VX-680 elicits apoptosis and autophagy in cells that overexpress Myc. (A) Cell killing by VX-680. RPE-NEO cells (□ and ■) and RPE-MYC cells (○ and ●) were treated with 300 nM VX-680 for 3 d and then either transferred into drug-free medium (○ and □) or maintained in fresh medium containing VX-680 (● and ■). Cells were harvested at daily intervals and then assayed for viability by the trypan blue exclusion assay. The data are expressed as mean ± SD for three independent experiments. The arrow indicates the point at which VX-680 treatment was terminated. (B) Reversible inhibition of aurora-B kinase by VX-680. RPE-MYC cells were treated for 3 d with 300 nM VX-680 and then transferred into drug-free medium. Cells were fixed either before (i and iii) or 3 h after (ii and iv) the transfer and stained for DNA in blue (iii and iv) and phosphorylation of histone H3 at Ser-10 in green (i and ii), a surrogate assay for the activity of aurora-B kinase. (C) Activation of caspase-9. RPE-MYC cells were treated for 3 d with either 0.03% DMSO or 300 nM VX-680, fixed, and stained for DNA (blue) and active caspase-9 (red). (D) Ultrastructural analysis of cell morphology. RPE-MYC cells were treated with 300 nM VX-680 for 3 d and then transferred into drug-free medium. Representative electron micrographs of RPE-MYC cells either before (a) or 7 d after (b–d) administration of VX-680 are shown. M, mitochondria; N, nucleus. White arrows denote vacuoles containing electron-dense material in b, multimembrane vesicles in c, and localized cytoplasmic degeneration in d. Black arrowheads in c and d point to double-membrane vesicles sequestering a portion of cytoplasm.

VX-680 elicits polyploidy in cells that overexpress Myc. (A) The effect of VX-680 on DNA synthesis. RPE-NEO cells (□) and RPE-MYC cells (■) were treated with 300 nM VX-680 for 3 d and then transferred into drug-free medium. BrdU incorporation assays were performed at the indicated times after initiation of VX-680 treatment. Each column, here and in B, represents the average of three independent experiments, and each experiment was done in triplicate. Error bars represent 1 SD. ^#Cells below the limit of detection. (B) The effect of VX-680 on the mitotic index. RPE-NEO cells (□) and RPE-MYC cells (■) were treated with 300 nM VX-680 for 0 or 3 d, fixed, and stained for DNA. The mitotic index was expressed as the percentage of cells in mitosis. (C) VX-680 elicits abnormalities in centrosome number, centromere number, and the mitotic spindle. RPE-MYC was treated with 300 nM VX-680 for 3 d and then stained for centrosomes with anti-pericentrin antibodies in green (a, an untreated RPE-MYC cell; b, a treated RPE-MYC cell), centromeres with the CREST human autoserum in red (c), microtubules with anti–α-tubulin antibodies in red (d), and DNA with DAPI in blue (a–d). A representative cell is shown in each panel. (Scale bar: 5 μm.) (D) The effect of VX-680 on centrosome number. The percentage of mitotic RPE-MYC cells with the indicated number of centrosomes (numbered squares) was determined after various durations of treatment with 300 nM VX-680. A representative experiment is shown. (E) The effect of VX-680 on cell morphology. RPE-MYC cells were treated with either 0.03% DMSO (a and c) or 300 nM VX-680 (b and d) for 3 d and then photographed. Three-fold magnifications of a field of RPE-NEO cells and a single multinucleated RPE-MYC cell are shown in c and d, respectively. (Scale bar: 20 μm.) (F) Polyploidy elicited by VX-680 in cells that overexpress Myc. RPE-NEO cells and RPE-MYC cells were treated with either DMSO or 300 nM VX-680 for the indicated days, and DNA content was analyzed by FACS after staining DNA with 10 μg/mL of propidium iodide. (G) Overexpression of Myc prevents induction of the p53-dependent G1 checkpoint pathway. RPE-NEO cells and RPE-MYC cells were treated for the indicated time periods with 300 nM VX-680, and expression of the indicated proteins was assessed by Western blot analysis. Equal loading was confirmed by staining of total proteins on the transfer membrane with Ponceau S.

The autophagy genes ATG5 and ATG7 contribute to delayed cell death elicited by VX-680. (A) Depletion of Atg5 and Atg7 by their cognate RNAi. RPE-MYC cells were infected with either pLKO.1-puro control lentiviral particles or lentiviral particles encoding shRNA against either ATG5 or ATG7, selected with 0.5 μg/mL of puromycin for 2 d, and subjected to Western blot analysis for Atg7 and Atg5, which are found mainly as Atg5–Atg12 conjugates. Actin was used as a loading control. (B) Suppression of autophagy by depletion of either Atg5 or Atg7. RPE-MYC cells were stably transfected with Cherry-LC3, infected with the indicated RNAi viruses, treated with 300 nM VX-680 for 3 d, and then transferred into drug-free medium. Quantification of autophagy with Cherry-LC3 was performed 2 d after withdrawal of VX-680. Cells that had more than 40 Cherry-LC3 puncta per cell were scored as positive. Each column, here and in ensuing panels, represents the average of three independent experiments, and each experiment was done in triplicate. Error bars represent SD. (C and D) Depletion of autophagy proteins suppresses delayed cell death, but not early death. RPE-MYC cells were infected with the indicated RNAi lentiviruses, treated with 300 nM VX-680 for 3 d, and then transferred into drug-free medium. Cells were harvested at 0, 3, and 6 d after initiation of VX-680 administration and assayed for viability by the trypan blue exclusion assay. (C) Delayed death was calculated using the following formula: percentage of dead cells = [(live cell number at day 3 − live cell number at day 6)/(live cell number at day 3)] × 100. (D) Early death was expressed as the percentage of dead cells at day 3. (E and F) Absence of atg5 protects cells against delayed death, but not against early death. atg5^+/+ and atg5^−/− MEF cells were infected with either a control virus (pMig) or a Myc-expressing virus (pMigMyc). The infected cells were then treated with DMSO or 300 nM VX-680 for 3 d and transferred into drug-free medium. Cells were harvested at 0, 3, and 6 d after initiation of treatment with VX-680 and assayed for viability by the trypan blue exclusion assay. Early death (E) and delayed death (F) were calculated as described for C and D. ^#Cells below the limit of detection. In the experiment represented by the hatched column, 100 μM z-VAD-fmk was included in the medium during the delayed cell death assay.

Synthetic lethal interaction between disablement of the CPPC and overexpression of Myc. (A) Cell killing by AZD1152. RPE-NEO cells (□ and ■) and RPE-MYC cells (○ and ●) were treated with 50 nM AZD1152 for 3 d and then either transferred into drug-free medium (○ and □) or maintained in fresh medium containing VX-680 (● and ■). Cells were harvested at the indicated intervals and then assayed for viability by the trypan blue exclusion assay. The data are expressed as mean ± SD for three independent experiments. The arrow indicates the time at which VX-680 treatment was terminated. (B) Depletion of the chromosomal passenger proteins survivin, aurora-B, and INCENP by RNAi. Extracts from RPE-MYC cells treated with RNAi against luciferase, INCENP, surviving, or aurora-B were analyzed by immunoblotting for expression of survivin, aurora-B, and INCENP at 4 d after transfection with RNAi. (C) Early apoptosis elicited by depletion of chromosomal passenger proteins in cells that overexpress Myc. Apoptosis was assessed by DAPI staining of DNA at 4 d after transfection of RPE-NEO cells (□) and RPE-MYC cells (■) with RNAi against the indicated genes. Cells with fragmented nuclei were scored as positive for apoptosis. Each column, here and in D, represents the average of three independent experiments, and each experiment was done in triplicate. Error bars represent 1 SD. (D) Autophagy elicited by depletion of chromosomal passenger proteins in cells that overexpress Myc. Autophagy was assessed by immunostaining for LC3 at 7 d after transfection of RPE-NEO cells (□) and RPE-MYC cells (■) with RNAi against the indicated genes. Cells that had more than 40 LC3 puncta per cell were scored as positive. (E and F) Delayed cell death elicited by depletion of the chromosomal passenger proteins. RPE-MYC cells (E) and RPE-NEO cells (F) were replated at 5% and 20% confluence, respectively, at 4 d after transfection with RNAi against indicated genes. The depletions by RNAi were comparable to those illustrated in B. Cells were photographed after being fixed and stained with crystal violet at 4 d (E) or 7 d (F) after replating.

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.