On the basis of our previous identification of aberrant phosphatidylinositol-3-kinase (PI3K)/Akt signaling as a novel poor prognostic factor in neuroblastoma, we evaluated the dual PI3K/mTOR inhibitor BEZ235 in the present study. Here, BEZ235 acts in concert with the lysosomotropic agent chloroquine (CQ) to trigger apoptosis in neuroblastoma cells in a synergistic manner, as calculated by combination index (CI < 0.5). Surprisingly, inhibition of BEZ235-induced autophagy is unlikely the primary mechanism of this synergism as reported in other cancers, since neither inhibition of autophagosome formation by knockdown of Atg7 or Atg5 nor disruption of the autophagic flux by Bafilomycin A1 (BafA1) enhance BEZ235-induced apoptosis. BEZ235 stimulates enlargement of the lysosomal compartment and generation of reactive oxygen species (ROS), while CQ promotes lysosomal membrane permeabilization (LMP). In combination, BEZ235 and CQ cooperate to trigger LMP, Bax activation, loss of mitochondrial membrane potential (MMP) and caspase-dependent apoptosis. Lysosome-mediated apoptosis occurs in a ROS-dependent manner, as ROS scavengers significantly reduce BEZ235/CQ-induced loss of MMP, LMP and apoptosis. There is a mitochondrial-lysosomal cross-talk, since lysosomal enzyme inhibitors significantly decrease BEZ235-and CQ-induced drop of MMP and apoptosis. In conclusion, BEZ235 and CQ act in concert to trigger LMP and lysosome-mediated apoptosis via a mitochondrial-lysosomal cross-talk. These findings have important implications for the rational development of PI3K/mTOR inhibitor-based combination therapies.Defects in apoptosis (programmed cell death) can contribute to tumor formation and treatment resistance of cancers. 1 There are two major apoptosis signaling pathways, i.e. the death receptor (extrinsic) pathway that is initiated by death receptors on the cell surface 2 and the mitochondrial (intrinsic) pathway. 3 The latter involves permeabilization of the outer mitochondrial membrane, resulting in the release of cytochrome c into the cytosol and subsequently caspase activation. 3 Mitochondrial outer membrane permeabilization (MOMP) is tightly controlled, for example by proteins of the Bcl-2 family. 3 Activation of Bax via a change in its conformation constitutes a key initial event in MOMP, whereas the antiapoptotic protein Bcl-2 blocks mitochondrial apoptosis.Lysosomal cell death represents another form of cell death and is initiated by lysosomal membrane permeabilization (LMP), which leads to the release of cathepsins and other hydrolases from the lysosomal lumen to the cytosol. 4,5 Depending on the extent of LMP and the cell type, LMP can result in apoptosis featuring caspase activation and MOMP or necrosis-like programmed cell death. 4 There are various stimuli that can trigger LMP, e.g. lysosomotropic compounds with detergent-like activity such as chloroquine (CQ). 4,6 The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway integrates survival signals provided by extracellular and intra...

Polo-like kinase 1 (PLK1) is frequently overexpressed in cancer, which correlates with poor prognosis. Therefore, we investigated PLK1 as therapeutic target using rhabdomyosarcoma (RMS) as a model. Here, we identify a novel synthetic lethal interaction of PLK1 inhibitors and microtubule-destabilizing drugs in preclinical RMS models and elucidate the underlying molecular mechanisms of this synergism. PLK1 inhibitors (i.e., BI 2536 and BI 6727) synergistically induce apoptosis together with microtubule-destabilizing drugs (i.e., vincristine (VCR), vinblastine (VBL) and vinorelbine (VNR)) in several RMS cell lines (combination index o0.9) including a patient-derived primary RMS culture. Importantly, PLK1 inhibitors and VCR cooperate to significantly suppress RMS growth in two in vivo models, including a mouse xenograft model, without causing additive toxicity. In addition, no toxicity was observed in non-malignant fibroblast or myoblast cultures. Mechanistically, BI 2536/VCR co-treatment triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of antiapoptotic BCL-2 family proteins, followed by BAX/BAK activation, production of reactive oxygen species (ROS) and activation of caspase-dependent or caspase-independent effector pathways. This conclusion is supported by data showing that BI 2536/VCR-induced apoptosis is significantly inhibited by preventing cells to enter mitosis, by overexpression of BCL-2 or a non-degradable MCL-1 mutant, by BAK knockdown, ROS scavengers, caspase inhibition or endonuclease G silencing. This identification of a novel synthetic lethality of PLK1 inhibitors and microtubule-destabilizing drugs has important implications for developing PLK1 inhibitor-based combination treatments.