Chromatin bridges, not micronuclei, activate cGAS after drug-induced mitotic errors in human cells - PubMed (original) (raw)

Chromatin bridges, not micronuclei, activate cGAS after drug-induced mitotic errors in human cells

Patrick J Flynn et al. Proc Natl Acad Sci U S A. 2021.

Abstract

Mitotic errors can activate cyclic GMP-AMP synthase (cGAS) and induce type I interferon (IFN) signaling. Current models propose that chromosome segregation errors generate micronuclei whose rupture activates cGAS. We used a panel of antimitotic drugs to perturb mitosis in human fibroblasts and measured abnormal nuclear morphologies, cGAS localization, and IFN signaling in the subsequent interphase. Micronuclei consistently recruited cGAS without activating it. Instead, IFN signaling correlated with formation of cGAS-coated chromatin bridges that were selectively generated by microtubule stabilizers and MPS1 inhibitors. cGAS activation by chromatin bridges was suppressed by drugs that prevented cytokinesis. We confirmed cGAS activation by chromatin bridges in cancer lines that are unable to secrete IFN by measuring paracrine transfer of 2'3'-cGAMP to fibroblasts, and in mouse cells. We propose that cGAS is selectively activated by self-chromatin when it is stretched in chromatin bridges. Immunosurveillance of cells that fail mitosis, and antitumor actions of taxanes and MPS1 inhibitors, may depend on this effect.

Keywords: cGAS; cancer; chemotherapy; interferon; mitosis.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

Fig. 1.

MT stabilizers and MPS1 inhibitors induce IFN secretion through the cGAS–STING axis. (A) Schematic of the luciferase coculture assay for measuring secreted IFN. The assay is as follows: 1) The adherent line is seeded in a 96-well plate, 2) BJ cells are exposed to drug, 3) BJ cells are cultured in drug for multiple days, 4) STING−/− reporter monocytes which express L. luciferase protein under an IFN response element are coseeded with the BJ cells for 18 h, and 5) luciferase is assayed with a plate reader. Luminescence signal reports on paracrine IFN signaling which originates in the BJ cells. (B) Dose–response for IFN signaling measured with the coculture luciferase assay. DTX produces a bell-shaped curve centered around 10 nM. MPS1 inhibitors (MPS1i_1 and MPS1i_2) produce a hyperbolic response. AurkB inhibition did not induce detectable IFN. ΔRLU is the measured signal with the background luminescence detected in vehicle controls subtracted. Different-color markers represent independent experiments. (C) IFN response induced in BJ cells by a panel of A-Ms. The doses for each drug are reported in Table 1. Only MT stabilizers and MPS1 inhibitors induce IFN signaling. Markers represent independent experiments. (D) BJ cells that lack either cGAS or STING do not produce IFN in response to MT stabilizers and MPS1 inhibitors. Different-color markers represent independent experiments. (E) TBK1 inhibition (MRT67307; 500 nM) and JAK 1/2 inhibition (ruxlotinib; 500 nM) suppress IFN induction by MT stabilizers and MPS1 inhibitors. Markers represent independent experiments. (F) BJ cells were treated with A-M for 3 d and stained for phospho-STAT1 (pSTAT1), a marker of IFN signaling. pSTAT1 showed increased expression and nuclear localization after treatment of BJ cells with DTX (20 nM) or MPS1i_1 (20 nM) but not after AurkB inhibition (100 nM). All error bars denote SD. ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 2.

Fig. 2.

Quantification of aberrant chromatin structures and cGAS localization produced by A-Ms. (A) Example nuclear morphologies produced by MPS1 inhibitors or Aurora B kinase inhibitors and visualized with a DNA dye. Aberrant structures are classified as micronuclei, gross nuclei, or chromatin bridges. Chromatin bridges appear as the small thin strands connecting independent nuclear objects. (B) A heatmap that summarizes the observed results for abnormal nuclear structures and IFN signal across different A-Ms. Chromatin bridges, not micronuclei, correlate with IFN signaling. Data were normalized to the corresponding vehicle control value. (C) Images of cGAS-positive micronuclei and gross nuclei produced after mitotic failure. cGAS was visualized with immunofluorescence. (D) Examples of cGAS-positive chromatin bridges. Observed chromatin bridges were either intact (first row), broken on one end (second and third rows), or broken on both ends (fourth row). cGAS was visualized with immunofluorescence. (E) Quantification of cGAS positivity on different types of aberrant chromatin structures produced after either MPS1 inhibition (750 nM; MPS1i_2) or AurkB inhibition (250 nM; barasertib). cGAS-positive micronuclei and gross nuclei showed similar frequencies after treatment with either drug. cGAS-positive chromatin bridges distinguish the two types of A-Ms. Each marker represents an independent experiment in which three to five FOVs were scored. All errors bars denote SD.

Fig. 3.

Fig. 3.

cGAS activation requires cytokinesis and stretching forces on chromatin. (A) A-M combinations antagonize IFN induction. BJ cells were simultaneously treated with an inflammatory dose of MPS1 inhibitor (MPS1_2; 750 nM) and titrations of other A-Ms which interfere with cytokinesis and chromatin-bridge formation. The majority of tested A-Ms show dose-dependent suppression of the IFN signal produced by MPS1 inhibition. CenpE inhibition is not known to interfere with cytokinesis and is considered a negative control. The IFN fraction represents the measured signal relative to MPS1 inhibition alone. Different-color markers represent independent experiments. (B) CytoD, an actin inhibitor, suppressed IFN produced by MPS1 inhibition. Different-color markers represent independent experiments. (C) Cytokinesis interference does not suppress IFN signaling generally. BJ cells were simultaneously treated with an inflammatory dose of ATM inhibitor (KU-55933; 40 μM) and a titration of AurkBi. DNA damage caused by ATM inhibition activated the cGAS–STING–IFN axis irrespective of AurkBi dose. Different-color markers represent independent experiments. (D) Example images of cGAS-positive micronuclei observed after A-M combinations with MPS1i_2 (750 nM). Notably, only CenpE inhibition permitted cGAS-positive chromatin-bridge formation when combined with MPS1 inhibition. This combination also retained IFN signaling. (E) Frequencies of aberrant chromatin structures produced by A-M combinations. A-M combinations which suppressed IFN signal in A also suppressed chromatin-bridge formation. Doses for drug combinations are described in Table 1. Markers represent independent experiments. (F) Model for how A-Ms affect mitotic failure and IFN signal. Only low-dose MT stabilizers and MPS1 inhibitors produce stretched chromatin bridges because they cause chromosome missegregation events without preventing spindle formation or cytokinesis. All A-Ms tested produce cGAS-positive micronuclei. Chromatin-bridge formation is directly perturbed by many A-Ms through targeting spindle formation (AurkAi and KIF11) or cytokinesis (CytoD and AurkBi) or both (CA4). All error bars denote SD. ns, P > 0.05; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 4.

Fig. 4.

A-Ms activate cGAS in cancer lines through chromatin-bridge formation. (A) MDAMB231 and HeLa cells were exposed to either DTX or MPS1_2 for 3 d. IFN signaling was not detected for either cell line in the coculture assay. ΔRLU is the measured signal with the background luminescence detected in vehicle controls subtracted. Different-color markers represent independent experiments. (B) Schematic of the modified coculture luciferase assay, referred to as the triculture assay, which was designed to detect cGAS activation in cancer cells through paracrine cGAMP signaling. (C) MPS1 inhibitors and MT stabilizers activate cGAS in cancer lines. Both MDAMB231 and HeLa cells showed dose-dependent responses to DTX and MPS1 inhibition when measured with the triculture assay. The response curves mirror the shape of IFN induction in the BJ cells. The negative ΔRLU in the DTX MDAMB231 response represents cytotoxicity that reduced signal below the high basal signal. Different-color markers represent independent experiments. (D) MPS1 inhibitors and MT stabilizers activate cGAS across a panel of cancer cells. Cancer lines were treated with either 1 nM DTX, 750 nM MPS1i_2, or 250 nM AurkBi for 3 d. They showed variable levels of IFN response to A-M treatment. Of the cancer lines that showed evidence of cGAS activation in the triculture assay, MPS1 inhibition and DTX treatment induced larger responses than AurkB inhibition. Markers represent independent experiments. (E) AurkB inhibitors reduce cGAS activation in MPS1i-treated cancer cells. Cancer cells were treated with MPS1i_2 (750 nM) in combination with AurkBi (250 nM) for 3 d. Markers represent independent experiments. (F) HeLa cells treated with different A-Ms. cGAS-positive chromatin bridges are produced after treatment with MPS1i and DTX but not by AurkBi or the combination of AurkBi and MPS1i_2. Notably, conditions which exhibited minimal cGAS activation contained abundant cGAS-positive micronuclei. All error bars denote SD. **P ≤ 0.01, ****P ≤ 0.0001.

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