p38 MAPK as a therapeutic target in Myelodysplastic syn*

p38 MAPK 作为骨髓增生异常综合征的治疗靶点*

• 批准号: 7127625
• 负责人: Amit K. Verma
• 金额: $ 39.5万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7127625
• 关键词: G protein; SDS polyacrylamide gel electrophoresis; apoptosis; bone marrow; cell growth regulation; clinical research; dyserythropoietic anemia; flow cytometry; fluorescent in situ hybridization; hematopoiesis; human subject; immunocytochemistry; immunoprecipitation; interferon gamma; interleukin 6; leukocyte activation /transformation; mitogen activated protein kinase; pathologic process; phosphorylation; protein isoforms; protein structure function; small interfering RNA; stem cells; transfection; tumor necrosis factor alpha; vascular endothelial growth factors

DESCRIPTION (provided by applicant): Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis and decreased blood counts. We have shown that the p38 MARK pathway is constitutively activated in MDS bone marrows and plays a role in the increased apoptosis seen in hematopoietic progenitors. Most importantly, pharmacological inhibition of p38 leads to increased hematopoietic colony formation from primary MDS CD34+ hematopoietic progenitors and leads to enhanced hematopoiesis in a variety of MDS subtypes in vitro. This proposal will define the pathophysiological role that p38 plays in MDS, and will identify its molecular and cellular targets. Specific Aim 1 will determine whether constitutive p38 activation results in ineffective hematopoiesis in MDS. We will selectively inhibit the expression of the various p38 isoforms (alpha, beta, gamma, delta) in CD34+ cells from MDS bone marrows and examine the effects of such inhibition on hematopoietic progenitor colony formation and apoptosis. The effects of adenoviral-mediated overexpression of various isoform-specific dominant negative mutants on hematopoietic progenitor cell growth will also be assessed. The effects seen will be correlated with various MDS subtypes and clinical characteristics. Specific Aim 2 will study the mechanisms of constitutive activation of p38 in MDS and identify its downstream effectors. Biochemical, immunohistochemical and flow cytometric methodologies will be used to examine the activation of putative upstream and downstream effectors in bone marrow-derived hematopoietic progenitors. These will include evaluation of the activation status of the small G-protein Rac1 and the upstream Map kinase kinases, MKK3, MKK6 and MKK4, as well as the activation of the downstream effectors MapKapK-2, MapKapK-3, and Msk1. siRNA-mediated knockdown of kinases found constitutively activated will be subsequently used to determine the functional relevance of each one of them in MDS hematopoiesis. Bone marrow microenvironment can also contribute to the pathophysiology of MDS by being involved in cytokine secretion. Thus, Specific Aim 3 is to examine whether activation of p38 MARK mediates overproduction of myelosuppressive cytokines in MDS bone marrows. We will examine whether p38 inhibitors suppress the overproduction of TNFalpha and IFNgamma by infiltrating macrophages and lymphocytes in the bone marrows of MDS patients. We will also evaluate IL-6 and VEGF production by MDS marrow derived stromal cells as compared to normal stromal cells and assess the effects of p38 inhibitors on such production. Altogether, these studies should provide valuable information on the role of p38 MARK pathway in the pathogenesis of MDS. Moreover, the results of these studies should be of direct clinical-translational relevance and lead to the development of novel therapeutic approaches for the treatment of MDS, including clinical trials with clinically relevant pharmacological inhibitors of p38 pathway such as SCIO-469.

描述（由申请人提供）： 骨髓增生异常综合征 (MDS) 的特点是无效造血和血细胞计数减少。我们已经证明，p38 MARK 通路在 MDS 骨髓中被组成型激活，并在造血祖细胞中观察到的细胞凋亡增加中发挥作用。最重要的是，p38的药理学抑制导致原代MDS CD34+造血祖细胞的造血集落形成增加，并导致多种MDS亚型的体外造血能力增强。该提案将定义 p38 在 MDS 中发挥的病理生理学作用，并将确定其分子和细胞靶标。 具体目标 1 将确定组成型 p38 激活是否会导致 MDS 造血无效。 我们将选择性抑制来自 MDS 骨髓的 CD34+ 细胞中各种 p38 亚型（α、β、γ、δ）的表达，并检查这种抑制对造血祖细胞集落形成和凋亡的影响。还将评估腺病毒介导的各种亚型特异性显性失活突变体的过度表达对造血祖细胞生长的影响。所观察到的影响将与各种 MDS 亚型和临床特征相关。具体目标 2 将研究 MDS 中 p38 组成型激活的机制并确定其下游效应器。生物化学、免疫组织化学和流式细胞术方法将用于检查骨髓来源的造血祖细胞中假定的上游和下游效应器的激活。这些将包括评估小 G 蛋白 Rac1 和上游 Map 激酶激酶 MKK3、MKK6 和 MKK4 的激活状态，以及下游效应器 MapKapK-2、MapKapK-3 和 Msk1 的激活。 siRNA 介导的激酶敲低发现组成型激活，随后将用于确定它们中的每一种在 MDS 造血中的功能相关性。骨髓微环境还可以通过参与细胞因子分泌来促进MDS的病理生理学。因此，具体目标 3 是检查 p38 MARK 的激活是否介导 MDS 骨髓中骨髓抑制细胞因子的过量产生。我们将检查p38抑制剂是否通过浸润MDS患者骨髓中的巨噬细胞和淋巴细胞来抑制TNFα和IFNγ的过量产生。我们还将评估 MDS 骨髓来源的基质细胞与正常基质细胞相比的 IL-6 和 VEGF 产生，并评估 p38 抑制剂对此类产生的影响。总而言之，这些研究应该为 p38 MARK 通路在 MDS 发病机制中的作用提供有价值的信息。此外，这些研究的结果应该具有直接的临床转化相关性，并导致开发治疗 MDS 的新治疗方法，包括使用 p38 途径的临床相关药理学抑制剂（例如 SCIO-469）进行的临床试验。