The role of Pleckstrin-2 as a functional node in myeloid proliferation

项目概要 骨髓增生性肿瘤（MPN）是一组表现为骨髓细胞过多的骨髓疾病 产生血栓并发展为急性髓性白血病的风险增加。 V617F JAK2 驱动突变是 MPN 的主要原因之一。这种突变的发现导致 开发 JAK 抑制剂来治疗 MPN。然而，JAK 抑制剂没有治疗作用。此外，MPN 使用 JAK 抑制剂治疗的患者经常会出现耐药性和严重的副作用，因为 JAK2 在正常造血过程中发挥着不可或缺的作用。我们一直在研究治疗 MPN 的新方法， 特别关注 JAK2 通路的下游效应器。我们最近发表的研究资助 当前的 R01 揭示了 JAK2-STAT5 途径的新靶点 Pleckstrin-2 (Plek2) 的丢失， 改善 JAK2V617F 诱导的骨髓增殖表型，更重要的是恢复血管 JAK2V617F MPN 小鼠模型的闭塞和致死率。鉴于 Plek2 在 MPN 中的重要性 发病机制，我们已经确定了 Plek2 小分子抑制剂的先导化合物，用于基于硅基的高 通量筛选和基于细胞的测定。我们未发表的小说初步数据进一步表明 Plek2 与多个 PI3K 效应子结合，Plek2 的缺失会降低 Akt 激活。初步的体内证据也 表明 Plek2 对于 PI3K-Akt 通路至关重要，因为 Plek2 敲除可改善 骨髓增殖并显着延长 Pten 造血特异性基因敲除小鼠的生存期。这些 研究结果使我们假设 Plek2 充当连接 JAK2-STAT 和 PI3K- 的中央枢纽。 Akt 通路并促进骨髓增殖。为了验证这一假设并研究其机制 Plek2 的功能，我们将使用 1) 体外生化和分子研究来确定 Plek2 如何增强 PI3K-Akt 信号转导，2) Pten 造血特异性敲除小鼠模型揭示 Plek2 的功能 体内，3) 临床前 MPN 模型和 MPN 患者样本，以确定 Plek2 抑制剂的功效。 该项目的成功完成将为 Plek2 在疾病发病机制中的作用提供新的见解 MPNs 并为使用 Plek2 抑制剂作为单药或联合用药的临床试验奠定基础 其他治疗 MPN 的化合物。

PROJECT SUMMARY Myeloproliferative neoplasms (MPNs) are a group of bone marrow diseases that show excessive myeloid cell production with an increased risk of developing thrombosis and evolving to acute myeloid leukemia. V617F driver mutation of JAK2 is one of the leading causes of MPNs. The discovery of this mutation led to the development of JAK inhibitors to treat MPNs. However, JAK inhibitors are not curative. In addition, MPN patients treated with JAK inhibitor often develop drug resistance and severe side effects due to the indispensable roles of JAK2 in normal hematopoiesis. We have been studying new approaches to treat MPNs, especially focusing on the downstream effectors of the JAK2 pathway. Our recently published studies funded by the current R01 revealed that loss of Pleckstrin-2 (Plek2), a novel target of the JAK2-STAT5 pathway, ameliorated JAK2V617F-induced myeloproliferative phenotypes, and more importantly reverted vascular occlusions and lethality of the JAK2V617F MPN mouse model. Given the significance of Plek2 in MPN pathogenesis, we have identified lead compounds of Plek2 small molecule inhibitors using in silico-based high- throughput screenings and cell-based assays. Our novel unpublished preliminary data further reveal that Plek2 binds to several PI3K effectors and loss of Plek2 reduces Akt activation. Preliminary in vivo evidence also demonstrates that Plek2 is critical for the PI3K-Akt pathway in that Plek2 knockout ameliorated myeloproliferation and significantly extended the survival of Pten hematopoietic specific knockout mice. These findings lead us to hypothesize that Plek2 functions as a central hub to connect the JAK2-STAT and the PI3K- Akt pathways and promote myeloproliferation. To test this hypothesis and investigate the mechanism of function of Plek2, we will use 1) in vitro biochemical and molecular studies to determine how Plek2 enhances the PI3K-Akt signaling, 2) Pten hematopoietic specific knockout mouse model to reveal the functions of Plek2 in vivo, and 3) pre-clinical MPN models and MPN patient samples to establish the efficacy of Plek2 inhibitors. Successful completion of this project will lead to novel insights into the role of Plek2 in the pathogenesis of MPNs and lay the foundation for clinical trials using Plek2 inhibitors as single agents or in combination with other compounds to treat MPNs.