Translocation Of Calreticulin Research Articles

Combining Cisplatin with Pinellia pedatisecta Schott Lipid-Soluble Extract Induces Tumor Immunogenic Cell Death in Cervical Cancer

BackgroundPinellia pedatisecta Schott extract (PE) is extracted from Pinellia pedatisecta Schott (PPS), a traditional Chinese medicinal plant with the potential for direct anticancer effects or eliciting an anti-tumor response by activating the immune system. PurposeTo explore PE's ability and mechanism to reconstruct cisplatin's immunogenicity. MethodsCervical cancer cells were treated with cisplatin (CDDP) and/or PE. The exposure of calreticulin (CRT) on cell membrane was investigated by flow cytometry. The extracellular of ATP and HMGB1 was investigated by Western blot analysis, immunofluorescence and ELISA assay. Changes in immune profiles were using flow cytometry in vaccination and anti-tumor assays in vivo. Lastly, the mechanism of PE influenced the ROS/ERS pathway was examined by ROS assay kit, flow cytometry and Western blotting. ResultsPE treatment induced translocation of CRT from the endoplasmic reticulum to the cell membrane of tumor cells, concomitantly triggering immunogenic cell death (ICD). In terms of mechanisms, endoplasmic reticulum (ER) stress relievers could impede the ability of PE to induce immunogenicity. This indicates that PE is activated by ER stress, leading to subsequent induction of ICD. Upon analyzing RNA-seq data, it was observed that PE primarily induces programmed cell death in tumors by impeding upstream antioxidant mechanisms. Additionally, it transforms dying tumor cells into vaccines, activating a series of immune responses. ConclusionsThis study observed for the first time that PE-induced CRT exposure on the membrane of cervical cancer cells compensates for the defect of nonimmunogenic cell death inducer CDDP thereby stimulating potent ICD. This ability restores the immunogenicity of CDDP through ER stress induced by the ROS signal. ROS played a role in PE's ability to induce ICD, leading to increased expression of ER stress-related proteins, including ATF3 and IRE-1α. PE exerted anti-cancer effects by increasing the ROS levels, and ROS/ERS signaling may be a potential avenue for cervical cancer treatment. Hence, the synergistic use of PE and CDDP holds potential for enhancing immunochemotherapy in cancer treatment.

Abstract 489: Unlocking immune resistance and metastasis of PDAC via dual acting circularized nanodisc

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest forms of cancer. While major progress in immunotherapy has been made in recent years, it remains poorly effective in this malignancy. Two key players stand behind immune resistance of PDAC and inadequate efficacy of immunotherapy to suppress its metastasis. First, the immune-resistant pancreatic cancer stem cells (CSCs) evade immune surveillance acting as a source for tumor relapse and metastasis. This immune resistance stems from low immunogenicity of PDAC cells as well as the inaccessible CSCs that reside deep in the tumor core. Second, pancreatic cancer associated fibroblasts (CAFs) fuel metastasis of PDAC cells by suppressing their immune recognition and establishing pre-metastatic niche at distant organs to help their colonization. Our hypothesis is to overcome immune resistance of PDAC and inhibit early steps of its metastasis via simultaneous immunogenic eradication of immune resistant CSCs and switching immunosuppressive and pro-metastatic CAFs into quiescent ones. Therefore, we engineered ultrasmall circularized nanodiscs (cND) that encapsulate oncolytic peptide LTX-315 and fibroblast remodeling Notch signaling inhibitor. The engineered oncolytic LTX-315 loaded cND successfully induced immunogenic cell death (ICD) of both bulk PANC1 and pancreatic CSCs. Compared to non-treated and free LTX-315 treated cells, LTX-315 cND resulted in enhanced release of DAMPs including ATP and HMGB-1 as well as surface translocation of calreticulin. The ultrasmall size (9 nm) of cND enabled its deep tumor penetration to eradicate resistant CSCs, as revealed using 3D PDAC heterospheroids. In parallel, the Notch blocking cND promoted quiescence of the activated pancreatic stellate cells as denoted by downregulation of SMA, FAP and collagen. In KPC syngeneic PDAC mouse model, the dual oncolytic/Notch blocking cND combined with aPD-L1 therapy significantly suppressed tumor growth and prevented its metastasis to liver compared to vehicle-treated mice. Immunophenotyping study showed marked reduction in the abundance of CSCs and CAFs in the tumor treated with the cNDs. Moreover, the tumor infiltration of CD86+ MHCII+ dendritic cells and cytotoxic CD8+ T cells was enhanced after treatment with the cND. Overall, our dual strategy of targeting pancreatic CSCs and CAFs via oncolytic/Notch blocking cND boosted the anti-tumor and anti-metastatic efficacy of aPD-L1 therapy to eliminate primary PDAC tumor and suppress its metastasis. Citation Format: Ahmed Elzoghby, Hagar Emam, Seungbin Yim, Cuiyan Xin, Mahmoud Nasr. Unlocking immune resistance and metastasis of PDAC via dual acting circularized nanodisc [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 489.

Abstract 7513: Tumor treating fields (TTFields) can induce immunogenic cell death in GBM resulting in enhanced immune modulation

Abstract Glioblastoma (GBM) is the most common and aggressive form of malignant primary brain tumors. Despite current standard of care, the overall survival for GBM is ~15 months. While immunotherapy using immune checkpoint inhibitors such as anti-PD-1/PD-L1 have been efficacious for solid malignancies, very little progress has been made in a clinical setting for GBM. This is mainly due to GBM being known as a “cold” and non-immunogenic tumor. It has been previously established that a tumor may become “hot” if the T cell responses can be enhanced by increasing the presentation of tumor-specific antigens to the immune system. Tumor Treating Fields (TTFields) are alternating electric fields in a specific frequency range (100-500 kHz) delivered to the human body through transducer arrays. Treatment with TTFields have been approved and have shown efficacy in GBM. TTFields have induced immunogenic cell death in GBM, which in turn can elicit anti-tumoral immunity by inducing damage-associated molecular patterns (DAMP). These patterns are important for promoting engulfment of lung cancer cells by dendritic cells and recruitment of immune cells. These findings suggest that TTFields may enhance the immune response in GBM by increasing tumor specific antigens. In this study we show in vitro that TTFields enhanced the immune response to GBM. We initially demonstrated that GBM cells (GL261 or CT2A) treated with TTFields (200 kHz) can elicit DAMP emissions as evident by the increase in secretion of ATP and HMGB1 and the enhanced translocation of calreticulin to the cell surface. Additionally, TTFields stimulated the phagocytotic activity of antigen presenting cells (Raw264.7) and increased T-cell early activation (CD69/CD4+, CD69/CD8+) and activity (IFNγ and perforin). Since our findings in vitro indicate TTFields enhanced the immune response to GBM, we studied the therapeutic potential of using TTFields together with anti- PD1 in a syngeneic mouse model. We applied TTFields to GBM cells prior to intracranial injections. While our survival studies are preliminary, our findings suggest that systemic anti-PD1 and TTFields may result in a myeloid and T-cell mediated anti-tumor response and enhanced survival. Our findings indicate that TTFields treatment may help transform GBM into a “hot” tumor thereby making it an attractive target for immunotherapy. Citation Format: Ryan T. Nitta, Si Yeon Lee, Michael Lim, Gordon Li. Tumor treating fields (TTFields) can induce immunogenic cell death in GBM resulting in enhanced immune modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7513.

Abstract 5272: APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells

Abstract Introduction: Treatment approach combining cancer cell targeted therapy and immunotherapy is an effective strategy to trigger robust tumor-specific immune responses. The aim of this study is to determine the impact of APL-1202, a clinical-stage drug candidate (NCT04813107), on cellular stress-driven immunogenic outcomes in bladder cancer cells. Methods: RNA-seq analysis was performed using bladder cancer and endothelial cell lines (A56, J82, T24, HUVEC) treated with APL-1202. Key determinants of immunogenic cell death pathway were measured by cell-surface translocation of calreticulin (flow cytometry), extracellular ATP release assay (bioluminescence) and HMGB1 secretion (ELISA) in the supernatants of cancer cells after APL-1202 treatment. Effects on the innate immune effector function was measured by flow cytometry-based phagocytosis assay consisting of Far-Red dye-stained, pre-treated bladder cancer cell lines (UMUC3, T24) in coculture with THP1-derived macrophages/immature dendritic cells or human monocyte-derived dendritic cells. In vivo, tumor end-point immune cell analysis, tumor weight and mouse survival were compared in chemically induced (BBN: N-butyl-N-(4-hydroxybutyl)-nitrosamine) orthotopic bladder cancer mice treated with APL-1202 and anti-PD1 antibody. Results: Pathway analysis of RNA-seq data from APL-1202 treated cells suggested upregulation of cellular stress-related gene signatures (oxidative stress, autophagy, P53) and pro-inflammatory responses (IL-6, IL-8, HMGB1 signaling). Conversely, downregulation of cell cycle (E2F targets, G2-M checkpoint) and IL-10 signaling suggested increased genomic stress and inflammatory switching in the treated cancer cells. Acute exposure to APL-1202 increased cell-surface translocation of calreticulin as well as extracellular release of ATP and HMGB1 in the treated T24 and UMUC3 cells. Higher percentages of phagocytic activities were detected in both THP-1-derived macrophages/immature dendritic cells and human monocyte-derived dendritic cells after coculture with APL-1202 treated T24 and UMUC3 cells compared to coculture with DMSO treated control cells. Immune phenotyping in tumors derived from BBN bladder cancer mouse model revealed increased infiltration of NK cells and CD8+ T cells. Furthermore, APL-1202 enhanced the anti-tumor effects of a therapeutic anti-PD1 antibody in mice as reflected by significantly decreased tumor weights and increased survival in a combination group with APL-1202 treatment compared to the anti-PD-1 antibody alone treatment group. Conclusion: APL-1202 may improve the efficacy of immune-checkpoint blockade therapies in bladder cancer patients by enhancing cancer cell immunogenicity and subsequent anti-tumor effector immune cell processes, particularly in tumors with deficits in antigen-specific immunity. Citation Format: Rakesh BAM, Jingmin Guan, Neetha Nanoth Vellichirammal, Murli Manohar, David Mulholland, John Sfakianos, Qiaoling Sun, Zuoan Yi, Alice Chen. APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5272.

Abstract 7144: BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death

Abstract BPR0L075 (also known as SCB01A in phase II clinical trial) is a novel anti-microtubule drug with potent anti-tumor and anti-angiogenic activity in vitro and in vivo. BPR0L075 inhibits tubulin polymerization (microtubule de-stabilizer) by binding to the colchicine-binding site of tubulin. We previously reported that BPR0L075 overcomes paclitaxel (microtubule stabilizer) resistance and induces vascular-disruption in vitro and in vivo. We observed that breast and ovarian cancer cells treated with BPR0L075 in vitro induced key hallmarks of immunogenic cell death (ICD). In both human and murine, ovarian (OVCAR3, ID8) and breast cancer (MDA-MB-231, 4T1) cell lines, treatment with low nanomolar concentration of BPR0L075 significantly decreased the viability of cells evidenced by SRB assay. Using a known ICD inducer mitoxantrone as a positive control, both flow cytometric analysis and immunofluorescence staining assays demonstrated that BPR0L075 treatment led to a time- and dose-dependent translocation of calreticulin, an endoplasmic reticulum chaperone protein, on the plasma membrane of dying cells which sends an “eat me” signal. The BPR0L75 treatment also led to secretion of High Mobility Group Box 1 (HMGB1), a non-histone chromatin-binding nuclear protein, as well as adenosine triphosphate (ATP) from the cell into the extracellular space using ELISA assay. These damage-associated molecular patterns (DAMPs) are known to lead to antigen-presenting cell recruitment, phagocytosis, maturation of dendritic cells, and chemotaxis. The appearance of the ICD biomarkers (calreticulin exposure, HMGB1 and ATP release) supports BPR0L075 as an ICD-inducing antimitotic agent. Further in vivo studies are warranted to delineate the effect of BPR0L075 on immune activation and recruitment of immune cells to the tumor microenvironment. Citation Format: Ayesha Bano, Xinli Liu. BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7144.

Improving combination cancer immunotherapy by manipulating dual immunomodulatory signals with enzyme-triggered, cell-penetrating peptide-mediated biomodulators.

Immunosuppressive tumor microenvironments challenge the effectiveness of protein-based biopharmaceuticals in cancer immunotherapy. Reestablishing tumor cell immunogenicity by enhancing calreticulin (CRT) exposure is expected to improve tumor immunotherapy. Given that CRT translocation is inherently modulated by phosphorylated eIF2α, the selective inhibition of protein phosphatase 1 (PP1) emerges as an effective strategy to augment tumor immunogenicity. To harness the PP1-disrupting potential of GADD34-derived motifs and address their limited intracellular delivery, we integrated these sequences into an enzyme-triggered, cell-penetrating peptide-mediated chimeric protein scaffold. This design not only facilitates efficient cytoplasmic delivery of these immunostimulatory motifs to induce "eat-me" signaling, but also provides a versatile platform for combination immunotherapy. Fabrication of biomodulators with cytotoxic BLF1 provides additional "eat-me" signaling through phosphatidylserine exposure or that with an immunomodulatory designed ankyrin repeat protein disables "don't-find-me" signaling by antagonizing PD-L1. Notably, these bifunctional biomodulators exhibit remarkable ability to induce macrophage phagocytosis, dendritic cell maturation, and CD8+ T activation, ultimately substantially inhibiting tumor growth. This study presents a modular genetic coding strategy for PP1-centered therapies that enables seamless integration of immunostimulatory sequences into protein-based anti-tumor cocktail therapies, thereby offering novel alternatives for improving antitumor efficacy.

Membranal Expression of Calreticulin Induced by Unfolded Protein Response in Melanocytes: A Mechanism Underlying Oxidative Stress–Induced Autoimmunity in Vitiligo

Calreticulin (CRT), a damage-associated molecular pattern molecule, is reported to translocate from endoplasmic reticulum to membrane in melanocytes under oxidative stress. To investigate the potential role of CRT in the pathogenesis of vitiligo, we analyzed the correlation between CRT and ROS in serum and lesions of vitiligo, detected CRT and protein kinase RNA-like ER kinase (PERK) expression in vitiligo lesions, and studied the production of CRT and mediators of unfolded protein response (UPR) pathway, then tested the chemotactic migration of CD8+ T cells or CD11c+ CD86+ cells. Initially, we verified the overexpression of CRT in perilesional epidermis that was positively correlated with the disease severity of vitiligo. Furthermore, the PERK branch of UPR was confirmed to be responsible for the overexpression and membranal translocation of CRT in melanocytes under oxidative stress. We also found that oxidative stress-induced membranal translocation of CRT promoted the activation and migration of CD8+ T cells in vitiligo. Additionally, dendritic cells from vitiligo patients were also prone to maturation with the co-incubation of melanocytes harboring membranal CRT. CRT could be induced on the membrane of melanocytes via UPR and might play a role in oxidative stress-triggered CD8+ T cell response in vitiligo.

LW-213 induces immunogenic tumor cell death via ER stress mediated by lysosomal TRPML1

Dying tumor cells release biological signals that exhibit antigenicity, activate cytotoxic T lymphocytes, and induce immunogenic cell death (ICD), playing a key role in immune surveillance. We demonstrate that the flavonoid LW-213 activates endoplasmic reticulum stress (ERS) in different tumor cells and that the lysosomal calcium channel TRPML1 mediates the ERS process in human cellular lymphoma Hut-102 cells. Apoptotic tumor cells induced by ERS often possess immunogenicity. Tumor cells treated with LW-213 exhibit damage-associated molecular patterns (DAMPs), including calreticulin translocation to the plasma membrane and extracellular release of ATP and HMGB1. When co-cultured with antigen-presenting cells (APCs), LW-213-treated tumor cells activated APCs. Two groups of C57BL/6J mice were inoculated with Lewis cells: a “vaccine group”, which demonstrated that LW-213-treated tumor cells promote the maturation of dendritic cells and increase CD8+ T cells infiltration in the tumor microenvironment and a “pharmacodynamic group”, treated with a combination of LW-213 and PD1/PD-L1 inhibitor (BMS-1), which reduced tumor growth and significantly prolonged the survival time of mice in the “pharmacodynamic group”. Therefore, LW-213 can be developed as a novel ICD inducer, providing a new concept for antitumor immunotherapy.

Abstract 5038: Evaluation of anti-tumor immunity in response to [177Lu]Lu-PSMA in a mouse model of prostate cancer

Abstract [177Lu]Lu-PSMA improves progression-free and overall survival in metastatic castration resistant prostate cancer. However, the role of immune stimulation by molecularly targeted radionuclide therapy (MRT) is poorly understood. The aim of this study was to evaluate the anti-tumor immune response induced by [177Lu]Lu-PSMA. The markers of immunogenic cell death (ICD), translocation of calreticulin (CRT) to the cell membrane, HMGB1 release and ATP release, were evaluated in the RM1 cell line (PSMA-negative murine prostate carcinoma) and its derivative, RM1-PGLS (stably transduced with human PSMA and SFG-EGFp/Luc), using flow cytometry, ELISA and a luminescence detection assay respectively. The in vivo biodistribution of [177Lu]Lu-PSMA in RM1-PGLS tumor-bearing C57BL/6 mice was investigated using microSPECT-CT imaging, and the in vivo efficacy of [177Lu]Lu-PSMA was tested in RM1-PGLS tumor growth inhibition studies. Vaccination/rechallenge experiments were performed to elucidate whether [177Lu]Lu-PSMA elicits ICD in vivo. The Nanostring Pan Cancer Immune Profiling Platform was used to assess changes in the tumor immune microenvironment (TIME) in response to [177Lu]Lu-PSMA. External radiation (EBRT; 137Cs γ-radiation; 6-10 Gy) was used as a comparator treatment in most experiments. We observed a dose-dependent increase in cell surface CRT following [177Lu]Lu-PSMA and EBRT at 24 and 72 h and of HMGB1 release in response to [177Lu]Lu-PSMA and EBRT at 96 and 144 h. At 24 h following [177Lu]Lu-PSMA (60 MBq) there was a significant increase in ATP release compared to untreated samples. Collectively these data indicate [177Lu]Lu-PSMA causes ICD. A high RM1-PGLS tumor:muscle uptake ratio (210 ± 59) was noted in biodistribution experiments confirming highly specific tumor accumulation of [177Lu]Lu-PSMA. In tumor growth inhibition studies the time for tumors to reach a volume of 400 mm3 was delayed in mice treated with [177Lu]Lu-PSMA (60 MBq) compared to no treatment (30 versus 21 days; P<0.01). We showed that a subcutaneous inoculum of [177Lu]Lu-PSMA (60 MBq)-treated RM1-PGLS cells prevents tumor growth in approximately 50% of cases on rechallenge with viable RM1-PGLS cells one week later. This indicates that [177Lu]Lu-PSMA induces ICD in vivo. We observed a significant reduction in most tumor infiltrating lymphocyte (TIL) types at 2 days after in vivo exposure to [177Lu]Lu-PSMA (80 MBq), followed by an increase in TILs by Day 7. The observed pattern is similar to that after EBRT (6 Gy). There was overlap in the top 10 differentially expressed genes (treatment versus no treatment) for the [177Lu]Lu-PSMA and EBRT groups. The gene showing the greatest level of upregulation at day 7 in [177Lu]Lu-PSMA-treated tumors, IDO1, is known to be involved in immune tolerance and radioresistance. In conclusion, [177Lu]Lu-PSMA induces ICD in vitro and in vivo and modulates the TIME. Citation Format: Gemma Dias, Sarah Able, Irini Skaripa-Koukelli, Rachel Anderson, Gracie Wilson, Katherine A. Vallis. Evaluation of anti-tumor immunity in response to [177Lu]Lu-PSMA in a mouse model of prostate cancer. [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 5038.

The therapeutic mavericks: Potent immunomodulating chaperones capable of treating human diseases.

Two major chaperones, calreticulin (CRT) and binding immunoglobulin protein (GRP78/BiP) dependent on their location, have immunoregulatory or anti-inflammatory functions respectively. CRT induces pro-inflammatory cytokines, dendritic cell (DC) maturation and activates cytotoxic T cells against tumours. By contrast, GRP78/BiP induces anti-inflammatory cytokines, inhibits DC maturation and heightens T-regulatory cell responses. These latter functions rebalance immune homeostasis in inflammatory diseases, such as rheumatoid arthritis. Both chaperones are therapeutically relevant agents acting primarily on monocytes/DCs. Endogenous exposure of CRT on cancer cell surfaces acts as an 'eat-me' signal and facilitates improved elimination of stressed and dying tumour cells by DCs. Therefore, therapeutics that promote endogenous CRT translocation to the cell surface can improve the removal of cancer cells. However, infused recombinant CRT dampens this cancer cell eradication by binding directly to the DCs. Low levels of endogenous BiP appear as a surface biomarker of endoplasmic reticulum (ER) stress in some types of tumour cells, a reflection of cells undergoing proliferation, in which resulting hypoxia and nutrient deprivation perturb ER homeostasis triggering the unfolded protein response, leading to increased expression of GRP78/BiP and altered cellular location. Conversely, infusion of an analogue of GRP78/BiP (IRL201805) can lead to long-term immune resetting and restoration of immune homeostasis. The therapeutic potential of both chaperones relies on them being relocated from their intracellular ER environment. Ongoing clinical trials are employing therapeutic interventions to either enhance endogenous cell surface CRT or infuse IRL201805, thereby triggering several disease-relevant immune responses leading to a beneficial clinical outcome.

Shc Is Implicated in Calreticulin-Mediated Sterile Inflammation in Alcoholic Hepatitis.

Src homology and collagen (Shc) proteins are major adapters to extracellular signals, however, the regulatory role of Shc isoforms in sterile inflammatory responses in alcoholic hepatitis (AH) has not been fully investigated. We hypothesized that in an isoform-specific manner Shc modulates pre-apoptotic signals, calreticulin (CRT) membrane exposure, and recruitment of inflammatory cells. Liver biopsy samples from patients with AH vs healthy subjects were studied for Shc expression using DNA microarray data and immunohistochemistry. Shc knockdown (hypomorph) and age-matched wild-type mice were pair-fed according to the chronic-plus-binge alcohol diet. To analyze hepatocyte-specific effects, adeno-associated virus 8-thyroxine binding globulin-Cre (hepatocyte-specific Shc knockout)-mediated deletion was performed in flox/flox Shc mice. Lipid peroxidation, proinflammatory signals, redox radicals, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratio, as well as cleaved caspase 8, B-cell-receptor-associated protein 31 (BAP31), Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist killer (Bak), were assessed invivo. CRT translocation was studied in ethanol-exposed p46ShcẟSH2-transfected hepatocytes by membrane biotinylation in conjunction with phosphorylated-eukaryotic initiation factor 2 alpha, BAP31, caspase 8, and Bax/Bak. The effects of idebenone, a novel Shc inhibitor, was studied in alcohol/pair-fed mice. Shc was significantly induced in patients with AH (P< .01). Alanine aminotransferase, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratios, production of redox radicals, and lipid peroxidation improved (P < .05), and interleukin 1β, monocyte chemoattractant protein 1, and C-X-C chemokine ligand 10 were reduced in Shc knockdown and hepatocyte-specific Shc knockout mice. Invivo, Shc-dependent induction, and, in hepatocytes, a p46Shc-dependent increase in pre-apoptotic proteins Bax/Bak, caspase 8, BAP31 cleavage, and membrane translocation of CRT/endoplasmic reticulum-resident protein 57 were seen. Idebenone protected against alcohol-mediated liver injury. Alcohol induces p46Shc-dependent activation of pre-apoptotic pathways and translocation of CRT to the membrane, where it acts as a damage-associated molecular pattern, instigating immunogenicity. Shc inhibition could be a novel treatment strategy in AH.

GPC2 antibody–drug conjugate reprograms the neuroblastoma immune milieu to enhance macrophage-driven therapies

BackgroundAntibody–drug conjugates (ADCs) that deliver cytotoxic drugs to tumor cells have emerged as an effective and safe anticancer therapy. ADCs may induce immunogenic cell death (ICD) to promote additional endogenous...

CD47KO/CRT dual-bioengineered cell membrane-coated nanovaccine combined with anti-PD-L1 antibody for boosting tumor immunotherapy.

Tumor vaccines trigger tumor-specific immune responses to prevent or treat tumors by activating the hosts’ immune systems, and therefore, these vaccines have potential clinical applications. However, the low immunogenicity of the tumor antigen itself and the low efficiency of the vaccine delivery system hinder the efficacy of tumor vaccines that cannot produce high-efficiency and long-lasting antitumor immune effects. Here, we constructed a nanovaccine by integrating CD47KO/CRT dual-bioengineered B16F10 cancer cell membranes and the unmethylated cytosine-phosphate-guanine (CpG) adjuvant. Hyperbranched PEI25k was used to load unmethylated cytosine-phosphate-guanine (CpG) through electrostatic adsorption to prepare PEI25k/CpG nanoparticles (PEI25k/CpG-NPs). CD47KO/CRT dual-bioengineered cells were obtained by CRISPR-Cas9 gene editing technology, followed by the cell surface translocation of calreticulin (CRT) to induce immunogenic cell death (ICD) in vitro. Finally, the extracted cell membranes were coextruded with PEI25k/CpG-NPs to construct the CD47KO/CRT dual-bioengineered cancer cell membrane-coated nanoparticles (DBE@CCNPs). DBE@CCNPs could promote endocytosis of antigens and adjuvants in murine bone marrow derived dendritic cells (BMDCs) and induce their maturation and antigen cross-presentation. To avoid immune checkpoint molecule-induced T cell dysfunction, the immune checkpoint inhibitor, the anti-PD-L1 antibody, was introduced to boost tumor immunotherapy through a combination with the DBE@CCNPs nanovaccine. This combination therapy strategy can significantly alleviate tumor growth and may open up a potential strategy for clinical tumor immunotherapy.

The New Microtubule-Targeting Agent SIX2G Induces Immunogenic Cell Death in Multiple Myeloma.

Microtubule-targeting agents (MTAs) are effective drugs for cancer treatment. A novel diaryl [1,2]oxazole class of compounds binding the colchicine site was synthesized as cis-restricted-combretastatin-A-4-analogue and then chemically modified to have improved solubility and a wider therapeutic index as compared to vinca alkaloids and taxanes. On these bases, a new class of tricyclic compounds, containing the [1,2]oxazole ring and an isoindole moiety, has been synthetized, among which SIX2G emerged as improved MTA. Several findings highlighted the ability of some chemotherapeutics to induce immunogenic cell death (ICD), which is defined by the cell surface translocation of Calreticulin (CALR) via dissociation of the PP1/GADD34 complex. In this regard, we computationally predicted the ability of SIX2G to induce CALR exposure by interacting with the PP1 RVxF domain. We then assessed both the potential cytotoxic and immunogenic activity of SIX2G on in vitro models of multiple myeloma (MM), which is an incurable hematological malignancy characterized by an immunosuppressive milieu. We found that the treatment with SIX2G inhibited cell viability by inducing G2/M phase cell cycle arrest and apoptosis. Moreover, we observed the increase of hallmarks of ICD such as CALR exposure, ATP release and phospho-eIF2α protein level. Through co-culture experiments with immune cells, we demonstrated the increase of (i) CD86 maturation marker on dendritic cells, (ii) CD69 activation marker on cytotoxic T cells, and (iii) phagocytosis of tumor cells following treatment with SIX2G, confirming the onset of an immunogenic cascade. In conclusion, our findings provide a framework for further development of SIX2G as a new potential anti-MM agent.

ROS-triggered nanoinducer based on dermatan sulfate enhances immunogenic cell death in melanoma.

Due to its complexity, diversity and heterogeneity, melanoma is a kind of malignant tumor. It has been proved that the enhancement of anti-tumor immune response such as immunogenic cell death (ICD) is an important therapeutic strategy. In previous studies, we confirmed that dermatan sulfate (DS) from skin tissue could specifically homing to melanoma B16F10 cells. In this study, we propose a nanoinducer (DOX/ADS NP) based on a functional DS for melanoma. This nanosystem is composed of DS as framework, aromatic thioketal derivative (ATK) as functional grafting unit and doxorubicin (DOX) designed as an ICD inducer. Through the intermolecular interaction between DOX and ATK, DOX/ADS NP with specific-homing, high-loading and ROS-triggering release was obtained via self-assemble. Compared with free DOX and non-functionalized nanomedicine, DOX/ADS NP could release DOX into B16F10 cells better, and strongly induce the translocation of calreticulin (CRT) to the cell membrane. CRT is a marker of ICD, also as a "eat me" signal to stimulate the maturation and antigen presentation of dendritic cells. Therefore, a series of subsequent immune responses were activated: maturation of dendritic cells, T cells proliferation, increased tumor-infiltrating CTLs and the ratio of CTLs to Tregs, and up-regulated cytotoxic cytokine expression. In conclusion, DOX/ADS NP promoted ICD-associated immune response through more specific targeting effect and sensitive responsive DOX release, achieving better inhibitory effect on melanoma than free DOX and other nanoformulation. This biomimetic ICD nanoinducer based on DS is expected to provide new strategies and references for the treatment of melanoma.

Calreticulin nuclear translocalization alleviates CaM/CaMKII/CREB signaling pathway to enhance chemosensitivity in HDAC inhibitor-resistant hepatocellular carcinoma cells.

Calreticulin (CRT) is located in the endoplasmic reticulum (ER), it helps proteins fold correctly inside the ER, and acts as a modulator of Ca2+ homeostasis. Aberrant expression of CRT is implicated in several cancer types, qualifying CRT as a potential therapeutic target. However, it remains unclear how CRT affects specific oncogenic pathways. In this study, we used histone deacetylase inhibitors (HDACis) to establish drug-resistant liver cancer cells and further analyzed the molecular mechanism of development of drug resistance in those cells. The 2D gel electrophoresis and RT-PCR data showed that CRT was downregulated in HDACis-resistant cells by comparing with HA22T parental cells. We previously elucidated the development of drug-resistance in HCC cells via activation of PP1-eIF2α pathway, but not via ER stress pathway. Here, we show that thapsigargin induced ER stress through mechanism other than ER stress downstream protein GRP78-PERK to regulate CRT expression in HDACis-R cells. Moreover, the expression level of CRT was not the main cause of apoptosis in HDACis-resistant cells. Mechanistic studies identified the apoptosis factors in the nucleus—the HDACis-mediated overexpression of CRT, CRT translocation to the cell nucleus, and reduced CaM/CaMKII/CREB pathway—that led to chemosensitivity in HDACis-R HCC cells.

Abstract 1294: Platinum-based chemotherapy immunomodulatory effects and immunotherapy association in upper tract urothelial carcinomas

Abstract Upper Tract Urothelial Carcinomas (UTUC) are extremely aggressive tumors of ureter or renal pelvis. UTUC present less tumor mutational burden and low tumor immune infiltrate compared to bladder cancer. Despite this they are treated with the same protocol than bladder cancer with more than 50% of relapses justifying the need of new therapeutic options. To improve patient care, we suggest stimulating the immune system by platinum-based chemotherapies (Cisplatin-Gemcitabine (CisGem) or Carboplatin-Gemcitabine (CarboGem)) in order to potentiate the effect of an anti-PD-L1, the Durvalumab. We launched a phase II clinical trial called “iNDUCT” which studies these combinations in UTUC patients. In parallel, we conduct an in vitro project that aims to determine if chemotherapies could transform cold tumor into a hot tumor, and if so by which mechanisms? Using UTUC cell lines (UM-UC-14,UCC03,UCC17,UCC14,UCC47) we have evaluated the cytotoxicity effects of the chemotherapies combinations in 2D and 3D cell cultures. We have assessed their potential (i) to induce DNA damage using image cytometry, (ii) to induce PD-L1 expression using flow cytometry, (iii) to induce immune cell death using ELISA kits, (iv) to activate the cGAS/STING pathway using qPCR and Western-Blot, and finally (v) to attract immune cells by using heterotypic spheroids model (tumoral cells+PBMCs).Our results demonstrate that CisGem and CarboGem present synergistic effects in UTUC spheroid cultures. These treatments also induce DNA damage pathway demonstrated by an increase of γH2AX, P-ATM, P-CHK1 and P-CHK2 positive cells. We found an increase of PD-L1 membrane expression after treatment in UTUC cell lines. RNA Seq analyses indicates that the major pathways upregulated by these combinations are inflammatory pathways (TNF-α signaling via NFkB, interferon alpha response, inflammatory response, interferon gamma response). We could observe an immune cell death induction demonstrated by an increase of ATP and HMGB1 release and calreticulin translocation. We showed cGAS/STING pathway activation as evidenced by an increase of P-IRF3 and interferon stimulated genes (ISGs) expression. We demonstrated an inhibition of the ISGs induction after treatment by our chemotherapies when cells are treated with an ATM inhibitor or in UMUC-14 deleted for STING. And we finally showed that CisGem and CarboGem can increase immune infiltration in the heterotypic tumor spheroids. These results indicate that the combination of platinum salts + gemcitabine induces inflammatory pathways via a non-canonical STING pathway dependent on ATM activation in UTUC model. Furthermore, these combination induce an upregulation of PD-L1 expression and allow immune cells attraction at the tumor. All these data support that a combination CisGem or CarboGem with an anti-PD-L1 will be efficient for UTUC patients. Citation Format: Alexandra Fauvre, Nadia Vie, Mathilde Robin, Clara Taffoni, Nadine Laguette, Julien Faget, Laurent Gros, Aurélie Garcin, Celine Gongora, Nadine Houede. Platinum-based chemotherapy immunomodulatory effects and immunotherapy association in upper tract urothelial carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1294.

PARP-inhibition with IFNg in the ovarian tumor microenvironment induces immunogenic cancer cell death for sustained anti-tumor immunity.

Abstract Poly(ADP-ribose) polymerase (PARP) inhibitors have been a remarkable breakthrough in the treatment of ovarian cancers with BRCA1/2 mutations. We have previously reported that PARP-inhibition with immune checkpoint blockade extended survival of BRCA1-deficient ovarian cancer (ov ca) preclinical models. A significant increase in IFNg producing effector T cells in the tumor environment promoted tumor cytotoxicity in a caspase-independent manner. In this study, we hypothesized that in the presence of IFNg, PARPi may induce immunogenic cell death (ICD) rather than apoptosis and promote immune-mediated tumor control. In vitro, PARPi+IFNg treatment induces hallmarks of ICD in BRCAmt ov ca; such as cell surface translocation of calreticulin, ATP and HMGB-1 release but not by PARPi or IFNg single treatment. Additionally, the PARPi+IFNg treated Ov ca cells induced maturation and activation of dendritic cells with enhanced antigen-presentation to stimulate anti-tumor T cells. Evidence of immunogenicity was confirmed by the benefit of vaccination with PARPi+IFNg treated Ov ca cells in vivo. The vaccination established the long-lasting protective anti-tumor immune responses to clear sequential tumor inoculations. T cell-depletion and adoptive transfer experiments showed the ICD-induced tumor immunity was surprisingly, both CD4 and CD8 T cell-mediated. Currently, we are addressing whether anti-tumor antibodies contribute to tumor control. Our results identified a novel mechanism by which PARP-inhibition with IFNg induces ICD in the ovarian tumor environment. These effects can be leveraged to enhance tumor clearance in the combination with immune therapies to modulate TME by stimulating tumor-immune cycles. Supported by grants from NCI (R37 CA229221)

Local anesthetics elicit immune-dependent anticancer effects

BackgroundRetrospective clinical trials reported a reduced local relapse rate, as well as improved overall survival after injection of local anesthetics during cancer surgery. Here, we investigated the anticancer effects of...

Geranylgeranyl diphosphate synthase inhibitor and proteasome inhibitor combination therapy in multiple myeloma

BackgroundMultiple myeloma (MM) remains an incurable malignancy, despite the advent of therapies such as proteosome inhibitors (PIs) that disrupt protein homeostasis and induce ER stress. We have pursued inhibition of geranylgeranyl diphosphate synthase (GGDPS) as a novel mechanism by which to target protein homeostasis in MM cells. GGDPS inhibitors (GGSI) disrupt Rab geranylgeranylation, which in turn results in perturbation of Rab-mediated protein trafficking, leading to accumulation of intracellular monoclonal protein, induction of ER stress and apoptosis. Our lead GGSI, RAM2061, has demonstrated favorable pharmacokinetic properties and in vivo efficacy. Here we sought to evaluate if combination therapy with GGSI and PI would result in enhanced disruption of the unfolded protein response (UPR) and increase anti-MM efficacy.MethodsMTT assays were conducted to evaluate the cytotoxic effects of combining RAM2061 with bortezomib in human MM cells. The effects of RAM2061 and/or PI (bortezomib or carfilzomib) on markers of UPR and apoptosis were evaluated by a combination of immunoblot (ATF4, IRE1, p-eIF2a, cleaved caspases and PARP), RT-PCR (ATF4, ATF6, CHOP, PERK, IRE1) and flow cytometry (Annexin-V). Induction of immunogenic cell death (ICD) was assessed by immunoblot (HMGB1 release) and flow cytometry (calreticulin translocation). Cell assays were performed using both concurrent and sequential incubation with PIs. To evaluate the in vivo activity of GGSI/PI, a flank xenograft using MM.1S cells was performed.ResultsIsobologram analysis of cytotoxicity data revealed that sequential treatment of bortezomib with RAM2061 has a synergistic effect in MM cells, while concurrent treatment was primarily additive or mildly antagonistic. The effect of PIs on augmenting RAM2061-induced upregulation of UPR and apoptotic markers was dependent on timing of the PI exposure. Combination treatment with RAM2061 and bortezomib enhanced activation of ICD pathway markers. Lastly, combination treatment slowed MM tumor growth and lengthened survival in a MM xenograft model without evidence of off-target toxicity.ConclusionWe demonstrate that GGSI/PI treatment can potentiate activation of the UPR and apoptotic pathway, as well as induce upregulation of markers associated with the ICD pathway. Collectively, these findings lay the groundwork for future clinical studies evaluating combination GGSI and PI therapy in patients with MM.