Abstract
Imatinib (STI571), a tyrosine kinase inhibitor, is becoming the new standard of care for patients with chronic and advanced phase CML. However, the treatment for blast crisis (BC) CML is less effective. Resistance to Imatinib develops in all phases, particularly in BC CML emphasizing the need for alternative therapies. Eg5, a microtubule-associated motor protein plays an important role in establishing a bipolar spindle during mitosis and is essential for cell cycle progression. Eg5 was recently found to be highly expressed in BC CML by microarray analysis (Oncogene22:3952–3963, 2003). In this study, we examined the regulation of Eg5 by Bcr-Abl tyrosine kinase signaling and tested Eg5 as a potential therapeutic target in BC CML and Imatinib resistant CML. We found that Eg5 is expressed in all Philadelphia chromosome positive (Ph+) CML cell lines and in BC CML patient samples. Inhibition of Bcr-Abl activity by Imatinib downregulated Eg5 expression in Imatinib sensitive KBM5 cells (a cell line derived from the blasts of a BC CML patient) and HL-60p185 cells (HL-60 cells transfected with Bcr-Abl fusion protein p185), but not in Imatinib resistant KBM5-STI571 cells and Bcr-Abl negative HL-60 cells suggesting that Eg5 is a downstream effector of Bcr-Abl and is regulated by Bcr-Abl tyrosine kinase signaling in Ph+ cells. Blocking Eg5 expression by its antisense oligonucleotide (Eg5-AS) induced G2/M cell cycle block, and subsequent cell death in both Imatinib sensitive KBM5 cells and Imatinib resistant KBM5-STI571 cells. At 48 hrs, 15.8±5.5% of KBM5 cells and 22.7±10.7% of KBM5-STI571 cells were blocked in G2/M in Eg5-AS treated cells compared to 3.5±1.9% and 7.6±1.4%, respectively, of the mismatched oligonucleotide (Eg5-NS) treated cells. Induction of cell death was observed at 72 hrs (29.1±1.9% in KBM5 and 29.4±1.1 % in KBM5-STI571 cells in Eg5-AS treated compared to 12.5±0.28% and 13.7±1.6% of Eg5-NS treated cells). Metaphase arrest due to disruption of bipolar spindle formation, loss of mitochondrial membrane potential, and caspase activation were observed in both cell lines. Similarly, inhibition of Eg5 activity by a small molecular inhibitor, S-trityl-L-cysteine, induced cell cycle block and cell death indistinguishably in Imatinib sensitive KBM5 and Ba/F3Bcr-Ablwt cells and in Imatinib resistant KBM5-STI571, Ba/F3Bcr-AblE255K, and Ba/F3Bcr-AblT315I cells. Treatment of Scid mice starting 7 days after injection of KBM5 cells with Eg5-AS, 25 mg/kg, 3 times a week for 3 weeks, significantly prolonged the survival of the animals (64 days vs. 49 days of Eg5-NS treated mice, p=0.0344). The effect of Eg5 inhibition on survival of Scid mice harboring Imatinib resistant KBM5-STI571 cells is currently under investigation. Our studies suggest that Eg5 is a downstream target of Bcr-Abl tyrosine kinase. Inhibition of Eg5 expression or its activity blocks cell cycle progression and induces cell death regardless of cell response to Imatinib. Eg5 could be a potential new critical therapeutic target for the treatment of Imatinib resistant CML and BC CML.
The retinoblastoma tumor suppressor inhibits cellular proliferation through two distinct mechanisms: inhibition of cell cycle progression and induction of cell death