Bone morphogenetic protein inhibitors and mitochondria targeting agents synergistically induce apoptosis-inducing factor (AIF) caspase-independent cell death in lung cancer cells.

BACKGROUND: Bone morphogenetic proteins (BMP) are evolutionarily conserved morphogens that are reactivated in lung carcinomas. In lung cancer cells, BMP signaling suppresses AMP activated kinase (AMPK) by inhibiting LKB1. AMPK is activated by mitochondrial stress that inhibits ATP production, which is enhanced 100-fold when phosphorylated by LKB1. Activated AMPK can promote survival of cancer cells but its "hyperactivation" induces cell death. The studies here reveal novel cell death mechanisms induced by BMP inhibitors, together with agents targeting the mitochondria, which involves the "hyperactivation" of AMPK. METHODS: This study examines the synergistic effects of two BMP inhibitors together with mitochondrial targeting agents phenformin and Ym155, on cell death of lung cancer cells expressing LKB1 (H1299), LKB1 null (A549), and A549 cells transfected with LKB1 (A549-LKB1). Cell death mechanisms evaluated were the activation of caspases and the nuclear localization of apoptosis inducing factor (AIF). A769662 was used to allosterically activate AMPK. Knockdown of BMPR2 and LKB1 using siRNA was used to examine their effects on nuclear localization of AMPK. Validation studies were performed on five passage zero primary NSCLC. RESULTS: Both BMP inhibitors synergistically suppressed growth when combined with Ym155 or phenformin in cells expressing LKB1. The combination of BMP inhibitors with mitochondrial targeting agents enhanced the activation of AMPK in lung cancer cells expressing LKB1. Allosteric activation of AMPK with A769662 induced cell death in both H1299 and A549 cells. Cell death induced by the combination of BMP inhibitors and mitochondrial-targeting agents did not activate caspases. The combination of drugs induced nuclear localization of AIF in cells expressing LKB1, which was attenuated by knockdown of LKB1. Knockdown of BMPR2 together with Ym155 increased nuclear localization of AIF. Combination therapy also enhanced cell death and AIF nuclear localization in primary NSCLC. CONCLUSIONS: These studies demonstrate that inhibition of BMP signaling together with mitochondrial targeting agents induce AIF caspase-independent cell death, which involves the "hyperactivation" of AMPK. AIF caspase-independent cell death is an evolutionarily conserved cell death pathway that is infrequently studied in cancer. These studies provide novel insight into mechanisms inducing AIF caspase-independent cell death in cancer cells using BMP inhibitors. Video Abstract.

BMP receptor 2 inhibition regulates mitochondrial bioenergetics to induce synergistic cell death with BCL-2 inhibitors in leukemia and NSLC cells.

Background: Bone morphogenetic protein (BMP) signaling cascade is a phylogenetically conserved stem cell regulator that is aberrantly expressed in non-small cell lung cancer (NSLC) and leukemias. BMP signaling negatively regulates mitochondrial bioenergetics in lung cancer cells. The impact of inhibiting BMP signaling on mitochondrial bioenergetics and the effect this has on the survival of NSLC and leukemia cells are not known. Methods: Utilizing the BMP type 2 receptor (BMPR2) JL189, BMPR2 knockout (KO) in cancer cells, and BMP loss of function mutants in C elegans, we determined the effects of BMPR2 inhibition (BMPR2i) on TCA cycle metabolic intermediates, mitochondrial respiration, and the regulation of mitochondrial superoxide anion (SOA) and Ca++ levels. We also examined whether BMPR2i altered the threshold cancer therapeutics induce cell death in NSLC and leukemia cell lines. KO of the mitochondria uniporter (MCU) was used to determine the mechanism BMPR2i regulates the uptake of Ca++ into the mitochondria, mitochondrial bioenergetics, and cell death. Results: BMPR2i increases mtCa++ levels and enhances mitochondrial bioenergetics in both NSLC and leukemia cell lines that is conserved in C elegans. BMPR2i induced increase in mtCa++ levels is regulated through the MCU, effecting mitochondria mass and cell survival. BMPR2i synergistically induced cell death when combined with BCL-2 inhibitors or microtubule targeting agents in both NSLC and leukemia cells. Cell death is caused by synergistic increase in mitochondrial ROS and Ca++ levels. BMPR2i enhances Ca++ uptake into the mitochondria induced by reactive oxygen species (ROS) produced by cancer therapeutics. Both acute myeloid leukemia (AML) and T-cell lymphoblastic leukemia cells lines were more responsive to the JL189 alone and when combined with venetoclax or navitoclax compared to NSLC.