High-mobility Group Box Protein B1 Research Articles

P53-armed oncolytic virotherapy induces abscopal effect in osteosarcoma by promoting immunogenic cell death

Osteosarcoma (OS), the most frequent primary malignant tumor of bone in children and adolescents, is refractory to immune checkpoint inhibitors due to poor antitumor immune response. Chemotherapy and virotherapy induce immunogenic cell death (ICD) and antitumor immune responses, leading to the abscopal effect in untreated tumors. We previously demonstrated the antitumor activity of the telomerase-specific replication-competent oncolytic adenoviruses OBP-301 and p53-armed OBP-702 in human OS cells. Here, we show the therapeutic potential of chemotherapeutic drugs (doxorubicin, cisplatin) and telomerase-specific oncolytic adenoviruses (OBP-301, p53-armed OBP-702) to induce ICD in human OS cells (U2OS, MNNG/HOS, SaOS-2) and murine OS cells (NHOS). OBP-702 induced more profound ICD via secretion of adenosine triphosphate (ATP) and high-mobility group box protein B1 (HMGB1) compared with chemotherapy and OBP-301 in human OS cells. Murine NHOS cells were also more sensitive to OBP-702 than OBP-301. Subcutaneous NHOS tumor models demonstrated that intratumoral injection of OBP-702 significantly increased the tumor infiltration of cytotoxic CD8+ T cells and induced the abscopal effect against non-treated tumors compared with OBP-301. Our results suggest that OBP-702 is a promising antitumor reagent to induce ICD with secretion of ATP and HMGB1 and the abscopal effect against OS.

Magnolia biondii flower extract attenuates UVB-induced skin damage through high-mobility group box protein B1.

Magnolia biondii, a plant containing many magnolian-like compounds in its flowers or buds, exhibits anti-inflammatory and antiallergic effects; however, no study has addressed its effect on alleviating ultraviolet light (UV)-induced skin damage. We thus aimed at studying the effects of M. biondii flower extract (MB) on UVB-induced skin damage and determine the relationship between cell damage and damage-associated molecular patterns (DAMPs). Reconstructed epidermal models and foreskin samples were selected to detect cellular reactions after UVB irradiation and MB treatment. MTT, haematoxylin-eosin and immunofluorescence staining were used to examine total viability, sunburned cells and expression and migration of DAMPs at 16 or 48 h. Prostaglandin E2 (PGE-2) and interleukin 8 (IL-8) levels were measured using enzyme-linked immunosorbent assays. A clinical UVB-damaged test was carried out on human arms subjected to MB pre- or post-treatment. Human skin probes were used to measure erythema, melanin, ITA° and transepidermal water loss (TEWL), while skin photos were captured using the VISIA system. MB is rich in lignans such as magnolin, pinoresinol dimethyl ether and fargesin, and shows weak UV absorption at 280-320 nm. Coculturing with MB for 16 or 48 h after UVB irradiation improved the tissue viability and structure of Skinovo-Epi, and reduced the expression and migration of high mobility group box protein B1 (HMGB1) as well as the expression of IL-8 and PGE-2. In the excised foreskin treated with MB after UVB irradiation, the generation of 8-hidroxy-2-deoxyguanosine and nuclear transfer of HMGB1 were reduced. When pre-treated with MB for 3 days, UVB-induced skin erythema and ITA° were significantly decreased. When post-treated with MB for 5 days, a decrease in skin erythema, melanin and TEWL values and an increase in skin ITA° were observed. Treatment with MB attenuated UVB-induced skin damage, such as erythema, pigmentation and skin barrier function, by improving the tissue viability and structure and reducing sunburned cells and skin inflammation. This effect may be related to DNA damage, which causes the migration of HMGB1 from the nucleus to the outside of the cell to induce skin inflammation.

Rapid detection of two markers of acute aortic dissection based on a lateral flow assay of hollow CdTe/CdS nanospheres

Acute aortic dissection (AAD) is an emergency disease with a high mortality rate, and its characteristics is variable clinical symptoms, complex pathology, high misdiagnosis rate, leading to its frequent confusion with other chest pains of myocardial infarction and chronic emphysema in clinic; Therefore, early diagnosis and treatment of acute aortic dissection is the key to reduce its mortality and improve the prognosis of patients., and timely & rapid diagnosis is crucial for successful treatment. Conventional methods (such as PCR, MRI, and ELISA) cannot be implemented as a rapid point-of-care (POC) test for ADD. Highly fluorescent hollow CdTe/CdS nanospheres (NSs) with the inside size of ∼240 nm and shell thickness of ∼19 nm were prepared via the hydrothermal method, and three color NSs with green, yellow and red fluorescence of wavelength of 535 nm, 580 nm, and 620 nm, respectively, were obtained by changing the preparation temperature. Immunochromatographic (ICG) assay were developed with the prepared yellow CdTe/CdS NSs for the sensitive and qualitative detection of high mobility group box protein B1 (HMGB1). Visual limit of detection of ICG for HMGB1 antigen was 3.13 ng/mL within 15 min, lower than that of normal ELISA. Furthermore, a multichannel lateral flow assays (mLFA) was developed with green and yellow CdTe/CdS NSs for the simultaneous, specific, sensitive, and qualitative detection of HMGB1 and soluble growth stimulating gene 2 (sST2), and visual limits of detection of HMGB1 and sST2 in mLFA were 10 ng/mL and 1.25 ng/mL, respectively. This mLFA of CdTe/CdS NSs with two marker-antibodies first facilitates its application of POC in the early diagnosis on AAD.

Role of HMGB1 in Vitiligo: Current Perceptions and Future Perspectives.

Vitiligo is a chronic depigmenting disorder of the skin and mucosa caused by the destruction of epidermal melanocytes. Although the exact mechanism has not been elucidated, studies have shown that oxidative stress plays an important role in the pathogenesis of vitiligo. High mobility group box protein B1 (HMGB1) is a major nonhistone protein and an extracellular proinflammatory or chemotactic molecule that is actively secreted or passively released by necrotic cells. Recent data showed that HMGB1 is overexpressed in both blood and lesional specimens from vitiligo patients. Moreover, oxidative stress triggers the release of HMGB1 from keratinocytes and melanocytes, indicating that HMGB1 may participate in the pathological process of vitiligo. Overall, this review mainly focuses on the role of HMGB1 in the potential mechanisms underlying vitiligo depigmentation under oxidative stress. In this review, we hope to provide new insights into vitiligo pathogenesis and treatment strategies.

Oncolytic virus-mediated p53 overexpression promotes immunogenic cell death and efficacy of PD-1 blockade in pancreatic cancer

Immune checkpoint inhibitors, including anti-programmed cell death 1 (PD-1) antibody, provide improved clinical outcome in certain cancers. However, pancreatic ductal adenocarcinoma (PDAC) is refractory to PD-1 blockade therapy due to poor immune response. Oncolytic virotherapy is a novel approach for inducing immunogenic cell death (ICD). We demonstrated the therapeutic potential of p53-expressing telomerase-specific oncolytic adenovirus OBP-702 to induce ICD and anti-tumor immune responses in human PDAC cells with different p53 status (Capan-2, PK-59, PK-45H, Capan-1, MIA PaCa-2, BxPC-3) and murine PDAC cells (PAN02). OBP-702 significantly enhanced ICD with secretion of extracellular adenosine triphosphate and high-mobility group box protein B1 by inducing p53-mediated apoptosis and autophagy. OBP-702 significantly promoted the tumor infiltration of CD8+ T cells and the anti-tumor efficacy of PD-1 blockade in a subcutaneous PAN02 syngeneic tumor model. Our results suggest that oncolytic adenovirus-mediated p53 overexpression augments ICD and the efficacy of PD-1 blockade therapy against cold PDAC tumors. Further in vivo experiments would be warranted to evaluate the survival benefit of tumor-bearing mice in combination therapy with OBP-702 and PD-1 blockade.

Telomerase-specific oncolytic immunotherapy for promoting efficacy of PD-1 blockade in osteosarcoma.

Immune checkpoint inhibitors including anti-programmed cell death 1 (PD-1) antibody have recently improved clinical outcome in certain cancer patients; however, osteosarcoma (OS) patients are refractory to PD-1 blockade. Oncolytic virotherapy has emerged as novel immunogenic therapy to augment antitumor immune response. We developed a telomerase-specific replication-competent oncolytic adenovirus OBP-502 that induces lytic cell death via binding to integrins. In this study, we assessed the combined effect of PD-1 blockade and OBP-502 in OS cells. The expression of coxsackie and adenovirus receptor (CAR), integrins αvβ3 and αvβ5, and programmed cell death ligand 1 (PD-L1) was analyzed in two murine OS cells (K7M2, NHOS). The cytopathic activity of OBP-502 in both cells was analyzed using the XTT assay. OBP-502-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). Subcutaneous tumor models for K7M2 and NHOS cells were used to evaluate the antitumor effect and number of tumor-infiltrating CD8+ cells in combination therapy. K7M2 and NHOS cells showed high expression of integrins αvβ3 and αvβ5, but not CAR. OBP-502 significantly suppressed the viability of both cells, in which PD-L1 expression and the release of ATP and HMGB1 were significantly increased. Intratumoral injection of OBP-502 significantly augmented the efficacy of PD-1 blockade on subcutaneous K2M2 and NHOS tumor models via enhancement of tumor-infiltrating CD8+ T cells. Our results suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to promote the efficacy of PD-1 blockade in OS.

Abstract 2406: Novel thienopyrimidine analog TPH104 induces immunogenic cell death in TNBC cells

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high propensity for metastasis and recurrence, accounting for one-fourth of all breast cancer deaths. Despite being chemo-sensitive to anthracyclines and taxanes, TNBCs have poor prognosis due to development of resistance often due to the overexpression of efflux transporters, increased DNA repair, and occurrence of genetic mutations that decrease the likelihood of apoptosis. Recently, we discovered a novel class of thieno-pyrimidin-hydrazinyl (TPH) compounds that in the nanomolar range, selectively elicited a unique, caspase-independent cell death in TNBC cells. The lead compound, TPH104, selectively activates receptor interacting protein kinase 1 (RIPK1), thus inducing a necroptosis like cell death. Since necroptosis is known to release endogenous immunogenic markers, we wanted to study whether TPH104 can promote immunogenic cell death (ICD) in TNBC cells and activate cells in the tumor immune milieu. ICD is characterized by presence of damage-associated molecular patterns (DAMPs) such as cell surface exposure of calreticulin (CRT), secretion of ATP, and release of high-mobility group box protein B1 (HMGB1) from dying tumor cells. Methods: Human TNBC cell line MDA-MB-231 cells were treated with TPH104 and cell survival was examined by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT), clonogenic and live-cell imaging assays. Western blot analysis was used to assess death signal transducers. DAMPs were examined by ELISA, luminescence-based assay systems and immunofluorescence. Activation of mouse bone marrow derived dendritic cells (BMDCs) in response to conditioned media (CM) harvested after treatment of MDA-MB-231 cells with TPH104 (TPH104-CM) and/or mitoxantrone (MX, positive control) for 48h was evaluated using flow cytometry analysis of maturation markers (CD86, MHCII) expressed on CD11C+ BMDCs. Inflammatory gene expression was evaluated by q-PCR. Results: The results demonstrated that TPH104 reduced cell survival and induced death of MDA-MB-231 cells in a dose-dependent pattern. TPH104 induced ICD, as evidenced by increased release of ATP, as well as surface exposure of CRT. Flow cytometric analysis revealed TPH104-CM treatment increased CD11C+CD86+MHC+ BMDCs as robustly as MX-CM. Further, TPH104 posed no toxicity to mice in a single dose toxicity study. Conclusion: TPH104 induced selective ICD in MDA-MB-231 cells. TPH104 similar to MX primed cells to ICD and increased extracellular ATP levels and surface exposure of CRT. TPH104 promoted maturation of CD11C+ BMDCs and increased inflammatory gene expression. Our study demonstrated TPH104 as an ICD-inducer and immunomodulator in TNBC. Citation Format: Diwakar Bastihalli Tukaramrao, Siddharth Saraiya, Ross A. Hanely, Saloni Malla, Amit K. Tiwari. Novel thienopyrimidine analog TPH104 induces immunogenic cell death in TNBC cells [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 2406.

Expression of HMGB1 protein in breast cancer and its clinicopathological significance

Objective: To investigate the expression and clinicopathological significance of high mobility group box protein B1 (HMGB1) protein in breast cancer. Methods: The expression of HMGB1 protein in 26 normal breast tissues and 417 invasive breast cancer tissues diagnosed at Dongyang People's Hospital, Zhejiang Province from 2016 to 2018 were detected by immunohistochemical EnVision method. The relationship between nuclear and cytoplasmic HMGB1 protein expression and clinicopathologic features of breast cancer patients were analyzed. Results: The nuclear and cytoplasmic expression of HMGB1 protein was 80.8% (337/417) and 16.8% (70/417) respectively in breast cancer, and was 46.2%(12/26) and 0(0/26) respectively in normal breast tissue. Both nuclear and cytoplasmic expression of HMGB1 protein in breast cancer were significantly higher than normal breast tissue (P<0.001, P=0.046, respectively). The nuclear expression of HMGB1 protein was also higher in high grade, estrogen receptor (ER) negative, progesterone receptor (PR) negative (P=0.006, P=0.004, P<0.001, respectively); whereas the cytoplasmic expression of HMGB1 protein was also higher in high grade, estrogen receptor (ER) negative, progesterone receptor (PR) negative (P<0.001 in all) breast cancers. Multivariate logistic regression model showed that nuclear HMGB1 expression correlated with histologic grade (OR=2.188, 95%CI=1.078-4.443, P=0.030), while cytoplasmic HMGB1 expression correlated with histologic grade (OR=3.031, 95%CI=1.600-5.742, P=0.001), ER (OR=0.129, 95%CI=0.034-0.494, P=0.003) and TNM staging (OR=3.820, 95%CI=1.042-14.001, P=0.043). Multivariate analysis of Cox proportional hazard model showed that nuclear HMGB1 expression was an independent risk factor for the overall survival of breast cancer patients (HR=0.366, 95%CI=0.138-0.972, P=0.044). Conclusion: Nuclear and cytoplasmic HMGB1 proteins are related to multiple poor prognostic factors in breast cancer, and may be a potential biomarker for breast cancer treatment.

1234P - Anti-cancer and immunomodulatory effects of cobimetinib in triple negative breast cancer

BackgroundImmune check point inhibitor therapy has shown efficacy in Triple-negative breast cancer (TNBC), highlighting the immune-responsive microenvironment of TNBC. Recently, anti-cancer therapeutics have been shown to induce immunogenic cell death (ICD). MEK/ERK cascade mediates T cell receptor signaling and involves in immune response. MEK inhibitor cobimetinib is approved for advanced melanoma and is studied in several clinical trials including breast cancer. Here, we aimed to explore the anti-tumor and immunomodulatory effects of cobimetinib in TNBC. MethodsDamage-associated molecular patterns (DAMPs), cell-surface translocation of calreticulin (CRT), extracellular release of ATP, and increase in high-mobility group box protein B1 (HMGB1) release from dying tumor cells, were examined by immunoblotting and flow cytometric analysis. The pan-caspase inhibitor z-VAD-FMK was used to validate cobimetinib-induced signaling. The immunocompetent and immunodeficient mice were conducted to investigate the efficacy of cobimetinib. ResultsCobimetinib significantly induced cell apoptosis and DAMPs in TNBC cells (human MDA-MB-231, MDA-MB-468, and murine 4T1 cells). Cobimetinib downregulated p-ERK and activated caspase-8, in association with increased DAMPs such as HMGB1 and CRT. Cobimetinib-induced CRT was attenuated by ectopic ERK expression. Moreover, cobimetinib-induced cell apoptosis and increased CRT expressions were restored by the pan-caspase inhibitor. The anti-tumor activity of cobimetinib was superior in immunocompetent than in immunodeficient mice. DAMPs and caspase-8 cascade were upregulated in cobimetinib-treated 4T1 xenografts. Interestingly, the immunomodulation of cobimetinib in 4T1-bearing mice were different from that in non-tumor bearing immunocompetent mice. Importantly, cobimetinib-treated 4T1-bearing immunocompetent mice exhibited increased total number of CD8+ T cells, effector CD4+ T cells, regulatory T cells and reduced the number of myeloid-derived suppressor cells, compared to vehicle-treated mice. ConclusionsOur findings suggest that the MEK inhibitor cobimetinib acts as an ICD-inducer and exerts immunomodulatory effects in TNBC. Legal entity responsible for the studyChun-Yu Liu. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Abstract 4852: Boosting immunity against pancreatic cancer by OBP-702 (Pfifteloxin), telomerase-specific replicative adenovirus armed with wild-type p53 gene

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal disease with a 5-year survival rate of less than 10%. Although immune checkpoint blockade has recently emerged as a novel antitumor therapy, PDAC is less sensitive to immunotherapy due to small number of tumor-infiltrating T cells. Recently, oncolytic virotherapy has been shown to stimulate the immune system as an immunogenic antitumor therapy. Activation of p53 has been also known to enhance antitumor immunity. In this study, we investigated the potential of telomerases-specific p53-expressing oncolytic adenovirus (OBP-702, Pfifeteloxin) for inducing the immunogenic cell death in PDAC cells. Methods: OBP-702 (Pfifteloxin) is a telomerase-specific oncolytic adenovirus, in which the human telomerase reverse transcriptase (hTERT) promoter drives the expression of the viral E1 gene for tumor-specific replication, that expresses the wild-type p53 by inserting the human p53 cDNA at the deleted E3. OBP-301 (Telomelysin) is an original telomerase-specific adenovirus lacking p53. OBP-502 is an RGD mutant fiber-containing OBP-301. We used 4 human PDAC cell lines (MIA PaCa-2, Capan-1, BxPC-3, Panc-1) and mouse PDAC cell line (Pan02). In vitro antitumor effect of these viruses was evaluated using a XTT assay. The molecular mechanism of virus-mediated cell death was investigated by western blot analysis. The virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1) using ELISA assay. The comparative in vivo antitumor efficacy was also investigated using a syngeneic Pan02 subcutaneous tumor model. Results: Telomerase-specific oncolytic adenoviruses induced the antitumor effect in all PDAC cells in a dose-dependent manner. The antitumor effect of OBP-702 was superior to that of OBP-301 or OBP-502. OBP-301 and OBP-502 mainly induced autophagy, whereas OBP-702 induced autophagy and apoptosis at 72 h after infection. The concentration of extracellular ATP and HMGB1 was significantly increased in OBP-702-infected PDAC cells compared to OBP-301- or OBP-502-infected cells at 24 and 48 h after infection. In mice carrying subcutaneous Pan02 murine PDAC tumors, intratumoral injection of OBP-702 resulted in a significant inhibition of tumor growth. Moreover, the number of tumor-infiltrating CD8+ T cells was significantly increased in OBP-702-treated groups compared to mock-treated group. Conclusion: Our data suggest that telomerases-specific p53-expressing oncolytic adenovirus OBP-702 (Pfifteloxin) induces profound immunogenic cell death to boost the immune responses in PDAC. Clinical trials with OBP-702 and immune checkpoint inhibitors for PDAC are warranted. Citation Format: Hiroyuki Araki, Hiroshi Tazawa, Takuro Fushimi, Takeyoshi Nishiyama, Satotu Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Boosting immunity against pancreatic cancer by OBP-702 (Pfifteloxin), telomerase-specific replicative adenovirus armed with wild-type p53 gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4852.

Abstract 3231: Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma

Abstract Background: Osteosarcoma (OS) is the most frequent primary malignant tumor of bone in children and adolescents. Although immune checkpoint inhibitor, anti-programmed death protein 1 (PD-1) antibody, has dramatically improved the clinical outcome in some cancer patients, OS patients are less sensitive to PD-1 blockade due to poor immune responses. Recently, oncolytic virotherapy has been shown to stimulate the immune system through induction of immunogenic cell death (ICD). We recently developed a RGD fiber-modified telomerase-specific oncolytic adenovirus OBP-502, which can enter into tumor cells by binding to cell surface integrin and induce oncolytic cell death in a telomerase-dependent manner. In this study, we assessed the in vitro and in vivo antitumor efficacy of combination therapy with PD-1 blockade and OBP-502 in OS cells. Methods: We used 2 murine OS cell lines, K7M2 and NHOS. The expression of PD-L1, coxsackie and adenovirus receptor (CAR), and integrin on the cell surface was analyzed by flow cytometric analysis. We analyzed the in vitro antitumor effect of OBP-502 using XTT assay and western blot analysis. Virus-induced ICD was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). To evaluate the therapeutic potential of oncolytic immunotherapy, we investigated the in vivo antitumor effect of combination therapy with anti-PD-1 antibody and OBP-502 using a subcutaneous K7M2 xenograft tumor model. Moreover, the number of tumor-infiltrating CD8+, CD4+ and Foxp3+ T cells was analyzed by immunohistochemistry. Results: Flow cytometric analysis demonstrated that K7M2 and NHOS cells had the expression of PD-L1 and integrin, but not CAR. XTT assay showed that OBP-502 efficiently suppressed the viability of K7M2 and NHOS cells in a dose-dependent manner. Western blot analysis revealed that OBP-502 induced the apoptosis-related cell death in K7M2 and NHOS cells. ELISA assay demonstrated that OBP-502 significantly increased the level of extracellular ATP and HMGB1 in K7M2 and NHOS cells. In vivo experiment using a subcutaneous K7M2 xenograft tumor model showed that combination of anti-PD-1 antibody and OBP-502 significantly reduced tumor growth compared to mock treatment or monotherapy. Immunohistochemical analysis revealed that OBP-502 significantly increased the number of tumor-infiltrating CD8+ T cells compared to mock treatment or monotherapy. By contrast, OBP-502 did not affect the number of CD4+ and Foxp3+ T cells in tumor tissues. Conclusion: These results suggest that oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus is a promising antitumor strategy to promote the therapeutic potential of PD-1 blockade in OS through stimulation of antitumor immune response. Citation Format: Koji Demiya, Hiroshi Tazawa, Yusuke Mochizuki, Miho Kure, Joe Hasei, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3231.

Abstract P4-06-21: MEK inhibitor cobimetinib induces immunogenic cell death and immune-modulatory effects in triple negative breast cancer

Abstract Background Triple-negative breast cancer (TNBC) has been associated with a robust tumor immune infiltrate. Tumor-infiltrating lymphocytes (TILs) in TNBC have been demonstrated a prognostic value. The mitogen-activated protein kinase (MAPK) signaling pathway have been shown to regulate the immune response with the production of immunomodulatory cytokines, such as TNFα, interleukin (IL)-1, IL-10, and IL-12. Clinical studies have shown that the high expression level of Extracellular signal–related kinase (ERK), a member of the MAPK pathway, correlates with shorter survival in TNBC patients. Accordingly, ERK is a potential target for anti-tumor and cancer immunotherapy. In this study, we aimed to investigate a MEK inhibitor cobimetinib and elucidate whether the MEK/ERK pathway is implicated in immunogenic cell death (ICD) and/or other immunomodulatory effects. Methods Mouse TNBC cell line 4T1 was treated with cobimetinib. The cell viability and cell apoptosis were determined by MTT assay and flow cytometric analysis. Cobimeinib-induced damage-associated molecular patterns (DAMPs), such as such as cell-surface translocation of calreticulin (CRT), extracellular release of ATP, and increase in high-mobility group box protein B1 (HMGB1) release from dying tumor cells, were examined by immunoblotting and flow cytometric analysis. Further, the molecular mechanisms involving cobimetinib-induced cell death were examined by Immunoblotting. Last but not least, establishment of 4T1 animal model in immunocompetent and immunodeficient mice was conducted to investigate the efficacy of cobimetinib in restoration of immunosurveillance and cancer metastasis in vivo. Results The results showed that cobimetinib impaired cell proliferation and induced cell apoptosis in a dose-dependent manner in 4T1 cells. Importantly, cobimetinib treated 4T1 cells elicited ICD, by increasing HMGB1 and ATP release, and by promoting membrane exposure of CRT. The molecular mechanisms involved in cobimetinib-induced DAMPs were partially through the downregulation of p-ERK expression. Moreover, cobimetinib induced processing of procaspase-3 and -8 in 4T1 cells, thereby resulting in caspase activation. In addition, 4T1 tumor bearing immunocompetent mice treated with cobimetinib showed reduced size of primary tumors, fewer lung metastases and increased survival. In addition, we noticed that the tumor-suppressive effects of cobimetinib were much stronger in immunocompetent mice than in immunodeficient mice, evident by distinct mice survival in drug-treated mice. Flow cytometric analysis of murine splenocytes also revealed that cobimetinib treatment increased total number of CD8+ T cells, dendritic cell maturation, and suppressed the number of Myeloid-derived suppressor cells (MDSCs) in immunocompetent mice. Conclusions Our findings suggested that the MEK/ERK inhibitor cobimetinib induces ICD in vitro and exerts additional immune-modulatory effects in immunocompetent TNBC mice model. Our study highlights the possibility for the use of cobimetinib as an ICD inducer and immunomodulator for therapeutic intervention in TNBC. Keywords Cobimetinib, immunogenic cell death, MAPK, ERK phosphorylation, immune regulation Citation Format: Liu C-Y, Lau K-Y, Chen J-L, Chu P-Y, Huang C-T, Wang W-L, Lien P-J, Tseng L-M. MEK inhibitor cobimetinib induces immunogenic cell death and immune-modulatory effects in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-21.

Association of High Mobility Group Box Protein B1 Gene Polymorphisms with Pneumonia Susceptibility and Severity.

To investigate the relationship between the high mobility group box protein B1 (HMGB1) single nucleotide polymorphisms (SNPs) rs1412125, rs2249825, and rs1045411 with pneumonia in terms of susceptibility, severity, and inflammatory response. The genotypes of HMGB1 rs1412125 (-1615T > C), rs2249825 (3814C > G), and rs1045411 (2262C > T) loci in 328 patients with community-acquired pneumonia (CAP) and 317 healthy subjects were analyzed by Sanger sequencing. The expression and secretion of the inflammatory cytokines HMGB1, interleukin (IL)-10, tumor necrosis factor-alpha (TNF-α), and IL-6 were determined after lipopolysaccharide (LPS) stimulation of peripheral whole blood cells. The risk of CAP was higher in carriers of the mutant HMGB1 rs1412125 and rs2249825 alleles than those that had the wild type alleles (adjusted odds ratio [OR] = 1.241; 95% confidence interval [CI] = 1.061-1.448; p = 0.007; adjusted OR = 1.225; 95% CI = 1.038-1.427; p = 0.016, respectively). Moreover, the mutation-carrying patients with CAP were more likely to develop severe community-acquired pneumonia (SCAP). There was no correlation between the HMGB1 rs1045411 SNP alleles and CAP or SCAP (p > 0.05). The expression and secretion of the inflammatory cytokines HMGB1, IL-10, TNF-α, and IL-6 was significantly higher in LPS-stimulated peripheral blood among patients with mutations at the rs1412125 and rs2249825 loci compared with those with wild type alleles (p < 0.05). The 30-day mortality rates for CAP patients with mutations at the rs1412125 and rs2249825 loci of HMGB1 were significantly higher than those that had wild type alleles. The mortality rate difference between rs1045411 wild-type CAP patients and mutant was not significant (p = 0.789). SNPs at the rs1412125 and rs2249825 loci of HMGB1 are associated with pneumonia in terms of susceptibility, severity, and inflammatory response.

Abstract 4570: Telomerase-specific oncolytic virotherapy promotes therapeuticefficacy of PD-1 blockade in murine osteosarcoma

Abstract Background: Treatment with immune checkpoint inhibitor anti-programmed death protein 1 (PD-1) antibody has dramatically improved the clinical outcome in cancer patients. Then immunotherapy is the most exciting field for cancer treatment in recent years. However, PD-1 blockade therapy is limited to certain cancer types. Therefore, the enhancement of therapeutic efficacy in PD-1 blockade therapy is required. Recently, it has been suggested that the effect of PD-1 blockade therapy can be enhanced when combined with immunogenic antitumor therapy including chemotherapy and virotherapy. We recently developed a RGD fiber-modified telomerase-specific oncolytic adenovirus OBP-502, which can enter into tumor cells by binding to cell surface integrin and induce oncolytic cell death in a telomerase-dependent manner. In this study, we assessed the in vitro and in vivo antitumor efficacy of combination therapy with anti-PD-1 antibody and OBP-502 in murine osteosarcoma. Methods: We used 2 murine osteosarcoma cell lines, K7M2 and NHOS. The expression of PD-L1, coxsackie and adenovirus receptor (CAR), and integrin on the cell surface was analyzed by flow cytometric analysis. The in vitro antitumor effect of OBP-502 was evaluated using a XTT assay. Virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). To evaluate the therapeutic potential of anti-PD-1 antibody and OBP-502, we assessed the antitumor effect of combination therapy with anti-PD-1 antibody and OBP-502 using a subcutaneous K7M2 xenograft tumor model. Moreover, the number of tumor-infiltrating CD8+ and Foxp3+ T cells was analyzed by immunohistochemistry. Results: K7M2 and NHOS cells showed the expression of PD-L1 and integrin, but not CAR, in flow cytometric analysis. OBP-502 efficiently suppressed the viability of K7M2 and NHOS cells in a dose-dependent manner. OBP-502 significantly increased the release of HMGB1 in K7M2 cells and the secretion of ATP in NHOS cells. Intratumoral injection of OBP-502 significantly induced the number of tumor-infiltrating CD8+ T cells and enhanced the suppression of tumor growth by PD-1 blockade in a subcutaneous K7M2 xenograft tumor model. Moreover, tumor free was observed in 6 out of 7 mice treated with anti-PD-1 antibody and OBP-502 even at 4 weeks after treatment. Conclusion: These results suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to promote the therapeutic efficacy of PD-1 blockade therapy in osteosarcoma. Citation Format: Yusuke Mochizuki, Hiroshi Tazawa, Koji Demiya, Tadashi Komatsubara, Kazuhisa Sugiu, Joe Hasei, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Telomerase-specific oncolytic virotherapy promotes therapeuticefficacy of PD-1 blockade in murine osteosarcoma [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 4570.

Abstract 5915: Overexpression of tumor suppressor p53 gene by oncolytic adenovirus boosts immune responses in human pancreatic ductal adenocarcinoma cells

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the worst prognosis disease with an overall 5-year survival rate of less than 5%. Although immune checkpoint therapy has been emerged as a novel antitumor therapy, PDAC is less sensitive to immunotherapy due to small number of infiltrating T cells. Recently, oncolytic virotherapy has been shown to promote intratumoral T cell infiltration as an immunogenic antitumor therapy. In the current study, we explored whether overexpression of the wild-type p53 tumor suppressor gene could synergize with oncolysis for induction of immunogenic cell death in human PDAC cells. Methods: OBP-702 is a tumor-specific replication-competent oncolytic adenovirus, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific viral replication and the Egr-1 promoter induces wild-type p53 expression. We used 4 PDAC cell lines (MIA PaCa-2, Capan-1, BxPC-3, Panc-1). Molecular mechanisms of OBP-702-mediated cell death was investigated. Migration and invasion properties were assessed using transwell chamber assay. In addition, virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). In vivo antitumor effect of OBP-702 was assessed using an orthotopic BxPC-3 xenograft tumor model. Results: At 72 hours after virus infection, OBP-702 induced profound antitumor effect in all human PDAC cells. OBP-702 induced autophagy- and apoptosis-related cell deaths. In migration and invasion assays, MIA Paca-2 and Capan-1 cells were low-invasive type, and Panc-1 and BxPC-3 cells were high-invasive type. OBP-702 inhibited migration and invasion properties of high-invasive type PDAC cells At 24 and 48 hours after virus infection, the secretion of ATP was significantly higher in OBP-702-infected PDAC cells compared to p53-null virus-infected cells. Moreover, OBP-702 significantly induced the higher amount of extracellular HMGB1 in all PDAC cells. Finally, OBP-702 significantly suppressed tumor growth in an orthotopic BxPC-3 xenograft tumor model compared to p53-null virus. Conclusions: Our data suggest that oncolytic adenovirus-mediated p53 overexpression induces more profound immunogenic cell death to boost the immune responses in PDAC. This additional immunological function of p53 is quite attractive and warrants clinical trials combined with immune checkpoint inhibitors. Citation Format: Hiroyuki Araki, Hiroshi Tazawa, Takeshi Ieda, Takuro Fushimi, Shinji Kuroda, Ryuichi Yoshida, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Overexpression of tumor suppressor p53 gene by oncolytic adenovirus boosts immune responses in human pancreatic ductal adenocarcinoma cells [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 5915.

Abstract P3-05-19: Regorafenib induces immunogenic cell death via p-stat3 inhibition in triple negative breast cancer cells

Abstract Background Metastatic triple-negative breast cancer (TNBC), despite being chemo-sensitive, remains poor prognostic. Rising data have suggested some anti-cancer agents exert immunostimulatory activities through inducing immunogenic cell death (ICD). ICD can be detected by presence of damage-associated molecular patterns (DAMPs) such as cell surface exposure of calreticulin (CRT), secretion of ATP, and increase in high-mobility group box protein B1 (HMGB1) release from dying tumor cells. Signal transducer and activator of transcription 3 (STAT3) activity is activated in TNBC and regorafenib has been shown to suppress p-STAT3 signal. We tested whether regorafenib induces ICD in TNBC. Methods Mice TNBC cell line 4T1 cells were treated with regorafenib and cell survival was examined by MTT assay and flow cytometric analysis. DAMPs were examined by western blot analysis, immunofluorescence microscopy and luminescent assay. The correlation between regorafenib-mediated ICD and STAT3 inhibition were validated in ectopic STAT3 transfected 4T1 cells. Moreover, we investigated the combination strategies of regorafenib with immune checkpoint blockade in syngeneic 4T1 tumor bearing mice (TNBC animal model) with treatment of regorafenib or mPD1 treatment alone/ combination of mPD1 and regorafenib. Results The results demonstrated that regorafenib reduced cell survival and induced cell apoptosis in a dose-dependent pattern in 4T1 cell line. Regorafenib induced ICD, as evidenced by it triggered the release of HMGB1 and ATP, as well as the exposure of CRT on the cell surface. Moreover, regorafenib-induced ICD was attenuated by ectopic expression of STAT3 (thus increased phosphorylation of STAT3) in STAT3-overexpressed 4T1 cells. Last but not the least, we observed 4T1 tumor bearing mice with treatment of regorafenib alone, or anti-PD1 monoclonal antibody (mAb) alone, or in combination of regorafenib and anti–PD1 mAb resulted in reduced size of primary tumors, increased survival and fewer lung metastases. Flow cytometric analysis revealed regorafenib treatment increased activated CD8+ T cells, increased antigen-presenting dendritic cells, and suppressed regulatory T cells (Tregs) in mice spleen. Conclusions Regorafenib induced ICD in 4T1 cells via the inhibition of p-STAT3. Regorafenib also promoted CD8+ T cells activation and antigen presenting ability of dendritic cells, and suppressed Tregs. Our study demonstrated regorafenib as an ICD-inducer and immunomodulator and its potential combination with immune checkpoint blockade in TNBC. Citation Format: Liu C-Y, Lau K-Y, Huang T-T, Chen J-L, Chu P-Y, Huang C-T, Wang W-L, Tseng L-M. Regorafenib induces immunogenic cell death via p-stat3 inhibition in triple negative breast cancer cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-19.

Inhibition of Toll-like receptor 4 ameliorates experimental postischemic injury in the cholestatic liver through inhibition of high-mobility group box protein b1 (HMGB1) signaling

BackgroundThe objective of this study was to elucidate whether the inhibition of Toll-like receptor 4 attenuates liver injury ischemia/reperfusion in the cholestatic liver. MethodRats were assigned into sham, bile duct ligation, sham ischemia/reperfusion (ischemia/reperfusion after laparotomy), and bile duct ligation ischemia/reperfusion (ischemia/reperfusion after bile duct ligation) groups. In some rats, TAK-242, an inhibitor of Toll-like receptor 4, was administered 15 minutes before ischemia/reperfusion. We measured intrahepatic Toll-like receptor 4 expression, serum hepatic marker expression, liver necrosis, gene expression of inflammation-associated factors, and serum high-mobility group box protein b1 levels. ResultsIntrahepatic Toll-like receptor 4 expression was significantly greater in the bile duct ligation group than in the sham group. Toll-like receptor 4 expression was further increased after ischemia/reperfusion in bile duct ligation ischemia/reperfusion groups. The levels of serum hepatic markers were significantly greater in both the sham ischemia/reperfusion and bile duct ligation ischemia/reperfusion groups than in the groups without ischemia/reperfusion. Liver necrosis was greater in the bile duct ligation group than in the sham group and was further increased in the bile duct ligation ischemia/reperfusion group. Genomic expression of inflammation-associated factors was also significantly greater in the bile duct ligation ischemia/reperfusion group than in the sham group. Serum high-mobility groups box protein b1 levels were greater in the bile duct ligation ischemia/reperfusion group than in the sham group (28.1 ng/ml versus 9.2 ng/ml, P = .011) and the bile duct ligation group (28.1 ng/ml versus 10.6 ng/ml, P = .017). These changes in the bile duct ligation ischemia/reperfusion group were significantly attenuated by preconditioning with TAK242. ConclusionsToll-like receptor 4 inhibition has a potential to minimize severe injury after ischemia/reperfusion in the cholestatic liver through inhibition of high-mobility groups box protein b1.

Ascorbic Acid Attenuates Hyperoxia-Compromised Host Defense against Pulmonary Bacterial Infection.

Supraphysiological concentrations of oxygen (hyperoxia) can compromise host defense and increase susceptibility to bacterial infections, causing ventilator-associated pneumonia. The phagocytic activity of macrophages is impaired by hyperoxia-induced increases in the levels of reactive oxygen species (ROS) and extracellular high-mobility group box protein B1 (HMGB1). Ascorbic acid (AA), an essential nutrient and antioxidant, has been shown to be beneficial in various animal models of ROS-mediated diseases. The aim of this study was to determine whether AA could attenuate hyperoxia-compromised host defense and improve macrophage functions against bacterial infections. C57BL/6 male mice were exposed to hyperoxia (≥98% O2, 48 h), followed by intratracheal inoculation with Pseudomonas aeruginosa, and simultaneous intraperitoneal administration of AA. AA (50 mg/kg) significantly improved bacterial clearance in the lungs and airways, and significantly reduced HMGB1 accumulation in the airways. The incubation of RAW 264.7 cells (a macrophage-like cell line) with AA (0-1,000 μM) before hyperoxic exposure (95% O2) stabilized the phagocytic activity of macrophages in a concentration-dependent manner. The AA-enhanced macrophage function was associated with significantly decreased production of intracellular ROS and accumulation of extracellular HMGB1. These data suggest that AA supplementation can prevent or attenuate the development of ventilator-associated pneumonia in patients receiving oxygen support.

Liuweiwuling tablets attenuate acetaminophen-induced acute liver injury and promote liver regeneration in mice.

To explore the mechanism of protection against acetaminophen-induced acute liver injury by Liuweiwuling tablets. Intraperitoneal injections of acetaminophen (250 mg/kg) were used to induce acute liver injury in male C57BL/6 mice. A total of 24 healthy mice were randomly assigned to two groups: an acute liver injury group (control group) and a Liuweiwuling tablet group. Mice were given Liuweiwuling tablets or a vehicle (PBS) orally prior to the administration of acetaminophen. Serum alanine aminotransferase (ALT) and aspartate aminotransaminase (AST) levels were measured at different time points within one week, and pathological examinations of liver tissues were performed 36 h after induction of acute liver injury. Serum inflammatory cytokines, such as high mobility group box protein B1 (HMGB1), tumor necrosis factor (TNF)-α and interleukin IL-1β, were detected using an ELISA method according to the manufacturer's instructions. Hepatic morphological changes at 36 h were assessed by hematoxylin and eosin staining. Expression of proliferating cell nuclear antigen (PCNA) in liver tissue was determined by Western blot analysis. The mRNA levels of hepatocyte proliferation markers (PCNA, CyclinD1 and p21) were detected by real-time quantitative reverse transcription-polymerase chain reaction. The levels of ALT/AST in the Liuweiwuling tablet group were decreased significantly at 6, 12 and 24 h compared to that of the control group (654.38 ± 120.87 vs 1566.17 ± 421.64, 1154.18 ± 477.72 vs 4654.84 ± 913.71 and 935.13 ± 252.34 vs 4553.75 ± 727.37, P < 0.01). Serum HMGB1 levels at 6 and 12 h for the Liuweiwuling tablet group were significantly lower than those of the control group (23.49 ± 3.89 vs 58.6 ± 3.65, 61.62 ± 13.07 vs 27.32 ± 5.97, P < 0.01). Furthermore, serum TNF-α and IL-1β levels at 12 h in the Liuweiwuling tablet group were also significantly lower than those of the control group (299.35 ± 50.61 vs 439.03 ± 63.59, 57.42 ± 12.98 vs 160.07 ± 49.87, P < 0.01). Centrilobular necrosis was evident in liver tissue of mice with acetaminophen-induced acute liver injury, but was almost abolished in the Liuweiwuling tablet group. The expression levels of PCNA and CyclinD1 were up-regulated in liver tissue in the Liuweiwuling tablet group (321.08 ± 32.87 vs 157.91 ± 21.52, 196.37 ± 25.39 vs 68.72 ± 11.27, P < 0.01); however, expression of p21 in liver tissue was down-regulated compared to that of the control group (40.26 ± 9.97 vs 138.24 ± 13.66, P < 0.01). Liuweiwuling tablets can attenuate acute liver injury by decreasing inflammatory cytokine (HMGB1, TNF-α and IL-1β) levels and promoting liver regeneration.

Intra-amniotic LPS modulates expression of antimicrobial peptides in the fetal sheep lung.

BackgroundDamage-associated molecular patterns (DAMPs) and antimicrobial peptides (AMPs) are components of pulmonary innate immunity and tissue repair. We hypothesized that DAMPs and AMPs would increase in response to fetal pulmonary inflammation caused by chorioamnionitis in a time-dependent manner.MethodsFetal sheep were exposed to intra-amniotic saline or LPS (10mg) between 5 hours and 15 days prior to preterm delivery at 125±2 days. Lung tissue mRNAs for pro-inflammatory cytokines; AMPs: myeloid antimicrobial peptide-29 (MAP29), dodecapeptide, sheep beta-defensin-1 (SBD1), sheep beta-defensin-2 (SBD2); DAMPs: IL-1α, lactoferrin, heat-shock protein-70 (HSP70), high-mobility group box protein-B1 (HMGB1), receptor for advanced glycation endproducts (RAGE) were measured by RT-qPCR. Immunohistochemistry of DAMPs and in situ hybridization of AMPs was performed.ResultsIL-1α, IL-1β, IL-6, IL-8, IL-10, MCP-1, and TNF-α mRNA increased after LPS exposure. MAP29, dodecapeptide, SBD1 and SBD2 mRNA were suppressed at 24 hours. MAP29 and dodecapeptide mRNA then increased at 8 days. Lactoferrin increased at 24 hours. There were no changes for HMGB1, HSP70 or RAGE. MAP29 and dodecapeptide localized to alveolar cells, increased 8 days after exposure to LPS.ConclusionAMPs are initially suppressed in the fetal lung by LPS-induced chorioamnionitis. The late induction of MAP29 and Dodecapeptide may be related to lung repair.