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Dysregulation of the mitochondrial pathway of apoptosis, controlled by the BCL-2 family of proteins, leads to disease states including cancer. Rapid analysis of a cell's dependency on the BCL-2 family of proteins is hindered by the complex interactions of more than a dozen proteins. Transcript or even protein levels are therefore generally insufficient to predict a cell's response to perturbations like chemotherapy. Previously, we developed the JC-1 BH3 method to provide a same day functional assay to assess a cell's propensity to undergo apoptosis and demonstrated its utility in predicting response to chemotherapy. We have now improved upon these methods to create a robust assay amenable to high throughput platforms using cytochrome c retention in formaldehyde fixed cells to remove the time sensitivity of JC-1 potential measurements. BH3 profiling by intracellular staining (iBH3) is suitable for 96- and 384-well formats, and can be used to directly screen candidate BH3-mimetic compounds for activity. When used as the final component of dynamic BH3 profiling (DBP), which uses a drug pretreatment prior to iBH3 to assess the change in profile due to treatment, it can predict the response of cells to chemotherapy days before they show signs of death. [ABSTRACT FROM AUTHOR]

Highlights: [•] Functional assay reports mitochondrial response without knowledge of protein levels. [•] Plate reader assay is suitable for large sample numbers for homogenous samples. [•] FACS assay can determine priming in sub-populations from heterogeneous samples. [•] Suitable for any viable single cell suspension including primary samples. [ABSTRACT FROM AUTHOR]

It is well established that CD4+ CD8+ thymocytes are more sensitive to myriad death stimuli than CD4+ or CD8+ single positive (SP) thymocytes. The mechanism behind this hypersensitivity to apoptosis of CD4+ CD8+ thymocytes is not understood. To test whether the difference lay in the apôptotic preset of mitochondria, established by the BCL-2 family of proteins, we developed a method, FACS- based BH3 profiling. Using this tool, we could discriminate thymo- cyte subpopulations and demonstrate that mitochondria in double positive (DP) thymocytes are more primed for death than those in single positive counterparts. Loss of proapoptotic BIM, known to cause autoimmunity, also causes loss of "priming." Priming is a phe- notype with physiologic consequences, which can be measured at the single-cell level in complex samples using FACS-based BH3 profiling. [ABSTRACT FROM AUTHOR]

Interest in harnessing natural killer (NK) cells for cancer immunotherapy is rapidly growing. However, efficacy of NK cell-based immunotherapy remains limited in most trials. Strategies to augment the killing efficacy of NK cells are thus much needed. In the current study, we found that mitochondrial apoptosis (mtApoptosis) pathway is essential for efficient NK killing, especially at physiologically relevant effector-to-target ratios. Furthermore, NK cells can prime cancer cells for mtApoptosis and mitochondrial priming status affects cancer-cell susceptibility to NK-mediated killing. Interestingly, pre-activating NK cells confers on them resistance to BH3 mimetics. Combining BH3 mimetics with NK cells synergistically kills cancer cells in vitro and suppresses tumor growth in vivo. The ideal BH3 mimetic to use in such an approach can be predicted by BH3 profiling. We herein report a rational and precision strategy to augment NK-based immunotherapy, which may be adaptable to T cell-based immunotherapies as well. [Display omitted] • Mitochondrial readiness for apoptosis affects cancer cell sensitivity to NK cells • Pre-activated NK cells acquire resistance to BCL-2, MCL-1, and BCL-XL inhibitors • BH3 mimetics and NK cells synergize in killing cancer cells in vitro and in vivo • BH3 profiling of cancer cells identifies which BH3 mimetic augments NK killing NK cells are an attractive option for cell-based therapy but require optimization. BH3 mimetics chosen by BH3 profiling synergized with NK cell therapy to enhance cancer-cell killing by NK cells in an approach that could also be used for other cell therapy platforms. [ABSTRACT FROM AUTHOR]

One of the hallmarks of cancer is a resistance to the induction of programmed cell death that is mediated by selection of cells with elevated expression of anti-apoptotic members of the BCL-2 family. To counter this resistance, new therapeutic agents known as BH3-mimetic small molecules are in development with the goal of antagonizing the function of anti-apoptotic molecules and promoting the induction of apoptosis. To facilitate the testing and modeling of BH3-mimetic agents, we have developed a powerful system for evaluation and screening of agents both in culture and in immune competent animal models by engineering mouse leukemic cells and re-programming them to be dependent on exogenously expressed human anti-apoptotic BCL-2 family members. Here we demonstrate that this panel of cell lines can determine the specificity of BH3-mimetics to individual anti-apoptotic BCL-2 family members (BCL-2, BCL-XL, BCL-W, BFL-1, and MCL-1), demonstrate whether cell death is due to the induction of apoptosis (BAX and BAK-dependent), and faithfully assess the efficacy of BH3-mimetic small molecules in pre-clinical mouse models. These cells represent a robust and valuable pre-clinical screening tool for validating the efficacy, selectivity, and on-target action of BH3-mimetic agents.

Acquired resistance to BH3 mimetic antagonists of BCL-2 and MCL-1 is an important clinical problem. Using acute myelogenous leukemia (AML) patient-derived xenograft (PDX) models of acquired resistance to BCL-2 (venetoclax) and MCL-1 (S63845) antagonists, we identify common principles of resistance and persistent vulnerabilities to overcome resistance. BH3 mimetic resistance is characterized by decreased mitochondrial apoptotic priming as measured by BH3 profiling, both in PDX models and human clinical samples, due to alterations in BCL-2 family proteins that vary among cases, but not to acquired mutations in leukemia genes. BCL-2 inhibition drives sequestered pro-apoptotic proteins to MCL-1 and vice versa, explaining why in vivo combinations of BCL-2 and MCL-1 antagonists are more effective when concurrent rather than sequential. Finally, drug-induced mitochondrial priming measured by dynamic BH3 profiling (DBP) identifies drugs that are persistently active in BH3 mimetic-resistant myeloblasts, including FLT-3 inhibitors and SMAC mimetics. • Reduced mitochondrial apoptotic priming drives acquired resistance to BH3 mimetics • BH3 profiling predicts clinical response to venetoclax and HMA combinations • Simultaneous BCL-2 and MCL-1 antagonism outperforms alternating regimens • Dynamic BH3 profiling identifies drug vulnerabilities in BH3 mimetic-resistant AML Bhatt et al. demonstrate that resistance to BCL-2 and MCL-1 antagonists emerges via selection for reduced mitochondrial apoptotic priming. Rapid measurements of drug-induced apoptotic signaling measured by dynamic BH3 profiling identify targeted agents with in vivo efficacy. BCL-2 and MCL-1 antagonist combinations overcome resistance to either single agent. [ABSTRACT FROM AUTHOR]