Patient-derived induced pluripotent stem cells recapitulate hematopoietic abnormalities of juvenile myelomonocytic leukemia

Abstract

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children initiated by mutations that deregulate cytokine receptor signaling. Studies of JMML are constrained by limited access to patient tissues. We generated induced pluripotent stem cells (iPSCs) from malignant cells of two JMML patients with somatic heterozygous p.E76K missense mutations in PTPN11, which encodes SHP-2, a nonreceptor tyrosine phosphatase. In vitro differentiation of JMML iPSCs produced myeloid cells with increased proliferative capacity, constitutive activation of granulocyte macrophage colony-stimulating factor (GM-CSF), and enhanced STAT5/ERK phosphorylation, similar to primary JMML cells from patients. Pharmacological inhibition of MEK kinase in iPSC-derived JMML cells reduced their GM-CSF independence, providing rationale for a potential targeted therapy. Our studies offer renewable sources of biologically relevant human cells in which to explore the pathophysiology and treatment of JMML. More generally, we illustrate the utility of iPSCs for in vitro modeling of a human malignancy.

Figure 1

Increased myelopoiesis from PTPN11 p.E76K heterozygous iPSCs. (A) EBs generated from WT or PTPN11 p.E76K (SHP-2) heterozygous iPSCs were cultured in a multilineage cytokine cocktail to support myeloerythroid maturation (see supplemental Methods). The iPSC lines are numbered according to the individual donor followed by the clone from that individual. The relative proportion of myeloid (My; CD45⁺CD18⁺), erythroid (Ery; CD235⁺), and megakaryocytic (Meg; CD41⁺CD42⁺) cells produced by each clone in three independent experiments is shown. The PTPN11-mutant clones produced increased proportions of myeloid cells (P = .008) and reduced proportions of erythroid cells (P = .001). (B) Hematopoietic progenitors released from day 8 EBs were enumerated in methylcellulose colony assays. Each bar represents the results of 3 separate experiments, each with triplicate colony assays. Myeloid colonies produced by PTPN11 p.E76K iPSCs were increased (P = .004), and erythroid colonies were decreased (P = .001). (C) Myeloid colonies from methylcellulose assays. Scale bar represents 200 µm. (D) Hematopoietic progenitors from day 8 monolayer differentiation cultures of PTPN11 p.E76K or WT iPSCs were suspended at 25 000 cells/mL in medium containing stem cell factor, GM-CSF, and IL-3 and were quantified at the indicated time points. By day 18, all cells exhibited myeloid morphology, similar to those represented in panel (E). (E) May-Grünwald-Giemsa-stained cells from WT and PTPN11 p.E76K myeloid colonies represented in panels (B) and (C). Scale bars represent 20 µm.

Figure 2

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.