Comparative Studies of B Cell Precursor ALL Pathogenesis and Translational Biology Using a Conditional E2a-PBX1 Transgenic Mouse Model

Pediatric acute lymphoblastic leukemia (ALL) represents a collection of orphan diseases that are defined by their genomic abnormalities. Their genetic diversity and low prevalence serve as major barriers to investigations of their molecular pathogenesis and translational biology. To address this, we have engineered novel mouse strains that conditionally activate and express E2a-PBX1, the fusion oncogene derived from chromosomal translocation t(1;19), present in 5-7% of pediatric ALL.

Somatic activation of the oncogene is accomplished by Cre-recombinase expressed under the control of specific B-lineage promoters CD19 or Mb1, or in hematopoietic stem cells using the Mx1 interferon-inducible promoter. The three mouse strains show similar pre-leukemic and leukemic phenotypes. At the time of disease, mice exhibit leukocytosis, anemia and thrombocytopenia as well as lymphadenopathy and hepatosplenomegaly and infiltration of several organs including kidney, lung and central nervous system.

Leukemia cell phenotypes (CD117+, CD19+, CD43+, CD45+, CD25+, sIgM-, Bp-1+, CD24+, CD127+, CD79a+ and TdT+) correspond to the phenotypic fraction B-C’ (or pro-B/large pre-B II stage, Basel nomenclature) that is very similar to human E2a-PBX1+ pre-B-ALL. Hence, we detected productive VDJ rearrangements and cytoplasmic heavy chain in 12.5 % of cases, a characteristic of human E2a-PBX1 leukemias.

Leukemia incidence varies from 5-50% depending on the Cre driver gene and the median latency is about 10 months in E2a.PBX1/Mb1.Cre and Mx1.Cre lines, suggesting the need for secondary mutations. Whole exome sequencing detected secondary genetic aberrations, which were validated in a larger cohort of leukemias. Spontaneous deletions of Pax5, which are present in ~45% of pediatric ALLs with E2a-PBX1 gene fusions, were found in about 30 % of mouse E2a-PBX1 leukemias. Conditional deletion of Pax5 and E2a-PBX1 expression expanded progenitor B cell subpopulations in healthy 3-months old preleukemic mice. Consequently, Pax5 haplo-insufficiency in mice cooperates with E2a-PBX1 increasing the penetrance and shortening the latency of leukemia, providing the first evidence for cooperative oncogenic effects of Pax5 haplo-insufficiency.

Tumor suppressor genes as Trp53 and Cdkn2a/b were inactivated by secondary mutations and deletions, respectively. Additionally, secondary recurrent activating mutations were detected in key signaling pathways such as Ras/Mapk and Jak/Stat on which the leukemia cells are strongly dependent. Furthermore, leukemia cells displayed higher basal levels of phosphorylated pSTAT5 and pAKT, pERK1/2, and were hyper-sensitive to stimulation with IL-7 and thymic stromal lymphopoietin (TSLP) as seen by induction of pSTAT5 and supported growth in colony-forming assays. The JAK1/2 inhibitor ruxolitinib blocked the induction of pSTAT5 by IL-7 and TSLP, inhibited colony formation in vitro, and increased disease-free survival after in vivo treatment. Human E2a-PBX1 primary cells and cell lines showed hypersensitivity to IL-7/pSTAT5 activation compared to other ALL karyotypes and pre-treatment with ruxolitinib blocked induction of pSTAT5 by IL-7.

In summary, we have developed conditional transgenic E2a-PBX1 mouse models that consistently develop leukemias that resemble human pre-B-ALL carrying the t(1;19) and identified key cooperating oncogenic pathways. This model provides experimental validation of the multistep pathogenesis for a subset of ALL previously inferred from genomic analyses and provides a platform for comparative mechanistic and preclinical studies.