Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: CD163 + M2 Macrophages Promote Fibrosis in IgG4‐Related Disease Via Toll‐like Receptor 7/Interleukin‐1 Receptor–Associated Kinase 4/ NF‐κB Signaling

doi: 10.1002/art.42043

Figure Lengend Snippet: Identification and distribution of interleukin‐1 receptor–associated kinase 4 (IRAK4) in SMGs from transgenic mice and patients with IgG4‐RD. A–C , Distribution of TLR‐7–related candidate molecules in SMGs from representative transgenic and knockout mice ( A ), from healthy controls and from patients with CS, SS, or IgG4‐RD ( B ), and in tonsils and lymph nodes from healthy patients and SMGs from patients with IgG4‐related dacryoadenitis and sialadenitis (IgG4‐DS) ( C ). Outlined area indicates a germinal center (GC). Bars = 100 μm. D , Number of IRAK4‐positive cells in SGs from healthy controls and patients with CS, SS, or IgG4‐RD, measured using TissueQuest software. E , Serial sections stained with H&E or antibodies to IRAK4, CD80, CD163, CD11c, or CD123. Bars = 100 μm. F , Representative double immunostaining for IRAK4 (red), CD80 (green), CD163 (green), CD11c (green), and CD123 (green) in SMGs from patients with IgG4‐RD. Counterstaining was performed with DAPI (blue). G , Number of IRAK4‐expressing CD163+, CD123+, CD11c+, and CD80+ cells in SMGs from patients with IgG4‐RD (n = 12), measured using TissueQuest software. In D and G , data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. * = P < 0.05; ** = P < 0.01, by Kruskal–Wallis test. See Figure for other definitions.

Article Snippet: CD163+ M2 macrophages were isolated from healthy peripheral blood mononuclear cells as described in a previous report , and were then stimulated with 500 μ M of the TLR‐7 agonist loxoribine (catalog no. ALX‐480‐097‐M025; Enzo Life Sciences) and/or 1 μ M of the IRAK4 inhibitor CA‐4948 (Selleck) (Supplementary Figures 1 and 2, available on the Arthritis & Rheumatology website at https://onlinelibrary.wiley.com/doi/10.1002/art.42043 ).

Techniques: Transgenic Assay, Knock-Out, Software, Staining, Double Immunostaining, Expressing

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: CD163 + M2 Macrophages Promote Fibrosis in IgG4‐Related Disease Via Toll‐like Receptor 7/Interleukin‐1 Receptor–Associated Kinase 4/ NF‐κB Signaling

doi: 10.1002/art.42043

Figure Lengend Snippet: Expression of inflammatory cytokines and Toll‐like receptor 7 (TLR‐7)–related candidate genes in M2 macrophages. A , Schematic illustration of the extraction of human CD163+ M2 macrophages stimulated with the TLR‐7 agonist loxoribine and/or interleukin‐1 receptor–associated kinase 4 (IRAK4) inhibitor CA‐4948. B , Detection of CD163 and TLR‐7 before and after the differentiation and selection of M2 macrophages (day 0 and day 6) from ≥2 independent experiments, as determined by flow cytometric analysis. C , Schematic representation of TLR‐7 pathways. D , Expression levels of mRNA for TLR‐7–related genes in human CD163+ M2 macrophages cultured in the presence or absence of loxoribine and/or CA‐4948 (n = 6 per group). E , Production of inflammatory cytokines in CD163+ M2 macrophages stimulated in the presence or absence of loxoribine and/or CA‐4948, as determined by enzyme‐linked immunosorbent assay. In D and E , Data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. * = P < 0.05; ** = P < 0.01, by Mann–Whitney U ‐ test ( D ) or Kruskal–Wallis test ( E ). PBMC = peripheral blood mononuclear cells; MyD88 = myeloid differentiation factor 88; TRAF6 = tumor necrosis factor receptor–associated factor 6; IRF‐5 = interferon (IFN) regulatory factor 5; IL‐33 = interleukin‐33; TGFβ = transforming growth factor β; NS = not significant.

Article Snippet: CD163+ M2 macrophages were isolated from healthy peripheral blood mononuclear cells as described in a previous report , and were then stimulated with 500 μ M of the TLR‐7 agonist loxoribine (catalog no. ALX‐480‐097‐M025; Enzo Life Sciences) and/or 1 μ M of the IRAK4 inhibitor CA‐4948 (Selleck) (Supplementary Figures 1 and 2, available on the Arthritis & Rheumatology website at https://onlinelibrary.wiley.com/doi/10.1002/art.42043 ).

Techniques: Expressing, Extraction, Selection, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: CD163 + M2 Macrophages Promote Fibrosis in IgG4‐Related Disease Via Toll‐like Receptor 7/Interleukin‐1 Receptor–Associated Kinase 4/ NF‐κB Signaling

doi: 10.1002/art.42043

Figure Lengend Snippet: Correlation between fibrosis and M2 macrophages in SMGs from patients with IgG4‐RD. A , Serial sections stained with H&E, MT, or antibodies to CD163 or interleukin‐1 receptor–associated kinase 4 (IRAK4). Bars = 100 μm. B , Correlation between fibrosis score and the number of CD163+ or IRAK4‐positive cells in SMGs (n = 12 per group). The numbers of CD163+ cells and IRAK4‐positive cells were measured using TissueQuest software. Correlation coefficients and P values were determined using Spearman's rank correlation. See Figure for other definitions.

Article Snippet: CD163+ M2 macrophages were isolated from healthy peripheral blood mononuclear cells as described in a previous report , and were then stimulated with 500 μ M of the TLR‐7 agonist loxoribine (catalog no. ALX‐480‐097‐M025; Enzo Life Sciences) and/or 1 μ M of the IRAK4 inhibitor CA‐4948 (Selleck) (Supplementary Figures 1 and 2, available on the Arthritis & Rheumatology website at https://onlinelibrary.wiley.com/doi/10.1002/art.42043 ).

Techniques: Staining, Software

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: CD163 + M2 Macrophages Promote Fibrosis in IgG4‐Related Disease Via Toll‐like Receptor 7/Interleukin‐1 Receptor–Associated Kinase 4/ NF‐κB Signaling

doi: 10.1002/art.42043

Figure Lengend Snippet: Schematic model of TLR‐7 signaling in M2 macrophages leading to fibrosis in IgG4‐RD. TLR‐7 expressed on M2 macrophages recognizes some viral RNAs or self RNAs released from stressed or injured cells. Activated M2 macrophages secrete fibrotic cytokines, including interleukin‐1β (IL‐1β), transforming growth factor β (TGFβ), and IL‐33, via interleukin‐1 receptor–associated kinase 4 (IRAK4)/NF‐κB signaling, which leads to fibrosis. MyD88 = myeloid differentiation factor 88; TRAF6 = tumor necrosis factor receptor–associated factor 6; IRF‐7 = interferon regulatory factor 7 (see Figure for other definitions).

Article Snippet: CD163+ M2 macrophages were isolated from healthy peripheral blood mononuclear cells as described in a previous report , and were then stimulated with 500 μ M of the TLR‐7 agonist loxoribine (catalog no. ALX‐480‐097‐M025; Enzo Life Sciences) and/or 1 μ M of the IRAK4 inhibitor CA‐4948 (Selleck) (Supplementary Figures 1 and 2, available on the Arthritis & Rheumatology website at https://onlinelibrary.wiley.com/doi/10.1002/art.42043 ).

Techniques:

Journal: bioRxiv

Article Title: Targeting IL-1/IRAK1/4 signaling in Acute Myeloid Leukemia Stem Cells Following Treatment and Relapse

doi: 10.1101/2024.11.09.622796

Figure Lengend Snippet: (A) Schematic of UR241-2 design and synthesis. This figure illustrates the logarithm of partition coefficient (LogP), topological polar surface area (tPSA), calculated logarithm of partition coefficient (CLogP), and logarithm of solubility (LogS). (B) Computer-simulated docking analyses for UR241-2 and PF06650833 against IRAK1 and IRAK4 using MedusaDock program. (C) The half maximal inhibitory concentration (IC 50 ) of UR241-2 for IRAK1 and IRAK4 measured by kinase profiling assay with protein suspension. UR241-2 underwent testing in 10-dose IC 50 duplicate mode, starting at 20 µM with 3-fold serial dilution. Reactions were carried out at 1 µM ATP. (D) The cellular IC 50 of UR241-2 for IRAK1 and IRAK4 measured by NanoBRET Target Engagement Assay in HEK293 cells. HEK293 cells were transiently transfected with 1 µg IRAK1 and IRAK4 NanoLuc Fusion Vectors, along with 9 µg transfection carrier DNA. The transfected cells were treated with UR241-2, starting at 20 µM (10-dose with 3-fold dilution) for 1 hour. The target engagement was then quantified by the NanoBRET assay. (E) Pie chart depicting the kinome profiling of UR241-2. The chart highlights kinases with >25% inhibition by UR241-2 at a concentration of 5 nM. Only 9 kinases out of 682 kinases were affected. STE = Homologs of yeast Sterile 7, Sterile 11, and Sterile 20 kinases. TK = Tyrosine Kinase. TKL = Tyrosine Kinase-Like kinases. CK1 = Casein Kinase 1. AGC = PKA, PKG, and PKC families. CAMK = Calcium/calmodulin-dependent protein kinase. CMGC = CDK, MAPK, GSK3, and CLK families. (F) Cell viability assay of UR241-2 in THP-1 cells. Cells were treated with serial concentrations of UR241-2 (0.0013-20 μM) in the 96-well plate. After 48 hours, the cell viability was assessed using CellTiter-Glo 2.0 Cell Viability Assay. Vehicle = DMSO. (G) NF-κB reporter cell assay of UR241-2 in THP-1 NF-κB (Luc/GFP) reporter cells. Cells were pre-treated with serial concentrations of UR241-2 (0.032-4 μM) in the 96-well plate for 30 minutes and incubated with human IL-1β (10 ng/mL) for 6 hours. The activation levels of NF-κB were evaluated using ONE-Glo Luciferase Assay System. Vehicle = DMSO. IKKi = 20 μM IKK2 inhibitor (positive control). (H) Immunoblotting assay investigated IL-1 signaling in THP-1 cells treated with IRAK1/4 inhibitors. Cells were starved overnight in RPMI plus 0.5% FBS medium. Subsequently, cells were pre-treated with either vehicle (DMSO) or IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7; 4 μM) for 30 minutes and then incubated with human IL-1β (10 ng/mL) for 10 and 30 minutes. At each time point, cells were harvested and the phosphorylation and total levels of p65, p38, ERK, and Akt were analyzed. (I) CFU-C assays of MLL-AF9 cells treated with IRAK1/4 inhibitors (UR241-2, PF06650833, and IRAK4-IN-7). Live GFP + MLL-AF9 splenocytes were sorted and mixed with drugs (0.2 and 4 μM) in the methylcellulose-based medium. Colonies were counted after 7 days of culture. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (F) , (G) , and (I) . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.

Article Snippet: After serum-starvation, cells were pre-treated with UR241-2, PF06650833 (Selleck), and IRAK4-IN-7 (MCE) (4 µM) or vehicle (DMSO) for 30 minutes.

Techniques: Solubility, Concentration Assay, Suspension, Serial Dilution, Transfection, Inhibition, Sterility, Viability Assay, Incubation, Activation Assay, Luciferase, Positive Control, Western Blot

Journal: bioRxiv

Article Title: Targeting IL-1/IRAK1/4 signaling in Acute Myeloid Leukemia Stem Cells Following Treatment and Relapse

doi: 10.1101/2024.11.09.622796

Figure Lengend Snippet: (A) CFU-C assays of paired primary human AML cells at diagnosis and in relapse (AML25). Cells were treated with IRAK1/4 inhibitors UR241-2, PF06650833, and IRAK4-IN-7 (0.2 and 4 μM) in methylcellulose-based medium. (B) CFU-C assays of paired diagnostic and relapsed AML34 cells, as described in (A). (C) CFU-C assays of primary human NBM CD34 + cells treated with UR241-2, PF06650833, and IRAK4-IN-7 (0.2 and 4 μM) in methylcellulose-based medium. Mean ± SD. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test in (A-C) . (D) Schematic of UR241-2 ex vivo treatment and transplantation using MLL-AF9 AML model. MLL-AF9 GFP + splenocytes were sorted and treated with vehicle (DMSO), UR241-2, and PF06650833 (4 μM) ex vivo for three days. These cells were transplanted into recipient C57BL/6J mice following treatment. Mice were sacrificed and assessed after four weeks. (E) Engraftment levels of MLL-AF9 GFP + cells in the bone marrow in recipients at four weeks after transplantation as described in (D) . Mean ± SD. Two-tailed Mann-Whitney test. (F) Spleen weights in recipients at four weeks after transplantation as described in (D) . Mean ± SD. Two-tailed Mann-Whitney test. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, ns = not significant. Schematics were created with BioRender.com.

Article Snippet: After serum-starvation, cells were pre-treated with UR241-2, PF06650833 (Selleck), and IRAK4-IN-7 (MCE) (4 µM) or vehicle (DMSO) for 30 minutes.

Techniques: Diagnostic Assay, Ex Vivo, Transplantation Assay, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Clinical Medicine

Article Title: Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors

doi: 10.3390/jcm12020399

Figure Lengend Snippet: IRAK4 inhibitor emavusertib as single agent in MZL models. Dose–response curves (from three independent experiments), error bars and IC50 values of emavusertib in Karpas1718 (K1718) and VL51 parental cell lines or resistant models to idelalisib (IDE-RES) ibrutinib (IBR-RES) or copanlisib (COP-RES). Cells were exposed (72 h) to increasing doses of emavusertib as single agent followed by MTT assay. IC50 values were calculated using the four-parameter logistic regression.

Article Snippet: Emavusertib was kindly provided by Curis (Lexington, MA, USA).

Techniques: MTT Assay

Journal: Journal of Clinical Medicine

Article Title: Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors

doi: 10.3390/jcm12020399

Figure Lengend Snippet: IRAK4 inhibitor emavusertib in Karpas1718 parental (K1718) and VL51 parental (PAR) and ibrutinib-resistant line (IBR). Cells were exposed (72 h) to increasing doses of ibrutinib alone or in combination with emavusertib followed by MTT assay. ( A ) Chou-Talalay index (CI). Each dot represents the CI value for a single ratio of ibrutinib/emavusertib. syn, synergism (CI < 0.9); add, additive effect [0.9 < CI < 1.1]; ant, antagonism/no benefit (CI > 1.1). ( B ) Synergistic efficacy (syn eff) calculated according to MuSyC algorithm. Each dot represents the syn eff value for a single ratio of ibrutinib/emavusertib. syn, synergism (syn eff > 1); add, additive effect [-1 < syn eff < 1]; ant, antagonism/no benefit (syn eff < -1). ( C ) Summary plot of benefit in terms of additivity or synergy as described in the method section. ( D ) Drug-response curves in VL51 ibrutinib-resistant cells upon emavusertib combination with ibrutinib. ( E ) Chou-Talalay index (CI) and ( F ) Drug-response curves in K1718 parental line treated (72 h) with ibrutinib alone or in combination with increasing doses of emavusertib.

Article Snippet: Emavusertib was kindly provided by Curis (Lexington, MA, USA).

Techniques: MTT Assay

Journal: Journal of Clinical Medicine

Article Title: Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors

doi: 10.3390/jcm12020399

Figure Lengend Snippet: IRAK4 inhibitor emavusertib in VL51 and Karpas1718 (K1718) parental (PAR) and idelalisib-resistant (IDE) lines. Cells were exposed (72 h) to increasing doses of idelalisib alone or in combination with emavusertib followed by MTT assay. ( A ) Chou-Talalay index (CI). Each dot represents the CI value for a single ratio of idelalisib/emavusertib. syn, synergism (CI < 0.9); add, additive effect [0.9 < CI < 1.1]; ant, antagonism/no benefit (CI > 1.1). ( B ) Synergistic efficacy (syn eff) calculated according to MuSyC algorithm. Each dot represents the syn eff value for a single ratio of idelalisib/emavusertib. syn, synergism (syn eff > 1); add, additive effect [−1 < syn eff < 1]; ant, antagonism/no benefit (syn eff < −1). ( C ) Summary plot of benefit in terms of additivity or synergy as described in the method section. ( D ) Drug-response curves in VL51 idelalisib-resistant and parental lines upon emavusertib and idelalisib combination.

Article Snippet: Emavusertib was kindly provided by Curis (Lexington, MA, USA).

Techniques: MTT Assay

Journal: Journal of Clinical Medicine

Article Title: Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors

doi: 10.3390/jcm12020399

Figure Lengend Snippet: IRAK4 inhibitor emavusertib in combination with copanlisib and umbralisib in MZL cell lines. Upper panel: combination of emavusertib and copanlisib in VL51 parental (PAR) and copanlisib-resistant (COP13) line. Lower panel: combination of emavusertib and umbralisib in VL51 and K1718 parental (PAR) and resistant models to copanlisib (COP13), idelalisib (IDE) or ibrutinib (IBR). In both panels, cells were exposed (72 h) to increasing doses of PI3K inhibitors alone and in combination with emavusertib followed by MTT. ( A , D ): syn, synergism (CI < 0.9); add, additive effect [0.9 < CI < 1.1]; ant, antagonism/no benefit (CI > 1.1). ( B , E ): syn, synergism (syn eff > 1); add, additive effect [−1 < syn eff < 1]; ant, antagonism/no benefit (syn eff < −1). Each dot represents the CI value for a single ratio of copanlisib/emavusertib (upper panel) or umbralisib/emavusertib (lower panel). ( C , F ): Summary plot of benefit in terms of additivity or synergy, as described in the method section, for the combinations of emavusertib with copanlisib (upper panel) or umbralisib (lower panel).

Article Snippet: Emavusertib was kindly provided by Curis (Lexington, MA, USA).

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors

doi: 10.3390/jcm12020399

Figure Lengend Snippet: Combination of IRAK4 inhibitor emavusertib and BCR-signaling targeting agents is beneficial (synergistic or additive) in 12/14 models tested. Summary plots of all combinations tested in MZL parental and resistant lines. Cells were exposed (72 h) to increasing doses of idelalisib, ibrutinib, copanlisib or umbralisib alone or in combination with increasing doses of emavusertib followed by MTT assay. Synergy scores from Chou-Talalay ( A ) and MuSyc (Efficacy, B ) models identified beneficial effects on the addition of emavusertib to inhibitors of downstream BCR signaling. ( C ) Summary of synergy models for all combinations.

Article Snippet: Emavusertib was kindly provided by Curis (Lexington, MA, USA).

Techniques: MTT Assay

Journal: Journal of Clinical Medicine

Article Title: Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors

doi: 10.3390/jcm12020399

Figure Lengend Snippet: Emavusertib in combination with ibrutinib increases sub G0-G1 population. Representative histograms and cell-cycle distribution upon emavusertib (10 µM) alone or in combination with ibrutinib (72 h) in ( A ) K1718 parental and ( B ) VL51 ibrutinib resistant cell line. Ibrutinib doses were selected based on the IC50 values in the parental lines (50 nM in K1718, 1 µM in VL51). Data represent the average of two independent experiments. p for nominal p -value from t-test.

Article Snippet: Emavusertib was kindly provided by Curis (Lexington, MA, USA).

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors

doi: 10.3390/jcm12020399

Figure Lengend Snippet: Addition of emavusertib to ibrutinib induces apoptosis. Apoptosis induction after emavusertib (10 µM) alone or in combination with ibrutinib (72 h) in ( A ) K1718 and ( B ) VL51 ibrutinib resistant line. Ibrutinib doses were selected based on the IC50 values in the parental lines (50 nM in K1718, 1 µM in VL51). Data represent the average of two independent experiments. Error bars for standard deviation of the mean. p for nominal p -value from t-test comparing each treatment to control (DMSO).

Article Snippet: Emavusertib was kindly provided by Curis (Lexington, MA, USA).

Techniques: Standard Deviation, Control

Journal: Journal of Clinical Medicine

Article Title: Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors

doi: 10.3390/jcm12020399

Figure Lengend Snippet: Combination of emavusertib with ibrutinib reduces p65/REL-A. ( A ) Representative immunofluorescence pictures and ( B ) Violin plot showing mean fluorescence quantification by confocal microscopy using specific anti-RELA/p65 (Magenta) and DAPI (4′-6-diamidino-2-phenylindole). Images were acquired on a Leica SP5 with an objective with ×63 magnification. Emavusertib (10 µM) was used alone or in combination with ibrutinib (50 nM) (6 h) in K1718 parental model. Data represent the mean fluorescence values. Scales of grey represent different experiments.

Article Snippet: Emavusertib was kindly provided by Curis (Lexington, MA, USA).

Techniques: Immunofluorescence, Fluorescence, Confocal Microscopy