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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 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.

Simple Summary Most breast cancers express the estrogen, progesterone, and/or HER2 receptors and patients are treated with inhibitors targeting these receptors. Triple negative breast cancers (TNBCs) lack these receptors and thus patients with TNBC do not benefit from existing targeted therapies. There is a continued search for effective targets for the treatment of these heterogeneous tumors. We identified a class of plant-derived natural products, including yuanhuacine, that selectively kill cells that represent a molecularly defined subtype of TNBC. These compounds also promote expression of an immunological profile that is beneficial for engaging the immune system, which could provide an added benefit in TNBC. The mechanism of action of both the TNBC selectivity and immunological phenotypes is associated with activation of protein kinase C. Yuanhuacine has potent antitumor efficacy in a mouse model of TNBC, identifying a new therapeutic target for the treatment of this deadly disease. Abstract The heterogeneity of triple negative breast cancer (TNBC) has led to efforts to further subtype this disease with the hope of identifying new molecular liabilities and drug targets. Furthermore, the finding that TNBC is the most inherently immunogenic type of breast cancer provides the potential for effective treatment with immune checkpoint inhibitors and immune adjuvants. Thus, we devised a dual screen to identify compounds from natural product extracts with TNBC subtype selectivity that also promote the expression of cytokines associated with antitumor immunity. These efforts led to the identification of yuanhuacine (1) as a potent and highly selective inhibitor of the basal-like 2 (BL2) subtype of TNBC that also promoted an antitumor associated cytokine signature in immune cells. The mechanism of action of yuanhuacine for both phenotypes depends on activation of protein kinase C (PKC), defining a novel target for the treatment of this clinical TNBC subtype. Yuanhuacine showed potent antitumor efficacy in animals bearing BL2 tumors further demonstrating that PKC could function as a potential pharmacological target for the treatment of the BL2 subtype of TNBC.

Microtubule targeting agents (MTAs), including both microtubule stabilizers and destabilizers, are highly effective chemotherapeutic drugs used in the treatment of solid tumors and hematological malignancies. In addition to the shared ability of all MTAs to block cell cycle progression, growing evidence shows that different agents of this class can also have mechanistically distinct effects on non-mitotic microtubule-dependent cellular processes, including cellular signaling and transport. Herein, we test the biological hypothesis that MTAs used in the treatment of triple-negative breast cancer (TNBC) can differentially affect innate immune signaling pathways independent of their antimitotic effects. Our data demonstrate that the microtubule destabilizer eribulin, but not the microtubule stabilizer paclitaxel, induces cGAS-STING-dependent expression of interferon β in both myeloid and TNBC cells. Activation of the cGAS-STING pathway by eribulin was further found to be mediated by the accumulation of cytoplasmic mitochondrial DNA. Together, these findings provide mechanistic insight into how eribulin can induce innate immune signaling independent of its antimitotic or cytotoxic effects. Significance StatementMicrotubule targeted agents are often used in the treatment of breast cancer and, most recently, have been used in combination with immune checkpoint inhibitors to improve their efficacy. While all clinically approved MTAs share an antimitotic mechanism of action, their distinct effects on interphase microtubules can promote differential downstream signaling consequences. We show that the microtubule destabilizer eribulin, but not the microtubule stabilizer paclitaxel, activates the cGAS-STING innate immune signaling pathway through the accumulation of mitochondrial DNA in the cytoplasm.

35 p.-5 fig.-1 tab., Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/β-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/β-tubulin−GB1 complex., This research was supported by the NIH, National Cancer Institute Grants R01CA172310 (to H.L.) and R50CA211487 (to R.R.) and National Institute of General Medical Sciences Grant P41GM086210 (to H.L. and V.J.P.), Commercialization Fund Award from University of Florida (UF) Innovate (UF Office of Technology Licensing), and Debbie and Sylvia DeSantis Chair professorship (H.L.). The biochemical and the crystal structure work was supported by grants Ministerio de Ciencia e Innovación PID2019-10454RB-I00/AEI/10.13039/501100011033, Fondo de Investigaciones Sanitarias and COV20/01007E Proyecto Intramural Especial 201920E111 from Consejo Superior de Investigaciones Científicas (to J.F.D.) and European Union H2020-MSCA-ITN-2019 860070 TUBINTRAIN grant (to J.F.D. and A.E.P.).

Triple-negative breast cancer (TNBC) patients have a higher response rate to immune checkpoint inhibitors as compared to patients with other types of breast cancer. This higher response rate is thought to be due to higher mutational burdens, the prevalence of PD-L1/PD-1 expression, and immune cell infiltration in TNBC. However, less than half of TNBC patients respond to these drugs as single agents, which has prompted their use in combination with chemotherapy, including microtubule targeted agents (MTAs). Multiple clinical trials now show that the combination of PD-1/PD-L1-targeted immune checkpoint inhibitors with chemotherapy, including the MTAs paclitaxel or eribulin, can improve clinical responses in both late and early-stage TNBC. Previous studies have demonstrated that MTAs can activate innate immune sensing pathways that could potentially mediate their efficacy in combination with immunotherapies, including the ability of paclitaxel to promote TLR4 signaling. However, there has not been a direct comparison of the immunogenic effects elicited by each of the five MTAs that are used to treat TNBC to inform on how agents of this class could differentially alter the tumor immune microenvironment. In the current study, we determined the concentration and time-dependent effects of paclitaxel, docetaxel, ixabepilone, eribulin, and vinorelbine on the cytokine expression profiles in both immune and TNBC cells. Our results demonstrate that the microtubule destabilizers, eribulin, and vinorelbine, but not the microtubule stabilizers, paclitaxel, docetaxel, and ixabepilone, promote upregulation of type 1 interferons (IFNα/β) and downstream interferon-stimulated-genes. A time-course analysis in the human monocytic THP-1 cell line and primary murine bone-derived macrophages found that the induction of IFNβ by eribulin occurred within 2-6 hours and was independent of mitotic arrest. A pharmacological and genetic-based assessment of the signaling pathways responsible for the eribulin-mediated expression of IFNβ revealed a dependency on the cGAS-STING cytosolic DNA-sensing innate immune signaling pathway in both immune and TNBC cells. Moreover, we demonstrate that the mechanism for the promotion of cGAS-STING-dependent interferon induction by eribulin is due to the release of mitochondrial DNA (mtDNA) into the cytoplasm. Together, these results provide evidence that different MTAs have distinct immunomodulatory properties and highlight the ability of eribulin to promote the release of mtDNA to activate the cGAS-STING pathway, which has been previously shown to enhance the efficacy of immunotherapy in TNBC. These studies were funded by Eisai. Citation Format: Charles Steven Fermaintt, Leila Takahashi-Ruiz, Susan L Mooberry, April L Risinger. Eribulin-mediated release of mitochondrial DNA activates the cGAS-STING innate immune signaling pathway [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-18.

Microtubule targeted agents (MTAs) are a mainstay in the treatment of breast cancer and they are often used in combination with targeted agents, including immunotherapies. Although patients with triple-negative breast cancers (TNBCs) have historically had poorer clinical outcomes due in part to a lack of targeted therapeutic options, these cancers are often more immunogenic than other types of breast cancer. Recently, the combination of the PD-L1 inhibitor atezolizumab with the taxane nab-paclitaxel was found to prolong progression-free survival in patients with metastatic TNBC. Other combinations of MTAs and immune checkpoint inhibitors are actively being evaluated in clinical trials. There is a mechanistic rationale for this combination because paclitaxel is a direct TLR4 agonist that promotes activation of the innate immune system. To date, there has not been a comprehensive comparison of the immunomodulatory effects of different MTAs on immune cells or on or TNBC cells. We hypothesized that MTAs that differentially alter interphase microtubule structure and dynamics would have distinct effects on cell-intrinsic immunogenic pathways and that these effects could impact the efficacy of these drugs in combination with immunotherapies. The effects of 5 MTAs used for the treatment of TNBC, paclitaxel, docetaxel, ixabepilone, vinorelbine, and eribulin, on the expression of antitumor cytokines and other immunomodulatory genes in myeloid and TNBC cells was evaluated. Our results show that the TLR4-mediated induction of TNFα that occurs in response to paclitaxel was not observed with either eribulin or vinorelbine. Conversely, upregulation of type 1 interferons and interferon-stimulated genes (ISGs) was observed in monocytes and in bone-marrow derived macrophages within 6 h of treatment with eribulin or vinorelbine, but not with any of the microtubule stabilizers. Eribulin-mediated induction of type 1 interferons was also observed in a subset of eribulin-treated TNBC cells and was independent of its antimitotic effects. Importantly, type I interferon signaling has been shown to enhance the efficacy of immune checkpoint inhibitors, suggesting that microtubule destabilizers could have distinct advantages as compared to microtubule stabilizers in combination with these agents. Together, these results provide evidence that MTAs have different immune modulatory properties and suggest that the specific immune signatures initiated by different MTAs need to be considered for their optimal use with targeted agents, including checkpoint inhibitors. These studies were funded by Eisai. Citation Format: Charles Fermaintt, Susan Mooberry, April L. Risinger. Eribulin promotes a distinct immunogenic gene expression signature as compared with paclitaxel [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 965.

Oligosaccharyltransferases (OSTs) N-glycosylate proteins by transferring oligosaccharides from lipid-linked oligosaccharides (LLOs) to asparaginyl residues of Asn-Xaa-Ser/Thr acceptor sequons. Mammals have OST isoforms with STT3A or STT3B catalytic subunits for cotranslational or posttranslational N-glycosylation, respectively. OSTs also hydrolyze LLOs, forming free oligosaccharides (fOSs). It has been unclear whether hydrolysis is due to one or both OSTs, segregated from N-glycosylation, and/or regulated. Transfer and hydrolysis were assayed in permeabilized HEK293 kidney and Huh7.5.1 liver cells lacking STT3A or STT3B. Transfer by both STT3A-OST and STT3B-OST with synthetic acceptors was robust. LLO hydrolysis by STT3B-OST was readily detected and surprisingly modulated: Without acceptors, STT3B-OST hydrolyzed Glc 3 Man 9 GlcNAc 2 -LLO but not Man 9 GlcNAc 2 -LLO, yet it hydrolyzed both LLOs with acceptors present. In contrast, LLO hydrolysis by STT3A-OST was negligible. STT3A-OST however may be regulatory, because it suppressed STT3B-OST–dependent fOSs. TREX1, a negative innate immunity factor that diminishes immunogenic fOSs derived from LLOs, acted through STT3B-OST as well. In summary, only STT3B-OST hydrolyzes LLOs, depending upon LLO quality and acceptor site occupancy. TREX1 and STT3A suppress STT3B-OST–dependent fOSs. Without strict kinetic limitations during posttranslational N-glycosylation, STT3B-OST can thus moonlight for LLO hydrolysis. In contrast, the STT3A-OST/translocon complex preserves LLOs for temporally fastidious cotranslational N-glycosylation.

SummaryTREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mutations are associated with autoimmune and autoinflammatory diseases. Biallelic mutations abrogating DNase activity cause autoimmunity by allowing immunogenic self-DNA to accumulate, but it is unknown how dominant frameshift (fs) mutations that encode DNase-active but mislocalized proteins cause disease. We found that the TREX1 C terminus suppressed immune activation by interacting with the ER oligosaccharyltransferase (OST) complex and stabilizing its catalytic integrity. C-terminal truncation of TREX1 by fs mutations dysregulated the OST complex, leading to free glycan release from dolichol carriers, as well as immune activation and autoantibody production. A connection between OST dysregulation and immune disorders was demonstrated in Trex1−/− mice, TREX1-V235fs patient lymphoblasts, and TREX1-V235fs knock-in mice. Inhibiting OST with aclacinomycin corrects the glycan and immune defects associated with Trex1 deficiency or fs mutation. This function of the TREX1 C terminus suggests a potential therapeutic option for TREX1-fs mutant-associated diseases.

Carbohydrates, in addition to their metabolic functions, serve important roles as receptors, ligands, and structural molecules for diverse biological processes. Insight into carbohydrate biology and mechanisms has been aided by metabolic oligosaccharide engineering (MOE). In MOE, unnatural carbohydrate analogs with novel functional groups are incorporated into cellular glycoconjugates and used to probe biological systems. While MOE has expanded knowledge of carbohydrate biology, limited metabolism of unnatural carbohydrate analogs restricts its use. Here we assess metabolism of SiaDAz, a diazirine-modified analog of sialic acid, and its cell-permeable precursor, Ac4ManNDAz. We show that the efficiency of Ac4ManNDAz and SiaDAz metabolism depends on cell type. Our results indicate that different cell lines can have different metabolic roadblocks in the synthesis of cell surface SiaDAz. These findings point to roles for promiscuous intracellular esterases, kinases, and phosphatases during unnatural sugar metabolism and provide guidance for ways to improve MOE.

TREX1/DNASE III, the most abundant 3'-5' DNA exonuclease in mammalian cells, is tail-anchored on the endoplasmic reticulum (ER). Mutations at the N-terminus affecting TREX1 DNase activity are associated with autoimmune and inflammatory conditions such as Aicardi-Goutieres syndrome (AGS). Mutations in the C-terminus of TREX1 cause loss of localization to the ER and dysregulation of oligosaccharyltransferase (OST) activity, and are associated with retinal vasculopathy with cerebral leukodystrophy (RVCL) and in some cases with systemic lupus erythematosus (SLE). Here we investigate mice with conditional expression of the most common RVCL mutation, V235fs, and another mouse expressing a conditional C-terminal mutation, D272fs, associated with a case of human SLE. Mice homozygous for either mutant allele express the encoded human TREX1 truncations without endogenous mouse TREX1, and both remain DNase active in tissues. The two mouse strains are similar phenotypically without major signs of retinal, cerebral or renal disease but exhibit striking elevations of autoantibodies in the serum. The broad range of autoantibodies is primarily against non-nuclear antigens, in sharp contrast to the predominantly DNA-related autoantibodies produced by a TREX1-D18N mouse that specifically lacks DNase activity. We also found that treatment with an OST inhibitor, aclacinomycin, rapidly suppressed autoantibody production in the TREX1 frame-shift mutant mice. Together, our study presents two new mouse models based on TREX1 frame-shift mutations with a unique set of serologic autoimmune-like phenotypes.

Immune checkpoint inhibitors targeting the PD1/PD-L1 and CTLA4 signaling axes have made a significant impact in the treatment of some solid tumors, particularly melanoma and non-small-cell lung carcinoma. While triple-negative breast cancers (TNBC) typically have poorer clinical outcomes due in part to a lack of targeted therapies, they are often more immunogenic than other types of breast cancer.1 Some forms of chemotherapy, including microtubule-targeting agents, have been proposed to enhance the efficacy of immunotherapy,2 which has resulted in the evaluations of combinations of these drugs in preclinical and clinical studies. Most notably, the combination of the PD-L1 inhibitor atezolizumab with the taxane nab-paclitaxel was shown to prolong progression-free survival in patients with metastatic TNBC.3 Although it is known that paclitaxel can function as a TLR4 agonist leading to activation of the innate immune system, there has been little comparison of the immunogenic effects elicited by different clinically used MTAs for the treatment of TNBC. We evaluated the effects of 5 distinct MTAs used for the treatment of TNBC, paclitaxel, docetaxel, ixabepilone, vinorelbine, and eribulin, on the expression of antitumor cytokines and other immunomodulatory genes in myeloid and in TNBC cells. Our results show that the expression signature generated by the microtubule stabilizers, paclitaxel, docetaxel, and ixabepilone, was markedly different from the signature elicited by the microtubule destabilizers eribulin and vinorelbine. The microtubule destabilizers caused upregulation of type 1 interferons and interferon-stimulated-genes (ISGs), effects not seen with the microtubule stabilizers. A time course analysis revealed that the transcriptional upregulation of the ISG CXCL10 by eribulin occurred before the expression of interferons. Mechanistically, microtubule destabilizer-induced expression of c-Jun which could upregulate the early expression of CXCL10 through activation of the AP-1 complex. The eribulin-mediated induction of type 1 interferons is similar to the expression signature promoted by the cGAS-STING pathway that has been shown to enhance the efficacy of checkpoint inhibitors,4 suggesting that microtubule destabilizers, but not stabilizers, can promote signaling through this pathway. Together, these results provide evidence that distinct MTAs have different immune modulatory properties and suggest that the specific immune signatures initiated by the different MTAs need to be considered for their optimal use with checkpoint inhibitors. These studies were funded by Eisai. 1. Liu, Z., Li, M., Jiang, Z. & Wang, X. A Comprehensive Immunologic Portrait of Triple-Negative Breast Cancer. Transl Oncol 11, 311-329, (2018). 2. Emens, L. A. & Middleton, G. The interplay of immunotherapy and chemotherapy: harnessing potential synergies. Cancer Immunol Res 3, 436-443, (2015). 3. Schmid, P. et al. Atezolizumab and Nab-Paclitaxel in Advanced Triple-Negative Breast Cancer. N Engl J Med 379, 2108-2121, (2018). 4. Cheng, N. et al. A nanoparticle-incorporated STING activator enhances antitumor immunity in PD-L1-insensitive models of triple-negative breast cancer. JCI Insight 3, (2018). Citation Format: Charles S Fermaintt, Shayne Hastings, Susan L Mooberry, April L Risinger. Eribulin treatment activates type 1 IFNs to promote a gene expression signature associated with antitumor immunity [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-03.