The long FLIP splice form FLIP(L) can act as both an inhibitor and promoter of caspase-8 at death-inducing signalling complexes (DISCs) formed by death receptors such as TRAIL-R2 and related intracellular complexes such as the ripoptosome. Herein, we describe a revised DISC assembly model that explains how FLIP(L) can have these opposite effects by defining the stoichiometry (with respect to caspase-8) at which it converts from being anti- to pro-apoptotic at the DISC. We also show that in the complete absence of FLIP(L), procaspase-8 activation at the TRAIL-R2 DISC has significantly slower kinetics, although ultimately the extent of apoptosis is significantly greater. This revised model of DISC assembly also explains why FLIP's recruitment to the TRAIL-R2 DISC is impaired in the absence of caspase-8 despite showing that it can interact with the DISC adaptor protein FADD and why the short FLIP splice form FLIP(S) is the more potent inhibitor of DISC-mediated apoptosis.

Multivalent second-generation TRAIL-R2 agonists are currently in late preclinical development and early clinical trials. Herein, we use a representative second-generation agent, MEDI3039, to address two major clinical challenges facing these agents: lack of predictive biomarkers to enable patient selection and emergence of resistance. Genome-wide CRISPR knockout screens were notable for the lack of resistance mechanisms beyond the canonical TRAIL-R2 pathway (caspase-8, FADD, BID) as well as p53 and BAX in TP53 wild-type models, whereas a CRISPR activatory screen identified cell death inhibitors MCL-1 and BCL-XL as mechanisms to suppress MEDI3039-induced cell death. High-throughput drug screening failed to identify genomic alterations associated with response to MEDI3039; however, transcriptomics analysis revealed striking association between MEDI3039 sensitivity and expression of core components of the extrinsic apoptotic pathway, most notably its main apoptotic effector caspase-8 in solid tumor cell lines. Further analyses of colorectal cell lines and patient-derived xenografts identified caspase-8 expression ratio to its endogenous regulator FLIP(L) as predictive of sensitivity to MEDI3039 in several major solid tumor types and a further subset indicated by caspase-8:MCL-1 ratio. Subsequent MEDI3039 combination screening of TRAIL-R2, caspase-8, FADD, and BID knockout models with 60 compounds with varying mechanisms of action identified two inhibitor of apoptosis proteins (IAP) that exhibited strong synergy with MEDI3039 that could reverse resistance only in BID-deleted models. In summary, we identify the ratios of caspase-8:FLIP(L) and caspase-8:MCL-1 as potential predictive biomarkers for second-generation TRAIL-R2 agonists and loss of key effectors such as FADD and caspase-8 as likely drivers of clinical resistance in solid tumors.

Impaired PTEN function is a genetic hallmark of aggressive prostate cancers (CaP) and is associated with increased CXCL8 expression and signaling. The current aim was to further characterize biological responses and mechanisms underpinning CXCL8-promoted progression of PTEN-depleted prostate cancer, focusing on characterizing the potential interplay between CXCL8 and other disease-promoting chemokines resident within the prostate tumor microenvironment. Autocrine CXCL8-stimulation (i) increased expression of CXCR1 and CXCR2 in PTEN-deficient CaP cells suggesting a self-potentiating signaling axis and (ii) induced expression of CXCR4 and CCR2 in PTEN-wild-type and PTEN-depleted CaP cells. In contrast, paracrine CXCL8 signaling induced expression and secretion of the chemokines CCL2 and CXCL12 from prostate stromal WPMY-1 fibroblasts and monocytic macrophage-like THP-1 cells. In vitro studies demonstrated functional co-operation of tumor-derived CXCL8 with stromal-derived chemokines. CXCL12-induced migration of PC3 cells and CCL2-induced proliferation of prostate cancer cells were dependent upon intrinsic CXCL8 signaling within the prostate cancer cells. For example, in co-culture experiments, CXCL12/CXCR4 signaling but not CCL2/CCR2 signaling supported fibroblast-mediated migration of PC3 cells while CXCL12/CXCR4 and CCL2/CCR2 signaling underpinned monocyte-enhanced migration of PC3 cells. Combined inhibition of both CXCL8 and CXCL12 signaling was more effective in inhibiting fibroblast-promoted cell motility while repression of CXCL8 attenuated CCL2-promoted proliferation of prostate cancer cells. We conclude that tumor-derived CXCL8 signaling from PTEN-deficient tumor cells increases the sensitivity and responsiveness of CaP cells to stromal chemokines by concurrently upregulating receptor expression in cancer cells and inducing stromal chemokine synthesis. Combined chemokine targeting may be required to inhibit their multi-faceted actions in promoting the invasion and proliferation of aggressive CaP.

Inhibitors of apoptosis proteins (IAPs) are intracellular proteins, with important roles in regulating cell death, inflammation, and immunity. Here, we examined the clinical and therapeutic relevance of IAPs in colorectal cancer. We found that elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in patients with microsatellite stable (MSS) stage III colorectal cancer treated with 5-fluorouracil (5FU)-based adjuvant chemotherapy, suggesting their involvement in promoting chemoresistance. A novel IAP antagonist tolinapant (ASTX660) potently and rapidly downregulated cIAP1 in colorectal cancer models, demonstrating its robust on-target efficacy. In cells co-cultured with TNFα to mimic an inflammatory tumor microenvironment, tolinapant induced caspase-8-dependent apoptosis in colorectal cancer cell line models; however, the extent of apoptosis was limited because of inhibition by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinapant-induced apoptosis was augmented by FOLFOX in human colorectal cancer and murine organoid models in vitro and in vivo, due (at least in part) to FOLFOX-induced downregulation of class I histone deacetylases (HDAC), leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, the effects of FOLFOX could be phenocopied using the clinically relevant class I HDAC inhibitor, entinostat, which also induced acetylation of Ku70 and FLIP downregulation. Further analyses revealed that caspase-8 knockout RIPK3-positive colorectal cancer models were sensitive to tolinapant-induced necroptosis, an effect that could be exploited in caspase-8-proficient models using the clinically relevant caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX in poor prognosis MSS colorectal cancer with elevated cIAP1/2 expression.

Expression of tumor necrosis factor-α (TNFα) in the serum of prostate cancer patients is associated with poorer outcome and progression to castrate-resistant (CRPC) disease. TNFα promotes the activity of NFκB, which regulates a number of anti-apoptotic and proinflammatory genes, including those encoding the inhibitor of apoptosis proteins (IAPs); however, in the presence of IAP antagonists, TNFα can induce cell death. In the presence of recombinant or macrophage-derived TNFα, we found that IAP antagonists triggered degradation of cIAP1 and induced formation of Complex-IIb, consisting of caspase-8, FADD and RIPK1 in CRPC models; however, no, or modest levels of apoptosis were induced. This resistance was found to be mediated by both the long (L) and short (S) splice forms of the caspase-8 inhibitor, FLIP, another NFκB-regulated protein frequently overexpressed in CRPC. By decreasing FLIP expression at the post-transcriptional level in PC3 and DU145 cells (but not VCaP), the Class-I histone deacetylase (HDAC) inhibitor Entinostat promoted IAP antagonist-induced cell death in these models in a manner dependent on RIPK1, FADD and Caspase-8. Of note, Entinostat primarily targeted the nuclear rather than cytoplasmic pool of FLIP(L). While the cytoplasmic pool of FLIP(L) was highly stable, the nuclear pool was more labile and regulated by the Class-I HDAC target Ku70, which we have previously shown regulates FLIP stability. The efficacy of IAP antagonist (TL32711) and Entinostat combination and their effects on cIAP1 and FLIP respectively were confirmed in vivo, highlighting the therapeutic potential for targeting IAPs and FLIP in proinflammatory CRPC.

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

Ligand binding to death receptors activates apoptosis in cancer cells. Stimulation of death receptors results in the formation of intracellular multiprotein platforms that either activate the apoptotic initiator Caspase-8 to trigger cell death, or signal through kinases to initiate inflammatory and cell survival signalling. Two of these platforms, the Death-Inducing Signalling Complex (DISC) and the RIPoptosome, also initiate necroptosis by building filamentous scaffolds that lead to the activation of mixed lineage kinase domain-like pseudokinase. To explain cell decision making downstream of death receptor activation, we developed a semi-stochastic model of DISC/RIPoptosome formation. The model is a hybrid of a direct Gillespie stochastic simulation algorithm for slow assembly of the RIPoptosome and a deterministic model of downstream caspase activation. The model explains how alterations in the level of death receptor-ligand complexes, their clustering properties and intrinsic molecular fluctuations in RIPoptosome assembly drive heterogeneous dynamics of Caspase-8 activation. The model highlights how kinetic proofreading leads to heterogeneous cell responses and results in fractional cell killing at low levels of receptor stimulation. It reveals that the noise in Caspase-8 activation-exclusively caused by the stochastic molecular assembly of the DISC/RIPoptosome platform-has a key function in extrinsic apoptotic stimuli recognition.

Colorectal cancer is a molecularly heterogeneous disease. Responses to genotoxic chemotherapy in the adjuvant or palliative setting vary greatly between patients, and colorectal cancer cells often resist chemotherapy by evading apoptosis. Antagonists of an inhibitor of apoptosis proteins (IAPs) can restore defective apoptosis signaling by degrading cIAP1 and cIAP2 proteins and by inhibition of XIAP. Due to the multiple molecular mechanisms-of-action of these targets, responses to IAP antagonist may differ between molecularly distinct colon cancer cells. In this study, responses to the IAP antagonist Birinapant and oxaliplatin/5-fluorouracil (5-FU) were investigated in 14 colon cancer cell lines, representing the consensus molecular subtypes (CMS). Treatment with Birinapant alone did not result in a substantial increase in apoptotic cells in this cell line panel. Annexin-V/PI assays quantified by flow cytometry and high-content screening showed that Birinapant increased responses of CMS1 and partially CMS3 cell lines to oxaliplatin/5-FU, whereas CMS2 cells were not effectively sensitized. FRET-based imaging of caspase-8 and -3 activation validated these differences at the single-cell level, with CMS1 cells displaying sustained activation of caspase-8-like activity during Birinapant and oxaliplatin/5-FU co-treatment, ultimately activating the intrinsic mitochondrial apoptosis pathway. In CMS2 cell lines, Birinapant exhibited synergistic effects in combination with TNFα, suggesting that Birinapant can restore extrinsic apoptosis signaling in the context of inflammatory signals in this subtype. To explore this further, we co-cultured CMS2 and CMS1 colon cancer cells with peripheral blood mononuclear cells. We observed increased cell death during Birinapant single treatment in these co-cultures, which was abrogated by anti-TNFα-neutralizing antibodies. Collectively, our study demonstrates that IAP inhibition is a promising modulator of response to oxaliplatin/5-FU in colorectal cancers of the CMS1 subtype, and may show promise as in the CMS2 subtype, suggesting that molecular subtyping may aid as a patient stratification tool for IAP antagonists in this disease.

Inhibiting androgen signaling using androgen signaling inhibitors (ASI) remains the primary treatment for castrate-resistant prostate cancer. Acquired resistance to androgen receptor (AR)-targeted therapy represents a major impediment to durable clinical response. Understanding resistance mechanisms, including the role of AR expressed in other cell types within the tumor microenvironment, will extend the clinical benefit of AR-targeted therapy. Here, we show the ASI enzalutamide induces vascular catastrophe and promotes hypoxia and microenvironment adaptation. We characterize treatment-induced hypoxia, and subsequent induction of angiogenesis, as novel mechanisms of relapse to enzalutamide, highlighting the importance of two hypoxia-regulated cytokines in underpinning relapse. We confirmed AR expression in CD34+ vascular endothelium of biopsy tissue and human vascular endothelial cells (HVEC). Enzalutamide attenuated angiogenic tubule formation and induced cytotoxicity in HVECs in vitro, and rapidly induced sustained hypoxia in LNCaP xenografts. Subsequent reoxygenation, following prolonged enzalutamide treatment, was associated with increased tumor vessel density and accelerated tumor growth. Hypoxia increased AR expression and transcriptional activity in prostate cells in vitro. Coinhibition of IL8 and VEGF-A restored tumor response in the presence of enzalutamide, confirming the functional importance of their elevated expression in enzalutamide-resistant models. Moreover, coinhibition of IL8 and VEGF-A resulted in a durable, effective resolution of enzalutamide-sensitive prostate tumors. We conclude that concurrent inhibition of two hypoxia-induced factors, IL8 and VEGF-A, prolongs tumor sensitivity to enzalutamide in preclinical models and may delay the onset of enzalutamide resistance.

Functional impairment of the tumour suppressor PTEN is common in primary prostate cancer and has been linked to relapse post-radiotherapy (post-RT). Pre-clinical modelling supports elevated CXC chemokine signalling as a critical mediator of PTEN-depleted disease progression and therapeutic resistance. We assessed the correlation of PTEN deficiency with CXC chemokine signalling and its association with clinical outcomes. Gene expression analysis characterized a PTEN LOW/CXCR1HIGH/CXCR2HIGH cluster of tumours that associates with earlier time to biochemical recurrence [hazard ratio (HR) 5.87 and 2.65, respectively] and development of systemic metastasis (HR 3.51). In vitro, CXCL signalling was further amplified following exposure of PTEN-deficient prostate cancer cell lines to ionizing radiation (IR). Inhibition of CXCR1/2 signalling in PTEN-depleted cell-based models increased IR sensitivity. In vivo, administration of a CXCR1/2-targeted pepducin (x1/2pal-i3), or CXCR2-specific antagonist (AZD5069), in combination with IR to PTEN-deficient xenografts attenuated tumour growth and progression compared to control or IR alone. Post-mortem analysis confirmed that x1/2pal-i3 administration attenuated IR-induced CXCL signalling and anti-apoptotic protein expression. Interventions targeting CXC chemokine signalling may provide an effective strategy to combine with RT in locally advanced prostate cancer patients with known presence of PTEN-deficient foci.