Synergistic cooperation between ABT-263 and MEK1/2 inhibitor: effect on apoptosis and proliferation of acute myeloid leukemia cells

Abstract

In spite of intensive research to improve treatment of acute myeloid leukemia (AML) more than half of all patients continue to develop a refractory disease. Therefore there is need to improve AML treatment. The overexpression of the BCL-2 family anti-apoptotic members, like BCL-2 or BCL-xL has been largely reported in lymphoid tumors but also in AML and other tumors. To counteract the anti-apoptotic effect of BCL-2, BH3 mimetics have been developed to target cancer cells. An increase in activity of ERK1/2 mitogen activated protein (MAP) kinase has also been reported in AML and might be targeted by MEK1/2 inhibitors. Hence, in the current work, we investigated whether the association of a BH3 mimetic such ABT-263 and the MEK1/2 inhibitor pimasertib (MEKI), was efficient to target AML cells. A synergistic increasing of apoptosis was observed in AML cell lines and in primary cells without affecting normal bone marrow cells. Such cooperation was confirmed on tumor growth in a mouse xenograft model of AML. In addition we demonstrated that MEKI sensitized the cells to apoptosis through its ability to promote a G1 cell cycle arrest. So, this combination of a MAP Kinase pathway inhibitor and a BH3 mimetic could be a promising strategy to improve the treatment of AML.

Keywords: BH3 mimetic drugs; MEK inhibitors; acute myeloid leukemia.

Figure 1. MEKI and ABT-263 synergize to inhibit cell proliferation and induce apoptosis in AML cell lines

A. and C. HL-60, THP-1 or U-937 (A) and MV-4–11 or MOLM-13 (C) cells were incubated with increasing concentrations of ABT-263, MEKI or both at a constant ratio (Mix). The amount of viable cells was measured through their ATP content by chemoluminescence after a 72-h culture (left column). The percentage of apoptotic cells was determined by flow cytometry with a MMP probe after a 24-h incubation (right column). Combination indexes were calculated at ED 50, 75 and 90 using the Calcusyn software. B. HL-60, THP-1 or U-937 cells were treated for 24 h with 1 μM MEKI (black bars), 200 nM ABT-263 (light grey bars) or both (MIX, grey bars). Apoptosis was then measured by flow cytometry as described in Figure 1. Mean +/− SD of seven experiments is shown. The calculated additive effect of both drugs is also shown (white bars) and compared to the measured MIX effect using the Student paired t test. C. MV-4–11 or MOLM-13 (C) cells were incubated with increasing concentrations of ABT-263, MEKI or both at a constant ratio (Mix). The amount of viable cells was measured through their ATP content by chemoluminescence after a 72-h culture (left column). The percentage of apoptotic cells was determined by flow cytometry with a MMP probe after a 24-h incubation (right column).

Figure 1. MEKI and ABT-263 synergize to inhibit cell proliferation and induce apoptosis in AML cell lines

A. and C. HL-60, THP-1 or U-937 (A) and MV-4–11 or MOLM-13 (C) cells were incubated with increasing concentrations of ABT-263, MEKI or both at a constant ratio (Mix). The amount of viable cells was measured through their ATP content by chemoluminescence after a 72-h culture (left column). The percentage of apoptotic cells was determined by flow cytometry with a MMP probe after a 24-h incubation (right column). Combination indexes were calculated at ED 50, 75 and 90 using the Calcusyn software. B. HL-60, THP-1 or U-937 cells were treated for 24 h with 1 μM MEKI (black bars), 200 nM ABT-263 (light grey bars) or both (MIX, grey bars). Apoptosis was then measured by flow cytometry as described in Figure 1. Mean +/− SD of seven experiments is shown. The calculated additive effect of both drugs is also shown (white bars) and compared to the measured MIX effect using the Student paired t test. C. MV-4–11 or MOLM-13 (C) cells were incubated with increasing concentrations of ABT-263, MEKI or both at a constant ratio (Mix). The amount of viable cells was measured through their ATP content by chemoluminescence after a 72-h culture (left column). The percentage of apoptotic cells was determined by flow cytometry with a MMP probe after a 24-h incubation (right column).

Figure 2. Protein expression of the Bcl-2 family members following ABT-263 and MEKI treatment

HL-60, THP-1 or U-937 cells were analyzed by western blot for expression of the Bcl-2 family proteins. The three cell lines were treated for 24 h with 1 μM MEKI, 200 nM ABT-263 or both (Mix) and analyzed for Erk1/2 phosphorylation, BIM, Bcl-2, BAX, Bcl-XL, Mcl-1 and PUMA expression and Caspase 3 cleavage.

Figure 3. BIM accumulation does not participate in the cooperation between ABT-263 and MEKI to induce apoptosis in THP-1

THP-1 cells or THP-1 cells depleted for BIM by RNA interference were treated with 1 μM MEKI, 200 nM ABT-263 or both in combination (Mix) for 24 h and analyzed for BIM expression A. and apoptosis induction B.

Figure 4. ABT-263-induced apoptosis depends on the cell cycle block in G1 phase

A. THP-1 and U-937 cells were untreated or treated with 1 μM MEKI or a combination of 1 μM MEKI and 200 nM ABT-263 for 24 h. Cells were then fixed, permeabilized and stained with anti-cleaved caspase 3 antibody (Y axis) and Vibrant Violet Cycle probe (X axis). Cells were then analyzed by flow cytometry. B. HL-60, THP-1 or U-937 cells were treated for 24 h with 1 μM MEKI, 200 nM ABT-263 or both (Mix) and analyzed by western blot for p27kip1, p21cip1 and tubulin expression.

Figure 5. CHX induces a cell cycle block in G1 phase and synergizes with ABT-263 to increase apoptosis

A. THP-1 and U-937 cells were pre-treated or not with 3 μM CHX for 24 h and treated or not with 200 nM ABT-263 for 24 h. Cells were then fixed, permeabilized and stained with anti-cleaved caspase 3 antibody (Y axis) and Vibrant Violet Cycle probe (X axis) and analyzed by flow cytometry. B. THP-1 and U-937 cells were pre-treated or not with 3 μM CHX for 24 h and treated or not with 1 μM MEKI, 200 nM ABT-263 or both (Mix) for 24 h. Cells were then fixed, permeabilized and stained with anti-cleaved caspase 3 antibody and analysed by flow cytometry. Mean +/− SD of three experiments is shown. C. THP-1 (black squares) and U-937 (white diamonds) cells were pre-treated with MEKI (200 nM) or CHX (3 μM) for 24 h. Samples were then treated or not with 200 nM ABT-263 for 24 h and analyzed by flow cytometry for cell cycle and caspase 3 activation. The graph represents the correlation between the percentage of ABT-263-induced apoptosis and the increased amount of cells in G1 phase.

Figure 6. MEKI and ABT-263 synergize to induce apoptosis in leukemic stem cells

Mononuclear cells from AML or normal control bone marrows were labelled with APC-CD34, PC5-CD38, PE-CD123 and FITC-annexin V. A. The percentage of CD34+ cells, CD38- cells in the CD34+ population and CD123+ cells in the CD34+/CD38- population was plotted for normal control and AML samples. Results are expressed as mean +/–SD and are representative of 10 and 17 experiments for respectively control and AML bone marrows. P indicates the significance using the Mann-Whitney test. Annexin-V+ cells were considered as apoptotic and their percentage was evaluated in the CD34+/CD38+, CD34+/CD38-, and CD34+/CD38-/CD123+ populations. B. Apoptosis induction was evaluated in 9 AML samples as indicated in each of the three populations after treatment with MEKI (black bars), ABT-263 (white bars) or both (grey bars). C: Apoptosis induction was evaluated in 6 normal control samples in the CD34+/CD38+ and CD34+/CD38- populations.

Figure 7. MEKI and ABT-263 synergize to reduce tumor growth in vivo

A. HL60 cells were subcutaneously injected in NSG mice and mice were treated five days a week with vehicle control, MEKI (0.2 mg/kg), ABT-263 (1 mg/kg) or both. The growth rate was calculated from the measurement of the tumour volume twice a week and defined as the increase in mm3/day in each mouse. The graph represents the values of two independent experiments performed on 10 mice/group for each experiment. B. Histological sections of tumours stained with anti-activated caspase 3 antibody. A significant field of slides in tumours from either vehicle-, MEKI-, ABT-263- or MEKI + ABT-263-fed mice is shown as indicated.