Figure 2. PARPi induced STING signaling and interferon responses were alleviated in KRAS mutant cancer cells.

(A) Dox-inducible iKRAS cells were incubated with Dox (2μg/mL) for 48hours, then treated with vehicle or olaparib (5 μM) for another 24 hours with or withdraw Dox. Then cells were collected for western blot with indicated antibodies.

(B-C) Quantification of CCL5, CXCL10, ISG15 and IFI44 expression by RT-qPCR, and CXCL10 secretion levels by ELISA (C) in iKRAS cell in the presence or absence of Dox (2μg/mL) after treatment with vehicle or 5 μM olaparib for 24 hours.

(D and E) Expression of pERK, and STING pathway signaling (STING, pTBK1, pIRF3 and pSTAT1) in both KRAS ^WT (MC38) and KRAS^G12D (CT26) cell lines after vehicle or 5 μM olaparib treatment for 24 hours by Western blot (left panel). CCL5, CXCL10, ISG15 and IFI44 gene expressions were quantified by RT-qPCR (middle panel), and CXCL10 secretion in culture media was quantified by ELISA (right panel). Data are shown as mean±SEM from each of three independent experiments. n=3.

(F) Heatmap showed z-score of relative expression changes of CCL5, CXCL10, ISG15 and IFI44 expression by RT-qPCR in KRAS ^WT (A2780, MC38, NCI-H1793 and OVCAR3) and KRAS^Mut (TOV-21G, HOC7, LOVO, A549, CT26, Pa02C, Pa16C, MACS, HEYA8 and NCI-H441) cell lines treated with 5 μM olaparib for 24 hours (olaparib/vehicle).

(G) Western blot pERK, and STING pathway signaling (STING, pTBK1, pIRF3 and pSTAT1) activation levels in CT26, LOVO HOC7 and NCI-H441 after treatment with vehicle, or trametinib (500nM) for 24 hours.

(H) Quantification of CXCL10 secretion by ELISA in iKRAS cells induced by DOX for 48 hours and subsequently treated with or without 500nM trametinib for 24 hours.

(I) Heatmap showed relative expression changes of CCL5, CXCL10, ISG15 and IFI44 by RT-qPCR in KRAS ^WT (A2780, MC38, NCI-H1793 and OVCAR3) and KRAS^Mut (HOC7, LOVO, CT26, Pa02C, Pa16C, MACS, A549 HEYA8 and NCI-H441) cell lines treated with 500 nM trametinib for 24 hours.

(J) Gene Set Enrichment Analysis (GSEA) identified the IFN-stimulate genes (ISGs) significantly elevated after treating with MEK, ERK inhibitor and iKRAS depletion with Dox withdraw in indicated GEO datasets. The bar graph shows enrichment score (ES) in indicated datasets. The top symbol indicates the suppression methods of MAPK pathway. The p values were calculated by the clusterProfiler package of the R platform (version 3.5.1.; www.r-project.org).

In B-C, data represent mean ± SEM.*p<0.05, **p<0.01, ***p<0.001 calculated by One-way ANOVA. In D-E, H were analysis by Student’s t test.