Eribulin is a microtubule destabilizer used in the treatment of triple-negative breast cancer (TNBC). Eribulin and other microtubule targeted drugs, such as the taxanes, have shared antimitotic effects, but differ in their mechanism of microtubule disruption, leading to diverse effects on cellular signaling and trafficking. Herein, we demonstrate that eribulin is unique from paclitaxel in its ability to enhance expression of the immunogenic cytokine interferon beta (IFNβ) in combination with STING agonists in both immune cells and TNBC models, including profound synergism with ADU-S100 and E7766, which are currently undergoing clinical trials. The mechanism by which eribulin enhances STING signaling is downstream of microtubule disruption and independent of the eribulin-dependent release of mitochondrial DNA. Eribulin did not override the requirement of ER exit for STING activation and did not inhibit subsequent STING degradation; however, eribulin significantly enhanced IRF3 phosphorylation and IFNβ production downstream of the RNA sensing pathway that converges on this transcription factor. Additionally, we found that eribulin enhanced the population of activated CD4+ T-cells in vivo when combined with either a STING agonist or tumor, demonstrating the ability to function as an immune adjuvant. We further interrogated the combination of eribulin with ADU-S100 in the MMTV-PyVT spontaneous murine mammary tumor model where we observed significant antitumor efficacy with combination treatment. Together, our findings demonstrate that microtubule targeted chemotherapeutics have distinct immunological effects and that eribulin’s ability to enhance innate immune sensing pathways supports its use in combination with immunotherapies, such as STING agonists, for the more effective treatment of TNBC and other malignancies.

Triple-negative breast cancer (TNBC) is a heterogenous subtype of breast cancer that lacks effective targeted treatment options. However, TNBC typically has a higher mutational burden and greater degree of immunogenicity than other breast tumors, making immunotherapy a viable strategy for effective treatment of this disease. Strategies to improve the response of TNBC patients to immunotherapy include the upregulation of the cGAS-STING innate immune sensing pathway and STING agonists are in clinical development for the treatment of TNBC. We demonstrate that eribulin, a microtubule destabilizer currently used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm. Eribulin also enhances type I interferon expression induced by STING agonists through a second TBK1-dependent mechanism downstream of STING activation that is shared by the RIG/MAVS RNA sensing pathway. Both mechanisms of eribulin-mediated activation of interferon expression occur in immune and TNBC cells and are shared with other microtubule destabilizers, including vinorelbine, but not with the microtubule stabilizing agent paclitaxel. These effects of eribulin on innate immune sensing pathways in vitro prompted further evaluations of the impact of eribulin on the in vivo immunological response to mammary tumors. We found that eribulin, but not paclitaxel, promotes the activation of CD4+ T-cells in the spleens and draining lymph nodes of tumored animals. This activation required tumor priming but was independent of any direct effect on tumor growth inhibition, demonstrating a specific role of eribulin as a tumor immune modulator. These data contribute to accumulating evidence that there are important mechanistic differences between the microtubule targeted chemotherapeutics currently used in the treatment of TNBC and suggest that eribulin elicits a more favorable innate immunological signature than paclitaxel. Research supported by Eisai Inc. Citation Format: Leila Takahashi-Ruiz, Charles Fermaintt, Nancy Wilkinson, Susan Mooberry, April Risinger. Eribulin enhances STING-dependent induction of type I interferons in immune and triple-negative breast cancer cells [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 P4-07-19.

OBJECTIVES/GOALS: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks effective targeted treatment options. TNBC's greater degree of immunogenicity than other breast tumors makes immunotherapy a viable strategy. Strategies to improve the immunotherapy response includes targeting the cGAS-STING innate immune pathway with STING agonists. METHODS/STUDY POPULATION: We have previously shown in vitro that eribulin, a microtubule destabilizer currently used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm. Separately, eribulin also significantly enhances type I interferon expression induced by STING agonists measured by qRT-PCR through a second TBK1-dependent mechanism downstream of STING activation through detecting higher amounts of phosphorylated IRF-3 by western blot protein analysis. Mechanisms of eribulin-mediated interferon expression occur in immune and TNBC cells and are shared with other microtubule destabilizers but not with the microtubule stabilizing agent paclitaxel. RESULTS/ANTICIPATED RESULTS: We determine that the enhancement of type I interferon expression by eribulin is pharmacologically synergistic with multiple STING agonists. The significant enhancement by eribulin led us to evaluate the antitumor efficacy of eribulin in combination the STING agonist ADU-S100 in a challenging spontaneous mammary tumor model MMTV-PyVT. We show that the combination treatment significantly decreased tumor growth which allowed for longer survival compared to other groups. This is particularly interesting because of our previous studies showing that eribulin alone, but not paclitaxel, promotes the activation of CD4+ T-cells in the spleen and draining lymph nodes of BALB/c mice with 4T1 tumors through flow cytometric analysis. DISCUSSION/SIGNIFICANCE: These data contribute to accumulating evidence that there are important mechanistic differences between the microtubule targeted chemotherapeutics currently used in the treatment of TNBC and suggest that eribulin can act as an immune adjuvant in addition to its anti-mitotic effect.

Zonula occluden‐1 (ZO‐1) has been implicated as a major regulator of tight junction formation, functioning as a scaffolding protein and mediator between the junction and the actomyosin cytoskeleton. MDCK II cells that have ZO‐1 knocked‐down (ZO‐1 KD) or knocked‐out (ZO‐1 KO) have observable phenotypic changes, including increased apical actin accumulation, increased cell height, and linearized junctions. The apical actin accumulation, resulting from the dysregulation of myosin II, is suspected to cause an increase in membrane tension seen in the ZO‐1 KO, which may explain the phenotypic changes. The function of ZO‐1 as a regulator of myosin II was confirmed by exposing the ZO‐1 KO to blebbistatin, a myosin II inhibitor, which restored the cell height to that of wild‐type MDCK II cells (WT) and the curvature of the junctions. Flipper‐TR, which is a live cell fluorescent membrane tension probe, along with fluorescence‐lifetime imaging microscopy (FLIM) analysis, are being used to test the membrane tension of the ZO‐1 KO cells compared to WT cells. The Flipper‐TR probe works by inserting itself into a lipid membrane where it undergoes conformational changes induced by membrane tension. The relationship between membrane tension and actin was examined using latrunculin A (LatA), which causes the depolymerization of actin filaments. When both the ZO‐1 KO and WT cells were incubated with LatA, the membrane tension of both decreased to a similar extent. The preliminary results of this testing have shown an increased membrane tension in the ZO‐1 KO cells. To measure the response of ZO‐1 KO cells to increased tension, the expression of tension‐related genes is being measured using quantitative real‐time polymerase chain reaction (qRT‐PCR). These tension‐related genes include the ERM (ezrin, radixin, and moesin) proteins, with a primary interest in ezrin given its known activity as a major regulator of membrane tension in epithelial cells. This study aims to understand the mechanism between ZO‐1 loss and regulation of membrane tension.

Triple-negative breast cancer (TNBC) is a heterogenous subtype of breast cancer lacking effective targeted treatment options. However, TNBC typically has a higher mutational burden and greater degree of immunogenicity than other breast tumors, making immunotherapy a viable strategy for effective treatment. Strategies to improve the response of TNBC patients to immunotherapy include the upregulation of the cGAS-STING innate immune sensing pathway and STING agonists are in clinical development. We previously showed that eribulin, a microtubule destabilizer used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm. In separate studies, we demonstrated that eribulin also significantly enhances type I interferon expression induced by STING agonists through a second TBK1-dependent mechanism downstream of STING activation. Both mechanisms of eribulin-mediated activation of interferon expression occur in immune and TNBC cells and are shared with other microtubule destabilizers but not with the microtubule stabilizer paclitaxel. Our current studies demonstrate the in vivo immunological effects of the microtubule destabilizer eribulin with the STING agonist ADU-S100 that lead to a significant decrease in tumor growth in the spontaneous MMTV-PyVT mammary tumor model upon combination treatment. The immunostimulatory effects of eribulin with or without concurrent administration of a STING agonist were determined in the spleen, tumor draining lymph nodes, and tumors with distinct effects observed in the activation of innate and adaptive responses. We further demonstrate the immunological and antitumor efficacy of combinations of eribulin and ADU-S100 in the syngeneic 4T1 model that is resistant to the antimitotic effects of eribulin as a single agent. Even in the absence of direct antimitotic effects on the tumor, eribulin was sufficient to promote activation of CD4+CD25− effector T-cells in the spleen and lymph nodes specifically in tumored animals but not untumored controls. Additionally, eribulin was able to promote a similar increase in the activation of CD4+ effector T-cells even in untumored animals when administered in combination with a STING agonist. Together, these data contribute to accumulating evidence that there are important mechanistic differences between the microtubule targeted chemotherapeutics currently used in the treatment of TNBC that could inform on their more rational use in the clinic as single agents or as chemotherapeutic backbones in combination with targeted therapy, including immunotherapeutics. More specifically, our data demonstrate that eribulin can act as an immune adjuvant to promote STING signaling and T-cell activation in multiple in vivo TNBC models in addition to its well established anti-mitotic effects to enhance antitumor efficacy. Citation Format: Leila Takahashi-Ruiz, Charles S. Fermaintt, Nancy Wilkinson, Peter Y. Chan, Susan L. Mooberry, April L. Risinger. Eribulin acts as an immune adjuvant to enhance the antitumor efficacy of STING agonists in triple-negative breast cancer models. [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 6397.