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Original citation: J Clin Invest. 2022;132(22):e157101. https://doi.org/10.1172/JCn57101. During the preparation of Supplemental Figure 12A, an image for day 35 was inadvertently used instead of an image for day 28. The [...]

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

Background aimsHuman cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Because Notch signaling is critical for embryonic blood vessel formation in utero, we hypothesized that Notch pathway activation may enhance cultured ECFC vasculogenic properties in vivo.MethodsIn vitro ECFC stimulation with an immobilized chimeric Notch ligand (Delta-like1(ext-IgG)) led to significant increases in the mRNA and protein levels of Notch regulated Hey2 and EphrinB2 that were blocked by treatment with γ-secretase inhibitor addition. However, Notch stimulated preconditioning in vitro failed to enhance ECFC vasculogenesis in vivo. In contrast, in vivo co-implantation of ECFCs with OP9-Delta-like 1 stromal cells that constitutively expressed the Notch ligand delta-like 1 resulted in enhanced Notch activated ECFC-derived increased vessel density and enlarged vessel area in vivo, an effect not induced by OP9 control stromal implantation.ResultsThis Notch activation was associated with diminished apoptosis in the exposed ECFC.ConclusionsWe conclude that Notch pathway activation in ECFC in vivo via co-implanted stromal cells expressing delta-like 1 promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC.

Hematopoietic stem cells (HSCs) first emerge during embryonic development within vessels such as the dorsal aorta of the aorta-gonad-mesonephros (AGM) region, suggesting that signals from the vascular microenvironment are critical for HSC development. Here, we demonstrated that AGM-derived endothelial cells (ECs) engineered to constitutively express AKT (AGM AKT-ECs) can provide an in vitro niche that recapitulates embryonic HSC specification and amplification. Specifically, nonengrafting embryonic precursors, including the VE-[cadherin.sup.-]expressing population that lacks hematopoietic surface markers, cocultured with AGM AKT-ECs specified into long-term, adult-engrafting HSCs, establishing that a vascular niche is sufficient to induce the endothelial-to-HSC transition in vitro. Subsequent to hematopoietic induction, coculture with AGM AKT-ECs also substantially increased the numbers of HSCs derived from VE-[cadherin.sup.+][CD45.sup.+] AGM hematopoietic cells, consistent with a role in supporting further HSC maturation and self-renewal. We also identified conditions that included NOTCH activation with an immobilized NOTCH ligand that were sufficient to amplify AGM-derived HSCs following their specification in the absence of AGM AKT-ECs. Together, these studies begin to define the critical niche components and resident signals required for HSC induction and self-renewal ex vivo, and thus provide insight for development of defined in vitro systems targeted toward HSC generation for therapeutic applications., Introduction In the developing embryo, the earliest hematopoietic activity is characterized by lineage-restricted progenitors that arise initially in the yolk sac prior to and independent of hematopoietic stem cells (HSCs) [...]

HSCs either self-renew or differentiate to give rise to multipotent cells whose progeny provide blood cell precursors. However, surprisingly little is known about the factors that regulate this choice of self-renewal versus differentiation. One candidate is the Notch signaling pathway, with ex vivo studies suggesting that Notch regulates HSC differentiation, although a functional role for Notch in HSC self-renewal in vivo remains controversial. Here, we have shown that Notch2, and not Notch1, inhibits myeloid differentiation and enhances generation of primitive [Sca-1.sup.+]c-[kit.sup.+] progenitors following in vitro culture of enriched HSCs with purified Notch ligands. In mice, Notch2 enhanced the rate of formation of short-term repopulating multipotential progenitor cells (MPPs) as well as long-term repopulating HSCs, while delaying myeloid differentiation in BM following injury. However, consistent with previous reports, once homeostasis was achieved, neither Notch1 nor Notch2 affected repopulating cell self-renewal. These data indicate a Notch2-dependent role in assuring orderly repopulation by HSCs, MPPs, myeloid cells, and lymphoid cells during BM regeneration., Introduction HSCs either self-renew or give rise to multipotent cells whose progeny provide precursors committed to the lymphoid and myeloid lineages. Recent studies have identified factors that maintain HSCs in [...]

Delayed myeloid engraftment after cord blood transplantation (CBT) is thought to result from inadequate numbers of progenitor cells in the graft and is associated with increased early transplant-related morbidity and mortality. New culture strategies that increase the number of cord blood progenitors capable of rapid myeloid engraftment after CBT would allow more widespread use of this stem cell source for transplantation. Here we report the development of a clinically relevant Notch-mediated ex vivo expansion system for human [CD34.sup.+] cord blood progenitors that results in a marked increase in the absolute number of stem/progenitor cells, including those capable of enhanced repopulation in the marrow of immunodeficient nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Furthermore, when cord blood progenitors expanded ex vivo in the presence of Notch ligand were infused in a clinical setting after a myeloablative preparative regimen for stem cell transplantation, the time to neutrophil recovery was substantially shortened. To our knowledge, this is the first instance of rapid engraftment derived from ex vivo expanded stem/progenitor cells in humans., Cord blood is an effective and widely used source of stem cells for individuals undergoing hematopoietic stem cell transplantation. Most patients now receive two units of cord blood derived from [...]

Hematopoietic stem cells give rise to progeny that either self-renew in an undifferentiated state or lose self-renewal capabilities and commit to lymphoid or myeloid lineages. Here we evaluated whether hematopoietic stem cell self-renewal is affected by the Notch pathway. Notch signaling controls cell fate choices in both invertebrates and vertebrates by inhibiting certain differentiation pathways, thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Notch receptors are present in hematopoietic precursors and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursors, determines T- or B-cell lineage specification from a common lymphoid precursor and promotes expansion of CD8[sup.+] cells. Here, we demonstrate that constitutive Notch1 signaling in hematopoietic cells established immortalized, cytokine-dependent cell lines that generated progeny with either lymphoid or myeloid characteristics both in vitro and in vivo. These data support a role for Notch signaling in regulating hematopoietic stem cell self-renewal. Furthermore, the establishment of clonal, pluripotent cell lines provides the opportunity to assess mechanisms regulating stem cell commitment and demonstrates a general method for immortalizing stem cell populations for further analysis., Author(s): Barbara Varnum-Finney (corresponding author) [1]; Lanwei Xu [2]; Carolyn Brashem-Stein [1]; Cynthia Nourigat [1]; David Flowers [1]; Sonia Bakkour [2]; Warren S. Pear [2]; Irwin D. Bernstein [1, 3] [...]