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Somatic drivers of B-ALL in a model of ETV6-RUNX1; Pax5 +/− leukemia
- Louise van der Weyden†1,
- George Giotopoulos†2,
- Kim Wong1,
- Alistair G. Rust1,
- Carla Daniela Robles-Espinoza1,
- Hikari Osaki2,
- Brian J. Huntly2 and
- David J. Adams1, 3Email author
© van der Weyden et al. 2015
Received: 26 April 2015
Accepted: 27 July 2015
Published: 13 August 2015
B-cell precursor acute lymphoblastic leukemia (B-ALL) is amongst the leading causes of childhood cancer-related mortality. Its most common chromosomal aberration is the ETV6-RUNX1 fusion gene, with ~25 % of ETV6-RUNX1 patients also carrying PAX5 alterations.
We have recreated this mutation background by inter-crossing Etv6-RUNX1 (Etv6 RUNX1-SB ) and Pax5 +/− mice and performed an in vivo analysis to find driver genes using Sleeping Beauty transposon-mediated mutagenesis and also exome sequencing.
Combination of Etv6-RUNX1 and Pax5 +/− alleles generated a transplantable B220 + CD19+ B-ALL with a significant disease incidence. RNA-seq analysis showed a gene expression pattern consistent with arrest at the pre-B stage. Analysis of the transposon common insertion sites identified genes involved in B-cell development (Zfp423) and the JAK/STAT signaling pathway (Jak1, Stat5 and Il2rb), while exome sequencing revealed somatic hotspot mutations in Jak1 and Jak3 at residues analogous to those mutated in human leukemias, and also mutation of Trp53.
Powerful synergies exists in our model suggesting STAT pathway activation and mutation of Trp53 are potent drivers of B-ALL in the context of Etv6-RUNX1;Pax5 +/− .
B-cell precursor acute lymphoblastic leukemia (B-ALL) is the most common childhood tumor . The most common chromosomal rearrangement in B-ALL is the t(12;21)(p13;q22) translocation generating the ETV6-RUNX1 fusion gene . This fusion is necessary but insufficient for the development of B-ALL, as monozygotic twin studies, and the detection of the ETV6-RUNX1 fusion in fetal blood spots from patients who do not go on to develop B-ALL have shown [3, 4].
PAX5, a guardian of B-cell identity and function, is somatically mutated in ~40 % of cases of childhood B-ALL . Moreover, the most common recurrent focal deletion region in ETV6-RUNX1+ tumors involves PAX5 (9p13.2; 25 %) and these deletions are thought to be early events in leukemogenesis . Previously, we generated a knock-in mouse model of ETV6-RUNX1 ALL, in which expression of the fusion gene is driven from the endogenous Etv6 promoter, and is linked to expression of the Sleeping Beauty (SB) transposase allowing the identification of transposon gene mutations that co-operate with Etv6-RUNX1 in leukemia development . Given that PAX5 heterozygosity is a frequent event in ETV6-RUNX1 patients , we bred these mice onto a background of Pax5 heterozygosity and performed a SB transposon-mediated mutagenesis screen to explore the profile of co-operating drivers. We coupled this approach with targeted exome sequencing of tumors to find additional mutations, and in particular hotspot mutation events.
Generation and genotyping of Etv6-RUNX1, T2Onc  and Pax5  mice has been described previously. For secondary bone marrow transplants of tumors, 6–12 week old SCID mice were inoculated with 3.5-5 × 105 bone marrow or spleen cells by tail vein injection. Animal studies were approved by the Home Office UK. Flow cytometric analysis of CD antigen expression was performed on single-cell suspensions from spleen or bone marrow as described previously .
Identification and analysis of genes affected by SB mutagenesis
Isolation of the transposon insertion sites and Gaussian kernel convolution statistical methods to identify common insertion sites (CISs) have been described previously . Whole transcriptome sequencing (RNA-seq) was performed on splenic RNA using the mRNA Seq Sample Prep Kit (Illumina, San Diego, CA) to create libraries that were sequenced on the Illumina platform. HTSeq-counts (HTSeq framework; v0.54p5) were used as input to edgeR (v3.4.2). Genes with significant differential expression were defined based on an FDR of 5 %. Pathway and gene set enrichment analysis (GSEA) was performed using Ingenuity Pathway Analysis and GSEA (v2.0.14), respectively.
Exome sequencing and bait design
Spleen (‘tumor’) and tail (‘normal’) genomic DNA were extracted using the Gentra Puregene Cell Kit (Qiagen). Exon-coding sequences of genes previously found to be involved in cancer were captured using custom-designed baits (Additional file 1) and sequenced on an Illumina platform. For each tumor-normal pair, MuTect (v1.14) was used to identify somatic SNVs, which were annotated using the Variant Effect Predictor tool (Ensembl v74). The Jak1, Jak3 and Trp53 mutations were validated by capillary sequencing.
Results and discussion
Interestingly an Etv6+/RUNX1-SB, T2Onc +/+ , Pax5 +/− mouse (TAPJ23.1a), in which transposition was not occuring, developed transplantable B-ALL (Fig. 1e), suggesting a contribution of background somatic mutations to tumor development leading us to investigate the somatic mutation landscape by targeted exome sequencing of 404 established cancer genes and candidate B-ALL drivers in 17 B220 + CD19+ B-ALL cases (Fig. 2b; Additional file 1). Strikingly the most commonly mutated genes were Jak3 (6/17 mice, 35 %), Trp53 (4/17 mice, 23 %) and Jak1 (2/17 mice, 11 %) with the missense mutations in Jak1/3 predominantly located in the pseudokinase domain (Fig. 2d). This domain has been demonstrated to exert an important negative regulatory function on the kinase domain  with many of the amino acid changes we identified falling into positions of JAK1/3 reported as being mutated in human leukemias, and shown to confer gain-of-function or transforming activity (Fig. 2e) [16–18]. Somatic mutations in JAK1 and JAK3 and have been reported in adult B-ALL  and high-risk/poor prognosis pediatric B-ALL , respectively. The variant allele frequency of Jak1/3 mutations was around 25 % suggesting that cells with these mutations represent a major clonal fraction (Fig. 2c). Recurrent somatic mutations in Jak1/3 have recently been reported in B-ALL tumors from Pax5 +/- mice [PMID: 25855603], suggesting that the synergy with Jak mutations in our model is a result of the knockout allele for Pax5 rather than the presence of the Etv6-RUNX1 allele. We also observed somatic Trp53 mutations in our mouse tumors with copy number and/or sequence alterations of p53 being an independent risk predictor of inferior outcome/high risk of treatment failure in B-ALL patients [21, 22].
Collectively, our findings support a model in which multiple small defects in a network of factors that regulate B-cell maturation (such as Pax5, Cblb, Zfp423, Foxp1, and Stat5b) together with activation/inactivation of oncogenes/tumor suppressor genes (such as the JAK/STAT signaling pathway and p53) cooperate with Etv6-RUNX1; Pax5 +/− to result in the development B-ALL. Our transplantable B-ALL tumors represent a novel tool for assessing potential therapeutic intervention strategies in cases of high risk/poor outcome B-ALL.
The authors wish to thank the staff of the Research Support Facility at the Wellcome Trust Sanger Institute for looking after the mice.
L.v.d.W., K.W., A.G.R., C.D.R.-E., and D.J.A. were supported by Cancer Research UK and the Wellcome Trust (WT098051). C.D.R.-E. was also supported by the Consejo Nacional de Ciencia y Tecnología of Mexico. B.J.H. and G.G. were supported by Medical Research Council UK and Cancer Research UK.
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