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Fanconi贫血（FA）蛋白保护造血干细胞/祖细胞（HS/Ps）免于凋亡的机制，以便设计靶向治疗FA骨髓衰竭的预防/治疗。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细胞凋亡易感性的机制具有重要的临床意义。目前，关于单个FA蛋白在维持HS/Ps存活中的功能知之甚少。利用小鼠FA C型模型，我们之前发现，Fancc -/-干细胞的再生能力显著降低，Fame -/-祖细胞表现出增强的抑制性细胞因子诱导的凋亡。初步数据还表明，Fancc /-祖细胞对氧化剂过敏。我们的中心假设是，IIS/Ps通过抑制细胞因子和氧化凋亡信号的改变而丧失，在FA-C患者骨髓衰竭的发展中起着至关重要的作用。先前的细胞系结构-功能研究显示了2种FANCC功能，可通过FANCC突变体分离。一(1)功能是保护免受基因毒素的侵害，另一个功能是通过抑制双链rna依赖性激酶（PKR）介导的细胞凋亡来提高IFN-y/TNF-a治疗后的存活率。虽然这些研究开始阐明FANCC在细胞系中的不同功能，但一个关键但尚未解决的问题是，这两种功能是否在提高原发性HS/Ps的存活中发挥同样重要的作用。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 （ASK1）依赖途径保护细胞免受TNF-a和氧化剂诱导的凋亡。我们假设Fancc -/-细胞表现出ASK1和PKR凋亡信号的改变，这有助于Fancc -/ HS/Ps在氧化或抑制性细胞因子处理后的促凋亡表型。本应用的目的是：1)确定pkr依赖和不依赖的FANCC功能是否增强了FANCC -/-干细胞的再生能力，并在体内保护FANCC -/-干细胞免受抑制性细胞因子和基因毒素的处理；2)确定ASK1是否参与FANCC -/- HS/Ps对凋亡刺激的超敏反应和再生能力。3)研究原代Fancc -/-细胞的抑制性细胞因子超敏是否涉及ASK1和PKR凋亡信号的改变。

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.