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Multinucleated H2B-RFP Tub-GFP MDA-MB-231 Multipolar Division

Mononucleated H2B-RFP Tub-GFP MDA-MB-231 Bipolar Division

Taxanes remain one of the most effective medical treatments for breast cancer. Clinical trials have coupled taxanes with immune checkpoint inhibitors in patients with triple-negative breast cancer (TNBC) with promising results. However, the mechanism linking taxanes to immune activation is unclear. To determine if paclitaxel could elicit an antitumoral immune response, we sampled tumor tissues from patients with TNBC receiving weekly paclitaxel (80 mg/m2) and found increased stromal tumor-infiltrating lymphocytes and micronucleation over baseline in three of six samples. At clinically relevant concentrations, paclitaxel can induce chromosome missegregation on multipolar spindles during mitosis. Consequently, post-mitotic cells are multinucleated and contain micronuclei, which often activate cyclic GMP-AMP synthase (cGAS) and may induce a type I IFN response reliant on the stimulator of IFN genes (STING) pathway. Other microtubule-targeting agents, eribulin and vinorelbine, recapitulate this cGAS/STING response and increased the expression of immune checkpoint molecule, PD-L1, in TNBC cell lines. To test the possibility that microtubule-targeting agents sensitize tumors that express cGAS to immune checkpoint inhibitors, we identified 10 patients with TNBC treated with PD-L1 or PD-1, seven of whom also received microtubule-targeting agents. Elevated baseline cGAS expression significantly correlated with treatment response in patients receiving microtubule-targeting agents in combination with immune checkpoint inhibitors. Our study identifies a mechanism by which microtubule-targeting agents can potentiate an immune response in TNBC. Further, baseline cGAS expression may predict patient treatment response to therapies combining microtubule-targeting agents and immune checkpoint inhibitors.

Introduction: Paclitaxel (TaxolTM) is a blockbuster chemotherapy used for solid tumors including breast cancer. In triple-negative breast cancer (TNBC), nab-paclitaxel plus PD-L1 inhibitor, atezolizumab, is now approved based on increased survival compared with nab-paclitaxel alone. We hypothesize that there may be an interaction by which taxane therapy is immunogenic. Paclitaxel induces aberrant cell division on multipolar spindles, which often results in formation of multiple small nuclei. Some of these resemble micronuclei. Here, we test the hypothesis that taxane-induced micronuclei activate cGAS-STING to increase immunogenicity in TNBC. Methods: We evaluate the impact of clinically relevant doses of paclitaxel on TNBC cell lines MDA-MB-231, MDA-MB-453, MDA-MB-468, BT-549 and HCC-1806 on generating micronuclei by immunofluorescence microscopy. Using immunoblotting and qPCR, we evaluate the impact of paclitaxel on cGAS recruitment and activation, STING activation, and interferon-beta production. Furthermore, we co-culture MDA-MB-231 and THP-1 cells to evaluate the impact of paclitaxel on polarization of THP-1 macrophages. We employ CRISPR-mediated cGAS knockout to evaluate its role in mediating these effects. Finally, we test the effect of neoadjuvant paclitaxel (80 mg/m2 qw, 16 treatments/4 cycles) in 4 patients for formation of micronuclei and tumor-infiltrating lymphocytes 20 hours after the third treatment of paclitaxel by H&E. Results: We observe that post-mitotic cells often contain micronuclei. cGAS recruitment is detected in some micronuclei, more commonly in multinucleated cells. With three days of paclitaxel, the cGAS-positive cell population increases from 9% (+/- 1.3) to 34% (+/- 2.4) in MDA-MB-231. Similar trends are observed in other cell lines. In MDA-MB-231 cells, synthesis of 2'3'-cGAMP and IFN-beta is increased after paclitaxel. Upregulation of interferon stimulated genes and phospho-proteins also supports STING activation, which is abolished in cGAS knockout cells. Co-culture of THP-1 macrophages with MDA-MB-231 cells in paclitaxel, or conditioned media from MDA-MB-231 cells pre-treated with paclitaxel, polarizes THP-1 cells from an M0 to M1 phenotype. Furthermore, we observe an increase in micronuclei and TILs after paclitaxel for 3 of 4 patient samples with one patient converting from a plasma cell-rich to a lymphocyte-rich infiltrate. Conclusions: Physiologic levels of paclitaxel can generate micronuclei to activate the cGAS-STING pathway in breast cancer cell lines and to induce a pro-inflammatory interferon-beta driven response. This may present a post-mitotic mechanism for paclitaxel-mediated cancer cell death. These data may provide the mechanistic basis for the high efficacy seen in combining taxanes with immunotherapies. Citation Format: Yang Hu, Ning Jin, Baraa K. Manasrah, Stephanie M. McGregor, Christina Scribano, John B. Tucker, Rachel Yan, Robert F. Lera, Beth A. Weaver, Mark E. Burkard. Paclitaxel-induced micronucleation activates pro-inflammatory cGAS/STING signaling in triple negative breast cancer [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 2248.