FIGURE 4 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>Aurora kinase inhibitors activate IFN signaling via secretion of IFN triggered via the MAVS/RIG-I pathway. <b>A,</b> HCT116-<i>IFI27</i> reporter cells secrete primarily type III but not type I IFN in response to DNTMi or AURKi. HCT116 cells were treated for 24 hours or 5 days with indicated drugs and the conditioned medium collected; alternatively, drugs diluted in culture medium were added to empty wells on the same plates. Conditioned media or drugs-only media were then applied to the HCT116-<i>IFI27</i> reporter line and assayed 24 hours later. Significance shown versus the DMSO control for each condition. <b>B,</b> Autocrine signaling in HCT116-<i>IFI27</i> cells is mediated by both type I and type III IFNs, and requires IRF3. HCT116-<i>IFI27</i> reporter cells were generated with CRISPR-mediated KOs of the indicated target genes. They were then treated with 1 µmol/L alisertib or an equivalent volume of DMSO, then luciferase activity at 5 days was normalized to the NTC cell line with DMSO treatment. Significance shown comparing each KO versus the parental with the same treatment. <b>C,</b> IFN induction by AURKi depends on JAK1, NFkB, and MAVS but not TLR3 or STING. HCT116-<i>IFI27</i> reporter cell lines with CRISPR KO of the indicated genes were treated for 5 days with 1 µmol/L alisertib, then luciferase activity measured and normalized to the NTC cell line with DMSO treatment. Significance shown comparing each KO versus the parental control for the same treatment. <b>D,</b> TPM heat maps for all significant genes (<i>P</i>adj < 1e-10 in at least one condition) after alisertib treatments in ctrl and MAVS KO clones. Group of genes that are differentially regulated in MAVS KO clones with alisertib treatment are boxed in red. <b>E,</b> Expanded view of red-boxed region of heat map in D showing IFN signature genes. <b>F,</b> IFN induction by alisertib in the cGAS-deficient line SW1417 depends on MAVS but not STING. NTC, MAVS, or STING KO SW1417 lines were treated with 1 µmol/L alisertib or DMSO, and the resulting conditioned medium, or drugs in media incubated without cells (“no cells”), was collected after 5 days. The conditioned medium was then overlaid 1:1 on the HCT116-<i>IFI27</i> reporter line, then luciferase activity measured 24 hours later. Values are relative to the NTC control with DMSO treatment and significance is shown for each genotype versus the NTC for the same treatment. <b>G,</b> IFN induction by alisertib in the cGAS-deficient line Ls174T depends on MAVS but not STING. NTC, MAVS, or STING KO lines were treated with 1 µmol/L alisertib or DMSO. After 5 days, IFIT1 transcript induction was assessed by qPCR and normalized to NTC#1 treated with DMSO. Values are relative to NTC#1 with DMSO and significance is shown for each genotype versus NTC#1 for the same treatment. <b>H,</b> IFN induction in cGAS-proficient lines HT-29 depends on STING more than MAVS. NTC, MAVS, or STING KO HT-29 lines were treated with 1 µmol/L alisertib or DMSO, and the resulting conditioned medium, or drugs in media incubated without cells (“no cells”), was collected after 5 days. The conditioned medium was then overlaid 1:1 on the HCT116-<i>IFI27</i> reporter line, then luciferase activity measured 24 hours later. Values are relative to the NTC with DMSO treatment and significance is shown for each genotype versus nontargeting control for the same treatment. <b>I,</b> IFN induction in cGAS-proficient lines CT26 depends on STING. Nontargeting, MAVS or STING KO CT26 lines were treated with 1 µmol/L alisertib or DMSO, and after 5 days, <i>Ifnβ</i> transcript induction was assessed by qPCR and expressed relative to the Ntc#1 treated with DMSO, and significance is shown for each genotype versus Ntc#1 for the same treatment. <b>J,</b> IFN induction by AURKi depends on RIG-I but not MDA5. HCT116-<i>IFI27</i> reporter cell lines with CRISPR KOs of RIG-I or MDA5 were treated for 5 days with 1 µmol/L alisertib or DMSO, then nanoluciferase activity measured. Values are relative to the NTC control with DMSO treatment and significance is shown for each genotype versus NTC#1 for the same treatment.</p>