Distinct from other forms of acute lymphoblastic leukemia (ALL), infant ALL with mixed lineage leukemia (MLL) gene rearrangement, the most common leukemia occurring within the first year of life, might arise without the need for cooperating genetic lesions. Through Ig/TCR rearrangement analysis of MLL-AF4+ infant ALL at diagnosis and xenograft leukemias from mice transplanted with the same diagnostic samples, we established that MLL-AF4+ infant ALL is composed of a branching subclonal architecture already at diagnosis, frequently driven by an Ig/TCR-rearranged founder clone. Some MLL-AF4+ clones appear to be largely quiescent at diagnosis but can reactivate and dominate when serially transplanted into immunodeficient mice, whereas other dominant clones at diagnosis can become more quiescent, suggesting a dynamic competition between actively proliferating and quiescent subclones. Investigation of paired diagnostic and relapse samples suggested that relapses often occur from subclones already present but more quiescent at diagnosis. Copy-number alterations identified at relapse might contribute to the activation and expansion of previously quiescent subclones. Finally, each of the identified subclones is able to contribute to the diverse phenotypic pool of MLL-AF4+ leukemia-propagating cells. Unraveling of the subclonal architecture and dynamics in MLL+ infant ALL may provide possible explanations for the therapy resistance and frequent relapses observed in this group of poor prognosis ALL.

IntroductionWhereas considerable knowledge has been gained with regard to the identity and roles of extrinsic and intrinsic regulators of blood lineage development, much less is known about the molecular mechanisms regulating lineage commitment of hematopoietic stem cells (HSCs). 1,2 Unraveling the involved molecular determinants and mechanisms of lineage restriction will be facilitated by, and most likely depend on, a more complete understanding of the cellular pathways of the lineage restriction process from pluripotent HSCs to lineage-restricted progenitor cells.It remains unclear and debated 2-4 exactly how the lineage commitment process from pluripotent HSCs to lineage-restricted progenitors occurs in adult bone marrow (BM), and even unequivocal evidence for one such pathway would not exclude the existence of alternative routes for HSC lineage commitment. In the prevailing model of HSC lineage commitment, 1-3 HSCs (long-term and short-term) and multipotent progenitors (MPPs) distinguish themselves from each other, only through gradual loss of self-renewal potential while sustaining the same degree of pluripotentiality, with the first lineage commitment event resulting in a strict separation of myelopoiesis and lymphopoiesis. This model for HSC lineage commitment was supported by the identification of common myeloid and common lymphoid progenitors (CMPs and CLPs, respectively). 5,6 However, the degree to which the identified CMPs and CLPs represent obligatory or even main intermediates for myeloid and lymphoid development in adult hematopoiesis remains to be established.Although conclusively established for long-term HSCs (LTHSCs), 7,8 the existence of short-term HSCs (ST-HSCs) and MPPs in the BM Lin Ϫ Sca-1 ϩ Kit ϩ (LSK) stem and primitive progenitor cell compartment, with sustained pluripotentiality has yet to be demonstrated at the single cell level. 3 Rather, more recent studies have uncovered considerable heterogeneity in the LSK MPP compartment. Through the use of different but overlapping markers such as FMS-like tyrosine kinase 3 (Flt3), 9-11 vascular cell adhesion molecule-1 (Vcam-1), 12,13 and an Ikaros-reporter, 14 the existence of lymphoid-primed MPPs (LMPPs) with combined granulocyte/monocyte (GM) and lymphoid potentials, but little or no megakaryocyte (Mk)/erythroid (E) potentials has been proposed. 3,10,12,14,15 Further, molecular analysis of putative LMPPs show down-regulated transcriptional priming of genes specific for the MkE lineage, and up-regulation of lymphoid-specific genes, not yet expressed in HSCs. 15,16 The identification of a putative LMPP, representing the earliest lineage-restricted lympho-myeloid progenitor identified in adult hematopoiesis, has provided a potential avenue toward uncovering alternative HSC lineage commitment pathways. 2,3,17 However, the existence of the LMPP remains contentious, 2,3,[18][19][20] largely reflecting functional heterogeneity of phenotypically defined candidate LMPPs. 9,10,[12][13][14] While the evidence for a large fraction of LSKFlt3 hi BM cell...

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Key Points CSF1R is expressed on the earliest fetal B-cell progenitors, and CSF1R deficiency impairs fetal B-cell development. CSF1R+ fetal ProB cells have a B-myeloid gene signature and possess B and myeloid potential.

Within the hematopoietic system, the Notch pathway is critical for promoting thymic T cell development and suppressing the B and myeloid lineage fates; however, its impact on NK lymphopoiesis is less understood. To study the role of Notch during NK cell development in vivo, we investigated different NK cell compartments and function in Rbp-Jk fl/fl Vav-Cre tg/+ mice, in which Rbp-Jk, the major transcriptional effector of canonical Notch signaling, was specifically deleted in all hematopoietic cells. Peripheral conventional cytotoxic NK cells in Rbp-Jk-deleted mice were significantly reduced and had an activated phenotype. Furthermore, the pool of early NK cell progenitors in the bone marrow was decreased, whereas immature NK cells were increased, leading to a block in NK cell maturation. These changes were cell intrinsic as the hematopoietic chimeras generated after transplantation of Rbp-Jk-deficient bone marrow cells had the same NK cell phenotype as the Rbp-Jk-deleted donor mice, whereas the wild-type competitors did not. The expression of several crucial NK cell regulatory pathways was significantly altered after Rbp-Jk deletion. Together, these results demonstrate the involvement of canonical Notch signaling in regulation of multiple stages of NK cell development.

Summary Given that FLT 3 expression is highly restricted on lymphoid progenitors, it is possible that the established role of FLT 3 in the regulation of B and T lymphopoiesis reflects its high expression and role in regulation of lymphoid‐primed multipotent progenitors ( LMPP s) or common lymphoid progenitors ( CLP s). We generated a Flt3 conditional knock‐out ( Flt3 fl/fl ) mouse model to address the direct role of FLT 3 in regulation of lymphoid‐restricted progenitors, subsequent to turning on Rag1 expression, as well as potentially ontogeny‐specific roles in B and T lymphopoiesis. Our studies establish a prominent and direct role of FLT 3, independently of the established role of FLT 3 in regulation of LMPP s and CLP s, in regulation of fetal as well as adult early B cell progenitors, and the early thymic progenitors ( ETP s) in adult mice but not in the fetus. Our findings highlight the potential benefit of targeting poor prognosis acute B‐cell progenitor leukaemia and ETP leukaemia with recurrent FLT 3 mutations using clinical FLT 3 inhibitors.

1432 Poster Board I-455 Whether a stochastic or hierarchical model best reflects the identity of leukemia initiating cell is still an open debate. In the case of acute lymphoblastic leukemia (ALL) little is known about the identity and the origin of leukemic stem cells (LSCs). In this study we focused on infant ALL carrying the t(4;11)/MLL-AF4, the most frequent and aggressive form of leukemia occurring within the first year of life. Using fluorescence-activated cell sorting (FACS) combined with fluorescence in situ hybridization (FISH) analysis, in vivo repopulating assay and clonal analysis, we investigated whether different BM subsets, sorted from infant ALL diagnostic samples on the bases of their CD34/CD19/NG2 phenotype, have similar or distinct LSC properties. Our studies demonstrate that in t(4;11)/MLL-AF4 infant ALL the 34+19- hematopoietic stem/progenitor cell pool is intact (MLL-AF4 negative by FISH), and that the LSC is not CD19-, as CD19- cells lacked repopulating ability (CD34-) or gave rise exclusively to normal multilineage reconstitution (CD34+). In contrast, CD19+ LSCs rapidly engraft and establish leukemia in NOD/SCID mice, with features recapitulating the human disease, and possess self-renewal potential in serial transplantation, regardless of CD34 and NG2 expression. However, although multiple leukemic subsets possess some “stemness”, we did observe differences in the kinetics and immunophenotypes of engraftment between phenotypically distinct LSC subpopulations, suggesting that in t(4;11)/MLL-AF4 infant ALL the LSCs are diverse but distinct. These findings might reconcile conflicting results with regard to previously proposed stochastic versus hierarchical models for LSCs. By designing patient-specific primers and probes on the junctional regions, we analyzed by RQ-PCR the clonal contribution to the leukemic grafts and found that, as in infant patients, ALL grafts in mice were oligoclonal, as several independent clones were able to sustain engraftment in recipient mice. However, among the engrafting clones, we observed different behaviours: either major or minor clones in the patient dominate in the graft; conversely some other clones which were predominant in the diagnostic sample failed to sustain leukemia in vivo. Specifically we observed that dominant clones in primary recipients reconstituted with leukemia can either persist over time or extinguish (or become dormant) in serial passages; while other clones, which were quiescent in primary recipents, never-the-less persisted, reactivated and became dominating in secondary recipients. In conclusion we provided evidences that LSC in infant ALL with t(4;11) is restricted to the CD19+ fraction and, among this, multiple phenotypically distinct BM subsets can initiate leukemia in NOD/SCID mice, with different kinetics and immunophenotypes of engraftment. Moreover, our clonal analysis results point out a further heterogeneity within purified LSC subsets and implicate the co-existence and competition between multiple clones. Disclosures No relevant conflicts of interest to declare.