Abstract C058: ADORA2B Inhibition in Mesothelioma (MMe) cells affects PDL-1 expression, exerts an effective response on Hyppo and AKT signaling and elicits anti-tumor immune response

Abstract Background: The link between hypoxia and activation of the immunosuppressive purinergic/adenosine pathway in MMe is well established. MMe is a highly aggressive and hypoxic malignancy and here we investigate the therapeutic potential of targeting adenosine receptor signaling as a treatment strategy. We have previously shown that: 1) MMe cells release Adenosine; 2) Adenosine receptor 2 B (A2B) is inversely related to patients survival; and that, 3) A2B signaling induces cell proliferation in 2D and 3D models (Manuscript in preparation). The Aim of this study is to assess whether A2B inhibition plays a role in PD-L1 expression and whether A2B is an actionable target for treating mesothelioma. Methods: Proteomic analysis was conducted on MMe spheroids after 5-days of treatment with an Adenosine agonist (NECA; 100 nM) and an A2B antagonist (MRS1754; 10 nM). PD-L1 and AKt expression by epithelioid MMe cells was tested after 72-hour of treatment with specific A2B inhibitors TT4 (Portage Biotech; 10μM), MRS1754 or an AKT inhibitor (AKTiVII; 10μM), NECA was added 15 minutes after the start of each treatment. For the in vivo experiments, ADORA2B positive 105 AB1B-LUC MMe cells were injected intraperitoneally in immunocompetent BALB/C mice. Mice were treated with PBS (control) ; TT-4 (A2B Inhibitor Portage Biotech, 4.4 mpk); anti-murine PD-1 (3 mg/Kg ), or the combination of TT4 and anti murine PD-1. Disease was measured with an IVIS® Lumina XR in vivo imaging system, Results: Amid the proteins with a ≥ 1.5 fold change (p value ≤ 0.05) and reversed by MRS1754 after NECA treatment, we identified AKT and YAP1. The effects on the latter were by the increased expression of the YAP/TAZ/TEAD positive modulator 14-3-3 pathway linked to tumor progression. After NECA treatment, we validated AKT by WBlots on the epithelial REN MME cells and showed increased PDL-1 expression that was similarly and significantly inhibited by AKT or A2B adenosine receptor inhibitors (**** p<0.0001). In vivo experiments demonstrated that TT-4 treatment alone was more active than anti-PD treatment alone vs. the AB1B-Luc mesothelioma model, and that the combination with anti-PD1 was significantly more effective than either treatment alone (p<0.01). IHC of murine samples from mice treated with TT4 revealed infiltration of T cells whereas the combo showed higher T infiltration with onset of B cells Tertiary Lymphoid Structures Conclusions: We provide the first evidence ever that A2B inhibition has an anti-tumor effect on both epithelial and non-epithelial MMe cells, decreases PDL-1 expression via AKT inhibition and exerts a significant mixed effect on YAP signaling and immune-mediated MMe growth inhibition Citation Format: luciano Mutti, Alessandra Rossetti, caterina costa, Steven Gray, Laura Moro, Giulia Pinton, Rita Lombardi, Francesco Bruzzese, Paolo Ascierto, Robert Kramer, Giovanni Luca Gravina. ADORA2B Inhibition in Mesothelioma (MMe) cells affects PDL-1 expression, exerts an effective response on Hyppo and AKT signaling and elicits anti-tumor immune response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C058.