Abstract P4-06-21: MEK inhibitor cobimetinib induces immunogenic cell death and immune-modulatory effects in triple negative breast cancer
Authors: Liu C-Y , Lau K-Y , Chen J-L , Chu P-Y , Huang C-T , Wang W-L , Lien P-J , Tseng L-M 
Published: May 2019

Background

Triple-negative breast cancer (TNBC) has been associated with a robust tumor immune infiltrate. Tumor-infiltrating lymphocytes (TILs) in TNBC have been demonstrated a prognostic value. The mitogen-activated protein kinase (MAPK) signaling pathway have been shown to regulate the immune response with the production of immunomodulatory cytokines, such as TNFα, interleukin (IL)-1, IL-10, and IL-12. Clinical studies have shown that the high expression level of Extracellular signal–related kinase (ERK), a member of the MAPK pathway, correlates with shorter survival in TNBC patients. Accordingly, ERK is a potential target for anti-tumor and cancer immunotherapy. In this study, we aimed to investigate a MEK inhibitor cobimetinib and elucidate whether the MEK/ERK pathway is implicated in immunogenic cell death (ICD) and/or other immunomodulatory effects.

Methods

Mouse TNBC cell line 4T1 was treated with cobimetinib. The cell viability and cell apoptosis were determined by MTT assay and flow cytometric analysis. Cobimeinib-induced damage-associated molecular patterns (DAMPs), such as such as cell-surface translocation of calreticulin (CRT), extracellular release of ATP, and increase in high-mobility group box protein B1 (HMGB1) release from dying tumor cells, were examined by immunoblotting and flow cytometric analysis. Further, the molecular mechanisms involving cobimetinib-induced cell death were examined by Immunoblotting. Last but not least, establishment of 4T1 animal model in immunocompetent and immunodeficient mice was conducted to investigate the efficacy of cobimetinib in restoration of immunosurveillance and cancer metastasis in vivo.

Results

The results showed that cobimetinib impaired cell proliferation and induced cell apoptosis in a dose-dependent manner in 4T1 cells. Importantly, cobimetinib treated 4T1 cells elicited ICD, by increasing HMGB1 and ATP release, and by promoting membrane exposure of CRT. The molecular mechanisms involved in cobimetinib-induced DAMPs were partially through the downregulation of p-ERK expression. Moreover, cobimetinib induced processing of procaspase-3 and -8 in 4T1 cells, thereby resulting in caspase activation. In addition, 4T1 tumor bearing immunocompetent mice treated with cobimetinib showed reduced size of primary tumors, fewer lung metastases and increased survival. In addition, we noticed that the tumor-suppressive effects of cobimetinib were much stronger in immunocompetent mice than in immunodeficient mice, evident by distinct mice survival in drug-treated mice. Flow cytometric analysis of murine splenocytes also revealed that cobimetinib treatment increased total number of CD8+ T cells, dendritic cell maturation, and suppressed the number of Myeloid-derived suppressor cells (MDSCs) in immunocompetent mice.

Conclusions

Our findings suggested that the MEK/ERK inhibitor cobimetinib induces ICD in vitro and exerts additional immune-modulatory effects in immunocompetent TNBC mice model. Our study highlights the possibility for the use of cobimetinib as an ICD inducer and immunomodulator for therapeutic intervention in TNBC.

Keywords

Cobimetinib, immunogenic cell death, MAPK, ERK phosphorylation, immune regulation

Citation Format: Liu C-Y, Lau K-Y, Chen J-L, Chu P-Y, Huang C-T, Wang W-L, Lien P-J, Tseng L-M. MEK inhibitor cobimetinib induces immunogenic cell death and immune-modulatory effects in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-21.