Fig. 5.
VX-680 extends the survival of mice with lymphomas elicited by Myc. (A) Inhibition in vivo of aurora-B kinase in tumor cells. Tet-o-MYC/Eμ-SR-tTA transgenic mice maintained on a doxycycline-free diet were treated with 0 mg/kg (lane 1), 45 mg/kg (lane 2), 65 mg/kg (lane 3), and 75 mg/kg (lane 4) of VX-680 via i.p. injection when tumors became evident. Higher doses were precluded by limited solubility of VX-680. Single-cell suspensions were prepared from spleen at 6 h after injection and analyzed for histone H3 and its phosphorylation at Ser-10 by Western blot analysis. (B) Survival of mice bearing T-cell lymphomas. Isogenic naïve mice were transplanted with T-cell lymphoma cells (1 million cells per recipient) from a single Tet-o-MYC/Eμ-SR-tTA transgenic mouse. One week later, the recipients were treated with either vehicle (n = 20) or 75 mg/kg of VX-680 (n = 10) by daily i.p. injection at the times indicated by arrows (see SI Materials and Methods for details). Survival is displayed as a Kaplan-Meier plot. Survival curves for vehicle treatment and VX-680 treatment were compared using the log-rank test; P = 0.0001. (C) Survival of mice bearing B-cell lymphomas. Isogenic naïve mice underwent transplantation with B-cell lymphoma cells (50,000 per recipient) from a single Eμ-MYC mouse and treated as described in B. Survival curves for vehicle treatment and VX-680 treatment were compared using the log-rank test; P = 0.0001. (D and E) Tumor suppression by VX-680. Isogenic naïve mice underwent transplantation with T-cell lymphomas (1 million cells per animal) and treated with either vehicle or 75 mg/kg of VX-680 for 4 d by daily i.p. injection that was initiated 1 wk after transplantation. Six days after the final injection, the indicated internal organs from animals were harvested for photography (D) and H&E staining of fixed sections (E). (F) VX-680 elicits apoptosis and polyploidy in MYC-driven lymphomas in vivo. Tet-o-MYC/Eμ-tTA transgenic mice maintained on a doxycycline-free diet were treated with five daily i.p. injections of 75 mg/kg of VX-680 or vehicle when tumors became evident (Dox OFF). One day after the final injection, spleens were removed and fixed with 4% paraformaldehyde. Spleens for controls were obtained from Tet-o-MYC/Eu-tTA mice that had been kept tumor-free by the administration of doxycycline in the diet (Dox ON). Sections of spleens were analyzed by H&E staining, TUNEL assay, and propidium iodide staining. Black and white arrows indicate polyploid cells. (G) Induction of autophagy by VX-680 in vivo. Six Tet-o-MYC/Eμ-tTA transgenic mice were treated with vehicle (lanes 1 and 2) or five daily i.p. injections of 75 mg/kg of VX-680 (lanes 3–6) when tumors became evident. Single-cell suspensions were prepared from spleen at 24 h after the last injection and assessed by Western blot analysis with antibodies against LC3 and actin. The numbers indicate the ratio between LC3-II and LC3-I.