Identification of a cytokine-responsive pSTAT3⁺/pSTAT5⁺ population in CD34⁺ early progenitors from patients with LNK mutations. Peripheral blood (PB) samples from patients with the LNK DEL and E208Q mutations, as well as PMF patients with the JAK2 V617F and MPL W515L mutations, were compared with normal donor. CD3⁻/CD66⁻/CD33^mid immature myeloid cells are shown in panels A through C. (A) Samples were preincubated with DMSO or JAK inhibitor I (5 μM) for 30 minutes, and then stimulated with TPO (50 ng/mL) or G-CSF (20 ng/mL) for 15 minutes, before assessment of STAT3 and STAT5 activation by phospho-specific flow cytometry. (B) CD34 and CD38 surface staining for the cytokine-responsive pSTAT3⁺/5⁺ (“responsive”) cells from DEL, in comparison to the nonresponsive cells (includes all cells other than pSTAT3⁺/5⁺ cells) is displayed. Cytokine-responsive cells are shown in green (TPO) and red (G-CSF). In panels A and B, numbers depicted in each quadrant represent the percentage of total cells present in each quadrant gate. (C) The frequency of CD34⁺ cytokine-responsive cells was quantified and is displayed as fold-change versus normal donor. (D) PB cells from DEL were stimulated with G-CSF, and 6 subsets were sorted by fluorescence-activated cell sorting (FACS), as defined by the surface markers and phosphorylated STAT proteins shown in the table. Not applicable (n/a) denotes surface markers that were not used to delineate that specific subset. DNA was isolated from each subset, and allele-specific quantitative PCR for the DEL mutation was performed. Allele burden for each subset is displayed. Error bars represent the SD of 3 replicates. (E) Schematic showing role of LNK in regulation of JAK-STAT signaling, and model depicting hypothetical mechanisms for LNK dysfunction is shown. (Left) Cytokine/receptor binding (eg, TPO/MPL) results in JAK-STAT activation, which then leads to recruitment of a negative feedback pathway, in which LNK binds to MPL and JAK2, thereby inhibiting downstream STAT activation. (Right) LNK mutations affecting the PH domain (depicted as a yellow line) may lead to mislocalization of LNK in the cytoplasm, thereby disrupting the ability of LNK to inhibit JAK-STAT signaling. As the dimerization domain is retained, mutant LNK forms may also sequester WT LNK, potentially resulting in a dominant-negative effect.