Fig. 3: Mitochondrial priming and apoptotic dependencies of A549 TIS lung cancer cells.

A Top. Both the level of mitochondrial priming—the proximity to the mitochondrial apoptotic threshold that determines the ability of TIS mitochondria to initiate apoptosis in response to BH3 peptides—and the nature of apoptotic blockade in TIS cancer cells—the distinct patterns of dependence on pro-survival BCL-2 proteins in the BCL2/BH3 interactome—were assessed using the plate-based JC-1 BH3 profiling. After proliferative (untreated) and TIS cancer cells are permeabilized with digitonin to allow BH3 peptides to diffuse into the cells and interact with intact mitochondria, the loss of JC-1 red fluorescence caused by depolarization of the mitochondrial transmembrane potential (a surrogate for the endpoint MOMP) allows real-time kinetic measurements of the loss of mitochondrial integrity. Primed mitochondria respond more robustly to both activator and sensitizer BH3 peptides and are more susceptible to apoptosis. The pattern of response to BH3 peptides can also identify the functional dynamics between pro- and anti-apoptotic proteins in maintaining cell survival in the TIS phenotypes (e.g., defects in pro-apoptotic signaling or increased addiction to anti-apoptotic proteins). Cell viability analysis using a panel of BH3 mimetics (ABT-263, ABT-199, A13318512, S63845) was used to assess senolytic indexes (SI), which were defined as the ratio of IC₅₀ values of proliferative cancer cells to IC₅₀ values of TIS cancer cells. Bottom. Apoptotic blocks based on BH3 profiling. After exposure to individual activator or sensitizer BH3 peptides, BH3 profiling can distinguish three major blocks (stop signs) through which cells can avoid apoptosis, namely: primed, unprimed-competent, and unprimed-incompetent. Bottom. Patterns of interaction between the anti-apoptotic proteins present in cells (columns) and the pro-apoptotic synthetic peptides or drugs (rows) used in the BH3 profiling assay and the BH3 mimetics cell viability toolkit. BIM, BID, and PUMA inhibit all the inhibitors and are pan-sensitizers. PUMA (shown in red rows) can act as an activator of BAX and BAK. Orange colors highlight those peptides/drugs that inhibit the BCL-2 protein, including BMF and BAD, Blue rows highlight the dependency on mantle cell lymphoma (MCL)-1, whereas NOXA inhibits MCL-1 and BFL-1. Green indicates BCL-X_L dependency, as HRK is primarily a BCL-X_L inhibitor, but can also inhibit other anti-apoptotic proteins with lower affinity (A and B, created with Biorender.com). BH3 profiling was performed to measure mitochondrial depolarization in proliferative (untreated) and TIS A549 lung cancer cells exposed to activator (B) and sensitizer (C) BH3 peptides. Figure shows heat maps of % mitochondrial depolarization caused by increasing concentrations of the activator (BIM, BID, PUMA) and sensitizer (BMF, BAD, NOXA, HRK) peptides in proliferative (untreated) and palbociclib, doxorubicin, alisertib, and bleomycin TIS cells. Data shown are the mean of ≥3 independent experiments using three technical replicates for each peptide. Graphs represent the means (columns) ± S.E.M. (bars) of ≥3 independent experiments BH3 peptide EC_50s (μmol/L) in proliferative and palbociclib, doxorubicin, alisertib, and bleomycin TIS cells. Statistically significant differences (ANOVA analysis) between proliferative and TIS phenotypes means are shown. n.s. not statistically significant. D Global heat maps of % mitochondrial depolarization caused by selected concentrations of activator and sensitizer peptides in A549 lung cancer cells. Samples are ordered according to depolarization by the HRK peptide. Data shown are the mean of ≥3 independent experiments with three technical replicates for each peptide.