Fig. 5: Targeted kinase-inhibition enhances NAR agonist-induced cell death. | Nature Communications

Fig. 5: Targeted kinase-inhibition enhances NAR agonist-induced cell death.

From: MAPK-pathway inhibition mediates inflammatory reprogramming and sensitizes tumors to targeted activation of innate immunity sensor RIG-I

Fig. 5

a Flow-cytometric analysis of cell death induction following 24 h treatment of PC9 cells carrying CRISPRv2 e.v. or sgMAVS with DMSO/osimertinib (300 nM) and IVT4/IVT-GAC (1 ng/µL) (vertical axis displays the normalized percentage of AnnexinV and/or PI-positive cells = % non-viable cells) (mean ± SEM of independent experiments with n = 3 osi + IVT4, n = 4 other treatments). b CTG assay for cell lines pre-treated (48 h) with trametinib (tram, 100 nM) or vemurafenib (vem, 1 µM) and subsequent addition of IVT4/IVT-GAC (1 ng/µL) for 24 h. Viability was normalized to respective IVT-GAC controls (mean ± SEM log10 viability of independent experiments: n = 3 A549, vem A375, Colo205, and n = 4 otherwise). c Flow-cytometric analysis of cell death induction for cell lines pre-treated (48 h) with trametinib (100 nM) or vemurafenib (1 µM) and subsequent addition of IVT4/IVT-GAC (1 ng/µL for 24 h (vertical axis displays the normalized percentage of AnnexinV and/or PI-positive cells = % cell death) (mean ± SEM of independent biological replicates for A375 n = 4, A549 n = 5, Colo205 n = 3). d Flow-cytometric analysis of cell death induction in PC9 cells transfected for 48 h with IRF1 or e.v. followed by the addition of IVT4/IVT-GAC (1 ng/µL) for 24 h (vertical axis displays the percentage of AnnexinV and/or PI-positive cells = Cell death %) (mean ± SEM of n = 3 independent experiments). e Humanized PC9 xenografts treated with osimertinib or vehicle p.o. followed by IVT4 or control IVT-GAC i.t. as shown in the schematic (left, n = 5 mice per arm inoculated with 2 tumors per mouse). Relative tumor volumes are shown on the right (mean ± SEM of tumors treated with osi-IVT4 (n = 9), osi-IVT-GAC (n = 8), veh-IVT4 (n = 6), veh-IVT-GAC (n = 6)). f Flow-cytometry of tumor-infiltrating CD4 and CD8 lymphocytes for TIM3 and PD1 expression after 4 days osimertinib and 6 days IVT4/IVT-GAC. (each data point = one of n = 4 tumors per group, error bars represent mean ± SEM). g Tumor volumes at study end compared to treatment start in mice from (e) (each bar = one tumor). h GSEA on RNA-seq of xenograft tumors in humanized mice after pre-treatment with osimertinib and IVT4/IVT-GAC. (FDR-adj. q-values). Significance was calculated by t tests (a, e) and paired t tests (b) adjusted for multiple testing with Bonferroni–Holm method; ANOVA with Tukey post-hoc tests (c, f), two-way ANOVA adjusting for the mouse in osi or vehicle groups (f) and two-way ANOVA with Tukey post hoc tests (d, g). All tests are two-sided. Source data are provided as a Source Data file.

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