PI3 Kinase Inactivation in Myelodysplastic Syndrome

PI 3激酶对骨髓增生异常综合征自噬的调控 骨髓增生异常综合征（MDS）是一种血液疾病，由骨髓增生异常综合征（MDS）的分子异常驱动。 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。PI 3激酶（PI 3 K）通路被激活， 许多造血细胞因子和生长因子，对红细胞生成很重要。我们 产生了一种新的三重敲除（TKO）小鼠模型，其中PI 3 K基因的缺失 HSC中的Pik 3ca、Pik 3cb和Pik 3cd导致全血细胞减少和骨髓增生异常， HSC分化。我们观察到自噬降解在TKO HSC中受损， 用诱导自噬的药物治疗可以改善HSC的分化。我们假设 PI 3 K的失活使HSC中的自噬降解失调，导致HSC受损， 分化和红细胞生成，其促进MDS。与此一致，我们观察到， 在MDS的一个亚组中，磷酸酶PTEN，其抵消PI 3 K信号传导，被上调 患者为了更直接地说明PI 3 K在红细胞生成中的作用，我们计划分析 TKO; Mx 1-Cre骨髓移植小鼠模型中的红细胞生成 骨髓和脾脏上的CD 71/Ter 119流式细胞术。我们还将生成 TKO;SCL-Cre-ERT小鼠模型，其中PI 3 K基因可以在HSC中缺失， 他莫昔芬给药而不进行移植。在Cre介导的Pik 3ca、Pik 3cb、 和Pik 3cd，我们将分析血细胞计数，骨髓组织学，存活率，并将 对骨髓和脾脏进行CD 71/Ter 119流式细胞术分析红细胞生成。 为了确定失调的自噬是否在人类MDS中起作用，我们建议 检查来自四种不同IPSSR的MDS样品中干细胞区室中的自噬 类别（低、中1、中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马槟榔中的HSC和祖细胞的LC 3 II和P62 培养基，有和没有氯喹处理，以测量自噬通量。此外，我们将 对来自每个MDS样品的⑶ 34+细胞进行定量RT-PCR以用于PTEN表达。我们 还将对PTEN和磷酸化AKT（pAKT）进行Western分析，并将PTEN与 和pAKT水平与相同MDS样品中的自噬诱导。这些实验将 更好地表征由PI 3 K缺失和失调引起的红细胞生成缺陷 在小鼠HSC中的自噬，并将确定PI 3 K失活是否与 人MDS干细胞中受损的自噬

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