1Child Health and Development and 2Black Family Stem Cell Institutes, Mount Sinai School of Medicine, New York, NY, USA, 10029, 3Recinto de Ciencias Medicas, Universidad Puerto Rico, San Juan, Puerto Rico, 00936-5067, 4Departement Genetique Hopital Robert Debre AP-HP, Paris, France,5Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy, 00161
BACKGROUND: Noonan syndrome (NS) is a genetic developmental disorder caused by deregulation of the RAS/MAPK pathway. Germ-line mutations in PTPN11, which encodes SHP-2, a key component of the RAS/MAPK pathway, cause 50% of NS, while somatic mutations in this gene account for 35% of juvenile myelomonocytic leukemia (JMML). Children with NS and specific PTPN11 mutations are at increased risk for developing JMML, inferring that certain SHP-2 mutants result in abnormal differentiation and cell maturation in hematopoietic lineages. The molecular mechanisms resulting from SHP-2 dysregulation that lead to these abnormalities remain largely unexplored.
AIM: To elucidate signaling pathway alterations in myeloid progenitors in NS and NS/JMML using human induced pluripotent stem cells (hiPSC) derived from patients with those disorders.
DESIGN/METHODS: We established two human iPSC lines as control samples and eight iPSC lines with germ-line mutations in specific residues of PTPN11: Y63C and E76D in NS samples and D61H and G503C in NS/JMML samples. We differentiated these iPSCs into hematopoietic lineages using specific cytokines. Hematopoietic populations (surface markers: CD33, CD14, CD11b, CD71, CD235a, and CD41) in these samples were determined with flow cytometry. Proliferation and apoptosis were determined with Ki67 and Annexin V staining, respectively. To check clonogenic capacity, cells were plated on methylcellulose with specific cytokines to obtain CFU-GMs and CFU-Es. We assessed two clinical criteria used routinely for definitively diagnosing JMML: hypersensitivity to GM-CSF and absence of BCR-ABL fusion gene by FISH. Using RT-PCR and western blotting, we analyzed the levels of STAT5, BCL-XL and a panel of specific miRNAs (miR181, miR128a, miR20a, miR17, miR106 and miR223) in both the mixed hematopoietic population and CD33+ myeloid progenitors.
RESULTS: We observed an increase of myeloid progenitors (45%), monocytes (18%), erythrocytes (37%) and megakaryocytes (15%) in NS/JMML compared to controls (15%, 8%, 25% and 8% respectively). In addition, we observed an increase in the size and the total number of colonies in NS/JMML (600 vs 200 total colonies). The NS-JMML lines showed hypersensitivity to GM-CSF, responding at 0.1 ng/ml to which controls were not responsive. BCR-ABL fusion was absent. Apoptosis tended to be decreased in NS/JMML. While proliferation rate was marginally increased in mixed hematopoietic NS/JMML cells compared it controls, it was increased approximately 8 fold in CD33+ NS/JMML cells. These changes in proliferation and apoptosis in CD33+ NS/JMML cells correlated with increased expression of BCL-XL and STAT5 compared to control CD33+ myeloid cells. After screening of miRNAs associated with differentiation of hematopoietic cells using RT-PCR, we observed that the expression level of miR223, a specific regulator of granulocyte/monocyte precursors, was increased 500 fold in the mixed population and 20 fold in the myeloid population with mutations of PTPN11 while the expression levels of the other miRNAs assessed were comparable to controls.
CONCLUSIONS: This study provides the first model of leukemia using hematopoietic cells differentiated from human iPSCs. Moreover, these studies provide new insights about PTPN11-driven JMML, revealing up-regulation of STAT5 and miR223. These findings provide potential novel molecular targets for treating JMML, which remains a lethal disorder. Our future work will be directed at determining the upstream regulators and downstream effects of increased STAT5 and miR223. We are also attempting to use the NS/JMML iPSC-derived hematopoietic progenitors to develop a transplant mouse model of JMML.