Role of ASK1 and PKR in Fancc hematopoiesis

描述（由申请人提供）：我们的长期目标是确定范可尼贫血（FA）蛋白在保护造血干/祖细胞（HS/Ps）免于凋亡方面的机制，以便设计用于预防/治疗FA中骨髓衰竭的靶向疗法。80%的FA患者死亡是进行性骨髓衰竭的直接结果。因此，了解FA HS/Ps的凋亡倾向所涉及的机制是至关重要的和临床相关的。目前，关于单个FA蛋白在维持HS/Ps存活中的功能知之甚少。使用小鼠模型的FA型C，我们以前表明，Fancc -/-干细胞有一个显着的减少，在再增殖能力和Fame -/-祖细胞表现出增强的抑制性精氨酸诱导的细胞凋亡。初步数据还显示，Fancc /-祖细胞对氧化剂过敏。我们的中心假设是，通过改变抑制性细胞因子和氧化剂凋亡信号传导，IIS/Ps的丢失在FA-C患者骨髓衰竭的发展中起着至关重要的作用。先前在细胞系中的结构-功能研究证明了2种FANCC功能，可通过FANCC突变体分离。一（1）功能是保护免受遗传毒素，另一个功能是通过抑制双链RNA依赖性激酶（PKR）介导的细胞凋亡来增强IFN-γ/TNF-α处理后的存活率。虽然这些研究开始阐明细胞系中不同的FANCC功能，但一个关键但尚未回答的问题是这2种功能是否在增强原发性HS/Ps的存活中表现出同等重要的作用。此外，我们的初步数据表明，FANCC可能通过凋亡信号调节激酶1（ASK 1）依赖的途径保护细胞免受TNF-α和氧化剂诱导的凋亡。我们假设Fancc -/-细胞表现出改变的ASK 1和PKR凋亡信号传导，这有助于氧化剂或抑制性细胞因子处理后Fancc -/ HS/Ps的促凋亡表型。本申请的目的是，1）确定PKR依赖性和非依赖性FANCC功能是否增强Fancc -/-干细胞再增殖能力并在体内保护免受抑制性细胞因子和遗传毒素处理，2）确定ASK 1是否参与Fancc -/- HS/Ps对凋亡刺激的超敏反应和再增殖能力，和3）研究原代Fancc -/-细胞中的抑制性细胞因子超敏性是否涉及ASK 1和PKR凋亡信号传导的改变。

DESCRIPTION (provided by applicant): Our long-term goal is to identify the mechanism(s) that Fanconi anemia (FA) proteins have in protecting hematopoietic stem/progenitor cells (HS/Ps) from apoptosis in order to design targeted therapies for prevention/treatment of bone marrow failure in FA. 80% of FA patient deaths are a direct result of a progressive marrow failure. Thus, understanding mechanisms involved in the apoptotic predisposition of FA HS/Ps is of critical importance and clinically relevant. Currently, little is known regarding the function(s) that individual FA proteins have in maintaining HS/Ps survival. Using a murine model of FA type C, we previously showed that Fancc -/- stem cells have a marked reduction in repopulating ability and Fame -/- progenitors exhibit enhanced inhibitory cytokine-induced apoptosis. Preliminary data also show that Fancc /- progenitors are hypersensitive to oxidants. Our central hypothesis is that loss of IIS/Ps via altered inhibitory cytokine and oxidant apoptotic signaling has a crucial role in the development of marrow failure in FA-C patients. Previous structure-function studies in cell lines demonstrated 2 FANCC functions, separable by FANCC" mutants. One (1) function was to protect from genotoxins, and the other was to enhance survival after IFN-y/TNF-a treatment by inhibiting double-stranded RNA-dependent kinase (PKR)-mediated apoptosis. While these studies begin to clarify distinct FANCC functions in cell lines, a critical yet unanswered question is whether these 2 functions exhibit equally important roles in enhancing the survival of primary HS/Ps. Furthermore, our preliminary data suggest that FANCC may protect cells from TNF-a and oxidant induced apoptosis through an apoptosis signal-regulating kinase 1 (ASK1) dependent pathway. We hypothesize that Fancc -/- cells exhibit both altered ASK1 and PKR apoptotic signaling, which contribute to the pro-apoptotic phenotype of Fancc -/ HS/Ps after oxidant or inhibitory cytokine treatment. The goals of this application are, 1) to determine whether PKR-dependent and -independent FANCC functions enhance Fancc -/- stem cell repopulating ability and protect from inhibitory cytokine and genotoxin treatment in vivo, 2) to determine whether ASK1 participates in Fancc -/- HS/Ps hypersensitivity to apoptotic stimuli and repopulating ability, and 3) to investigate whether inhibitory cytokine hypersensitivity in primary Fancc -/- cells involves alterations in both ASK1 and PKR apoptotic signaling.