RORγt agonist synergizes with CTLA-4 antibody to inhibit tumor growth through inhibition of Treg cells via TGF-ß signaling in cancer.

To date, the overall response rate to checkpoint blockade remains unsatisfactory, partially due to the limited understanding of the tumor immune microenvironment. The retinoic acid-related orphan receptor γt (RORγt) is the key transcription factor of T helper cell 17 (Th17) cells and plays an essential role in tumor immunity. In this study, we used JG-1, a potent and selective small-molecule RORγt agonist to evaluate the therapeutic potential and mechanism of action of targeting RORγt in tumor immunity. JG-1 promotes Th17 cells differentiation and inhibition of regulatory T (Treg) cells differentiation. JG-1 demonstrates robust tumor growth inhibition in multiple syngeneic models and shows a synergic effect with the Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) antibody. In tumors, JG-1 not only promotes Th17 cells differentiation and increases C-C Motif Chemokine Receptor 6 (CCR6)- Chemokine (C-C motif) ligand 20 (CCL20) expression, but also inhibits both the expression of transforming growth factor-ß1 (TGF-ß1) and the differentiation and infiltration of Treg cells. In summary, JG-1 is a lead compound showing a potent activity in vitro and robust tumor growth inhibition in vivo with synergetic effects with anti-CTLA-4.

Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

Conventional type 1 dendritic cells (cDC1) are the essential antigen-presenting DC subset in antitumor immunity. Suppressing B-cell lymphoma 9 and B-cell lymphoma 9-like (BCL9/BCL9L) inhibits tumor growth and boosts immune responses against cancer. However, whether oncogenic BCL9/BCL9L impairs antigen presentation in tumors is still not completely understood. Here, we show that targeting BCL9/BCL9L enhanced antigen presentation by stimulating cDC1 activation and infiltration into tumor. Pharmacological inhibition of BCL9/BCL9L with a novel inhibitor hsBCL9z96 or Bcl9/Bcl9l knockout mice markedly delayed tumor growth and promoted antitumor CD8+ T cell responses. Mechanistically, targeting BCL9/BCL9L promoted antigen presentation in tumors. This is due to the increase of cDC1 activation and tumor infiltration by the XCL1-XCR1 axis. Importantly, using single-cell transcriptomics analysis, we found that Bcl9/Bcl9l deficient cDC1 were superior to wild-type (WT) cDC1 at activation and antigen presentation via NF-κB/IRF1 signaling. Together, we demonstrate that targeting BCL9/BCL9L plays a crucial role in cDC1-modulated antigen presentation of tumor-derived antigens, as well as CD8+ T cell activation and tumor infiltration. Targeting BCL9/BCL9L to regulate cDC1 function and directly orchestrate a positive feedback loop necessary for optimal antitumor immunity could serve as a potential strategy to counter immune suppression and enhance cancer immunotherapy.

RORγt agonist enhances anti-PD-1 therapy by promoting monocyte-derived dendritic cells through CXCL10 in cancers.

BACKGROUND: The overall response rate to checkpoint blockade remains unsatisfactory, partially due to the immune-suppressive tumor microenvironment. A retinoic acid-related orphan receptor γt (RORγt) agonist (LYC-55716) is currently used in clinical trials combined with anti-PD-1, but how the Th17 cell transcription factor RORγt enhances antitumor immunity of PD-1 in the tumor microenvironment remains elusive. METHODS: The expression of mRNA was analyzed using qPCR assays. Flow cytometry was used to sort and profile cells. Cell migration was analyzed using Transwell assays. Biacore was used to determine the binding affinity to the RORγt protein. The RORγt GAL4 cell-based reporter gene assay was used to measure activity in the RORγt driven luciferase reporter gene expression. RESULTS: We designed a potent and selective small-molecule RORγt agonist (8-074) that shows robust antitumor efficacy in syngeneic tumor models and improves the efficacy of anti­PD­1 in a murine lung cancer model. RORγt agonist treatment increased intratumoral CD8+ T cells, which were correlated with CXCL10 and monocyte-derived dendritic cells (MoDCs). In addition, the RORγt agonist promoted Type 17 T cell migration by upregulating CCL20 and CCR6 expression, and Type 17 T cell tumor infiltration. CCL20 induces MoDCs migration, and CXCL10 derived from MoDCs promotes CD8+ T cell migration. CONCLUSION: Our results revealed that the RORγt agonist improved the efficacy of anti-PD-1. The RORγt agonist increased the migration of MoDCs, which increased the local levels of CXCL10, thus promoting CD8+ T cell tumor infiltration. Our findings provide the mechanistic insights implicating the RORγt agonist in immunotherapy and offer a strategy for targeting the RORγt agonist to improve PD-1 antibody efficacy in cancers.

Targeting Wnt Signaling in the Tumor Immune Microenvironment to Enhancing EpCAM CAR T-Cell therapy.

Colorectal cancer (CRC) patients are still lacking viable treatments. Chimeric antigen receptor (CAR) T cells have shown promise in hematologic malignancies, but their efficacy in solid tumors has been limited due to the immunosuppressive tumor microenvironment. We found that cancer antigen- EpCAM expression increased in the metastatic stage compared with the primary stage in cancers and the activation of Wnt and TGFß pathways was positively correlated with EpCAM expression in multiple cancers, including colorectal cancer. We constructed CAR T cells targeting EpCAM that successfully showed selective cytotoxicity in highly EpCAM-expressing cancer cell lines. The combination of EpCAM CAR-T with the Wnt inhibitor-hsBCL9CT-24 displayed synergetic effect against EpCAM-positive colon cells in vitro and also in vivo. A mechanistic study showed that hsBCL9CT-24 treatment could modulate the tumor environment and improve infiltration of T cells, while possibly promoting the effector T cells at the early stages and postponing the exhaustion of CAR T cells at advanced stages. Overall, these results demonstrated that the combination of EpCAM CAR T-cell therapy with the Wnt inhibitor can overcome the limitations of CAR T cells in treating solid tumors.

BCL9 regulates CD226 and CD96 checkpoints in CD8+ T cells to improve PD-1 response in cancer.

To date, the overall response rate of PD-1 blockade remains unsatisfactory, partially due to limited understanding of tumor immune microenvironment (TIME). B-cell lymphoma 9 (BCL9), a key transcription co-activator of the Wnt pathway, is highly expressed in cancers. By genetic depletion and pharmacological inhibition of BCL9 in tumors, we found that BCL9 suppression reduced tumor growth, promoted CD8+ T cell tumor infiltration, and enhanced response to anti-PD-1 treatment in mouse colon cancer models. To determine the underlying mechanism of BCL9's role in TIME regulation, single-cell RNA-seq was applied to reveal cellular landscape and transcription differences in the tumor immune microenvironment upon BCL9 inhibition. CD155-CD226 and CD155-CD96 checkpoints play key roles in cancer cell/CD8+ T cell interaction. BCL9 suppression induces phosphorylation of VAV1 in CD8+ T cells and increases GLI1 and PATCH expression to promote CD155 expression in cancer cells. In The Cancer Genome Atlas database analysis, we found that BCL9 expression is positively associated with CD155 and negatively associated with CD226 expression. BCL9 is also linked to adenomatous polyposis coli (APC) mutation involved in patient survival following anti-PD-1 treatment. This study points to cellular diversity within the tumor immune microenvironment affected by BCL9 inhibition and provides new insights into the role of BCL9 in regulating CD226 and CD96 checkpoints.

The Effect of Rotigotine Extended-Release Microspheres Alone or With Celecoxib on the Inflammatory Pain.

Clinical trials of rotigotine extended-release microspheres (RTGT-MS), which provides a sustained release of rotigotine for near 2 weeks in vivo, have been conducted in the treatment of Parkinson's disease (PD). This study was to investigate the analgesic effect of RTGT-MS, and to know whether RTGT-MS have synergistic interaction with non-steroidal anti-inflammatory drug, celecoxib. The inflammatory pain model of rats was prepared by carrageenan-induced paw edema. The thermal and mechanical stimuli were applied and the hindpaw withdrawal latency (HWL) response was evaluated. Treatment with RTGT-MS increased the HWL in a dose-dependent manner. The ED50 of RTGT-MS was 24.68 ± 1.02 mg/kg. Isobolographic analysis shows that the combination of RTGT-MS and celecoxib resulted in a synergistic antinociceptive effect. Further results demonstrated that antinociceptive effect of RTGT-MS was accompanied with that PKA, cAMP, COX-2, and PGE2 levels were decreased. Chlorpromazine, a dopamine receptor blocker, not only weakened the analgesic effect of RTGT-MS, but also increased the levels of cAMP, PKA, COX-2, and PGE2. These findings provide a rationale for the combination of RTGT-MS and celecoxib in the treatment of PD, which may reduce the dose of celecoxib, thereby lowering the incidence of adverse effects and improving the pain management in PD patients.