Shabnam Kharazi

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.

Lymphomyeloid Contribution of an Immune-Restricted Progenitor Emerging Prior to Definitive Hematopoietic Stem Cells.

In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis.

FLT3 ligand and not TSLP is the key regulator of IL-7–independent B-1 and B-2 B lymphopoiesis

Phenotypically and functionally distinct progenitors and developmental pathways have been proposed to exist for fetally derived B-1 and conventional B-2 cells. Although IL-7 appears to be the primary cytokine regulator of fetal and adult B lymphopoiesis in mice, considerable fetal B lymphopoiesis and postnatal B cells are sustained in the absence of IL-7; in humans, B-cell generation is suggested to be largely IL-7-independent, as severe combined immune-deficient patients with IL-7 deficiency appear to have normal B-cell numbers. However, the role of other cytokines in IL-7-independent B lymphopoiesis remains to be established. Although thymic stromal lymphopoietin (TSLP) has been proposed to be the main factor driving IL-7-independent B lymphopoiesis and to distinguish fetal from adult B-cell progenitor development in mice, recent studies failed to support a primary role of TSLP in IL-7-independent fetal B-cell development. However, the role of TSLP in IL-7-independent adult B lymphopoiesis and in particular in regulation of B-1 cells remains to be established. Here we demonstrate that, rather than TSLP, IL-7 and FLT3 ligand are combined responsible for all B-cell generation in mice, including recently identified B-1-specified cell progenitors. Thus, the same IL-7- and FLT3 ligand-mediated signal-ing regulates alternative pathways of fetal and adult B-1 and B-2 lymphopoiesis.

Downregulation of Mpl marks the transition to lymphoid-primed multipotent progenitors with gradual loss of granulocyte-monocyte potential.

Evidence for a novel route of adult hematopoietic stem-cell lineage commitment through Lin-Sca-1+Kit+Flt3hi (LSKFlt3hi) lymphoid-primed multipotent progenitors (LMPPs) with granulocyte/monocyte (GM) and lymphoid but little or no megakaryocyte/erythroid (MkE) potential was recently challenged, as LSKFlt3hi cells were reported to possess MkE potential. Herein, residual (1%-2%) MkE potential segregated almost entirely with LSKFlt3hi cells expressing the thrombopoietin receptor (Mpl), whereas LSKFlt3hiMpl- LMPPs lacked significant MkE potential in vitro and in vivo, but sustained combined GM and lymphoid potentials, and coexpressed GM and lymphoid but not MkE transcriptional lineage programs. Gradually increased transcriptional lymphoid priming in single LMPPs from Rag1GFP mice was shown to occur in the presence of maintained GM lineage priming, but gradually reduced GM lineage potential. These functional and molecular findings reinforce the existence of GM/lymphoid-restricted progenitors with dramatically down-regulated probability for committing toward MkE fates, and support that lineage restriction occurs through gradual rather than abrupt changes in specific lineage potentials.

FLT3-ITDs Instruct a Myeloid Differentiation and Transformation Bias in Lymphomyeloid Multipotent Progenitors.

Summary Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies.

Impact of gene dosage, loss of wild-type allele, and FLT3 ligand on Flt3-ITD-induced myeloproliferation

Acquisition of homozygous activating growth factor receptor mutations might accelerate cancer progression through a simple gene-dosage effect. Internal tandem duplications (ITDs) of FLT3 occur in approximately 25% cases of acute myeloid leukemia and induce ligand-independent constitutive signaling. Homozygous FLT3-ITDs confer an adverse prognosis and are frequently detected at relapse. Using a mouse knockin model of Flt3-internal tandem duplication (Flt3-ITD)-induced myeloproliferation, we herein demonstrate that the enhanced myeloid phenotype and expansion of granulocyte-monocyte and primitive Lin(-)Sca1(+)c-Kit(+) progenitors in Flt3-ITD homozygous mice can in part be mediated through the loss of the second wild-type allele. Further, whereas autocrine FLT3 ligand production has been implicated in FLT3-ITD myeloid malignancies and resistance to FLT3 inhibitors, we demonstrate here that the mouse Flt3(ITD/ITD) myeloid phenotype is FLT3 ligand-independent.

Expression and role of FLT3 in regulation of the earliest stage of normal granulocyte-monocyte progenitor development

Mice deficient in c-fms-like tyrosine kinase 3 (FLT3) signaling have reductions in early multipotent and lymphoid progenitors, whereas no evident myeloid phenotype has been reported. However, activating mutations of Flt3 are among the most common genetic events in acute myeloid leukemia and mice harboring internal tandem duplications within Flt3 (Flt3-ITD) develop myeloproliferative disease, with characteristic expansion of granulocyte-monocyte (GM) progenitors (GMP), possibly compatible with FLT3-ITD promoting a myeloid fate of multipotent progenitors. Alternatively, FLT3 might be expressed at the earliest stages of GM development. Herein, we investigated the expression, function, and role of FLT3 in recently identified early GMPs. Flt3-cre fate-mapping established that most progenitors and mature progeny of the GM lineage are derived from Flt3-expressing progenitors. A higher expression of FLT3 was found in preGMP compared with GMP, and preGMPs were more responsive to stimulation with FLT3 ligand (FL). Whereas preGMPs and GMPs were reduced in Fl(-/-) mice, megakaryocyte-erythroid progenitors were unaffected and lacked FLT3 expression. Notably, mice deficient in both thrombopoietin (THPO) and FL had a more pronounced GMP phenotype than Thpo(-/-) mice, establishing a role of FL in THPO-dependent and -independent regulation of GMPs, of likely significance for myeloid malignancies with Flt3-ITD mutations.

Molecular and cellular effects of oncogene cooperation in a genetically accurate AML mouse model

Biallelic CEBPA mutations and FMS-like tyrosine kinase receptor 3 (FLT3) length mutations are frequently identified in human acute myeloid leukemia (AML) with normal cytogenetics. However, the molecular and cellular mechanisms of oncogene cooperation remain unclear because of a lack of disease models. We have generated an AML mouse model using knockin mouse strains to study cooperation of an internal tandem duplication (ITD) mutation in the Flt3 gene with commonly observed CCAAT/enhancer binding protein alpha (C/EBPα) mutations. This study provides evidence that FLT3 ITD cooperates in leukemogenesis by enhancing the generation of leukemia-initiating granulocyte-monocyte progenitors (GMPs) otherwise prevented by a block in differentiation and skewed lineage priming induced by biallelic C/EBPα mutations. These cellular changes are accompanied by an upregulation of hematopoietic stem cell and STAT5 target genes. By gene expression analysis in premalignant populations, we further show a role of FLT3 ITD in activating genes involved in survival/transformation and chemoresistance. Both multipotent progenitors and GMP cells contain the potential to induce AML similar to corresponding cells in human AML samples showing that this model resembles human disease.

Niche-mediated depletion of the normal hematopoietic stem cell reservoir by Flt3-ITD-induced myeloproliferation.

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD–driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.

TSLP-mediated fetal B lymphopoiesis?

To the editor: In the August 2003 issue of Nature Immunology1, as well as in later studies2, Voβhenrich et al. presented experimental data on the cytokine requirements of developing B cells, which led them to conclude that “TSLP [thymic stromal lymphopoietin] is the factor responsible for most of the fetal and perinatal B cell production that takes place when the IL-7–γc [interleukin 7–common γ-chain] signaling pathway is disrupted.”1 Although the data reported were technically sound and compatible with such a conclusion, the authors did not provide direct evidence to support (or exclude) the idea of a critical function for TSLP in IL-7-independent fetal B lymphopoiesis. The conclusions of Voβhenrich et al. were based on the demonstration that B lymphopoiesis was much more affected (tenfold more) in mice deficient in IL-7 receptor α-chain, essential for IL-7 as well as TSLP signaling, than in mice deficient in the common γ-chain (γc), required for IL-7 but not TSLP-mediated signaling3,4. However, these data could at best be considered strong indirect support for the idea of TSLP as the main cytokine driving IL-7-independent fetal B lymphopoiesis, as there could be other reasons for a difference in the phenotypes of γcdeficient mice and those deficient in the IL-7 receptor α-chain. Furthermore, Voβhenrich et al. used bone marrow of mice 4–12 weeks of age, not fetal liver, for their comparative in vivo analysis of B lymphopoiesis in these mice1,2. Instead, the extrapolation to the idea that TSLP is key to the fetal stages of B lymphopoiesis was based on the finding that fetal but not adult pro–B cells were responsive to TSLP in vitro1,2. In contrast, a lack of an important function for TSLP in adult B lymphopoiesis has been indicated by studies of TSLP receptor–deficient (Tpte2–/–) mice5,6. As fetal lymphopoiesis had not been examined in singly deficient Tslp–/– or Tpte2–/– mice, we investigated B lymphopoiesis in the livers of Tpte2–/– mice at embryonic day 17.5 but found no deficiency in Tpte2–/– fetuses at any stage of B cell development (Fig. 1a and Supplementary Fig. 1 online). Furthermore, when comparing B lymphopoiesis in the fetal livers of Il7–/– and Il7–/–Tpte2–/– mice, we obtained no evidence for substantial involvement of TSLP in IL-7independent regulation of fetal pro–B cells or pre–B cells, whereas we noted a slight additional reduction in the number of immature B cells in Il7–/–Tpte2–/– fetuses relative to that in Il7–/– fetuses (Fig. 1b and Supplementary Fig. 1). Thus, although Voβhenrich et al. provided compelling evidence that fetal pro–B cells are highly responsive to TSLP1, our studies of Tpte2–/– and Il7–/–Tpte2–/– fetuses fail to support their claim that TSLP is the most important cytokine promoting IL-7independent fetal B lymphopoiesis. Instead, although Voβhenrich et al. also concluded that “Flk-2 is involved, but TSLP is the main factor driving IL-7-independent fetal and perinatal lymphopoiesis,”1 we have done additional studies of mice deficient in the cytokine Flt3L (also called Flk-2 ligand) and IL-7 (Flt3l–/–Il7–/– mice) and of Flt3l–/–Tpte2–/– mice and have found that the reported complete loss of B-1 as well as B-2 B lymphopoiesis in Flt3l–/–Il7r–/– mice7 and Flk2–/–Il7r–/– mice1 is entirely due to the simultaneous loss of function of IL-7 and Flt3L (C.T.J. and S.E.W.J., unpublished observations). Collectively, our findings suggest that Flt3L rather than TSLP is the key regulator of IL-7-independent B lymphopoiesis and that intact TSLP function is insufficient to restore any detectable B lymphopoiesis in the absence of these two critical regulators of B cell progenitors.