CENP-E inhibition induces chromosomal instability and synergizes with diverse microtubule-targeting agents in breast cancer.

Drugs that perturb microtubules are commonly used to treat breast cancers of all subtypes in both early stage and metastatic disease, but they are only effective in approximately 50% of patients. High concentrations of microtubule-targeting agents can elicit mitotic arrest in cell culture models; however, recent evidence from primary and metastatic breast cancers revealed that they only accumulate at intratumoral levels capable of inducing abnormal multipolar mitotic spindles, not mitotic arrest. While maintenance of multipolar spindles can generate cytotoxic rates of chromosomal instability (CIN), focusing of aberrant multipolar spindles into normal bipolar spindles dramatically reduces CIN and confers resistance to microtubule poisons. Here, we showed that inhibition of the mitotic kinesin CENP-E overcomes resistance caused by focusing multipolar spindles. Clinically relevant microtubule-targeting agents used a mechanistically conserved pathway to induce multipolar spindles without requiring centrosome amplification. Focusing could occur at any point in mitosis, with earlier focusing conferring greater resistance to anti-microtubule agents. CENP-E inhibition increased CIN on focused spindles by generating chromosomes that remained misaligned at spindle poles during anaphase, which substantially increased death in the resulting daughter cells. CENP-E inhibition synergized with diverse, clinically relevant microtubule poisons to potentiate cell death in cell lines and suppress tumor growth in orthotopic tumor models. These results suggest that primary resistance to microtubule-targeting drugs can be overcome by simultaneous inhibition of CENP-E.

Label-free fluorescence lifetime imaging for the assessment of cell viability in living tumor fragments.

Significance: To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM). Aim: Using murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death. Approach: We use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays. Results: Through a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected. Conclusions: These data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments.

Diverse microtubule-targeted anticancer agents kill cells by inducing chromosome missegregation on multipolar spindles.

Microtubule-targeted agents are commonly used for cancer treatment, though many patients do not benefit. Microtubule-targeted drugs were assumed to elicit anticancer activity via mitotic arrest because they cause cell death following mitotic arrest in cell culture. However, we recently demonstrated that intratumoral paclitaxel concentrations are insufficient to induce mitotic arrest and rather induce chromosomal instability (CIN) via multipolar mitotic spindles. Here, we show in metastatic breast cancer and relevant human cellular models that this mechanism is conserved among clinically useful microtubule poisons. While multipolar divisions typically produce inviable progeny, multipolar spindles can be focused into near-normal bipolar spindles at any stage of mitosis. Using a novel method to quantify the rate of CIN, we demonstrate that cell death positively correlates with net loss of DNA. Spindle focusing decreases CIN and causes resistance to diverse microtubule poisons, which can be counteracted by addition of a drug that increases CIN without affecting spindle polarity. These results demonstrate conserved mechanisms of action and resistance for diverse microtubule-targeted agents. Trial registration: clinicaltrials.gov, NCT03393741.

Paclitaxel Induces Micronucleation and Activates Pro-Inflammatory cGAS-STING Signaling in Triple-Negative Breast Cancer.

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.

Chromosomal instability sensitizes patient breast tumors to multipolar divisions induced by paclitaxel.

Paclitaxel (Taxol) is a cornerstone of cancer treatment. However, its mechanism of cytotoxicity is incompletely understood and not all patients benefit from treatment. We show that patients with breast cancer did not accumulate sufficient intratumoral paclitaxel to induce mitotic arrest in tumor cells. Instead, clinically relevant concentrations induced multipolar mitotic spindle formation. However, the extent of early multipolarity did not predict patient response. Whereas multipolar divisions frequently led to cell death, multipolar spindles focused into bipolar spindles before division at variable frequency, and maintaining multipolarity throughout mitosis was critical to induce the high rates of chromosomal instability necessary for paclitaxel to elicit cell death. Increasing multipolar divisions in paclitaxel resulted in improved cytotoxicity. Conversely, decreasing paclitaxel-induced multipolar divisions reduced paclitaxel efficacy. Moreover, we found that preexisting chromosomal instability sensitized breast cancer cells to paclitaxel. Both genetic and pharmacological methods of inducing chromosomal instability were sufficient to increase paclitaxel efficacy. In patients, the amount of pretreatment chromosomal instability directly correlated with taxane response in metastatic breast cancer such that patients with a higher rate of preexisting chromosomal instability showed improved response to taxanes. Together, these results support the use of baseline rates of chromosomal instability as a predictive biomarker for paclitaxel response. Furthermore, they suggest that agents that increase chromosomal instability or maintain multipolar spindles throughout mitosis will improve the clinical utility of paclitaxel.

Mad1 destabilizes p53 by preventing PML from sequestering MDM2.

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.