Abstract PS4-07-05: JNK1 and JNK2 isoforms promote triple-negative breast cancer aggressiveness by fostering an M2 macrophage-driven immunosuppressive tumor microenvironment

Abstract Background: Triple-negative breast cancer (TNBC) is characterized by an immunologicallycold tumor microenvironment (TME), which facilitates immune evasion and tumorprogression. Our recent study showed that c-Jun N-terminal kinase (JNK) signaling promotesan immunosuppressive TME via macrophage-derived CCL2, thereby driving TNBC growthand metastasis. JNK has three isoforms, among which JNK1 and JNK2 have been implicatedin the development of various cancers. However, the distinct roles of JNK1 and JNK2 inregulating the TME immune landscape and aggressiveness in TNBC remain poorly understood.Building on our previous findings, in this study, we tested our hypothesis that both JNK1 andJNK2 promote TNBC aggressiveness by fostering an M2 macrophage–drivenimmunosuppressive TME. Methods: The effect of JNK singling inhibition by the pan-JNK inhibitor JNK-IN-8 and theeffect of JNK1 or JNK2 knockout (KO) on the growth of murine PyMT-M TNBC cells wereassessed in vitro using CellTiter-Blue cell viability and clonogenic assays. The roles ofintratumoral JNK1 and JNK2 in regulating the TME immune landscape and tumor growth wereexamined using an immunocompetent syngeneic C57BL/6 mouse model bearing PyMT-Mtumors derived from JNK1 or JNK2 KO cells. The contributions of JNK1 and JNK2 in theTME was examined using jnk1 -/- and jnk2 -/- immunocompetent syngeneic C57BL/6 mousemodels bearing PyMT-M tumors. The effects of the loss of JNK1 and JNK2 in tumors or in theTME on immune cell tumor infiltration were analyzed by flow cytometry. Key cytokinesinvolved in JNK-mediated modulation of the TME immune landscape were identified bycytokine array analysis. Results: Inhibition of JNK signaling by JNK-IN-8 and KO of either JNK1 or JNK2significantly suppressed the proliferation of murine PyMT-M TNBC cells in vitro, suggestingthat both JNK1 and JNK2 are essential for TNBC cell growth. However, compared to the Cas9control cells, intratumoral KO of either JNK1 or JNK2 in PyMT-M tumor cells did not affecttumor initiation and growth or immune cell tumor infiltration in the wild-typeimmunocompetent syngeneic C57BL/6 mice, demonstrating that intratumoral JNK1 and JNK2are not required for PyMT-M tumor initiation and progression. In contrast, regardless of theabsence or presence of JNK1 and JNK2 in tumor cells, tumor initiation was markedly delayedand tumor growth was significantly reduced in jnk1 -/- and jnk2 -/- immunocompetent syngeneicC57BL/6 mice, with a more pronounced effect observed in jnk1 -/- mice. This result highlightsthat both JNK1 and JNK2 in the TME play a critical role in PyMT-M tumor initiation andprogression. Interestingly, compared to tumors from wild-type mice, those from both jnk1 -/- and jnk2 -/- mice exhibited a marked increase in M1 macrophages and a reduction in M2macrophages, with a greater effect observed in jnk1 -/- mice. This result indicates that JNKdeficiency in the TME induces the reprogramming of M2 macrophages toward an M1phenotype. Indeed, cytokine array analysis revealed elevated expression of CCL12, CCL5, andCXCL9 in JNK1 KO tumors from jnk1 -/- mice, as well as increased CXCL9 expression in JNK2KO tumors from jnk2 -/- mice. These cytokines play critical roles in macrophage recruitmentand polarization. Taken together, these findings support our hypothesis that both JNK1 andJNK2 promote TNBC aggressiveness via fostering an M2 macrophage–drivenimmunosuppressive TME. Conclusion: Our findings demonstrate that both JNK1 and JNK2 in the TME drive TNBCinitiation and progression via promoting an M2 macrophage–driven immunosuppressive TMEin the macrophage-enriched TNBC mouse model. These findings establish JNK1 and JNK2 askey regulators of the immunosuppressive TME in TNBC, highlighting their therapeuticimportance in treating TNBC. Citation Format: B. Kuntal, X. Stern, T. Semba, K. Nakaoka, M. Fujimoto, M. William, K. Leonard, D. Rampa, J. Lee, N. Ueno, X. Xie. JNK1 and JNK2 isoforms promote triple-negative breast cancer aggressiveness by fostering an M2 macrophage-driven immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-07-05.