Immunogenic Form Of Cell Death Research Articles

Conditionally Activatable Pyroptosis‐Inducing Agents for Cancer Therapy

Pyroptosis is a recently defined form of immunogenic cell death that shows great promise in cancer immunotherapy. However, almost all small‐molecule pyroptosis‐inducing agents (PyAs) reported to date indiscriminately induce pyroptosis in multiple cell types, leading to off‐target pyroptotic death of normal and immune cells. One promising approach to addressing this biosafety issue is the design of conditionally activatable PyAs that specifically respond to disease biomarkers. Herein, a general solution for facilely tailoring and synthesizing activatable PyAs based on a newly developed class of photoactive PyAs, termed PyPSs, is reported. The unique structurally encoded properties of PyPSs, including endo reticulum targeting, hypoxia tolerance, and sensing properties, excitingly meet a demanding set of performance requirements for constructing conditionally activatable PyAs for cancer immunotherapy. Based on PyPS‐1 scaffold, hypoxia‐activatable PyPS‐NF as a proof‐of‐concept example is prepared, demonstrating specific hypoxia‐activated pyroptotic cell death and favorable immunotherapeutic efficacy of solid tumors. Herein, a general design strategy for tailoring activatable PyAs to precisely control GSDME‐mediated cell pyroptosis is established, with great potential to advance cancer immunotherapy.

Enhancing immunogenic responses through CDK4/6 and HIF2α inhibition in Merkel cell carcinoma

Approximately 50% of Merkel cell carcinoma (MCC) patients facing this highly aggressive skin cancer initially respond positively to PD-1-based immunotherapy. Nevertheless, the recurrence of MCC post-immunotherapy emphasizes the pressing need for more effective treatments. Recent research has highlighted Cyclin-dependent kinases 4 and 6 (CDK4/6) as pivotal cell cycle regulators gaining prominence in cancer studies. This study reveals that the CDK4/6 inhibitor, palbociclib can enhance PD-L1 gene transcription and surface expression in MCC cells by activating HIF2α. Inhibiting HIF2α with TC-S7009 effectively counteracts palbociclib-induced PD-L1 transcription and significantly intensifies cell death in MCC. Simultaneously, co-targeting CDK4/6 and HIF2α boosts ROS levels while suppressing SLC7A11, a key regulator of cellular redox balance, promoting ferroptosis- a form of immunogenic cell death linked to iron. Considering the rising importance of immunogenic cell death in immunotherapy, this strategy holds promise for improving future MCC treatments, markedly increasing immunogenic cell death across various MCC cell lines, thus advancing cancer immunotherapy.

Epigenetic Priming By Hypomethylation Enhances the Immunogenic Potential of Tolinapant in T-Cell Lymphoma

Background:Durable single-agent responses with the IAP antagonist, tolinapant (ASTX660), have been reported in peripheral T-cell lymphoma (PTCL) and cutaneous TCL (CTCL) patients (NCT02503423, Michot et al., EHA 2022). We have found that tolinapant has the potential to drive immunogenic form of cell death (ICD), such as necroptosis, in preclinical models of TCL (Ferrari et al., Blood Adv. 2021). Here, we further explored this by modulating key necrosome components in TCL cell lines and analysing changes in cell death signalling. We hypothesised the epigenetic mechanisms that limit induction of ICD could be reversed by combining tolinapant and hypomethylating agents, such as decitabine. Our biomarker-led approach demonstrated that this novel combination could overcome inherent resistance to a form of cell death that can engage a strong immune response. Materials and Methods: Key necrosome components were modified in human and mouse TCL cell lines by CRISPR knockout and activation, and effects of tolinapant on their viability measured in vitro. Promoter methylation was assessed by pyrosequencing following bisulfite treatment (EpigenDx). Effects of tolinapant and decitabine, as single-agents and in combination, on cytokine/chemokine release were assessed using Meso Scale Discovery (MSD) or Luminex assay. ICD biomarkers in cells and tumor lysates were analysed by ELISAs, MSD assays, qPCR and Western blotting. In vivo activity of tolinapant and decitabine were tested in caspase-8 knockout EL4 cells in a syngeneic setting. PTCL patient plasma samples were analysed by Human ExplorerMAP™ v. 1.0 (Luminex xMAP® technology). Results: Loss of necrosome components or necrosome inactivation led to reduced sensitivity of TCL cells to tolinapant. We confirmed decitabine treatment resulted in re-expression of necrosome proteins (e.g., RIPK3) and cancer testis antigens. Combination of decitabine and tolinapant led to increased lytic cell death over monotherapies, and this was accompanied by increased levels of interferon signalling and ICD biomarkers (e.g., HMGB1). Tolinapant treatment alone, or in combination with decitabine, led to elevated chemokine release from dying TCL cells. These observations indicate activation of necroptosis. In vivo, the combination treatment prolonged survival of mice bearing caspase-8 knockout EL4 tumors while monotherapies had limited effects. We also report that IP-10 (CXCL10) levels were increased in plasma samples of tolinapant-treated PTCL patients, as well as other plasma proteins, implying immune activation. Conclusions: We demonstrate that tolinapant acts as an immune modulator via promoting ICD, and this can be enhanced by epigenetic reprogramming with decitabine. These findings provide a rationale to support the on-going combination study of tolinapant with ASTX727 (oral decitabine and cedazuridine) that recently started in PTCL (ASCERTAIN-P, NCT05403450). It has also identified putative biomarkers of immunomodulatory activity that could be utilized in clinic.

Pyroptosis induction synergized with IDO inhibition of ternary biomedicine for photodynamic activated tumor immunotherapy

The development of immunotherapy is seriously limited by the insufficient tumor immunogenicity and immunosuppressive microenvironment. Pyroptosis is a new form of immunogenic cell death (ICD) associated with the gasdermin protein family, which has received increasing attention as a promising approach to activate antitumor immunity. Herein, we construct a ternary biomedicine (CNS) based on the self-assembly of photosensitizer chlorine e6 (Ce6), NLG919 (NLG) and simvastatin (Sim), which has a fairly high drug content, good stability and uniform morphology. Interestingly, the photodynamic therapy of CNS can generate reactive oxygen species (ROS) to induce intracellular oxidative stress, activating NOD-like receptor thermal protein domain associated protein 3 (NLRP3)/Caspase-1 (Cas-1)/gasdermin D N-terminal domain (GSDMD-N) dependent pyroptosis. Further, Sim-mediated NLRP3 upregulation will also promote cell pyroptosis to activate ICD and stimulate immune responses. Benefiting from NLG-induced indoleamine 2,3-dioxygenase (IDO) inhibition, CNS is capable of improving the immunosuppressive microenvironment to enhance immunotherapy. Consequently, the pyroptosis induction synergized with IDO inhibition of CNS exhibits excellent antitumor immune activities, as evidenced by enhancing DCs maturation and effector memory T cells frequency, which significantly inhibit tumor growth and metastasis. This proposal of self-delivery ternary nanomedicine might open a new window to construct drug delivery systems for pyroptosis activated immunotherapy.

Cytotoxicity as a form of immunogenic cell death leading to efficient tumor antigen cross-priming.

Antigen cross-priming of CD8+ T cells is a critical process necessary for the effective expansion and activation of CD8+ T cells endowed with the ability to recognize and destroy tumor cells. The cross-presentation of tumor antigens to cross-prime CD8+ T cells is mainly mediated, if not only, by a subset of professional antigen-presenting cells termed type-1 conventional dendritic cells (cDC1). The demise of malignant cells can be immunogenic if it occurs in the context of premortem stress. These ways of dying are termed immunogenic cell death (ICD) and are associated with biochemical features favoring cDC1 for the efficient cross-priming of tumor antigens. Immunosurveillance and the success of immunotherapies heavily rely on the ability of cytotoxic immune cells, primarily CD8+ T cells and NK cells, to detect and eliminate tumor cells through mechanisms collectively known as cytotoxicity. Recent studies have revealed the significance of NK- and CTL-mediated cytotoxicity as a prominent form of immunogenic cell death, resulting in mechanisms that promote and sustain antigen-specific immune responses. This review focuses on the mechanisms underlying the cross-presentation of antigens released during tumor cell killing by cytotoxic immune cells, with an emphasis on the role of cDC1 cells. Indeed, cDC1s are instrumental in the effectiveness of most immunotherapies, underscoring the significance of tumor antigen cross-priming in contexts of immunogenic cell death. The notion of the potent immunogenicity of cell death resulting from NK or cytotoxic T lymphocyte (CTL)-mediated cytotoxicity has far-reaching implications for cancer immunotherapy.

Inhibition of CDC20 potentiates anti-tumor immunity through facilitating GSDME-mediated pyroptosis in prostate cancer

BackgroundIncreasing evidence suggests that immunotherapy, especially immune checkpoint inhibitors (ICIs), has the potential to facilitate long-term survival in various cancer besides prostate cancer. Emerging evidence indicated that pyroptosis, an immunogenic form of cell death, could trigger an anti-tumor immune microenvironment and enhance the effectiveness of immunotherapy. Nevertheless, the mechanism underlying the regulation of pyroptosis signaling in prostate cancer remains unclear.MethodsThe differential expression of human E3 ligases in prostate cancer was integratedly analyzed from five independent public datasets. Moreover, the immunohistochemistry analysis of a tissue microarray derived from prostate cancer patients confirmed the results from the bioinformatic analysis. Furthermore, prostate cancer cell lines were evaluated via the next-generation RNA sequencing to assess transcriptomic profile upon CDC20 depletion. Next, qRT-PCR, Western blotting, cycloheximide assay, immunoprecipitation, and ubiquitination assay were employed to explore the correlation and interaction between CDC20 and GSDME. Both immune-deficient and immune-competent murine models were utilized to examine the anti-tumor efficacy of CDC20 inhibition with or without the anti-PD1 antibodies, respectively. To analyze the immune microenvironment of the xenografts, the tumor tissues were examined by immunohistochemistry and flow cytometry.ResultsThe analysis of multiple prostate cancer cohorts suggested that CDC20 was the most significantly over-expressed E3 ligase. In addition, CDC20 exerted a negative regulatory effect on the pyroptosis pathway by targeting GSDME for ubiquitination-mediated proteolysis in a degron-dependent manner. Knockdown of CDC20 leads to increased GSDME abundance and a transition from apoptosis to pyroptosis in response to death signals. Furthermore, in our syngeneic murine models, we found that depletion of CDC20 significantly enhances the anti-tumor immunity by promoting the infiltration of CD8+ T lymphocytes dependent on the existence of GSDME, as well as reducing myeloid immune cells. More importantly, Apcin, a small molecular inhibitor that targets CDC20, exhibited synergistic effects with anti-PD1-based immunotherapy in murine models of prostate cancer.ConclusionsOverall, these findings provide new insights into the upstream regulation of GSDME-mediated pyroptosis by CDC20, which specifically interacts with GSDME and facilitates its ubiquitination in a degron-dependent manner. Importantly, our data highlight novel molecular pathways for targeting cellular pyroptosis and enhancing the effectiveness of anti-PD1-based immunotherapy.

#4967 GASDERMIN D IS REQUIRED FOR CONTROL OF NECROPTOTIC CELL DEATH IN AKI

Abstract Background and Aims Contradicting previous reasoning, it is now established that necrotic rather than apoptotic cell death pathophysiologically drives acute tubular necrosis during AKI. Whereas involvement of necroptosis and ferroptosis has been demonstrated by various groups, the role of pyroptosis, a highly immunogenic form of cell death requiring proteolytic activation of gasdermin protein family members, remains uncertain upon AKI. Thus, we aimed to investigate the role of pyroptosis and its mechanism of action in AKI. Method We set to detect gasdermin D (GSDMD) expression in murine renal samples after ischemia/reperfusion injury (AKI) by immunohistochemistry. Furthermore, we investigated GSDMD-deficient mice in IRI and cisplatin-induced AKI. To conclude on mechanisms, we isolated fresh murine renal tubules and measured LDH release over time. We generated MLKL/GSDMDdko mice to investigate the interplay of these caspase-dependent forms of regulated necrosis in AKI. Results After IRI, we detected a specific GSDMD signal in areas surrounding necrotic tubules, whereas no such signal could be detected within necrotic areas. Whereas interference with other forms of regulated necrosis leads to reduced injury, GSDMD-deficient mice demonstrated higher levels of serum creatinine and urea as well as more severe tubular injury compared to wildtypes. In line with the findings of immunohistochemistry, we only found GSDMD protein expression in whole kidney lysates but not isolated renal tubules; and no difference in spontaneous cell death propagation was detectable. Co-deletion of MLKL (effector protein of necroptosis) reversed the sensitization to AKI by GSDMD-deficiency. These results could be reproduced in cisplatin-induced AKI. Conclusion Here, we found an unexpected protective role for GSDMD, the effector protein of pyroptosis, in two different models of AKI. Mechanistically, our studies indicate the effect of GSDMD to function outside the tubular compartment, specifically surrounding areas of tubular necrosis. These infiltrating innate immune cells appear to interfere with tubular necroptosis in a non-cell autonomous manner. Alongside with these mechanistic insights, our data urge caution when inhibition of pyroptosis is therapeutically considered.

Brincidofovir induces potent anti‐tumor activity in MYC‐driven Natural Killer/T‐cell Lymphoma with loss of transcriptional repressor TLE1

Introduction: Natural killer/T-cell lymphoma (NKTCL) is an aggressive Epstein-Barr virus (EBV)-associated malignancy which is prevalent in Asian countries. Brincidofovir (BCV), a lipid conjugated form of cidofovir, is a biologically-active acyclic nucleoside phosphonate recently shown to exhibit both antiviral and antitumor properties. Methods: We investigated BCV in NKTCL cell-lines (n = 11) and mouse xenograft models. Whole exome sequencing, bulk RNAseq and single cell RNAseq (scRNAseq) were employed to examine mechanisms of action, and validated using orthogonal assays. Results: BCV inhibited viability in all NKTCL cell-lines in a dose- and time-dependent manner. Highest sensitivity was demonstrated in four cell-lines KAI-3, NK-S1, NK-92 and KHYG-1 (IC50 36.0 to 303.6 ng/ml). Intraperitoneal BCV (40 mg/kg, 2X per week) inhibited tumor growth in NOD/SCID mice NK-S1 xenografts, compared with vehicle alone (p = 0.0005, two-tailed t-test). Whole exome sequencing of the cell-lines revealed widespread mutations in the JAK/STAT and DNA repair pathways, with no clear correlation observed with BCV sensitivity. RNAseq and Hallmark gene set enrichment analysis showed that MYC target pathways (FDR q < 0.001) were prominently upregulated in the four sensitive cell-lines compared to the rest. RNAseq of BCV-treated cell-lines (KAI-3 and NK-S1) revealed striking downregulation of MYC target pathways. scRNAseq revealed distinct temporal cell states evoked by BCV, including appearance of cell clusters enriched for p53 and apoptosis pathways, cell cycle and DNA repair pathways, as well as type I/II interferon and cytokine signaling. Western blot and quantitative PCR showed that markers of replication stress, DNA damage and STING signaling were potently upregulated, implicating an immunogenic form of cell death. Notably, TLE1, a known transcriptional repressor of the MYC oncogene, was the topmost downregulated gene (log2FoldChange -7.39, adjusted p = 3.51E-31) in the four BCV-sensitive cell-lines on RNAseq. This finding was verified on qPCR (p = 0.0061, Mann-Whitney test) and Western blot. In keeping with this result, RNAseq on NKTCL patient samples (n = 36) showed that TLE1-low tumors were enriched for MYC target pathways, which conferred worse progression-free survival (HR 3.44, 95% CI: 1.23–10.49, p = 0.0301). Conclusions: Taken together, these results suggest that BCV may play a novel role in the treatment of MYC-driven NKTCL. Loss of TLE1, a putative predictive biomarker, implies that BCV may favour patients in this poor prognostic subgroup. The research was funded by: Symbio Pharmaceuticals, Tanoto Foundation Professorship in Medical Oncology, New Century Foundation Limited, Ling Foundation, Singapore Ministry of Health’s National Medical Research Council Transition Award (TA21jun-0005), Research Training Fellowship Seed Fund (SEEDFD21jun-0002), Large Collaborative Grant (OFLCG18May-0028), and Collaborative Centre Grant (TETRAD II). Keywords: Diagnostic and Prognostic Biomarkers, Extranodal non-Hodgkin lymphoma, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. J. Y. Chan Consultant or advisory role: Symbio Pharmaceuticals Research funding: Symbio Pharmaceuticals M. Hazama Employment or leadership position: SymBio Pharmaceuticals Limited K. Fukushima Employment or leadership position: SymBio Pharmaceuticals Limited C. K. Ong Research funding: SymBio Pharmaceuticals Limited

Lysosomal lipid peroxidation regulates tumor immunity.

Lysosomal inhibition elicited by palmitoyl protein transferase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC), but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12, was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naïve T cells, and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells, engendered adaptive immunity and tumor rejection in "immune hot" tumors but not in "immune cold" tumors. These findings demonstrate LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

Abstract 6144: Polyploidization enables initial survival of chemotherapy but a progressively increased susceptibility to ferroptosis

Abstract Once cancer has metastasized it remains incurable owing to evolved resistance to nearly all systemic therapies. Classical views of therapeutic resistance include tumor heterogeneity and genetic instability, but our group and others have identified an unappreciated cancer cell phenotype, the polyaneuploid cancer cell (PACC) state, that provides an alternative model. The PACC state is induced upon exposure to a variety of applied stressors, including multiple different classes of chemotherapy, and is characterized by polyploidization of the aneuploid cancer genome. We have shown that this polyploidization is accomplished through endocycling that leads to an enlarged cell size with increased transcriptomic and protein content, higher oxidative buffer capacity, and increased autophagy. We have demonstrated that PACCs are present in a variety of cell lines, arise in response to multiple stressors in vitro, and are resistant to subsequent treatment with several classes of chemotherapy. Thus, we hypothesize that the PACC state is driving therapeutic resistance to current treatment regimens. Elimination of this overlooked phenotype is critical for sustained anticancer therapy response. Cancer cells, including those in the PACC state, control canonical cell cycle and cell death programs to evade apoptosis from external stressors including chemotherapy, highlighting an opportunity to leverage cell cycle-independent death pathways as an alternative therapeutic strategy1. Ferroptosis is a cell cycle agnostic immunogenic form of cell death that is an ideal pathway for elimination of cells in the PACC state. Characterization of ROS levels, glutathione content, labile iron homeostasis, and lipid content indicate that cells in the PACC state have a mounting susceptibility to ferroptosis. Our data suggests that cells in the PACC state keep ferroptosis in check through elevated levels of reduced glutathione and proficient redox activity, including turn over of GPX4, the sole enzyme counteracting lipid peroxidation. Direct inhibition of GPX4 is highly toxic to cells in the PACC state, particularly at later timepoints post therapy removal when the immediate stress response is largely resolved. These studies will directly lead to development of a therapeutic strategy specifically targeting the PACC state with the intention of eliminating cancer resistance. 1Loftus, L.V.; Amend, S.R.; Pienta, K.J. Interplay between Cell Death and Cell Proliferation Reveals New Strategies for Cancer Therapy. Int. J. Mol. Sci. 2022, 23, 4723. https://doi.org/10.3390/ijms23094723 Citation Format: Luke Loftus. Polyploidization enables initial survival of chemotherapy but a progressively increased susceptibility to ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6144.

Abstract 632: Exploiting pyroptosis in melanoma brain metastasis

Abstract Melanoma is the deadliest form of skin cancer due to its frequent metastasis to the brain. Melanoma brain metastases (MBM) are a significant clinical challenge, and current model systems and therapeutic strategies are poor. Inhibitors targeting the most common oncogenic driver of melanoma, BRAFV600E (BRAFi), disrupt mitogenic signaling and promote caspase-3 associated cell death. Though caspase-3 is a well-known apoptotic driver, it can also cleave Gasdermin E (GSDME) to create pore-forming N-terminal fragments that oligomerize at plasma and mitochondrial membranes and trigger pyroptosis - a form of immunogenic cell death (ICD). Our recent work illustrates the importance of GSDME mediated pyroptosis and associated immunogenicity in providing a durable therapeutic response against melanoma. Since pyroptosis can be activated by GSDME cleavage via caspase-3, and is prominent in various neuropathologies, a better understanding of the interplay between central nervous system (CNS) immune cells and BRAFi-mediated ICD is critical for designing more effective therapies against MBM. Here, we engineered a panel of inducible pyroptotic melanoma cells to model BRAFi-mediated pyroptosis. Using ex vivo CNS slice co-cultures, we found that inducible N-GSDME expression in human and murine melanoma cells drives propidium iodide uptake and release of inflammatory activators like HMGB1 and IL-1 - hallmarks of pyroptosis. Importantly, conditioned media from these pyroptotic cells stimulates a more robust phagocytic response from resident macrophages of the CNS and pyroptotic melanoma elicit stronger cytotoxicity from syngeneic CD8+ T cells, further highlighting the benefits of BRAFi-mediated ICD. Our findings provide evidence that resident CNS immune cells and peripheral T cells respond to pyroptotic melanoma, suggesting that induction of ICD should be a consideration in the development of anti-MBM therapies. Citation Format: Maria R. Cavallo. Exploiting pyroptosis in melanoma brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 632.

RIG-I immunotherapy overcomes radioresistance in p53-positive malignant melanoma.

Radiotherapy induces DNA damage, resulting in cell-cycle arrest and activation of cell-intrinsic death pathways. However, the radioresistance of some tumour entities such as malignant melanoma limits its clinical application. The innate immune sensing receptor retinoic acid-inducible gene I (RIG-I) is ubiquitously expressed and upon activation triggers an immunogenic form of cell death in a variety of tumour cell types including melanoma. To date, the potential of RIG-I ligands to overcome radioresistance of tumour cells has not been investigated. Here, we demonstrate that RIG-I activation enhanced the extent and immunogenicity of irradiation-induced tumour cell death in human and murine melanoma cells in vitro and improved survival in the murine B16 melanoma model invivo. Transcriptome analysis pointed to a central role for p53, which was confirmed using p53-/- B16 cells. In vivo, the additional effect of RIG-I in combination with irradiation on tumour growth was absent in mice carrying p53-/- B16 tumours, while the antitumoural response to RIG-I stimulation alone was maintained. Our results identify p53 as a pivotal checkpoint that is triggered by RIG-I resulting in enhanced irradiation-induced tumour cell death. Thus, the combined administration of RIG-I ligands and radiotherapy is a promising approach to treating radioresistant tumours with a functional p53 pathway, such as melanoma.

Pyroptosis Remodeling Tumor Microenvironment to Enhance Pancreatic Cancer Immunotherapy Driven by Membrane Anchoring Photosensitizer.

Immunotherapy, the most promising strategy of cancer treatment, has achieved promising outcomes, but its clinical efficacy in pancreatic cancer is limited mainly due to the complicated tumor immunosuppressive microenvironment. As a highly inflammatory form of immunogenic cell death (ICD), pyroptosis provides a great opportunity to alleviate immunosuppression and promote systemic immune responses in solid tumors. Herein, membrane‐targeted photosensitizer TBD‐3C with aggregation‐induced emission (AIE) feature to trigger pyroptosis‐aroused cancer immunotherapy via photodynamic therapy (PDT) is applied. The results reveal that pyroptotic cells induced by TBD‐3C could stimulate M1‐polarization of macrophages, cause maturation of dendritic cells (DCs), and activation of CD8+ cytotoxic T‐lymphocytes (CTLs). Pyroptosis‐aroused immunological responses could convert immunosuppressive “cold” tumor microenvironment (TME) to immunogenic “hot” TME, which not only inhibits primary pancreatic cancer growth but also attacks the distant tumor. This work establishes a platform with high biocompatibility for light‐controlled antitumor immunity and solid tumor immunotherapy aroused by cell pyroptosis.

Photocatalytic Superoxide Radical Generator that Induces Pyroptosis in Cancer Cells

Pyroptosis, a newly characterized form of immunogenic cell death, is attracting increasing attention as a promising approach to cancer immunotherapy. However, biocompatible strategies to activate pyroptosis remain rare. Here, we show that a photocatalytic superoxide radical (O2-•) generator, NI-TA, triggers pyroptosis in cancer cells. NI-TA was designed to take advantage of an intramolecular triplet-ground state splitting energy modulation approach. Detailed studies revealed that the pyroptosis triggered by NI-TA under conditions of photoexcitation proceeds through a caspase-3/gasdermin E (GSDME) pathway rather than via canonical processes involving caspase-1/gasdermin-D (GSDMD). NI-TA was found to function via a partial-O2-recycling mode of action and to trigger cell pyroptosis and provide for effective cancer cell ablation even under conditions of hypoxia (≤2% O2). In the case of T47D 3D multicellular spheroids, good antitumor efficiency and stemness inhibition are achieved. This work highlights how photocatalytic chemistry may be leveraged to develop effective pyroptosis-inducing agents.

Abstract 1443: CHKing melanoma: CHK1 inhibitor +low dose hydroxyurea triggers immunogenic cell death and immunostimulatory cytokine expression to drive an anti-tumor immune response

Abstract About half of melanomas respond to immunotherapy, but we currently lack reliable markers to identify likely responders. Therefore, a large number of patients receive this expensive therapy for no benefit. We have previously shown that inhibition of the cell cycle regulator, Checkpoint kinase 1 (CHK1), with subclinical dose of the replication stress inducer hydroxyurea is cytotoxic in a high proportion of melanomas tested. We now report that CHK1 inhibitor + low dose hydroxyurea (LDHU) treatment triggers immunostimulatory cytokine and chemokine expression, and an immunogenic form of cell death. In xenograft models, the drug combination blocks tumor growth for extended periods after the completion of treatment. In syngeneic mouse melanoma models, the drug combination also effectively controls tumor growth and induces regression in a proportion of cases. It also increases immune cell infiltration and activation of a tumor directed cytotoxic T cell response. We also demonstrate that the drug combination does not adversely affect an adaptive immune response or rapidly proliferating human T cells. The mechanism by which drug treatment promotes cytokine expression is unlikely to involve the cGAS-STING signalling pathway which is commonly defective in melanomas. Using two engineered mouse melanoma models we have found that CHK1 inhibitor + LDHU increases cytotoxic CD8+ T cells and NK cell infiltration and activation, but we have also found increased CD4+ Treg tumor infiltration and increased CD8+ T cell PD-1 expression. We have also found increased expression of other immune checkpoint regulators. This indicates a strong anti-tumor immune response is initiated but immune suppression and T cell exhaustion have limited the extent of the response of the tumor infiltrating T cells. However, circulating T cells retained strong anti-tumor activity. The harvested tumors from the drug combination treated mice that do increase in size after treatment show strong evidence of cytotoxic activity. These data provide evidence that CHK1 inhibitors + LDHU is well tolerated and triggers a strong anti-cancer immune response that should combine with immune therapy, although the selection of the target of the immunotherapy will be critical for successfully overcoming the immune suppression triggered. Citation Format: Brian G. Gabrielli, Martina Proctor, Jazmina Gonzalez Cruz, Bijun Zeng, Riccardo Dolcetti, Colm Keane, Nikolas Haass, James Wells. CHKing melanoma: CHK1 inhibitor +low dose hydroxyurea triggers immunogenic cell death and immunostimulatory cytokine expression to drive an anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1443.

Emerging roles of ferroptosis in the tumor immune landscape: from danger signals to anti-tumor immunity.

Regulated cell death (RCD) is a vital event in various physiological and pathological processes. Ferroptosis is a newly described RCD, which is driven by iron accumulation and unrestricted lipid peroxidation. The interaction between ferroptosis and immunity has been a topic of substantial interest since its discovery in 2012. It has become increasingly evident that ferroptosis is critically involved in the regulation of antitumor immunity and may provide potential strategies in immunotherapy. Ferroptosis could release various damage-associated molecular patterns (DAMPs) or lipid metabolites to regulate the cellular immune response, validating its role as a form of immunogenic cell death (ICD). Specifically, the oxygenated membrane lipids on ferroptotic cells could mediate the phagocytosis by macrophages to maintain the immune responses. Additionally, immune checkpoint inhibitor therapy may sensitize cancer cells to ferroptosis, while ferroptosis might contribute to tumor immune evasion by directly interfering with the function of various immune cells. Based on these insights, we provided a comprehensive review on the interaction patterns between ferroptosis and immunity, which may not only offer insight into the underlying regulatory mechanisms but also facilitating the development of ferroptosis-based antitumor therapeutics.

Pouring petrol on the flames: Using oncolytic virotherapies to enhance tumour immunogenicity.

Oncolytic viruses possess the ability to infect, replicate and lyse malignantly transformed tumour cells. This oncolytic activity amplifies the therapeutic advantage and induces a form of immunogenic cell death, characterized by increased CD8 + T‐cell infiltration into the tumour microenvironment. This important feature of oncolytic viruses can result in the warming up of immunologically ‘cold’ tumour types, presenting the enticing possibility that oncolytic virus treatment combined with immunotherapies may enhance efficacy. In this review, we assess some of the most promising candidates that might be used for oncolytic virotherapy: immunotherapy combinations. We assess their potential as separate agents or as agents combined into a single therapy, where the immunotherapy is encoded within the genome of the oncolytic virus. The development of such advanced agents will require increasingly sophisticated model systems for their preclinical assessment and evaluation. In vivo rodent model systems are fraught with limitations in this regard. Oncolytic viruses replicate selectively within human cells and therefore require human xenografts in immune‐deficient mice for their evaluation. However, the use of immune‐deficient rodent models hinders the ability to study immune responses against any immunomodulatory transgenes engineered within the viral genome and expressed within the tumour microenvironment. There has therefore been a shift towards the use of more sophisticated ex vivo patient‐derived model systems based on organoids and explant co‐cultures with immune cells, which may be more predictive of efficacy than contrived and artificial animal models. We review the best of those model systems here.

Carbon ion triggered immunogenic necroptosis of nasopharyngeal carcinoma cells involving necroptotic inhibitor BCL-x.

To explore the potential and mechanisms of necroptosis, a form of immunogenic cell death, induced by carbon ion as compared to photon beams in established photon resistant- (PR-) and sensitive nasopharyngeal carcinoma (NPC) cells. MLKL is considered a central executor of necroptosis and phosphorylation of MLKL (p-MLKL) was a critical event of necroptosis. The clonogenic survival and DNA microarray demonstrated that after repeated photon irradiation, radiosensitive NPC cells became apoptosis-resistant but could be effectively inhibited by carbon ion irradiation. The relative biologic effectiveness (RBE) at D10 and D37 were 2.15 and 2.78 for PR-NPC cells. Carbon ion induced delayed DNA damage repair, cell cycle arrest, cytogenetic damage, morphological change and cell necrosis, indicating the possibility of necroptosis in both PR- and sensitive NPC cell types. The lower expression of necroptotic inhibitors (caspase-8 and Bcl-x) and higher level of MLKL in PR-NPC cells showed it was relatively more predisposed to necroptosis compared to the sensitive cells. Subsequent experiments demonstrated the significant upregulation of p-MLKL in the PR-NPC cells treated by carbon ion (4 Gy) compared with photon irradiation at both physical (4 Gy) and RBE (10 Gy) doses (P≤0.0001). Moreover, carbon ion induced a robust (up to 28 folds) p-MLKL in the PR-NPC cells as well as sensitive cells (up to 6-fold) coupled with a lower level of BCL-x expression and increased GM-CSF implicated in resculputure of immune system. These results suggested that carbon ion could induce necroptosis of NPC cells, especially in PR-NPC cells, and its mechanisms involve BCL-x.

The effect of calcium electroporation on viability, phenotype and function of melanoma conditioned macrophages

Electroporation in combination with chemotherapy is an established treatment used on solid malignancies that results in enhanced chemotherapeutic uptake. Recent advances have begun to transition to the use of non-toxic compounds, such as calcium, in lieu of chemotherapy, which can also induce tumour cell death. While the effect of treatment on tumour cell death has been well characterized and has been shown to induce an immunogenic form of cell death, the effect of treatment on intratumoural immune cells has not been investigated. Here we present data showing the effect of calcium electroporation on immune cells, using melanoma-conditioned bone marrow-derived macrophages. Similar to tumour cells, macrophage cell membranes are susceptible to poration following treatment and subsequently reseal. Macrophages are less susceptible to calcium electroporation induced cell death in comparison to B16F10 melanoma cells. However treatment with electroporation with or without bleomycin or calcium was shown to affect macrophage phenotype and function. Coculture of calcium electroporated macrophages revealed that both the capacity of macrophages to stimulate and direct T cell responses are affected following exposure to treatment. We conclude that calcium electroporation has the potential to boost the immunogenic capacity of exposed tumour associated macrophages, and further research is warranted to determine if calcium electroporation can be optimised to generate systemic anti-cancer immune responses.

Abstract 1698: Cellular cytotoxicity is a form of immunogenic cell death

Abstract Background: The immune response to cancer is often conceptualized with the Cancer Immunity Cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic cells to naive or memory T cells in the tumor-draining lymph nodes. Whether tumor cell death resulting from cytotoxicity, as mediated by T cells or NK lymphocytes, is actually immunogenic currently remains unknown. Methods: In this study, tumor cells were killed by antigen-specific T-cell receptor (TCR) transgenic CD8 T cells or activated natural killer cells. Immunogenic cell death was studied analyzing the membrane exposure of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells. Furthermore, the potential immunogenicity of the tumor cell debris was evaluated in immunocompetent mice challenged with an unrelated tumor sharing only one tumor-associated antigen and by class I MHC-multimer stainings. Mice deficient in BATF-3, IFNAR and STING were used to study mechanistic requirements. Results: We observe in co-cultures of tumor cells and effector cytotoxic cells, the presence of markers of immunogenic cell death such as calreticulin exposure and soluble HMGB1 protein. OVA-transfected MC38 colon cancer cells, exogenously pulsed to present the gp100 epitope are killed in culture by mouse gp100 specific TCR transgenic CD8 T cells. Immunization of mice with the resulting destroyed cells induces epitope spreading as observed by detection of OVA-specific T cells by MHC multimer staining and rejection of OVA+ EG7 lymphoma cells. Similar results were observed in mice immunized with cell debris generated by NK-cell mediated cytotoxicity. Mice deficient in BATF3-dependent dendritic cells (cDC1) fail to develop an anti-OVA response when immunized with tumor cells killed by cytotoxic lymphocytes. In line with this, cultured cDC1 dendritic cells uptake and can readily cross-present antigen from cytotoxicity-killed tumor cells to cognate CD8+ T lymphocytes. Conclusion: These results support that an ongoing cytotoxic antitumor immune response can lead to immunogenic tumor cell death. Citation Format: Luna Minute, Alvaro Teijeira, Alfonso Rodriguez Sánchez-Paulete, Maria del Carmen Ochoa, Maite Alvarez, Itziar Otano, Iñaki Etxeberria, Elixabet Bolaños, Arantza Azpilikueta, Saray Garasa, Noelia Casares, José Luiz Pérez-Gracia, Maria Esperanza Rodríguez-Ruiz, Pedro Berraondo, Ignacio Melero. Cellular cytotoxicity is a form of immunogenic cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1698.