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

<b>Rationale:</b> In the era of precision medicine, there is a growing need for rapid reliable <i>ex vivo</i> 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 <i>ex vivo</i> 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. <b>Methods:</b> 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. <b>Results:</b> 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 <i>ex vivo</i> drug treatments. We also compared BH3 toolkit baseline results on cell lines with those obtained using conventional BH3 profiling. <b>Conclusion:</b> Overall, our data validates this streamlined BH3 drug toolkit, allowing for a more extensive use of the BH3 profiling technique.