PTPN11 p.E76K iPSC-derived myeloid cells exhibit GM-CSF hypersensitivity that is ameliorated by MEK inhibition. (A) GM-CSF dose-response for myeloid colony formation from WT and PTPN11 p.E76K iPSCs. Day 8 EB-derived hematopoietic progenitors (3 × 10³) were seeded into methylcellulose cultures with the indicated doses of GM-CSF. Representative photographs of myeloid colonies from one experiment are shown. Scale bar indicates 200 µm. (B) GM-CSF dose-response for myeloid colony formation from WT and PTPN11 p.E76K iPSCs, summarizing the results from multiple clones. Colonies were generated as described in panel (A). Myeloid colonies containing >50 cells were scored. The resultant colony numbers were normalized to the maximum colony number obtained at saturating GM-CSF concentration (10 ng/mL). Three independent experiments were performed with each clone. **P < .01 for PTPN11 p.E76K sample vs controls at 0 and 0.01 ng/mL GM-CSF. *At 0.1 ng/mL GM-CSF, P < .01 for PTPN11 p.E76K samples vs WT2.1 and P ≤ .05 vs WT1.1. (C) GM-CSF dose-responses for STAT5 activation. EB-derived hematopoietic progenitors were cultured for 4 days in GM-CSF, IL-3, and stem cell factor to generate myeloid cells. The cells were rested in cytokine- and serum-free medium for 16 hours and then stimulated with the specified concentrations of GM-CSF for 15 minutes. Cells were then fixed, permeabilized, stained with antibodies against surface markers and intracellular pSTAT5, and analyzed by phosphoflow cytometry. Cells are gated upon the CD45⁺CD18⁺ myeloid population for pSTAT5 analysis (not shown). (D) Summary of data from panel (C). Results at each GM-CSF concentration are normalized to the maximal response obtained at 10 ng/mL. (E) GM-CSF dose-response for myeloid colony formation from WT and PTPN11 p.E76K iPSCs with 100 nM PD0325901 (PD901) or 100 nM ruxolitinib (RUXO), representing the ED₅₀ for each drug (supplemental Figure 2E). Representative experiments are shown for 1 clone of each genotype; similar results were obtained from additional clones (supplemental Figure 2F). **P < .01; *P ≤ .05 for dimethylsulfoxide (DMSO) vs PD901. (F) Average number of cells per myeloid colonies generated from WT and PTPN11 p.E76K iPSC clones at 10 ng/mL GM-CSF with PD0325901 (PD901) or ruxolitinib (RUXO), as depicted in panel (E). Methylcellulose cultures with myeloid colonies were solubilized in growth medium and individual cells were harvested and enumerated. Graphs show the results of three pooled dishes for each sample. Results are shown for multiple iPSC clones depicted in Figure 2E and supplemental Figure 2F. **P < .01; *P = .02. (G) Phosphoflow cytometric analysis of JAK-inhibited and MEK-inhibited iPSCs. iPSCs were rested for 1 hour in serum-free medium, incubated with 1 μM RUXO or 100 nM PD901, then fixed, permeabilized, stained, and analyzed as in Figure 2C. Basal levels of pSTAT5 are similarly increased in WT and PTPN11-mutant iPSCs (in comparison with isotype staining control; not shown), and RUXO modestly inhibits pSTAT5 in both samples. pERK is constitutively activated only in PTPN11-mutant iPSCs and inhibited by PD901. Cells are gated on the CD45⁺CD18⁺ population (not shown). Representative data from 1 experiment are shown.