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

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

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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

PI3激酶在骨髓增生综合征中对自噬的调节骨髓增生综合征（MDS）是由分子异常驱动的血液疾病造血干细胞（HSC）。贫血是MDS患者发病率的常见原因。但是，HSC分化和红系成熟的机械基础导致MDS患者贫血的情况尚不清楚。 PI3激酶（PI3K）途径被激活许多造血细胞因子和生长因子，对促红细胞生成很重要。我们生成了一个新的三重敲除（TKO）小鼠模型，其中删除了PI3K基因的删除 HSC中的PIK3CA，PIK3CB和PIK3CD会导致全年型和骨髓增生性，受损 HSC分化。我们观察到自噬降解在TKO HSC中受到损害，并且用自噬诱导药物治疗可改善HSC分化。我们假设 PI3K的失活失调会在HSC中自噬降解，从而导致HSC受损促进MD的分化和红细胞生成。与此一致，我们观察到磷酸酶PTEN抵抗PI3K信号传导，在MDS的子集中被上调患者。为了更直接地解决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骨髓和脾脏上的流式细胞仪。我们还将生成 tko; scl-cre-ert小鼠模型，其中可以在HSC中删除PI3K基因他莫昔芬给药没有移植。在Cre介导的PIK3CA切除后，Pik3CB，和HSC中的PIK3CD，我们将分析血液计数，骨髓组织学，生存和将会分析在骨髓和脾脏上执行CD71/TER119流式细胞仪，以分析红细胞生成。为了确定失调的自噬是否在人类MD中起作用，我们建议检查来自四种不同IPSSR的MDS样品中的干细胞室中的自噬类别（低，中级1，中级2和高风险）。我们将测量自噬 HSC和祖细胞使用LC3II的细胞内流式细胞术和无细胞因子中的p62 培养基，有和没有氯喹治疗以测量自噬通量。此外，我们将对CD34+细胞进行定量RT-PCR，以从每个MDS样品中进行PTEN表达。我们还将对PTEN和PTEN-AKT（PAKT）进行西方分析，并将与PTEN相关在同一MDS样品中具有自噬诱导的PAKT水平。这些实验会更好地表征由PI3K缺失和失调而导致的红细胞生成缺陷小鼠HSC中的自噬，将确定PI3K失活是否与人类MDS干细胞自噬受损