HCK is hyperactive in MYD88-mutated cells, and its activation is triggered by IL-6. (A) Results of Phosflow analysis for phosphorylated (p) HCK (pHCK^Tyr411) in MYD88-mutated WM (BCWM.1 and MWCL-1) and ABC DLBCL (TMD-8 and OCI-Ly3) cells; MYD88 WT GCB DLBCL (OCI-Ly7 and OCI-Ly19), Ramos Burkitt, and RPMI-8226 myeloma cells. (B) Results of Phosflow analysis for pHCK^Tyr411 in primary LPCs (CD20⁺) from 3 representative MYD88 L265P expressing WM patients, as well as nonmemory (CD20⁺CD27⁻) and memory (CD20⁺CD27⁺) B cells from HDs. Percentage of cells gating for pHCK^Tyr411 expression are shown in (A-B). Histogram depicts the results of pHCK^Tyr411 expression in LPC derived from 20 WM patients, as well as nonmemory and memory B cells from 5 HDs. P < .01 for comparison of pHCK expression in WM LPC vs either HD nonmemory or memory B cells. The number of WM patients that were evaluated for LPC (open circles), as well as the number of healthy donors that were evaluated for nonmemory (open diamonds) and memory (open diamonds) B cells are shown. (C) pHCK^Tyr411 expression in the presence or absence of IL-6 (1 ng/mL) for 5 minutes in MYD88-mutated (BCWM.1, MWCL-1, TMD-8, HBL-1, and OCI-Ly3) and MYD88 WT (OCI-Ly7, OCI-Ly19, Ramos, and RPMI 8226) (top) cell lines, as well as primary WM LPCs. Peak pHCK^Tyr411 activation was determined by Phosflow analysis after 3 MYD88 mutated cell lines were cultured with IL-6 (1 ng/mL) for 2, 5, and 15 minutes (bottom). *P < .05; **P < .01; ***P < .001 vs untreated controls. (D) Phosflow analysis of pHCK^Tyr411 in BCWM.1 cells transduced with scrambled control vector or IL-6ST knockdown vector (shRNA2) in the presence or absence of 1 ng/mL of IL-6. Ab, antibody; MFI, mean fluorescence intensity; PBMCs, peripheral blood mononuclear cells; SSC, side scatter.