Enforced expression of MLL-AF4 fusion in cord blood CD34+ cells enhances the hematopoietic repopulating cell function and clonogenic potential but is not sufficient to initiate leukemia

Abstract

Infant acute lymphoblastic leukemia harboring the fusion mixed-lineage leukemia (MLL)-AF4 is associated with a dismal prognosis and very brief latency. Our limited understanding of transformation by MLL-AF4 is reflected in murine models, which do not accurately recapitulate the human disease. Human models for MLL-AF4 disease do not exist. Hematopoietic stem or progenitor cells (HSPCs) represent probable targets for transformation. Here, we explored in vitro and in vivo the impact of the enforced expression of MLL-AF4 in human cord blood-derived CD34(+) HSPCs. Intrabone marrow transplantation into NOD/SCID-IL2Rγ(-/-) mice revealed an enhanced multilineage hematopoietic engraftment, efficiency, and homing to other hematopoietic sites on enforced expression of MLL-AF4. Lentiviral transduction of MLL-AF4 into CD34(+) HSPCs increased the in vitro clonogenic potential of CD34(+) progenitors and promoted their proliferation. Consequently, cell cycle and apoptosis analyses suggest that MLL-AF4 conveys a selective proliferation coupled to a survival advantage, which correlates with changes in the expression of genes involved in apoptosis, sensing DNA damage and DNA repair. However, MLL-AF4 expression was insufficient to initiate leukemogenesis on its own, indicating that either additional hits (or reciprocal AF4-MLL product) may be required to initiate ALL or that cord blood-derived CD34(+) HSPCs are not the appropriate cellular target for MLL-AF4-mediated ALL.

A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification

“…Although hESC-derived hematopoietic cells have been extensively reported to barely repopulate immunodeficient mice, we finally attempted to ensure that MLL-AF4 does not affect the in vivo behavior of hEB-derived hematopoietic cells. Expectedly, MLL-AF4 did not confer in vivo function and engraftment potential to hESC-derived hematopoietic cells (Figure 6C), confirming that MLL-AF4 expression does not transform hESC-derived hematopoietic cells in vitro or in vivo, similar to that reported in neonatal CD34 + HSPCs [9]. …”

“…Mounting evidence indicates that MLL-AF4 is the initiating leukemogenic event with an in utero origin [5,25]. However, an understanding of potential changes in early hematopoietic development mediated by MLL-AF4 is lacking, despite of the recent advances by Krivtsov et al [8] and Bursen et al [26], current mouse models do not accurately recapitulate either the disease phenotype or latency [6,8,9]. Furthermore, studies using primary cells from MLL-AF4 patients are incapable of addressing the developmental genesis of the hematopoietic system.…”

“…Postnatal (CB-derived) or prenatal (hESC-derived) stem cells represent ontogenically primitive target cells to address the developmental impact of MLL-AF4. Very recently, Montes et al [9] explored for the first time the effect of MLL-AF4 on the fate of human neonatal HSPCs. MLL-AF4-expressing CB-HSPCs displayed an enhanced in vivo hematopoietic engraftment and in vitro clonogenic potential, but MLL-AF4 expression was not sufficient for leukemogenesis, indicating that either additional hits are required to develop leukemia or that CB-derived HSPCs do not represent the appropriate target for MLL-AF4 to induce transformation.…”

“…Among these, postnatal (cord blood (CB)-derived) CD34 + hematopoietic stem/progenitor cells (HSPCs) or prenatal (fetal-or embryonic-derived) cells represent potential ontogenically early target cells in MLL-AF4 pathogenesis. Very recently, Montes et al [9] explored for the first time the in vitro and in vivo developmental impact of MLL-AF4 on the fate of human neonatal CD34 + HSPCs. The expression of MLL-AF4 in human CB-derived HSPCs augmented the in vivo multilineage hematopoietic engraftment and homing, the in vitro clonogenic potential and enhanced their proliferation.…”

Prognostic significance of FLT3 mutational status and expression levels in MLL-AF4+ and MLL-germline acute lymphoblastic leukemia

“…Most of our understanding of transformation by MLL-AF4 has come from murine models [2][3][4]26,27 and human stem cell systems. 1,[28][29][30][31][32][33] These have provided important insights into the likely mode of action of MLL-AF4, but the in vivo leukemias produced in these studies do not recapitulate the actual human disease phenotype and latency, and therefore MLL-AF4 leukemogenesis remains particularly difficult to model. This suggests that in order to develop a bona fide MLL-AF4 model by which to further understand the disease pathogenesis and to screen novel small-molecule compounds, additional oncogenic events such as FLT3 constitutive activation 8 or the derivative AF4-MLL seem to be required to develop ALL.…”