CD155 on Multiple Myeloma Cells Is Epigenetically Regulated, Reduce Patients Survival and Compromises the Action of Novel Immunotherapies
Authors: Martinez-Verbo Laura , Villanueva Lorea , Llinàs-Arias Pere , García-Prieto Carlos Antonio , Piñeyro David , Oliver-Caldes Aina , García-Ortiz Almudena , Valeri Antonio , Fernández de Larrea Carlos , Martinez-Lopez Joaquin , Ferrer Gerardo , Esteller Manel 
Published: December 2023

Epigenetics, especially DNA methylation, is a key molecular regulator of gene expression. This regulation is important for cytotoxic cell activation (i.e., T-cells and NK cells) and is crucial for the clearance of malignant cells. Immune checkpoint (IC) events mediate cytotoxicity, one IC receptor involved in the process is CD155. Such receptor interacts primarily with TIGIT on cytotoxic cells and induces the inhibition of cytotoxicity. Multiple myeloma (MM) represents the 10% of all hematological malignancies and it has been described that MM cells hinder cytotoxic cell activation by inhibition and exhaustion. Furthermore, novel immunotherapeutic strategies such as Bispecific T-cell Engagers (BiTE) and Chimeric Antigen Receptor (CAR) therapies are being evaluated in MM treatment, with promising results. Nevertheless, they will confront a hostile environment. This is a very active field with frequent descriptions of new targets and therapeutic approaches being studied each day. We investigated whether CD155 is epigenetically regulated in MM and its importance in inducing inhibition of anti-tumor response in the context of novel immunotherapies through the CD155/TIGIT axis.

To achieve our objectives, we focused on the promoter region of CD155 (the region up to 1500 base pairs upstream of the TSS) and characterized it at DNA, RNA and protein levels through bisulfite sequencing, quantitative PCR, and flow cytometry. We confirmed CD155 as an epigenetically regulated gene, thus demonstrating an existing correlation between CD155 promoter methylation and gene expression. In vitro treatment with azacytidine was performed to assess the active repression of CD155 expression by DNA methylation. Six different MM cell lines were characterized (two not expressing: AMO-1, KMS-12-BM, four expressing: RPMI-8226, MM.1S, EJM and JJN-3).

From there, we created CD155 depletion models on expressing cell lines by short hairpin RNA interference and developed an in vitro coculture system with T-cells (from healthy donors). We observed a loss of expression between the 60% to 90% (when compared with controls). Depletion of CD155 did not significantly impact cell growth, apoptosis, or cell cycle. On the coculture with healthy T-cells, more cytotoxicity was detected against CD155-depleted cells (in RPMI-8226 Mann-Whitney P=0.020; JJN-3 and EJM P&lt;0.001). To incorporate novel immunotherapeutic strategies, we carried out the same cocultures with 100pg/mL of anti-BCMA/CD3 BiTE, against anti-BCMA CAR-T or anti-BCMA CAR-NK cells (for RPMI-8226 and JJN-3 Mann-Whitney P=0.008). Coculture combinations were also performed in the presence of 10μg/ml of neutralizing αTIGIT (RPMI-8226, Mann-Whitney P =0.005; JJN-3 P=0.004) and/or αPD1 antibody. When novel immunotherapy approaches were included in the system, we observed how the depletion of CD155 was related to a cytotoxicity increase in all cases. In the presence of neutralizing αTIGIT, cytotoxicity was increased against CD155-expressing cells, while we observed no difference against CD155-depleted cells. In the presence of αPD1, we saw a significant restoration of T-cell cytotoxicity against both models and the antibody combination showed a synergic effect in CD155 expressing cells.

Finally, we analyzed the existence of a correlation between CD155 gene expression and survival in newly diagnosed MM samples from CoMMpass project public data (N= 793). These data supported our results as the newly diagnosed MM patients with higher expression of CD155 had shorter overall survival than lower expressing ones (Logrank test P&lt;0.001).

In summary, in the context of MM, the expression of CD155 is regulated by the methylation status of its promoter region. Expression of CD155 effectively reduces cytotoxic cell response, which is mediated by the interaction of CD155 and TIGIT. The addition of αTIGIT and αPD1 validated that the CD155 cytotoxic cell inhibition is mediated in part by TIGIT interaction, independently of PD1/PD-L1 axis. This interaction seems to impact patients treated with current therapies, since their outcome correlates with CD155 expression. Based on our results, novel immunotherapies focused on enhancing immune cytotoxicity will benefit from CD155/TIGIT axis blockade. These results warrant further investigation on CD155 as a biomarker and target for novel immunotherapies for MM and other malignancies.