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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.

During mitosis, sister chromatids attach to microtubules which generate ~ 700 pN pulling force focused on the centromere. We report that chromatin-localized signals generated by Polo-like kinase 1 (Plk1) maintain the integrity of the kinetochore and centromere against this force. Without sufficient Plk1 activity, chromosomes become misaligned after normal condensation and congression. These chromosomes are silent to the mitotic checkpoint, and many lag and mis-segregate in anaphase. Their centromeres and kinetochores lack CENP-A, CENP-C, CENP-T, Hec1, Nuf2, and Knl1; however, CENP-B is retained. CENP-A loss occurs coincident with secondary misalignment and anaphase onset. This disruption occurs asymmetrically prior to anaphase and requires tension generated by microtubules. Mechanistically, centromeres highly recruit PICH DNA helicase and PICH depletion restores kinetochore disruption in pre-anaphase cells. Furthermore, anaphase defects are significantly reduced by tethering Plk1 to chromatin, including H2B, and INCENP, but not to CENP-A. Taken as a whole, this demonstrates that Plk1 signals are crucial for stabilizing centromeric architecture against tension.