Fig 2: GSEA analysis on PIM kinase family in DLBCL. A–C Relationship of PIM kinase family with NK cell-mediated toxicity, antigen presentation, B cell receptor signaling pathway, and T cell receptor signaling pathway in DLBCL

Fig 3: IRAK4/PIM1 dual inhibition by KIC-0101 effectively suppresses NF-κB and JAK/STAT pathways with apoptotic effect on ibrutinib-resistant TMD8 cells. (A) Immunoblots showing protein levels of PIM1 in parental (Par) and ibrutinib-resistant (Res) TMD8 cells. GAPDH was used as total protein loading control. (B) Relative normalized mRNA expression levels of PIM1 in indicated cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed and mRNA levels of GAPDH were used as normalized control. Data are expressed in three independent experiments. (C, D) Immunoblots showing degraded protein levels of PIM1 in parental (C) and resistant (D) cells treated with cycloheximide (CHX) during indicated time points. The density of each PIM1 bands was calculated using iBright analysis software and was normalized to the GAPDH band. Data are expressed in three independent experiments. (E, F) Immunoblots showing protein levels of phosphorylated IRAK4, total IRAK4, phosphorylated STAT3, total STAT3, PIM1 and GAPDH in parental (E) and resistant (F) cells treated with indicated conditions for 24 h. (G, H) Immunofluorescence images showing subcellular localization of p65 in parental (G) and resistant (H) cells treated with indicated conditions. Cells were treated with 10 μmol/L of compounds for 4 h (parental) or 6 h (resistant). Bar graphs show the percentage of cells with nuclear p65. Data are expressed in three independent experiments. Scale bars, 10 μm. (I, J) Levels of IL-6 secretion in parental (I) and resistant (J) cells treated with indicated conditions for 24 h. Data are expressed in four independent experiments. (K, L) Immunoblots showing protein levels of phosphorylated BAD, total BAD, phosphorylated BCL2, total BCL2, cleaved caspase 3, c-MYC and GAPDH in parental (K) and resistant (L) cells treated with indicated conditions for 24 h. KIC; KIC-0101, AZD; AZD-1208, PF; PF-06650833. All quantitative data are presented as the mean ± SEM. ∗∗∗P < 0.001. (M) Schematic diagrams showing intracellular mechanisms and expected therapeutic effects of KIC-0101 on RA and ABC-DLBCL which exhibit activation of the NF-κB and JAK/STAT pathways.

Fig 4: Relationship between PIM kinase family and the immune-infiltrating cells of DLBCL. A Correlation coefficients between PIM1 and B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and DC cells, as well as mRNA expression of PIM1 were 0.237, − 0.155, − 0.172, − 0.194, 0.234, and 0.22, respectively. B Correlation coefficients between PIM1 and B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and DC cells, as well as mRNA expression of PIM2 were 0.283, 0.173, 0.007, − 0.15, 0.093, and 0.088, respectively. C Correlation coefficients between PIM1 and B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and DC cells, as well as mRNA expression of PIM3 were 0.36, − 0.228, − 0.13, − 0.164, 0.534, and 0.083, respectively

Fig 5: DLBCL patients with high expression of PIM kinase family have a poor prognosis. A–C Relationship between the expression level of PIM kinase family and OS of DLBCL patients. D, E Univariate and multivariate cox regression analyses to verify the independent prognostic value of PIM kinase family. F–H TIMER online database to visualize the relationship between the expression levels of PIM kinase family and OS of DLBCL patients