aim100  (MedChemExpress)

Journal: Nature Communications

Article Title: Targeting TNK2/ACK1 reverses the immunosuppressive tumor microenvironment and synergizes with immunochemotherapy in pancreatic cancer

doi: 10.1038/s41467-025-67197-3

Figure Lengend Snippet: a Representative images of TNK2 IHC staining and CT scans in PDAC patients treated with neoadjuvant therapy. b Association between TNK2 expression level and neoadjuvant chemotherapy response. Counts of PR, SD and PD are shown for TNK2-low ( n = 22) and TNK2-high ( n = 23) tumors (total n = 45 independent experiments). The overall association across the three response categories was tested by Pearson’s χ² test (two-sided); p = 0.0046. For the binary summary, NPD = PR + SD versus PD (comparison indicated in the panel). PR partial remission, SD stable disease, PD progressive disease, NPD non-progressive disease. c The synergy score of GEM, AIM100 and αPD-1 were analyzed through Synergy finder for the treatment of orthotopic transplantation mouse model. The representative tumor images ( d up) and the representative PET-CT images ( d down, e ) of the KPC GEMM mouse which treated with control, AG, AIM100 and AG, AG and aPD-1, or AG, AIM100 and αPD-1, at the experimental endpoint before the therapy ending stage ( n = 3 independent experiments). f The pancreas weights of the mouse groups described in ( d ) ( n = 3 independent experiments). g Kaplan–Meier curves for tumor free survival in the mouse groups in ( d ). Flow cytometry analysis showing the proportions of CD8 + T cells ( h ), TNFα + CD8 + T cells ( i ), and PD-1 + CD8 + T ( j ) cells in the mouse groups in ( d ). k The representative mIHC images of tumor issues (CK19 in cyan, Ki67 in red, and nuclei in blue) from the mouse groups in ( d ). l The representative mIHC images of tumor issues from the mouse groups in ( d ) at the experimental endpoint, with TNK2 stained in blue, CD8 in red, CK19 in purple, and nuclei in blue. Statistical significance was determined by log-rank (Mantel–Cox) test in survival analysis ( g ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( e , f , h – l ). All tests were two sided ( e – l ). Data represent mean ± s.d. Source data are provided as a Source Data file.

Article Snippet: Additionally, an anti-PD-1 antibody (200 μg/100 μl per mouse, Bio X Cell, #BE0146) was injected on the 4th, 7th, and 10th days, along with AIM100 (6 mg/kg, MCE, HY-15290) every four days or (R)-9b (25 mg/kg, Sigma-Aldrich, SML2073) every five days through intraperitoneal injection.

Techniques: Immunohistochemistry, Expressing, Comparison, Transplantation Assay, Positron Emission Tomography-Computed Tomography, Control, Flow Cytometry, Staining

Journal: eLife

Article Title: ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis

doi: 10.7554/eLife.96085

Figure Lengend Snippet: ( A ) Immunoprecipitation (IP) kinase assay. ACK1 (left panel) was immunoprecipitated from 293T cells expressing Flag-tagged ACK1 wild type (WT), ACK1 A156T, or ACK1 K161Q with anti-Flag Ab. Immunoprecipitated proteins were used for duplicate in vitro kinase reactions with WASP synthetic peptide. Samples of the immunoprecipitates were also probed with anti-Flag Ab. BRK (right panel) was immunoprecipitated as above from 293T cells expressing Flag-tagged BRK WT and mutants with anti-Flag Ab. Kinase reactions were performed with peptide AEEEIYGEFEAKKKG, and represented as above, and samples of the immunoprecipitates probed with anti-Flag Ab. n=2 per condition. p-values were calculated using an Anova test (Tukey’s multiple comparisons test). ( B ) Western blot analyses of lysates from 293T cells expressing (left panel) Flag-tagged WT or mutant forms ACK1 probed with anti-ACK1 Tyr(P) 284 (PY284), anti-Flag and anti-tubulin antibodies (Ab), and expressing (right panel) Flag-tagged WT or mutant BRK probed with anti-BRK Tyr(P) 342 (PY342), anti-Flag and anti-tubulin antibodies. For BRK, 293T cells were starved overnight, and stimulated with 100 ng/ml EGF for 10 min. The lysate from WT BRK indicated as low was from cells transfected with one-tenth the amount of WT DNA. ( C ) Western blot analyses of lysates from 293T cells expressing ACK1-Flag or BRK-Flag treated with AIM100 or Cpd4f and probed with anti-ACK1 Tyr(P)284 (PY284) or anti-BRK Tyr(P)342 (PY342) and anti-Flag antibodies. ( D ) May-Grunwald-Giemsa staining of iPSC-derived macrophages from familial controls and ACK1 and BRK patients. Scale bar 10 μm, 100 X objective. Representative images from over 50 observed cells per line. ( E ) Immunoprecipitation (IP) kinase assay in patients’ macrophages. (Left panel) ACK1 was immunoprecipitated from BRK WT/G321R , ACK1 WT/K161Q and ACK1 A156T/K161Q iPSC-derived macrophages with anti-ACK1 Ab. The immunoprecipitated proteins were used in duplicate for in vitro kinase reactions with WASP synthetic peptide. Samples of the immunoprecipitates were also probed with anti-ACK1 Ab and anti-tubulin Ab. (Right panel) BRK was immunoprecipitated from ACK1 WT/K161Q , ACK1 A156T/K161Q , BRK WT/G321R and BRK G257A/G321R iPSCs-derived macrophages with anti-BRK Ab. The immunoprecipitated proteins were probed with anti-BRK Tyr(P) 342 (PY342) and anti-BRK antibodies. Figure 2—source data 1. : Immunoprecipitation (IP) kinase activity assay of WT and mutant ACK1 and BRK kinases in HEK293T cells. Figure 2—source data 2. : Immunoprecipitation (IP) kinase activity assay in patients’ macrophages. Figure 2—source data 3. Uncropped and labeled gels for . Figure 2—source data 4. Raw unedited gels for .

Article Snippet: 24 hr after plating, the macrophages were pretreated for 30 min with AIM100 (Tocris, 4946; 2 μM) and/or Cpd4f (Thermo Fisher Scientific, 53-100-00001; 0.5 μM) in RPMI without FBS prior to incubation with beads, E. coli or C. albicans .

Techniques: Immunoprecipitation, IP-Kinase Assay, Expressing, In Vitro, Western Blot, Mutagenesis, Transfection, Staining, Derivative Assay, Kinase Assay, Labeling

Journal: eLife

Article Title: ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis

doi: 10.7554/eLife.96085

Figure Lengend Snippet: ( A ) Heatmaps comparing the levels of IgG autoantibodies detected in serum of mice treated with inhibitors. Heatmaps show autoantigen microarray panels performed on serum from 4-month-old BALB/cByJ female mice which received a weekly intra-peritoneal injection of DMSO (vehicle, 20 µl/mice), AIM100 (25 mg/kg in 20 µl), or Cpd4f (20 mg/kg in 20 µl) since the age of 5 weeks. Top panel depicts results for mice that did not receive a pristane injection. Bottom panel represents results for the top differentially produced auto-antibodies in inhibitor treated or control mice that received a Pristane injection at the age of 5 weeks. Plotted values represent Ab Scores (Log 2 [antigen net fluorescence intensity (NFI) x signal-to-noise ratio (SNR) +1]). Heatmap columns represent serum analysis of independent mice (n=4–5 for each of the 3 conditions). Heatmap rows sorted top to bottom starting with most significantly increased Ab Score in Cpd4f and AIM100 mice in comparison to DMSO-treated mice. p-Values were calculated using a Wilcoxon matched-pairs signed rank tests. Hierarchical clustering is based on one minus Pearson correlation with complete linkage method. K-means clustering is based on Euclidean distance, with two clusters, with 10,000 maximum iterations. ( B,C ) Immunofluorescence for mouse IgG on kidney sections. Representative micrographs ( B ) displaying glomeruli on kidney sections from 4-month-old BALB/cByJ female mice treated as in ( A ) and stained with Hoechst 33342, anti-mouse IgG, and anti-mouse Podoplanin antibody. In the quantification plot ( C ) each symbol represents the IgG mean fluorescence intensity (MFI) in a single glomerulus, of mice treated with designated inhibitors, in the presence or absence of pristane. Approximately 250 glomeruli we analyzed per section/mouse (>95% of all glomeruli in an entire longitudinal kidney section). n=4–5 mice per condition. p-Values were obtained using a Kruskal-Wallis test with multiple comparisons. Figure 3—source data 1. : Heatmaps comparing the levels of IgG autoantibodies detected in serum of control and inhibitor treated mice. Figure 3—source data 2. : Quantification of glomerular IgG in kidney sections of control and inhibitor treated mice.

Article Snippet: 24 hr after plating, the macrophages were pretreated for 30 min with AIM100 (Tocris, 4946; 2 μM) and/or Cpd4f (Thermo Fisher Scientific, 53-100-00001; 0.5 μM) in RPMI without FBS prior to incubation with beads, E. coli or C. albicans .

Techniques: Microarray, Injection, Produced, Control, Fluorescence, Comparison, Immunofluorescence, Staining

Journal: eLife

Article Title: ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis

doi: 10.7554/eLife.96085

Figure Lengend Snippet: ( A ) Heatmaps showing a comparison of serum autoantibodies detected in 4-month-old BALB/cByJ female mice. Mice received an intraperitoneal pristane injection and a weekly intraperitoneal injection of DMSO (vehicle, 20 µl/mice), AIM100 (25 mg/kg in 20 µl), or Cpd4f (20 mg/kg in 20 µl/) since the age of 5 weeks. Plotted values represent Ab Scores (Log 2 [antigen net fluorescence intensity (NFI) x signal-to-noise ratio (SNR) +1]). Heatmap columns represent serum analysis of independent mice (n=4–5 for each of the 3 conditions). Heatmap rows sorted top to bottom starting with most significantly increased Ab Score in Cpd4f and AIM100 mice in comparison to DMSO-treated mice. p-Values were calculated using a Wilcoxon matched-pairs signed rank tests. ( B ) Additional immunofluorescence images for mouse IgG on kidney sections. Representative images of glomeruli displaying the average observed signal on kidney sections from 4-month-old BALB/cByJ female mice which were left untreated or received a single intraperitoneal injection of pristane, and received a weekly intraperitoneal injection of DMSO (vehicle, 20 µµ/mice), AIM100 (25 mg/kg in 20 ul), or Cpd4f (20 mg/kg in 20 µl/) since the age of 5 weeks. Kidney sections were stained with Hoechst 33342, anti-mouse IgG, and anti-mouse Podoplanin antibody. Representative images from 250 glomeruli analyzed per kidney section/mouse (>95% of all glomeruli in an entire longitudinal kidney section) where n=4–5 mice per condition. Figure 3—figure supplement 1—source data 1. Related to : Heatmap comparing the levels of select IgG autoantibodies detected in serum of control and inhibitor treated mice.

Article Snippet: 24 hr after plating, the macrophages were pretreated for 30 min with AIM100 (Tocris, 4946; 2 μM) and/or Cpd4f (Thermo Fisher Scientific, 53-100-00001; 0.5 μM) in RPMI without FBS prior to incubation with beads, E. coli or C. albicans .

Techniques: Comparison, Injection, Fluorescence, Immunofluorescence, Staining, Control

Journal: eLife

Article Title: ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis

doi: 10.7554/eLife.96085

Figure Lengend Snippet: ( A ) Western blot analysis for AKT phosphorylation by ACK1 and BRK. Cell lysates from 293T cells were incubated with anti-AKT. Immunoprecipitated proteins were probed with anti-phosphotyrosine and anti-AKT antibodies. ( B ) Western blot analysis for STAT3 phosphorylation by ACK1 and BRK. Lysates from 293T cells coexpressing STAT3 and Flag-tagged WT or mutant forms (A156T and K161Q) of ACK1 or mutant forms (G257A and G321R) of BRK were probed with anti-phospho-STAT3 (Tyr705), anti-STAT3 and anti-Flag antibodies. For analysis of BRK, cells were treated with 100 ng/ml EGF for 10 min. ( C ) RAC activation by WT ACK1 and BRK. Cell lysates from 293T cells expressing WT or mutant forms of ACK1 ( left ) and lysates from 293T cells expressing WT or mutant forms of BRK ( right ) were incubated with GST-PAK CRIB sepharose beads, and the level of RAC1 GTP was determined by immunoblotting with anti-Rac1 antibody. Lysates were also probed with anti-Rac1, anti-FLAG and anti-tubulin antibody. For analysis of BRK, 293T cells were cotransfected with CAS and stimulated with 100 ng/ml EGF for 10 min. ( D ) MERTK increases kinase activity of BRK and ACK1. IP kinase assay. ACK1 (left) was immunoprecipitated from 293T cells co-transfected with Flag-tagged ACK1 WT, ACK1 A156T, or ACK1 K161Q and MERTK with anti-Flag Ab. Immunoprecipitated proteins were used in duplicate in vitro for kinase reactions with WASP synthetic peptide and results represented as pmol phosphate transferred. BRK (right) was immunoprecipitated as above from 293T cells co-transfected with Flag-tagged BRK WT or mutants and MERTK with anti-Flag Ab. Kinase reactions was performed with peptide AEEEIYGEFEAKKKG, and represented as above. p-Values were calculated using an Anova test (Tukey’s multiple comparison test). ( E ) Regulation of inflammatory response. Significant normalized enrichment scores (NES) for GO ‘positive regulation of acute inflammation’ gene set, GO ‘negative regulation of inflammatory response’ gene set, and GO ‘AKT_UP.V1_UP’ gene set in WT and mutant macrophages, and WT treated with AIM100 (2 µM) or Cpd4f (0.5 µM), exposed to apoptotic cells, with three replicates per experimental condition. Significant enrichment (p-value <0.05 and FDR (q-value) <0.25) are calculated as reported in Materials and methods. ( F ) Table of the top 10 differentially regulated genes by apoptotic cells in WT macrophages are not differentially expressed in mutant macrophages and WT macrophages treated with AIM100 or Cpd4f (treated as in E). Numbers indicate FDR (q-value). Known target genes of STAT3 and AKT are labeled in blue and red respectively ( G ) TNF mRNA production by WT macrophages treated with AIM100 (2 µM) 4 hr after exposure to apoptotic cells. n=6, from two independent experiments. ( H,I ) TNF and IL1β production by macrophages, as measured by ELISA on media collected from mutant and isogenic WT macrophages (C12.1) incubated with mouse apoptotic thymocytes for 90 min, then stimulated with LPS (1 ng/ml) for 18 hr. n≥4, from ≥2 independent experiments. p-Values in H were calculated by Wilcoxon matched-pairs signed rank tests for data that is not normally distributed, while p-values in G and I were calculated using an Anova test with Tukey’s correction for multiple comparisons. Figure 4—source data 1. : Immunoprecipitation (IP) kinase activity assay of WT and mutant ACK1 and BRK kinases in HEK293T cells in the presence of MERTK and/or GAS6. Figure 4—source data 2. : GSEA of control, inhibitor treated, and mutant iPSC-derived macrophages in the presence or absence of apoptotic cells. Figure 4—source data 3. : TNF mRNA production by WT AIM100 treated macrophages 4 hr after exposure to apoptotic cells. Figure 4—source data 4. : TNF protein production by WT and mutant iPSC-derived macrophages in the presence or absence of LPS and/or apoptotic cells. Figure 4—source data 5. : IL1b protein production by WT and mutant iPSC-derived macrophages in the presence of LPS and/or apoptotic cells. Figure 4—source data 6. Uncropped and labeled gels for . Figure 4—source data 7. Raw unedited gels for .

Article Snippet: 24 hr after plating, the macrophages were pretreated for 30 min with AIM100 (Tocris, 4946; 2 μM) and/or Cpd4f (Thermo Fisher Scientific, 53-100-00001; 0.5 μM) in RPMI without FBS prior to incubation with beads, E. coli or C. albicans .

Techniques: Western Blot, Phospho-proteomics, Incubation, Immunoprecipitation, Mutagenesis, Activation Assay, Expressing, Activity Assay, IP-Kinase Assay, Transfection, In Vitro, Comparison, Labeling, Enzyme-linked Immunosorbent Assay, Kinase Assay, Control, Derivative Assay

Journal: eLife

Article Title: ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis

doi: 10.7554/eLife.96085

Figure Lengend Snippet: ( A ) Actin remodeling in macrophages. Schematic and representative images of of F-actin by TIRF microscopy in macrophages of indicated genotype, deposited on PtdSer-coated plates for 20 min. ( B ) Quantification of actin clearance factor for macrophages of the indicated genotypes. Actin remodeling (actin clearance factor) was calculated as a ratio of F-actin staining intensity at cell border divided by F-actin staining intensity at cell center. The actin clearance factor ratios were normalized to the mean value of WT control. Each replicate indicates actin clearance factor fold change from WT mean in single cells. n>20, from two independent experiments. Red lines denote the mean. ( C, D ) Actin remodeling quantification (as in A,B) and representative TIRF images of WT macrophages (C12.1 line) pretreated with DMSO, AIM100 (2 µM) or Cpd4f (0.5 µM). n>24, from three independent experiments. p-Values in B-D were obtained using a Mann-Whitney test. ( E, F ) Uptake of apoptotic cells. ( E ) Schematic depicts uptake of apoptotic mouse thymocytes treated with Fiz-shFASL and labeled with the pH-sensitive dye pHrodo by iPSC-derived macrophages. Isogenic WT (C12.1 line) and isogenic ACK and BRK point mutant macrophages were incubated with pHrodo-labeled mouse apoptotic thymocytes for 90 min and analyzed by flow cytometry. Graph represents mean pHrodo fluorescence Intensity (MFI) fold change calculated by dividing total pHrodo MFI (610/20 nm) of individual samples by the average MFI of isogenic WT macrophages. n≥3, from three independent experiments. p-Values were obtained using an Anova test with Tukey’s correction for multiple comparisons. ( F ) Uptake of apoptotic cells as in ( E ) with WT macrophages (C12.1 line) pretreated with AIM100 (2 µM), R-9b (4 µM), Cpd4f (0.5 µM), or DMSO. n≥8, from ≥4 independent experiments. ( G ) Uptake of opsonized sheep red blood cells. WT macrophages (C12.1 line) are pretreated as in ( F ) and incubated with opsonized pHrodo sheep red blood cells for 90 min. Graphs represent mean fluorescence Intensity (MFI) fold change calculated by dividing total pHrodo MFI (610/20 nm) of individual samples by the average MFI of WT macrophages. n≥2, from two independent experiments. p-Values are obtained using an Anova test with Tukey’s correction for multiple comparisons. ( H ) Schematic representation of ACK1 and BRK proposed function in efferocytosis. Figure 5—source data 1. : Frustrated engulfment assays for actin remodeling quantification in response to PtdSer in WT and mutant iPSC-derived macrophages. Figure 5—source data 2. : Frustrated engulfment assay for actin remodeling quantification in response to PtdSer in AIM100 treated WT macrophages. Figure 5—source data 3. : Frustrated engulfment assay for actin remodeling quantification in response to PtdSer in Cpd4f treated WT macrophages. Figure 5—source data 4. : Quantification of apoptotic cell uptake by WT and mutant iPSC-derived macrophages. Figure 5—source data 5. : Quantification of apoptotic cell uptake by inhibitor-treated and control WT iPSC-derived macrophages. Figure 5—source data 6. : Quantification of opsonized red blood cell uptake by inhibitor-treated and control WT and mutant iPSC-derived macrophages.

Article Snippet: 24 hr after plating, the macrophages were pretreated for 30 min with AIM100 (Tocris, 4946; 2 μM) and/or Cpd4f (Thermo Fisher Scientific, 53-100-00001; 0.5 μM) in RPMI without FBS prior to incubation with beads, E. coli or C. albicans .

Techniques: Microscopy, Staining, Control, MANN-WHITNEY, Labeling, Derivative Assay, Mutagenesis, Incubation, Flow Cytometry, Fluorescence

Journal: eLife

Article Title: ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis

doi: 10.7554/eLife.96085

Figure Lengend Snippet: ( A ) Phagocytosis of 2 µm beads. iPSC-macrophages (C12 line) pretreated with DMSO, AIM100 (2 µM), Cpd4f (0.5 µM), or both, were incubated for 60 min at 37°C or 4 °C with red fluorescent 2 µm beads. Quantification of uptake is represented as fold change in mean fluorescence intensity (MFI) (670/30 nm) between DMSO-treated and inhibitor-treated macrophages (left panels). Quantification of uptake as percent red fluorescent (bead) positive macrophages (right panels). n=2 per experimental condition. ( B–C ) Phagocytosis of E. coli and C. albicans . Phagocytosis quantification of pHrodo labeled E. coli ( B ) or tdTomato fluorescent C. albicans ( C ) by iPSC-macrophages (C12 line) pretreated with DMSO, AIM100 (2 µM), Cpd4f (0.5 µM), or both, after a 60 min incubation at 37 °C or 4 °C. MFI fold change determined by dividing total MFI (585/15 nm) of individual samples by the average MFI of DMSO treated macrophages. n=2 per experimental condition. p-Values in all plots were calculated using an Anova test (Tukey’s multiple comparisons test).

Article Snippet: 24 hr after plating, the macrophages were pretreated for 30 min with AIM100 (Tocris, 4946; 2 μM) and/or Cpd4f (Thermo Fisher Scientific, 53-100-00001; 0.5 μM) in RPMI without FBS prior to incubation with beads, E. coli or C. albicans .

Techniques: Incubation, Fluorescence, Labeling

Journal: eLife

Article Title: ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis

doi: 10.7554/eLife.96085

Figure Lengend Snippet: RNA-Seq analysis FASTQ files.

Article Snippet: 24 hr after plating, the macrophages were pretreated for 30 min with AIM100 (Tocris, 4946; 2 μM) and/or Cpd4f (Thermo Fisher Scientific, 53-100-00001; 0.5 μM) in RPMI without FBS prior to incubation with beads, E. coli or C. albicans .

Techniques: Inhibition