Synergistic activity of rapamycin and dexamethasone in vitro and in vivo in acute lymphoblastic leukemia via cell-cycle arrest and apoptosis

Abstract

Activation of the mTOR pathway subsequent to phosphatase and tensin homolog (PTEN) mutation may be associated with glucocorticoid (GC) resistance in acute lymphoblastic leukemia (ALL). The combination activity of rapamycin and dexamethasone in cell lines and xenograft models of ALL was determined. Compared with either drug alone, dexamethasone+rapamycin showed significantly greater apoptosis and cell cycle arrest in some cell lines, and was more frequently seen in T-lineage cell lines with PTEN mutation. The combination significantly extended the event-free survival of mice carrying PTEN mutated xenografts. Our data suggest that PI3K/mTOR pathway inhibitors could benefit patients with PTEN mutated T-ALL.

Figure 1

Combination cytotoxicity of DEX and RAP in T-ALL (A) and B-ALL cell lines (B). Cytotoxicity was evaluated after treating the cells with DEX, RAP, or DEX+RAP (D+P) for 72h by DIMSCAN system. The fractional survival was determined by mean fluorescence of the treated cells/mean fluorescence of vehicle control cells. Each point represents the mean value for 12 replicates, error bars correspond to standard deviations. * and †: Cell lines with the same symbol were established from the same patient. For B-C the points/columns are the mean and error/vertical bars are standard deviation from experiments performed twice (n=12).

Figure 2

DEX + RAP on apoptosis. (A) COG-LL-317h cells were incubated with vehicle control, DEX, RAP (10 ng/ml), or DEX+RAP for 36 hours, and subjected to flow cytometry after staining. (B) The percentages of cells undergoing apoptosis (black bars) and the cells with mitochondrial depolarization (white bar) were measured by flow cytometry after treating the cells with vehicle control, DEX, RAP, or DEX+RAP for 36h. (C) Cytochrome c and Smac release into cytosol was assessed at 36h of incubation with the agents. (D) Whole-cell extracts from cells incubated with DEX, RAP, or DEX+RAP for 48h were immunoblotted with caspase-9 and caspase-3 antibodies. β-actin was used for loading control. Experiments were performed twice and were consistently repeatable; for simplicity, one representative experiment for each condition is shown.

Figure 3

DEX+RAP on cell cycle progression. (A) Cells (COG-LL-317h) were treated with DEX, RAP or DEX+RAP for 24h and then fixed in 70% ethanol overnight and stained with PI for subsequent analysis by flow cytometry (n=3). Experiments were performed twice and were consistently repeatable; one representative experiment for each condition is shown. (B) COG-LL-317h cells were treated with DEX, RAP, and DEX+RAP for 12, 24 and 36h. Cells were lysed and the lysates were immunobloted with antibodies to CDK2, 4, 6, cyclin D2, cyclin E, p27, retinoblastoma (Rb), p-Rb, and E2F1.

Figure 4

Changes in PI3K pathway proteins by DEX + RAP. Cells were treated with DEX (50 nM), RAP (10 ng/ml), or DEX+RAP for 24 and 36h. Cells were lysed and the extracts were subjected to analysis by western blotting using antibodies specific for p85, AKT, p-AKT, PTEN, p-PTEN, S6K1, p-S6K1, S6, and p-S6 (A). Changes in S6K1 enzyme activity after the treatment (B) were assessed in COG-LL-317 cell line as described under Methods (*, p < 0.05; **, p < 0.01). The data represent the mean values for triplicate measurements from two independent experiments, and the values are normalized to vehicle control; error bars represent standard deviation (n=3). The knockdown efficiency (C) was assessed. Although S6K1 protein level was low up to 72 hours of transfection, p-S6 levels increased by 72 hours. Therefore, the cytotoxicity assay after the transfection of siRNAs against S6K1 was performed at 48h after the treatment (D). The cytotoxicity of DEX+RAP after transfecting scrambled siRNA (●) or siRNAs against S6K1 (○) was assessed at 48h after the treatment. Bars represent standard deviation of 12 samples. Experiments were performed twice and were consistently repeatable; one representative experiment for each condition is shown.

Figure 5

In vivo activity of DEX, RAP, DEX+RAP with ALL and lymphoma xenograft models. NOD/SCID mice (n=8 per group) were inoculated with COG-LL-317x (A) or RS4;11 (B) through tail vein injection within 8h of irradiation. Mice were treated with vehicle control (Con, black), DEX (green), RAP (blue), or DEX+RAP (red) by intraperitoneal injection. The rapamycin dose was 5mg/kg/day, and dexamethasone 30mg/kg/day as a single agent or 7.5mg/kg/day in combination with rapamycin. The EFS of mice was quantified as the time from the initiation of treatment until mice were killed due to treatment-related toxicity or disease. Each line represents the proportion of mice remaining event-free over time.