Prominent IL-12 production and tumor reduction in athymic nude mice after Toxoplasma gondii lysate antigen treatment.

Toxoplasma gondii is an intracellular protozoan parasite that causes a Th1 cellular immunity. Our previous study showed that T. gondii lysate antigen (TLA) treatment in S180 tumor-bearing mice resulted in tumor reduction by suppressing CD31 expression, a marker of angiogenesis. In the present study, to investigate tumor suppressive effect of TLA under the absence of T lymphocytes, athymic nude mice were compared with euthymic mice in the anti-tumorigenic effect triggered by TLA in CT26 tumors. According to the results, intratumorally injected TLA reduced tumor growth and TIMP-1 level, a metastatic marker, in both euthymic and athymic mice. TLA treatment led to a sharp increase in IL-12 expression in serum cytokine profiling of athymic mice, and increased MyD88 signals in macrophages derived from the bone marrow, implying the activation of innate immunity. The selective induction of IL-12 by TLA treatment had an anti-tumorigenic effect.

Discovery of a Novel Potent EGFR Inhibitor Against EGFR Activating Mutations and On-Target Resistance in NSCLC.

PURPOSE: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) serve as the standard first-line therapy for EGFR-mutated non-small cell lung cancer (NSCLC). Despite the sustained clinical benefits achieved through optimal EGFR-TKI treatments, including the third-generation EGFR-TKI osimertinib, resistance inevitably develops. Currently, there are no targeted therapeutic options available postprogression on osimertinib. Here, we assessed the preclinical efficacy of BI-4732, a novel fourth-generation EGFR-TKI, using patient-derived preclinical models reflecting various clinical scenarios. EXPERIMENTAL DESIGN: The antitumor activity of BI-4732 was evaluated using Ba/F3 cells and patient-derived cell/organoid/xenograft models with diverse EGFR mutations. Intracranial antitumor activity of BI-4732 was evaluated in a brain-metastasis mouse model. RESULTS: We demonstrated the remarkable antitumor efficacy of BI-4732 as a single agent in various patient-derived models with EGFR_C797S-mediated osimertinib resistance. Moreover, BI-4732 exhibited activity comparable to osimertinib in inhibiting EGFR-activating (E19del and L858R) and T790M mutations. In a combination treatment strategy with osimertinib, BI-4732 exhibited a synergistic effect at significantly lower concentrations than those used in monotherapy. Importantly, BI-4732 displayed potent antitumor activity in an intracranial model, with low efflux at the blood-brain barrier. CONCLUSIONS: Our findings highlight the potential of BI-4732, a selective EGFR-TKI with high blood-brain barrier penetration, targeting a broad range of EGFR mutations, including C797S, warranting clinical development.

Preclinical Study of a Biparatopic METxMET Antibody-Drug Conjugate, REGN5093-M114, Overcomes MET-driven Acquired Resistance to EGFR TKIs in EGFR-mutant NSCLC.

PURPOSE: MET amplification is a frequent mechanism of resistance to EGFR tyrosine kinase inhibitors (TKI) in patients with EGFR-mutated non-small cell lung cancer (NSCLC), and combined treatment with EGFR TKIs and MET TKIs has been explored as a strategy to overcome resistance. However, durable response is invariably limited by the emergence of acquired resistance. Here, we investigated the preclinical activity of REGN5093-M114, a novel antibody-drug conjugate targeting MET in MET-driven patient-derived models. EXPERIMENTAL DESIGN: Patient-derived organoids, patient-derived cells, or ATCC cell lines were used to investigate the in vitro/in vivo activity of REGN5093-M114. RESULTS: REGN5093-M114 exhibited significant antitumor efficacy compared with MET TKI or unconjugated METxMET biparatopic antibody (REGN5093). Regardless of MET gene copy number, MET-overexpressed TKI-naïve EGFR-mutant NSCLC cells responded to REGN5093-M114 treatment. Cell surface MET expression had the most predictive power in determining the efficacy of REGN5093-M114. REGN5093-M114 potently reduced tumor growth of EGFR-mutant NSCLC with PTEN loss or MET Y1230C mutation after progression on prior osimertinib and savolitinib treatment. CONCLUSIONS: Altogether, REGN5093-M114 is a promising candidate to overcome the challenges facing functional MET pathway blockade.

Primary Tumor Suppression and Systemic Immune Activation of Macrophages through the Sting Pathway in Metastatic Skin Tumor.

PURPOSE: Agonists of the stimulator of interferon genes (STING) play a key role in activating the STING pathway by promoting the production of cytokines. In this study, we investigated the antitumor effects and activation of the systemic immune response of treatment with DMXAA (5,6-dimethylxanthenone-4-acetic acid), a STING agonist, in EML4-ALK lung cancer and CT26 colon cancer. MATERIALS AND METHODS: The abscopal effects of DMXAA in the treatment of metastatic skin nodules were assessed. EML4-ALK lung cancer and CT26 colon cancer models were used to evaluate these effects after DMXAA treatment. To evaluate the expression of macrophages and T cells, we sacrificed the tumor-bearing mice after DMXAA treatment and obtained the formalin-fixed paraffin-embedded (FFPE) tissue and tumor cells. Immunohistochemistry and flow cytometry were performed to analyze the expression of each FFPE and tumor cell. RESULTS: We observed that highly infiltrating immune cells downstream of the STING pathway had increased levels of chemokines after DMXAA treatment. In addition, the levels of CD80 and CD86 in antigen-presenting cells were significantly increased after STING activation. Furthermore, innate immune activation altered the systemic T cell-mediated immune responses, induced proliferation of macrophages, inhibited tumor growth, and increased numbers of cytotoxic memory T cells. Tumor-specific lymphocytes also increased in number after treatment with DMXAA. CONCLUSION: The abscopal effect of DMXAA treatment on the skin strongly reduced the spread of EML4-ALK lung cancer and CT26 colon cancer through the STING pathway and induced the presentation of antigens.

SKI-G-801, an AXL kinase inhibitor, blocks metastasis through inducing anti-tumor immune responses and potentiates anti-PD-1 therapy in mouse cancer models.

OBJECTIVES: AXL-mediated activation of aberrant tyrosine kinase drives various oncogenic processes and facilitates an immunosuppressive microenvironment. We evaluated the anti-tumor and anti-metastatic activities of SKI-G-801, a small-molecule inhibitor of AXL, alone and in combination with anti-PD-1 therapy. METHODS: In vitro pAXL inhibition by SKI-G-801 was performed in both human and mouse cancer cell lines. Immunocompetent mouse models of tumor were established to measure anti-metastatic potential of SKI-G-801. Furthermore, SKI-G-801, anti-PD-1 or their combination was administered as an adjuvant or neoadjuvant in the 4T1 tumor model to assess their potential for clinical application. RESULTS: SKI-G-801 robustly inhibited pAXL expression in various cell lines. SKI-G-801 alone or in combination with anti-PD-1 potently inhibited metastasis in B16F10 melanoma, CT26 colon and 4T1 breast models. SKI-G-801 inhibited the growth of B16F10 and 4T1 tumor-bearing mice but not immune-deficient mice. An antibody depletion assay revealed that CD8+ T cells significantly contributed to SKI-G-801-mediated survival. Anti-PD-1 and combination group were observed the increased CD8+Ki67+ and effector T cells and M1 macrophage and decreased M2 macrophage, and granulocytic myeloid-derived suppressor cell (G-MDSC) compared to the control group. The neoadjuvant combination of SKI-G-801 and anti-PD-1 therapy achieved superior survival benefits by inducing more profound T-cell responses in the 4T1 syngeneic mouse model. CONCLUSION: SKI-G-801 significantly suppressed tumor metastasis and growth by enhancing anti-tumor immune responses. Our results suggest that SKI-G-801 has the potential to overcome anti-PD-1 therapy resistance and allow more patients to benefit from anti-PD-1 therapy.

Machine learning model for predicting malaria using clinical information.

BACKGROUND: Rapid diagnosing is crucial for controlling malaria. Various studies have aimed at developing machine learning models to diagnose malaria using blood smear images; however, this approach has many limitations. This study developed a machine learning model for malaria diagnosis using patient information. METHODS: To construct datasets, we extracted patient information from the PubMed abstracts from 1956 to 2019. We used two datasets: a solely parasitic disease dataset and total dataset by adding information about other diseases. We compared six machine learning models: support vector machine, random forest (RF), multilayered perceptron, AdaBoost, gradient boosting (GB), and CatBoost. In addition, a synthetic minority oversampling technique (SMOTE) was employed to address the data imbalance problem. RESULTS: Concerning the solely parasitic disease dataset, RF was found to be the best model regardless of using SMOTE. Concerning the total dataset, GB was found to be the best. However, after applying SMOTE, RF performed the best. Considering the imbalanced data, nationality was found to be the most important feature in malaria prediction. In case of the balanced data with SMOTE, the most important feature was symptom. CONCLUSIONS: The results demonstrated that machine learning techniques can be successfully applied to predict malaria using patient information.

Repotrectinib Exhibits Potent Antitumor Activity in Treatment-Naïve and Solvent-Front-Mutant ROS1-Rearranged Non-Small Cell Lung Cancer.

PURPOSE: Although first-line crizotinib treatment leads to clinical benefit in ROS1+ lung cancer, high prevalence of crizotinib-resistant ROS1-G2032R (ROS1G2032R) mutation and progression in the central nervous system (CNS) represents a therapeutic challenge. Here, we investigated the antitumor activity of repotrectinib, a novel next-generation ROS1/TRK/ALK-tyrosine kinase inhibitor (TKI) in ROS1+ patient-derived preclinical models. EXPERIMENTAL DESIGN: Antitumor activity of repotrectinib was evaluated in ROS1+ patient-derived preclinical models including treatment-naïve and ROS1G2032R models and was further demonstrated in patients enrolled in an on-going phase I/II clinical trial (NCT03093116). Intracranial antitumor activity of repotrectinib was evaluated in a brain-metastasis mouse model. RESULTS: Repotrectinib potently inhibited in vitro and in vivo tumor growth and ROS1 downstream signal in treatment-naïve YU1078 compared with clinically available crizotinib, ceritinib, and entrectinib. Despite comparable tumor regression between repotrectinib and lorlatinib in YU1078-derived xenograft model, repotrectinib markedly delayed the onset of tumor recurrence following drug withdrawal. Moreover, repotrectinib induced profound antitumor activity in the CNS with efficient blood-brain barrier penetrating properties. Notably, repotrectinib showed selective and potent in vitro and in vivo activity against ROS1G2032R. These findings were supported by systemic and intracranial activity of repotrectinib observed in patients enrolled in the on-going clinical trial. CONCLUSIONS: Repotrectinib is a novel next-generation ROS1-TKI with improved potency and selectivity against treatment-naïve and ROS1G2032R with efficient CNS penetration. Our findings suggest that repotrectinib can be effective both as first-line and after progression to prior ROS1-TKI.

Targeting YAP to overcome acquired resistance to ALK inhibitors in ALK-rearranged lung cancer.

Clinical benefit of ALK tyrosine kinase inhibitors (ALK-TKIs) in ALK-rearranged lung cancer has been limited by the inevitable development of acquired resistance, and bypass-molecular resistance mechanisms remain poorly understood. We investigated a novel therapeutic target through screening FDA-approved drugs in ALK-TKI-resistant models. Cerivastatin, the rate-limiting enzyme inhibitor of the mevalonate pathway, showed anti-cancer activity against ALK-TKI resistance in vitro/in vivo, accompanied by cytoplasmic retention and subsequent inactivation of transcriptional co-regulator YAP. The marked induction of YAP-targeted oncogenes (EGFR, AXL, CYR61, and TGFßR2) in resistant cells was abolished by cerivastatin. YAP silencing suppressed tumor growth in resistant cells, patient-derived xenografts, and EML4-ALK transgenic mice, whereas YAP overexpression decreased the responsiveness of parental cells to ALK inhibitor. In matched patient samples before/after ALK inhibitor treatment, nuclear accumulation of YAP was mainly detected in post-treatment samples. High expression of YAP in pretreatment samples was correlated with poor response to ALK-TKIs. Our findings highlight a crucial role of YAP in ALK-TKI resistance and provide a rationale for targeting YAP as a potential treatment option for ALK-rearranged patients with acquired resistance to ALK inhibitors.

Enhancer Remodeling and MicroRNA Alterations Are Associated with Acquired Resistance to ALK Inhibitors.

Anaplastic lymphoma kinase (ALK) inhibitors are highly effective in patients with ALK fusion-positive lung cancer, but acquired resistance invariably emerges. Identification of secondary mutations has received considerable attention, but most cases cannot be explained by genetic causes alone, raising the possibility of epigenetic mechanisms in acquired drug resistance. Here, we investigated the dynamic changes in the transcriptome and enhancer landscape during development of acquired resistance to ALK inhibitors. Histone H3 lysine 27 acetylation (H3K27ac) was profoundly altered during acquisition of resistance, and enhancer remodeling induced expression changes in both miRNAs and mRNAs. Decreased H3K27ac levels and reduced miR-34a expression associated with the activation of target genes such as AXL. Panobinostat, a pan-histone deacetylase inhibitor, altered the H3K27ac profile and activated tumor-suppressor miRNAs such as miR-449, another member of the miR-34 family, and synergistically induced antiproliferative effects with ALK inhibitors on resistant cells, xenografts, and EML4-ALK transgenic mice. Paired analysis of patient samples before and after treatment with ALK inhibitors revealed that repression of miR-34a or miR-449a and activation of AXL were mutually exclusive of secondary mutations in ALK. Our findings indicate that enhancer remodeling and altered expression of miRNAs play key roles in cancer drug resistance and suggest that strategies targeting epigenetic pathways represent a potentially effective method for overcoming acquired resistance to cancer therapy.Significance: Epigenetic deregulation drives acquired resistance to ALK inhibitors in ALK-positive lung cancer. Cancer Res; 78(12); 3350-62. ©2018 AACR.

Preventive Effects of Resveratrol-enriched Extract of Peanut Sprout on Bacteria- and Estradiol-induced Prostatitis in Mice.

The present study investigated the effect of peanut sprout extract (PSE) as a natural resveratrol supplement on chronic bacterial prostatitis (CBP) and estradiol- induced benign prostatic hyperplasia (BPH). PSE contained a high level of resveratrol (148.51 ± 3.05 µg/g), and was tested on the mouse models of CBP . (induced by Escherichia coli 292 infection) and BPH (induced by treatment with ß-estradiol and dihydrotestosterdne). PSE toxicity was assessed on the basis of changes in body weight, alanine aminotransferase activity (an indicator of hepatotoxicity), and expression of the kidney injury marker KIM-1. The effects of PSE on the histopathology of prostate tissue, the proportion of neutrophils, and immune cell profiles in the blood and spleen were examined. PSE administration did not result in any toxicity but reduced the bacterial burden and histopathological changes in the prostate. In addition, lymphocytes (CD4⁺, CD8⁺, and CD 19⁺) in the spleen were significantly increased after PSE administration in CBP mice, suggesting immune enhancement. PSE treatment of bone Snarrow-derived macrophages increased the expression of CD40, which is related to the pro-inflammatory function and host defense against pathogens. It is concluded that PSE would be a good supplement for the mitigation of prostate hyperplasia and prostatitis.

Development of a Multiplex PCR System for the Simultaneous Detection of the Shrimp Species Fenneropenaeus chinensis, Litopenaeus vannamei, and Penaeus monodon.

Due to mixed use of shrimp in foodstuffs, it is important to distinguish Fenneropenaeus chinensis from other shrimp species, such as Litopenaeus vannamei and Penaeus monodon. For this purpose, we have developed a multiplex PCR method to detect the simultaneous presence of all three species. Universal primers specific to the 16S ribosomal RNA subunit were used to sequence and ascertain the species in the samples. In this study, specific primers were designed to result in a unique PCR product size (143, 260, and 348 bp for L. vannamei, F. chinensis and P. monodon, respectively). In addition, duplex and triplex PCR protocols were developed to concurrently and rapidly detect these three shrimp species without nonspecific gene amplification. This multiplex PCR system is expected to be widely used to distinguish shrimp species that should be monitored to prevent their mixed use in food.

Immune adjuvant effect of a Toxoplasma gondii profilin-like protein in autologous whole-tumor-cell vaccination in mice.

Profilin-like protein in Toxoplasma gondii (TgPLP) is a Toll-like receptor (TLR) agonist. In this study, we investigated whether TgPLP has an adjuvant effect on immune function in autologous whole-tumor-cell vaccine (AWV) treatment. Mice vaccinated with AWV together with recombinant TgPLP protein had smaller CT26 tumors and increased survival. TgPLP treatment strongly increased the production of IL-12 through MyD88 signaling and several chemokines, including CCL5, CCL12, and XCL1, in bone marrow-derived macrophages (BMMs). In addition, TgPLP increased the phagocytosis of tumor cells by BMMs and promoted immune cell mobility on a tumor-matrigel scaffold. TgPLP triggered immune responses as demonstrated by increased expression of antigen presenting cell markers (MHC class I and II, B7.1, and B7.2) in BMMs and increased IL-12 and IFN-γ expression in mice. Mice vaccinated with AWV and TgPLP had more immune cells (CD4+ and CD8+ T cells, natural killer cells, and macrophages) in the spleen and higher total IgG and IgG2a concentrations in the blood than mice vaccinated with AWV alone. These findings suggest that TgPLP is a TLR-based vaccine adjuvant that enhances antitumor immune responses during vaccination with AWV.

In vitro inhibition of Toxoplasma gondii by the anti-malarial candidate, 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol.

An anti-malarial candidate, 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251), was studied to characterize its potential as a novel anti-Toxoplasma gondii drug. In the present study, IC50 and LC50 of N-251 on host cells and T. gondii were compared to those of artemisinin and sulfadiazine. The IC50 on Huh-7 cells was 10.19µg/ml, 67.69µg/ml and 310.17µg/ml for N-251, artemisinin, and sulfadiazine, respectively. The LC50 for anti-T. gondii effect was shown to be 1.11µg/ml, 5.79µg/ml, and 5.45µg/ml for N-251, artemisinin and sulfadiazine, respectively. N-251 concentration causing complete parasiticidal effect with minimal cytotoxicity on host cells was determined to be 5µg/ml. Additionally, the anti-T. gondii effect of N-251 was confirmed by ultrastructural changes, loss of organelles, degenerated morphology and the increase of amylopectin as detected by transmission electron microscope (TEM). Accordingly, the present study suggests that the anti-malarial synthetic endoperoxide, N-251, is an emerging drug candidate more effective than artemisinin and sulfadiazine.