Abstract
Targeting BCL-2, a key regulator of survival in B-cell malignancies including precursor B-cell acute lymphoblastic leukemia, has become a promising treatment strategy. However, given the redundancy of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1), single targeting may not be sufficient. When analyzing the effects of BH3-mimetics selectively targeting BCL-XL and MCL-1 alone or in combination with the BCL-2 inhibitor venetoclax, heterogeneous sensitivity to either of these inhibitors was found in ALL cell lines and in patient-derived xenografts. Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. Consequently, co-inhibition of BCL-2 and MCL-1 or BCL-XL resulted in synergistic apoptosis induction. Functional analysis by BH3-profiling and analysis of protein complexes revealed that venetoclax-treated ALL cells are dependent on MCL-1 and BCL-XL, indicating that MCL-1 or BCL-XL provide an Achilles heel in BCL-2-inhibited cells. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies.
Highlights
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells are characterized by an imbalance of cell death and survival pathways
We systematically investigated the effects of venetoclax (BCL-2), S63845 (MCL-1), and A-1331852 (BCL-XL) sideby-side in BCP-ALL, identifying heterogeneous sensitivities of all three inhibitors
Shuttling of BIM from B-cell lymphoma 2 (BCL-2) to MCL-1 and vice versa upon venetoclax and S63845 can be blocked by co-inhibition of BCL-2 and MCL-1 To further elucidate these mechanisms of varying functional dependencies of ALL cells on anti-apoptotic apoptosis regulators, we studied the interaction of BCL-2 family proteins with BIM, the most important pro-apoptotic activator of BAX [28] which showed high expression in BCP-ALL cells (Supplementary Fig. 7A)
Summary
BCP-ALL cell lines inhibitors selectively antagonizing BCL-2 (venetoclax), MCL-1. RS4;11, KOPN-8, REH, EU-3, RCH-ACV, and NALM-6 cells were purchased (DSMZ, Germany), UoCB6 cells were kindly provided by J. Cell viability assays To assess half maximal effective concentration (EC50) values, cells were exposed to venetoclax, S63845 and A-1331852 and cell death was analyzed either according to propidium iodide (PI) positivity (cell lines) or forward/side scatter criteria (PDX samples). Comparison of both methods three compounds analyzing the EC50 values of cell lines did not result in a significant difference (Fig. 1H). Leukemia characteristics of the cell lines were not found to be associated with inhibitor sensitivities (Supplementary Fig. 3). As observed for the cell lines, the different BH3-mimetics showed heterogeneous activities in the individual primary PDX samples (Fig. 2A–C, Supplementary Fig. 4). We found an association of venetoclax sensitivity (low EC50 values) with high BCL-2 protein expression and with low MCL-1
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