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Runx1 deficiency permits granulocyte lineage commitment but impairs subsequent maturation.

ABSTRACT: First-hits in the multi-hit process of leukemogenesis originate in germline or hematopoietic stem cells (HSCs), yet leukemia-initiating cells (LICs) usually have a lineage-committed phenotype. The molecular mechanisms underlying this compartment shift during leukemia evolution have not been a major focus of investigation and remain poorly understood. Here a mechanism underlying this shift was examined in the context of Runx1 deficiency, a frequent leukemia-initiating event. Lineage-negative cells isolated from the bone marrow of Runx1-haploinsufficient and wild-type control mice were cultured in granulocyte-colony-stimulating factor to force lineage commitment. Runx1-haploinsufficient cells demonstrated significantly greater and persistent exponential cell growth than wild-type controls. Not surprisingly, the Runx1-haploinsufficient cells were differentiation-impaired, by morphology and by flow-cytometric evaluation for granulocyte differentiation markers. Interestingly, however, this impaired differentiation was not because of decreased granulocyte lineage commitment, as RNA and protein upregulation of the master granulocyte lineage-commitment transcription factor Cebpa, and Hoxb4 repression, was similar in wild-type and Runx1-haploinsufficient cells. Instead, RNA and protein expression of Cebpe, a key driver of progressive maturation after lineage commitment, were significantly decreased in Runx1-haploinsufficient cells. Primary acute myeloid leukemia cells with normal cytogenetics and RUNX1 mutation also demonstrated this phenotype of very high CEBPA mRNA expression but paradoxically low expression of CEBPE, a CEBPA target gene. Chromatin-immunoprecipitation analyses suggested a molecular mechanism for this phenotype: in wild-type cells, Runx1 binding was substantially greater at the Cebpe than at the Cebpa enhancer. Furthermore, Runx1 deficiency substantially diminished high-level Runx1 binding at the Cebpe enhancer, but lower-level binding at the Cebpa enhancer was relatively preserved. Thus, Runx1-deficiency permits Cebpa upregulation and the exponential cell growth that accompanies lineage commitment, but by impairing activation of Cebpe, a key proliferation-terminating maturation gene, extends this exponential growth. These mechanisms facilitate germline cell or HSC of origin, yet evolution into LIC with lineage-committed phenotype.

SUBMITTER: Ng KP

PROVIDER: S-EPMC3849692 | biostudies-other | 2013 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundComplex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role.MethodsTo identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls.ResultsGene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues.ConclusionsOur results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

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Runx1 deficiency permits granulocyte lineage commitment but impairs subsequent maturation.

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