FIGURE 2 from Inhibition of Aurora Kinase Induces Endogenous Retroelements to Induce a Type I/III IFN Response via RIG-I
Authors: Choy Lisa , Norris Stephen , Wu Xiumin , Kolumam Ganesh , Firestone Ari , Settleman Jeffrey , Stokoe David 
Published: February 2024

<p>Activation of IFN is mediated through inhibition of Aurora B. <b>A,</b> Western blot analysis of the HCT116-<i>IFI27</i> 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>B,</b> qPCR analysis showing induction of IFN and target genes <i>IFNβ</i>, <i>IFNλ1</i>, <i>CXCL10</i>, <i>IFI27</i>, <i>IFIT1</i>, <i>IFIT3</i> after 5 days treatment of the HCT116-<i>IFI27</i> reporter line with DMSO, 100 nmol/L decitabine or 1 µmol/L alisertib. Significance is shown versus the DMSO control for each gene. <b>C,</b> Dose–response titration of Aurora A, Aurora B selective drugs on the <i>IFI27</i> 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-<i>IFI27</i> reporter line for 5 days, then luciferase activity measured. Significance is shown for each drug versus the DMSO control at each timepoint. <b>D,</b> Western blot analysis of p-S10-H3 after treatment with alisertib, barasertib, or the Aurora A selective inhibitor MK8745 suggest that induction of the <i>IFI27</i> reporter coincides with inhibition of P-S10-H3, a biomarker of Aurora B activity. <b>E,</b> Ruxolitinib inhibition of <i>IFI27</i> 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. <b>F,</b> siRNA against Aurora A versus Aurora B shows that Aurora B inhibition is critical for induction of the IFN response. HCT116-<i>IFI27</i> 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. <b>G,</b> 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. <b>H,</b> Western blot analysis showing phospho-Aurora A/B and total Aurora A and B after siRNA knockdown. HCT116-<i>IFI27</i> 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. <b>I,</b> Individual siRNAs against Aurora A and Aurora B show that siRNAs against Aurora B activate <i>IFI27</i> reporter. HCT116-<i>IFI27</i> reporter cells were transfected with indicated siRNAs and reporter activity measured 72 hours later. Significance shown is relative to the siPPIB transfection.</p>