Dual inhibition of BTK and IRAK leads to synergistic killing in MYD88 L265P-expressing WM cells, and more robust suppression of NF-κB pathway activity. (A,C) BCWM.1 and MWCL-1 cells were treated with an inhibitor of BTK (ibrutinib; PCI 32765), IRAK 1 and 4, or both. Cell death was assessed by high-throughput CellTiter-Glo Luminescent cell viability assay. (B,D) Synergism was assessed by CI analysis, with the heat maps depicting the CI values at varying dosimetry for ibrutinib and the IRAK 1 and 4 kinase inhibitor. CI, combination index. (E) For these experiments, CI values <1 denote synergistic interactions. p65-NF-κB activity was assessed by a luciferase promoter assay at 6 hours in BCWM.1 cells. The doses were used as follows: ibrutinib (lane a, 5.000; lane b, 1.580; lane c, 0.500; lane d, 0.158; lane e, 0.050; lane f, 0.016 µM) and IRAK1/4 kinase inhibitor (lane g, 20.000; lane h, 6.325; lane i, 2.000; lane j, 0.633; lane k, 0.200; lane l, 0.063 µM). Experiments were performed in triplicate. A representative experimental set is shown. (F) The inhibition of NF-κB activity was also assessed by examination of phospho-IkBα following treatment with ibrutinib, IRAK 1 and 4 kinase inhibitor, or both using western blot analysis in BCWM.1 cells. (G) Primary BMMCs from 4 WM patients genotyped for MYD88 L265P patients, and PBMCs from 4 healthy donors genotyped for MYD88 WT were cultured with ibrutinib (4.0, 2.0, 1.0 μM) or IRAK 1 and 4 inhibitor (20.0, 10.0, 5.0 μM) or in combination for 24 hours. Cell apoptosis was assessed in CD19⁺-expressing cells following annexin V staining.