Synergistic effects of Smac mimetic APG-1387 with anti-PD-1 antibody are attributed to increased CD3 + NK1.1 + cell recruitment secondary to induction of cytokines from tumor cells.

BACKGROUND: Immune checkpoint inhibitors are approved for the treatment of various tumors, but the response rate is not satisfactory in certain malignancies. Inhibitor of apoptosis proteins (IAP) ubiquitin-E3 ligase activity is involved in the regulation of immune responses. APG-1387 is a novel second mitochondria-derived activator of caspase (Smac) mimetic IAP inhibitor. The aim of this study was to explore the synergistic effect of APG-1387 when combined with anti-PD-1 antibody in a preclinical setting. METHODS: We utilized syngeneic mouse models of ovarian cancer (ID8), colon cancer (MC38), malignant melanoma (B16), and liver cancer (Hepa1-6) to assess the combination effect of APG-1387 and anti-PD-1 antibody, including immune-related factors, tumor growth, and survival. MSD V-PLEX validated assays were used to measure in vitro and in vivo cytokine release. RESULTS: In ID8 ovarian cancer and MC38 colon cancer models, APG-1387 and anti-PD1 antibody had synergistic antitumor effects. In the MC38 model, the combination of APG-1387 and anti-PD-1 antibody significantly inhibited tumor growth (P < 0.0001) and increased the survival rate of tumor-bearing animals (P < 0.001). Moreover, we found that APG-1387 upregulated tumor-infiltrating CD3 + NK1.1 + cells by nearly 2-fold, by promoting tumor cell secretion of IL-12. Blocking IL-12 secretion abrogated the synergistic effects of APG-1387 and anti-PD-1 antibody in both MC38 and ID8 models. CONCLUSIONS: APG-1387 has the potential to turn "cold tumors" into hot ones by recruiting more CD3 + NK1.1 + cells into certain tumors. Based on these and other data, the safety and therapeutic effect of this combination will be investigated in a phase 1/2 trial in patients with advanced solid tumors or hematologic malignancies (NCT03386526).

Sea Buckthorn Polyphenols Alleviate High-Fat-Diet-Induced Metabolic Disorders in Mice via Reprograming Hepatic Lipid Homeostasis Owing to Directly Targeting Fatty Acid Synthase.

Our previous studies found that Sea Buckthorn polyphenols (SBP) extract inhibits fatty acid synthase (FAS) in vitro. Thus, we continued to explore possible effects and underlying mechanisms of SBP on complicated metabolic disorders in long-term high-fat-diet (HFD)-fed mice. To reveal that, an integrated approach was developed in this study. Targeted quantitative lipidomics with a total of 904 unique lipids mapping contributes to profiling the comprehensive features of disarranged hepatic lipid homeostasis and discovering a set of newfound lipid-based biomarkers to predict the occurrence and indicate the progression of metabolic disorders beyond current indicators. On the other hand, technologies of intermolecular interactions characterization, especially surface plasmon resonance (SPR) assay, contribute to recognizing targeted bioactive constituents present in SBP. Our findings highlight hepatic lipid homeostasis maintenance and constituent-FAS enzyme interactions, to provide new insights that SBP as a functional food alleviates HFD-induced metabolic disorders in mice via reprograming hepatic lipid homeostasis caused by targeting FAS, owing to four polyphenols directly interacting with FAS and cinaroside binding to FAS with good affinity.

The atypical ß-blocker S-oxprenolol reduces cachexia and improves survival in a rat cancer cachexia model.

BACKGROUND: Beta-blockers and selected stereoisomers of beta-blockers, like bisoprolol and S-pindolol (ACM-001), have been shown to be effective in preclinical cancer cachexia models. Here, we tested the efficacy of stereoisomers of oxprenolol in two preclinical models of cancer cachexia-the Yoshida AH-130 rat model and the Lewis lung carcinoma (LLC) mouse model. METHODS AND RESULTS: In the Yoshida AH130 hepatoma rat cancer cachexia model and compared with placebo, 50 mg/kg/d S-oxprenolol (HR: 0.49, 95% CI: 0.28-0.85, P = 0.012) was superior to 50 mg/kg/d R-oxprenolol (HR: 0.83, 95% CI 0.38-1.45, P = 0.51) in reducing mortality (= reaching ethical endpoints). Combination of the three doses (12.5, 25 and 50 mg/kg/d) that had a significant effect on body weight loss in the S-oxprenolol groups vs the same combination of the R-oxprenolol groups lead to a significantly improved survival of S-oxprenolol vs R-oxprenolol (HR: 1.61, 95% CI: 1.08-2.39, P = 0.0185). Interestingly, there is a clear dose dependency in S-oxprenolol-treated (5, 12.5, 25 and 50 mg/kg/d) groups, which was not observed in groups treated with R-oxprenolol. A dose-dependent attenuation of weight and lean mass loss by S-oxprenolol was seen in the Yoshida rat model, whereas R-oxprenolol had only had a significant effect on fat mass. S-oxprenolol also non-significantly reduced weight loss in the LLC model and also improved muscle function (grip strength 428 ± 25 and 539 ± 37 g/100 g body weight for placebo and S-oxprenolol, respectively). However, there was only a minor effect on quality of life indicators food intake and spontaneous activity in the Yoshida model (25 mg/kg/S-oxprenolol: 11.9 ± 2.5 g vs placebo: 4.9 ± 0.8 g, P = 0.013 and also vs 25 mg/kg/d R-oxprenolol: 7.5 ± 2.6 g, P = 0.025). Both enantiomers had no effects on cardiac dimensions and function at the doses used in this study. Western blotting of proteins involved in the anabolic/catabolic homoeostasis suggest that anabolic signalling is persevered (IGF-1 receptor, Akt) and catabolic signalling is inhibited (FXBO-10, TRAF-6) by S-pindolol, but not he R-enantiomer. Expression of glucose transporters Glut1 and Glut 4 was similar in all groups, as was AMPK. CONCLUSIONS: S-oxprenolol is superior to R-oxprenolol in cancer cachexia animal models and shows promise for a human application in cancer cachexia.

An update on inhibitors targeting RNA-dependent RNA polymerase for COVID-19 treatment: Promises and challenges.

The highly transmissible variants of SARS-CoV-2, the causative pathogen of the COVID-19 pandemic, bring new waves of infection worldwide. Identification of effective therapeutic drugs to combat the COVID-19 pandemic is an urgent global need. RNA-dependent RNA polymerase (RdRp), an essential enzyme for viral RNA replication, is the most promising target for antiviral drug research since it has no counterpart in human cells and shows the highest conservation across coronaviruses. This review summarizes recent progress in studies of RdRp inhibitors, focusing on interactions between these inhibitors and the enzyme complex, based on structural analysis, and their effectiveness. In addition, we propose new possible strategies to address the shortcomings of current inhibitors, which may guide the development of novel efficient inhibitors to combat COVID-19.

Comparison of Immune Microenvironment Between Colon and Liver Metastatic Tissue in Colon Cancer Patients with Liver Metastasis.

BACKGROUND: Liver metastasis is an indicator of unfavorable responses to immunotherapy in colorectal cancer patients. However, the difference of immune microenvironment between primary tumors and liver metastases has not been well understood. PATIENTS AND METHODS: Fifty-four colon cancer with liver metastasis patients who received resection of both primary and metastasis lesions have been analyzed. The immune score is based on the density of infiltrating immune cells (CD3+ cell, CD8+ cell, CD11b+ cell, CD11c+ cell, and CD33+ cell) in the center and margin of the tumor. The expression of immune markers between the primary tumor and hepatic metastases was analyzed using Wilcoxon's signed rank test. RESULTS: All the five markers had higher expression in tumor margins than center tumor in both primary tumor and hepatic metastases lesions. The expression of CD11c and CD11b had no difference between metastatic lesions and primary tumor. In tumor margins, except CD11b, all the other 4 markers expressed significantly higher in hepatic metastases than in primary tumor. Intra-tumor, CD3 had higher expression in primary tumor than in hepatic metastases, while CD33 had higher expression in hepatic metastases than in primary tumor. CD8+ CD3+ cells of the total CD8+ cell population in primary tumor was significantly higher than in hepatic metastases (36.42% vs. 24.88%, p = 0.0069). CONCLUSIONS: The immune microenvironment between primary tumor and hepatic metastasis is different. More immunosuppressing cells in liver may partially explain why immunotherapy in colon cancer is less effective with liver metastatic disease.

Bcl-2/Bcl-xl inhibitor APG-1252-M1 is a promising therapeutic strategy for gastric carcinoma.

Gastric carcinoma is the third major cause of cancer-related death in China. Bcl-2 and other BH3 family proteins are critically important in the process of apoptosis pathway, which may be a promising target. APG-1252-M1 specifically connects to Bcl-2 and Bcl-xl. The antitumor effect of APG-1252-M1 in six gastric cancer cells was identified by the Cell Counting Kit-8 assay. The expression level of proapoptotic proteins was evaluated by Western blot. Meanwhile, the cell cycle and apoptosis distributions were analyzed by flow cytometry and JC-1. Xenograft models were used to investigate the roles of APG-1252-M1 in suppressing the growth of tumors and enhancing the chemotherapy antitumor effect. The antitumor effect of APG-1252-M1 was time- and dose-dependent and acted by initiating apoptosis. The change of cell cycle distribution was not discovered in gastric cancer cells treated with APG-1252-M1. APG-1252-M1 also exhibited synergy with chemotherapy in vivo. The combined group inhibited xenograft tumor growth more obviously than the other groups. Moreover, Ki-67 was remarkably decreased in the combination group compared to other groups. In conclusion, APG-1252-M1 had a strong antitumor effect by inducing apoptosis and was synergistic with chemotherapy.

A Novel BCL-2 Inhibitor APG-2575 Exerts Synthetic Lethality With BTK or MDM2-p53 Inhibitor in Diffuse Large B-Cell Lymphoma.

Despite therapeutic advances, the effective treatment for relapsed or refractory diffuse large B-cell lymphoma (DLBCL) remains a major clinical challenge. Evasion of apoptosis through upregulating antiapoptotic B-cell lymphoma-2 (BCL-2) family members and p53 inactivation, and abnormal activation of B-cell receptor signaling pathway are two important pathogenic factors for DLBCL. In this study, our aim is to explore a rational combination of BCL-2 inhibitor plus Brutons tyrosine kinase (BTK) blockade or p53 activation for treating DLBCL with the above characteristics. We demonstrated that a novel BCL-2 selective inhibitor APG-2575 effectively suppressed DLBCL with BCL-2 high expression via activating the mitochondrial apoptosis pathway. BTK inhibitor ibrutinib combined with BCL-2 inhibitors showed synergistic antitumor effect in DLBCL with mean expression of BCL-2 and myeloid cell leukemia-1 (MCL-1) through upregulating the expression level of BIM and modulating MCL-1 and p-Akt expression. For p53 wild-type DLBCL with high expression of BCL-2, APG-2575 showed strong synergic effect with mouse double minute 2 (MDM2)p53 inhibitor APG-115 that can achieve potent antitumor effect and markedly prolong survival in animal models. Collectively, our data provide an effective and precise therapeutic strategy through rational combination of BCL-2 and BTK or MDM2p53 inhibitors for DLBCL, which deserves further clinical investigation.

More attention should be paid to adult gastric cancer patients younger than 35 years old: extremely poor prognosis was found.

Purpose: Several studies have reported controversial results about prognosis of gastric cancer in young age patients. The difference may partially result from variable definitions of young age. The aim of this study was to find out the relation between age and prognosis of gastric cancer patients, and to analyze the clinicopathological features and prognostic factors in young gastric cancer patients. Methods: Data queried for this analysis included GC patients from the Surveillance, Epidemiology, and End Results Program database from 1973 to 2014. Gastric cancer patients (N=79,505) diagnosed with an age≥18 were included. By combining patients with similar prognosis, we figured out 3 cutoff values of age, 35 years old, 65 years old and 75 years old. We divided patients into 4 groups: young age patients: 18-34 years; middle-age patients: 35-64 years; elderly patients: 65-74 years; extremely elderly patients: >74 years. GC patients from Sun Yat-sen University Cancer Center (SYSUCC) were used as external validation data. Results: The clinicopathological features of young age gastric cancer patients included: poor-differentiated, diffuse type of cancer, and advanced stage at diagnosis. The median survival of patients <35 years old was significantly lower than middle-age patients (35-64 years) and elderly patients (65-74 years) (12 months vs. 15 and 16 months, respectively, both p <0.001). Location of tumor, ethnicity, tumor size, surgery and TNM stage were independent prognostic factors by multivariate analysis in young patients. The poor prognosis for young patients remained valid in the SYSUCC database. Conclusions: Our results demonstrated that gastric cancer patients <35 years old had an extremely poor prognosis. Early detection of gastric cancer is of paramount importance in young age people.

A novel small molecule inhibitor of MDM2-p53 (APG-115) enhances radiosensitivity of gastric adenocarcinoma.

BACKGROUND: Gastric cancer is the leading cause of cancer related death worldwide. Radiation alone or combined with chemotherapy plays important role in locally advanced and metastatic gastric adenocarcinoma. MDM2-p53 interaction and downstream signaling affect cellular response to DNA damage which leads to cell cycle arrest and apoptosis. Therefore, restoring p53 function by inhibiting its interaction with MDM2 is a promising therapeutic strategy for cancer. APG-115 is a novel small molecule inhibitor which blocks the interaction of MDM2 and p53. In this study, we investigated that the radiosensitivity of APG-115 in gastric adenocarcinoma in vitro and in vivo. METHODS: The role of APG-115 in six gastric cancer cells viability in vitro was determined by CCK-8 assay. The expression level of MDM2, p21, PUMA and BAX in AGS and MKN45 cell lines was measured via real-time PCR (RT-PCR). The function of treatment groups on cell cycle and cell apoptosis were detected through Flow Cytometry assay. Clonogenic assays were used to measure the radiosensitivity of APG-115 in p53 wild type gastric cancer cell lines. Western blot was conducted to detect the protein expressions of mdm2-p53 signal pathway. Xenograft models in nude mice were established to explore the radiosensitivity role of APG-115 in gastric cancer cells in vivo. RESULTS: We found that radiosensitization by APG-115 occurred in p53 wild-type gastric cancer cells. Increasing apoptosis and cell cycle arrest was observed after administration of APG-115 and radiation. Radiosensitivity of APG-115 was mainly dependent on MDM2-p53 signal pathway. In vivo, APG-115 combined with radiation decreased xenograft tumor growth much more significantly than either single treatment. Moreover, the number of proliferating cells (Ki-67) significantly decreased in combination group compared with single treatment group. CONCLUSIONS: In summary, we found that combination of MDM2-p53 inhibitor (APG-115) and radiotherapy can enhance antitumor effect both in vitro and in vivo. This is the first report on radiosensitivity of APG-115 which shed light on clinical trial of the combination therapy of radiation with APG-115 in gastric adenocarcinoma.

Correction to: Novel smac mimetic APG-1387 elicits ovarian cancer cell killing through TNF-alpha, Ripoptosome and autophagy mediated cell death pathway.

In the publication of this article [1], there was an error in Figs. 2, 3 and 6.

A novel SMAC mimetic APG-1387 exhibits dual antitumor effect on HBV-positive hepatocellular carcinoma with high expression of cIAP2 by inducing apoptosis and enhancing innate anti-tumor immunity.

Check point inhibitor anti-PD1 antibody produced some efficacy in Hepatocellular Carcinoma (HCC) patients previously treated with sorafenib. Unfortunately, HCC patients with hepatitis B virus (HBV) infection did not respond as well as uninfected patients. Previously, Second mitochondria-derived activator of caspases (SMAC) mimetics-the antagonist for inhibitor of apoptosis proteins (IAPs) can rapidly reduce serum hepatitis B virus DNA in animal model. APG-1387 is a novel SMAC-mimetic, small molecule inhibitor targeting inhibitor of apoptosis proteins (IAPs). In our study, firstly, we found that HCC patients with copy number alteration of cIAP1, cIAP2, and XIAP had a dismal prognosis. Then, we discovered that APG-1387 alone could induce apoptosis of PLC/PRF/5 which was HBV positive both in-vitro and in-vivo. Furthermore, we found that APG-1387 significantly up-regulated the expression of calreticulin and HLA-DR in PLC/PRF/5 via activating non-classic NF-κB pathway. Also, compared to vehicle group, APG-1387 increased NK cell counts by 5 folds in PLC/PRF/5 xenograft model. In-vitro, APG-1387 positively regulated T cells by reducing Treg differentiation and down-regulating PD1 expression in CD4 T cell. Moreover, APG-1387 had no impact on memory T cells. Consequently, our results suggest that APG1387 could be a good candidate to combine with anti-PD1 antibody treatment to overcome low responds of check point inhibitors in HBV positive HCC.

Novel smac mimetic APG-1387 elicits ovarian cancer cell killing through TNF-alpha, Ripoptosome and autophagy mediated cell death pathway.

BACKGROUND: Ovarian cancer is a deadly disease. Inhibitors of apoptosis proteins (IAPs) are key regulators of apoptosis and are frequently dysregulated in ovarian cancer. Overexpression of IAPs proteins has been correlated with tumorigenesis, treatment resistance and poor prognosis. Reinstalling functional cell death machinery by pharmacological inhibition of IAPs proteins may represent an attractive therapeutic strategy for treatment of ovarian cancer. METHODS: CCK-8 and colony formation assay was performed to examine cytotoxic activity. Apoptosis was analyzed by fluorescence microscopy, flow cytometry and TUNEL assay. Elisa assay was used to determine TNFα protein. Caspase activity assay was used for caspase activation evaluation. Immunoprecipitation and siRNA interference were carried out for functional analysis. Western blotting analysis were carried out to test protein expression. Ovarian cancer cell xenograft nude mice model was used for in vivo efficacy evaluation. RESULTS: APG-1387 demonstrated potent inhibitory effect on ovarian cancer cell growth and clonogenic cell survival. APG-1387 induced RIP1- and TNFα-dependent apoptotic cell death in ovarian cancer through downregulation of IAPs proteins and induction of caspase-8/FADD/RIP1 complex, which drives caspase-8 activation. NF-κB signaling pathway was activated upon APG-1387 treatment and RIP1 contributed to NF-κB activation. APG-1387 induced cytoprotective autophagy while triggering apoptosis in ovarian cancer cells and inhibition of autophagy enhanced APG-1387-induced apoptotic cell death. APG-1387 exhibited potent antitumor activity against established human ovarian cancer xenografts. CONCLUSIONS: Our results demonstrate that APG-1387 targets IAPs proteins to potently elicit apoptotic cell death in vitro and in vivo, and provide mechanistic and applicable rationale for future clinical evaluation of APG-1387 in ovarian cancer.

Minicircle-oriP-miR-31 as a Novel EBNA1-Specific miRNA Therapy Approach for Nasopharyngeal Carcinoma.

MicroRNAs (miRNAs) are important post-transcriptional regulators that control cancer development and progression. However, the application of miRNA therapy in cancer has been hampered by a lack of an efficient and targeted delivery system. In our previous studies, an oriP promoter-based minicircle system successfully mediated targeted foreign gene expression in EBNA1-positive nasopharyngeal carcinoma (NPC). However, it remains to be evaluated whether this system can be applied for tumor miRNA therapy. miR-31-5p, a tumor suppressive miRNA involved in the tumorigenesis of EBV-positive NPC, was selected as the therapeutic miRNA to be transferred. In this work, we constructed a novel EBNA1-specific miRNA expression system, minicircle-oriP-miR-31. The results indicated that mc-oriP-miR-31 mediated selective miR-31-5p expression in EBNA1-positive NPC cells. Both the proliferation and migration of EBNA1-positive NPC cell lines were inhibited by mc-oriP-miR-31 treatment in vitro. Furthermore, mc-oriP-miR-31 treatment inhibited xenograft growth and lung metastasis in vivo. We also identified WDR5 as a novel miR-31-5p target. Knockdown of WDR5 inhibited NPC cell proliferation and migration and was associated with downregulation of Notch1. Reintroduction of WDR5 partially abrogated the suppressive effects induced by miR-31-5p. In conclusion, we demonstrate for the first time that targeted expression of miR-31-5p using a nonviral minicircle vector can serve as a novel approach for tumor miRNA therapy. Moreover, WDR5 may be a promising therapeutic target for NPC treatment.

IL-17 induces antitumor immunity by promoting beneficial neutrophil recruitment and activation in esophageal squamous cell carcinoma.

Interleukin (IL)-17 has been reported to play a controversial role in tumor immunity. Our previous studies showed that infiltration of IL-17-producing cells in esophageal squamous cell carcinoma (ESCC) induced tumor protective immunity by recruiting CD8+T lymphocytes, natural killer (NK) cells, and B lymphocytes into the tumor microenvironment. However, the mechanism of IL-17 regulation of tumor-associated neutrophils remains elusive in ESCC. In this study, we therefore evaluated the accumulation of myeloperoxidase (MPO)+ neutrophils and its association with IL-17-producing cells within ESCC tumor nests. We also investigated the effects of IL-17 on the recruitment and antitumor activity of neutrophils. MPO+ neutrophil infiltration was found to predict a favorable prognosis in ESCC patients and was positively correlated with IL-17+ cell density. IL-17 stimulated ESCC tumor cells to release more of the CXC chemokines CXCL2 and CXCL3, which are involved in neutrophil migration. Furthermore, IL-17 potentiates the direct killing capability of neutrophils by enhancing the production of cytotoxic molecules, including reactive oxygen species (ROS), MPO, TNF-related apoptosis-inducing ligand (TRAIL), and IFN-γ. Experiments in mice suggested that IL-17 alone might not affect tumor progression in the tumor-bearing host, but IL-17 can inhibit tumor growth by promoting beneficial neutrophil infiltration and activation at tumor sites. As emerging evidence indicates that targeting tumor-associated neutrophils is a strategy for antitumor therapy, our findings reveal a positive contribution of IL-17 to the modulation of neutrophil-mediated antitumor immunity in ESCC. This study provides further understanding of the mechanisms that selectively regulate functional activities of neutrophils, which may be critical for developing new tumor immunotherapy.

HIV Screening and Awareness Survey for Pregnant Women in a Remote Area in Xinjiang Uyghur Autonomous Region of China.

OBJECTIVE: The number of people infected with human immunodeficiency virus (HIV) in China has increased in recent years. HIV screening for pregnant women was performed in a remote area in Xinjiang, as an effort to promote universal HIV screening in pregnant women and to help prevention of mother-to-child transmission. METHODS: Pregnant women in Burqin and Jeminay Counties in Xinjiang were offered free voluntary HIV screening. Local mid-level medical workers were trained to use Determine® HIV-1/2 kit for HIV screening. All the tested pregnant women signed a consent form, received HIV education material, and participated in an HIV knowledge survey. RESULTS: All the 890 pregnant women receiving HIV test had negative result. Among these women, 67.6% were Kazakh and 40.9% were farmers. Survey of HIV knowledge showed that these women's awareness about mother-to-child transmission was limited. The levels of HIV knowledge were related with ethnic background, age, education and profession of the pregnant women. CONCLUSION: The results suggested that HIV infection had not become a significant problem among the pregnant women in this remote area of Xinjiang, but continued efforts to improve the awareness of HIV, especially the knowledge about mother-to-child transmission of HIV, in pregnant women were needed.