In this study, the MCL-1-specific inhibitor S63845 and BCL-XL-specific inhibitor A-1331852 were investigated side-by-side with the BCL-2-specific inhibitor Venetoclax in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines and a cohort of 27 BCP-ALL patient-derived xenograft (PDX) samples. Drug sensitivities were assessed by cell viability assays and estimation of half-maximal effective concentrations. Combinatorial effects were analyzed using the Chou-Talalay method. Additionally, aiming to identify predictive biomarkers for patient selection, patient- and leukemia characteristics were investigated along with functional analyses and mechanistic studies of apoptosis regulators, using BH3 Profiling, intracellular staining of BCL-2 family proteins and a Cytochrome C release assay. Single-drug EC50 evaluation demonstrated heterogeneous sensitivities for all three inhibitors in BCP-ALL cell lines and similarly in PDXs. Sensitivities of Venetoclax and S63845 significantly overlapped, whereas Venetoclax and A-1331852 showed greater differences, highlighting BCL-XL more than MCL-1 as an alternative target in selected Venetoclax -resistant samples. If combining Venetoclax treatment with S63845 or A-1331852, synergistic effects were identified for both inhibitors in cell lines and in Venetoclax-insensitive PDXs. Functional analysis via dynamic BH3 Profiling showed rapidly risen dependence on both MCL-1 and BCL-XL upon Venetoclax treatment. Testing predictive biomarkers showed Venetoclax sensitivity to be associated with different biomarkers tested, demonstrating significant correlations with high levels of its target protein BCL-2, BCL-2-dependence evaluated via BH3 Profiling and early assessment of drug response via Cytochrome C release assay, both in vitro and in vivo. By side-by side investigation of drug response in permeabilized and non-permeabilized cells, the cell membrane barrier seems to play a role in mediating Venetoclax sensitivity besides BCL-2 family dependence. S63845 showed activity in P2RY8-CRLF2 fusion-positive samples and was associated with low expression levels of its target protein MCL-1. However, protein levels alike BH3 Profiling parameters were not found to qualify as predictive markers. A-1331852 sensitivity was found in the majority of PDX samples from patients with later-on relapse. However, neither BH3 Profiling, BCL-2 family protein expression nor early Cytochrome C release was significantly associated with A-1331852 response. Taken together, the results of this study highlight MCL-1 and BCL-XL as important potential therapeutic targets besides BCL-2 in pediatric BCP-ALL.
