Activity of the kinesin spindle protein inhibitor ispinesib (SB-715992) in models of breast cancer

Abstract

Purpose: Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein, a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have shown a 9% response rate in patients with locally advanced or metastatic breast cancer and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities, or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer, we explored the activity of ispinesib alone and in combination with several therapies approved for the treatment of breast cancer.

Experimental design: We measured the ispinesib sensitivity and pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo and tested the ability of ispinesib to enhance the antitumor activity of approved therapies.

Results: In vitro, ispinesib displayed broad antiproliferative activity against a panel of 53 breast cell lines. In vivo, ispinesib produced regressions in each of five breast cancer models and tumor-free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the antitumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine and exhibited activity comparable with paclitaxel and ixabepilone.

Conclusions: These findings support further clinical exploration of kinesin spindle protein inhibitors for the treatment of breast cancer.

Fig. 1

Antitumor activity of ispinesib in vitro against models of breast cancer. A, growth inhibition at 50% (GI₅₀) induced by ispinesib was determined for 53 breast cell lines of luminal, basal A, basal B, and noncancerous origin. B, differences in cell cycle profiles in MDA-MB-468, a cell line sensitive to ispinesib, compared with a less sensitive cell line, BT-474, after treatment with 150 nmol/L ispinesib (3- to 7-fold GI₅₀). C, expression of cell cycle markers (cyclin A, cyclin B, and cyclin E) and apoptotic proteins [Bax, Bid, phospho-Bcl2 (p-Bcl2), Bcl2, and Bcl-X_L] was analyzed by Western blotting in MDA-MB-468 and BT-474 cells following treatment with 150 nmol/L ispinesib.

Fig. 2

Antitumor activity of ispinesib in vivo in preclinical models of breast cancer. Vehicle control (black) and ispinesib (green) at its MTD (10 mg/kg in nude mice, 8 mg/kg in SCID mice) were dosed i.p. q4d×3 in models of ER-positive (MCF7), HER2-positive (KPL4, HCC1954, and BT-474), and triple-negative (MDA-MB-468) breast cancer. All cell lines were grown in nude mice, except BT-474 and MDA-MB-468, which were grown in SCID mice. Arrows indicate the days on which ispinesib was administered.

Fig. 3

Mitotic arrest and apoptosis induced in the MDA-MB-468 ispinesib-hypersensitive xenograft model. A, antitumor activity of ispinesib compared with paclitaxel and ixabepilone in MDA-MB-468 xenografts in SCID mice. Arrows indicate the days on which the respective drugs were administered. B, mice with MDA-MB-468 and BT-474 xenografts were treated with a single 10 mg/kg i.p. dose of ispinesib, and tumor sections were stained for the mitotic antigen PH3 (green) and nuclear 4′,6-diamidino-2-phenylindole (blue). PH3 images were taken at ×10 magnification. C, quantification of ispinesib-induced PH3 staining in MDA-MB-468 and BT-474 xenografts (calculated as the area of PH3-positive signal relative to the area of DNA-positive signal). D, Ki67 (brown) was used as a marker of cellular proliferation and cleaved caspase-3 (pink) as a marker of apoptosis; images were taken at ×20 magnification.

Fig. 4

Ispinesib enhances the antitumor activity of therapies approved for the treatment of breast cancer. Combination of ispinesib with trastuzumab in BT-474 (A) and KPL4 (B) xenografts. Ispinesib was dosed i.p. q4d×3 at its MTD (10 mg/kg) in nu/nu mice (KPL4 xenografts) and 8 mg/kg in Fox Chase SCID mice (BT-474 xenografts). Trastuzumab was dosed i.p. twice weekly for 4 wk at 10 mg/kg. C, combination of ispinesib with the anthracycline doxorubicin. nu/nu mice with MCF7 xenografts were treated i.p q4d×3 with ispinesib (6 mg/kg) and i.v. q4d×3 with doxorubicin (2.5 mg/kg). D, combination of ispinesib with capecitabine in KPL4 xenografts in nu/nu mice. Ispinesib was dosed q4d×3 i.p. at 5 mg/kg (0.5× MTD), and capecitabine was dosed at 450 mg/kg (MTD) orally daily for 14 d (qdx14). Arrows indicate the days on which ispinesib was administered.