Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients

Abstract

Somatic mutation of RUNX1 is implicated in various hematological malignancies, including myelodysplastic syndrome and acute myeloid leukemia (AML), and previous studies using mouse models disclosed its critical roles in hematopoiesis. However, the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder (FPD)/AML is an autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. By in vitro differentiation assays, FPD-iPSCs were clearly defective in the emergence of hematopoietic progenitors and differentiation of megakaryocytes, and overexpression of wild-type (WT)-RUNX1 reversed most of these phenotypes. We further demonstrated that overexpression of mutant-RUNX1 in WT-iPSCs did not recapitulate the phenotype of FPD-iPSCs, showing that the mutations were of loss-of-function type. Taken together, this study demonstrated that haploinsufficient RUNX1 allele imposed cell-intrinsic defects on hematopoietic differentiation in human experimental settings and revealed differential impacts of RUNX1 dosage on human and murine megakaryopoiesis. FPD-iPSCs will be a useful tool to investigate mutant RUNX1-mediated molecular processes in hematopoiesis and leukemogenesis.

Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia

“…Furthermore, the importance of NR4A3 dosage was already highlighted in the induction of myeloproliferative neoplasms and AML in NR4A3 KO mouse models. [36][37][38] Contrary to our results showing the amplification of GM lineage after almost complete knockdown of RUNX1 or with DN-like R174Q mutation, this amplification was not observed with N233fsX283 mutation, 30 which is highly associated with AML. However, this last mutation induces a deep defect in the generation of hematopoietic progenitors, which could in some way also be a preleukemic state similar to that in some aplastic anemias.…”

“…47 In conclusion, whereas analysis of mouse models failed to identify any defect in primitive hematopoiesis, we show for the first time that RUNX1 germline alterations affect human embryonic hematopoiesis, particularly the generation of primitive erythroid and MK cells. As already described by others, 30,35 defects in the generation of mature MK cells do not depend on the type of RUNX1 variants. Conversely, a DN R174Q mutant of RUNX1 increases the proliferative rate and induces a genomic instability of GM progenitors, thereby contributing to development of a preleukemic state.…”

“…28,29 The number of human hematopoietic progenitors generated from FPD/AML iPSCs depends on the type of RUNX1 alteration. 30 Here, we show that the knockdown of RUNX1 in ESCs limits the commitment to hematopoietic lineage at the clonal level.…”

Recurrent CDC25C mutations drive malignant transformation in FPD/AML

“…Regarding the three missense variants in our study (p.Ser140Asn in pedigree 54, p.Gly172Glu in pedigree 57 and p.Leu445Pro in pedigree 32), Ser140 and Gly172 have been reported to be mutated in sporadic AML and/or MDS cases 25,26 . In addition, induced pluripotent stem cells from a FPD/AML pedigree with p.Gly172Glu recapitulate the phenotype of FPD/AML after hematopoietic differentiation 27 . Ser140 has been also shown to be important for RUNX1 conformation, and a mutation of this site affects hydrogen bonds and results in functional loss 28,29 .…”

Myeloid neoplasms with germ line RUNX1 mutation

“…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]. Gene correction of RUNX1 mutations might also hold the promise of establishing a new therapeutic strategy for FPD/ AML patients [54,55].…”