emavusertib  (MedChemExpress)

Journal: bioRxiv

Article Title: Cross-species graph-embedding unmasks the ageing microenvironment as a key determinant of pancreatic cancer malignant cell biology and therapy response

doi: 10.64898/2026.02.02.703350

Figure Lengend Snippet: (A) Left: Graphical representation of the prioritization strategy for candidate drug targets within the inflammatory communities on GN moPDAC-old and GN huPDAC-old . Right: Graphical representation of the prioritization strategy for candidate drug targets within inflammatory-related communities on GN moMalign-old and GN huMalign-old . IRAK4 was the only common gene to both prioritization strategies. DGIdb, drug-gene interaction database. CPAT, canSAR.ai protein annotation tool. (B) Representative phospho-IRAK4 (p-IRAK4) stains in moPDAC-young and moPDAC-old. Scale bars, 50 μm. (C) Quantification of p-IRAK4 stain in moPDAC-young and moPDAC-old. Results show mean ± SEM. ***, P < 0.001, Mann-Whitney test. (D) Representative p-IRAK4 stains in huPDAC-young and huPDAC-old. Scale bars, 100 μm. (E) Quantification of p-IRAK4 stain in huPDAC-young and huPDAC-old. Results show mean ± SEM. *, P < 0.05, Mann-Whitney test. (F) Schematic of 2-week study in moPDAC-young and moPDAC-old tumour-bearing orthotopically grafted KPC PDAC organoid-derived mouse models with 100 mg/Kg IRAK4 inhibitor (IRAK4i, emavusertib, CA-4948) or vehicle by daily oral gavage. (G) Representative p-IRAK4 stains in vehicle- and IRAK4i- treated moPDAC-old. Scale bars, 50 μm. (H) Quantification of p-IRAK4 stain in vehicle- and IRAK4i- treated moPDAC-old. Results show mean ± SEM. **, P < 0.01, Mann-Whitney test. (I) Significantly upregulated and downregulated pathways identified by GSEA of IRAK4i-treated moPDAC-old (n=8) compared to vehicle-treated moPDAC-old (n=9). (J) Representative cleaved caspase 3 (CC3) stains in vehicle- and IRAK4i-treated moPDAC-old. Scale bars, 50 μm. (K) Quantification of CC3 stain in vehicle- and IRAK4i-treated moPDAC-old. Results show mean ± SEM. *, P < 0.05, Mann-Whitney test. (L) Number of diaphragm metastases of vehicle- and IRAK4i- treated moPDAC-old. Results show mean ± SEM. *, P < 0.05, Mann-Whitney test. (M) Tumour growth, as measured by ultrasound-based imaging, shown as ratio of tumour volumes at day 14 (d14) over tumour volumes at day -1 (d-1) of vehicle- and IRAK4i- moPDAC-old. Results show mean ± SEM. *, P < 0.05, Mann-Whitney test. (N) Body weight change in vehicle- and IRAK4i- moPDAC-old at day 14 compared to day -1. Results show mean ± SEM. *, P < 0.05, Mann-Whitney test. Body weight at endpoint was calculated by removing the tumour weight. (O) Number of diaphragm metastases of vehicle- and IRAK4i- treated moPDAC-young. Results show mean ± SEM. No significant difference was observed, as assessed by Mann-Whitney test. (P) Tumour growth, as measured by ultrasound-based imaging, shown as ratio of tumour volumes at day 14 (d14) over tumour volumes at day -1 (d-1) of vehicle- and IRAK4i- moPDAC-young. Results show mean ± SEM. No significant difference was observed, as assessed by Mann-Whitney test. (Q) Body weight change in vehicle- and IRAK4i- moPDAC-young at day 14 compared to day -1. Results show mean ± SEM. No significant difference was observed, as assessed by Mann-Whitney test. Body weight at endpoint was calculated by removing the tumour weight.

Article Snippet: Mice were administered vehicle or 100 mg/Kg of emavusertib (CA-4948; HY-135317, MedChem Express) for 14 days, once a day (in the AM) via oral gavage , .

Techniques: Staining, MANN-WHITNEY, Derivative Assay, Imaging

Journal: Current Issues in Molecular Biology

Article Title: FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia

doi: 10.3390/cimb46040184

Figure Lengend Snippet: In vitro dose response to emavusertib (CA4948) treatment in AML cell lines. AML cells were treated with the dual IRAK4 and FLT3 inhibitor CA4948 at the indicated dosages for 20 h ( A ). Susceptibility of AML cells to CA4948 under exposure to lipopolysaccharides ( B , C ). Cell viability was determined in AML cells after exposure to 50–250 pg/mL LPS and 50 to 150 nM CA4948 in FLT3-ITD positive MOLM-13 ( B ) and FLT3 wild-type ML-2 ( C ) cells. Cell viability data are average values of multiple repeat measurements per dosage. The standard deviation was 3–6%. Significance of differences denoted for p < 0.01 (**); and p < 0.0001 (****).

Article Snippet: The IRAK4 inhibitor Emavusertib (CA4948), the MCL1 inhibitor S63845, the HSP90 inhibitor PU-H71, and lipopolysaccharides (LPS) were purchased from MedChem-Express (Monmouth Junction, NJ, USA).

Techniques: In Vitro, Standard Deviation

Journal: Current Issues in Molecular Biology

Article Title: FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia

doi: 10.3390/cimb46040184

Figure Lengend Snippet: Schematic presentation of the FLT3 and SCFR (CD117, c-KIT) as well as IL1R and TLR4 signaling pathways and downstream effects. FLT3-ITD and FLT3-TKD are constitutively active growth factor receptors. SCFR (c-KIT) is an inducible growth factor receptor activated by stem cell factor (SCF). Both tyrosine receptor kinases activate PI3K-AKT, RAS-MEK-ERK, and STAT5, leading to cell growth and proliferation via inhibition of the tumor suppressor TP53 and induction of the apoptosis regulators MCL1 and BCL2. IL1R and TLR4 are inducible receptors signaling via MyD88 and IRAK4. HSP90 protein can bind and stabilize client proteins, including AKT, BCL2, FLT3, JAK, MDM2, STAT5, SHP2, and BRAF. Hsp90 proteins are indicated in yellow, oncogenic protein functions in red, tumor suppressor functions in green ovals, and targeted inhibitors in blue rectangles. Sharp arrows and blunt arrows indicate target induction and inhibition, respectively.

Article Snippet: The IRAK4 inhibitor Emavusertib (CA4948), the MCL1 inhibitor S63845, the HSP90 inhibitor PU-H71, and lipopolysaccharides (LPS) were purchased from MedChem-Express (Monmouth Junction, NJ, USA).

Techniques: Inhibition

Journal: Current Issues in Molecular Biology

Article Title: FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia

doi: 10.3390/cimb46040184

Figure Lengend Snippet: Susceptibility of AML cell lines to various treatment combinations. Cell viability was determined in AML cell lines MOLM-13, ML-2, and OCI-AML3 after 20 h of treatment with single compounds and in combination with 50–150 nM CA4948 (CA) and 10–100 nM venetoclax (VC100), 100–200 nM PU-H71 (PU), or 50–100 nM S63845 (S100). Susceptibility of MOLM-13 cells to various treatment combinations in the absence or presence of HS-5 bone marrow stroma ( B ). Cell viability data are average values of multiple repeat measurements per dosage. Synergistic effects of combination treatments are depicted in MOLM-13 ( A ). Oppositional effects in ML-2 ( C ) and OCI-AML3 ( D ) cells. Cell viability data are average values of multiple repeat measurements per dosage. Significance of differences denoted for p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); and p < 0.0001 (****).

Article Snippet: The IRAK4 inhibitor Emavusertib (CA4948), the MCL1 inhibitor S63845, the HSP90 inhibitor PU-H71, and lipopolysaccharides (LPS) were purchased from MedChem-Express (Monmouth Junction, NJ, USA).

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia

doi: 10.3390/cimb46040184

Figure Lengend Snippet: Induction of cell cycle arrest, apoptosis, and cell death in AML cells treated with CA4948 in combination with PU-H71, S63845, or venetoclax. Cytometric analysis of MOLM-13 cells treated with 100 nM PU-H71 (PU), 50 nM S63845 (S), or 25 nM venetoclax (VC) and stained with Annexin-V and PI ( A , C ) or DAPI ( B , D ). According to Annexin V and PI staining intensity, cells were classified as vital (Ann lo, PI lo), early apoptotic (Ann hi, PI lo), late apoptotic (Ann hi, PI hi), or necrotic (Ann lo, PI hi). According to DAPI staining intensity, cells were classified as subG1 (<2 N), G0/G1 (2 N), S phase (2–4 N), or G2 phase (4 N). Significance of differences denoted for p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); and p < 0.0001 (****).

Article Snippet: The IRAK4 inhibitor Emavusertib (CA4948), the MCL1 inhibitor S63845, the HSP90 inhibitor PU-H71, and lipopolysaccharides (LPS) were purchased from MedChem-Express (Monmouth Junction, NJ, USA).

Techniques: Staining

Journal: Current Issues in Molecular Biology

Article Title: FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia

doi: 10.3390/cimb46040184

Figure Lengend Snippet: Primary AML cells’ in vitro response to Emavusertib and combination treatments. Cell viability was determined in mononuclear cells isolated from the peripheral blood or bone marrow of 23 AML patients (A1 to A23) after 20 h of treatment. ( A ) Primary AML cells treated with 100 nM CA4948 (CA100) and 100 nM S63845 (S100), alone and in combination. ( B ) Primary AML cells treated with 100 nM CA4948 (CA100) and 100 nM venetoclax (VC100), alone and in combination. ( C ) Primary AML cells treated with 100 nM CA4948 (CA100) and 100 nM PU-H71 (PU100), alone and in combination. Treatment for 20 h with 100 nM CA4948 and 100 nM S63845 ( D ), 100 nM CA4948 and 100 nM venetoclax ( E ), and 100 nM CA4948 and 100 nM PU-H71 ( F ). Significance was calculated by paired t -test. The patient samples were sorted into two groups, susceptible (S) and resistant (R), in a single compound treatment with CA4948 ( G ), S63845 ( H ), venetoclax ( I ), and PU-H71 ( J ), as well as in different combination treatments with CA4948 and S63845 ( K ), CA4948 and venetoclax ( L ), and CA4948 and PU-H71 ( M ). Significance of differences in median values was calculated using the Mann–Whitney test. Significance is denoted for p < 0.001 (***) and p < 0.0001 (****).

Article Snippet: The IRAK4 inhibitor Emavusertib (CA4948), the MCL1 inhibitor S63845, the HSP90 inhibitor PU-H71, and lipopolysaccharides (LPS) were purchased from MedChem-Express (Monmouth Junction, NJ, USA).

Techniques: In Vitro, Isolation, MANN-WHITNEY

Journal: Current Issues in Molecular Biology

Article Title: FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia

doi: 10.3390/cimb46040184

Figure Lengend Snippet: Biomarkers of response to emavusertib and combination treatments. Cell viability was determined in mononuclear cells isolated from AML patients’ peripheral blood or bone marrow after 20 h of treatment with 100 nM CA4948 (CA100), 100 nM S63845 (S100), 100 nM venetoclax (VC100), and 100 nM PU-H71 (PU100), alone and in combination. Response was correlated to FLT3 gene mutation status ( A ), NPM1 gene mutation status ( B ), blast cell percentage ( C ), bone marrow infiltration ( D ), CD11b ( E ), CD34 ( F ), CD64 ( G ), and CD117 ( H ). Best fit lines are according to simple linear regression analysis. Association of response and biomarkers was analyzed by unpaired t -test ( I ). Significance denoted for p < 0.05 (*); p < 0.01 (**); p < 0.001 (***). Legend for treatment combinations ( J ).

Article Snippet: The IRAK4 inhibitor Emavusertib (CA4948), the MCL1 inhibitor S63845, the HSP90 inhibitor PU-H71, and lipopolysaccharides (LPS) were purchased from MedChem-Express (Monmouth Junction, NJ, USA).

Techniques: Isolation, Mutagenesis

Journal: Blood

Article Title: Paralog-specific signaling by IRAK1/4 maintains MyD88-independent functions in MDS/AML

doi: 10.1182/blood.2022018718

Figure Lengend Snippet: IRAK4 inhibition causes the activation of IRAK1. (A) Colony formation in a panel of MDS/AML cell lines and patient-derived samples treated with the indicated concentrations of CA-4948 (2 independent experiments). (B) Immunoblots for IRAK4 in WT and IRAK4 KO AML cell lines and patient-derived samples. (C) Colony formation of WT and IRAK4 KO AML cell lines and patient-derived samples. (D) Experimental overview: RNA sequencing was performed using WT and IRAK4 KO THP1 cells, and THP1 cells were treated for 24 hours with the indicated inhibitors. Genes upregulated upon IRAK4 deficiency or chemical inhibition were used to annotate compensatory pathways. (E) Venn diagrams of overlapping upregulated genes upon IRAK4 deficiency or IRAK4 chemical inhibition. (F) Pathway enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) data sets using overlapping genes increased upon the treatment with IRAK4 inhibitors. (G) Pathway enrichment of KEGG data sets using overlapping genes increased upon treatment with IRAK4 degrader-1 or after the deletion of IRAK4. (H) Overview of canonical Myd88-dependent signaling: upon TLR ligation, MyD88 nucleates a complex with IRAK4, which signals through IRAK1 and/or IRAK2 and then TRAF6 to activate the NF-κB and MAPK pathways. (I) Immunoblots for IRAK1, IRAK2, TRAF6, and MyD88 in WT and IRAK4 KO AML cell lines and patient-derived samples. (J) Immunoblots for phoshpo-IRAK1, total IRAK1, and IRAK4 in WT and IRAK4 KO cell lines. (K) Immunoblots for phoshpo-IRAK1, total IRAK1, and IRAK4 in MDSL and THP1 cells treated for 24 hours with IRAK4 degrader-1. (L) Immunoblots for phospho-IRAK1, total IRAK1, IRAK2, and IRAK4 in MDSL and AML (1714) treated for 24 hours with CA-4948 (10 μM). Significance was determined with a Student t test (∗ P < .05). Error bars represent the standard deviation.

Article Snippet: CA-4948 and IRAK4 degrader-1 were purchased from MedChemExpress.

Techniques: Inhibition, Activation Assay, Derivative Assay, Western Blot, RNA Sequencing Assay, Ligation, Standard Deviation

Journal: Blood

Article Title: Paralog-specific signaling by IRAK1/4 maintains MyD88-independent functions in MDS/AML

doi: 10.1182/blood.2022018718

Figure Lengend Snippet: The inhibition of IRAK1 confers an exaggerated leukemic defect to IRAK4-deficient AML. (A) Growth curves of WT and IRAK1 KO MDSL and THP1 cells treated with CA-4948 (10 μM) or vehicle (2 independent experiments). (B) Colony formation of WT and IRAK1 KO MDSL and THP1 cells treated with CA-4948 (30 μM) or vehicle (3 independent experiments). (C) Colony formation of WT and IRAK1 KO MDSL and THP1 cells treated with IRAK4 degrader-1 (MDSL, 5 μM; THP1, 10 μM) or vehicle. (D) Immunoblots for IRAK1 and IRAK4 in WT and IRAK4 KO cell lines transduced with nontargeting control short hairpin RNA (shRNA; shControl) or shIRAK1. (E) Colony formation of WT and IRAK4 KO AML cell lines transduced with nontargeting control shRNA (shControl) or shIRAK1. (F) Representative colony images of WT and IRAK4 KO AML (1294) cells transduced with nontarget control shRNA (shControl) or shIRAK1 (original magnification ×40). (G) Immunoblots for IRAK1 and IRAK4 in WT, IRAK4 KO , IRAK1 KO , and IRAK1/4 dKO THP1 cells. (H) Colony formation of isogenic THP1 cells. (I) Kaplan-Meier survival analysis of NSGS mice (n = 7 mice per group) that received engraftment with WT, IRAK4 KO , IRAK1 KO , and IRAK1/4 dKO THP1 cells (Data represent 1 of 2 independent experiments with similar trends). (J) Bone marrow engraftment of WT (n = 4), IRAK4 KO (n = 5), IRAK1 KO (n = 5), and IRAK1/4 dKO (n = 5) THP1 cells in NSGS mice that underwent xenograftment at the time of death. Leukemic engraftment was determined as the percentage of huCD45 + huCD33 + cells. (K) Liver engraftment of WT (n = 4), IRAK4 KO (n = 5), IRAK1 KO (n = 5), and IRAK1/4 dKO (n = 5) THP1 cells in NSGS mice that underwent xenograftment at the time of death. Leukemic engraftment was determined as the percentage of huCD45 + huCD33 + cells normalized to the number of days. (L) Representative images of livers collected from NSGS mice that underwent xenograftment with WT, IRAK4 KO , IRAK1 KO , and IRAK1/4 dKO THP1 cells. Arrows indicate examples of AML cell infiltration. Significance was determined with a Student t test (∗ P < .05). Error bars represent the standard deviation.

Article Snippet: CA-4948 and IRAK4 degrader-1 were purchased from MedChemExpress.

Techniques: Inhibition, Western Blot, Transduction, Control, shRNA, Standard Deviation

Journal: Blood

Article Title: Paralog-specific signaling by IRAK1/4 maintains MyD88-independent functions in MDS/AML

doi: 10.1182/blood.2022018718

Figure Lengend Snippet: MyD88 is dispensable for MDS/AML LSPCs. (A) Immunoblots for MyD88 and the activation of downstream pathways (phospho-p38, phospho-JNK, phospho-IKK, and phospho- extracellular signal-regulated kinase) in WT and MYD88 KO THP1 cells upon a 30-minute treatment with IL-1β (10 ng/µL) or the TLR1/2 ligand PAM3CSK4 (1 µg/mL) as compared with DMSO. (B) Immunoblots for IRAK4 and MyD88 in WT and MYD88 KO THP1 and MDSL cells transduced with nontargeting shControl or shIRAK4. (C) Colony formation of WT and MYD88 KO THP1 and MDSL cells transduced with nontargeting shControl or shIRAK4. (D) Representative colony images of WT and MyD88 KO MDSL cells transduced with nontargeting shControl or shIRAK4 (original magnification ×40). Significance was determined with a Student t test (∗ P < .05). Error bars represent the standard deviation. DMSO, dimethyl sulfoxide.

Article Snippet: CA-4948 and IRAK4 degrader-1 were purchased from MedChemExpress.

Techniques: Western Blot, Activation Assay, Transduction, Standard Deviation

Journal: Blood

Article Title: Paralog-specific signaling by IRAK1/4 maintains MyD88-independent functions in MDS/AML

doi: 10.1182/blood.2022018718

Figure Lengend Snippet: Noncanonical IRAK1/4 signaling is essential for maintaining MDS/AML LSPCs. (A) Principal component analysis of gene expression profiles of WT, IRAK4 KO , IRAK1 KO , and IRAK1/4 dKO THP1 cells. (B) Volcano plots of differentially expressed genes in IRAK4 KO , IRAK1 KO , and IRAK1/4 dKO THP1 cells relative to WT THP1 cells (>2-fold change; ∗ P < .05). (C) Heatmap of differentially expressed genes in IRAK4 KO , IRAK1 KO , and IRAK1/4 dKO THP1 relative to WT. Bars on the right denote differentially expressed genes attributed to the deficiency of IRAK4 (red) or IRAK1 (orange), or are unique to IRAK1/4 dKO (blue). (D) Venn diagrams of overlapping upregulated or downregulated genes in IRAK4 KO , IRAK1 KO , and IRAK1/4 dKO relative to WT THP1. (E) Pathway enrichment of KEGG data sets of upregulated and downregulated genes in IRAK4 KO , IRAK1 KO , and IRAK1/4 dKO THP1 cells. (F) Gene set enrichment analysis of genes dysregulated in IRAK1/4 dKO vs WT THP1 cells. Absolute normalized enrichment score and the corresponding P value is shown for each pathway. (G) Representative Wright-Giemsa stains of WT and IRAK4 KO cells expressing nontargeting shControl and shIRAK1, respectively (original magnification ×40). (H) Immunophenotyping of the indicated cells for CD34 expression.

Article Snippet: CA-4948 and IRAK4 degrader-1 were purchased from MedChemExpress.

Techniques: Expressing

Journal: Blood

Article Title: Paralog-specific signaling by IRAK1/4 maintains MyD88-independent functions in MDS/AML

doi: 10.1182/blood.2022018718

Figure Lengend Snippet: IRAK1 and IRAK4 interactomes reveal noncanonical signaling in AML. (A) Experimental overview of IRAK1 and IRAK4 proximity labeling in THP1 cells: Doxycycline-inducible IRAK4- and IRAK1-APEX2 fusion constructs were transduced into IRAK4 KO and IRAK1 KO THP1 cells, respectively. Functional rescue of canonical signaling was confirmed in NF-κB assays. Biotin phenol was added to induce proximity labeling with biotin. Biotinylated proteins were isolated and identified via mass spectrometry. (B) Venn diagram of unique and overlapping proteins in the IRAK4 and IRAK1 proximal proteins. (C) Pathway enrichment using IRAK4-specific proximal proteins. Bars represent the number of IRAK4 interacting proteins that appear in the designated pathway. Dots represent the −log(q value) of the pathway enrichment. (D) Pathway enrichment using IRAK1-specific proximal proteins. Bars represent the number of IRAK1 interacting proteins that appear in the designated pathway. Dots represent the −log(q value) of the pathway enrichment. (E) Pathway enrichment using proximal proteins common to IRAK1 and IRAK4. Bars represent the number of interacting proteins that appear in the designated pathway. Dots represent the −log(q value) of the pathway enrichment. (F) Interaction map highlighting IRAK4 interactors in the PRC2 complex and IRAK1 interactors in JAK/STAT/interferon signaling. Circle sizes indicate the adjusted P value for the identified interaction with IRAK1 or IRAK4. (G) Immunoblots for IRAK1 and IRAK4 in the nuclear (Nuc) and cytoplasmic (Cyto) fractions isolated from the indicated cells. (H) Immunoprecipitation of IRAK4 (or immunoglobulin G control) followed by immunoblotting of IRAK4 and EZH2 from THP1 cells. (I) Immunoblots for phospho-STAT5 and STAT5 in WT, IRAK4 KO , and IRAK1 KO THP1 cells. (J) Colony formation of MyD88 KO , IRAK1 KO , and IRAK4 KO THP1 cells treated with DMSO or BBI608 (STAT3 inhibitor) (500 nM). Error bars represent the standard error of the mean. DMSO, dimethyl sulfoxide.

Article Snippet: CA-4948 and IRAK4 degrader-1 were purchased from MedChemExpress.

Techniques: Labeling, Construct, Functional Assay, Isolation, Mass Spectrometry, Western Blot, Immunoprecipitation, Control

Journal: Blood

Article Title: Paralog-specific signaling by IRAK1/4 maintains MyD88-independent functions in MDS/AML

doi: 10.1182/blood.2022018718

Figure Lengend Snippet: IRAK1/4 maintains undifferentiated leukemic cell states through chromatin and transcription factor networks. (A) Heatmap of chromatin accessibility (assay for transposase-accessible chromatin sequencing) peaks within a 3 kb distance of transcription start sites of genes in WT, IRAK1 KO , IRAK4 KO , and IRAK1/4 dKO THP1 cells. (B) The total number of accessibility peaks lost and acquired in IRAK1 KO , IRAK4 KO , and IRAK1/4 dKO THP1 relative to WT cells. (C) Venn diagrams of overlap genes that are associated with both differential expression (RNA sequencing) and concordant changes in chromatin accessibility (assay for transposase-accessible chromatin sequencing) in IRAK1 KO , IRAK4 KO , and IRAK1/4 dKO THP1 cells. (D-E) Heatmaps of transcription factor enrichment among genes associated with the downregulation and loss of chromatin peaks (D) or the upregulation and acquisition of open chromatin peaks (E) in IRAK1 KO , IRAK4 KO , and IRAK1/4 dKO THP1 cells relative to WT cells. Enrichment of transcription factor signatures was determined with the Chromatin Immunoprecipitation (ChIP) Enrichment Analysis (ChEA) 2022 library. Color intensity reflects the Log ( P value) of the enrichment score. (F) Heatmap of differential gene expression in patients with AML (relative to healthy controls) using gene expression data curated from the Beat AML data set. The heatmap represents a subset of genes that are downregulated and associated with the loss of chromatin accessibility in IRAK1/4 dKO THP1 (IRAK1/4 gene signature). Unsupervised hierarchical clustering analysis resolved distinct cohorts of IRAK1/4-high signature (Group 1) and IRAK1/4-low/intermediate signature (Groups 2 and 3) patients with AML. (G) Enrichment of AML-associated mutations in IRAK1/4-high signature (Group 1) and IRAK1/4-low/intermediate signature (Groups 2 and 3) Patients with AML (from panel F) based on hypergeometric testing. (H) Schematic diagram of the CRISPR activation screen. WT and IRAK1/4 dKO THP1 cells were transduced with the pooled sgRNA library targeting more than 18 000 coding isoforms. After 3 weeks, deep sequencing was performed to identify candidate genes. (I) Average Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) score for candidate genes from WT and IRAK1/4 dKO THP1 replicate samples. Blue circles represent genes selectively enriched in IRAK1/4 dKO THP1 cells. (J) Most significant pathways (KEGG analysis) selectively enriched in IRAK1/4 dKO THP1 cells among the top 438 candidate genes (based on fold change and P value). (K) Most significant transcription factors (ENCODE/ChEA analysis) selectively enriched in IRAK1/4 dKO THP1 cells among the top 438 candidate genes.

Article Snippet: CA-4948 and IRAK4 degrader-1 were purchased from MedChemExpress.

Techniques: Sequencing, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, CRISPR, Activation Assay, Transduction, Genome Wide, Knock-Out

Journal: Blood

Article Title: Paralog-specific signaling by IRAK1/4 maintains MyD88-independent functions in MDS/AML

doi: 10.1182/blood.2022018718

Figure Lengend Snippet: A dual IRAK1/4 inhibitor is more effective at suppressing MDS/AML as compared with a selective IRAK4 inhibitor. (A) Chemical structures of KME-3859 (IRAK4-inh) and KME-2780 (dual IRAK1/4-inh) with IC 50 and K d values for IRAK1 and IRAK4. (B) Heatmap of differentially expressed genes downregulated by both KME-3859 and KME-2780 (IRAK4-dependent), genes downregulated by KME-2780 (“IRAK1-dependent”), and genes representing the IRAK1/4 AML signature (from Figure 6 F). (C) Pathway enrichment using KEGG of downregulated genes upon treatment with both KME-3859 and KME-2780 (IRAK4-dependent genes) as compared with vehicle control (top). Pathway enrichment using KEGG of downregulated genes upon the treatment with KME-2780 (IRAK1-dependent genes) as compared with KME-3859 and vehicle control (bottom). (D) Colony formation of MDSL (250 nM), THP1 (1 μM), OCIAML3 (1 μM), AML (1714) (1 μM), AML (1294) (1 μM), AML (08) (250 nM), and MDS (3328) (250 nM) cells treated with DMSO, KME-3859, or KME-2780. (E) Representative Wright-Giemsa stains of cells treated with vehicle (DMSO), KME-3859 (500 nM), or KME-2780 (500 nM) for 12 days (original magnification ×40). (F) Experimental overview: AML (1714) cells derived from patients were treated in vitro with vehicle (DMSO), KME-3859 (500 nM), or KME-2780 (500 nM) for 21 days. After the treatment, live cells were evaluated for colony formation in mice that received xenografts. (G) Bone marrow engraftment of AML (1714) cells in NSGS mice that received xenografts on day 36. Leukemic engraftment was determined as the percentage of huCD45 + huCD33 + cells. (H) Kaplan-Meier survival analysis of NSGS mice (n = 10 mice per group) engrafted with AML (1714) cells pretreated with the indicated inhibitors. (I) Experimental overview: AML (64519), AML (0169), and MDS (76960) cells derived from patients were engrafted into NSGS mice. Two weeks after the engraftment, mice were randomly assigned to groups and treated orally daily with vehicle (PBS), KME-3859 (30 mg/kg), or KME-2780 (100 mg/kg). These concentrations were selected to equilibrate the free drug concentrations ( supplemental Table 14 ). (J-L) Peripheral blood engraftment of AML (64519) (J), MDS (76960) (K), and AML (0169) (L) cells in NSGS mice that received xenografts on day 40, 48, and 29 after the treatment. Leukemic engraftment was determined as the percentage of huCD45 + huCD33 + cells. (M) Kaplan-Meier survival analysis of NSGS mice (n = 8 mice/group) engrafted with AML (0169) cells and treated with the indicated inhibitors. Significance was determined with a Student t test (∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001). Error bars represent the standard error of the mean or standard deviation.

Article Snippet: CA-4948 and IRAK4 degrader-1 were purchased from MedChemExpress.

Techniques: Control, Derivative Assay, In Vitro, Standard Deviation