Development of a robust BH3 drug toolkit for precision medicine in hematologic malignancies.

Abstract

Rationale: In the era of precision medicine, there is a growing need for rapid reliable ex vivo functional assays capable of predicting treatment efficacy. One drug class that may particularly benefit from such assays is BH3 mimetics. These small molecules antagonize anti-apoptotic proteins such as BCL-2, MCL-1, or BCL-XL, on which cancer cells depend for their survival. A functional assay known as BH3 profiling was previously developed to measure those dependencies through the use of specific BH3-only peptides. A variation of this technique, dynamic BH3 profiling (DBP), allows for measuring changes in those dependencies, after ex vivo treatment with a drug of interest. Though well-validated to predict clinical response in hematologic malignancies, BH3 profiling technique requires the use of specialized BH3-only peptides and requires significant optimization to achieve reproducible results. Methods: We used a toolkit of BH3 mimetics drugs as probes instead of BH3-only peptides. This technique reduces the complexity and cost by using Annexin V/7AAD staining instead of cytochrome c release as a functional readout for apoptosis. We also used cell lines as internal controls for a representative response to BH3 mimetics that allow us to easily compare and stratify patients according to their profile. Results: We demonstrate that our new protocol enables apoptotic dependencies to be measured efficiently across different hematologic malignancies. In addition to a detailed description of the assay, we describe the results in several models including cell lines and primary tumor cells, both at baseline and dynamically after ex vivo drug treatments. We also compared BH3 toolkit baseline results on cell lines with those obtained using conventional BH3 profiling. Conclusion: Overall, our data validates this streamlined BH3 drug toolkit, allowing for a more extensive use of the BH3 profiling technique.