4864

Inosine monophosphate dehydrogenase (IMPDH) is a pivotal enzyme in the de novo pathway of guanine nucleotide biosynthesis. Inhibitors of this enzyme have been demonstrated to reduce intracellular guanine nucleotide levels both in vitro and in vivo and have potential as anti-neoplastic agents. Both mycophenolic acid and a new drug, AVN-944, are highly specific inhibitors of IMPDH and AVN-944 is currently in clinical trials for the treatment of refractory hematologic malignances. Both compounds induce depletion of total intracellular GTP by 50-80% and induce cell cycle arrest or apoptosis in a number of tumor cell lines. We have compared these two compounds for efficacy of growth inhibition on several cell lines and have investigated their mechanism(s) of action. Both MPA and AVN-944 inhibit the growth of human myeloid progenitor K562 cells, and induce apoptosis in the Burkitt lymphoma Raji cell line, and human T cell leukemia CCRF-CEM cell line. AVN-944 is more potent with ID50 concentrations ranging from 0.13 to 0.16 μM as compared with 0.40 to 0.58 μM for MPA. Both compounds induce striking inhibition of RNA synthesis within 2 h of exposure. We have demonstrated that PAF53, a component of RNA polymerase I (Pol I) and TIF-IA, a Pol I associated transcription initiation factor, rapidly translocate to the peripheral region of the nucleolus (nucleolar cap) in response to GTP depletion; a similar translocation occurs with Pol I inhibition by low dose actinomycin-D (Act-D, 5 nM). Depletion of guanine nucleotides by MPA and AVN-944 also causes an early and near-complete reduction in levels of the 45S precursor rRNA synthesis, as measured by PCR, in K562, CCRF-CEM, and U2OS cells. Consequent to the putative inhibition of Pol I, other nucleolar proteins including nucleolin, nucleophosmin, and nucleostemin translocate from the nucleolus to the nucleoplasm. This shift correlates temporally with the sustained induction of p53. We conclude that inhibition of IMPDH and consequent depletion of guanine nucleotides leads to inhibition of Pol I activity, a reduction in rRNA synthesis, translocation of nucleolar proteins and, hence, to the up-regulation of p53 expression. The ability of AVN-944 to induce apoptosis in a number of leukemic cell lines supports its potential utility in the treatment of hematologic malignancies.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA