The Role of PKR in a Novel IL-3 Signal Transduction Pathway

说明(申请人提供)：白介素3(IL-3)是一种多潜能的造血生长因子，能激活细胞信号通路，调节骨髓前体细胞的生长和分化。从依赖因子的细胞中去除IL-3会诱导细胞周期停滞和细胞凋亡。我们的实验室发现，细胞应激，包括IL-3从因子依赖的造血细胞中撤出、炎性细胞因子、化疗或病毒感染，都会通过其细胞激活蛋白RAX促进双链RNA依赖的蛋白激酶PKR的激活。一旦被激活，PKR通过磷酸化eIF2的阿尔法亚单位来抑制蛋白质的合成，从而启动细胞凋亡。值得注意的是，我们的发现表明，丝氨酸18上的Rax磷酸化是由不同的细胞应激启动的PKR激活、翻译抑制和凋亡所必需的。此外，我们发现了一种新的RAX依赖机制，用于调节细胞应激过程中抑癌基因P53的转录活性，并发现内源性RAX水平的降低或显性负性RAX突变体的强制表达将促进细胞的异常生长。值得注意的是，我们的初步数据表明，与低危MDS患者样本或正常造血祖细胞相比，PKR在IPSS高危患者样本中有不同的定位和激活。综上所述，这些发现表明，Rax-PKR应激信号通路可能是维持正确的骨髓细胞组成和启动对外来病原体感染的反应的关键。我们推测，在应激过程中，PKR被RAX激活，从而促进eIF2的磷酸化和P53的激活，从而导致翻译抑制、基因表达变化、细胞周期停滞和细胞凋亡。因此，在某些情况下，抑制或激活PKR活性可能在治疗上有用。具体地说，PKR激活剂可能有助于促进PKR水平降低的白血病细胞的凋亡，而能够抑制PKR活性的小分子可能有助于治疗与骨髓前体细胞异常凋亡相关的活化PKR水平增加的造血疾病，例如进展为AML的范可尼贫血(FA)和骨髓增生异常综合征(MDS)。为了验证这些假设，我们将1)确定RAX-PKR信号转导调节细胞周期并在细胞应激后启动造血细胞凋亡的机制(S)；2)确定PKR在MDS进展为急性白血病中的意义。对于这些研究，我们将使用分子生化和细胞培养方法，并使用NUP98-Hoxd13转基因小鼠模型，该模型忠实地模拟在人类中观察到的MDS和进展为AML。通过实现这些特定的目标，我们将获得新的和重要的见解，填补关于细胞应激如何激活Rax-PKR信号轴以调节细胞生长和凋亡的知识空白。一种治疗MDS和AML患者的新治疗策略有望成为结果。公共卫生相关性：这项研究的结果将增加我们对PKR促进细胞应激反应的分子机制(S)以及PKR在MDS向AML演变中的作用的理解。研究结果将为发现新的抗白血病疗法指明方向并提供基础。

DESCRIPTION (provided by applicant): Interleukin 3 (IL-3) is a multi-potential hematopoietic growth factor that activates cellular signaling pathways to mediate growth and differentiation of bone marrow progenitor cells. Removal of IL-3 from factor- dependent cells induces cell cycle arrest and apoptosis. Our laboratory has discovered that cellular stresses, including IL-3 withdrawal from factor-dependent hematopoietic cells, inflammatory cytokines, chemotherapy treatment or viral infection promotes activation of the double-stranded RNA dependent protein kinase, PKR, by its cellular activator protein, RAX. Once activated, PKR inhibits protein synthesis by phosphorylating the alpha subunit of eIF2 to initiate apoptosis. Significantly, our findings reveal that RAX phosphorylation on serine 18 is required for PKR activation, translation inhibition and apoptosis initiated by diverse cellular stresses. In addition, we identified a novel RAX-dependent mechanism for regulation of tumor suppressor p53 transcriptional activity during cellular stress and discovered that reduced levels of endogenous RAX or forced expression of a dominant negative RAX mutant will promote aberrant cell growth. Significantly, our preliminary data indicate that PKR is differentially localized and activated in IPSS high risk compared to low risk MDS patient samples or normal hematopoietic progenitors. Taken together these findings suggest that the RAX- PKR stress signaling pathway may be critical for maintaining the correct composition of bone marrow cells and for initiating the response to infection from foreign agents. We hypothesize that PKR is activated by RAX during stress to promote both eIF2 phosphorylation and p53 activation that leads to translation inhibition, changes in gene expression, cell cycle arrest and apoptosis. Thus, inhibition or activation of PKR activity may be therapeutically useful in certain conditions. Specifically, PKR activators may be useful for promoting apoptosis of leukemic cells that have reduced levels of PKR, while small molecules that can inhibit PKR activity may be useful for treating hematopoietic disorders that display increased levels of activated PKR associated with aberrant apoptosis of bone marrow progenitor cells, such as in Fanconi anemia (FA) and Myelodysplastic Syndrome (MDS) that progress to AML. To test these hypotheses we will 1) determine the mechanism(s) by which RAX-PKR signal transduction regulates the cell cycle and initiates apoptosis following cellular stress in hematopoietic cells and 2) determine the significance of PKR in MDS progression to acute leukemia. For these studies we will use molecular biochemical and cell culture approaches and make use of the NUP98- HOXD13 transgenic mouse model that faithfully mimics MDS and progression to AML observed in humans. By achieving these specific aims we will gain new and important insights that fill knowledge gaps about how cellular stress activates the RAX-PKR signaling axis to regulate cell growth and apoptosis. A novel therapeutic strategy for treating patients with MDS and AML is expected to be the outcome. PUBLIC HEALTH RELEVANCE: The results generated by this research will increase our understanding of both the molecular mechanism(s) by which PKR promotes the cellular stress response and PKR's role in MDS evolution to AML. Results will point the way to and provide a basis for discovering novel anti-leukemia therapies.