Inhibition of Hypusination of Eukaryotic Initiation Factor 5a (eIF-5A) as a Novel a Synergistic Treatment Strategy in Imatinib-Treated BCR-ABL Positive Leukemias Identified by a Global Proteomics Approach.

Imatinib (IM) is highly effective in the treatment of CML and remains the gold standard non-transplant therapeutic option. However, resistance to IM develops frequently, particularly in late stage disease. The aim of this study was to discover novel cellular Bcr-Abl targets leading to the identification of potential novel treatment options that would synergise with IM. Protein expression analysis of K562 leukemic cells after IM treatment was performed using 2-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Differential protein expression of selected proteins was confirmed by Western blot analysis. Among others, we detected the IM-dependent down-regulation of the eukaryotic translation initiation factor 5A (eIF-5A), which is essential for cell proliferation. EIF-5a represents the only known eukaryotic protein activated by a unique, 2-step post-translational modification called hypusination, a process that can be specifically inhibited by hypusination inhibitors (HI), e.g. ciclopirox and GC7. We demonstrated that selective HI elicit a strong synergistic effect with IM on both cellular cytotoxicity (using MTT assay) and apoptosis (using PI staining) of Bcr-Abl positive leukemic cell lines and even more strikingly on primary CD34⁺ cells from CML patients at diagnosis. The effect of HI on CD34⁺ cells from healthy donors and CML patients was compared by short-term expansion in serum free medium using high resolution cell cycle analysis and carboxy-fluorescein succinimidyl ester (CFSE) labelling to track cell division. We observed a substantial anti-proliferative effect of the drug combination on Bcr-Abl positive as compared to Bcr-Abl negative cells. However, we were unable to demonstrate a significant cytotoxic effect of the combination on the quiescent Ph+ stem cell population. Despite this, HI exerted strong antiprolifertive effects not only on Ba/Fp210 but also on resistant Ba/F3p210 mutants T315I, E255V and M351T.

In conclusion, IM induced significant changes of global cellular protein expression in K562 cells, including down-regulation of eIF-5A. Inhibition of eIF-5A hypusination represents a promising target for combined therapy in Bcr-Abl positive leukemias. Ongoing studies are investigating whether novel, more specific, HI in combination with IM (and/or 2^nd generation Bcr-Abl inihibitors e.g AMN107, BMS-354825) will be able to exert these effects on the quiescent Bcr-Abl⁺ stem cell compartment, and if these novel combinations are effective in IM-resistant cells.