Inosine monophosphate dehydrogenase (IMPDH) catalyzes the first step in the de novo synthesis of guanine nucleotides and this enzyme activity is essential for cell proliferation. IMPDH inhibitors are in current use and under development as immunosuppressive agents and have potential for the treatment of hematologic malignancies. It is the overall goal of this proposal to define the mechanisms by which loss of IMPDH activity and depletion of guanine nucleotides inhibit immune responses and result in apoptosis in leukemic cells. To meet the first objective, IMPDH type II will be selectively targeted for homozygous deletion in immature T cells of a mouse model using a Cre-Lox system. The effects of IMPDH II deficiency on T cell maturation and function will be examined. Particular attention will be paid to the development of the CD4 Th2 subpopulation and to the function of CD8 cytolytic T lymphocytes, both of which are mildly impaired with partial IMPDH inactivation. The effects of IMPDH II loss on thymocyte transformation by the large T antigen of SV40 virus will also be determined. Based on evidence that inhibition of IMPDH results in phosphorylation of the IMPDH protein and dramatic alterations in its intracellular localization, we will define the phosphorylation site(s) and determine whether phosphorylation is responsible for protein targeting by mutating each site. The effect of protein phosphorylation on inhibition of IMPDH activity and on its intracellular distribution will be determined. The ability of IMPDH II and its post-translationally modified form to bind to intracellular proteins will be examined and those proteins identified to more fully understand the role of IMPDH in cell physiology. Finally, the effects of guanine nucleotide depletion on intracellular signaling molecules of the Ras-Raf-MAPK cascade and on the mTOR pathway will be determined. Constitutively active intermediates of these pathways will be used in an attempt to define the molecular mechanism by which guanine nucleotide depletion is """"""""sensed"""""""" by malignant hematopoietic cells, leading to inhibition of cell cycle progression and/or apoptosis. The susceptibility of human and murine leukemic cell lines to apoptosis through IMPDH inhibition will be determined and the effects of combinations of IMPDH inhibitors with other signal transduction inhibitors, including farnesyltransferase inhibitors, will be examined. These studies should (1) greatly improve our understanding of the molecular sequelae of guanine nucleotide depletion for T lymphocyte development and for cellular biology in general, and (2) expand the indications for use of IMPDH inhibitors into the treatment of malignant disease.
Showing the most recent 10 out of 14 publications
