Inhibition of KSP by ARRY-520 Induces Cell Cycle Block and Cell Death Via the Mitochondrial Pathway in AML Cells.

Abstract

Kinesin spindle protein (KSP or Eg5), a microtubule-associated motor protein, plays an important role in establishing a bipolar spindle during mitosis and is essential for cell cycle progression. It has been demonstrated that inhibition of KSP prevents bipolar spindle formation leading to mitotic arrest and cell death. As such, a KSP inhibitor may have anti-tumor potential without toxicities associated with anti-microtubule agents such as taxanes. We found that KSP is highly expressed in various acute leukemic cells. Inhibition of KSP by a specific inhibitor, ARRY-520, at low nanomolar concentrations, blocked cell cycle progression and led to subsequent cell death in OCI-AML3, Molm13, HL-60, U937, and Jurkat cells. Knocking down p53 by p53shRNA in OCI-AML3 cells did not alter the effectiveness of ARRY-520. U937 cells overexpressing XIAP (U937XIAP) and Jurkat cells lacking caspase 8 (JurkatI2.1) showed unchanged sensitivities to ARRY-520 compared with their respective control cells, suggesting that cell cycle block and cell death induced by KSP inhibition is independent of p53 status, XIAP levels, and the activation of the extrinsic apoptotic pathway. However, although ARRY-520 blocked cell growth and induced mitotic arrest in both HL-60 cells and HL-60 cells overexpressing Bcl-2 (HL-60Bcl-2), cell death (determined by annexin V staining and changes in mitochondrial potential) was significantly abolished in HL-60Bcl-2 cells. These results suggest that cell death following cell cycle blockade by KSP inhibition is mediated through the intrinsic mitochondrial pathway. Furthermore, ARRY-520 induced the protein level of Bim, a proapoptotic BH3-only Bcl-2 family protein, prior to the activation of caspases in HL-60 cells. Although KSP inhibition by ARRY-520 had no effect on the survival of non-dividing AML blasts in vitro, ARRY-520 significantly inhibited the colony formation capacities of AML blasts, further supporting the critical role of KSP in cell proliferation. Our studies demonstrated that inhibition of KSP by ARRY-520 potently induces blockage of cell cycle progression which leads to cell death of various leukemic cells via the mitochondrial pathway and has the potential to eradicate AML progenitor cells.