Mcl-1 Stability Determines Mitotic Cell Fate of Human Multiple Myeloma Tumor Cells Treated with the Kinesin Spindle Protein Inhibitor ARRY-520

Brian J Tunquist,Richard D Woessner,Duncan H Walker

doi:10.1158/1535-7163.mct-10-0033

Brian J Tunquist, Richard D Woessner + Show 1 more

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https://doi.org/10.1158/1535-7163.mct-10-0033

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Abstract

Kinesin spindle protein (KSP/Eg5) inhibitors are novel anticancer agents that have thus far shown only modest activity in the clinic. Understanding how to identify patients who may be most sensitive to treatment is clearly needed to improve the development of these molecules. We studied four multiple myeloma cell lines treated with the KSP inhibitor ARRY-520 to identify factors important for initiating apoptosis while cells are arrested in mitosis. The majority (three of four) of cell lines underwent mitotic arrest, with apoptosis occurring in mitosis within 24 to 30 hours. The remaining line (NCI H929) is temporally refractory to ARRY-520 treatment, undergoing mitotic slippage and subsequently peaking in apoptotic markers after 72 hours of treatment, while most cells are in interphase. Interestingly, loss of the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) coincided with mitotic cell death. Stabilization of Mcl-1 resulted in a delayed onset of apoptosis, whereas enforced downregulation of Mcl-1 increased cell death in response to KSP inhibition. Thus, variation in responses to KSP inhibition is governed by a balance between survival proteins and spindle checkpoint integrity. Cells relying on short-lived survival proteins during mitosis are more likely to undergo apoptosis in response to KSP inhibition. We propose that patients with hematologic malignancies, which rely on Mcl-1, would therefore be good candidates for treatment with KSP inhibitors.

Highlights

Antimitotic microtubule inhibitors are among the most active and broadly used cancer drugs
Three of the cell lines (RPMI 8226, JJN3, and U266) exposed to ARRY520 exhibited a rapid onset of apoptosis as defined by a peak in caspase-3/7 activity that occurred ∼24 hours after compound treatment (Fig. 1A)
NOXA knockdown correlated with greater stabilization of myeloid cell leukemia 1 (Mcl-1) protein 24 hours after ARRY-520 treatment, and a delay in detectable levels of cleavedPARP (Fig. 4D) compared with control-transfected cells (Fig. 4C). These results suggest that Mcl-1 is capable of maintaining survival signals in multiple myeloma cell lines arrested in mitosis by ARRY520, and that only once Mcl-1 levels decrease below a threshold level for survival can cell death pathways become active

Summary

Introduction

Antimitotic microtubule inhibitors are among the most active and broadly used cancer drugs. These drugs, which include taxanes, epothilones, and Vinca alkaloids, directly perturb microtubule function, resulting in a defective mitotic spindle and mitotic arrest. As microtubules perform critical functions in postmitotic cells, these drugs exhibit unwanted side effects, including peripheral neuropathies [1]. Inhibitors of mitosis-specific targets that lack side effects in postmitotic cells are desired. One such target is kinesin spindle protein (KSP, Eg5). KSP is a mitosis-specific kinesin essential for the assembly of a bipolar spindle and equal segregation of sister chromatids [2]. Inhibition of KSP prevents centrosome separation, resulting in the formation of a monopolar spindle and activation of the mitotic spindle assembly checkpoint (SAC), causing metaphase

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