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

描述（由申请人提供）：白细胞介素3 （IL-3）是一种多潜能造血生长因子，可激活细胞信号通路，介导骨髓祖细胞的生长和分化。从因子依赖性细胞中去除IL-3可诱导细胞周期阻滞和凋亡。我们的实验室已经发现，细胞应激，包括IL-3从因子依赖性造血细胞、炎症细胞因子、化疗治疗或病毒感染中撤出，通过其细胞激活蛋白RAX促进双链RNA依赖性蛋白激酶PKR的激活。一旦被激活，PKR通过磷酸化eIF2的α亚基来抑制蛋白质合成，从而引发细胞凋亡。值得注意的是，我们的研究结果表明，RAX在丝氨酸18上的磷酸化是PKR激活、翻译抑制和多种细胞应激引发的细胞凋亡所必需的。此外，我们发现了一种新的依赖于RAX的机制来调节肿瘤抑制因子p53在细胞应激期间的转录活性，并发现内源性RAX水平的降低或RAX显性阴性突变体的强制表达将促进异常细胞生长。值得注意的是，我们的初步数据表明，与低风险MDS患者样本或正常造血祖细胞相比，PKR在IPSS高风险患者样本中存在差异定位和激活。综上所述，这些发现表明RAX- PKR应激信号通路可能对维持骨髓细胞的正确组成和启动对外来病原体感染的反应至关重要。我们假设PKR在应激时被RAX激活，促进eIF2磷酸化和p53激活，从而导致翻译抑制、基因表达改变、细胞周期阻滞和细胞凋亡。因此，在某些情况下，抑制或激活PKR活性可能在治疗上有用。具体来说，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，而抑制PKR活性的小分子可能有助于治疗与骨髓祖细胞异常凋亡相关的激活PKR水平升高的造血疾病，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信号转导在造血细胞细胞应激后调控细胞周期和启动细胞凋亡的机制，2)确定PKR在MDS进展为急性白血病中的意义。在这些研究中，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，该模型忠实地模拟了在人类中观察到的MDS和AML进展。通过实现这些特定目标，我们将获得新的重要见解，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：本研究产生的结果将增加我们对PKR促进细胞应激反应的分子机制和PKR在MDS向AML进化中的作用的理解。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.