Characterization of a myeloid leukemia cell line resistant to inosine monophosphate dehydrogenase (IMPDH) inhibition: correlation of molecular pathway markers and GTP depletion with clinical biomarkers.
Authors: Jeffrey Strovel, Tammy Lawrence, Martin Korade, Pachai Natarajan, Karina Zuck, Stephen Glanowski, Thomas Genna, Simon Katz, Stephen Horrigan, J. Michael Hamilton, David Bol
Journal: Cancer Research (2008)

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA

226 

IMPDH2 is highly up-regulated, and is required for the de novo production of guanine nucleotides in many malignancies, including hematologic cancers. As GTP is an essential cellular factor for proliferation, most cancer cell lines are sensitive to IMPDH inhibition by molecular targeted agents. In order to model potential future clinical resistance to IMPDH inhibitors in development, we cultured K562 chronic myeloid leukemia cells in the presence of IMPDH inhibitors to create a resistant K562 clone to use as a tool for identification of molecular characteristics of resistance to these drugs. Cells were treated for a total of 52 weeks on a schedule using 5µM AVN944 or 0.1% DMSO vehicle for three consecutive days followed by four day incubation in drug-free media. After one year of high selective pressure a resistant sub-line emerged more than 25-fold resistant to AVN944 compared to the parental line. Parental (untreated) K562 cells were significantly growth inhibited at an IC50 of 0.20µM, whereas the resistant line displayed an IC50 of 6.0uM after 72 hours of drug treatment. GTP measurements in the parent and resistant K562 cells revealed a 50% decrease in GTP in the resistant line compared with parent. Additionally, treatment with 0.5uM AVN944 resulted in an elevation of GTP in the resistant line whereas the same treatment in the parent line resulted in ~75% depletion of GTP. Interestingly, the resistant cell line showed no cross resistance to a second inhibitor of IMPDH, mycophenolic acid (MPA), with regards to inhibition of cellular proliferation or depletion of GTP levels. The resistant and parent lines were subject to microarray gene expression profiling, and differences in gene expression in the two cell lines were correlated to GTP levels in AVN944 and MPA treated samples. Concurrently, we have isolated peripheral blood samples from AML patients in a phase I trial both before and after receiving AVN944. These samples were analyzed to determine the effects of the drug on GTP pools, and to correlate these changes to biomarkers examined in circulating cells using gene expression detection technologies. In this study, we show the similarities in gene expression signatures between a population of AVN944-treated AML patient samples and the resistant and parent cell lines. Furthermore, we show how gene expression biomarkers (qPCR) that report on the activity of AVN944 only respond in the parental K562 cell line and in AML samples from patients known to have benefited from AVN944 therapy. This information will contribute to the design of patient enrichment biomarker approaches to identify patients more likely to respond to the drug, as well as monitoring the activity of the drug in future clinical trials.