p38 MAPK--Therapeutic target in Myelodysplastic syndrome

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计数下降。我们已经表明，p38 MARK通路在MDS骨髓中被组成性激活，并在造血祖细胞中观察到的细胞凋亡增加中起作用。最重要的是，p38的药理学抑制导致来自原代MDS CD 34+造血祖细胞的造血集落形成增加，并导致体外多种MDS亚型的造血增强。该建议将定义p38在MDS中发挥的病理生理作用，并将确定其分子和细胞靶点。 具体目标1将确定组成性p38激活是否导致MDS中无效的造血。 我们将选择性抑制MDS骨髓CD 34+细胞中各种p38亚型（α、β、γ、δ）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亚型特异性显性失活突变体的过表达对造血祖细胞生长的影响。观察到的效果将与各种MDS亚型和临床特征相关。具体目标2将研究MDS中p38组成性激活的机制并鉴定其下游效应物。将使用生物化学、免疫组织化学和流式细胞术方法来检查骨髓来源的造血祖细胞中推定的上游和下游效应物的活化。这些将包括评价小G蛋白Rac 1和上游Map激酶激酶MKK 3、MKK 6和MKK 4的活化状态，以及下游效应物MapKapK-2、MapKapK-3和Msk 1的活化。发现组成型激活的激酶的siRNA介导的敲低随后将用于确定它们中的每一个在MDS造血中的功能相关性。骨髓微环境也可以通过参与细胞因子分泌而促进MDS的病理生理学。因此，具体目标3是检查p38 MARK的活化是否介导MDS骨髓中骨髓抑制细胞因子的过度产生。我们将研究p38抑制剂是否通过浸润MDS患者骨髓中的巨噬细胞和淋巴细胞来抑制TNF α和IFN γ的过度产生。我们还将评估MDS骨髓源性基质细胞与正常基质细胞相比的IL-6和VEGF产生，并评估p38抑制剂对这种产生的影响。总之，这些研究为p38 MARK通路在MDS发病机制中的作用提供了有价值的信息。此外，这些研究的结果应具有直接的临床转化相关性，并导致开发用于治疗MDS的新治疗方法，包括使用临床相关的p38通路药理学抑制剂（如Scio-469）的临床试验。

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.