Abstract PD9-07: Role of immunosuppressive JNK pathway in the tumor microenvironment among Triple Negative Breast Cancer subtypes in IBCSG Trial 22-00
Authors: Garcia Andrea Joaquin , Semba Takashi , Rediti Mattia , McGrail Daniel J. , Xie Xuemei , Wang Xiaoping , Rampa Dileep R. , Venet David , Majjaj Samira , Kammler Roswitha , Colleoni Marco , Loi Sherene , Viale Giuseppe , Regan Meredith , Rothé Françoise , Sotiriou Christos , Ueno Naoto T. 
Published: March 2023

Abstract
BACKGROUND: Although the triple-negative breast cancer (TNBC) tumor microenvironment (TME) has been deeply characterized, much remains unknown about pathways attributing to an immunosuppressive TME in this disease. The phosphorylation of JNK (c-Jun N-terminal kinase) pathway has been associated with promoting an immunosuppressive phenotype, enhancing TNBC aggressiveness. Here, we aimed to explore the role of the JNK pathway in TNBC using a gene signature inferring the level of JNK phosphorylation. For this purpose, we used the TNBC cohort from the phase III adjuvant IBCSG 22-00 trial, which evaluated low-dose cyclophosphamide and methotrexate (CM) chemotherapy showing no clinical benefit in the overall population. METHODS: JNK gene signature was developed in TNBC samples by integrating RNA-seq gene expression data and phospho-JNK-targeted proteomic data in The Cancer Genome Atlas. Stochastic subsampling was performed to select the candidate markers. LASSO regression was performed to determine the final transcriptional signature to reflect JNK phosphorylation. The signature was then applied in a cohort composed of 498 TNBCs selected using stratified 1:3 relapse cases and non-relapse subcohort ratio from the IBCSG 22-00 trial. RNA-seq data from FFPE tumor samples were available for 347 patients. The JNK signature was calculated as the mean of the products between gene expression and signature coefficients, defining high and low levels using the median cut-off. Immune hot tumors were defined according to TNBC molecular subtypes or tumor-infiltrating lymphocytes (TILs) levels higher than 30%. Multivariable Cox models were used for disease-free survival (DFS) analysis. Wilcoxon test was used to evaluate the association between gene signatures and the levels of JNK. RESULTS: Tumors with either immunomodulatory (IM) phenotype or high TILs showed better DFS when presenting low levels of JNK compared to high levels (HR = 0.75, 95% CI, 0.57 to 0.99; P-value = 0.024 and HR = 0.62, 95% CI, 0.43 to 0.89, P-value = 0.0013). No significant differences in DFS were observed in other TNBC subtypes or tumors with low TILs, further highlighting the relevance of JNK signaling pathway in tumors presenting immune infiltration. Moreover, immune hot tumors with high levels of JNK were enriched for angiogenesis and eosinophils signatures and linked to immunosuppression. The immune targets B7-H3, CSF1R, GITR, and GARP were significantly associated with high levels of JNK. Of note, these genes are involved in the immune escape, activation of macrophages, and regulation of Tregs population. On the other hand, low levels of JNK were associated with higher levels of activated CD8+ T cells, pointing to an anti-tumor immune response, as well as with higher levels of the immune targets PDL-1, CTLA4, CD47, DCIR, and TIGIT. Of interest, a significant DFS benefit was found in IM tumors with high levels of JNK when treated with low-dose CM, compared to those who were not (HR = 0.52, 95% CI, 0.28 to 0.99; P interaction = 0.045). CONCLUSIONS: We developed a JNK gene signature to estimate the phosphorylation level of JNK from gene expression data in TNBC, and validated the associated biological and prognostic value in the IBCSG 22-00 trial. Our results are in line with the immunosuppressive effect described by the JNK gene signature and highlight the heterogeneity of immune response in immune hot TNBCs. Of note, high levels of JNK were associated with worse DFS, as well as with a benefit from low-dose CM potentially related to the immunomodulatory effect described for metronomic regimens. Overall, our findings suggest a potential role of the JNK signature in identifying TNBCs with an immunosuppressive TME and provide the rationale to explore its role as a biomarker for immunotherapy. Further validation of these findings is required.
Citation Format: Andrea Joaquin Garcia, Takashi Semba, Mattia Rediti, Daniel J. McGrail, Xuemei Xie, Xiaoping Wang, Dileep R. Rampa, David Venet, Samira Majjaj, Roswitha Kammler, Marco Colleoni, Sherene Loi, Giuseppe Viale, Meredith Regan, Françoise Rothé, Christos Sotiriou, Naoto T. Ueno. Role of immunosuppressive JNK pathway in the tumor microenvironment among Triple Negative Breast Cancer subtypes in IBCSG Trial 22-00 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-07.