AhR diminishes the efficacy of chemotherapy via suppressing STING dependent type-I interferon in bladder cancer

The induction of type-I interferons (IFN-Is) is important for the efficacy of chemotherapy. By investigating the role of amino acids in regulation of IFN-I production under chemo-drug treatment in bladder cancer (BC) cells, we find an inherent AhR-dependent negative feedback to restrain STING signaling and IFN-I production. Mechanistically, in a ligand dependent manner, AhR bridges STING and CUL4B/RBX1 E3 ligase complex, facilitating STING degradation through ubiquitin-proteasome pathway. Inhibition of AhR increases STING levels and reduces tumor growth under cisplatin or STING agonist treatment. Endogenous AhR ligands are mainly consisted of tryptophan (Trp) metabolites; dietary Trp restriction, blocking the key Trp metabolism rate-limiting enzyme IDO1 or inhibition of cellular Trp importation also show similar effect as AhR inhibition. Clinically, BC patients with higher intratumoral expression of AhR or stronger intratumoral Trp metabolism (higher IDO1 or Kyn levels) that lead to higher AhR activation show worse response rate to neoadjuvant chemotherapy (NAC).

Supplementary Fig. 6 AhR worked as an adaptor to facilitate STING ubiquitination through CUL4B-RBX1 E3 complex, related to Fig. 6.  (A-C, E, F, I, K-N).All p-value< 0.05 as statistic difference.Error bars represent Mean ± SD, unless otherwise indicated.(A-H, J).Source data are provided as a Source Data file.

B
Diagram shows Trp metabolites and corresponding metabolic enzymes.C mRNA expression of Trp catabolic enzymes after treated with RT.D Immunoassay for IDO1 expression after treated with cisplatin or RT.E HPLC-MS detection of Kyn levels after treated with cisplatin or RT under indicated medium.F ELISA for IFN-β content in the supernatant after treated with cisplatin or RT combined with Kyn.G Immunoassay for pSTING/STING expression after treated with cGAMP or cGAMP combined Kyn.H Immunoassay for IDO1 knocked-out efficiency.I Immunoassay for representative cGAS-STING related genes expression after treated with oxaliplatin combined with 1-MT.J Immunoassay for STAT1 knockedout efficiency.K Immunoassay for IDO1, pSTING, and STING expression of STAT1 knocked-out cells after indicated treatment.L Immunoassay for IDO1, pSTING, and STING expression of STAT1 knockedout cells or STAT1 knocked-out with overexpress IDO1 cells after treated with cGAMP.M Immunoassay for pSTING and STING expression and IFN-β content in MB49 bearing mice after indicated treatment (n=6).N Effect of diABZI combined with 1-MT on MB49 growth (n=6) (Mean ± SEM).O Immunoassay of pSTING and STING expression in MB49 bearing mice after 1-MT combined indicated treatment (n=6).P Percentage of CD8 + T cells, IFNγ + CD8 + T cells, and IL2 + CD8 + T cells in MB49 bearing mice after treated with cisplatin combined with 1-MT (n=6).P-value by two-tailed t-test (E, F).P-value by two-tailed Wilcoxon (N, P).P-value by One-way ANOVA (M, O).All p-value< 0.05 as statistic difference.Error bars represent Mean ± SD, unless otherwise indicated.Three biologically independent experiments were performed (A, C-L).Source data are provided as a Source Data file.Supplementary Fig.4 Trp-IDO1-Kyn metabolic pathway regulated STING stability through AhR in a ubiquitin-proteasome-dependent manner, related to Fig.4.A mRNA expression of STING after indicated treatment and medium.B Immunoassay for STING expression after Trp-free medium cultured and treated with cisplatin in different time points.C Immunoassay for STING expression after treated with Kyn combined with CQ or MG132.D Coimmunoprecipitation and immunoassay for extracts of cell lysate of IDO1 knocked-out cells by anti-STING antibodies after treated with Kyn and cGAMP.E Coimmunoprecipitation and immunoassay for extracts of cell lysate after cultured with complete medium or Trp-free medium by anti-STING antibodies after treated with Kyn and cGAMP.F Immunoassay for STING expression after indicated treatment.G Immunoassay for STING expression after indicated treatment combined with digitonin.H Coimmunoprecipitation and immunoassay for extracts of cell lysate by anti-STING antibodies after treated with Kyn combined SR1 or CH223191.I Immunoassay for AhR knocked-out efficiency.J mRNA expression of STING remaining after treated with actinomycin (10μM).K Coimmunoprecipitation and immunoassay for extracts of HEK293T cells under indicated treatment and transfected with HA-AhR, MYC-STING by anti-MYC beads.L Immunoassay for pSTING and STING expression after treated with SR1 and CH223191 combined with cGAMP.M Immunoassay for representative cGAS-STING related genes expression after treated with oxaliplatin combined with CH223191.N-O Effect of CH223191 combined with diABZI on MB49 growth (n=6) (Mean ± SEM) (N), pSTING and STING expression and IFN-β production (O) in tumor.P Kaplan-Meier survival curves of MB49 bearing mice after treated with CH223191 combined with diABZI (n=6).Q Kaplan-Meier survival curves of MB49 bearing mice after treated with CH223191 combined with RT (n=6).R Effect of RT combined with CH223191 on MB49 growth (n=6) (Mean ± SEM).S Percentage of CD8 + T cells, IFNγ + CD8 + T cells, and IL2 + CD8 + T cells in MB49 bearing mice after treated with cisplatin combined with CH223191 (n=6).P-value by One-way ANOVA (A, N, O, R).P-value by two-tailed t-test (B, J).P-value by two-tailed Wilcoxon (O, S).P-value by Kaplan-Meier survival analysis (P, Q).All p-value< 0.05 as statistic difference.Error bars represent Mean ± SD, unless otherwise indicated.Three biologically independent experiments were performed (A-M).Source data are provided as a Source Data file.Supplementary Fig.5 STING was ubiquitinated on lysine 236 with K48 linkage by AhR, related to Fig.5.A, B RMSF plot for AhR-STING, AhR-STING-KYN, AhR-STING (dimer), and AhR-STING-KYN (dimer) of the simulation.C The interaction between KYN and AhR for AhR-STING-KYN in the simulation.AhR is colored with marine, STING with magenta, KYN with yellow.The key residues in AhR are shown as marine stick.The orange dashes represent hydrogen bond interaction.The yellow dashes represent π-π conjugation interaction.D, E Schematic presentation of UB and its mutants.K0 denotes UB with all lysine residues mutated to arginine.F, G Coimmunoprecipitation and immunoassay for extracts of cells lysate after treated with Kyn and transfected with HA-AhR (WT) and MYC-STING (WT) by anti-MYC beads.H Alignment of STING amino acid sequences.Highlighted amino acids indicate conserved lysine (K) of STING.I Immunoassay and mRNA expression for STING and IFNB1 after transfected with HA-AhR (WT), and MYC-STING (WT), STING (K236R) and treated with cisplatin.P-value< 0.05 as statistic difference by One-way ANOVA (Mean ± SD).J Sequencing of parental and individual clones of parental SYBC1 cells with knock-in expression of STING (K236R) mutants.Black arrows indicate mutated nucleotides.A mutated amino acid and its wild-type counterpart are indicated by the solid red box.K mRNA expression of IFNB1 in K236R cells after treated with cisplatin or cGAMP (Mean ± SD).P-value< 0.05 as statistic difference by two-tailed t-test.Three biologically independent experiments were performed (F, G, I-K).Source data are provided as a Source Data file.

A- C
Immunoassay for CUL4B (A), Cul4b (B) and Sting/Cul4b (C) knocked-out efficiency.Three biologically independent experiments were performed (A-C).Source data are provided as a Source Data file.Supplementary Fig.7 SLC7A5 acted as critical Trp transporter to regulate STING stability, related to Fig.7.A mRNA expression for indicated genes knocked-down efficiency.B ELISA for IFN-β content in the supernatant after knocked-down indicated transporters and treated with cGAMP.C HPLC-MS detection of Kyn after knocked-down indicated genes and treated with cGAMP or cisplatin.D Immunoassay for SLC7A5 knocked-out efficiency.E ELISA for IFN-β content in the supernatant after knocked-out SLC7A5 and treated with cGAMP or cisplatin.F HPLC-MS detection of Trp remaining in the supernatant after knocked-out SLC7A5 and treated with cGAMP or cisplatin.G Coimmunoprecipitation and immunoassay for extracts of cell lysate in SLC7A5 knocked-out cells and treated with cGAMP by anti-STING antibodies.H Immunoassay for Slc7a5 knocked-out efficiency.I Effect of Slc7a5 knocked-out on MB49 growth (n=6) (Mean ± SEM).J Immunoassay for Sting/Slc7a5 knocked-out efficiency.K, L Effect of diABZI and BCH on MB49 growth (n=6) (Mean ± SEM) (K) and IFN-β production (L) in tumor.M Effect of cisplatin combined with BCH on MB49 growth (n=6) (Mean ± SEM).N Effect of BCH, 1-MT, and CH223191 combined with cisplatin on orthotopic MB49 growth (n=7) (Mean ± SEM).P-value by One-way ANOVA

Table 6 .
Used signature and pathways in this paper.

Table 7 .
Simulation Model Set.