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.