Identification of Hypusination Inhibitors as a Novel Therapuetical Option in BCR-ABL Positive Leukemias Using a Proteomics Approach.

Introduction: BCR-ABL has been shown to be both necessary and sufficient for the induction of chronic myeloid leukemia (CML) in experimental models. The selective tyrosine kinase inhibitor imatinib has been shown to induce major cytogenetic remissions in >80% of CML patients in chronic phase. However, resistance to imatinib develops frequently especially in later stages of the disease. Therefore, investigations on potential synergistic treatment strategies are required in order to be able to overcome clinical resistance.

Materials and Methods: Protein expression analysis of K562 leukemic cells was performed using 2-dimensional gel electrophoresis after treatment with imatinib (4 μM) or DMSO (as a solvent control) for 24 hours and 48 hours. Resulting differentially expressed prominent proteins were analyzed using matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Differential expression was confirmed by western blot analysis for some candidate proteins. Cytotoxicity was measured in K562 cells and primary CD34+ cells from patients with CML at diagnosis using MTT-assay, the fraction of apoptotic cells was measured by flow cytometry using propydium iodide (PI).

Results: Using 2-dimensional gel electrophoresis, thirteen candidate proteins were identified to be down-regulated whereas 6 proteins were up-regulated by cell treatment with imatinib. Interestingly, we detected the down-regulation of the eukaryotic translation initiation factor 5A (eif5A), which is essential for cell proliferation. Eif5a represents the only known eukaryotic protein activated by posttranslational hypusination. Formation of hypusine is one of the most specific polyamine-dependent biochemical reactions and its biosynthesis involves two enzymatic steps which can be inhibited with the compounds ciclopirox and GC-7. To assess the effects of the hypusination inhibitors as single agent and in combination with imatinib on Bcr-Abl positive or negative cells, we performed cytotoxicity- and apoptosis-assays. Hypusination inhibition resulted in substantial and dose-dependent cytotoxicity and in an increasing fraction of apoptotic cells in K562, HL-60 and primary Bcr-Abl positiv CD34+ cells. Interestingly, synergistic effects of imatinib and ciclopirox or GC-7 could be detected on cellular cytotoxicity and apoptosis in both Bcr-Abl positive K562 cells and Bcr-Abl positive primary CD34+ cells from four newly diagnosed CML patients.

Discussion: We could detect significant differences in protein expression levels in the BCR-ABL-dependent cell line K562 upon treatment with imatinib. Furthermore, we observed down-regulation of eif5a which so far has not been shown to be involved in proliferation control of Bcr-Abl positive leukemias. Eif5a suggests to be a promising target for imatinib-based synergistic treatment strategies. Considering that a number of hypusination inhibitors are clinically approved drugs with acceptable toxicity profiles, our results might have important implications for the design of novel synergistic treatment strategies in patients with Bcr-Abl-positive leukemias and other imatinib-responsive diseases.