Investigating the role of the JAK/STAT3 Pathway in Clonal Proliferation and Immune Dysfunction in Myelodysplastic Syndrome

项目摘要 骨髓增生异常综合征（MDS）是一组异质性白血病前骨髓衰竭疾病， 在美国，每年的发病率约为13，000例。约三分之一的MDS患者将继续 发展为急性髓性白血病（AML），存活率为5- 10%。标准治疗仅限于 在需要治疗的患者中，平均寿命为9个月，并且在干细胞外无法治愈疾病 细胞移植随着人口老龄化，这种疾病只会变得更加普遍，因此迫切需要 需要更有效的治疗MDS。MDS发病机制的特点是克隆性骨髓增生 和骨髓免疫功能障碍。在MDS骨髓中，抑制性免疫微环境 在最初的炎症反应中发展，并有助于发展中的胚细胞的增殖， 不受免疫系统的控制而自由生长。事实上，髓源性抑制细胞（MDSC）， 其抑制T细胞功能，与疾病的发病机制和进展有关。的 JAK/STAT 3通路在髓样分化中是关键的，并且在成髓细胞和成纤维细胞中被异常激活。 MDSC，两者都随着MDS进展而增加。为了更好地理解MDS中的JAK/STAT 3信号传导， 我们将使用单细胞和功能基因组分析来分析原发性患者样本中的STAT 3， 重点关注成髓细胞和MDSC-MDS白血病进展中两种独立但重要的细胞类型。给定 根据我们在AML中研究单细胞表型和细胞内信号传导的经验，我们将调整我们的方法， 研究MDS，特别关注p-STAT 3。这种方法的一个优点是能够测量所有细胞 并为每个样品创建单细胞信号传导谱，并响应于 抑制作用我们提出了三个具体的目标来研究JAK/STAT 3信号在MDS。在目标I中，基底和 将在储存的MDS样品中测量精氨酸诱导的STAT 3信号传导应答，以了解其在MDS中的作用。 STAT 3信号在白血病进展中的作用。Aim II将测试对各种 JAK/STAT 3通路的抑制剂。最后，目标III将确定标准治疗对JAK/STAT 3的影响。 信号转导和STAT 3靶点。染色质免疫沉淀（ChIP-seq）将在这里使用， 目的研究STAT 3在MDS中的靶基因。这些研究将大大有助于我们的基础 对MDS的理解和JAK/STAT 3通路在这种疾病中的治疗潜力。 该提案的最终目的是让Ferrell博士进一步发展质谱细胞术、磷酸化细胞计数和细胞计数技术。 流动，免疫学技术和ChIP，并为他提供必要的关键指导和培训， 成为一名独立的科学家。

Project Summary Myelodysplastic syndrome (MDS) is a heterogeneous group of pre-leukemic bone marrow failure disorders with a yearly incidence of approximately 13,000 in the U.S. Approximately one third of MDS patients will go on to develop acute myeloid leukemia (AML) with a dismal survival of 5-10%. Standard of care therapy only extends life by a median of 9 months in those requiring treatment and there is no cure for the disease outside of stem cell transplant. As the population ages, this disease will only become more common, thus there is an urgent need for more effective treatments in MDS. MDS pathogenesis is marked by both clonal myeloid proliferation and bone marrow immune dysfunction. In MDS bone marrow, a suppressive immune microenvironment develops in response to initial inflammation and contributes to the proliferation of developing blasts, which are free to grow unchecked by the immune system. In fact, myeloid-derived suppressor cells (MDSCs), myeloid cells which are suppressive to T cell function, have been implicated in disease pathogenesis and progression. The JAK/STAT3 pathway is critical in myeloid differentiation and is aberrantly activated in both myeloblasts and MDSCs, both of which increase as MDS progresses. In order to better understand JAK/STAT3 signaling in MDS, we will use single cell and functional genomic assays to profile STAT3 in primary patient samples, with particular focus on myeloblasts and MDSCs – two separate but central cells types in leukemic progression of MDS. Given our experience studying single cell phenotype and intracellular signaling in AML, we will adapt our methods to study MDS, with particular focus on p-STAT3. An advantage of this approach is the ability to measure all cells within a sample simultaneously and create single cell signaling profiles for each sample and in response to inhibition. We propose three specific aims to investigate JAK/STAT3 signaling in MDS. In Aim I, the basal and cytokine-induced responses of STAT3 signaling will be measured in banked MDS samples to understand the role of STAT3 signaling in leukemic progression. Aim II will test functional and signaling responses to various inhibitors of the JAK/STAT3 pathway. Finally, Aim III will determine the impact of standard therapy on JAK/STAT3 signaling and STAT3 targets in resistant MDS. Chromatin immunoprecipitation (ChIP-seq) will be used here in order to profile the gene targets of STAT3 in MDS. These studies will significantly contribute to our basic understanding of MDS and the therapeutic potential of the JAK/STAT3 pathway moving forward in this disease. This proposal is ultimately designed to allow Dr. Ferrell to develop further skills in mass cytometry, phospho- flow, immunology techniques and ChIP and to provide him with the critical mentoring and training necessary to become an independent physician-scientist.