplastic Syndrome Myelodysplastic Syndrome (MDS) is a blood disease driven by molecular abnormalities in hematopoietic stem cells (HSCs). Anemia is a common cause of morbidity in MDS patients. However, the mechanistic basis for the impaired HSC differentiation and erythroid maturation that leads to anemia in MDS patients is unclear. The PI3 kinase (PI3K) pathway is activated by many hematopoietic cytokines and growth factors, and is important for erythropoiesis. We generated a new triple knockout (TKO) mouse model in which deletion of the PI3K genes Pik3ca, Pik3cb, and Pik3cd in HSCs causes pancytopenia and myelodysplasia with impaired HSC differentiation. We observed that autophagic degradation is impaired in TKO HSCs, and that treatment with autophagy-inducing drugs improves HSC differentiation. We hypothesize that inactivation of PI3K dysregulates autophagic degradation in HSCs, leading to impaired HSC differentiation and erythropoiesis, which promotes MDS. Consistent with this, we observed that the phosphatase PTEN, which counteracts PI3K signaling, is upregulated in a subset of MDS patients. To more directly address the roles of PI3K in erythropoiesis, we plan to analyze erythropoiesis in the TKO;Mx1-Cre bone marrow transplant mouse model by performing CD71/Ter119 flow cytometry on the bone marrow and spleen. We will also generate the TKO;SCL-Cre-ERT mouse model, in which the PI3K genes can be deleted in HSCs after tamoxifen administration without transplantation. After Cre-mediated excision of Pik3ca, Pik3cb, and Pik3cd in HSCs, we will analyze blood counts, bone marrow histology, survival, and will perform CD71/Ter119 flow cytometry on the bone marrow and spleen to analyze erythropoiesis. To determine whether dysregulated autophagy plays a role in human MDS, we propose to examine autophagy in the stem cell compartment in MDS samples from the four different IPSSR categories (low, intermediate-1, intermediate-2, and high-risk). We will measure autophagy in HSCs and progenitors using intracellular flow cytometry for LC3II and P62 in cytokine-free media, with and without chloroquine treatment to measure autophagic flux. In addition, we will perform quantitative RT-PCR on CD34+ cells for PTEN expression from each MDS sample. We will also perform Western analysis for PTEN and phospho-AKT (pAKT), and will correlate PTEN and pAKT levels with autophagy induction in the same MDS samples. These experiments will better characterize the erythropoiesis defects that result from PI3K deletion and dysregulated autophagy in mouse HSCs, and will determine whether PI3K inactivation correlates with impaired autophagy in human MDS stem cells

骨髓增生异常综合征中 PI3 激酶对自噬的调节骨髓增生异常综合征 (MDS) 是一种由骨髓中的分子异常引起的血液疾病造血干细胞（HSC）。贫血是MDS患者发病的常见原因。然而，HSC 分化和红系成熟受损的机制基础导致 MDS 患者贫血的原因尚不清楚。 PI3 激酶 (PI3K) 通路由以下物质激活许多造血细胞因子和生长因子，对红细胞生成很重要。我们生成了一个新的三重敲除（TKO）小鼠模型，其中 PI3K 基因被删除 HSC 中的 Pik3ca、Pik3cb 和 Pik3cd 导致全血细胞减少和骨髓增生异常，并伴有受损 HSC分化。我们观察到 TKO HSC 中的自噬降解受到损害，并且自噬诱导药物治疗可改善 HSC 分化。我们假设 PI3K 失活会导致 HSC 中的自噬降解失调，导致 HSC 受损分化和红细胞生成，促进MDS。与此相一致的是，我们观察到磷酸酶 PTEN 可以抵消 PI3K 信号传导，在 MDS 的子集中上调患者。为了更直接地阐明 PI3K 在红细胞生成中的作用，我们计划分析 TKO;Mx1-Cre 骨髓移植小鼠模型中的红细胞生成 CD71/Ter119 流式细胞术对骨髓和脾脏的影响。我们还将生成 TKO;SCL-Cre-ERT 小鼠模型，其中 PI3K 基因可以在 HSC 中删除后他莫昔芬给药无需移植。 Cre 介导的 Pik3ca、Pik3cb 切除后，和 HSC 中的 Pik3cd，我们将分析血细胞计数、骨髓组织学、存活率和意志对骨髓和脾脏进行 CD71/Ter119 流式细胞术分析红细胞生成。为了确定自噬失调是否在人类 MDS 中发挥作用，我们建议检查来自四种不同 IPSSR 的 MDS 样本中干细胞区室的自噬类别（低风险、中风险 1、中风险 2 和高风险）。我们将测量自噬使用细胞内流式细胞术检测无细胞因子的 LC3II 和 P62 的 HSC 和祖细胞培养基，经过或未经氯喹处理来测量自噬通量。此外，我们将对 CD34+ 细胞进行定量 RT-PCR，以检测每个 MDS 样本中的 PTEN 表达。我们还将对 PTEN 和磷酸化 AKT (pAKT) 进行 Western 分析，并将 PTEN 关联起来在相同的 MDS 样本中，自噬诱导后的 pAKT 水平和 pAKT 水平。这些实验将更好地表征 PI3K 缺失和失调导致的红细胞生成缺陷小鼠 HSC 中的自噬，并将确定 PI3K 失活是否与人类MDS干细胞自噬受损