Paclitaxel cytotoxicity in breast tumors depends on multipolar spindle maintenance and preexisting chromosomal instability.

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.

Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the mitotic checkpoint. However, interphase roles of Mad1 that do not impact mitotic checkpoint function remain largely uncharacterized. Here we show that upregulation of Mad1, which is common in human breast cancer, prevents stress-induced stabilization of the tumor suppressor p53 in multiple cell types. Upregulated Mad1 localizes to ProMyelocytic Leukemia (PML) nuclear bodies in breast cancer and cultured cells. The C-terminus of Mad1 directly interacts with PML, and this interaction is enhanced by sumoylation. PML stabilizes p53 by sequestering MDM2, an E3 ubiquitin ligase that targets p53 for degradation, to the nucleolus. Upregulated Mad1 displaces MDM2 from PML, freeing it to ubiquitinate p53. Upregulation of Mad1 accelerates growth of orthotopic mammary tumors, which show decreased levels of p53 and its downstream effector p21. These results demonstrate an unexpected interphase role for Mad1 in tumor promotion via p53 destabilization.

<p>Multinucleated H2B-RFP Tub-GFP MDA-MB-231 Multipolar Division</p>