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FIGURE 4 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

Aurora kinase inhibitors activate IFN signaling via secretion of IFN triggered via the MAVS/RIG-I pathway. A, HCT116-IFI27 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-IFI27 reporter line and assayed 24 hours later. Significance shown versus the DMSO control for each condition. B, Autocrine signaling in HCT116-IFI27 cells is mediated by both type I and type III IFNs, and requires IRF3. HCT116-IFI27 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. C, IFN induction by AURKi depends on JAK1, NFkB, and MAVS but not TLR3 or STING. HCT116-IFI27 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. D, TPM heat maps for all significant genes (Padj < 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. E, Expanded view of red-boxed region of heat map in D showing IFN signature genes. F, 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-IFI27 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. G, 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. H, 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-IFI27 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. I, 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, Ifnβ 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. J, IFN induction by AURKi depends on RIG-I but not MDA5. HCT116-IFI27 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.

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.