mTOR regulates cell survival after etoposide treatment in primary AML cells

Abstract

Acute myeloid leukemia cells have constitutive activation of phosphatidylinositol 3(PI3) kinase and require PI3 kinase activation for survival; however, the function of the PI3 kinase pathway in the survival of leukemic cells is poorly defined. We have studied the role of one PI3 kinase substrate, mTOR (mammalian target of rapamycin), in primary leukemic cells. In initial experiments, we have defined a novel growth medium that improves survival of acute myeloid leukemia (AML) blasts in long-term suspension culture and the survival of leukemic stem cells in short-term cultures. Inhibition of mTOR using rapamycin leads to a modest decrease in cell survival after 2 days of incubation with more significant decrease in survival after 7 days of culture. However, when rapamycin is added to etoposide in 2-day cultures, there is a dramatic increase in the cytotoxicity of etoposide against AML blasts. Furthermore, etoposide consistently decreased the engraftment of AML cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals, and this effect was enhanced by coincubation with rapamycin, demonstrating that mTOR regulates survival of AML stem cells after etoposide treatment. These results suggest that rapamycin in combination with etoposide-based chemotherapy may be efficacious in the treatment of AML.

Figures

Figure 1.

EGM and EGM2 media support the survival of leukemic stem cells. (A-C) Primary AML cells were thawed and plated at 1 × 106 cells/mL in indicated medium. Viable cell number was counted by trypan blue exclusion. (D) Primary AML cells were thawed and allowed to recover for 2 hours. Viable cells were counted, and 5 million cells per animal were injected immediately (day 0) or after 1 to 2 days of incubation in EGM2 medium. No adjustments to cell numbers were made after incubation. Cells were injected into sublethally irradiated NOD/SCID mice, and animals were monitored for 7 weeks. Animals were humanely killed and bilateral femurs were harvested. Percentage shown is average of human CD45+/CD33+ cells in the bone marrow. Each dot indicates 1 animal, and the central bar is the mean of the average engraftment.

Figure 2.

AML cells require mTOR for long-term survival in vitro. (A) Samples were thawed and immediately lysed for analysis. Samples were lysed and analyzed by SDS-PAGE and Western blotting for the indicated proteins. U937 cells treated with rapamycin (lane 1, +) or not treated were used as control samples. Note that in U937 samples, inhibition of phosphorylation of 4EBP-1 is not complete after treatment with rapamycin. (B) Samples were thawed, allowed to recover for 2 hours, counted, and plated at 2 × 106 cells/mL in 96-well plates. Cells were incubated in EGM2 medium for 48 hours in indicted concentrations of rapamycin, and relative cell survival was measured using the XTT assay. Results were standardized so that untreated samples were assigned a value of 1.0. (C) Cells were processed and cultured as for panel B, but plates were incubated for 7 days before analysis by XTT assay. (D) Normal human CD34 cells were cultured for 2 or 7 days in serum-free medium with hematopoietic cytokines, and relative cell survival was measured using the XTT assay described under “Materials and methods.”

Figure 3.

AML cells require mTOR for survival of genotoxic stress. (A-C) Primary patient samples were thawed and plated in 96-well plates at 2 × 106 cells/mL in increasing concentrations of etoposide for 48 hours. Relative cell survival was measured using an XTT assay. All values are expressed as a fraction of the untreated controls. Cells were either incubated in increasing concentrations of etoposide alone (□) or increasing concentrations of etoposide with 10 μM rapamycin present (▪). (D) Normal purified CD34+ hematopoietic stem cells were thawed, plated at 1 × 106 cells/mL in 96-well dishes, and analyzed as described under “Materials and methods.” Cells were incubated in different concentrations of etoposide in the absence (□) or presence (▪) of rapamycin.

Figure 4.

Rapamycin inhibits mTOR in primary AML samples. (A-B) Patient samples were thawed and incubated for 16 hours in different concentrations of rapamycin as shown. As controls, U937 cells were similarly incubated. After incubation, cells were harvested, lysed, and analyzed for expression and phosphorylation of p70S6 kinase by Western blotting. (C-D) Patient samples were thawed and incubated for 16 hours in nothing (-), 0.1% DMSO (D), 10 μM etoposide (E), 10 μM rapamycin (R), or a combination of etoposide and rapamycin at the same concentrations (E + R). Cells were harvested, lysed, and analyzed by Western blotting.

Figure 5.

Rapamycin enhances apoptosis in primary AML cells. Primary patient samples were thawed and incubated as for Figure 3. Cells were harvested, pelleted, and incubated with anti-annexin V-FITC and PI. Single-cell suspensions were analyzed by FACScan (Becton Dickinson). Early apoptotic cells were scored as annexin V positive, PI negative to exclude necrotic cells. (A) FACS data from a representative sample. (B) Graph of percentage early apoptotic cells for 3 different patient samples indicated by white, hatched, or black bars.

Figure 6.

AML stem cells require mTOR for survival of genotoxic stress. Cells were thawed, and 5 × 106 cells per animal were injected immediately or incubated overnight in the presence of indicated compound. Etoposide was used at 5 μM and rapamycin at 10 μM. Cells were injected into sublethally irradiated mice and monitored for 7 weeks. Animals were humanely killed, bilateral femurs were harvested and analyzed for the presence of human cells. Percentage engraftment is indicated as percentage of human CD45+/CD33+ cells present. (C) Purified CD34 cells (1 × 106) per animal were incubated overnight in the indicated conditions. Cells were injected in NOD/SCID mice as above and analyzed as above. In this case, analysis confirmed expression of CD45+CD33+ cells and CD45+CD19- cells, indicating myeloid and lymphoid engraftment. For comparison, values shown are the percentage of CD45+CD33+ cells, but similar effects were seen comparing CD19+ cells or total CD45+ populations. Note that because of morbidity, some conditions show results of only 2 animals, and standard deviations in this experiment could not be calculated.