Fig 2: Frequency of RIG-I+ tumor-infiltrating CD8+ T cells associates with the poor prognosis for patients with colon cancer.(A–F) Publicly available scRNA-Seq data from 12 individuals with CRC were analyzed, focusing on tumor-infiltrating CD8+ T cells and CD8+ T cells from adjacent nonmalignant tissues. Data are from GSE108989. Box plot showing the expression of RIG-I in CD8+ T cells from adjacent nonmalignant tissues (Normal) or tumor tissues (Tumor) (A). Violin plot showing the expression of RIG-I in indicated subsets of CD8+ T cells (B). GSEA plots showing enriched T cell activation (C), IFN-γ response (D), apoptosis by serum deprivation (E) and STAT5a target gene signatures (F) in tumor-infiltrating CD8+ T cells with high versus low RIG-I expression. (G and H) Slides of tumor tissue from colorectal carcinoma patients were costained by RIG-I antibody (green) and CD8 antibody (red). DAPI was used to visualize the nuclei (blue). Representative images (G) and the proportion of CD8+RIG-I+ cells of CD8+ cells from stage I–III or stage IV patients were shown (H). (I) The percentages of CD8+ T cells in nucleated cells for RIG-I+ CD8+ low group or RIG-I+ CD8+ high group are shown. (J) Kaplan-Meier plot depicting OS of patients from RIG-I+CD8+–low group or RIG-I+CD8+–high group are shown. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, by unpaired Student’s t test (A, B, H, and I) or log-rank test (J).

Fig 3: RIG-I restrains CD8+ T cell survival and cytotoxicity by dampening HSP90-mediated protective effect on STAT5.(A) Indicated protein levels of splenic naive CD8+ T cells after 48 hours anti-CD3/CD28 stimulation were analyzed by Western blotting. (B) GSEA of RNA-Seq (same as Figure 6, D–G) comparing in vitro activated CD8+ T cells as indicated are shown. (C) Empirical cumulative distribution function for change in expression of all genes (black) expressed in in vitro activated Rig-I–/– CD8+ T cells (change relative to that in Rig-I+/+ CD8+ T cells) and for subsets of genes upregulated (red) or downregulated (blue) by overexpression of constitutively active STAT5 in CD8+ T cells. (D and E) Splenic naive CD8+ T cells were stimulated by anti-CD3/CD28 and STAT5-IN-1 (10 µM) for 2 days and analyzed by flow cytometry. (F and G) Cell lysates of in vitro stimulated CD8+ T cells were immunoprecipitated with dynabeads-coupled control IgG or RIG-I antibody (F). Cell lysates of naive Rig-I+/+ or Rig-I–/– CD8+ T cells stimulated with anti-CD3/CD28 for 2 days were immunoprecipitated with dynabeads-coupled control IgG or STAT5 antibody (G). The precipitates were then probed with indicated antibodies. (H) Lysates of 293T cells cotransfected with indicated plasmids were immunoprecipitated with HA antibody and analyzed by immunoblot with indicated antibodies. (I) Indicated protein levels of MC38 tumor-infiltrating CD45+CD8+ T cells were analyzed by Western blotting. (J) Mice s.c. inoculated with MC38 cells were treated with STAT5-IN-1. Schematic diagram and tumor growth curve are shown (n = 5 per group). Data are representative of at least 2 independent experiments and expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by 1-way ANOVA (D and E) or 2-way ANOVA (J). The same batch of samples were run contemporaneously in different gels for A and I.

Fig 4: Sitravatinib exerts potent anti-tumor activities against FLT3-ITD AML cell lines. A The IC50 values of each drug for FLT3-WT (THP1, HL60, U937, OCI-AML2, OCI-AML3) or FLT3-ITD (MV4-11 and MOLM13) AML cell lines. Data are mean ± standard error from three independent experiments. B The percentage of cells in different cell cycle phases after 24-h treatment with various concentrations of sitravatinib (S) or gilteritinib (G). Error bars indicate mean ± standard error, n = 3 independent experiments for each cell line. C Apoptotic cell populations of MV4-11 and MOLM13 cell lines after 48-h treatment with sitravatinib (S) or gilteritinib (G). Error bars indicate mean ± standard error, n = 3 independent experiments for each cell line. ***P < 0.001, ****P < 0.0001. D Western blot analysis of PARP1, caspase 8, cleaved-PARP1 (C-PARP1) and cleaved-caspase8 (C-caspase8) expression in MV4–11 and MOLM13 cells after treatment with different doses of sitravatinib for 48 h. E Western blot analysis of p-FLT3, p-STAT5, p-AKT and p-ERK 1/2 in MV4-11 and MOLM13 cells after treatment with sitravatinib at the indicated doses for 4 h

Fig 5: Sitravatinib is efficient to eliminate FLT3-ITD-F691L cells in vitro and in vivo. A IC50 values of BaF3-FLT3-ITD and BaF3-FLT3-ITD-F691L cells treated with each drug for 48 h. Error bars indicate mean ± standard error, n = 3 independent experiments. B Fold changes of IC50 values of FLT3 inhibitors for BaF3-FLT3-ITD cells with or without secondary TKD mutations. Error bars indicate mean ± standard error, n = 3 independent experiments. C Western blot analysis of p-FLT3, p-STAT5, p-AKT and p-ERK 1/2 in BaF3-FLT3-ITD-F691L cells after treatment with sitravatinib at the indicated concentrations for 4 h. D BALB/c mice were intravenously inoculated with BaF3-FLT3-ITD-F691L cells. From 2 days after the injection, mice were administrated with vehicle, sitravatinib (20 mg/kg/day), gilteritinib (30 mg/kg/day) or quizartinib (10 mg/kg/day) until the first mouse died in the vehicle group. The percentage of leukemia cells in PB of BaF3-FLT3-ITD-F691L-diseased mice on day 10 was detected by flow cytometry (n = 6 mice per group). E The percentage of leukemia cells in BM and SP of BaF3-FLT3-ITD-F691L-diseased mice on day 10 (n = 3 mice per group). F Top: The spleen image and weight of mice in (E); Bottom: One mouse of each group in (E) was randomly selected for the H&E staining of spleens. A normal mouse used as control. Scale bars: 1500 μm. Green arrows: typical spleen lymph nodules; red arrows: fuzzy lymph nodules. G Body weight measurements of the mice during drug administration. H The survival curves of BaF3-FLT3-ITD-F691L-diseased mice treated with vehicle (n = 10), sitravatinib (n = 10), gilteritinib (n = 12) or quizartinib (n = 9). Error bars indicate mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001