Abstract 6382: Activation of transcription factors, p65 and NFATc1, mediated by WNT5a, induces up-regulation of novel immune checkpoint CNTN4 expression
Authors: Cha Mi Young , Jeon Bu-Nam , Kang Hyeokgu , Park Kyung Mi , Park Hansoo 
Published: April 2023

We previously confirmed that contactin 4 (CNTN4) is highly expressed in the various human tumor tissues and inhibits mouse and human T cell activity by suppressing a cascade of signaling processes associated with T cell activation through binding with amyloid precursor protein (APP) of T cells. However, the molecular mechanism for the expression of CNTN4 in tumors has yet to be elucidated. So, we investigated whether oncogenic ligands regulate the expression of CNTN4 through CNTN4 promoter (- 2,000 bp ~ + 100 bp)-based reporter gene assays in HEK293 cells using oncogenic ligands. Here, we identified that WNT5a significantly increased the expression of CNTN4 in a dose-dependent manner. Furthermore, the treatment of the WNT5a ligand in the human colorectal cancer cell line SW480, which expresses CNTN4 at a low level, increased the expression of CNTN4. In addition, CNTN4 expression was decreased when the WNT5a antagonist (Box5, a WNT5a-derived N-butyloxycarbonyl hexapeptide) was treated in the human osteosarcoma cell line U2OS expressing CNTN4 endogenously. The changes in the expression of CNTN4 were observed by RT-PCR and FACS. Treatment of the WNT5a ligand or WNT5a antagonist changed the transcriptional expression of CNTN4 in each cell line and affected the cell surface expression of CNTN4. Since WNT5a ultimately increases the activity of transcription factors such as NFATc1 or p65 to increase the transcriptional expression of the target gene, we further investigated the effect of NFATc1 or p65 on the expression of CNTN4. We found that NFATc1 or p65 binds to the promoter of CNTN4 and increases the expression of transcription. Moreover, it was recently reported that anti-PD-1-refractory melanoma exhibits elevated WNT ligand signaling activity, especially upregulated WNT7b and WNT5a in the WNT ligands. Following this background, the expression level of CNTN4 can be increased in the refractory environment to immunotherapy induced by elevated WNT signaling. Therefore, we constructed B16F10 murine melanoma cells overexpressing WNT5a (B16F10/WNT5a) to assess the anti-tumor efficacy by administering GENA-104A16, the anti-CNTN4 humanized monoclonal antibody, in the syngeneic mice model with increased expression of CNTN4 induced by WNT5a, and in vivo efficacy by treatment of GENA-104A16 is under assessment in the B16F10/WNT5a syngeneic mice model. Through the investigation, we expect to support the rationale and strategy for a combination therapy approach of GENA-104A16 for non-responding patients to immunotherapy.

Citation Format: Mi Young Cha, Bu-Nam Jeon, Hyeokgu Kang, Kyung Mi Park, Hansoo Park. Activation of transcription factors, p65 and NFATc1, mediated by WNT5a, induces up-regulation of novel immune checkpoint CNTN4 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6382.