p38 MAPK as a therapeutic target in Myelodysplastic syn*

描述(由申请人提供)： 骨髓增生异常综合征(MDS)的特征是无效的造血和血细胞减少。我们已经证明，p38Mark通路在MDS骨髓中被结构性激活，并在造血祖细胞中增加的凋亡中发挥作用。最重要的是，对p38的药理抑制可增加原代MDS CD34+造血祖细胞的造血祖细胞集落形成，并在体外增强多种MDS亚型的造血力。这项提议将定义p38在MDS中所起的病理生理学作用，并将确定其分子和细胞靶点。具体目标1将确定结构性p38激活是否会导致MDS的无效造血。我们将选择性地抑制MDS骨髓CD34+细胞中各种p38亚型(α、β、γ、Delta)的表达，并检测这种抑制对造血祖细胞集落形成和细胞凋亡的影响。还将评估腺病毒介导的各种异构体特异性显性负性突变体对造血祖细胞生长的影响。所看到的效果将与不同的MDS亚型和临床特征相关。具体目标2将研究p38在MDS中结构性激活的机制，并确定其下游效应因子。生物化学、免疫组织化学和流式细胞术方法将被用来检测骨髓来源的造血祖细胞中可能的上游和下游效应因子的激活。这些将包括评估小G蛋白rac1和上游MAPKK3、MKK6和MKK4的激活状态，以及下游效应器MapKapK-2、MapKapK-3和Msk1的激活。随后，将使用siRNA介导的对被发现的结构性激活的激酶的敲除来确定它们中的每一个在MDS造血中的功能相关性。骨髓微环境也可能通过参与细胞因子的分泌参与MDS的病理生理过程。因此，特定的目的3是检查p38Mark的激活是否介导了MDS骨髓中骨髓抑制细胞因子的过度产生。我们将研究p38抑制剂是否通过渗透MDS患者骨髓中的巨噬细胞和淋巴细胞来抑制TNFpha和IFNGamma的过度产生。我们还将评估MDS骨髓基质细胞与正常基质细胞相比产生IL-6和VEGF的情况，并评估p38抑制剂对这种产生的影响。综上所述，这些研究将为p38Mark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-翻译相关性，并导致开发治疗MDS的新的治疗方法，包括临床相关的p38途径药物抑制剂的临床试验，如SCIO-469。

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.