Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax

Abstract

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2's hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca²⁺ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP₃R). The cell-permeable Bcl-2/IP₃R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2's BH4 domain, unleashing pro-apoptotic Ca²⁺-release events. We compared eight "primed to death" diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC₅₀ using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP₃R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP₃R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca²⁺ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca²⁺-dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca²⁺-signaling events via its BH4 domain.

Keywords: B-cell lymphoma; BIRD-2; anti-apoptotic Bcl-2; apoptosis; venetoclax.

Figure 1. The apoptotic response of eight different DLBCL cell lines towards venetoclax treatment

(A) Representative dot plots from flow cytometric analysis of Annexin V-FITC/7-AAD stained SU-DHL-4, PFEIFFER, KARPAS-422, SU-DHL-6, TOLEDO, OCI-LY-18, OCI-LY-1, and Ri-1 cells, treated with venetoclax at a concentration (indicated in the left top corner of the dot plot) around its IC₅₀ value during 24h (10 000 cells per analysis). (B) Concentration-response curves of the 8 different DLBCL cell lines after incubation with increasing concentrations of venetoclax for 24h. The apoptotic population was defined as the Annexin V-FITC/7-AAD-positive fraction. Data represented are average ± SD (N≥3).

Figure 2. Positive correlation between the IC₅₀ values for venetoclax determined in this project and previously published IC₅₀ values

Linear regression analysis of the IC₅₀ values obtained for venetoclax from the concentration-response curves of Figure 1 against the previously published results obtained by Souers et al. [11] for respectively six different DLBCL cell lines.

Figure 3. Heterogeneity in the apoptotic response of eight different DLBCL cell lines towards BIRD-2 treatment

(A) Representative dot plots from flow cytometric analysis of Annexin V-FITC/7-AAD-stained SU-DHL-4, KARPAS-422, SU-DHL-6, TOLEDO, OCI-LY-18, Ri-1, PFEIFFER and OCI-LY-1 cells, treated with BIRD-2 at a concentration (indicated in the left top corner of the dot plot) around its IC₅₀ value, during 24h (10 000 cells per analysis). (B) Concentration-response curves of the different DLBCL cell lines after incubation with increasing concentrations of BIRD-2 for 24h. The apoptotic population was identified as the Annexin V-FITC/7-AAD-positive fraction. Data represented are average ± SD (N≥3).

Figure 4. The IC₅₀ values of the analysed DLBCL cell lines indicate an opposite response to BIRD-2 versus venetoclax

The different IC₅₀ values were determined via the concentration-response curves (Figures 1 and 3) and the log IC₅₀ values for BIRD-2 were plotted as a function of the log IC₅₀ values for venetoclax. These IC₅₀ values were subjected to statistical analysis via linear regression.

Figure 5. Positive correlation between the IP₃R2 levels and the BIRD-2 sensitivity

(A-B) Representative western blots of the IP₃R2 (A) and Bim_EL (B) expression levels. GAPDH was used as loading control. (C-D) Quantification of IP₃R2 (C) and Bim_EL (D) expression levels. Data are represented as the average ± SD of N≥3 with ^** p<0.01 and ^***p<0.001 obtained via a repeated measure ANOVA with a Bonferroni’s post-hoc test versus SU-DHL-4. (E-F) Correlation between the expression levels of IP₃R2 (E) and Bim_EL (F) with the IC₅₀ values of BIRD-2, subjected to statistical analysis via linear regression.

Figure 6. Intracellular Ca²⁺ has a primary role in the BIRD-2-induced cell death

Analysis of Annexin V-FITC/7-AAD-negative cells (living cells (%), A) and caspase-3-negative cells (B) obtained using flow-cytometric analysis of KARPAS-422, OCI-LY-18, Ri-1 and SU-DHL-4 cells treated with or without BIRD-2 and 10 μM BAPTA-AM for 2h. Data are represented as mean ± SEM >3 independent experiments. Significance was obtained using a two-tailed paired t-test with ^* p< 0.05, ^**p<0.01.

Figure 7. Intracellular Ca²⁺ does not contribute to the venetoclax-induced cell death

Analysis of Annexin V-FITC/7-AAD-negative cells (living cells (%), A) and caspase-3-negative cells (B) obtained using flow-cytometric analysis of KARPAS-422, OCI-LY-1, OCI-LY-18, Ri-1 and SU-DHL-4 cells treated with or without venetoclax and 10 μM BAPTA-AM for 4h. Data are represented as mean ± SEM >3 independent experiments. Significance was obtained using a two-tailed paired t-test with ^* p< 0.05, ^**p<0.01, ^***p<0.001.

Figure 8. Synergistic effect of BIRD-2 and venetoclax in SU-DHL-4 cells, correlating to Bim upregulation in BIRD-2-treated SU-DHL-4 cells

(A) SU-DHL-4 cells were treated for 24h with various concentrations of venetoclax alone (black bars), BIRD-2 alone (blue bars) or a combination of venetoclax/DMSO Ctrl (0.03%) and BIRD-2 (red bars). Cell death was measured using flow cytometry of Annexin V-FITC/7-AAD-stained cells and plotted as the BIRD-2- or venetoclax-induced apoptotic fraction. The combination index, calculated using the response additive method (CI= (E_venetoclax+ E_BIRD-2)/E_{venetoclax+BIRD-2}) was measured as indication for synergistic or additive cell killing. Data are represented as average ± SEM of N=5. Statistical significance was determined with a two-way ANOVA with a Bonferroni post-hoc test comparing E_venetoclax or E_BIRD-2 with E_{venetoclax+BIRD-2} for the different venetoclax concentrations. (B) Representative western blots showing the Bim_EL-expression levels after 24h treatment of SU-DHL-4 cells with 3 μM BIRD-2 in the presence or absence of 10 μM BAPTA-AM. Vinculin was used as loading control. Data are represented as the average ± SD of N≥3 with ^* p<0.05 obtained via Wilcoxon signed rank test.