Deregulation of STING Signaling in Colorectal Carcinoma Constrains DNA Damage Responses and Correlates With Tumorigenesis

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1 Cell Reports Supplemental Information Deregulation of STING Signaling in Colorectal Carcinoma Constrains DNA Damage Responses and Correlates With Tumorigenesis Tianli Xia, Hiroyasu Konno, Jeonghyun Ahn, and Glen N. Barber

2 Supplemental Figures htert FHC SW1116 LS13 LS17T SW8 SW98 SW117 LoVo SW8 Figure S1, Related to Figure 1. Transfection efficiency into Colon Cancer Cell Lines were monitor with FITC 3 hours post fectamine transfection under fluorescent microscopy. Images shown are at 6X. Bar size, 5µm.

3 A B 8 mock polyi:c C mock polyi:c 7 CXCL1 Fold Changes IFNB Fold Changes 3 FHC SW1116 LS13 SW8 SW117 LOVO SW8 CTRL htert polyi:c IRF3 translocation CTRL D polyi:c p65 translocation 1µm Figure S, Related to Figure 1 and. Normal and colon cancer cells were transfected with 3µg/ml polyic or or mock transfected for 3 hour followed by qpcr analysis of human IFNB (A) and CXCL1 (B) expression. Data is representative of at least two independent experiments. Cells similarly treated as A were subjected to Immunofluorescence Microscopy analysis of IRF3 translocation (C) and p65 translocation (D). Representative images are shown at original magnification, 16X. Bar size, 1µm.

4 untreated HSV1γ3.5 untreated HSV1γ3.5 untreated HSV1γ3.5 untreated HSV1γ3.5 untreated HSV1γ3.5 IFNB Fold Changes CXCL1 Fold Changes IFNB Fold Changes A htert FHC SW1116 LoVo sirna: NS ST NS ST NS ST NS ST NS ST STING β-actin B si-nt si-sting htert FHC SW1116 LoVo C si-nt si-sting htert FHC SW1116 LoVo D si-nt si-sting htert FHC SW1116 LoVo Figure S3, Related to Figure 1 and. (A) Normal and colon cancer cells were treated with non-specific sirna (si-nt) or STING sirna (si-sting) for 3 days. Cells were then analyzed for STING sirna efficiency by immunoblot. (B) sirna treated Cells same as A were transfected with at 3µg/ml for 3 hours followed by qpcr analysis for IFNB expression. (C) qpcr analysis for CXCL1 expression in cells same as B. (D) sirna treated Cells same as A were infected with HSVγ3.5 at M.O. I. 5 for 3 hours. Cells were then analyzed by qpcr for IFNB expression. Data is representative of at least two independent experiments. Error bars indicate s.d., p<.5;, p<.1; Student s t-test.

5 Relative Level of cgas 8 Normal Adenocarcinoma of Colon ST. I ST. II ST. III ST. IV Figure S, Related to Figure 3. cdna from 5 normal human colon tissues and 3 human colon cancers of various stages were analyzed by qpcr for cgas expression.

Relative cgas Expression Level hifnb Fold Changes IL1B Fold Changes A CpG Islands in MB1D1 Promoter -3 - -1-6 + CGI1: start=1113, end=1759, %GC=51.8, Obs/Exp=.

6 Relative cgas Expression Level hifnb Fold Changes IL1B Fold Changes A CpG Islands in MB1D1 Promoter CGI1: start=1113, end=1759, %GC=51.8, Obs/Exp=.699, Length=67 CGI: start=9, end=1, %GC=59.8, Obs/Exp=.773, Length=193 B htert FHC SW SW8 LS17T SW Unmethylated Methylated Not Sequenced C untreated 5AZADC SAHA BIX19 D E htert SW8 LS17T htert SW8 LS17T Figure S5, Related to Figure 3. (A) Schematic representation of CpG Islands located in the proximal promoter regions of cgas. (B) Bisulfite sequencing analysis of cgas promoter region. Each box represent s one CpG dinucleotide located within the promoter region indicated by the position marker at the bottom. Red represents methylated CpG; Blue represents unmethylated CpG; White, not sequenced). (C) Colon cancer cells were treated with 5AZADC (DNA methyltransferase inhibitor), SAHA (histone deacetylase inhibitor) and BIX19 (histone-lysine methyltransferase inhibitor) at 1µM for 5 days. cgas expression were then examined by qpcr. (D) IFNB qpcr analysis of cells (same as in Figure 3C) treated with 5AZADC followed by dsdna transfection at 3µg/ml for 3 hours. (E) IL1B qpcr analysis of cells same as D. Data is representative of at least two independent experiments. Error bars indicate s.d., p<.5;, p<.1; Student s t-test.

7 Average Tumor Diameter (mm) Average Tumor Diameter (mm) htert SW1116 LS13 LS17T SW8 SW117 SW8 htert SW1116 LS13 LS17T SW8 SW117 SW8 # of Viable Cells X1e5 # of Viable Cells X1e5 htert SW1116 LS13 LS17T SW8 SW117 SW8 htert SW1116 LS13 LS17T SW8 SW117 SW8 Vaccinia(1E6 PFU/ml) Vaccinia(1E6 PFU/ml) A M.O.I. 1 B M.O.I. 1 cgas positive cgas negative STING STING positive negative cgas positive cgas negative STING STING positive negative C 6 5 M.O.I. 1 hr hr 8hr D 6 5 M.O.I. 1 hr hr 8hr cgas positive cgas negative STING STING positive negative cgas positive cgas negative STING STING positive negative E PBS (n=) Vaccinia (n=3) (cgas pos.) F PBS (n=) Vaccinia (n=3) (cgas neg.) p= Days p= Days Figure S6, Related to Figure and 7. Normal and Colon Cancer Cells were infected with Vaccinia Virus at M.O.I. 1 (A) or M.O.I. 1 (B) for hours, and viral replication was analyzed by standard plaque assay in Vero cells. Cells were infected with Vaccinia Virus at M.O.I. 1 (C) or M.O.I. 1 (D), and cell viability was analyzed by trypan blue staining hours and 8 hours later. Colon Cancer Cell (E) or (F) xenograft tumors were generated in the right flank of nude Balb/c mice. When tumors had reached approximately.5 cm in diameter, tumors were injected every other day a total of three times (arrows) with 1E7 PFU Vaccinia Virus in 5 µl PBS (N=3) or 5 µl PBS only (N=) and tumor growth measured every other day. Statistical analysis was carried out comparing the two treatment groups at the last time point using the unpaired Student s t-test. P values are as indicated.

8 IFNβ(IU/ml) A hsting B β-actin hsting β-actin C 5 5 Transformed vs Primary Cells mock polyic Figure S7, Related to Figure 6. STING expression and dsdna induced Interferon β production in human cancer cell lines. (A) Immunoblot of STING in various transformed or cancer derived human cell lines (as indicated in the top row). Normal HUVEC cells was used as positive control. (B) Northern blot analysis of STING mrna expression in cell lines same as A. HUVEC was used as positive control. (C), ELISA analysis of Interferon β production in the media of cells (same as A) transfected with 3µg/ml polyic or or mock transfected for 16 hours. Normal human cells (PASMC, NHDF-ad and htert) were included as positive controls. Data is representative of at least two independent experiments. Error bars indicate s.d.

9 SIINFEKL specific IFNg (pg/ml) Tumor volume (mm3) A WT-PBS WT-HSVg3.5 SKO-PBS SKO-HSVg Days B WT SKO PBS HSV-g3.5 Figure S8, Related to Figure 7. (A) B16-OVA cells (5X1 5 /mouse) were subcutaneously injected into C57/BL6 WT and Sting knockout (SKO) mice on day. On days 7, 9, 11, mice were injected intratumorally with HSV1γ3.5 (5X1 5 PFU/mouse) and tumor volume was monitored every other day. (B) ELISA of IFNγ in 1X1 6 splenocytes from mice same as A and stimulated with SIINFEKL peptides for 8 hours. Data presented is the mean SD of to 6 mice. Error bars indicate s.d.;, p<.5; Student s t-test.

10 Table S1, Related to Figure 1. Sequencing of STING in Colon Cancer Cell Lines Sample Name Age Gender Ethnicity Colon Cancer Grade Target Region Position in Target Ref. Base Variant Found Variant Name Variant Class Variant Function Reference Codon Variant Codon FHC normal TMEM173_Exon3 1 G S rs7797 SNP Coding, Synon. GTG=Val GTC=Val SW male Caucasian Dukes' type A, grade III TMEM173_Exon3 1 G C rs7797 SNP Coding, Synon. GTG=Val GTC=Val LS13 65 female Caucasian Dukes' type B TMEM173_Exon3 1 G C rs7797 SNP Coding, Synon. GTG=Val GTC=Val TMEM173_Intron1 16 C T rs85911 SNP UTR LS17T 58 female Caucasian Dukes' type B TMEM173_Exon3 1 G C rs7797 SNP Coding, Synon. GTG=Val GTC=Val TMEM173_Exon3 1 G S rs7797 SNP Coding, Synon. GTG=Val GTC=Val TMEM173_Exon3 1 G R rs SNP Coding, Non-Synon. CGC=Arg CAC=His TMEM173_Intron 1 G R rs7387 SNP Intronic SW8 5 male Caucasian Dukes' type B TMEM173_Exon6 169 G S rs SNP Coding, Non-Synon. GGT=Gly GCT=Ala TMEM173_Intron6_3 3 A R rs75766 SNP Intronic TMEM173_Exon7 119 G R rs7388 SNP Coding, Non-Synon. CGG=Arg CAG=Gln TMEM173_Intron7_ 5 G S rs SNP Intronic TMEM173_Intron1 16 C Y rs85911 SNP UTR SW98 81 female Caucasian Dukes' type C, grade III TMEM173_Exon3 1 G S rs7797 SNP Coding, Synon. GTG=Val GTC=Val TMEM173_Exon6 175 A R rs SNP Coding, Non-Synon. CAT=His CGT=Arg SW female Caucasian Dukes' type C, grade III TMEM173_Exon3 1 G S rs7797 SNP Coding, Synon. GTG=Val GTC=Val TMEM173_Exon6 175 A R rs SNP Coding, Non-Synon. CAT=His CGT=Arg TMEM173_Exon3 1 G S rs7797 SNP Coding, Synon. GTG=Val GTC=Val TMEM173_Exon3 1 G R rs SNP Coding, Non-Synon. CGC=Arg CAC=His TMEM173_Intron 1 G R rs7387 SNP Intronic LOVO 56 male Dukes' type C, grade IV TMEM173_Exon6 169 G S rs SNP Coding, Non-Synon. GGT=Gly GCT=Ala TMEM173_Intron6_3 3 A R rs75766 SNP Intronic TMEM173_Exon7 119 G R rs7388 SNP Coding, Non-Synon. CGG=Arg CAG=Gln TMEM173_Intron7_ 5 G S rs SNP Intronic TMEM173_Intron1 16 C Y rs85911 SNP UTR SW8 8 female Caucasian Dukes' type C, grade IV TMEM173_Exon3 1 G C rs7797 SNP Coding, Synon. GTG=Val GTC=Val TMEM173_Exon3 6 A R Novel SNP Coding, Non-Synon. AG=Arg GG=His female Caucasian colorectal adenocarcinoma TMEM173_Exon3 1 G C rs7797 SNP Coding, Synon. GTG=Val GTC=Val adult male colorectal carcinoma TMEM173_Exon3 1 G C rs7797 SNP Coding, Synon. GTG=Val GTC=Val Colo5 7 male Caucasian Dukes' type D TMEM173_Exon3 1 G S rs7797 SNP Coding, Synon. GTG=Val GTC=Val TMEM173_Exon6 175 A R rs SNP Coding, Non-Synon. CAT=His CGT=Arg

11 Table S, Related to Figure 3. Sequencing of cgas in Colon Cancer Cell Lines Sample Name Age Gender Ethnicity Colon Cancer Grade Target Region Position in Target Ref. Base Variant Found Variant Name Variant Class Variant Function Reference Codon Variant Codon MB1D1_Exon5_ 7 G A rs SNP Coding, Synon. AAG=Lys AAA=Lys MB1D1_Exon1_1 3 C A rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn FHC normal MB1D1_Exon1_3 11 T C rs969 SNP Coding, Synon. CCT=Pro CCC=Pro MB1D1_Exon 15 C M rs61913 SNP Coding, Non-Synon. CCT=Pro CAT=His MB1D1_Exon 3 C Y rs SNP Coding, Synon. ATC=Ile ATT=Ile MB1D1_Exon1_1 3 C A rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn SW male Caucasian Dukes' type A, grade III MB1D1_Exon1_3 11 T C rs969 SNP Coding, Synon. CCT=Pro CCC=Pro MB1D1_Exon 15 C A rs61913 SNP Coding, Non-Synon. CCT=Pro CAT=His MB1D1_Exon5_ 7 G A rs SNP Coding, Synon. AAG=Lys AAA=Lys MB1D1_Exon1_1 3 C A rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn MB1D1_Exon1_ 11 C M rs SNP Coding, Non-Synon. GCG=Ala GAG=Glu LS13 65 female Caucasian Dukes' type B MB1D1_Exon1_3 11 T C rs969 SNP Coding, Synon. CCT=Pro CCC=Pro MB1D1_Exon 15 C A rs61913 SNP Coding, Non-Synon. CCT=Pro CAT=His MB1D1_Exon5_ 7 G R rs SNP Coding, Synon. AAG=Lys AAA=Lys MB1D1_Exon1_1 3 C A rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn LS17T 58 female Caucasian Dukes' type B MB1D1_Exon1_3 11 T C rs969 SNP Coding, Synon. CCT=Pro CCC=Pro MB1D1_Exon 15 C A rs61913 SNP Coding, Non-Synon. CCT=Pro CAT=His MB1D1_Exon5_ 7 G A rs SNP Coding, Synon. AAG=Lys AAA=Lys MB1D1_Exon1_1 33 T - rs31338 Deletion UTR SW8 5 male Caucasian Dukes' type B MB1D1_Exon1_1 3 C M rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn MB1D1_Exon1_3 11 T Y rs969 SNP Coding, Synon. CCT=Pro CCC=Pro MB1D1_Exon5_ 7 G R rs SNP Coding, Synon. AAG=Lys AAA=Lys MB1D1_Exon1_1 3 C M rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn SW98 81 female Caucasian Dukes' type C, grade III MB1D1_Exon1_3 11 T Y rs969 SNP Coding, Synon. CCT=Pro CCC=Pro MB1D1_Exon 15 C M rs61913 SNP Coding, Non-Synon. CCT=Pro CAT=His MB1D1_Exon5_ 7 G A rs SNP Coding, Synon. AAG=Lys AAA=Lys MB1D1_Exon1_1 3 C A rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn SW female Caucasian Dukes' type C, grade III MB1D1_Exon1_3 11 T C rs969 SNP Coding, Synon. CCT=Pro CCC=Pro MB1D1_Exon 15 C A rs61913 SNP Coding, Non-Synon. CCT=Pro CAT=His MB1D1_Exon5_ 7 G A rs SNP Coding, Synon. AAG=Lys AAA=Lys MB1D1_Exon1_1 33 T HD rs31338 Deletion UTR LOVO 56 male Dukes' type C, grade IV MB1D1_Exon1_1 3 C M rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn MB1D1_Exon1_3 11 T Y rs969 SNP Coding, Synon. CCT=Pro CCC=Pro MB1D1_Exon5_ 7 G R rs SNP Coding, Synon. AAG=Lys AAA=Lys MB1D1_Exon1_1 33 T HD rs31338 Deletion UTR SW8 8 female Caucasian Dukes' type C, grade IV MB1D1_Exon1_1 3 C M rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn MB1D1_Exon5_ 7 G R rs SNP Coding, Synon. AAG=Lys AAA=Lys female Caucasian colorectal adenocarcinoma MB1D1_Exon1_1 3 C M rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn MB1D1_Exon1_1 33 T HD rs31338 Deletion UTR MB1D1_Exon1_1 3 C A rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn adult male colorectal carcinoma MB1D1_Exon1_3 11 T C rs969 SNP Coding, Synon. CCT=Pro CCC=Pro MB1D1_Exon 15 C M rs61913 SNP Coding, Non-Synon. CCT=Pro CAT=His MB1D1_Exon 15 C S Novel SNP Coding, Non-Synon. CTA=Leu GTA=Val MB1D1_Exon5_ 7 G R rs SNP Coding, Synon. AAG=Lys AAA=Lys MB1D1_Exon1_1 3 C A rs935 SNP Coding, Non-Synon. ACC=Thr AAC=Asn Colo5 7 male Caucasian Dukes' type D MB1D1_Exon1_3 11 T C rs969 SNP Coding, Synon. CCT=Pro CCC=Pro MB1D1_Exon 15 C A rs61913 SNP Coding, Non-Synon. CCT=Pro CAT=His MB1D1_Exon5_ 7 G A rs SNP Coding, Synon. AAG=Lys AAA=Lys

12 Table S3, related to Figure 6. IHC analysis of STING and cgas in Colon Cancer Tissue Microarray Type Stage Position Age Sex Diagnose STING cgas Area Intensity H-Score Status Area Intensity H-Score Status I E5 59 Female Adenocarcinoma of colon IIA B 68 Male Adenocarcinoma of colon IIA C 69 Female Adenocarcinoma of colon, mucinous IIA C 66 Male Adenocarcinoma of colon IIA D1 75 Male Adenocarcinoma of rectum IIA D5 83 Female Adenocarcinoma of colon IIA F 57 Female Adenocarcinoma of colon IIA H3 7 Female Adenocarcinoma of colon IIB G5 58 Female Adenocarcinoma of colon IIB H5 75 Male Adenocarcinoma of colon IIIA H1 71 Female Adenocarcinoma of colon, metastatic IIIB B1 79 Male Adenocarcinoma of colon IIIB C5 Female Adenocarcinoma of colon IIIB E 5 Male Adenocarcinoma of colon IIIB F1 6 Female Adenocarcinoma of colon IIIB F5 78 Male Adenocarcinoma of colon, mucinous IIIB G 89 Female Adenocarcinoma of colon IIIC C1 55 Female Adenocarcinoma of colon IIIC D3 38 Male Adenocarcinoma of colon Tumor IIIC F 5 Male Adenocarcinoma of colon IIIC G1 6 Male Adenocarcinoma of colon IIIC G 76 Female Adenocarcinoma of colon IIIC G3 56 Male Adenocarcinoma of colon, mucinous IIIC H 6 Female Adenocarcinoma of colon IV A1 7 Male Adenocarcinoma of colon, metastatic IV A3 36 Male Adenocarcinoma of colon IV A 57 Female Adenocarcinoma of colon, metastatic IV A5 66 Female Adenocarcinoma of colon, metastatic IV B3 77 Male Adenocarcinoma of colon, metastatic IV B 7 Male Adenocarcinoma of colon, metastatic IV B5 9 Female Adenocarcinoma of colon, metastatic IV D 7 Male Adenocarcinoma of colon IV D 6 Male Adenocarcinoma, metastatic Not Reported A Adenocarcinoma of colon Not Reported E1 Adenocarcinoma of colon Not Reported E3 Adenocarcinoma of colon Not Reported E Adenocarcinoma of colon Not Reported F3 Adenocarcinoma of colon Not Reported H Adenocarcinoma of colon I1 71 Female Within normal limits I3 7 Female Within normal limits I5 75 Male Within normal limits Normal I 6 Female Within normal limits J1 3 Female Within normal limits J 71 Female Within normal limits J 7 Male Within normal limits J5 31 Female Within normal limits Statistical analysis of STING/cGAS expression in TMA-IHC STAGE STING expression cgas expression H-Score p-value H-Score p-value Normal 8.5±1. (n=8) N/A 7.1±1.6 (n=8) N/A I 9 (n=1) N/A 9 (n=1) N/A II 7±1.5 (n=9).96.9±3. (n=9).5 III 6.3±3. (n=1).119.±3.7 (n=1).6 IV 5.8±. (n=9).1 3.6±3.9 (n=9).3 Unkown Stage 6.8±.8 (n=6).33 5.±3 (n=6).136

Irf1 fold changes (D) 24h 48h. p-p65. t-p65. p-irf3. t-irf3. β-actin SKO TKO 100% 80% 60% 40% 20%

Irf1 fold changes (D) 24h 48h. p-p65. t-p65. p-irf3. t-irf3. β-actin SKO TKO 100% 80% 60% 40% 20% Irf7 Fold changes 3 1 Irf1 fold changes 3 1 8h h 8h 8h h 8h p-p6 p-p6 t-p6 p-irf3 β-actin p-irf3 t-irf3 β-actin TKO TKO STKO (E) (F) TKO TKO % of p6 nuclear translocation % % 1% 1% % % p6 TKO % of IRF3