Fig 2: Inhibiting deISGylase activity is not sufficient to confer IFN sensitivityIndividual unedited control (unedited), mUSP18 C61S KI (C61S), and mUsp18 KO (KO) clones were compared to parental CT26 cells (WT). Unedited control clones underwent same electroporation conditions as C61S KI and KO clones, but no editing was observed at the endogenous mUsp18 locus.(A) Cells were treated with 1000 U/mL murine IFN-α for 24 h prior to cell lysis and whole-cell lysates were analyzed by western blot for levels of mouse ISGylated proteins and free mISG15 (top) or vinculin (bottom).(B) Cell viability (left) and confluence (right) were measured after 72 h or 84 h treatment with the indicated concentration of murine IFN-α, respectively. Viability and confluence were normalized to 0 U/mL IFN control for each cell line. Each data point denotes a mean of n = 2 replicates ± SEM. EC50 determinations for each cell line are presented in Table S1.(C and D) Tumor growth curves of WT, C61S, or KO CT26 tumors injected into wild-type BALB/c mice. For each genotype, 3 individual clones were injected with n = 10 animals per clone (n = 30 animals per genotype). Data are represented as (C) individual animals and (D) mean tumor growth was first determined for each clone, and data for each genotype are represented as the mean of 3 clones ± SEM. Results from nonlinear regression analysis are presented in Table S1.(E) Wild-type USP18 can negatively regulate Type I IFN responses through its catalytic function to deISGylate ISG15-conjuagated proteins and its scaffold function to repress IFNAR signaling by displacing JAK1. Negative IFN regulation by wild-type USP18 protects cancer cells from Type I IFN sensitivity. Complete loss-of-function mutations in USP18 confer sensitivity to Type I IFN, resulting in enhanced ISG induction, ISGylation, and cancer cell growth inhibition. Mutations in USP18 C64S (human) or C61S (mouse) disrupt deISGylase activity, but are not sufficient to confer IFN sensitivity across cancer lineages. However, partial impairments in scaffold function in human USP18 I60N mutants results in partial IFN sensitivity and an intermediate phenotype between WT and hUSP18 KO. Model created with BioRender.com. See also Figures S5–S7.

Fig 3: Disrupting the ISGylation pathway does not rescue IFN sensitivityUBA7 KO pools and UBA7/USP18 double KO (dKO) pools were compared to USP18 KO clone and parental HAP1 cells (WT).(A) Whole-cell lysates were analyzed by western blot for levels of ISGylated proteins and ISG15 (top) or vinculin (bottom). Cells were treated with 1000 U/mL human IFN-β for 24 h prior to cell lysis.(B) Schematic of the enzymatic cascade required for ISGylation (UBA7, UBCH8/UBE2L6, and HERC5) and deISGylation (USP18), created with BioRender.com.(C) Cell confluence and (D) relative mRNA expression of IFIT1 (left) or ISG15 (right) to GAPDH measured by RT-qPCR after 48 h treatment with the indicated concentration of human IFN-β. Each data point denotes mean of n = 3 replicates ± SEM. EC50 determinations for each cell line are presented in Table S1. (C) Confluence was normalized to 0 U/mL IFN control from each cell line.

Fig 4: mUSP18 loss in CT26 tumors inhibits tumor growth in vivo(A and B) In vitro characterization of mUsp18−/− and Rosa26−/− cancer cells prior to implantation. (A) Relative mRNA expression of Ifit1 (left) and Isg15 (right) to Gapdh measured by RT-qPCR after 72 h treatment with the indicated concentration of murine IFN-β. Each data point denotes the mean of n = 3 replicates ± SEM. (B) Confluence was normalized to 0 U/mL IFN control from each cell line.(C–E) Tumor growth curves of (C,E) control Rosa26−/− and (D and E) mUsp18−/− CT26 tumors injected into wild-type BALB/c mice. Data are represented as (C and D) individual animals and (E) mean, n = 12 animals per genotype. (B) EC50 determinations for each cell line are presented in Table S1. (E) Results from nonlinear regression analysis are presented in Table S1 (slopes significantly different for each dataset, p < 0.0001). See also Figures S1 and S2.