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).

Survival benefit of surgery with postoperative radiotherapy in locally advanced cervical adenocarcinoma: a population-based analysis.

BACKGROUND: The incidence of cervical adenocarcinoma (AC) has experienced a considerable increase in recent decades. Despite this, our understanding of the optimal management of locally advanced cervical AC remains limited. The present study sought to compare the clinical outcomes of radical hysterectomy with postoperative radiotherapy (PORT) and primary radiotherapy (RT) in patients with locally advanced cervical AC using the Surveillance, Epidemiology, and End Results (SEER) database. METHODS: The data were extracted from the SEER database utilizing the SEER ∗ STAT software (version 8.4.0.1). The study included patients diagnosed with locally advanced cervical AC between 2004 and 2017 with adequate information available for analysis. Patients were assigned to either the Surgery + PORT or Primary RT group based on treatment modality, and their clinical characteristics were compared. Propensity score matching (PSM) was utilized to adjust for differences in baseline characteristics between groups. The primary endpoints of the study were overall survival (OS) and cancer-specific survival (CSS). RESULTS: Of the 1363 patients who met the inclusion criteria, 302 (22.16%) underwent Surgery + PORT, while 1061 patients received Primary RT. The two groups differed significantly in terms of age, year of diagnosis, tumor size, grade, stage, T/N stage, and chemotherapy. PSM was performed to balance the baseline characteristics between the two groups, resulting in 594 patients being analyzed. After PSM, the Surgery + PORT group exhibited significantly improved survival rates. The 5-year OS rates were 69.7% (95% CI: 63.3%-76.9%) for the Surgery + PORT group and 60.9% (95% CI: 56.0%-66.3%) for the group receiving Primary RT (p = 0.002). The 5-year CSS rates for the two groups were 70.7% (95% CI: 64.3%-77.8%) and 66.2% (95% CI: 61.3%-71.5%), respectively (p = 0.049). Multivariate analysis revealed that Surgery + PORT was an independent favorable prognostic factor for OS (HR = 0.60, p = 0.001) and CSS (HR = 0.69, p = 0.022). Although the combined approach of surgery and PORT resulted in a favorable impact on OS in patients aged 65 years or older (HR = 0.57, p = 0.048), it did not result in a statistically significant improvement in CSS in the same age group (HR = 0.56, p = 0.087). Similarly, the combined treatment did not yield a statistically significant increase in either OS (HR = 0.78, p = 0.344) or CSS (HR = 0.89, p = 0.668) in patients with tumors larger than 60 mm. CONCLUSION: The present study demonstrated that Surgery + PORT was associated with improved OS and CSS in patients with locally advanced cervical AC when compared to Primary RT. As such, Surgery + PORT may be a preferable therapeutic option for carefully selected patients with cervical AC. These findings offer valuable insight into the management of locally advanced cervical AC and may assist in personalized treatment decisions.

Prognostic impact of the combination of HIF­1α and GLUT1 in patients with oesophageal squamous cell carcinoma.

Oesophageal squamous cell carcinoma (ESCC) is a common type of carcinoma. Hypoxia is associated with chemo- and radio-resistance, which may lead to a poor prognosis. Hypoxia-inducible factor-1α (HIF-1α) is the main transcriptional regulator of the cellular response to low oxygen levels. Moreover, it can trigger the expression of critical genes, including glucose transporter protein type 1 (GLUT1). The aim of the present study was to evaluate the roles of HIF-1α and GLUT1 in ESCC and their usefulness as prognostic markers. HIF-1α and GLUT1 were measured in four ESCC cell lines, namely Eca109, KYSE150, TE-1 and TE-10, by western blotting following culture under normoxic and hypoxic conditions. In addition, xenograft tumors were established in mice using normoxic and hypoxic Eca109 cells and the chemosensitivity of the xenografts to 5-fluorouracil (5-FU) was evaluated. Furthermore, HIF-1α and GLUT1 were analysed by immunochemistry in the tumor tissues of patients with ESCC and the associations of their expression levels with clinicopathological parameters were investigated. The results revealed that HIF-1α and GLUT1 protein expression was weak in all four cell lines under a normoxic atmosphere but increased following culture in a hypoxic environment. In vivo, 5-FU inhibited tumor growth more strongly in normoxic Eca109 ×enografts than hypoxic Eca109 ×enografts. Higher levels of apoptosis were also detected in the normoxic Eca109 ×enografts via western blotting and TUNEL analysis. In patients with ESCC, HIF-1α expression was associated with advanced ESCC while GLUT1 expression was associated with the sex of the patients. Multivariate analysis demonstrated that HIF-1α and GLUT1 were negatively associated with progression-free survival (PFS) and overall survival (OS). Additionally, a combination of HIF-1α and GLUT1 expression was a predictor of RFS and OS in patients with ESCC without lymph node metastasis but not those with lymph node metastasis. The study demonstrated that HIF-1α and GLUT1 were strongly expressed in vitro and in xenograft models when cells were exposed to hypoxia. The simultaneous high expression of HIF-1α and GLUT1 was associated with poorer survival, and may play an important role in ESCC chemoresistance and the prognosis of ESCC.

Circular RNA circ_0004488 Increases Cervical Cancer Paclitaxel Resistance via the miR-136/MEX3C Signaling Pathway.

Circular RNAs have been proven to play a pivotal role in cervical cancer development, progression, and treatment resistance. However, it is unclear how these RNAs influence chemoresistance in cervical cancer, particularly cancer stem cell (CSC)-like properties. In this study, we found that circRNA circ_0004488 was highly expressed in CSC-enriched subsets of cervical cancer cell lines. The expression of circ_0004488 was upregulated in cervical cancer cells that were resistant to paclitaxel. When circ_0004488 expression was high, the prognosis was poor. Specifically, we discovered that knocking down circ_0004488 greatly decreased the development of cervical cancer cells in vivo by decreasing cell proliferation, invasion, and sphere formation. By blocking cir_0004488, cervical cancer cells become more sensitive to paclitaxel. In cervical cancer cells, circ_0004488 acted as a microRNA-136 (miR-136) sponge, increasing the expression of MEX3C (a direct target gene of miR-136) using dual-luciferase reporter assays. Moreover, MEX3C downregulation significantly reduced cell proliferation, invasion, sphere formation, and paclitaxel resistance. In conclusion, circ_0004488 was shown to induce CSC-like features and paclitaxel resistance through the miR-136/MEX3C axis. Therefore, circ_0004488 might be a good therapeutic target for treating cervical cancer.

Distant organ metastasis patterns and prognosis of neuroendocrine cervical carcinoma: a population-based retrospective study.

Background: Neuroendocrine carcinoma of the cervix (NECC) is a rare pathological form of cervical cancer. The prognosis of NECC with distant organ metastases is unclear. In our study, the patterns and prognosis of distant organ metastasis of NECC were investigated. Methods: Data were obtained from the surveillance epidemiology and end results (SEER) database from 2000 to 2018. Cox regression, Kaplan-Meier and log-rank analyses were conducted. Results: NECC was prone to single and multi-site metastases. The median overall survival (OS) was greatly decreased in patients with distant metastasis (P < 0.0001). Other characteristics such as age ≥60 years, poorer grade, higher T stage, those without surgery, no radiotherapy, and no chemotherapy were predictors of poor prognosis. Conclusions: Metastasis is an independent prognostic factor for patients with NECC. Surgery, radiotherapy, and chemotherapy give an overall survival advantage for patients with distant organ metastases.

Oncogenic circular RNA circ_0007534 contributes to paclitaxel resistance in endometrial cancer by sponging miR-625 and promoting ZEB2 expression.

Circular RNAs (circRNAs) and epithelial to mesenchymal transition (EMT) have been implicated in the development of human cancer and paclitaxel resistance. CircRNA circ_0007534 has been described as a key oncogenic circular RNA that is upregulated in a variety of cancer tissues. However, whether circ_0007534 causes EMT and paclitaxel resistance in endometrial cancer is still unknown. In this work, we revealed that circ_0007534 levels were significantly higher in endometrial cancer tissues, and that high circ_0007534 expression was associated with poor differentiation, advanced tumor stage, cancer invasion, cancer metastasis, and poor prognosis in endometrial cancer patients. Overexpression of circ_0007534 boosted endometrial cancer cell proliferation, invasion, EMT, and paclitaxel resistance. Knockdown of circ_0007534 restored paclitaxel sensitivity and reversed EMT in endometrial cancer cells. We also showed that circ_0007534 enhanced endometrial cancer aggressiveness, progression, and paclitaxel resistance by sponging microRNA-625 (miR-625) and subsequently increasing the expression of the miR-625 target gene ZEB2. Our cell functional studies demonstrated that inhibiting miR-625 or increasing ZEB2 mimicked the effects of circ_0007534 overexpression. Consequently, our data show that circ_0007534 plays a crucial role in EMT and paclitaxel resistance through miR-625/ZEB2 signaling. Targeting the circ_0007534/miR-625/ZEB2 pathway might be an effective strategy for overcoming paclitaxel resistance in endometrial cancer.

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.

Gefitinib induces non-small cell lung cancer H1650 cell apoptosis through downregulating tumor necrosis factor-related apoptosis-inducing ligand expression levels.

Non-small cell lung cancer (NSCLC) presents severe threats to the lives of patients. Gefitinib is one of the first-line drugs available for the treatment of NSCLC in the clinical setting. The present study investigated the effects of gefitinib on NSCLC H1650 cell viability and apoptosis via MTT assays and flow cytometry. Western blot analysis was employed to detect tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression levels in H1650 cells. In the present study, H1650 cells were treated with TRAIL siRNA or an empty plasmid vector control, followed by gefitinib treatment to investigate apoptosis. Gefitinib treatment markedly inhibited H1650 cell viability, induced apoptosis and reduced TRAIL expression levels. TRAIL interference enhanced H1650 cell apoptosis induced by gefitinib. TRAIL overexpression suppressed gefitinib-induced H1650 cell apoptosis. In addition, gefitinib induced NSCLC H1650 cell apoptosis by downregulating TRAIL expression levels.

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 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.

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.

APG-1252-12A induces mitochondria-dependent apoptosis through inhibiting the antiapoptotic proteins Bcl-2/Bcl-xl in HL-60 cells.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Despite improved remission rates, current treatment regimens for AML are often associated with a very poor prognosis and adverse effects, necessitating more effective and safer agents. B-cell leukemia/lymphoma 2 (Bcl-2) family proteins regulate apoptotic pathway that can be targeted with small molecule inhibitors. APG-1252-12A is a Bcl-2 homology (BH)-3 mimetic that specifically binds to Bcl-2 and Bcl-xl, which has shown efficacy in some Bcl-2 dependent hematological cancers. In this study, we investigated whether APG-1252-12A inhibits the growth of five leukemia cell lines in a concentration- or time-dependent manner by MTS assay. Following treatment of AML cell line HL-60 with this compound, cell apoptosis was detected using flow cytometry and nuclear condensation was observed after Hoechst 33258 dye. Immunoblotting for cytochrome c, cleaved caspase-3 and PARP-1 cleavage was used to demonstrate the mechanism of inducing mitochondria-dependent apoptosis by APG-1252-12A. Our findings showed that this new compound inhibited cell proliferation in five leukemia cell lines and induced apoptotic death. There was a link between the level of Bcl-2 protein and IC50. APG-1252-12A targeted mitochondria and induced caspase-dependent apoptosis by inducing the HL-60 cell cytochrome c released, PARP cleavage and caspase activation. These data suggested that APG-1252-12A is a candidate drug for the in vivo analysis and clinical evaluation in AML.

Restoration of p53 using the novel MDM2-p53 antagonist APG115 suppresses dedifferentiated papillary thyroid cancer cells.

Dedifferentiated papillary thyroid cancer (DePTC) is characterized by aggressive growth, recurrence, distant metastasis, and resistance to radioactive iodine (RAI) therapy. DePTC is also accompanied by poor prognosis and high early-mortality. Nevertheless, most DePTC cells show intact p53 downstream functionality. In cells with wild-type p53, the murine double minute2 (MDM2) protein interacts with p53 and abrogates its activity. Inhibition of the MDM2-p53 interaction restores p53 activity and leads to cell cycle arrest and apoptosis. Restoring p53 function by inhibiting its interaction with p53 suppressors such as MDM2 is thus a promising therapeutic strategy for the treatment of DePTC. The novel MDM2-p53 interaction antagonist APG115 is an analogue of SAR405838, and is being tested in a phase I clinical trial. In this study, we evaluated the efficacy of APG115 as a single-agent to treat DePTC. APG115 diminished the viability of p53 wild-type DePTC cells and induced cell cycle arrest and apoptosis. In a human xenograft mouse model, APG115 elicited robust tumor regression and cell apoptosis. These data demonstrate that further research is warranted to determine whether APG115 can be used to effectively treat DePTC patients.