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FIGURE 2 from Inhibition of Aurora Kinase Induces Endogenous Retroelements to Induce a Type I/III IFN Response via RIG-I

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posted on 2024-02-26, 14:20 authored by Lisa Choy, Stephen Norris, Xiumin Wu, Ganesh Kolumam, Ari Firestone, Jeffrey Settleman, David Stokoe

Activation of IFN is mediated through inhibition of Aurora B. A, Western blot analysis of the HCT116-IFI27 reporter line treated with either DMSO, 1,000 U/mL IFNα, 1 µmol/L alisertib, or 100 nmol/L decitabine for the indicated amount of time. A total of 20 µg of whole cell lysates per lane were blotted with the indicated antibodies, with vinculin blotted as a loading control. B, qPCR analysis showing induction of IFN and target genes IFNβ, IFNλ1, CXCL10, IFI27, IFIT1, IFIT3 after 5 days treatment of the HCT116-IFI27 reporter line with DMSO, 100 nmol/L decitabine or 1 µmol/L alisertib. Significance is shown versus the DMSO control for each gene. C, Dose–response titration of Aurora A, Aurora B selective drugs on the IFI27 reporter suggests that IFN induction occurs preferentially after inhibition of Aurora B. The Aurora A selective drugs TC-7010 and MK8745, the weakly Aurora A selective drug alisertib, and the Aurora B selective drugs barasertib and GSK1070916 were dosed at 12, 37, 110, 330, 1,000, or 2,500 nmol/L on the HCT116-IFI27 reporter line for 5 days, then luciferase activity measured. Significance is shown for each drug versus the DMSO control at each timepoint. D, Western blot analysis of p-S10-H3 after treatment with alisertib, barasertib, or the Aurora A selective inhibitor MK8745 suggest that induction of the IFI27 reporter coincides with inhibition of P-S10-H3, a biomarker of Aurora B activity. E, Ruxolitinib inhibition of IFI27 reporter induction by Aurora kinase inhibitors demonstrates dependence of IFN induction by Aurora inhibition on JAK signaling. Cells were treated with DMSO, 1 µmol/L alisertib, 830 nmol/L TC-7010, 280 nmol/L barasertib, or 280 nmol/L GSK1070916, without or with 10 µmol/L ruxolitinib for 5 days, then luciferase activity measured. Significance shown for each treatment compared with DMSO alone. F, siRNA against Aurora A versus Aurora B shows that Aurora B inhibition is critical for induction of the IFN response. HCT116-IFI27 reporter cells were transfected with siRNAs to PPIB, or pooled siRNAs (4 per target) to Aurora A or Aurora B, and luciferase activity was measured 5 days later. Significance is shown for each Aurora siRNA versus the PPIB siRNA condition for each dose. G, GFP fluorescent and phase imaging of cells shown in F, with characteristic morphologic changes accompanying siRNA transfection of either Aurora A or Aurora B, but induction of the reporter only with knockdown of Aurora B. H, Western blot analysis showing phospho-Aurora A/B and total Aurora A and B after siRNA knockdown. HCT116-IFI27 reporter cells were transfected with the indicated siRNAs and 48 hours later additionally treated with nocodazole, then 24 hours later whole cell lysates prepared and blotted with Aurora A, Aurora B, or p-T288-Aurora A/T232-B antibodies. Vinculin blotting was also performed as a loading control. I, Individual siRNAs against Aurora A and Aurora B show that siRNAs against Aurora B activate IFI27 reporter. HCT116-IFI27 reporter cells were transfected with indicated siRNAs and reporter activity measured 72 hours later. Significance shown is relative to the siPPIB transfection.

Funding

Calico LLC

History

ARTICLE ABSTRACT

Type I IFN signaling is a crucial component of antiviral immunity that has been linked to promoting the efficacy of some chemotherapeutic drugs. We developed a reporter system in HCT116 cells that detects activation of the endogenous IFI27 locus, an IFN target gene. We screened a library of annotated compounds in these cells and discovered Aurora kinase inhibitors (AURKi) as strong hits. Type I IFN signaling was found to be the most enriched gene signature after AURKi treatment in HCT116, and this signature was also strongly enriched in other colorectal cancer cell lines. The ability of AURKi to activate IFN in HCT116 was dependent on MAVS and RIG-I, but independent of STING, whose signaling is deficient in these cells. MAVS dependence was recapitulated in other colorectal cancer lines with STING pathway deficiency, whereas in cells with intact STING signaling, the STING pathway was required for IFN induction by AURKi. AURKis were found to induce expression of endogenous retroviruses (ERV). These ERVs were distinct from those induced by the DNA methyltransferase inhibitors (DNMTi), which can induce IFN signaling via ERV induction, suggesting a novel mechanism of action. The antitumor effect of alisertib in mice was accompanied by an induction of IFN expression in HCT116 or CT26 tumors. CT26 tumor growth inhibition by alisertib was absent in NSG mice versus wildtype (WT) mice, and tumors from WT mice with alisertib treatment showed increased in CD8+ T-cell infiltration, suggesting that antitumor efficacy of AURKi depends, at least in part, on an intact immune response. Some cancers deactivate STING signaling to avoid consequences of DNA damage from aberrant cell division. The surprising activation of MAVS/RIG-I signaling by AURKi might represent a vulnerability in STING signaling deficient cancers.