Acquired resistance to venetoclax (ABT-199) in t(14;18) positive lymphoma cells

Abstract

The chromosomal translocation t(14;18) in follicular lymphoma (FL) is a primary oncogenic event resulting in BCL-2 over-expression. This study investigates activity of the BH3 mimetic venetoclax (ABT-199), which targets BCL-2, and mechanisms of acquired resistance in FL.The sensitivity of FL cells to venetoclax treatment correlated with BCL-2/BIM ratio. Cells with similar expression of anti-apoptotic proteins, but with higher levels of BIM were more sensitive to the treatment. Venetoclax induced dissociation of BCL-2/ BIM complex and a decrease in mitochondrial potential. Interestingly the population of cells that survived venetoclax treatment showed increased p-ERK1/2 and p-BIM (S69), as well as a decrease in total BIM levels. Venetoclax resistant cells initially showed elevated levels of p-AKT and p-Foxo1/3a, a dissociation of BIM/BCL-2/BECLIN1 complex, and a decrease in SQSTM1/p62 level (indicating increased autophagy) together with a slight decline in BIM expression. After stable resistant cell lines were established, a significant reduction of BCL-2 levels and almost total absence of BIM was observed.The acquisition of these resistance phenotypes could be prevented via selective ERK/AKT inhibition or anti-CD20 antibody treatment, thus highlighting possible combination therapies for FL patients.

Keywords: follicular lymphoma; resistance; venetoclax.

Figure 1. Venetoclax induces proliferation inhibition and apoptosis in t(14;18) positive cells

(A) Apoptosis induction in primary FL samples after venetoclax treatment. Primary cells were treated with venetoclax for 4 H and Annexin-V/7-AAD based flow cytometry assay was performed to determine the percentage of apoptotic/necrotic cells. (B) An example (sample LY74) of quantitative flow cytometry analysis of BCL-2 and BIM expression (C) Correlation between BCL-2/BIM ratio and IC₅₀ values of venetoclax. BCL-2 and BIM expression (molecule number/cell) was analyzed by quantitative flow cytometry assay. IC₅₀ of venetoclax was calculated using data collected in 1a. (D) Cytotoxicity of venetoclax in primary FL samples treated for 72 H and analyzed with WST-1 assay. (E) A comparison of BCL-2, MCL-1, BIM, and cleaved caspase-3 protein expressions in primary FL samples.

Figure 2. The effect of venetoclax on t(14;18) positive cell lines

(A) Cytotoxicity of venetoclax in FL cell lines treated for 72 H and analyzed with WST-1 assay. (B) A comparison of pro- and anti-apoptotic proteins expression in untreated WSU-FSCCL (FS) and FC-TxFL2 (FC) cell lines. (C) Annexin-V/7-AAD analysis of FL cell lines treated with 100 nM venetoclax for 24 H. (D) WB analysis of cleaved PARP in FL cell lines after 24 H venetoclax treatment.

Figure 3. Cellular events proceeding and accompanying venetoclax induced apoptosis in FC-TxFL2 cell line

(A) BIM protein immunoprecipitation followed by BCL-2, MCL-1 and BCL-XL WB detection of lysates of FC-TxFL2 cells treated with 100 nM venetoclax for 2 H. (WL – whole cell lysate) (B) Decrease of mitochondrial potential after 1 H venetoclax treatment analyzed by JC-1 assay. (C) Cell cycle analysis cells treated with venetoclax for 2 H and analysis of subG0/G1 apoptotic cells treated with venetoclax for 10, 30, 60 and 120 minutes. (D) Inhibition of caspase-3 activation, PARP and BID cleavage (t-BID) and decrease of JNK1/2 phosphorylation with pan caspase inhibitor Q-VD-OPH after 2 H venetoclax treatment.

Figure 4. Activation of ERK1/2 and JNK1/2 in FC-TxFL2 cells treated with venetoclax

(A) Expression of p-ERK1/2, p-BIM and BIM in viable FC-TxFL2 cells isolated using dead cell removal kit (DCR) after 2 H venetoclax treatment. (B) Inhibition of ERK1/2 and p-BIM using ERK inhibitor SCH772984 in whole cell population after 2 H treatment. (C) Potentiation of venetoclax – induced apoptosis analyzed as subG0/G1 cell population by ERK inhibitor (*p < 0.05, ***p < 0.001). Cells were exposed to SCH772984 for 24 H followed by 2 H treatment with venetoclax. (D) Detection of BCL-2 and BIM proteins after immunoprecipitation with p-JNK1/2 antibody after 2 H venetoclax treatment. (E) Detection of autophagy using SQSTM1/p62 marker and Beclin1 (BCLN1) immunoprecipitation. Cells were treated with the increasing concentrations of venetoclax (up to 25 nM) for 16 days. (F) Analysis of apoptosis induction in FC-TxFL2 cells after SP600125/venetoclax treatment for 24 H (***p < 0.001).

Figure 5. AKT pathway inhibition potentiates venetoclax induced apoptosis and proliferation inhibition

(A) Western Blot analysis of p-AKT, p-Foxo1/3a and BIM in cells after 16 days' cultivation in the presence of increasing concentrations of venetoclax (up to 25 nM). (B) BKM120 potentiates venetoclax-induced apoptosis in FC-TxFL2 cells (** p<0.01, *** p<0.001). (C) Analysis of p-AKT, p-Foxo1/3a and BIM protein levels in cells treated with pan-PI3k inhibitor BKM120/venetoclax combination for 2 H. (D) Analysis of combination effect on proliferation inhibition using WST-1 assay in FC-TxFL2 cells after 72 H BKM120 (0.2 − 1 μM)/venetoclax (1 − 5 nM) (constant ratio 200:1) treatment. Combination index (CI) values were used to determine the combined effect as synergistic (<1), additive (=1), or antagonistic (>1) using Calcusyn software.

Figure 6. Acquired resistance to venetoclax in FL cells

(A) Venetoclax delays tumor growth of FC-TxFL2 xenograft in vivo. Venetoclax was administered at the indicated doses once daily by gavage starting 5 days after the cell injection for 21 days. When tumors reached the maximum allowed size or at day 27, they were frozen or disintegrated for further experiments. Time refers to days from the injection of cells. (B) Increased resistance to venetoclax of FC-TxFL2 cells isolated from tumors previously treated with 50 mg/kg/day venetoclax in comparison to cells treated with vehicle (**p < 0.01, ***p < 0.001). Mice were treated until tumors reached maximum allowed size. Then the tumors were harvested, dissociated and the effect of 20 H venetoclax treatment on isolated cells in vitro was determined by flow cytometric analysis of annexin-v/7-AAD staining. (C) Comparison of sensitivity of FC-TxFL2 cells isolated from tumors treated with 100 mg/kg/day venetoclax to cells treated with vehicle one or two weeks after the isolation. Cells were treated with venetoclax for 72 H and analyzed by WST-1 assay. (D) Effect of venetoclax on viability of FS-R (WSU-FSCCL) and FC-R (FC-TxFL2) cells with acquired resistance to venetoclax. (E) Comparison of proteins regulating apoptosis and autophagy in original FL cell lines with cell lines with acquired venetoclax resistance. (F) Cumulative proportion surviving curve of NSG mice after combination venetoclax/rituximab treatment (p < 0.05). Mice were treated with vehicle, 8 mg/kg rituximab i.p. (only one dose) at day 14 and/or then daily with 50 mg/kg venetoclax by gavage. Venetoclax treatment continued until the tumor reached maximum allowed size. Survival curves were calculated when tumor size reached 1000 mm³. Six mice per group per experiment were used.