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. 2012 Mar;10(3):347-59.
doi: 10.1158/1541-7786.MCR-11-0394. Epub 2012 Jan 12.

Phosphoinositide 3-kinase/AKT/mTORC1/2 signaling determines sensitivity of Burkitt's lymphoma cells to BH3 mimetics

Lindsay C Spender  1 , Gareth J Inman
Affiliations

Phosphoinositide 3-kinase/AKT/mTORC1/2 signaling determines sensitivity of Burkitt's lymphoma cells to BH3 mimetics

Lindsay C Spender et al. Mol Cancer Res. 2012 Mar.

Abstract

Burkitt's lymphoma (BL), driven by translocation and overexpression of the c-MYC gene, is an aggressive, highly proliferative lymphoma, and novel therapeutic strategies are required to overcome drug resistance following conventional treatments. The importance of the prosurvival BCL-2 family member BCL-X(L) in BL cell survival suggests that antagonistic BH3-mimetic compounds may have therapeutic potential. Here, we show that treatment of BL cell lines with ABT-737 induces caspase-3/7 activation and apoptosis with varying potency. Using selective inhibitors, we identify phosphoinositide 3-kinase (PI3K) as a proproliferative/survival pathway in BL cells and investigate the potential of combined pharmacologic inhibition of both the BCL-2 family and PI3K signaling pathway. PI3K/AKT inhibition and ABT-737 treatment induced synergistic caspase activation, augmented BL cell apoptosis, and rendered chemoresistant cells sensitive. Targeting mTORC1/2 with PP242 was also effective, either as a monotherapy or, more generally, in combination with ABT-737. The combined use of a dual specificity PI3K/mTOR inhibitor (PI 103) with ABT-737 proved highly efficacious. PI 103 treatment of BL cells was associated with an increase in BIM/MCL-1 expression ratios and loss of c-MYC expression. Furthermore, blocking c-MYC function using the inhibitor 10058-F4 also induced apoptosis synergistically with ABT-737, suggesting that maintenance of expression of BCL-2 family members and/or c-MYC by the PI3K/AKT/mTOR pathway could contribute to BL cell survival and resistance to ABT-737. The combined use of BH3 mimetics and selective mTORC1/2 inhibitors may therefore be a useful novel therapeutic approach for the treatment of B-cell malignancy, including chemoresistant lymphomas.

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Figures

Figure 1
Figure 1. Sensitivity of human BL cells to apoptosis induced by ABT-737
Cells were either left untreated or treated with a two-fold dilution series of ABT-737. (A) After 48 hours, the mean (± s.d, n=3) increase in the percentage of cells with sub-G1 DNA content above background was determined by PI staining. (B) The (mean ± s.d, n=3) percent increase in intracellular caspase 3/7 positive cells induced following 24 hours ABT-737 treatment was determined flow cytometry using PhiPhiLux. (C) Cells treated for 24 hours were lysed and analysed by western blotting for cleavage of the caspase substrate PARP. The proportion of cleaved PARP is indicated above each lane as a percentage of the total amount of PARP determined by densitometry and ImageJ software. A western blot for actin is shown as a loading control.
Figure 2
Figure 2. PI3K signaling contributes to BL cell proliferation and/or survival
(A) BL cells were treated for 2 hours with the pan PI3K inhibitor LY-294002 (10μM), lysed and analysed by western blotting for phosphorylation of the PI3K substrate AKT/PKB (ser473). (B, C) BL cells treated for 48 hours with LY-294002 (0, 5 and 10μM) were fixed, stained with PI and analysed by flow cytometry and CellQuest Pro for G1 arrest (B), and for cell death (C). (D) Ramos and BL2 cells were treated for 2 hours with DMSO (vehicle) or with 1 or 0.25μM of the AKT1/2 inhibitor (AKTiVIII). Cell lysates were analysed by western blot using phospho-specific and total AKT and S6 ribosomal protein antibodies as indicated. (E) BL cells were treated for 48 hours with LY-294002 (5μM) or AKTiVIII (1μM) and the amount of apoptosis above background determined by PI. Shown is the mean apoptosis induction above background from at least two experiments each performed in triplicate.
Figure 3
Figure 3. mTOR inhibitors as monotherapies for Burkitt’s Lymphoma
(A) Ramos cells were treated for 2 hours with LY-294002 (LY), the dual PI3K/mTOR inhibitor PI 103 or selective inhibitors of mTOR, Rapamycin (Rap) and the active site inhibitor PP242. Equal amounts of cell lysate were analysed by western blot. (B) BL cell lines were treated for 48 hours with LY-294002 (5μM), PI 103 (1μM), Rapamycin (2nM) or PP242 (1.25μM). Cells were analysed by PI staining for percent (mean ± s.d., n=3) cell death above background. (C) BL2 cells were pretreated with the pan-caspase inhibitor ZVAD-fmk (25μM) for 1 hour prior to PI 103 (1μM) addition for the indicated time periods. Cell lysates were analysed by western blot.
Figure 4
Figure 4. Synergistic effects of PI3K/AKT inhibitors and ABT-737
The combined effect of PI3K/AKT inhibitors and ABT-737 on apoptosis in BL cells was assessed by caspase 3/7 assay (A-D), and by PI staining (E, F). Ramos cells (A, C) or BL40 cells (B, D) were left untreated, or treated with concentrations of ABT-737 (ABT) the presence or absence of LY-294002 (LY) or AKTiVIII in equimolar ratios as indicated. Caspase activity was analysed by caspase 3/7-glo assay after 24 hours and potential synergism determined by the Chou-Talalay equation for dose effect analysis of drug combinations. Combination Indices (CI) with respect to the fraction affected (Fa-CI) plots and the values determined at ED50, ED75 and ED90 are shown directly below the relevant line graph. CI values of >1, =1 and <1 represent antagonism, additive effect and synergism respectively. (E, F) Ramos (E) and BL40 cells (F) were treated with solvent or ABT-737 (500nM) in the presence or absence of LY-294002 (5μM) or AKT inhibitor VIII (1μM). Cells were harvested after 48 hours, and the percent apoptosis (mean ± s.d., n=3) determined by PI staining.
Figure 5
Figure 5. Synergistic effects of the mTOR inhibitor PP242 and ABT-737
The combined effect of the active site mTOR inhibitor PP242 and ABT-737 on apoptosis in BL cells was assessed by caspase 3/7 activation assay (A, B), and by PI staining (C, D). Ramos (A) and BL40 cells (B) were left untreated or treated with concentrations of ABT-737 (ABT) in the presence or absence of PP242 in equimolar ratios. Increases in caspase activity were analysed by caspase 3/7-glo assay after 24 hours. Potential synergism was determined as described in Figure 4. (C) Ramos and (D) BL40 cells were pre-treated for 15 minutes with vehicle, PP242 (1.25μM) or LY-294002 (5μM), followed by addition of ABT-737 (500nM) or vehicle control. Cells were analysed by PI staining after 48 hours and the percent apoptotic cells above background (mean ± s.d., n=3) determined.
Figure 6
Figure 6. The dual PI3K/mTOR inhibitor PI 103 increases BIM:MCL-1 ratios and enhances ABT-737 sensitivity
The combined effect of PI 103 and ABT-737 on apoptosis in BL cells was assessed by caspase 3/7 activation assay (A, B), and by PI staining (C, D). Ramos (A) and BL40 cells (B) were left untreated or treated with concentrations of ABT-737 in the presence or absence of PI 103 in equimolar ratios. The potential synergistic induction of caspase activity was determined as described in Figure 4. (C) Ramos and (D) BL40 cells were pre-treated for 15 minutes with vehicle, PI 103 (1μM) or LY-294002 (5μM), followed by addition of ABT-737 (500nM) or vehicle control. The percent apoptotic cells above background (mean ± s.d., n=3) was determined by PI staining after 48 hours. (E) Ramos cells were treated with PI 103 (1μM) and cell lysates analysed by western blot. (F) Western blots (representative blots shown in (E)) were analysed by densitometry and ImageJ software and the ratio of BIM to MCL-1 protein expression determined over the time course of PI 103 treatment. BIM:MCL-1 ratios determined after 24 hours for untreated versus PI 103 treated cells were 0.78±0.03 and 3.2± 1.05 respectively (n=3). (G) Western blot analysis of the indicated proteins in Ramos cells treated with PI 103 (1μM) in the presence or absence of zVAD-fmk (25μM).
Figure 7
Figure 7
Inhibition of c-MYC synergises with ABT-737 to induce BL cell apoptosis. (A) Cells were treated for 48 hours with a two-fold dilution series of the Myc inhibitor 10058-F4 (Myc i), fixed, stained with propidium iodide and analysed by flow cytometry. The mean (± s.d, n=3) increase in the percentage of cells with sub-G1 DNA content above background is shown. (B) The combined effect of the MYC inhibitor and ABT-737 on apoptosis in BL cells was assessed by caspase 3/7 assay. BL cells were treated with solvent as a control, or treated with concentrations of ABT-737 (ABT) the presence or absence of 10058-F4 in equimolar ratios as indicated. Caspase activity was analysed by caspase 3/7-glo assay after 24 hours and potential synergism determined by the Chou-Talalay equation for dose effect analysis of drug combinations. Combination Indices (CI) were determined at ED50, ED75 and ED90. CI values of <1 represent synergism. (C) The effect of combining ABT-737 (500nM) with the MYC inhibitor (25μM) on apoptosis of BL cells was assessed by propidium iodide staining and flow cytometry after 48 hours treatment. The drug combination of ABT and LY-294002 was included as a positive control for augmented apoptosis.
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