Dexamethasone treatment promotes Bcl-2 dependence in multiple myeloma resulting in sensitivity to venetoclax

Abstract

Venetoclax (ABT-199), a specific inhibitor of the anti-apoptotic protein Bcl-2, is currently in phase I clinical trials for multiple myeloma. The results suggest that venetoclax is only active in a small cohort of patients therefore we wanted to determine its efficacy when used in combination. Combining venetoclax with melphalan or carfilzomib produced additive or better cell death in four of the five cell lines tested. The most striking results were seen with dexamethasone (Dex). Co-treatment of human myeloma cell lines and primary patient samples, with Dex and venetoclax, significantly increased cell death over venetoclax alone in four of the five cell lines, and in all patient samples tested. The mechanism by which this occurs is an increase in the expression of both Bcl-2 and Bim upon addition of Dex. This results in alterations in Bim binding to anti-apoptotic proteins. Dex shifts Bim binding towards Bcl-2 resulting in increased sensitivity to venetoclax. These data suggest that knowledge of drug-induced alterations of Bim-binding patterns may help inform better combination drug regimens. Furthermore, the data indicate combining this novel therapeutic with Dex could be an effective therapy for a broader range of patients than would be predicted by single-agent activity.

Trial registration: ClinicalTrials.gov NCT01794507.

Figure 1. Multiple myeloma cell lines are insensitive to venetoclax

A. KMS11, KMS18, MM.1s, 8226, and OPM2 were treated for 24 h with indicated concentrations of venetoclax. B. 8226 cells were transfected with the indicated siRNA. 12 h post transfection cells were treated with venetoclax for 24 h. C. KMS18 was treated with venetoclax for 24 h and protein lysates were prepared and subjected to co-immunoprecipitation with anti-Mcl-1, anti-Bcl-x_L, and anti-Bcl-2 antibodies. Resulting protein complexes were subjected to Western blot analysis using anti- Mcl-1, Bcl-x_L, and Bcl-2. Additionally, 20 μg of the protein lysates were subjected to Western blotting using the above antibodies to determine the protein input for each treatment. Cell viability in A. and B. was determined by flow cytometry following annexin-V-FITC/PI staining, Annexin = Annexin-V. The data in A and B are presented as the means (±SE) of 3 independent experiments. The Western blot in C is representative of four experiments.

Figure 2. Venetoclax is highly active in combination therapy

KMS18, 8226, MM.1s, KMS11, and OPM2 were treated with indicated concentrations of venetoclax alone or in combination with the indicated concentration of A. Carfilzomib, or C. Dexamethasone for 24 h. B. KMS18 was treated with Carfilzomib for 24 h and protein lysates subjected to input analysis and co-immunoprecipitation as described in Figure 1. Densitometry measurements of the Bim bound to Mcl-1, Bcl-x_L, and Bcl-2 under each treatment condition is represented as pie charts and the size of the pie is proportional to the total amount of bound Bim present under each treatment, relative to the control (1.0). Carfilzomib = 1.05. The data in A and C are presented as the means (±SE) of 3 independent experiments. The blot in B is representative of three experiments and densitometry average of two experiments.

Figure 3. Dexamethasone synergizes with venetoclax through the induction of Bcl-2 and Bim

A. MM.1s and KMS18 were treated with the indicated concentrations of venetoclax (Ven) and dexamethasone alone or in combination for 24 h. Real-time PCR was performed on cDNA generated from RNA isolated from control and treated samples. The data are presented as the means (±SE) of 3 independent experiments. B. Protein lysates were obtained from MM.1s and KMS18 6 and 24 h following treatment in A. Twenty μg of proteins were subjected to Western blotting and membranes probed with anti-Mcl-1, anti-Bcl-x_L, anti-Bcl-2, anti-Bim, anti-Noxa, and anti-β-Actin antibodies. Blots are representative of two experiments.

Figure 4. Co-treatment of MM.1s and KMS18 cell lines with venetoclax and dexamethasone induces changes in the pattern of Bim binding to anti-apoptotic proteins, resulting in apoptosis

Lysates were obtained from A. MM.1s or B. KMS18 and protein subjected to co-immunoprecipitation and input Western blot analysis as described in Fig. 1. Densitometry measurements of the Bim bound to Mcl-1, Bcl-x_L, and Bcl-2 under each treatment condition, as described in Fig. 2. A. MM.1s control = 1.00, Dex = 1.48, Venetoclax = 1.07, Combination = 0.89. B. KMS18 control = 1.00, Dex = 1.2, Venetoclax = 1.09, Combination = 0.65. Blots are representative of at least four experiments, densitometry average of two experiments.

Figure 5. A synergistic response to the combination of venetoclax and dexamethasone in plasma cells from myeloma patients

Two million total cells from buffy coats were re-suspended in medium for culture, while the remaining cells were subjected to CD138+ cell isolation. Cells were treated with the indicated concentrations of venetoclax either alone or in combination with 0.5 μM dexamethasone for 24 h. Apoptosis was determined by staining with antibodies against CD38, CD45, and Annexin-V-FITC. Lysates were obtained from isolated CD138+ cells and protein subjected to co-immunoprecipitation and Western blot analysis as described in Fig. 1.

Figure 6

Model of Dexamethasone-induced Bcl-2 priming.