Bruton's tyrosine kinase inhibition increases BCL-2 dependence and enhances sensitivity to venetoclax in chronic lymphocytic leukemia

Abstract

Although the BTK inhibitor ibrutinib has transformed the management of patients with chronic lymphocytic leukemia (CLL), it does not induce substantial apoptosis in vitro, and as such the mechanisms underlying its ability to kill CLL cells are not well understood. Acalabrutinib, a more specific BTK inhibitor now in development, also appears to be highly effective in CLL, but the connection of its mechanism with CLL cell death is also unclear. Using dynamic BH3 profiling, we analyzed alterations in the function of the mitochondrial apoptotic pathway induced by ibrutinib and acalabrutinib. We studied CLL patient samples treated ex vivo with both drugs, as well as primary samples from CLL patients on clinical trials of both drugs. We found that BTK inhibition enhances mitochondrial BCL-2 dependence without significantly altering overall mitochondrial priming. Enhancement of BCL-2 dependence was accompanied by an increase in the pro-apoptotic protein BIM. In contrast, treatment with the selective BCL-2 inhibitor venetoclax enhanced overall mitochondrial priming without increasing BCL-2 dependence. Pre-treatment of CLL cells with either BTK inhibitor, whether ex vivo or in vivo in patients, enhanced killing by venetoclax. Our data suggest that BTK inhibition enhances mitochondrial BCL-2 dependence, supporting the ongoing development of clinical trials combining BTK and BCL-2 inhibition.

Figure 1. Ex vivo BTK inhibition increases CLL cell BCL-2 dependence as shown by dynamic BH3 profiling

Primary CLL cells were co-cultured with the NKTert stromal cell line, and subjected to single drug or combination treatment with ibrutinib alone (panel A), venetoclax alone (panel B), or the ibrutinib + venetoclax combination (panel C). Analogous experiments were performed with acalabrutinib alone (panel D), venetoclax alone (panel E), or the acalabrutinib + venetoclax combination (panel F). Ibrutinib and acalabrutinib were both used at 1 uM for 72 hours, while venetoclax at 1.5 nM was added only for the last 20 hours. Cells were then harvested for BH3 profiling. Delta priming is the difference in mitochondrial response to a BH3 peptide between DMSO- and drug treated samples (Y-axis). The response from BAD peptide, a specific indicator for BCL2 dependence, is shown side-by-side with BIM peptide response. Means are depicted as horizontal bars +/− SD error bars. p values were calculated using a one sample t test to validate if delta priming is different from 0. Ib, ibrutinib; ACP, acalabrutinib. * and ** indicate statistical significance.

Figure 2. Pre-treatment with ibrutinib or acalabrutinib increases CLL cell sensitivity to venetoclax

Primary CLL cells were seeded on top of the NKTert stromal cells and treated with 1 uM ibrutinib or acalabrutinib for 72 hours. Venetoclax was added for the last 1 hour, and viability was measured by flow cytometry analysis using FITC conjugated Annexin V and propidium iodide staining. Panel A and B top panels show representative dose curves for ibrutinib and acalabrutinib experiments, respectively. Panel A and B bottom panels show the maximal decrease of viability from each CLL primary sample calculated from dose curve experiments shown in the supplemental data. The statistical analysis used paired t-test (two-tailed) for a BTK inhibitor alone or in combination with venetoclax was shown in the bottom panels. Means are depicted as horizontal bars +/− SD error bars.

Figure 3. Ex vivo BTK inhibition increases expression of the pro-apoptotic BH3-only proteins BIM

Primary CLL cells were treated with ibrutinib or acalabrutinib alone or in combination with venetoclax under identical conditions as in Figure 2. Cells were collected at the end of treatment to prepare protein lysates for SDS-PAGE electrophoresis. Specific proteins were detected by the antibodies indicated in the figure. Densitometry analysis was performed for images shown in panel A and B. Ratio of BIM-EL or BAD protein to ACTIN was compared within different treatments and shown in panel C. The concentrations used in the experiments were 1 uM for ibrutinib and acalabrutinib; 1.5 nM for venetoclax. In patients 1 and 2 in panel A, the BIM antibody was run before the BAD antibody, resulting in multiple bands in the box labeled BAD. For all other patients BAD antibody was run after BIM antibody. * leftover BIM protein, ** BAD protein.

Figure 4. In vivo BTK inhibition increases BCL-2 dependence in cells from CLL patients

CLL patient samples were collected before and after ibrutinib or acalabrutinib therapy in the respective clinical trials. BH3 profiling was performed under identical conditions as the ex vivo samples shown in Figure 1. Delta priming was calculated from pre- and post-treatment mitochondrial responses to BIM or BAD peptides and low-dose venetoclax (used like a peptide in the BH3 profiling assay). Ibrutinib samples were obtained 6 hours (panel A) and 15 days (panel B) after the first dose. Acalabrutinib samples were obtained 4 weeks (panel C) and 12 weeks (panel D) after the first dose. Means are depicted as horizontal bars +/− SD error bars. One sample t test was used to validate if the delta priming is different from 0 and p values are indicated with * and ** indicating statistical significance. Normality was confirmed in all experiments with the exception of BIM 0.3 uM in panel (A), which was also subjected to a Wilcoxon rank test and was not significant (0.0625), suggesting that delta-priming for this peptide is not significantly changed.

Figure 5. In vivo BTK inhibition sensitizes CLL cells to venetoclax

CLL patient samples obtained from the BTK inhibitor clinical trials for the BH3 profiling assays were also aliquoted for viability assessment. Cells were subjected to ex vivo venetoclax treatment for 1 hour and viability was measured by flow cytometry analysis using FITC conjugated AnnexinV and PI staining. Panel A and B top panels show representative dose curves for ibrutinib and acalabrutinib treated patients, respectively. Panel A and B bottom panels show the maximal decrease of viability from each CLL patient sample calculated from dose curve experiments shown in the supplemental data. The statistical analysis used paired t-test (two-tailed) for viability decrease resulting from a BTK inhibitor alone or in combination with venetoclax is shown in bottom panels. Means are depicted as horizontal bars +/− SD error bars. Normality was confirmed in all experiments with the exception of the data for ibrutinib at day 15 and acalabrutinib at week 12. N/A: sample not available.

Figure 6. BIM expression is increased in CLL cells treated in vivo with BTK inhibition

Protein lysates were prepared using aliquots of primary CLL cells obtained from clinical trials of ibrutinib (panel A) or acalabrutinib (panel B), and analyzed by SDS-PAGE electrophoresis. Antibodies were used to detect BCL-2 family proteins as indicated in the figure. Densitometry analysis was performed for BIM-EL and ACTIN, and their ratio is shown in panel C. One-way ANOVA for BIM-EL/ACTIN ratios in acalabrutinib pretreated and week 4 samples showed a p<0.05. Pretx=pre-treatment samples. Other specified time points refer to the time after initial dosing of the respective drug. * underloaded samples not calculated for BIM-EL/ACTIN ratio.

Figure 7. Schema summarizing the complementary effects of BTK and BCL-2 inhibition on CLL cell mitochondria

At baseline (top left), CLL cells are BCL-2 dependent and primed for apoptosis, with the pro-apoptotic protein BIM occupying some, but not all of the binding sites on BCL-2. Treatment with venetoclax (bottom left) leads to apoptosis in most CLL cells by displacing BIM from BCL-2, allowing it to bind to the pro-apoptotic effector proteins BAX/BAK, which can then homo-oligomerize leading to mitochondrial outer membrane permeabilization (MOMP), cytochrome C release, and subsequent caspase-mediated cell death. BTK inhibition with ibrutinib or acalabrutinib (right side) can increase BIM protein thereby saturating essentially all of the binding sites on BCL-2 and increasing the BCL-2 dependence of the CLL cell even further. Subsequently adding venetoclax (lower right) rapidly triggers MOMP and subsequent CLL cell death.