Fig. 1.

Targeting the DNA damage pathway induced the expression of immune stimulatory and immune suppressive molecules via cGAS- and IRF3-dependent cytosolic DNA response. (A) LY2603618 and VE822 induced cytosolic DNA accumulation in 786-O and RCC4 cells. Double-stranded DNA was detected with the fluorescent Picogreen reagent. (B) Heatmap showing LY2603618 induced gene expression that both positively and negatively modulate tumor immune microenvironment. 786-O cells were untreated (Control) or treated with 25nM LY2603618 for 48 hrs. Samples from two independent experiments (Exp1 and Exp2) were subjected to RNAseq. (C) LY2603618 and VE822 induced the expression of inflammatory cytokines (CCL5, CXCL10, and IFNB1) and immune checkpoint ligands (CD274 and PDCD1LG2) in 786-O and RCC4 cells. (D) LY2603618 and VE822 induced PD-L1 protein expression in 786-O and RCC4 cells. (E) cGAS knockdown reduced IRF3 phosphorylation, γH2AX, and PD-L1 expression in response to LY2603618 and VE822. cGAS knockdown 786-O stable cell line #06 was used. (F) IRF3 knockdown reduced γH2AX and PD-L1 expression in response to LY2603618 and VE822. IRF3 knockdown 786-O stable cell line #19 was used. (G) cGAS knockdown reduced CCL5, CXCL10, CD274, and PDCD1LG2 mRNA expression in response to LY2603618 and VE822. (H) IRF3 knockdown reduced CCL5, CXCL10, CD274, and PDCD1LG2 mRNA expression in response to LY2603618 and VE822. RCC parental cells (786-O cells, RCC4 cells), 786-O cells stably expressing control shRNA, cGAS shRNA, or IRF3 shRNA were treated with 25nM LY2603618 or 2.5μM VE822 for 48 hrs. Con, Untreated control; LY, LY2603618; VE, VE822. Protein expression was analyzed by immunoblot using antibodies against cGAS, IRF3, P-IRF3, γH2AX, PD-L1, and GAPDH. CCL5, CXCL10, IFNB1, CD274, and PDCD1LG2 mRNA levels were detected using real-time PCR. Data represent mean±s.d., n= 3. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001, compared with untreated control in C, with control knockdown cells in each treatment condition in G and H.