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. 2015 Jan 15;6(1):e1593.
doi: 10.1038/cddis.2014.525.

MCL-1 and BCL-xL-dependent resistance to the BCL-2 inhibitor ABT-199 can be overcome by preventing PI3K/AKT/mTOR activation in lymphoid malignancies

G S Choudhary  1 , S Al-Harbi  2 , S Mazumder  2 , B T Hill  3 , M R Smith  3 , J Bodo  4 , E D Hsi  4 , A Almasan  2
Affiliations

MCL-1 and BCL-xL-dependent resistance to the BCL-2 inhibitor ABT-199 can be overcome by preventing PI3K/AKT/mTOR activation in lymphoid malignancies

G S Choudhary et al. Cell Death Dis. .

Erratum in

Abstract

Overexpression of anti-apoptotic BCL-2 family members is a hallmark of many lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) that can be targeted with small molecule inhibitors. ABT-199 is a rationally designed BCL-2 homology (BH)-3 mimetic that specifically binds to BCL-2, but not to MCL-1 and BCL-xL. Although the thrombocytopenia that occurs with navitoclax treatment has not been a problem with ABT-199, clinical trials in CLL could benefit by lowering the ABT-199 concentration through targeting other survival pathways. In this study, we investigated the mechanisms of resistance that develops to ABT-199 therapy by generating ABT-199-resistant (ABT199-R) cell lines via chronic exposure of NHL cell lines to ABT-199. Acquired resistance resulted in substantial AKT activation and upregulation of MCL-1 and BCL-xL levels that sequestered BIM. ABT199-R cells exhibited increased MCL-1 stability and failed to activate BAX in response to ABT-199. The ABT-199 acquired and inherent resistant cells were sensitized to treatment with ABT-199 by inhibitors of the PI3K, AKT, and mTOR pathways, NVP-BEZ235 and GS-1101. NVP-BEZ235, a dual inhibitor of p-AKT and mTOR, reduced MCL-1 levels causing BIM release from MCL-1 and BCL-xL, thus leading to cell death by BAX activation. The PI3Kδ inhibitor GS-1101 (idelalisib) downregulated MCL-1 and sensitized ABT199-R cells through AKT-mediated BAX activation. A genetic approach, through siRNA-mediated down-regulation of AKT, MCL-1, and BCL-xL, significantly decreased cell survival, demonstrating the importance of these cell survival factors for ABT-199 resistance. Our findings suggest a novel mechanism that modulates the expression and activity of pro-survival proteins to confer treatment resistance that could be exploited by a rational combination therapeutic regimen that could be effective for treating lymphoid malignancies.

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Figures

Figure 1
Figure 1
DLBCL cells with low MCL-1 and/or BCL-xL expression are sensitive to ABT-199 and develop resistance after chronic ABT-199 exposure by upregulating MCL-1, BCL-xL, and p-AKT levels. (a) Expression levels of the pro-survival proteins MCL-1, BCL-2, and BCL-xL in DLBCL (SU-DHL-6, OCL-LY-19, SU-DHL-16), follicular lymphoma (DOHH2), acute lymphoblastic leukemia (Nalm6, Reh), and CLL (Mec2, MO1040) cell lines. β-actin was used as the loading control. (b) The indicated cell lines were treated with 50 nM ABT-199 for 48 h. The viability shown represents the percentage of live cells relative to control cells treated with dimethyl sulfoxide. Parental and ABT-199-R (c) SU-DHL-6 and (d) OCl-Ly-19 cells were treated with the indicated concentrations of ABT-199 for 48 h and cell viability was determined by Annexin V-PI staining. Control cells were treated with dimethyl sulfoxide. (e) Expression levels of: (i) pro-survival proteins MCL-1, BCL-2, and BCL-xL, (ii) pro-apoptotic proteins BIM, NOXA, and PUMA, and (iii) total AKT and p-AKT (Ser473) in untreated parental and ABT199-R-derivative DLBCL cell lines at the indicated time. SU-DHL-6 parental and resistant cells. Both panels represent one experiment with BCL-2 and β-actin serving as loading controls. (f) Cells were treated with ABT-199 for the indicated time. MCL-1, BCL-2, BCL-xL, BIM, NOXA, PUMA, and cleaved caspase-3 were determined by immunoblotting. β-actin was used as a loading control. Standard deviation (S.D.) is indicated in b–d as error bars (N=3). The experiments in a, e, and f are representative of three independent experiments. Numbers below blots indicate increase in protein levels as determined by ImageJ quantification, which in case of pAKT was normalized to total AKT levels
Figure 2
Figure 2
ABT-199-resistance is associated with the upregulation of Mcl-1 and Bcl-xL mRNA and increased MCL-1 protein stability. RNA was extracted from parental and ABT199-R SU-DHL-6 and OCl-LY-19 cells after culture in the absence of ABT-199 for 72 h. (a) Mcl-1, (b) Bcl-xL, and (c) Bcl-2 fold change was analyzed by qRT-PCR. RNA levels of parental cells were set to 1 for analysis (*P<0.04, **P<0.05). MCL-1 protein half-life was determined by treating parental and ABT199-R (d) SU-DHL-6 and (f) OCl-LY-19 cells with cycloheximide (10 μg/ml) for the indicated time, followed by immunoblotting. β-actin was used as the loading control. Data in e and g were quantified by ImageJ. The experiments from a to g are representative of three independent experiments
Figure 3
Figure 3
BIM associates with increased MCL-1 and BCL-xL protein in ABT199-R cells. MCL-1 and BCL-xL were immunoprecipitated with (a) MCL-1- and (b) BCL-xL-specific antibodies and their association with BIM and NOXA was probed by immunoblotting with specific antibodies. Reciprocal immunoprecipitation-western blotting for BIM was performed in (c) SU-DHL-6 (d) OCL-LY-19 parental and ABT199-R cell lines and its association with MCL-1, BCL-xL, and BCL-2 was analyzed. (e) Parental and resistant cells were treated with ABT-199 at the indicated time and BIM immunoprecipitates were examined by immunoblotting for association with MCL-1, BCL-xL, and BCL-2 in SU-DHL-6 parental and ABT199-R cells. (f) SU-DHL-6 ABT199-R cells were transfected with siAKT, siMcl-1, siBcl-xL or siControl and treated with the indicated concentration of ABT-199 for 24 h. Cell viability was determined by Annexin V-PI staining. Control cells were treated with dimethyl sulfoxide (*P<0.02, **P<0.001) (N=2). β-actin was used as loading control. The experiments from a to e are representative of three independent experiments. L.E and H.E: low and high exposure, respectively. #, non-specific band
Figure 4
Figure 4
ABT-199 in combination with NVP-BEZ235 sensitizes ABT199-R cells. Parental and resistant derivative DLBCL cell lines (a) SU-DHL-6 (b) SU-DHL-6 ABT199-R (c) OCl-Ly-19 ABT-199R (e) SU-DHL-16, and (d) FL cell line DOHH2 were treated with the indicated concentration of ABT-199 and NVP-BEZ235, alone and in combination for 24 h. Cell viability was determined by Annexin V-PI staining represented as percentage relative to control cells treated with dimethyl sulfoxide. Standard deviation (S.D.) is indicated by the error bars (N=3)
Figure 5
Figure 5
NVP-BEZ235 downregulates MCL-1 through p-AKT and mTOR inhibition and causes BAX activation following BIM release from MCL-1. (a) Expression of MCL-1, BCL-xL, BCL-2, BIM, p-AKT (Ser473), AKT, p-p70S6 kinase (Thr389), p-70S6 kinase, p-4EBP1 (Ser65), and cleaved caspase-3 in SU-DHL-6 ABT199-R and OCL-LY-19 ABT199-R cells treated with ABT-199 (400 nM), NVP-BEZ235 (40 nM) or in combination for 6 h. Both panels represent one experiment with BCL-2 and β-actin serving as loading controls. * denotes a non-specific band. SU-DHL-6 ABT199-R cells were treated with ABT-199R or NVP-BEZ235, alone or in combination, in concentrations as in a for 6 h and cells were lysed with 2% CHAPS buffer. (b) MCL-1 and (c) BIM were immunoprecipiated and their corresponding binding partners MCL-1, BIM, NOXA, BCL-2, or BCL-xL were analyzed by western blotting with specific antibodies. (d) Parental and resistant SU-DHL-6 cells treated with ABT-199R, NVP-BEZ235, or their combination (concentration as in a) were lysed with 1% CHAPS buffer and active BAX was immunoprecipated with BAX 6A7 and probed by BAX N20 antibodies by immunoblotting. The experiments in a to d are representative of three independent experiments
Figure 6
Figure 6
GS-1101 in combination with ABT-199 targets the PI3K pathway, sensitizing ABT-199-R cells by MCL-1 downregulation and BAX activation. Parental and ABT-199-R-derivative DLBCL cell lines SU-DHL-6 (a–d), OCL-LY-19 ABT199-R (e) and the FL cell line DOHH2 (f) were treated for 24 h with the indicated concentration of ABT-199, RAD001, and GS-1101, alone or in combination. Cell viability was determined by staining with Annexin V-PI, and represented as the percentage relative to control cells treated with dimethyl sulfoxide. Standard deviation (S.D.) is indicated in a–e by error bars (N=3). (g) Expression levels of MCL-1, BCL-xL, BCL-2, BIM, p-AKT (Ser473), p-AKT (Thr308), AKT, and cleaved caspase-3 in SU-DHL-6 ABT199-R cells treated with ABT-199 (400 nM) and GS-1101 (4 μM) alone or in combination, and OCL-LY-19 ABT199-R treated with ABT-199 (400 nM) and GS-1101 (8 μM), alone or in combination, for 24 h. β-actin was used as the loading control. (h) ABT-199-R SU-DHL-6 cells treated with ABT-199R (400 nM), GS-1101 (4 μM), alone or in combination, were lysed with 1% CHAPS buffer, and active BAX was immunoprecipated with BAX 6A7 and detected using BAX N20 antibodies following immunoblotting. The experiments in g and h are representative of three independent experiments
Figure 7
Figure 7
Model for ABT-199-resistance and PI3K-mTOR-mediated targeting of ABT-199-R cells. (a) ABT-199 targets BCL-2 in sensitive cells and displaces BIM to cause apoptosis through BAX activation. (b) ABT-199 does not target MCL-1 and BCL-xL, which confers resistance by sequestering BIM displaced from BCL-2. (c) NVP-BEZ235 inhibits the PI3K and mTOR pathways, which interfere with MCL-1 stability, thereby freeing BIM, which then activates BAX, leading to release of cytochrome c to cause cell death
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