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. 2023 May 16;142(11):989–1007. doi: 10.1182/blood.2022018718

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Figure 5. — 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.

Figure 5. — 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.