Role of ASK1 and PKR in Fancc hematopoiesis

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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.