A novel IRAK4/PIM1 inhibitor ameliorates rheumatoid arthritis and lymphoid malignancy by blocking the TLR/MYD88-mediated NF- κ B pathway

Abstract

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a pivotal enzyme in the Toll-like receptor (TLR)/MYD88 dependent signaling pathway, which is highly activated in rheumatoid arthritis tissues and activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). Inflammatory responses followed by IRAK4 activation promote B-cell proliferation and aggressiveness of lymphoma. Moreover, proviral integration site for Moloney murine leukemia virus 1 (PIM1) functions as an anti-apoptotic kinase in propagation of ABC-DLBCL with ibrutinib resistance. We developed a dual IRAK4/PIM1 inhibitor KIC-0101 that potently suppresses the NF-κB pathway and proinflammatory cytokine induction in vitro and in vivo. In rheumatoid arthritis mouse models, treatment with KIC-0101 significantly ameliorated cartilage damage and inflammation. KIC-0101 inhibited the nuclear translocation of NF-κB and activation of JAK/STAT pathway in ABC-DLBCLs. In addition, KIC-0101 exhibited an anti-tumor effect on ibrutinib-resistant cells by synergistic dual suppression of TLR/MYD88-mediated NF-κB pathway and PIM1 kinase. Our results suggest that KIC-0101 is a promising drug candidate for autoimmune diseases and ibrutinib-resistant B-cell lymphomas.

Keywords: ABC-DLBCL; Drug discovery; Dual inhibitor; IRAK4; Ibrutinib resistance; NF-κB pathway; PIM1; Rheumatoid arthritis.

Graphical abstract

Figure 1

KIC-compounds are selective inhibitors of both IRAK4 and PIM1 kinases. (A, B) Chemical structures of KIC-compounds and a comparative drug, PF-06650833. (C) IRAK4 and PIM1 kinase activity in the indicated condition of KIC-compounds and ATP. (D–G) Predicted binding poses of KIC-0096 (green), KIC-0099 (blue) and KIC-0101 (magenta) in IRAK4 (D, E) and PIM1 (F, G) kinase. Major residues are exhibited in thin sticks and obstructing flap regions are omitted for clarity. (H) Correlation plot of Prime MM-GBSA scores and experimental activity of the IRAK4 (blue) and PIM1 (orange) kinase.

Figure 2

KIC-0101 properly suppresses LPS-mediated proinflammation in vitro and in vivo. (A) Effects of KIC-compounds on LPS-induced NF-κB promotor activity in THP1-Lucia NF-κB cells. Cells were treated with indicated compounds after LPS (100 ng/mL) treatment and incubated for 24 h. (B, C) Effects of KIC-compounds on LPS-induced IL-6 secretion in THP1 cells and TNFα secretion in PBMCs. Data are presented as the mean ± SEM of three independent experiments, ∗∗∗P < 0.001 and N.D.; not determined. (D) Effects of KIC-compounds on LPS-induced TNFα secretion in serum of C57BL/6 mice (n = 3–6). KIC-compounds and PF-06650833 were orally administered to mice 2 h before LPS injection. Data are expressed as the mean ± SEM. PF; PF-06650833. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. (E) Pharmacokinetic profiles of the KIC-0101 and PF-06650833 in mice. Time to maximum plasma concentration (T_max), maximum serum concentration (C_max), half-life (T_1/2), area under the curve (AUC_0–t), clearance (CL), volume of distribution at steady state (V_ss) and % bioavailability (F_0–t, %) are shown, ∗F_0–t (%) = (oral AUC_0–t/oral dose)/(i.v. AUC_0–t/i.v. dose) × 100, ∗∗NA; not applicable. (F) Kinome maps show kinase selectivity of KIC-0101 at 1 μmol/L concentration in 59 kinases. Percent activity was normalized by un-treated control.

Figure 3

KIC-0101 shows the improvement of rheumatoid arthritis in collagen-induced arthritis models. (A) Schematic diagram of collagen-induced arthritis (CIA) mouse model development. (B) The graph of arthritis scores (0–4 per limbs, max 16) measured by three independent observers in indicated conditions. Mice of normal (n = 5) and other groups (n = 10) were used. Data are presented as the mean ± SEM. (C) Hematoxylin and eosin (H&E) staining of joint tissues in each group after 13 weeks of the first immunization. KIC; KIC-0101, Tofa; tofacitinib. (D, E) Scores of the bone damage (D) and the inflammation (E) measured by three individuals. H&E images of normal (n = 3) and other groups (n = 5) in 13 weeks were used. (F) Safranin O staining of the ankle sections in CIA mouse models. (G) Scores of the cartilage damage on the tibia and navicular bone measured by three individuals. Images of normal (n = 6) and other groups (n = 10) in 13 weeks were used. (H–K) Quantifications of IL-1β (H), IL-17 (I), IL-6 (J) and TNFα (K) positive cells (⁺) on the ankle section of all groups. Data are presented as the mean ± SEM (n = 3–5), ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. Scale bar: 200 μm (C) and 100 μm (F).

Figure 4

Inhibition of NF-κB pathway by KIC-0101 causes anti-tumor effect of TMD8 cells. (A) Inhibitory effects of KIC-0101 on the cell viability of ABC-DLBCLs. Viability of lymphoma cells was analyzed by WST-1 assay in different concentrations of KIC-0101, PF-06650833 and ibrutinib for 72 h. Data are expressed in three independent experiments. (B) Immunoblots showing protein levels of phosphorylated IRAK4, total IRAK4 and GAPDH in TMD8 cells. Cells were treated with indicated conditions for 24 h. GAPDH was used as a total protein loading control. (C) Levels of IL-6 secretion in TMD8 cells which were treated with KIC-0101 and PF-06650833 (PF) in indicated conditions. Data are expressed in four independent experiments. (D) Immunoblots showing cytoplasmic and nucleus fractions of proteins in TMD8 cells. Cells were treated with KIC-0101 and PF-06650833 in indicated conditions and protein levels of p65, α-Tubulin and Lamin A/C were analyzed in fractionated lysates. α-Tubulin and Lamin A/C served as loading controls for the cytosolic and nuclear fractions, respectively. The density of p65 bands was calculated using iBright analysis software (ThermoFisher Scientific, US) and was normalized to each loading control band. Data are expressed in three independent experiments. (E) Immunofluorescence images showing subcellular localization of p65 in indicated conditions. TMD8 cells were treated with 10 μmol/L of KIC-0101 or PF-06650833 for 4 h. Scale bar, 10 μm. The nucleus (blue) was labeled with DAPI. The bar graph shows the percentage of cells with nuclear p65. Data are expressed in three independent experiments. (F) Immunoblots showing protein levels of phosphorylated STAT3, total STAT3 and GAPDH in TMD8 cells. (G) Tumor growth curves for xenografts of TMD8 cells in SCID mice (n = 8) with treatment of indicated conditions. Mice were orally treated with 50 mg/kg of KIC-0101, ibrutinib and their combination once a day for 14 days. Data are expressed as the mean (mm³) ± SEM, ∗∗∗P < 0.001.

Figure 5

Ibrutinib resistance affects expression of upstream regulators and downstream targets of NF-κB and JAK/STAT pathways. (A) Schematic diagrams of the ibrutinib-resistant TMD8 cell development. (B, C) Combination effects of KIC-0101 on ibrutinib-mediated cell death in parental (B) and ibrutinib-resistant TMD8 cells (C). (D) Principal component analysis (PCA) plot showing transcriptomic distance among the samples. Parental (Par) and resistant (Res) cells are indicated by blue and red dots, respectively (n = 3). (E) Volcano plot showing fold change (FC) and statistical significance (P-value) of each gene calculated by differentially expressed gene (DEG) analysis (Res vs. Par). (F) Dot plot with clusters showing representative gene ontology (GO) terms (dots) and their clusters (colored eclipses) of significant DEGs (adjusted P-value < 0.05 and |fold change| > 2). Size and color of dot indicate number of significant DEGs and statistical significance of the GO term (color gradient from red to blue corresponds to P-value range from 0 to 0.05). (G) Cnetplot showing gene networks of DEGs between NF-κB-related GO terms. Distance and thickness of line between dots are proportional to dissimilarity and number of the shared DEGs between GO terms, respectively. Size of a large dot is proportional to the number of the DEGs associated with the GO term. Color of small dot indicates logarithm based on 2 of FC (Log₂FC) of the DEG. Colored lines between small and large dots represent association of the DEGs with the GO terms. (H, I) Heatmaps showing expression of NF-κB (H) and JAK/STAT (I) signaling pathway-related genes in each sample. Color of blocks indicate scaled logarithm based on 2 of normalized expressions (Scaled Log₂Exp).

Figure 6

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.