Tankyrase-mediated ADP-ribosylation is a regulator of TNF-induced death.

Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose-binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

25 years of research put RIPK1 in the clinic.

Receptor Interacting Protein Kinase 1 (RIPK1) is a key regulator of inflammation. To warrant cell survival and appropriate immune responses, RIPK1 is post-translationally regulated by ubiquitylations, phosphorylations and caspase-8-mediated cleavage. Dysregulations of these post-translational modifications switch on the pro-death function of RIPK1 and can cause inflammatory diseases in humans. Conversely, activation of RIPK1 cytotoxicity can be advantageous for cancer treatment. Small molecules targeting RIPK1 are under development for the treatment of cancer, inflammatory and neurogenerative disorders. We will discuss the molecular mechanisms controlling the functions of RIPK1, its pathologic role in humans and the therapeutic opportunities in targeting RIPK1, specifically in the context of inflammatory diseases and cancers.

Targeting triple-negative breast cancers with the Smac-mimetic birinapant.

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER+) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER+ breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER+ cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.

MK2 Inhibition Induces p53-Dependent Senescence in Glioblastoma Cells.

MAPK-activated protein kinase 2 (MK2) has diverse roles in cancer. In response to chemotherapy, MK2 inhibition is synthetically lethal to p53-deficiency. While TP53 deletion is rare in glioblastomas, these tumors often carry TP53 mutations. Here, we show that MK2 inhibition strongly attenuated glioblastoma cell proliferation through p53wt stabilization and senescence. The senescence-inducing efficacy of MK2 inhibition was particularly strong when cells were co-treated with the standard-of-care temozolomide. However, MK2 inhibition also increased the stability of p53 mutants and enhanced the proliferation of p53-mutant stem cells. These observations reveal that in response to DNA damaging chemotherapy, targeting MK2 in p53-mutated cells produces a phenotype that is distinct from the p53-deficient phenotype. Thus, MK2 represents a novel drug target in 70% glioblastomas harboring intact TP53 gene. However, targeting MK2 in tumors with TP53 mutations may accelerate disease progression. These findings are highly relevant since TP53 mutations occur in over 50% of all cancers.

Antagonism of IAPs Enhances CAR T-cell Efficacy.

Chimeric antigen receptor (CAR) T-cell therapy has proven successful in the treatment of hematological malignancies, notably acute lymphoblastic leukemia and B-cell lymphoma. However, the efficacy of CAR T cells against solid tumors is poor, likely due to tumor-associated immunosuppression. Here, we demonstrated that antagonizing the "inhibitor of apoptosis proteins" with the clinical smac-mimetic, birinapant, significantly enhanced the antitumor activity of CAR T cells in a tumor necrosis factor (TNF)-dependent manner. Enhanced tumor cell death occurred independently of the perforin-mediated granule exocytosis pathway, underscoring the cytotoxic potential of CAR T-cell-derived TNF. Combining CAR T-cell therapy with birinapant significantly reduced established tumor growth in vivo, where either therapy alone was relatively ineffective. Using patient biopsy-derived tumoroids, we demonstrated the synergistic potential of combining CAR T-cell therapy with smac-mimetics. Taken together, we identified CAR T-cell-derived TNF as a potent antitumor effector, which can be further harnessed by smac-mimetics.

Tumor immune evasion arises through loss of TNF sensitivity.

Immunotherapy has revolutionized outcomes for cancer patients, but the mechanisms of resistance remain poorly defined. We used a series of whole-genome clustered regularly interspaced short palindromic repeat (CRISPR)-based screens performed in vitro and in vivo to identify mechanisms of tumor immune evasion from cytotoxic lymphocytes [CD8+ T cells and natural killer (NK) cells]. Deletion of key genes within the tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways provided protection of tumor cells from CD8+ T cell-mediated killing and blunted antitumor immune responses in vivo. Deletion of a number of genes in the TNF pathway also emerged as the key mechanism of immune evasion from primary NK cells. Our screens also identified that the metabolic protein 2-aminoethanethiol dioxygenase (Ado) modulates sensitivity to TNF-mediated killing by cytotoxic lymphocytes and is required for optimal control of tumors in vivo. Remarkably, we found that tumors delete the same genes when exposed to perforin-deficient CD8+ T cells, demonstrating that the dominant immune evasion strategy used by tumor cells is acquired resistance to T cell-derived cytokine-mediated antitumor effects. We demonstrate that TNF-mediated bystander killing is a potent T cell effector mechanism capable of killing antigen-negative tumor cells. In addition to highlighting the importance of TNF in CD8+ T cell- and NK cell-mediated killing of tumor cells, our study also provides a comprehensive picture of the roles of the TNF, IFN, and antigen presentation pathways in immune-mediated tumor surveillance.

Recent advances in understanding inhibitor of apoptosis proteins.

The inhibitor of apoptosis proteins (IAPs) are a family of proteins that were chiefly known for their ability to inhibit apoptosis by blocking caspase activation or activity. Recent research has shown that cellular IAP1 (cIAP1), cIAP2, and X-linked IAP (XIAP) also regulate signaling by receptors of the innate immune system by ubiquitylating their substrates. These IAPs thereby act at the intersection of pathways leading to cell death and inflammation. Mutation of IAP genes can impair tissue homeostasis and is linked to several human diseases. Small-molecule IAP antagonists have been developed to treat certain malignant, infectious, and inflammatory diseases. Here, we will discuss recent advances in our understanding of the functions of cIAP1, cIAP2, and XIAP; the consequences of their mutation or dysregulation; and the therapeutic potential of IAP antagonist drugs.

The TNF Receptor Superfamily-NF-κB Axis Is Critical to Maintain Effector Regulatory T Cells in Lymphoid and Non-lymphoid Tissues.

After exiting the thymus, Foxp3+ regulatory T (Treg) cells undergo further differentiation in the periphery, resulting in the generation of mature, fully suppressive effector (e)Treg cells in a process dependent on TCR signaling and the transcription factor IRF4. Here, we show that tumor necrosis factor receptor superfamily (TNFRSF) signaling plays a crucial role in the development and maintenance of eTreg cells. TNFRSF signaling activated the NF-κB transcription factor RelA, which was required to maintain eTreg cells in lymphoid and non-lymphoid tissues, including RORγt+ Treg cells in the small intestine. In response to TNFRSF signaling, RelA regulated basic cellular processes, including cell survival and proliferation, but was dispensable for IRF4 expression or DNA binding, indicating that both pathways operated independently. Importantly, mutations in the RelA binding partner NF-κB1 compromised eTreg cells in humans, suggesting that the TNFRSF-NF-κB axis was required in a non-redundant manner to maintain eTreg cells in mice and humans.

Synergistic action of the MCL-1 inhibitor S63845 with current therapies in preclinical models of triple-negative and HER2-amplified breast cancer.

The development of BH3 mimetics, which antagonize prosurvival proteins of the BCL-2 family, represents a potential breakthrough in cancer therapy. Targeting the prosurvival member MCL-1 has been an area of intense interest because it is frequently deregulated in cancer. In breast cancer, MCL-1 is often amplified, and high expression predicts poor patient outcome. We tested the MCL-1 inhibitor S63845 in breast cancer cell lines and patient-derived xenografts with high expression of MCL-1. S63845 displayed synergistic activity with docetaxel in triple-negative breast cancer and with trastuzumab or lapatinib in HER2-amplified breast cancer. Using S63845-resistant cells combined with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) technology, we identified deletion of BAK and up-regulation of prosurvival proteins as potential mechanisms that confer resistance to S63845 in breast cancer. Collectively, our findings provide a strong rationale for the clinical evaluation of MCL-1 inhibitors in breast cancer.

PD-L1 and IAPs co-operate to protect tumors from cytotoxic lymphocyte-derived TNF.

Smac-mimetics are emerging as promising anti-cancer agents and are being evaluated in clinical trials for a variety of malignancies. Smac-mimetics can induce TNF production from a subset of tumor cells and simultaneously sensitize them to TNF-induced apoptosis. However, TNF derived from other cellular sources, such as cytotoxic lymphocytes (CLs) within the tumor, may also contribute to the anti-tumor activity of SMs. Here, we show that CD8+ T cells and NK cells potently kill tumor cells in the presence of the SM, birinapant. Enhanced CL killing occurred through TNF secretion upon tumor antigen recognition or NK-activating receptor ligation. Importantly, the perforin/granzyme route to CL-mediated tumor cell killing was dispensable for the efficacy of birinapant, emphasizing the importance of the TNF-mediated apoptosis pathway. Time-lapse microscopy revealed that birinapant sensitized tumor cells to apoptosis as bystanders and to membrane-bound TNF delivered to tumor cells within the immunological synapse. Furthermore, PD-L1 expression on tumor cells suppressed antigen-driven TNF production by CD8+ T cells, which could be antagonized through PD-1 blockade. Importantly, the elevated levels of TNF produced upon PD-1 blockade further enhanced tumor cell killing when combined with birinapant. The combined anti-tumor activity of IAP antagonism and PD-1 blockade occurred independently of perforin-mediated tumor cell death. Taken together, we identify CL-derived TNF as a potent effector of birinapant mediated anti-tumor immunity and opportunity for combination therapy through co-inhibition of immune checkpoints.

Inhibitor of Apoptosis Proteins (IAPs) Limit RIPK1-Mediated Skin Inflammation.

Inhibitor of apoptosis proteins (IAPs) are critical regulators of cell death and survival pathways. Mice lacking cIAP1 and either cIAP2 or XIAP die in utero, and myeloid lineage-specific deletion of all IAPs causes sterile inflammation, but their role in the skin is unknown. We generated epidermal-specific IAP-deficient mice and found that combined genetic deletion of cIAP1 (epidermal knockout [EKO]) in keratinocytes and ubiquitous cIAP2 deletion (cIap1EKO/EKO.cIap2-/-) caused profound skin inflammation and keratinocyte death, lethal by postpartum day 10. To investigate their role in skin homeostasis, we injected an IAP antagonist compound subcutaneously into wild-type and knockout mice. This induced a toxic epidermal necrolysis-like local inflammation, which mirrored the phenotype seen in cIap1EKO/EKO.cIap2-/- mice. Loss of one Ripk1 allele limited lesion formation and significantly extended the lifespan of cIap1EKO/EKO.cIap2-/- mice. cIAP activities are important for recruitment of LUBAC to signaling complexes, and loss of LUBAC component SHARPIN, induces dermatitis in mice. Consistent with this relationship between cIAPs and LUBAC, Ripk1 heterozygosity also protected against development of dermatitis in Sharpin-deficient mice. This work therefore refines our molecular understanding of inflammatory signaling in the skin and defines potential targets for treating skin inflammation.

MK2 Phosphorylates RIPK1 to Prevent TNF-Induced Cell Death.

TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-κB, p38α, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production.

'Did He Who Made the Lamb Make Thee?' New Developments in Treating the 'Fearful Symmetry' of Acute Myeloid Leukemia.

Malignant cells must circumvent endogenous cell death pathways to survive and develop into cancers. Acquired cell death resistance also sets up malignant cells to survive anticancer therapies. Acute Myeloid Leukemia (AML) is an aggressive blood cancer characterized by high relapse rate and resistance to cytotoxic therapies. Recent collaborative profiling projects have led to a greater understanding of the 'fearful symmetry' of the genomic landscape of AML, and point to the development of novel potential therapies that can overcome factors linked to chemoresistance. We review here the most recent research in the genetics of AML and how these discoveries have led, or might lead, to therapies that specifically activate cell death pathways to substantially challenge this 'fearful' disease.

Jekyll & Hyde: The Other Life of the Death Ligand TRAIL.

Henry and Martin (2017) and Hartwig et al. (2017) provide more insights into the non-apoptotic function of the FADD/caspase-8 duo in TRAIL signaling.

Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways.

Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF- and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SM-induced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents.

Relevance of necroptosis in cancer.

Resistance to caspase-dependent apoptosis is often responsible for treatment failures in cancer. Finding novel therapeutic strategies that can activate alternative cell death programs appears to be appealing. Necroptosis is a form of programmed necrosis that occurs under caspase-deficient conditions. This alternative form of cell death has recently emerged as a potential anticancer therapy that could overcome apoptosis resistance. A growing understanding of the molecular events triggering necroptosis helped to examine its implication in cancer development and to define new therapeutic strategies. Genetic and proteomic analysis suggest that necroptosis is deregulated in many cancers. Various preclinical and clinical compounds induced necroptosis and have demonstrated significant therapeutic efficacy. Moreover, accumulating evidence has shown that necroptosis promotes anticancer immune response. A better knowledge of the cascade of events regulating necroptosis is expected to assess the feasibility of its therapeutic exploitation for cancer therapy.

The caspase-8 inhibitor emricasan combines with the SMAC mimetic birinapant to induce necroptosis and treat acute myeloid leukemia.

Resistance to chemotherapy is a major problem in cancer treatment, and it is frequently associated with failure of tumor cells to undergo apoptosis. Birinapant, a clinical SMAC mimetic, had been designed to mimic the interaction between inhibitor of apoptosis proteins (IAPs) and SMAC/Diablo, thereby relieving IAP-mediated caspase inhibition and promoting apoptosis of cancer cells. We show that acute myeloid leukemia (AML) cells are sensitive to birinapant-induced death and that the clinical caspase inhibitor emricasan/IDN-6556 augments, rather than prevents, killing by birinapant. Deletion of caspase-8 sensitized AML to birinapant, whereas combined loss of caspase-8 and the necroptosis effector MLKL (mixed lineage kinase domain-like) prevented birinapant/IDN-6556-induced death, showing that inhibition of caspase-8 sensitizes AML cells to birinapant-induced necroptosis. However, loss of MLKL alone did not prevent a caspase-dependent birinapant/IDN-6556-induced death, implying that AML will be less likely to acquire resistance to this drug combination. A therapeutic breakthrough in AML has eluded researchers for decades. Demonstrated antileukemic efficacy and safety of the birinapant/emricasan combination in vivo suggest that induction of necroptosis warrants clinical investigation as a therapeutic opportunity in AML.

Targeting p38 or MK2 Enhances the Anti-Leukemic Activity of Smac-Mimetics.

Birinapant is a smac-mimetic (SM) in clinical trials for treating cancer. SM antagonize inhibitor of apoptosis (IAP) proteins and simultaneously induce tumor necrosis factor (TNF) secretion to render cancers sensitive to TNF-induced killing. To enhance SM efficacy, we screened kinase inhibitors for their ability to increase TNF production of SM-treated cells. We showed that p38 inhibitors increased TNF induced by SM. Unexpectedly, even though p38 is required for Toll-like receptors to induce TNF, loss of p38 or its downstream kinase MK2 increased induction of TNF by SM. Hence, we show that the p38/MK2 axis can inhibit or promote TNF production, depending on the stimulus. Importantly, clinical p38 inhibitors overcame resistance of primary acute myeloid leukemia to birinapant.

Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells.

Ezrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells.