Synergism between Anti-Leukemia Therapeutics and Isoprenoid Pathway Inhibitors in K562 Leukemia Cells.

Chronic myeloid leukemia (CML) is a consequence of the BCR-ABL oncogene which mediates a phosphorylation cascade of downstream effectors including those of the Ras family. Drugs that target the isoprenoid pathway, such as the farnesyl transferase (FT) inhibitors have shown promise as novel anti-cancer therapies for some hematologic malignancies. Leukemias are treated with specific inhibitors, like imatinib mesylate in the case of CML, and less-specific chemotherapeutic drugs such as the pyrimidine analog cytarabine (ara-C). We systematically evaluated combinations of these and other agents with isoprenoid pathway inhibitors for synergistic anti-leukemia effects. Toxicity was measured in the BCR-ABL-expressing K562 cells using an MTT assay, and drug synergy was assessed using a modified isobologram method. Cell cycle distribution, Annexin-V apoptosis detection, and Western blot analyses were performed on cells treated with synergistic drug combinations. Imatinib mesylate exhibited significant synergy with the squalene synthase inhibitor zaragozic acid, but not with inhibitors for HMG coenzyme A reductase (lovastatin), FPP synthase (zoledronic acid), GGPP synthase (digeranyl bisphosphonate), and FT (alpha-hydroxyfarnesyl phosphonate). Western blot analysis revealed a moderate decrease in Ras protein levels upon treatment with zaragozic acid. This zaragozic acid-mediated decrease in Ras protein may explain the synergy seen between the drugs since imatinib mesylate also affects Ras by decreasing its phosphorylation through inhibition of the BCR-ABL protein. Apoptosis detection and cell cycle distribution did not yield insight into the mechanism underlying this synergy. Ara-C displayed synergy with digeranyl bisphosphonate in the MTT assay. Subsequent cell cycle analysis showed a concentration-dependent G0–G1 arrest by digeranyl bisphosphonate which was enhanced by ara-C. Annexin-V/propidium iodide staining revealed increased cellular apoptosis in response to either digeranyl bisphosphonate or ara-C with synergistic apoptosis to combination treatments. Western blot analysis for anti-apoptotic proteins showed a decrease in Mcl-1 and Bcl-X_L protein levels in the presence of digeranyl bisphosphonate. Ara-C has been shown to alter pro-apoptotic and anti-apoptotic proteins in a manner that induces apoptosis. These modifications in pro- and anti-apoptotic proteins and alterations in cell cycling may constitute a mechanism for the synergy seen between digeranyl bisphosphonate and ara-C. Discovery of these synergistic combinations is important because translation of these cell culture results to in vivo testing and human clinical trials may enhance treatment efficacy and diversify available treatment options. Future work will focus on further determining the mechanisms for these synergistic interactions, finding new synergistic combinations, and applying these regimens to in vivo models and clinical trials.