HCK activity is blocked by ibrutinib and impacts the survival of MYD88-mutated WM cells. (A) Phosflow analysis showing changes in pHCK^Tyr411 following treatment of MYD88-mutated WM (BCWM.1 and MWCL-1) and ABC DLBCL (TMD-8, OCI-Ly3, and HBL-1) cells and MYD88-mutated primary LPCs from 4 WM patients with 0.5 μM of A419259, ibrutinib, or CC-292 for 30 minutes. (B) Dose-dependent survival determined by CellTiter-Glo Luminescent Cell Viability Assay for mutated (BCWM.1, MWCL-1, TMD-8, HBL-1, and OCI-LY3) and WT (OCI-LY7, OCI-LY19, Ramos, and RPMI 8226) MYD88 cells following treatment with ibrutinib (top), A419259 (middle), or CC-292 (bottom) for 72 hours. (C) Apoptotic changes using PI and Annexin V (FITC-A) staining following treatment of mutated and WT MYD88 cell lines, and primary LPCs from 3 WM patients with 0.5 μM of A419259, ibrutinib, or CC-292 for 18 hours. DMSO denotes vehicle control only treated cells. Cell line results are from experiments performed in triplicate. Primary LPC data are from results obtained from 6 WM patients. *P ≤ .05 and **P ≤ .01 vs DMSO controls. (D) Dose-dependent tumor cell survival of MYD88-mutated BCWM.1 cells transduced with control vector or vectors expressing WT BTK (BTK WT); BTK-expressing mutated site for ibrutinib binding (BTK C481S); WT HCK (HCK WT); or HCK-expressing the gatekeeper mutation (HCK^T333M; HCK^T338M based on c-SRC numbering)¹⁵  and treated with ibrutinib, A419259, or CC-292. Protein levels following transduction with control, WT, or mutated BTK or HCK vectors are also shown at the bottom of (D). FITC-A, fluorescein isothiocyanate A; RLU, relative luminescence units.