Differential response of tumor cell lines to inhibition of the mitotic checkpoint regulator and mitotic kinesin, CENP-E

A114 Centromere-associated protein E (CENP-E) is a kinesin motor protein functioning exclusively in mitosis to integrate mitotic spindle mechanics with mitotic checkpoint signaling. Disruption of CENP-E function using a variety of methods, including small molecule inhibition of CENP-E kinesin motor domain function, causes cell cycle arrest in mitosis with a bipolar mitotic spindle with misaligned chromosomes, often followed by cell death. GSK923295A is a novel and selective inhibitor of CENP-E motor domain enzymatic activity currently in Phase I clinical trial. To aid in identification of molecular determinants of sensitivity to GSK923295A, 216 solid tumor cell lines were classified with respect to their sensitivity to GSK923295A in culture. Classification was based on the maximal extent of tumor cell growth achieved (Y min ) relative to the number of input cells (T 0 ). To understand molecular differences underlying differential response, six breast cancer cell lines were selected for detailed characterization of cell cycle and apoptotic response: three relatively sensitive lines HCC1954, EFM19 and SK-BR-3 (Y min /T 0 ≤ 0.5) and three relatively refractory lines MDA-MB-231, MX-1 and BT474 (Y min /T 0 ≥ 1.3).
 Treatment with CENP-E inhibitor (CENP-Ei) induced mitotic arrest in each of the three sensitive lines and in two refractory lines. In sensitive lines, CENP-Ei-induced accumulation of markers of cell death coincided with appearance of mitotic markers, beginning as early as 24 hours after exposure to inhibitor and persisting until 72 hours. Treatment with the pan-caspase inhibitor zvad-fmk antagonized appearance of markers of cell death, consistent with activation of an apoptotic program.
 In contrast, CENP-Ei-refractory lines either did not begin to die during the course of the experiment, or expressed markers of cell death weakly only after prolonged treatment with CENP-Ei. BT474 failed to arrest in response to treatment, despite the appearance among mitotic cells of mitotic figures with misaligned chromosomes characteristic of CENP-E inhibition. CENP-Ei induced MX-1 cells to arrest briefly in mitosis but did not initiate cell death, and MDA-MB-231 arrested for a prolonged period losing mitochondrial membrane potential only at later time points. In all refractory lines, the abundance of full-length PARP decreased without appearance of the cleavage fragment apparent in the three sensitive lines.
 Two sensitive and two refractory lines are wild-type for the tumor suppressor p53. Treatment of each of these four lines with CENP-Ei induced the transient appearance of p53 protein over a period of time coincident with mitotic arrest. However, p21 WAF1/CIP1 was induced in only one these lines; MX-1. MX-1 cells appeared to complete mitosis after a short period of mitotic arrest and subsequently accumulated elevated levels of cyclin E protein, possibly due to p21 WAF1/CIP -mediated arrest in the G 1 phase of the cell cycle.
 Correlation of cell line sensitivity to CENP-E inhibitor with functional defects in cell cycle and cell death pathways may identify one or more biomarkers for identification of patients who would most benefit from treatment with a CENP-E targeted therapy.