LNK mutations cause dysregulated TPO-dependent growth and JAK2-STAT3/5 activation. (A) Schematic of WT and the LNK DEL, E208Q, and R392E mutants is shown. WT LNK includes an N-terminal proline-rich dimerization domain (Pro/DD), a PH domain, an SH2 domain, and a C-terminal conserved tyrosine residue (Y). The DEL mutation leads to a frameshift and premature stop codon, resulting in loss of the PH and SH2 domains. The E208Q mutation localizes to the PH domain; R392E is a synthetic point mutation that disrupts the SH2 domain. In B through D, BaF3-MPL cells were transduced with EV, WT LNK, or mutant LNK (DEL, E208Q, R392E). Reproducible results were obtained for at least 3 independent experiments. (B) BaF3-MPL cells were washed in cytokine-free media and replated in the presence of concentrations of TPO ranging from 0 to 10 ng/mL. Cumulative growth of BaF3-MPL cells over 4 days is shown. (C) BaF3-MPL cells were starved overnight in cytokine-free media and stimulated with TPO (1 ng/mL) for the durations indicated, followed by measurement of JAK2, STAT3, and STAT5 activation via phospho-specific flow cytometry. Histograms for phosphorylated forms of JAK2 (pJAK2), STAT3 (pSTAT3), and STAT5 (pSTAT5) are displayed, with internal color representing fold-change in median fluorescence intensities (MFI) compared with unstimulated cells (Unstim) for each cell line. Red lines denote the MFI of EV unstimulated cells for comparison. (D) BaF3-MPL cells were cultured in the presence of TPO (10 ng/mL) and dimethyl sulfoxide (DMSO) or concentrations of JAK inhibitor I ranging from 0.2μM to 5μM. Cumulative growth at 4 days (normalized to maximal growth for each cell line) is shown. Error bars represent the SD of 2 replicates per sample in panels B and D.