Molecular mechanisms of ATP secretion during immunogenic cell death

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.

VB6-845d is a Targeted Protein Therapeutic, TPT, that comprises a Fab fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to deBouganin via a furin protease sensitive linker. DeBouganin (deB) is a de-immunized variant of bouganin, a ribosome inactivating protein (RIP), that when internalized blocks protein synthesis thereby leading to cell death. While all cytotoxic molecules induce tumor cell killing, only some are capable of inducing biological manifestations indicative of immunogenic cell death (ICD). ICD is characterized by the collective appearance of distinct cellular changes termed damage associated molecular patterns, or DAMPs, which play a role in immune cell activation by triggering pro-inflammatory processes. Key DAMPs necessary for defining ICD are cell surface translocation of calreticulin, an endoplasmic reticulum chaperone protein, ATP secretion and release of HMGB1 (high mobility group box 1). To evaluate whether VB6-845d cell killing elicits the hallmark features of ICD, VB6-845d treated tumor cell lines were assessed for the presence of these distinct signaling molecules. In vitro studies showed that VB6-845d cytotoxicity induces the translocation of calreticulin to the cell surface as well as the release of ATP and HMGB1. The expression of other potential immune regulators following VB6-845d treatment was also examined. In summary, the data presented suggests that VB6-845d mediates tumor cell killing by an ICD pathway. The potential cross-priming effect initiated by VB6-845d-induced ICD suggests the use of VB6-845d in combination with immune checkpoint inhibitors may enhance their effectiveness in EpCAM-positive epithelial cancers. Citation Format: Rachelle L. Dillon, Shilpa Chooniedass, Arjune Premsukh, Glen C. MacDonald, Jeannick Cizeau, Gregory P. Adams. VB6-845d tumor cell killing elicits biologic features of immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2788.

Oncolytic Adenovirus With Temozolomide Induces Autophagy and Antitumor Immune Responses in Cancer Patients

ABSTRACTChemotherapeutics, including the platinum compounds oxaliplatin (OXP) and cisplatin (CDDP), are standard care of treatment for cancer. Although chemotherapy has long been considered immunosuppressive, evidence now suggests that certain cytotoxic agents can efficiently stimulate antitumor responses, through the induction of a form of apoptosis, called immunogenic cell death (ICD). ICD is characterized by exposure of calreticulin and heat shock proteins (HSPs), secretion of ATP and release of high-mobility group box 1 (HMGB1). Proper activation of the immune system relies on the integration of these signals by dendritic cells (DCs). Studies on the crucial role of DCs, in the context of ICD, have been performed using mouse models or human in vitro-generated monocyte-derived DCs (moDCs), which do not fully recapitulate the in vivo situation.Here, we explore the effect of platinum-induced ICD on phenotype and function of human blood circulating DCs. Tumor cells were treated with OXP or CDDP and induction of ICD was investigated. We show that both platinum drugs triggered translocation of calreticulin and HSP70, as well as the release of ATP and HMGB1. Platinum treatment increased phagocytosis of tumor fragments by human blood DCs and enhanced phenotypic maturation of blood myeloid and plasmacytoid DCs. Moreover, upon interaction with platinum-treated tumor cells, CD1c+ DCs efficiently stimulated allogeneic proliferation of T lymphocytes. Together, our observations indicate that platinum-treated tumor cells may exert an active stimulatory effect on human blood DCs. In particular, these data suggest that CD1c+ DCs are critical mediators of immune responses induced by ICD.

SY03-01: The desirable death of the cancer cell: Immunogenic cell death for optimal chemotherapy

EMBO J 31 5, 1062–1079 (2012); published online January172012 In this issue of The EMBO Journal, Garg et al (2012) delineate a signalling pathway that leads to calreticulin (CRT) exposure and ATP release by cancer cells that succumb to photodynamic therapy (PTD), thereby providing fresh insights into the molecular regulation of immunogenic cell death (ICD).

High Linear Energy Transfer Particle Irradiation Leads to Increased Expression of In Vitro Markers of Immunogenic Cell Death

Stearoyl lysophosphatidylcholine (LPC) has recently been proven protective against lethal sepsis by stimulating neutrophils to eliminate invading pathogens through an H2O2-dependent mechanism. Here, we demonstrate that stearoyl LPC, but not caproyl LPC, significantly attenuates circulating high-mobility group box 1 (HMGB1) levels in endotoxemia and sepsis by suppressing endotoxin-induced HMGB1 release from macrophages/monocytes. Neutralizing antibodies against G2A, a potential cell surface receptor for LPC, partially abrogated stearoyl LPC-mediated suppression of HMGB1 release. Thus, stearoyl LPC confers protection against lethal experimental sepsis partly by facilitating the elimination of the invading pathogens and partly by inhibiting endotoxin-induced release of a late proinflammatory cytokine, HMGB1.

The extracellular levels of damage-associated molecular patterns (DAMPs) released during immunogenic cell death (ICD) are positively correlated with the magnitude and efficacy of the resulting in vivo immune response. Therefore, extracellular ATP (eATP) and high mobility group box 1 (HMGB1) have been identified as key biomarkers for their predictive capacity during in vitro ICD screening activities. Current methods for identifying eATP and HMGB1 inducers are laborious, costly, and hampered by poor throughput. To overcome these challenges, we developed easy-to-use, homogeneous, bioluminescent assays that measure dose-dependent release of these immunostimulatory agents directly in cell culture. The eATP assay utilizes an optimized ATP detection chemistry that can be employed directly to assess live-cell kinetic responses for up to 24 h, with even longer exposures supported by a staggered reagent addition approach. The HMGB1 assay measures the protein's concentration at exposure endpoint in the same sample well using complementary luciferase fragment-labelled monoclonal antibodies. We tested the utility of the assays using U20S, Jurkat and U937 cells dosed with serial dilutions of known ICD inducers (doxorubicin, idarubicin, mitoxantrone, and bortezomib). The resulting data suggest the potency of eATP and HMGB1 release is dependent upon cell model and agent but can be reproducibly and robustly measured in 96 and 384 well environments. Further, the eATP assay produced remarkable early dose-dependent response resolution whereas the HMGB1 data provided a confirmatory post-mortem ICD parameter. This new ICD biomarker detection workflow may help to efficiently define and rank-order the capacity of new chemical entities to induce apoptosis and immunogenic cell death. Citation Format: Andrew L. Niles, Kevin R. Kupcho, Dan F. Lazar, James J. Cali. Improved DAMP assays for the in vitro assessment of immunogenic cell death [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 1933.

Methods for measuring HMGB1 release during immunogenic cell death.

Objective To investigate the effects of adenosine monophosphate-activated protein kinase(AMPK)on high-mobility group box 1(HMGB1)release from PC12 cells after oxygen-glucose deprivation and reoxygenation(OGD/R)and its mediated inflammatory response in BV2 cells. Methods PC12 and BV2 cells were cultured, respectively. The PC12 cells were used to induce a model of oxygen glucose deprivation for 12 h and reoxygenation for 24 h. After giving 5-aminoimidazole-4-carboxamide(AICAR)5, 50 and 100 μmol/L as well as Compound C 0.1, 1 and 10 μmol/L activation or inhibition of AMPK phosphorylation, respectively, methyl thiazolyl tetrazolium(MTT)was used to detect the PC12 cell activity. Enzyme-linked immunosorbent assay was used to detect the HMGB1 release level in the PC12 cell culture media. After OGD/R in each group, the PC12 culture media were acted on normal cultured BV2 cells for 24 h respectively. Western blotting and Enzyme-linked immunosorbent assay were used to detect the NFκB inhibitory protein(inhibitor of NFκB, IκB)phosphorylation level and TNF-α release level in BV2 cells, respectively. Results After OGD/R, the PC12 cell activity was decreased significantly(68.84%±6.60% vs. 100.04%±8.82%; P<0.01); the AMPK phosphorylation level was increased significantly(1.95±0.39 vs. 1.00±0.20; P<0.05), and the extracellular HMGB1 release was increased significantly(287.66± 26.42 pg/μl vs. 53.05±9.11 pg/μl; P<0.01). Compared with the OGD/R group, AICAR 100 μmol/L significantly increased the survival rate of PC12 cell after OGD/R(78.6%±3.75% vs. 68.84%±6.60%; P<0.05), promoted AMPK phosphorylation(3.32±0.66 vs. 1.95±0.39; P<0.01), and reduce the release of extracellular HMGB1(164.06±12.77 pg/μl vs. 287.66±26.42 pg/μl; P<0.01). In contrast, Compound C 10 μmol/L significantly reduced the cell survival rate of PC12(40.44%±3.79% vs. 68.84%±6.60%; P<0.01), inhibited AMPK phosphorylation(1.07±0.21 vs. 1.95±0.39; P<0.05), and increased the release of HMGB1(337.97±18.9 pg/μl vs. 287.66±26.42 pg/μl; P<0.01). The conditioned medium from the AICAR 100 μmol/L group significantly inhibited IκB phosphorylation(1.68±0.51 vs. 3.09±0.10; P<0.05)and reduced the release of TNF-α(669.53±38.58 pg/μl vs. 841.76±45.82 pg/μl; P<0.05)in BV2 cells. The conditioned medium from the compound C 10 μmol/L group significantly promoted IκB phosphorylation(4.98±1.24 vs. 3.09±0.10; P<0.01)and increased the release of TNF-α(1 035.32±128.06 pg/μl vs. 841.76±45.82 pg/μl; P<0.05)in BV2 cells. Conclusions Promoting AMPK phosphorylation activation may reduce the release of HMGB1 from PC12 cells after OGD/R, and inhibit its mediated NF-κB inflammatory pathway and reduce the release of TNF-αin BV2 cells, and thus reducing neuroinflammatory injury. On the contrary, inhibiting AMPK phosphorylation may promote the release of HMGB1 from PC12 cells after OGD/R and aggravate its mediated inflammatory reaction in BV2 cells. Key words: AMP-Activated Protein Kinases; HMGB1 Protein; Cells, Cultured; Neurons; Microglia; Oxygen; Glucose; Inflammation

High mobility group box 1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic injury, initiates inflammatory response and aggravates brain tissue damage. Acetylpuerarin (AP), an acetylated derivative of puerarin, was reported to protect against cerebrovascular ischemia-reperfusion injury in rats through anti-inflammation. In the present study, we aim to investigate whether AP inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated BV2 microglia. BV2 microglia viability after OGD with or without AP was measured by CCK-8 assay, apoptosis of BV2 microglia was determined by Hoechst 33258 staining and FITC-Annexin V/7-AAD staining. HMGB1 protein level and release was detected by western blotting and immunofluorescent FITC-staining. The results demonstrated that AP significantly rescued OGD-induced cell death and apoptosis in a dose-dependent manner. AP inhibited OGD-induced HMGB1secretion at the level of nuclear to cytoplasmic translocation, decreased cytoplasmic HMGB1 at protein level, and the effects showed dose-dependent. The findings suggest that AP can protect against OGD-induced cellular injury in BV2 microglia by inhibition of HMGB1 release.

The translocation of the protein high mobility group box 1 (HMGB1) from the nucleus to the cytoplasm and its secretion or passive release through the permeabilized plasma membrane, constitutes a major cellular danger signal. Extracellular HMGB1 can interact with pattern recognition receptors to stimulate pro-inflammatory and immunostimulatory pathways. Here, we developed a screening assay to identify pharmacological agents endowed with HMGB1 releasing properties. For this, we took advantage of the “retention using selective hooks” (RUSH) system in which a streptavidin-NLS3 fusion protein was used as a nuclear hook to sequestrate streptavidin-binding peptide (SBP) fused with HMGB1 and green fluorescent protein (GFP). When combined with biotin, which competitively disrupts the interaction between streptavidin-NLS3 and HMGB1-SBP-GFP, immunogenic cell death (ICD) inducers such as anthracyclines were able to cause the nucleo-cytoplasmic translocation of HMGB1-SBP-GFP. This system, was used in a high-content screening (HCS) campaign for the identification of HMGB1 releasing agents. Hits fell into three functional categories: known ICD inducers, microtubule inhibitors and epigenetic modifiers. These agents induced ICD through a panoply of distinct mechanisms. Their effective action was confirmed by multiple methods monitoring nuclear, cytoplasmic and extracellular HMGB1 pools, both in cultured human or murine cells, as well as in mouse plasma.

Gastric cancer (GC) often exhibits resistance to anti-programmed death-1 (PD-1) immunotherapy. Immunogenic cell death (ICD) enhances antitumor immunity, and trifluridine/tipiracil (FTD/TPI) induces ICD and modulates immunity. Radiation therapy (RT) may trigger abscopal effects. We investigated whether FTD/TPI combined with RT enhances tumor immunity in GC and its impact with PD-1 blockade. ICD induction by FTD and RT in YTN16 mouse GC cells was assessed by calreticulin, high-mobility group box 1 (HMGB1), and ATP evaluation. A dual subcutaneous YTN16 tumor model was established in C57BL/6J mice; mice were treated with FTD/TPI and RT (with irradiation of the first tumor). ICD was induced in vitro by FTD or RT and enhanced by the combination, as indicated by increased calreticulin surface expression and HMGB1 and ATP release. ICD induction in the first tumor was confirmed by HMGB1 release. FTD/TPI + RT suppressed growth of the second tumor and enhanced antitumor immunity by increasing CD8⁺ T-cell infiltration and depleting M2 macrophages. PD-1 expression on CD8⁺ T cells increased after FTD/TPI + RT; adding anti-PD-1 further suppressed the second tumor growth. Our findings support clinical trials of this triple-combination strategy in advanced GC, particularly in subgroups refractory to immune checkpoint blockade.

Neuropeptide Modulators of High Mobility Group Box 1 Secretion as Potential Therapeutic Agents for Severe Sepsis