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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.

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 Flt3ITD/ITD myeloid phenotype is FLT3 ligand-independent.

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.

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.

Abstract 1380 Although the growth factor receptor (GFR) FLT3 has a crucial role in normal early B- and T-lymphoid development, constitutively activating internal tandem duplications (ITDs) of FLT3 are almost entirely restricted to patients with adverse-risk acute myeloid leukemia. We used a murine knock-in model of FLT3-ITD myeloproliferative disease (MPD) to gain a better understanding of the cellular and molecular basis for the myeloid-bias of FLT3-ITD-induced hematological malignancies. As Flt3 cell surface expression is lacking in homozygous Flt3-itd mice, we used CD48 and CD150 expression to investigate the distribution of multipotent progenitors (MPPs) and hematopoietic stem cells (HSCs) within the primitive Lin-Sca1+Kit+ (LSK) compartment. Notably, phenotypic (LSKCD150+CD48-) and functional HSCs were markedly reduced in adult Flt3-itd mice. Competitive transplantation experiments using fetal liver confirmed that HSC numbers were reduced (20-fold reduction) by Flt3-ITDs, in a cell-extrinsic manner. Rather, LSKCD48+150- cells (MPPs) were expanded 2.7-fold in Flt3-itd mice comprising >90% of LSK cells. Similarly to Flt3high wild type (WT) lymphoid-primed multipotent progenitors (LMPPs), nanofluidic gene-expression analysis demonstrated that WT MPPs and Flt3-itd MPPs were myeloid-primed (Csf1r, Csf2r, Cebpa, Mpo) with loss of megakaryocyte and erythroid (MkE) priming (Eklf, Epor, Vwf, Gata1). In contrast, the lymphoid (Il7r, Rag1, sIgH) transcriptional priming of WT MPPs was downregulated in Flt3-itd MPPs. In agreement with this, Flt3-itd MPPs sustained extensive GM potential in vitro, with no MkE potential and, unlike WT MPPs, considerably reduced lymphoid potential. Furthermore, microarray analysis demonstrated global upregulation of the myeloid program in Flt3-itd MPPs. These findings demonstrate that primitive lympho-myeloid MPPs, are expanded and biased towards myeloid development by Flt3-ITDs. In agreement with reduced lymphoid-priming of Flt3-itd MPPs, analysis of early thymic development demonstrated a 10-fold reduction of early thymic progenitors (DN1 Kit+) in Flt3-itd mice. Subsequent stages of thymic development were also reduced, as was overall thymic cellularity. Interestingly, expression of the chemokine receptor CCR9 was 5.5-fold reduced in Flt3-itd MPPs suggesting that thymic seeding progenitors in the bone marrow are suppressed by FLT3-ITDs. Previous studies have suggested that the earliest stage of B-cell development, pre-pro-B cells, retain both B-cell and myeloid potential. Lin-CD19-CD24-AA4.1+CD43+B220+ pre-pro-B cells were expanded 13.7-fold in Flt3-itd mice, whereas subsequent stages of CD19+ B-lymphopoiesis were all reduced. The expanded pre-pro-B cells in Flt3-itd mice were myeloid biased at the transcriptional level with markedly reduced expression of lymphoid genes. Pu1 is a master-regulator of myeloid commitment in early hematopoiesis and a STAT3 target gene. As FLT3-ITDs are known to activate STAT3, unlike WT FLT3, we therefore investigated Pu1 expression in Flt3-itd mice using a Pu1-YFP reporter. Expression of Pu1 was significantly increased in LSK cells (1.4 fold) and in pre-pro-B cells (2.6 fold) in Flt3-itd mice. Furthermore, other STAT3 target genes (Cish, Id1, Pim1, Socs1, Junb) were also upregulated in these cell populations in Flt3-itd mice. Moreover, gene-set enrichment analysis in MPPs demonstrated upregulation of Pu1 target genes in Flt3-itd mice, thus providing a link between aberrant ITD signaling and the observed myeloid bias. In order to determine the functional relevance of this myeloid-bias of Flt3-itd MPPs for disease transformation, we targeted a conditional Aml1-ETO fusion-gene to the earliest B-cell progenitors in Flt3-itd mice using Mb1-Cre. Expression of AML1-ETO in WT mice did not induce any phenotype. However, Mb1-Cre induced AML1-ETO expression in Flt3-itd mice led to a high-penetrance, short latency acute leukaemia. All leukaemias expressed myeloid markers (Mac1 and Gr1) but lacked CD19 and B220 expression. These data demonstrate that Flt3-ITDs expand primitive MPPs with a myeloid lineage bias at the molecular and cellular level, at the expense of HSCs and early lymphoid development. This provides insight into the mechanisms by which mutations resulting in activation of a GFR introduce a lineage bias of resulting hematological malignancies. Disclosures: No relevant conflicts of interest to declare.