A New ETV6-RUNX1 In Vivo Model Produces a Phenocopy of the Human Pb-ALL
Authors: rodriguez-Hernandez Guillermo , Hauer Julia , Martín-Lorenzo Alberto , Schaefer Daniel , Bartenhagen Christoph , Garcia-Ramirez Idoia , Auer Franziska , Gonzalez-Herrero Ines , Ruiz-Roca Lucia , Okpanyi Vera , Fischer Ute , Dugas Martin , Bhatia Sanil , Gombert Michael , Krell Pina , Chen Cai , Garcia Suquia Marta , Rascon Trincado Maria , Garcia Sanchez Angel , Blanco Oscar , garcia Cenador Maria , Garcia-criado Francisco , Cobaleda Cesar , Alonso-Lopez Diego , de Las Rivas Javier , vicente-Duenas Carolina , Sanchez-Gracia Isidro , Borkhardt Arndt 
Published: October 2019

Introduction

The ETV6-RUNX1 fusion gene,the most common subtype of childhood pB-ALL, is acquired in utero, producing a persistent and hidden preleukemic clone. However, the underlying mechanism explaining how the preleukemic clone evolves to pB-ALL remains to be identified. The lack of genetically engineered human-like ETV6-RUNX1 pB-ALL models has hampered our understanding of the pathogenesis of this disease.

Methods

We have used a novel experimental approach to generate a murine strain that mimics the human ETV6-RUNX1 pB-ALL. We expressed ETV6-RUNX1 specifically in hematopoietic stem cells (HSC) of C57BL/6 x CBA mice by placing ETV6-RUNX1 under the control of the Sca1 promoter. Two founder mice were obtained for the Sca1-ETV6-RUNX1 transgene, which had normal gestation, were viable and developed normally. Sca1-ETV6-RUNX1 transgenic mice were characterized with respect to clinical, immunephenotypic and genetic characteristics. For the detection of shared secondary genomic alterations we analyzed three murine Sca1-ETV6-RUNX1 and 11 ETV6-RUNX1 positive human pB-ALL and corresponding germline by whole-exome (WES) and whole-genome sequencing using a HiSeq 2500 (Illumina) platform.

Results

In our transgenic murine model Sca1-ETV6-RUNX1 transgene expression was detected in HSCs, while there was no detectable expression in pro B cells or later stages of B-cell development, which mimics human ETV6-RUNX1 preleukemic biology. Sca1-ETV6-RUNX1 mice developed exclusively pB-ALL at a low penetrance (7.5%; 3 out of 40) with a CD19+ B220+ IgM- cell surface phenotype. Overall survival was not significantly reduced compared to wild-type mice (P value = 0.7901). pB-ALL in Sca1-ETV6-RUNX1 mice manifested with splenomegaly, disruption of splenic architecture, and appearance of blast cells in the peripheral blood (PB). All leukemic cells displayed clonal immature BCR rearrangement. Tumor pro B cells grew independent of IL-7 and were able to propagate the disease when transplanted into sub-lethally irradiated syngeneic recipient mice. Whole-exome sequencing of murine pB-ALL revealed in one mouse a deletion of three amino acids in the B-cell differentiation factor EBF1, which is well known in the context of human ETV6-RUNX1 leukemia. Additionally we found mutations in genesimplicated in histone modification, i.e. in KDM5C causing a premature translation stop.

We compared the genomic alterations detected in the mouse model to published genomic data of pediatric ETV6 -RUNX1 pB-ALL and identified multiple copy number variations, which are shared between the murine and human ETV6 -RUNX1 pB-ALL. Among them were copy number gains and losses including i.e. the tumorsuppressor locus CDKN2A/B with a well-known role in human and mouse pB-ALL. A high proportion of genes implicated in histone modification was also mutated in published data of human ETV6-RUNX1 positive pB-ALL. We validated this novel finding of recurrent alterations of histone modifying genes in both the murine model and the human disease using an independent human ETV6-RUNX1 cohort of 11 patients. In this cohort were able to reproduce this finding. Similar to the murine model, we also detected a missense mutation in the methyltransferase KDM5C in one patient of our cohort of ETV6-RUNX1 positive patients.

Conclusion

In summary, we have characterized a new Sca1-ETV6-RUNX1 mouse model and this is, to our knowledge the first model, which represents a phenocopy of the human pB-ALL. Sca1-ETV6-RUNX1 mice develop exclusively pB-ALL at a very low penetrance as it is the case in human ETV6-RUNX1 positive pB-ALL. The acquisition of secondary mutations in pB-ALL with a high proportion in histone modifying genes confers the second hit for the conversion of a preleukemic clone into the clinically overt ETV6-RUNX1 positive pB-ALL disease. These findings are important for encouraging novel interventions that might help to prevent or treat ETV6-RUNX1 positive childhood leukemias.

Disclosures

No relevant conflicts of interest to declare.