Dasatinib suppression of medulloblastoma survival and migration is markedly enhanced by combining treatment with the aurora kinase inhibitor AT9283

Abstract

Medulloblastoma (MB) expresses Src kinase, while aurora kinase A overexpression correlates with poor survival. We thus investigated novel combination treatment with dasatinib and AT9283, inhibitors of Src and aurora kinase, respectively, on MB growth in vitro and in vivo. Treatment with each drug significantly reduced cell viability and combined treatment markedly potentiated this response. AT9283 induced p53 expression, autophagy, and G2/M cell-cycle arrest, while combined treatment induced S phase arrest. Dasatinib treatment caused tumor regression in vivo. Activated Src was detected in 44% MB analyzed. We conclude that further evaluation of this combination therapy for MB is highly warranted.

Keywords: Aurora kinase; Dasatinib; Medulloblastoma; Survival.

Figure 1

Dasatinib treatment abolishes Src phosphorylation, but induces histone H3 phosphorylation (p-HH3), while AT9283 treatment inhibits dasatinib-induced p-HH3. DAOY and D556 cells grown in culture were treated with dasatinib and AT9283 and then the level of the respective phosphorylated effector proteins, p-Src and p-HH3, were detected by Western blot of whole cell lysates. Total Src and GAPDH served as internal protein loading controls for each target, respectively. A) p-Src levels are shown at 1 hour after treatment with increasing concentrations of dasatinib (upper panels) and at the indicated timepoints after treatment with 1 nM dasatinib (lower panels). B) p-HH3 levels are shown at 1 hour after treatment with increasing concentrations of AT9283 (upper panels) and at the indicated timepoints after treatment with 10 nM AT9283 (lower panels). C) p-HH3 levels are shown at 1 hour after treatment with increasing concentrations of dasatinib alone (upper panels) and in combination with 10 nM AT9283 (lower panels).

Figure 2

Dasatinib or AT9283 treatment decreases cell proliferation and combined treatment with dasatinib and AT9283 potentiates the individual drug responses. A) DAOY, B) D556, and C) murine PS125 cells were grown in culture and treated with a single dose of dasatinib (1nM for DAOY and D556; 3nM or 10nM for PS125 cells), AT9283 (10nM), dasatinib and AT9283 combined, or vehicle control, and then proliferation was measured daily by the MTT assay (optical density, OD, measured at 570nm) for 3–5 days after treatment. Graphs show the average daily OD per treatment condition. *, p-value < or = 0.05.

Figure 3

Dasatinib or AT9283 treatment induces apoptosis and autophagy, while combined treatment with dasatinib and AT9283 markedly potentiates apoptosis. DAOY and D556 cells were grown in culture and treated for 1 hour with dasatinib (1nM), AT9283 (10nM), dasatinib and AT9283 combined, or vehicle control, and then cells were assayed for apoptosis by annexin V labeling at 72 hours after treatment, and for evidence of autophagy by Western blot for the autophagy marker LC3-II at 1 hour and 24 hours after treatment. For the autophagy assays, rapamycin (10nM) treatment for 16 hours was used for a positive control. A) Graphs show the average fold-change in the percentage of annexin V-positive apoptotic cells per treatment condition compared to pre-treatment for DAOY and D556 cells. B) Representative changes in the levels of the autophagy protein marker LC3-II are shown at 1 hour and 24 hours after the individual drug treatments.

Figure 4

Dasatinib and AT9283 combined treatment induces G2/M and S phase cell-cycle arrest. DAOY and D556 cells were serum-starved overnight and then treated with a single dose of dasatinib (1nM), AT9283 (10nM), dasatinib and AT9283 combined, or vehicle control, with 10% FBS, and then cell cycle analysis was performed by flow cytometry of PI-labeled cells at 24, 48, and 72 hours after treatment, without drug replenishment after the initial treatment at hour 0. Results shown are representative cell-cycle plots of D556 cells at 72 hours after the respective treatments, with the mean percentage (%) of D556 cells in each phase of the cell cycle at 72 hours after the treatments indicated, as measured using the FlowJo program.

Figure 5

Dasatinib or AT9283 treatment inhibits migration and combined treatment markedly potentiates the individual drug treatment responses. DAOY and D556 cells were grown to confluency on culture plates, a scratch wound was made, and the cells were treated with a single dose of dasatinib (1nM), AT9283 (10nM), dasatinib and AT9283 combined, or vehicle control, and then cell movement across the scratch was measured at 24 and 48 hours following treatment at hour 0. Drug treatment was not replenished after hour 0. Results of the scratch assays are shown for A) DAOY and B) D556 cells by their corresponding representative photomicrographs displayed in the upper panels and bar graphs demonstrating the relative ratio of the mean cumulative movement relative to hour 0, as displayed in the lower panels, for each of the treatment conditions and indicated time points after treatment. *p-value < 0.05.

Figure 5

Dasatinib or AT9283 treatment inhibits migration and combined treatment markedly potentiates the individual drug treatment responses. DAOY and D556 cells were grown to confluency on culture plates, a scratch wound was made, and the cells were treated with a single dose of dasatinib (1nM), AT9283 (10nM), dasatinib and AT9283 combined, or vehicle control, and then cell movement across the scratch was measured at 24 and 48 hours following treatment at hour 0. Drug treatment was not replenished after hour 0. Results of the scratch assays are shown for A) DAOY and B) D556 cells by their corresponding representative photomicrographs displayed in the upper panels and bar graphs demonstrating the relative ratio of the mean cumulative movement relative to hour 0, as displayed in the lower panels, for each of the treatment conditions and indicated time points after treatment. *p-value < 0.05.

Figure 6

Dasatinib treatment induces tumor regression in a mouse syngeneic MB flank model, and MB tumors ubiquitously express Src, AurkA and AurkB, and exhibit Src activation. A) Plot shows the effect of dasatinib treatment on MB tumor growth over time in mice bearing flank syngeneic MB tumor implants. Cohorts of 6 mice per group were treated with dasatinib or vehicle control by oral gavage once daily for 5 consecutive days at a dose of 15 mg/kg/day for a total of 20 days of treatment. Tumor volume (mm³) over the course of the treatment was measured at the indicated timepoints for each mouse and then averaged for each cohort. *p-value < 0.05. B) Representative photomicrographs show the positive (+) and negative (−) immunohistochemistry staining results for childhood MB tumor and normal fetal cerebellum tissue specimens stained for activated Src, p-Src (Y416), and inactivated Src, p-Src (Y529).