Selective CDK9 inhibition overcomes TRAIL resistance by concomitant suppression of cFlip and Mcl-1.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many cancer cells without causing toxicity in vivo. However, to date, TRAIL-receptor agonists have only shown limited therapeutic benefit in clinical trials. This can, most likely, be attributed to the fact that 50% of all cancer cell lines and most primary human cancers are TRAIL resistant. Consequently, future TRAIL-based therapies will require the addition of sensitizing agents that remove crucial blocks in the TRAIL apoptosis pathway. Here, we identify PIK-75, a small molecule inhibitor of the p110α isoform of phosphoinositide-3 kinase (PI3K), as an exceptionally potent TRAIL apoptosis sensitizer. Surprisingly, PI3K inhibition was not responsible for this activity. A kinome-wide in vitro screen revealed that PIK-75 strongly inhibits a panel of 27 kinases in addition to p110α. Within this panel, we identified cyclin-dependent kinase 9 (CDK9) as responsible for TRAIL resistance of cancer cells. Combination of CDK9 inhibition with TRAIL effectively induced apoptosis even in highly TRAIL-resistant cancer cells. Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels. Concomitant cFlip and Mcl-1 downregulation was required and sufficient for TRAIL sensitization by CDK9 inhibition. When evaluating cancer selectivity of TRAIL combined with SNS-032, the most selective and clinically used inhibitor of CDK9, we found that a panel of mostly TRAIL-resistant non-small cell lung cancer cell lines was readily killed, even at low concentrations of TRAIL. Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window. Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo. Based on the high potency of CDK9 inhibition as a cancer cell-selective TRAIL-sensitizing strategy, we envisage the development of new, highly effective cancer therapies.Cell Death and Differentiation advance online publication, 20 December 2013; doi:10.1038/cdd.2013.179

Ferroptosis spreads to neighboring cells via plasma membrane contacts

Ferroptosis is a lytic, iron-dependent form of regulated cell death characterized by excessive lipid peroxidation and associated with necrosis spread in diseased tissues through unknown mechanisms. Using a novel optogenetic system for light-driven ferroptosis induction via degradation of the anti-ferroptotic protein GPX4, we show that lipid peroxidation and ferroptotic death can spread to neighboring cells through their closely adjacent plasma membranes. Ferroptosis propagation is dependent on cell distance and completely abolished by disruption of α-catenin-dependent intercellular contacts or by chelation of extracellular iron. Remarkably, bridging cells with a lipid bilayer or increasing contacts between neighboring cells enhances ferroptosis spread. Reconstitution of iron-dependent spread of lipid peroxidation between pure lipid, contacting liposomes provides evidence for the physicochemical mechanism involved. Our findings support a model in which iron-dependent lipid peroxidation propagates across proximal plasma membranes of neighboring cells, thereby promoting the transmission of ferroptotic cell death with consequences for pathological tissue necrosis spread

Ferroptotic pores induce Ca²⁺ fluxes and ESCRT-III activation to modulate cell death kinetics

Ferroptosis is an iron-dependent form of regulated necrosis associated with lipid peroxidation. Despite its key role in the inflammatory outcome of ferroptosis, little is known about the molecular events leading to the disruption of the plasma membrane during this type of cell death. Here we show that a sustained increase in cytosolic Ca2+ is a hallmark of ferroptosis that precedes complete bursting of the cell. We report that plasma membrane damage leading to ferroptosis is associated with membrane nanopores of a few nanometers in radius and that ferroptosis, but not lipid peroxidation, can be delayed by osmoprotectants. Importantly, Ca2+ fluxes during ferroptosis induce the activation of the ESCRT-III-dependent membrane repair machinery, which counterbalances the kinetics of cell death and modulates the immunological signature of ferroptosis. Our findings with ferroptosis provide a unifying concept that sustained increase of cytosolic Ca2+ prior to plasma membrane rupture is a common feature of regulated types of necrosis and position ESCRT-III activation as a general protective mechanism in these lytic cell death pathways

TRAIL-receptor 2-a novel negative regulator of p53

TNF-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2) can induce apoptosis in cancer cells upon crosslinking by TRAIL. However, TRAIL-R2 is highly expressed by many cancers suggesting pro-tumor functions. Indeed, TRAIL/TRAIL-R2 also activate pro-inflammatory pathways enhancing tumor cell invasion, migration, and proliferation. In addition, nuclear TRAIL-R2 (nTRAIL-R2) promotes malignancy by inhibiting miRNA let-7-maturation. Here, we show that TRAIL-R2 interacts with the tumor suppressor protein p53 in the nucleus, assigning a novel pro-tumor function to TRAIL-R2. Knockdown of TRAIL-R2 in p53 wild-type cells increases the half-life of p53 and the expression of its target genes, whereas its re-expression decreases p53 protein levels. Interestingly, TRAIL-R2 also interacts with promyelocytic leukemia protein (PML), a major regulator of p53 stability. PML-nuclear bodies are also the main sites of TRAIL-R2/p53 co-localization. Notably, knockdown or destruction of PML abolishes the TRAIL-R2-mediated regulation of p53 levels. In summary, our finding that nTRAIL-R2 facilitates p53 degradation and thereby negatively regulates p53 target gene expression provides insight into an oncogenic role of TRAIL-R2 in tumorigenesis that particularly manifests in p53 wild-type tumors

Ferroptosis triggers mitochondrial fragmentation via Drp1 activation

Constitutive mitochondrial dynamics ensure quality control and metabolic fitness of cells, and their dysregulation has been implicated in various human diseases. The large GTPase Dynamin-related protein 1 (Drp1) is intimately involved in mediating constitutive mitochondrial fission and has been implicated in mitochondrial cell death pathways. During ferroptosis, a recently identified type of regulated necrosis driven by excessive lipid peroxidation, mitochondrial fragmentation has been observed. Yet, how this is regulated and whether it is involved in ferroptotic cell death has remained unexplored. Here, we provide evidence that Drp1 is activated upon experimental induction of ferroptosis and promotes cell death execution and mitochondrial fragmentation. Using time-lapse microscopy, we found that ferroptosis induced mitochondrial fragmentation and loss of mitochondrial membrane potential, but not mitochondrial outer membrane permeabilization. Importantly, Drp1 accelerated ferroptotic cell death kinetics. Notably, this function was mediated by the regulation of mitochondrial dynamics, as overexpression of Mitofusin 2 phenocopied the effect of Drp1 deficiency in delaying ferroptosis cell death kinetics. Mechanistically, we found that Drp1 is phosphorylated and activated after induction of ferroptosis and that it translocates to mitochondria. Further activation at mitochondria through the phosphatase PGAM5 promoted ferroptotic cell death. Remarkably, Drp1 depletion delayed mitochondrial and plasma membrane lipid peroxidation. These data provide evidence for a functional role of Drp1 activation and mitochondrial fragmentation in the acceleration of ferroptotic cell death, with important implications for targeting mitochondrial dynamics in diseases associated with ferroptosis

Potent pro-apoptotic combination therapy is highly effective in a broad range of cancers

Primary or acquired therapy resistance is a major obstacle to the effective treatment of cancer. Resistance to apoptosis has long been thought to contribute to therapy resistance. We show here that recombinant TRAIL and CDK9 inhibition cooperate in killing cells derived from a broad range of cancers, importantly without inducing detectable adverse events. Remarkably, the combination of TRAIL with CDK9 inhibition was also highly effective on cancers resistant to both, standard-of-care chemotherapy and various targeted therapeutic approaches. Dynamic BH3 profiling revealed that, mechanistically, combining TRAIL with CDK9 inhibition induced a drastic increase in the mitochondrial priming of cancer cells. Intriguingly, this increase occurred irrespective of whether the cancer cells were sensitive or resistant to chemo- or targeted therapy. We conclude that this pro-apoptotic combination therapy has the potential to serve as a highly effective new treatment option for a variety of different cancers. Notably, this includes cancers that are resistant to currently available treatment modalities

Potent pro-apoptotic combination therapy is highly effective in a broad range of cancers

Primary or acquired therapy resistance is a major obstacle to the effective treatment of cancer. Resistance to apoptosis has long been thought to contribute to therapy resistance. We show here that recombinant TRAIL and CDK9 inhibition cooperate in killing cells derived from a broad range of cancers, importantly without inducing detectable adverse events. Remarkably, the combination of TRAIL with CDK9 inhibition was also highly effective on cancers resistant to both, standard-of-care chemotherapy and various targeted therapeutic approaches. Dynamic BH3 profiling revealed that, mechanistically, combining TRAIL with CDK9 inhibition induced a drastic increase in the mitochondrial priming of cancer cells. Intriguingly, this increase occurred irrespective of whether the cancer cells were sensitive or resistant to chemo- or targeted therapy. We conclude that this pro-apoptotic combination therapy has the potential to serve as a highly effective new treatment option for a variety of different cancers. Notably, this includes cancers that are resistant to currently available treatment modalities

Ferroptosis in health and disease.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis

MAPK-pathway inhibition mediates inflammatory reprogramming and sensitizes tumors to targeted activation of innate immunity sensor RIG-I

Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8(+) T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients

MAPK-pathway inhibition mediates inflammatory reprogramming and sensitizes tumors to targeted activation of innate immunity sensor RIG-I.

Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8+ T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients