The RIPK1 death domain restrains ZBP1- and TRIF-mediated cell death and inflammation.

RIPK1 is a multi-functional kinase that regulates cell death and inflammation and has been implicated in the pathogenesis of inflammatory diseases. RIPK1 acts in a kinase-dependent and kinase-independent manner to promote or suppress apoptosis and necroptosis, but the underlying mechanisms remain poorly understood. Here, we show that a mutation (R588E) disrupting the RIPK1 death domain (DD) caused perinatal lethality induced by ZBP1-mediated necroptosis. Additionally, these mice developed postnatal inflammatory pathology, which was mediated by necroptosis-independent TNFR1, TRADD, and TRIF signaling, partially requiring RIPK3. Our biochemical mechanistic studies revealed that ZBP1- and TRIF-mediated activation of RIPK3 required RIPK1 kinase activity in wild-type cells but not in Ripk1R588E/R588E cells, suggesting that DD-dependent oligomerization of RIPK1 and its interaction with FADD determine the mechanisms of RIPK3 activation by ZBP1 and TRIF. Collectively, these findings revealed a critical physiological role of DD-dependent RIPK1 signaling that is important for the regulation of tissue homeostasis and inflammation.

Identification of FasL as a crucial host factor driving COVID-19 pathology and lethality.

The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL.

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.

RIPK1-mediated immunogenic cell death promotes anti-tumour immunity against soft-tissue sarcoma.

Drugs that mobilise the immune system against cancer are dramatically improving care for many people. Dying cancer cells play an active role in inducing anti-tumour immunity but not every form of death can elicit an immune response. Moreover, resistance to apoptosis is a major problem in cancer treatment and disease control. While the term "immunogenic cell death" is not fully defined, activation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) can induce a type of death that mobilises the immune system against cancer. However, no clinical treatment protocols have yet been established that would harness the immunogenic potential of RIPK1. Here, we report the first pre-clinical application of an in vivo treatment protocol for soft-tissue sarcoma that directly engages RIPK1-mediated immunogenic cell death. We find that RIPK1-mediated cell death significantly improves local disease control, increases activation of CD8+ T cells as well as NK cells, and enhances the survival benefit of immune checkpoint blockade. Our findings warrant a clinical trial to assess the survival benefit of RIPK1-induced cell death in patients with advanced disease at limb extremities.

RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

LUBAC determines chemotherapy resistance in squamous cell lung cancer.

Lung squamous cell carcinoma (LSCC) and adenocarcinoma (LADC) are the most common lung cancer subtypes. Molecular targeted treatments have improved LADC patient survival but are largely ineffective in LSCC. The tumor suppressor FBW7 is commonly mutated or down-regulated in human LSCC, and oncogenic KRasG12D activation combined with Fbxw7 inactivation in mice (KF model) caused both LSCC and LADC. Lineage-tracing experiments showed that CC10+, but not basal, cells are the cells of origin of LSCC in KF mice. KF LSCC tumors recapitulated human LSCC resistance to cisplatin-based chemotherapy, and we identified LUBAC-mediated NF-κB signaling as a determinant of chemotherapy resistance in human and mouse. Inhibition of NF-κB activation using TAK1 or LUBAC inhibitors resensitized LSCC tumors to cisplatin, suggesting a future avenue for LSCC patient treatment.

RIPK1 prevents TRADD-driven, but TNFR1 independent, apoptosis during development.

RIPK1 is an essential downstream component of many pattern recognition and death receptors. RIPK1 can promote the activation of caspase-8 induced apoptosis and RIPK3-MLKL-mediated necroptosis, however, during development RIPK1 limits both forms of cell death. Accordingly, Ripk1-/- mice present with systemic cell death and consequent multi-organ inflammation, which is driven through the activation of both FADD-caspase-8 and RIPK3-MLKL signaling pathways causing perinatal lethality. TRADD is a death domain (DD) containing molecule that mediates signaling downstream of TNFR1 and the TLRs. Following the disassembly of the upstream receptor complexes either RIPK1 or TRADD can form a complex with FADD-caspase-8-cFLIP, via DD-DD interactions with FADD, facilitating the activation of caspase-8. We show that genetic deletion of Ripk1 licenses TRADD to complex with FADD-caspase-8 and activates caspase-8 during development. Deletion of Tradd provided no survival advantage to Ripk1-/- animals and yet was sufficient to reduce the systemic cell death and inflammation, rescue the intestinal and thymic histopathologies, reduce cleaved caspases in most tissues and rescue the anemia observed in Ripk1-/- neonates. Furthermore, deletion of Ripk3 is sufficient to rescue the neonatal lethality of Ripk1-/-Tradd-/- animals and delays but does not completely prevent early mortality. Although Ripk3 deletion provides a significant survival advantage, Ripk1-/-Tradd-/-Ripk3-/- animals die between 22 and 49 days, are runty compared to littermate controls and present with splenomegaly. These findings reveal a new mechanism by which RIPK1 limits apoptosis through blocking TRADD recruitment to FADD and preventing aberrant activation of caspase-8.

Abiraterone shows alternate activity in models of endocrine resistant and sensitive disease.

BACKGROUND: Resistance to endocrine therapy remains a major clinical problem in the treatment of oestrogen-receptor positive (ER+) breast cancer. Studies show androgen-receptor (AR) remains present in 80-90% of metastatic breast cancers providing support for blockade of AR-signalling. However, clinical studies with abiraterone, which blocks cytochrome P450 17A1 (CYP17A1) showed limited benefit. METHODS: In order to address this, we assessed the impact of abiraterone on cell-viability, cell-death, ER-mediated transactivation and recruitment to target promoters. together with ligand-binding assays in a panel of ER+ breast cancer cell lines that were either oestrogen-dependent, modelling endocrine-sensitive disease, or oestrogen-independent modelling relapse on an aromatase inhibitor. The latter, harboured wild-type (wt) or naturally occurring ESR1 mutations. RESULTS: Similar to oestrogen, abiraterone showed paradoxical impact on proliferation by stimulating cell growth or death, depending on whether the cells are hormone-dependent or have undergone prolonged oestrogen-deprivation, respectively. Abiraterone increased ER-turnover, induced ER-mediated transactivation and ER-degradation via the proteasome. CONCLUSIONS: Our study confirms the oestrogenic activity of abiraterone and highlights its differential impact on cells dependent on oestrogen for their proliferation vs. those that are ligand-independent and harbour wt or mutant ESR1. These properties could impact the clinical efficacy of abiraterone in breast cancer.

Mind Bomb Regulates Cell Death during TNF Signaling by Suppressing RIPK1's Cytotoxic Potential.

Tumor necrosis factor (TNF) is an inflammatory cytokine that can signal cell survival or cell death. The mechanisms that switch between these distinct outcomes remain poorly defined. Here, we show that the E3 ubiquitin ligase Mind Bomb-2 (MIB2) regulates TNF-induced cell death by inactivating RIPK1 via inhibitory ubiquitylation. Although depletion of MIB2 has little effect on NF-κB activation, it sensitizes cells to RIPK1- and caspase-8-dependent cell death. We find that MIB2 represses the cytotoxic potential of RIPK1 by ubiquitylating lysine residues in the C-terminal portion of RIPK1. Our data suggest that ubiquitin conjugation of RIPK1 interferes with RIPK1 oligomerization and RIPK1-FADD association. Disruption of MIB2-mediated ubiquitylation, either by mutation of MIB2's E3 activity or RIPK1's ubiquitin-acceptor lysines, sensitizes cells to RIPK1-mediated cell death. Together, our findings demonstrate that Mind Bomb E3 ubiquitin ligases can function as additional checkpoint of cytokine-induced cell death, selectively protecting cells from the cytotoxic effects of TNF.

Ubiquitin-Mediated Regulation of RIPK1 Kinase Activity Independent of IKK and MK2.

Tumor necrosis factor (TNF) can drive inflammation, cell survival, and death. While ubiquitylation-, phosphorylation-, and nuclear factor κB (NF-κB)-dependent checkpoints suppress the cytotoxic potential of TNF, it remains unclear whether ubiquitylation can directly repress TNF-induced death. Here, we show that ubiquitylation regulates RIPK1's cytotoxic potential not only via activation of downstream kinases and NF-kB transcriptional responses, but also by directly repressing RIPK1 kinase activity via ubiquitin-dependent inactivation. We find that the ubiquitin-associated (UBA) domain of cellular inhibitor of apoptosis (cIAP)1 is required for optimal ubiquitin-lysine occupancy and K48 ubiquitylation of RIPK1. Independently of IKK and MK2, cIAP1-mediated and UBA-assisted ubiquitylation suppresses RIPK1 kinase auto-activation and, in addition, marks it for proteasomal degradation. In the absence of a functional UBA domain of cIAP1, more active RIPK1 kinase accumulates in response to TNF, causing RIPK1 kinase-mediated cell death and systemic inflammatory response syndrome. These results reveal a direct role for cIAP-mediated ubiquitylation in controlling RIPK1 kinase activity and preventing TNF-mediated cytotoxicity.

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.

PIM1 kinase regulates cell death, tumor growth and chemotherapy response in triple-negative breast cancer.

Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1. Treatment with the pan-PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.

The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles.

Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-ß to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.

AKT Antagonist AZD5363 Influences Estrogen Receptor Function in Endocrine-Resistant Breast Cancer and Synergizes with Fulvestrant (ICI182780) In Vivo.

PI3K/AKT/mTOR signaling plays an important role in breast cancer. Its interaction with estrogen receptor (ER) signaling becomes more complex and interdependent with acquired endocrine resistance. Targeting mTOR combined with endocrine therapy has shown clinical utility; however, a negative feedback loop exists downstream of PI3K/AKT/mTOR. Direct blockade of AKT together with endocrine therapy may improve breast cancer treatment. AZD5363, a novel pan-AKT kinase catalytic inhibitor, was examined in a panel of ER(+) breast cancer cell lines (MCF7, HCC1428, T47D, ZR75.1) adapted to long-term estrogen deprivation (LTED) or tamoxifen (TamR). AZD5363 caused a dose-dependent decrease in proliferation in all cell lines tested (GI50 < 500 nmol/L) except HCC1428 and HCC1428-LTED. T47D-LTED and ZR75-LTED were the most sensitive of the lines (GI50 ∼ 100 nmol/L). AZD5363 resensitized TamR cells to tamoxifen and acted synergistically with fulvestrant. AZD5363 decreased p-AKT/mTOR targets leading to a reduction in ERα-mediated transcription in a context-specific manner and concomitant decrease in recruitment of ER and CREB-binding protein (CBP) to estrogen response elements located on the TFF1, PGR, and GREB1 promoters. Furthermore, AZD5363 reduced expression of cell-cycle-regulatory proteins. Global gene expression highlighted ERBB2-ERBB3, ERK5, and IGFI signaling pathways driven by MYC as potential feedback-loops. Combined treatment with AZD5363 and fulvestrant showed synergy in an ER(+) patient-derived xenograft and delayed tumor progression after cessation of therapy. These data support the combination of AZD5363 with fulvestrant as a potential therapy for breast cancer that is sensitive or resistant to E-deprivation or tamoxifen and that activated AKT is a determinant of response, supporting the need for clinical evaluation.

Importance of EGFR/ERCC1 interaction following radiation-induced DNA damage.

PURPOSE: The epidermal growth factor receptor (EGFR) plays an important role in cellular response to chemotherapy and radiotherapy through modulation of DNA repair. EGFR activates DNA-dependent protein kinase (DNA-PK) stimulating repair of DNA strand breaks (SB) and interstrand crosslinks (ICL). We investigated the role of EGFR in repair of ionizing radiation (IR)-induced SB independently of DNA-PK. EXPERIMENTAL DESIGN: The EGFR interactome was investigated via mass spectrometry. IR-induced EGFR-ERCC1 binding was validated biochemically and via proximity ligation assay in different cell lines including the M059K and M059J glioma cell lines, proficient and deficient for the expression of DNAPKcs, respectively. EGFR-ERCC1 functional significance following IR-induced SB was investigated in knockdown experiments with the Comet and γH2AX foci assays. The effect of this interaction was tested with EGFR-ERCC1 knockdown in combination with gefitinib and NU7026 using the MTT and apoptosis assays. RESULTS: This study demonstrates that EGFR inhibition further impairs IR-induced DNA repair in cells lacking expression of DNAPKcs or in combination with the DNAPK inhibitor NU7026. Our data suggest a role for EGFR in DNA repair independent of DNAPKcs but dependent on ERCC1. Alkaline comet and γH2AX foci assays in cells depleted of EGFR, ERCC1, or EGFR-ERCC1 expression demonstrated involvement of this interaction in DNA repair. Cellular survival and apoptosis data correlate with levels of residual DNA damage underlying the importance of this complex following SB. CONCLUSION: These data emphasize the importance of understanding the various mechanisms by which EGFR modulates DNA repair to optimize targeted therapy for patients with cancer.

EGFR nuclear translocation modulates DNA repair following cisplatin and ionizing radiation treatment.

Epidermal growth factor receptor (EGFR) overexpression is associated with resistance to chemotherapy and radiotherapy. It modulates DNA repair after radiation-induced damage through association with the catalytic subunit of DNA protein kinase (DNA-PKcs). We investigated the role of EGFR nuclear import and its association with DNA-PKcs on DNA repair after exposure to cisplatin or ionizing radiation (IR). The model system was based on EGFR-null murine NIH3T3 fibroblasts in which EGFR expression was restored with isoforms that were wild-type (wt), derived from human cancers (L858R, EGFRvIII), or mutated in the nuclear localization signal (NLS) sequence. In cells expressing wtEGFR or EGFRvIII, there was complete unhooking of cisplatin-induced interstrand cross-links and repair of IR-induced strand breaks. In contrast, cells expressing L858R or NLS mutations showed reduced unhooking of interstrand cross-links and repair of strand breaks. Immunoprecipitation showed wtEGFR and EGFRvIII binding to DNA-PKcs, increasing 2-fold 18 hours after cisplatin therapy. Confocal microscopy and proximity ligation assay showed that this interaction in the cytoplasm and nucleus was associated with increased DNA protein kinase complex (DNA-PK) activity. Cells expressing the EGFR L858R mutation, which has constitutive kinase activity, exhibited reduced DNA repair without nuclear localization. EGFR-NLS mutants showed impaired nuclear localization and DNA-PKcs association with reduced DNA repair and DNA-PK kinase activity. In summary, EGFR nuclear localization was required for modulation of cisplatin and IR-induced repair of DNA damage. EGFR-DNA-PKcs binding was induced by cisplatin or IR but not by EGFR nuclear translocation per se. Our findings show that EGFR subcellular distribution can modulate DNA repair kinetics, with implications for design of EGFR-targeted combinational therapies.