Targeted correction of RUNX1 mutation in FPD patient-specific induced pluripotent stem cells rescues megakaryopoietic defects

Abstract

• Successful generation of iPSC lines from FPD patient fibroblasts.• Restoration of megakaryopoiesis after RUNX1 mutation correction.Familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease of the hematopoietic system that is caused by heterozygous mutations in RUNX1. FPD/AML patients have a bleeding disorder characterized by thrombocytopenia with reduced platelet numbers and functions, and a tendency to develop AML. No suitable animal models exist for FPD/AML, as Runx1 1/2 mice and zebra fish do not develop bleeding disorders or leukemia. Here we derived induced pluripotent stem cells (iPSCs) from 2 patients in a family with FPD/AML, and found that the FPD iPSCs display defects in megakaryocytic differentiation in vitro. We corrected the RUNX1 mutation in 1 FPD iPSC line through gene targeting, which led to normalization of megakaryopoiesis of the iPSCs in culture. Our results demonstrate successful in vitro modeling of FPD with patient-specific iPSCs and confirm that RUNX1 mutations are responsible for megakaryopoietic defects in FPD patients. (Blood. 2014;124(12):1926-1930 IntroductionFamilial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) is a rare autosomal dominant disease. Patients are characterized by mild to moderate bleeding disorder, thrombocytopenia, and defective platelet functions.1-4 Notably, .35% of patients develop AML during their life. The disease is caused by heterozygous mutations in RUNX1, 5 which encodes a transcription factor required for definitive hematopoiesis and is frequently involved in leukemogenesis.6-10 To date, .30 FPD mutations have been identified in RUNX1, 11 which can be dominant-negative, haploinsufficient, or hypermorphic. 12,13 Existing animal models do not recapitulate all features of FPD/ AML, because Runx1 1/2 animals (both mouse and zebra fish) have no defects in megakaryopoiesis and do not develop leukemia. 14,15On the other hand, induced pluripotent stem cells (iPSCs) have the potential to differentiate into many cell types of the human body so that they may be used to model many human diseases as well as to serve as a valuable resource in the field of regenerative medicine. 16-18If disease-causing mutations can be corrected in patient-specific iPSCs, differentiated to therapeutically relevant cell types, and transplanted back to the patients, the symptoms of numerous diseases might be alleviated and the patients might be cured. Here, we derived iPSCs from 2 FPD/AML patients and demonstrated that these iPSCs have a megakaryopoietic defect in culture. It is important to note that we were able to rescue the megakaryopoietic defect by correcting the RUNX1 mutation with a gene-targeting strategy enhanced by zinc finger nucleases (ZFNs). Study designPatient skin biopsies were performed at the National Institutes of Health Clinical Center, and the fibroblast cultures were established at the Tissue Culture Shared Resource, Georgetown University. Skin biopsies and consent for iPSC derivat...

Myeloid neoplasms with germ line RUNX1 mutation

“…To overcome this problem, studies using human cells were conducted. [54,55]. Importantly, this megakaryocyte phenotype was able to be rescued by gene-targeting correction of RUNX1 mutation [54,55].…”

“…[54,55]. Importantly, this megakaryocyte phenotype was able to be rescued by gene-targeting correction of RUNX1 mutation [54,55]. Patient-derived iPSCs would be a useful tool to dissect the pathogenesis of FPD/AML and the process of leukemic transformation in human hematopoietic cells [53].…”

“…Patient-derived iPSCs would be a useful tool to dissect the pathogenesis of FPD/AML and the process of leukemic transformation in human hematopoietic cells [53]. Gene correction of RUNX1 mutations might also hold the promise of establishing a new therapeutic strategy for FPD/ AML patients [54,55].…”

iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations

“…We will from here on refer to both fibroblast subclones and iPSC lines as "daughter lines." Parental fibroblast line 1 (PF1) was obtained from a patient with familial platelet disorder (FPD), who carried a Y260X mutation in the RUNX1 gene (17), and the second parental fibroblast line (PF2) was obtained from a clinically healthy donor (CTRL-1) (18). Early passage fibroblasts (passage 4) were single-cell sorted into 96-well plates and clonally expanded to establish the subclones and to obtain sufficient amounts of genomic DNA (gDNA) for whole exome sequencing and SNP genotyping analyses (Fig.…”

RUNX1 deficiency (familial platelet disorder with predisposition to myeloid leukemia, FPDMM)

“…Introducing a wild-type copy of RUNX1 into iPSC lines from patients with FPDMM reversed most of the hematopoietic differentiation defects and resulted in a significant increase of both the number of megakaryocytic progenitors and of erythroid progenitors. In parallel, the number of granulomonocytic progenitors decreased to control levels indicating that the leukemic potential may also be reduced [54]. Alternative approaches used transcription activator-like effector nucleases (TALEN) and a plasmid containing wild type RUNX1 cDNA sequences in patient-derived iPS cells [55].…”