FLT3 receptor in normal and malignant hematopoiesis

All hematopoietic cells originate from hematopoietic stem cells (HSCs) residing in the bone marrow (BM). The classical model of hematopoietic lineage commitment, that has been the prevailing model for hematopoiesis, proposes that the first lineage commitment step of HSCs and progenitors results in a strict separation of myelopoiesis and lymphopoiesis. However, an alternative model has already been suggested by introducing the lymphoid-primed multipotent progenitors (LMPPs) which highly express the receptor tyrosine kinase FLT3, and have sustained lymphoid and myeloid potentials, but little or no megakaryocyte/erythroid (MkE) potential. In this work, we further characterized LMPPs by applying clonal single cell culture assay, global gene expression analysis and multiplex single cell PCR assay. We demonstrated that LMPPs could generate myeloid, B- and T- cells at very high frequencies in culture but had very low MkE potential, and that MkE transcriptional priming was downregulated in LMPPs whereas lymphoid priming was upregulated, and lymphoid genes were coexpressed with myeloid genes. We could also show that the MkE potential of LMPP segregated almost entirely with the expression of thrombopoietin receptor (THPOR) in this population. Besides, using RAG1-GFP reporter mice, we could demonstrate that the GM potential is sustained but gradually reduced with increasing levels of lymphoid transcriptional priming within the LMPP compartment, suggesting the lineage restriction process from HSCs to LMPPs occurs as a gradual process. FLT3 plays an important role in normal hematopoiesis. Moreover, gain of function mutations of FLT3 has been reported in about one third of acute myeloid leukemia (AML) patients. The most common mutation of FLT3 is the internal tandem duplication (ITD) of its juxtamembrane domain, which confers a poor clinical prognosis. The clinical outcome is even worse in those patients who lose the FLT3 wild type (WT) gene. Using an Flt3-ITD knockin mouse model crossed with FLT3 receptor knockout mice, we could demonstrate that the development of a myeloproliferative disease in these mice is dependent both on ITD gene dosage as well as the absence of the WT allele. Further, we found that the development of the myeloproliferative disease in Flt3-ITD homozygous mice was FLT3 ligand independent

The Concerted Action of E2-2 and HEB Is Critical for Early Lymphoid Specification

The apparition of adaptive immunity in Gnathostomata correlates with the expansion of the E-protein family to encompass E2-2, HEB, and E2A. Within the family, E2-2 and HEB are more closely evolutionarily related but their concerted action in hematopoiesis remains to be explored. Here we show that the combined disruption of E2-2 and HEB results in failure to express the early lymphoid program in Common lymphoid precursors (CLPs) and a near complete block in B-cell development. In the thymus, Early T-cell progenitors (ETPs) were reduced and T-cell development perturbed, resulting in reduced CD4 T- and increased γδ T-cell numbers. In contrast, hematopoietic stem cells (HSCs), erythro-myeloid progenitors, and innate immune cells were unaffected showing that E2-2 and HEB are dispensable for the ancestral hematopoietic lineages. Taken together, this E-protein dependence suggests that the appearance of the full Gnathostomata E-protein repertoire was critical to reinforce the gene regulatory circuits that drove the emergence and expansion of the lineages constituting humoral immunity

DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction. Nat Genet. 2009; 41(11):1207–1215. [PubMed: 19801979

2 0 7 A r t i c l e s The crucial molecular mechanisms that control stem cell fate have received widespread attention because these mechanisms could potentially be manipulated to engineer stem cell biology for therapeutic interventions or tissue repair. Moreover, increasing evidence indicates that many tumors are sustained by cancer stem cells (CSCs) whose self-renewal may be controlled by mechanisms similar to those that control normal stem cells 1,2 . The hematopoietic system provides a paradigm for studying molecular mechanisms controlling stem cell function 3,4 . Lifelong replenishment of all hematopoietic cells is maintained by HSCs, which in a tightly controlled process give rise to a hierarchy of multipotent and lineage-committed progenitors 5 . Regulation of the diverse functional repertoire of HSCs requires the coordinated action of transcription factors 6 . The activity of most transcription factors relies on the recruitment of cofactors, many of which control gene expression by catalyzing epigenetic modifications of chromatin 7 . However, the functional impact of epigenetic modification mechanisms on coordination of stem cell fate programs is still poorly understood. Methylation of CpG dinucleotides within the DNA is a major epigenetic modification, which in mammals is controlled by at least three different DNA methyltransferases (DNMTs): DNMT3a and DNMT3b for de novo methylation, and DNMT1 for methylation maintenance 8 . The impact of methylation on stem cell features has been studied in embryonic stem cells, but little is known about its function in somatic stem cells in vivo Here we address this issue using mice with gradually diminished Dnmt1 expression. We show that distinct methylation threshold levels are required for alternative fate decisions of both HSCs and CSCs. The data suggest that competing stem cell programs require different methylation dosage-dependent control mechanisms and identify CpG methylation as a shared epigenetic program in the control of normal and neoplastic stem cells. RESULTS DNMT1 is indispensable for cell-autonomous survival of HSCs HSCs express high levels of Dnmt1, the major methyltransferase of postnatal mammalian cells 10 . To investigate the role of DNA methylation in HSCs, we bred mice in which exons 4 and 5 of Dnmt1 were flanked by loxP sites 12 with mice expressing Cre recombinase under the control of the type I interferon-inducible Mx1 promoter 13 (transgene officially named Tg(Mx1-cre); referred to here as MxCre). This strategy allowed inducible deletion of the catalytic Dnmt1 domain DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but dispensable for homing, cell cycle control and suppression of apoptosis. Notably, HSCs from mice with reduced DNA methyltransferase 1 activity cannot suppress key myeloerythroid regulators and thus can differentiate into myeloerythroid, but not lymphoid, progeny. A similar methylation dosage effect controls stem cell function in leukemia. These data identify DNA methylation as an essential epigenetic mechanism to protect stem cells from premature activation of predominant differentiation programs and suggest that methylation dynamics determine stem cell functions in tissue homeostasis and cancer

FOXO Dictates Initiation of B Cell Development and Myeloid Restriction in Common Lymphoid Progenitors

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs

FOXO1 and FOXO3 Cooperatively Regulate Innate Lymphoid Cell Development

Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15R beta (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment

Permissive roles of hematopoietin and cytokine tyrosine kinase receptors in early T-cell development.

Although several cytokines have been demonstrated to be critical regulators of development of multiple blood cell lineages, it remains disputed to what degree they act through instructive or permissive mechanisms. Signaling through the FMS-like tyrosine kinase 3 (FLT3) receptor and the hematopoietin IL-7 receptor alpha (IL-7Ralpha) has been demonstrated to be of critical importance for sustained thymopoiesis. Signaling triggered by IL-7 and thymic stromal lymphopoietin (TSLP) is dependent on IL-7Ralpha, and both ligands have been implicated in T-cell development. However, we demonstrate that, whereas thymopoiesis is abolished in adult mice doubly deficient in IL-7 and FLT3 ligand (FLT3L), TSLP does not play a key role in IL-7-independent or FLT3L-independent T lymphopoiesis. Furthermore, whereas previous studies implicated that the role of other cytokine tyrosine kinase receptors in T lymphopoiesis might not involve permissive actions, we demonstrate that ectopic expression of BCL2 is sufficient not only to partially correct the T-cell phenotype of Flt3l(-/-) mice but also to rescue the virtually complete loss of all discernable stages of early T lymphopoiesis in Flt3l(-/-)Il7r(-/-) mice. These findings implicate a permissive role of cytokine receptors of the hematopoietin and tyrosine kinase families in early T lymphopoiesis