Patient-Derived Induced Pluripotent Stem Cells Recapitulate Hematopoietic Abnormalities of Juvenile Myelomonocytic Leukemia

Abstract 637

Juvenile Myelomonocytic Leukemia (JMML) is an aggressive childhood myeloproliferative disorder caused by NF1, NRAS, KRAS, PTPN11 or CBL gene mutations that induce Ras pathway activation with associated hypersensitivity to cytokine stimulation in myeloid progenitor cells. Understanding the pathophysiology of JMML and developing new treatments is constrained by limited access to relevant patient material. To address this problem, we generated induced pluripotent stem cells (iPSCs) from normal neonatal umbilical cord blood and two JMML patients with different mutations: 1) a somatic heterozygous E76K substitution in PTPN11 and 2) a CBL Y371H substitution arising from a germline mutation. We created iPSCs from the patient's heterozygous CBL Y371H newborn cord blood cells prior to the diagnosis of JMML and then from peripheral blood at age 7 months, when JMML ensued with outgrowth of CBL Y371H homozygous malignant cells. We reprogrammed control and malignant cells using the STEMCCA lentiviral vector system that expresses OCT4, KLF4, MYC and SOX2 in a doxycyline-inducible fashion. Resultant iPSCs exhibited hallmark features after more than twenty passages, including characteristic morphology, expression of endogenous pluripotency markers, silencing of viral reprogramming genes in the absence of doxycycline, normal karyotype and formation of endoderm-, ectoderm- and mesoderm-derived tissues in teratoma assays. Relevant PTPN11 and CBL genotypes of these clones were confirmed by DNA sequencing.

Selected iPSC clones were differentiated into blood by inducing the formation of embryoid bodies in serum free medium with defined cytokines. By day 8–9, hematopoietic progenitors (CD43+, CD235+, CD41+) with erythroid, megakaryocytic and myeloid potential developed from both control (n = 2 lines) and JMML iPSCs (n= 2 lines from each patient). In methylcellulose colony assays, JMML progenitors exhibited relative hypersensitivity to GM-CSF, as reflected by increased numbers and larger size of myeloid colonies at limiting GM-CSF concentrations. In liquid cultures containing SCF, TPO, EPO, IL-3, IL-11 and IGF-1, with or without GM-CSF, the JMML progenitors produced increased proportions of CD33+CD14+ myelomonocytic cells compared to controls. Moreover, in GM-CSF dose-response assays, single cell phospho-flow cytometry analysis showed sustained STAT5 activation in JMML myeloid cells vs. controls. Thus, key pathological features of JMML, including propensity to myelomonocytic cell expansion and GM-CSF hypersensitivity, are recapitulated by in vitro differentiation of JMML iPSCs. Our findings illustrate the utility of iPSCs for modeling human blood disorders and more specifically, provide renewable sources of biologically relevant, patient-derived cells in which to explore the pathophysiology and therapy of JMML.