Mouse-human co-clinical trials demonstrate superior anti-tumour effects of buparlisib (BKM120) and cetuximab combination in squamous cell carcinoma of head and neck.

BACKGROUND: Recurrent and/or metastatic squamous cell carcinoma of head and neck (R/M SCCHN) is a common cancer with high recurrence and mortality. Current treatments have low response rates (RRs). METHODS: Fifty-three patients with R/M SCCHN received continuous oral buparlisib. In parallel, patient-derived xenografts (PDXs) were established in mice to evaluate resistance mechanisms and efficacy of buparlisib/cetuximab combination. Baseline and on-treatment tumour genomes and transcriptomes were sequenced. Based on the integrated clinical and PDX data, 11 patients with progression under buparlisib monotherapy were treated with a combination of buparlisib and cetuximab. RESULTS: For buparlisib monotherapy, disease control rate (DCR) was 49%, RR was 3% and median progression-free survival (PFS) and overall survival (OS) were 63 and 143 days, respectively. For combination therapy, DCR was 91%, RR was 18% and median PFS and OS were 111 and 206 days, respectively. Four PDX models were originated from patients enrolled in the current clinical trial. While buparlisib alone did not inhibit tumour growth, combination therapy achieved tumour inhibition in three of seven PDXs. Genes associated with apoptosis and cell-cycle arrest were expressed at higher levels with combination treatment than with buparlisib or cetuximab alone. CONCLUSIONS: The buparlisib/cetuximab combination has significant promise as a treatment strategy for R/M SCCHN. CLINICAL TRIAL REGISTRATION: NCT01527877.

Establishment and characterization of patient-derived xenografts as paraclinical models for head and neck cancer.

BACKGROUND: We investigated whether head and neck squamous cell carcinoma (HNSCC) patient-derived xenografts (PDXs) reaffirm patient responses to anti-cancer therapeutics. METHODS: Tumors from HNSCC patients were transplanted into immunodeficient mice and propagated via subsequent implantation. We evaluated established PDXs by histology, genomic profiling, and in vivo anti-cancer efficacy testing to confirm them as the authentic in vivo platform. RESULTS: From 62 HNSCCs, 15 (24%) PDXs were established. The primary cancer types were tongue (8), oropharynx (3), hypopharynx (1), ethmoid sinus cancer (1), supraglottic cancer (1), and parotid gland (1); six PDXs (40%) were established from biopsy specimens from advanced HNSCC. PDXs mostly retained donor characteristics and remained stable across passages. PIK3CA (H1047R), HRAS (G12D), and TP53 mutations (H193R, I195T, R248W, R273H, E298X) and EGFR, CCND1, MYC, and PIK3CA amplifications were identified. Using the acquisition method, biopsy showed a significantly higher engraftment rate when compared with that of surgical resection (100% [6/6] vs. 16.1% [9/56], P < 0.001). Specimens obtained from metastatic sites showed a significantly higher engraftment rate than did those from primary sites (100% [9/9] vs. 11.3% [6/53], P < 0.001). Three PDX models from HPV-positive tumors were established, as compared to 12 from HPV-negative (15.8% [3/19] and 27.9% [12/43] respectively, P = 0.311), suggesting that HPV positivity tends to show a low engraftment rate. Drug responses in PDX recapitulated the clinical responses of the matching patients with pan-HER inhibitors and pan-PI3K inhibitor. CONCLUSIONS: Genetically and clinically annotated HNSCC PDXs could be useful preclinical tools for evaluating biomarkers, therapeutic targets, and new drug discovery.

Phylogenetic relationships of 3 Korean Neodiplostomum species (Digenea: Neodiplostomidae) based on partial CO1 gene.

The phylogenetic relationships of the 3 Neodiplostomum spp. (Digenea: Neodiplostomidae) occurring in Korea (N. seoulense, N. leei, and N. boryongense) were analyzed using the partial mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. The adult flukes were recovered from Sprague-Dawley rats (N. seoulense) and newborn chicks (N. leei and N. boryongense) experimentally infected with the neodiplostomula from the grass snake, Rhabdophis tigrinus tigrinus. The genomic DNA was amplified using specific primers, and the sequence of CO1 was obtained. According to the results, the pairwise similarity was 96.1% between N. boryongense and N. seoulense, but was 95.0% between N. boryongense and N. leei and 94.2% between N. leei and N. seoulense. The results demonstrated a closer phylogenetic relationship between N. seoulense and N. boryongense. This high relationship of N. seoulense and N. boryongense may be related to their similar morphologic features including the limited distribution of vitellaria and the presence of a genital cone. N. leei is distinct on the other hand with an extensive distribution of vitellaria and the absence of a genital cone.

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.

Exploring the Expression and Function of T Cell Surface Markers Identified through Cellular Indexing of Transcriptomes and Epitopes by Sequencing.

PURPOSE: By utilizing both protein and mRNA expression patterns, we can identify more detailed and diverse immune cells, providing insights into understanding the complex immune landscape in cancer ecosystems. MATERIALS AND METHODS: This study was performed by obtaining publicly available Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) data of peripheral blood mononuclear cells (PBMCs) from the Gene Expression Omnibus database. A total of 94674 total cells were analyzed, of which 32412 were T cells. There were 228 protein features and 16262 mRNA features in the data. The Seurat package was used for quality control and preprocessing, principal component analysis was performed, and Uniform Manifold Approximation and Projection was used to visualize the clusters. Protein and mRNA levels in the CITE-seq were analyzed. RESULTS: We observed that a subset of T cells in the clusters generated at the protein level divided better. By identifying mRNA markers that were highly correlated with the CD4 and CD8 proteins and cross-validating CD26 and CD99 markers using flow cytometry, we found that CD4+ and CD8+ T cells were better discriminated in PBMCs. Weighted Nearest Neighbor clustering results identified a previously unobserved T cell subset. CONCLUSION: In this study, we used CITE-seq data to confirm that protein expression patterns could be used to identify cells more precisely. These findings will improve our understanding of the heterogeneity of immune cells in the future and provide valuable insights into the complexity of the immune response in health and disease.

Successful expansion and cryopreservation of human natural killer cell line NK-92 for clinical manufacturing.

Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Despite this promise, NK cell manufacturing workflows remain largely manual, open, and disconnected, and depend on feeders, as well as outdated unit operations or processes, often utilizing research-grade reagents. Successful scale-up of NK cells critically depends on the availability and performance of nutrient-rich expansion media and cryopreservation conditions that are conducive to high cell viability and recovery post-thaw. In this paper we used Cytiva hardware and media to expand the NK92 cell line in a model process that is suitable for GMP and clinical manufacturing of NK cells. We tested a range of cryopreservation factors including cooling rate, a range of DMSO-containing and DMSO-free cryoprotectants, ice nucleation, and cell density. Higher post-thaw recovery was seen in cryobags over cryovials cooled in identical conditions, and cooling rates of 1°C/min or 2°C/min optimal for cryopreservation in DMSO-containing and DMSO-free cryoprotectants respectively. Higher cell densities of 5x107 cells/ml gave higher post-thaw viability than those cryopreserved at either 1x106 or 5x106 cells/ml. This enabled us to automate, close and connect unit operations within the workflow while demonstrating superior expansion and cryopreservation of NK92 cells. Cellular outputs and performance were conducive to clinical dosing regimens, serving as a proof-of-concept for future clinical and commercial manufacturing.

Exploration of Immune-Modulatory Effects of Amivantamab in Combination with Pembrolizumab in Lung and Head and Neck Squamous Cell Carcinoma.

Immune checkpoint inhibitors are effective first-line therapy for solid cancers. However, low response rate and acquired resistance over time has led to the need for additional therapeutic options. Here, we evaluated synergistic antitumor efficacy of EGFR × MET targeting bispecific antibody, amivantamab with PD-L1 immunotherapy, pembrolizumab in head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma tumor-bearing humanized patient-derived xenograft (PDX) models. We demonstrated that pembrolizumab or amivantamab alone was ineffective and that combination treatment induced a significant reduction of tumor growth in both models (P < 0.0001 and P < 0.01, respectively). It appeared that combination of amivantamab and pembrolizumab significantly enhanced infiltration of granzyme B-producing CD8 T cells was in the TME of HNSCC PDX (P < 0.01) and enhanced neoantigen-associated central memory CD8 T cells in circulating immune cells. Analysis of single-cell RNA transcriptomics suggested that the tumor cells dramatically upregulated EGFR and MET in response to PD-L1 immunotherapy, potentially creating a metabolic state fit for tumor persistence in the tumor microenvironment (TME) and rendered pembrolizumab ineffective. We demonstrated that EGFRHIGHMETHIGH subcluster displayed an increased expression of genes implicated in production of lactate [SLC16A3 and lactate dehydrogenase A (LDHA)] compared to the EGFRLOWMETLOW cluster. Accumulation of lactate in the TME has been associated with immunosuppression by hindering the infiltration of tumor killing CD8 T and NK cells. This study proved that amivantamab reduced glycolytic markers in the EGFRHIGHMETHIGH subcluster including SLC16A3 and LDHA and highlighted remodeling of the TME by combination treatment, providing rationale for additional therapy of amivantamab with PD-1 immunotherapy. SIGNIFICANCE: Amivantamab in synergy with pembrolizumab effectively eradicated EGFRHIGHMETHIGH tumor subcluster in the tumor microenvironment of head and neck squamous cell carcinoma and overcame resistance against anti-PD-1 immunotherapy.

Exploring aryl hydrocarbon receptor expression and distribution in the tumor microenvironment, with a focus on immune cells, in various solid cancer types.

Introduction: Aryl hydrocarbon receptor (AhR) is a transcription factor that performs various functions upon ligand activation. Several studies have explored the role of AhR expression in tumor progression and immune surveillance. Nevertheless, investigations on the distribution of AhR expression, specifically in cancer or immune cells in the tumor microenvironment (TME), remain limited. Examining the AhR expression and distribution in the TME is crucial for gaining insights into the mechanism of action of AhR-targeting anticancer agents and their potential as biomarkers. Methods: Here, we used multiplexed immunohistochemistry (mIHC) and image cytometry to investigate the AhR expression and distribution in 513 patient samples, of which 292 are patients with one of five solid cancer types. Additionally, we analyzed the nuclear and cytosolic distribution of AhR expression. Results: Our findings reveal that AhR expression was primarily localized in cancer cells, followed by stromal T cells and macrophages. Furthermore, we observed a positive correlation between the nuclear and cytosolic expression of AhR, indicating that the expression of AhR as a biomarker is independent of its localization. Interestingly, the expression patterns of AhR were categorized into three clusters based on the cancer type, with high AhR expression levels being found in regulatory T cells (Tregs) in non-small cell lung cancer (NSCLC). Discussion: These findings are anticipated to serve as pivotal evidence for the design of clinical trials and the analysis of the anticancer mechanisms of AhR-targeting therapies.

CD81 and CD82 expressing tumor-infiltrating lymphocytes in the NSCLC tumor microenvironment play a crucial role in T-cell activation and cytokine production.

Introduction: To understand the immune system within the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC), it is crucial to elucidate the characteristics of molecules associated with T cell activation. Methods: We conducted an in-depth analysis using single-cell RNA sequencing data obtained from tissue samples of 19 NSCLC patients. T cells were classified based on the Tumor Proportion Score (TPS) within the tumor region, and molecular markers associated with activation and exhaustion were analyzed in T cells from high TPS areas. Results: Notably, tetraspanins CD81 and CD82, belonging to the tetraspanin protein family, were found to be expressed in activated T cells, particularly in cytotoxic T cells. These tetraspanins showed strong correlations with activation and exhaustion markers. In vitro experiments confirmed increased expression of CD81 and CD82 in IL-2-stimulated T cells. T cells were categorized into CD81highCD82high and CD81lowCD82low groups based on their expression levels, with CD81highCD82high T cells exhibiting elevated activation markers such as CD25 and CD69 compared to CD81lowCD82low T cells. This trend was consistent across CD3+, CD8+, and CD4+ T cell subsets. Moreover, CD81highCD82high T cells, when stimulated with anti-CD3, demonstrated enhanced secretion of cytokines such as IFN-γ, TNF-α, and IL-2, along with an increase in the proportion of memory T cells. Bulk RNA sequencing results after sorting CD81highCD82high and CD81lowCD82low T cells consistently supported the roles of CD81 and CD82. Experiments with overexpressed CD81 and CD82 showed increased cytotoxicity against target cells. Discussion: These findings highlight the multifaceted roles of CD81 and CD82 in T cell activation, cytokine production, memory subset accumulation, and target cell cytolysis. Therefore, these findings suggest the potential of CD81 and CD82 as promising candidates for co-stimulatory molecules in immune therapeutic strategies for cancer treatment within the intricate TME.

Species identification of medically important trematodes in aquatic food samples using PCR-RFLP targeting 18S rRNA.

The authors describe a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method targeting the 18S rRNA gene for the species detection of medically important trematode infecting fish and oysters, and suggest that this PCR-RFLP method based on a specific Tre-18 primer and the restriction enzymes, Acc1, Ava2, Msp1, and Hinf1, is useful for the detection of parasites in aquatic food.

STAT6 expression and IL-13 production in association with goblet cell hyperplasia and worm expulsion of Gymnophalloides seoi from C57BL/6 mice.

In intestinal helminth infections, Th2 immune respones are generally associated with mucin secretion for worm expulsion from the host intestine. In particular, IL-4 and IL-13 are the important cytokines related with intestinal mucus production via STAT6 signalling in nematode infections. However, this perspective has never been studied in Gymnophalloides seoi infection. The present study aimed to observe the STAT6 signalling and cytokine responses in C57BL/6 mice, a mouse strain resistant to infection with this trematode. The results showed that worm expulsion occurred actively during days 1-2 post-infection (PI), when goblet cells began to proliferate in the small intestine. The STAT6 gene expression in the mouse spleen became remarkable from day 2 PI. Moreover, G. seoi infection induced a significant increase of IL-13 from day 4 PI in the spleen of infected mice. Our results suggested that goblet cell hyperplasia and worm expulsion in G. seoi-infected mice should be induced by STAT6 signalling, in which IL-13 may be involved as a dominant triggering cytokine.

Artificial intelligence-based non-small cell lung cancer transcriptome RNA-sequence analysis technology selection guide.

The incidence and mortality rates of lung cancer are high worldwide, where non-small cell lung cancer (NSCLC) accounts for more than 85% of lung cancer cases. Recent non-small cell lung cancer research has been focused on analyzing patient prognosis after surgery and identifying mechanisms in connection with clinical cohort and ribonucleic acid (RNA) sequencing data, including single-cell ribonucleic acid (scRNA) sequencing data. This paper investigates statistical techniques and artificial intelligence (AI) based non-small cell lung cancer transcriptome data analysis methods divided into target and analysis technology groups. The methodologies of transcriptome data were schematically categorized so researchers can easily match analysis methods according to their goals. The most widely known and frequently utilized transcriptome analysis goal is to find essential biomarkers and classify carcinomas and cluster NSCLC subtypes. Transcriptome analysis methods are divided into three major categories: Statistical analysis, machine learning, and deep learning. Specific models and ensemble techniques typically used in NSCLC analysis are summarized in this paper, with the intent to lay a foundation for advanced research by converging and linking the various analysis methods available.

Polo-like Kinase 4: A Multifaceted Marker Linking Tumor Aggressiveness and Unfavorable Prognosis, and Insights into Therapeutic Strategies.

(1) Background: This study investigated whether polo-like kinase 4 (PLK4) is a suitable therapeutic target or biomarker for lung adenocarcinoma (LUAD). (2) Methods: We acquired LUAD data from The Cancer Genome Atlas (TCGA) database through the UCSC Xena data portal. Gene expression, clinical, survival, and mutation data from multiple samples were analyzed. Gene enrichment analysis, unsupervised clustering of PLK4-related pathways, and differential gene expression analyses were performed. Additionally, correlations, t-tests, survival analyses, and statistical analyses were performed. (3) Results: PLK4 expression was higher in LUAD tissues than in normal tissues and was associated with poor prognosis for both overall and progression-free survival in LUAD. PLK4 was highly correlated with cell-proliferation-related pathways using Gene Ontology (GO) biological process terms. PLK4 expression and pathways that were highly correlated with PLK4 expression levels were upregulated in patients with LUAD with the TP53 mutation. (4) Conclusions: PLK4 expression affects the survival of patients with LUAD and is a potential therapeutic target for LUAD with TP53 mutations.

Depressed neuronal growth associated protein (GAP)-43 expression in the small intestines of mice experimentally infected with Neodiplostomum seoulense.

Neodiplostomum seoulense (Digenea: Neodiplostomidae) is an intestinal trematode that can cause severe mucosal pathology in the small intestines of mice and even mortality of the infected mice within 28 days after infection. We observed neuronal growth associated protein-43 (GAP-43) expression in the myenteric plexus of the small intestinal wall of N. seoulense-infected mice until day 35 post-infection (PI). BALB/c mice were infected with 200 or 500 N. seoulense metacercariae isolated from naturally infected snakes and were killed every 7 days for immunohistochemical demonstration of GAP-43 in the small intestines. N. seoulense-infected mice showed remarkable dilatation of intestinal loops compared with control mice through days 7-28 PI. Conversely, GAP-43 expression in the mucosal myenteric plexus was markedly (P<0.05) reduced in the small intestines of N. seoulense-infected mice during days 7-28 PI and was slightly normalized at day 35 PI. From this study, it is evident that neuronal damage occurs in the intestinal mucosa of N. seoulense-infected mice. However, the correlation between intestinal pathology, including the loop dilatation, and depressed GAP-43 expression remains to be elucidated.

Effect of temperature on embryonation of Ascaris suum eggs in an environmental chamber.

The influence of temperature on the development and embryonation of Ascaris suum eggs was studied using coarse sand medium in an environmental chamber with 50% humidity. The time required for development and embryonation of eggs was examined under 3 different temperature conditions, 5°C, 25°C, and 35°C. A. suum eggs did not develop over 1 month at the temperature of 5°C. However, other temperature conditions, 25°C and 35°C, induced egg development to the 8-cell-stage at days 5-6 after incubation. All eggs examined developed to the 8-cell stage at day 6 after incubation in the sand medium at 25°C. The higher temperature, 35°C, slightly accelerated the A. suum egg development compared to 25°C, and the development to the 8-cell stage occurred within day 5 after incubation. The formation of larvae in A. suum eggs at temperatures of 35° and 25°C appeared at days 17 and 19 after incubation, respectively. These findings show that 35° condition shortens the time for the development of A. suum eggs to the 8-cell-stage in comparison to 25°C, and suggest the possibility of accelerated transmission of this parasite, resulting from global warming and ecosystem changes.

Molecular landscape of osimertinib resistance in patients and patient-derived preclinical models.

INTRODUCTION: Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) that is approved for the use of EGFR-mutant non-small cell lung cancer (NSCLC) patients. In this study, we investigated the acquired resistance mechanisms in NSCLC patients and patient-derived preclinical models. METHODS: Formalin-fixed paraffin-embedded tumor samples and plasma samples from 55 NSCLC patients who were treated with osimertinib were collected at baseline and at progressive disease (PD). Next-generation sequencing was performed in tumor and plasma samples using a 600-gene hybrid capture panel designed by AstraZeneca. Osimertinib-resistant cell lines and patient-derived xenografts and cells were generated and whole exome sequencing and RNA sequencing were performed. In vitro experiments were performed to functionally study the acquired mutations identified. RESULTS: A total of 55 patients and a total of 149 samples (57 tumor samples and 92 plasma samples) were analyzed, and among them 36 patients had matched pre- and post-treatment samples. EGFR C797S (14%) mutation was the most frequent EGFR-dependent mechanism identified in all available progression samples, followed by EGFR G824D (6%), V726M (3%), and V843I (3%). Matched pre- and post-treatment sample analysis revealed in-depth acquired mechanisms of resistance. EGFR C797S was still most frequent (11%) among EGFR-dependent mechanism, while among EGFR-independent mechanisms, PIK3CA, ALK, BRAF, EP300, KRAS, and RAF1 mutations were detected. Among Osimertinib-resistant cell lines and patient-derived models, we noted acquired mutations which were potentially targetable such as NRAS p.Q61K, in which resistance could be overcome with combination of osimertinib and trametinib. A patient-derived xenograft established from osimertinib-resistant patient revealed KRAS p.G12D mutation which could be overcome with combination of osimertinib, trametinib, and buparlisib. CONCLUSION: In this study, we explored the genetic profiles of osimertinib-resistant NSCLC patient samples using targeted deep sequencing. In vitro and in vivo models harboring osimertinib resistance revealed potential novel treatment strategies after osimertinib failure.

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.

YH29407 with anti-PD-1 ameliorates anti-tumor effects via increased T cell functionality and antigen presenting machinery in the tumor microenvironment.

Among cancer cells, indoleamine 2, 3-dioxygenase1 (IDO1) activity has been implicated in improving the proliferation and growth of cancer cells and suppressing immune cell activity. IDO1 is also responsible for the catabolism of tryptophan to kynurenine. Depletion of tryptophan and an increase in kynurenine exert important immunosuppressive functions by activating regulatory T cells and suppressing CD8+ T and natural killer (NK) cells. In this study, we compared the anti-tumor effects of YH29407, the best-in-class IDO1 inhibitor with improved pharmacodynamics and pharmacokinetics, with first and second-generation IDO1 inhibitors (epacadostat and BMS-986205, respectively). YH29407 treatment alone and anti-PD-1 (aPD-1) combination treatment induced significant tumor suppression compared with competing drugs. In particular, combination treatment showed the best anti-tumor effects, with most tumors reduced and complete responses. Our observations suggest that improved anti-tumor effects were caused by an increase in T cell infiltration and activity after YH29407 treatment. Notably, an immune depletion assay confirmed that YH29407 is closely related to CD8+ T cells. RNA-seq results showed that treatment with YH29407 increased the expression of genes involved in T cell function and antigen presentation in tumors expressing ZAP70, LCK, NFATC2, B2M, and MYD88 genes. Our results suggest that an IDO1 inhibitor, YH29407, has enhanced PK/PD compared to previous IDO1 inhibitors by causing a change in the population of CD8+ T cells including infiltrating T cells into the tumor. Ultimately, YH29407 overcame the limitations of the competing drugs and displayed potential as an immunotherapy strategy in combination with aPD-1.

Incorporation of SKI-G-801, a Novel AXL Inhibitor, With Anti-PD-1 Plus Chemotherapy Improves Anti-Tumor Activity and Survival by Enhancing T Cell Immunity.

A recently developed treatment strategy for lung cancer that combines immune checkpoint inhibitors with chemotherapy has been applied as a standard treatment for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), and it has improved the outcomes of chemotherapy. Maintenance treatment with anti-PD-1 antibody (aPD-1) enhances the effect of immunochemical combination therapy and improves therapeutic efficacy, which contributes toward a significant improvement in patient survival rates. The AXL receptor tyrosine kinase (AXL), which is expressed in tumor cells, plays an essential role in the resistance of cancers to chemotherapy and immunotherapy, and stimulates signaling associated with epithelial-mesenchymal transition (EMT) in metastatic cancer. AXL is thus an attractive target for controlling resistance to anti-tumor therapies. In this study, we examined the effect of AXL inhibitors on immune activation and tumor growth in TC1 and C3PQ mouse tumor models, in the context of clinical immunotherapy/chemotherapy and maintenance treatment, using an aPD-1 with/without pemetrexed. To determine the optimal timing for administration of SKI-G-801, an AXL inhibitor, we investigated its anti-tumor effects based on inclusion at the immunochemotherapy and maintenance therapy stages. We also performed flow cytometry-based immune profiling of myeloid cells and lymphoid cells at different points in the treatment schedule, to investigate the immune activation and anti-tumor effects of the AXL inhibitor. The addition of SKI-G-801 to the immune checkpoint inhibitor and chemotherapy stage, as well as the maintenance therapy stage, produced the best anti-tumor results, and significant tumor growth inhibition was observed in both the TC1 and C3PQ models. Both models also exhibited increased proportion of effector memory helper T cells and increased expression of CD86+ macrophages. Especially, regulatory T cells were significantly reduced in the TC1 tumor model and there was an increase in central memory cytotoxic T cell infiltration and an increased proportion of macrophages with high CD80 expression in the C3PQ tumor model. These results suggest increased infiltration of T cells, consistent with previous studies using AXL inhibitors. It is expected that the results from this study will serve as a stepping stone for clinical research to improve the existing standard of care.

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.