Modeling of Pediatric Acute Megakaryoblastic Leukemia Using Cord Blood Stem/Progenitor Cells
Authors: Cardin Sophie , Laramee Louise , MacRae Tara , Chagraoui Jalila , Sauvageau Guy , Humphries R. Keith , Hébert Josée , Wilhelm Brian T. , Cellot Sonia 
Published: October 2019

Abstract
Pediatric acute megakaryoblastic leukemia (AMKL) accounts for 10% of childhood acute myeloid leukemia (AML) cases and remains a high fatality cancer. CBFA2T3-GLIS2, NUP98-KDM5A, RBM15-MKL1 and MLL gene rearrangements are recurrent aberrations that are mutually exclusive and found at similar frequencies in half the cases of pediatric AMKL. The recently identified CBFA2T3-GLIS2 and NUP98-KDM5A chimeric oncogenes are associated with inferior outcomes (overall survival, OS: ~30%) compared to patients harboring the RBM15-MKL1 gene fusion (OS ~70%). To investigate NUP98-KDM5A driven leukemogenesis, human cell lines and mouse models were engineered using overexpression of the chimeric oncogene in CD34+cord blood (CB) stem/progenitor cells.
To this end, the cDNA of the NUP98-KDM5A fusion, consisting of the 5' portion of the nuclear pore protein nucleoporin 98 (NUP98) gene fused to the 3' portion of the histone lysine demethylase 5A (KDM5A) encoding gene, was cloned into a MNDU lentiviral vector carrying a GFP reporter gene. Using optimized culture conditions, 10,000 freshly isolated CB-CD34+ (day 0) cells were seeded in multiple wells in vitro and transduced with either NUP98-KDM5A or control (CTL) vectors. Initial gene transfer rates were 8.2 ± 0.95% (expt.1, n=3) and 22.9 ± 4.16% (expt.2, n=12) for NUP98-KDM5A and 48 ± 0.04% (expt.1, n=3) and 76.9 ± 6.36% (expt.2, n=4) for CTL conditions. Overexpression (OE) of NUP98-KDM5A led to maturation block and increased cell proliferation in vitro as compared to CTL, as assessed by cytological examination of cells, serial tracking of increasing CD34+GFP+ cell fractions and lack of a c-KIThi differentiated mast cell population. This was observed by day 25 of culture in 66% of the wells in a first experiment (2/3), and in 92% (11/12) of the wells in a confirmatory study. An overriding NUP98-KDM5A CD34+GFP+ immature cell population was present by day 42 of culture (up to 94% of cells), while CD34+GFP+cells were significantly decreased in the CTL conditions (0.3 to 8%), where mast cells predominated. Cell lines were maintained in culture for 180 days in the first experiment (day 145 in ongoing expt.2) and aliquoted at regular intervals for immunophenotypic assessment, ARN/DNA extraction, cryopreservation, and cytogenetics studies. Clonogenic activity was markedly increased in NUP98-KDM5A-OE cells (colony forming cell frequency of 1:3 vs 1:200 in CTL, at day 88 of culture). Transcriptomic profiling of selected cell lines closely matched that of NUP98-KDM5A bearing pediatric AMKL samples. Notably, there was an up-regulation of HOXA and HOXB gene clusters, as well as other transcription factors, epigenetic regulators, cell surface receptors and kinases. No additional driver mutations were detected in the cell lines or patient samples (n=2). Epigenetic characterisation of 2 cell lines is ongoing (H3K4, H3K9, H3K27 and H3K79 methylation status) as the chimeric oncogene encodes for the C-terminal portion of a H3K4 histone demethylase (KDM5A).
Xenotransplantation of 75% of day 7 cells in immunodeficient mice resulted in the development of overt AMKL in 1 of 3 mice after 32 weeks. Recipient mouse bones were white and brittle, and the marrow cavity infiltrated by 30% hCD45loCD61+GFP+ leukemic blasts, with typical megakaryoblastic morphology. The leukemic blasts were also detected in blood (5%), and in enlarged spleen (0.2%). Secondary transplantation of isolated AMKL cells (from bone marrow and spleen) was performed, along with expression profiling by RNA sequencing. In an ongoing confirmatory experiment, 4 out of 12 mice displayed hCD45+NUP98-KDM5A-GFP+ cells (0.1, 0.2, 0.5, and 1.4%) in blood at 16 weeks, while 1 out of 4 CTL mice displayed 0.4% hCD45+-GFP+ engraftment at the same time point. Analysis of lentiviral integration patterns of genomic DNA of both cell lines and xenografts by southern blot is ongoing to determine clonality.
Overexpression of the NUP98-KDM5A chimeric oncogene in cord blood CD34+ cells can thus be used to engineer human cell lines and xenograft models to study this poor prognostic subgroup of pediatric AMKL. By crossing expression profiles of patient samples, cell lines and xenograft models, a list of potential NUP98-KDM5A direct targets was elaborated. The engineered cell lines pave the way to genetic and chemical screens, to identify leukemia specific functional dependencies and drug targets, to ultimately improve patient outcome.

Disclosures
No relevant conflicts of interest to declare.