Abstract
DNA promoter methylation acts as a key regulator for gene expression and is deeply altered in tumorigenesis. In the context of tumor clearance, cytotoxic cells (i.e. T and NK cells) play a critical role. Their function is mainly controlled by immune checkpoint (IC) synapse events, where known inhibitory markers are detrimental for cytotoxic activation. TIGIT, an inhibitory receptor on immune cells, interacts with PVR on malignant cells, precluding recognition and clearance. Multiple Myeloma (MM) remains and incurable disease and represents 10% of all hematological malignancies. In this project, we have proved PVR is regulated by promoter methylation and the importance of PVR/TIGIT axis inhibition on current immunotherapeutic options for MM. Studying newly diagnosed MM patients (n=793) we observed that higher expression of PVR is associated with shorter overall survival (median overall survival of PVR high: 56 months; PVR low: not reached; Log-rank P<0.001). In silico analysis of the promoter region of PVR, suggested its regulation by DNA methylation, so we characterized the epigenetic regulation of PVR at DNA, RNA and protein levels on a panel of MM cell lines (two methylated: AMO-1, KMS-12_BM and four unmethylated: EJM, JJN-3, MM.1S and RPMI-8226) and validated its epigenetic regulation. We cocultured cytotoxic cells with PVR unmethylated (expressing) models as well as PVR depleted ones. This depletion was obtained by shRNA interference and at least four different donors were evaluated in all the coculture experiments. Three different cell lines (EJM, JJN-3 and RPMI-8226) were tested and all expressing controls showed resistance to cytotoxicity (Mann-Whitney P<0.05). To validate the PVR/TIGIT role, we incorporated 10μg/ml of neutralizing anti-TIGIT into the system. We observed an increase in cytotoxicity in the expressing models as observed in the depleted models (Mann-Whitney P<0.05). We then tested PVR/TIGIT effect on anti-BCMA/CD3 BiTE, anti-BCMA (ARI0002h) CAR-T, and anti-BCMA (ARI0002h) CAR-NK cells. In the presence of BiTE, PVR depletion had a significant impact on cytotoxicity (Mann-Whitney P<0.05) when compared to controls. Coculturing CAR-T cells and MM models showed similar effects (Mann-Whitney P<0.05). As for CAR-NK coculture, only depleting PVR on RPMI-8226 showed a significant increase in cytotoxicity (Mann-Whitney P=0.01) when compared with the control, whereas JJN-3 remained unaffected. In summary, in the context of MM, PVR's expression is regulated by its promoter region's methylation status. Expression of PVR effectively reduces cytotoxic cell response, and the addition of anti-TIGIT validated that the PVR cytotoxic cell inhibition is mediated by TIGIT interaction. Furthermore, immunotherapies benefit from PVR depletion. Finally, PVR/TIGIT interaction seems to impact patients treated with current therapies since their outcome correlates with PVR expression. These results warrant further investigation on PVR as a biomarker and target for novel immunotherapies.
Citation Format: Laura Martinez-Verbo, Lorea Villanueva, Pere Llinàs-Arias, Carlos A. García-Prieto, David Piñeyro, Aina Oliver-Caldes, Almudena García-Ortiz, Antonio Valeri, Carlos Fernández de Larrea, Joaquín Martínez-López, Gerardo Ferrer, Manel Esteller. PVR (CD155) epigenetic status mediates adoptive cell therapy and anti-TIGIT response in Multiple Myeloma [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P22.
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