Sequencing a mouse acute promyelocytic leukemia genome reveals genetic events relevant for disease progression

Abstract

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). It is characterized by the t(15;17)(q22;q11.2) chromosomal translocation that creates the promyelocytic leukemia-retinoic acid receptor α (PML-RARA) fusion oncogene. Although this fusion oncogene is known to initiate APL in mice, other cooperating mutations, as yet ill defined, are important for disease pathogenesis. To identify these, we used a mouse model of APL, whereby PML-RARA expressed in myeloid cells leads to a myeloproliferative disease that ultimately evolves into APL. Sequencing of a mouse APL genome revealed 3 somatic, nonsynonymous mutations relevant to APL pathogenesis, of which 1 (Jak1 V657F) was found to be recurrent in other affected mice. This mutation was identical to the JAK1 V658F mutation previously found in human APL and acute lymphoblastic leukemia samples. Further analysis showed that JAK1 V658F cooperated in vivo with PML-RARA, causing a rapidly fatal leukemia in mice. We also discovered a somatic 150-kb deletion involving the lysine (K)-specific demethylase 6A (Kdm6a, also known as Utx) gene, in the mouse APL genome. Similar deletions were observed in 3 out of 14 additional mouse APL samples and 1 out of 150 human AML samples. In conclusion, whole genome sequencing of mouse cancer genomes can provide an unbiased and comprehensive approach for discovering functionally relevant mutations that are also present in human leukemias.

Figure 1. Flow chart for the identification of SNVs in the mouse 9500 APL genome.

Tier 1 contains all changes in amino acid coding regions of annotated exons and consensus splice-site regions.

Figure 2. Heat map of residual 129/SvJ SNPs by chromosomal location.

A heat map was generated to show the number of SvJ/129 SNPs present in the tumor 9500 genome per 1-Mb window divided by the number total SvJ/129 SNPs present in the same 1-Mb window, aligned to the mouse chromosome ideogram. The location of cathepsin G (Ctsg) is highlighted by an arrow. The mouse 9500 breeding strategy placed selective pressure to retain the genetically modified Ctsg locus on chromosome 14 (the PML-RARA cDNA was knocked into the 5ι untranslated region of the Ctsg gene). The experimentally derived heat map shows a skewing of retained 129/SvJ SNPs surrounding the Ctsg locus, as expected, with a random distribution of 129/SvJ SNPs throughout the remaining chromosomes. Min., minimum.

Figure 3. Functional validation of the recurrent Jak1 mutation identified in the sequenced mouse APL genome.

(A) The mouse Jak1 V657F (mJAK1) mutation is orthologous to the human JAK1 V658F (hJAK1) and human JAK2 V617F (hJAK2) mutations. B41, band 4.1 domain; SH2, src-homology domain; JH2, pseudokinase domain; JH1, kinase domain. (B) Mouse APL Jak1 V657F mutation allele frequency by 454 sequencing. The variant allele frequencies of the V657F mutation from tumor 9500 and the 6 other tumors with V657F mutations are shown. On the far right are the read counts for the controls of a WT 129/SvJ DNA pool (n = 6) and a WT B6/T DNA pool (n = 6). F, female; M, male. (C) Functional validation with human JAK1 cDNA MSCV retroviral constructs. IRES-GFP (WT/GFP) alone versus JAK1 WT-IRES-GFP (WT/JAK1 WT) versus JAK1 V658F (WT/JAK1 V658F) mutant-IRES-GFP retroviral constructs were transduced into WT B6/T or mCG-PR bone marrow from 6-week-old mice. The transduced marrow was transplanted into lethally irradiated WT B6/T mice. The JAK1 V658F mutant cooperates with PML-RARA (PR) in a mouse model of APL and causes a fatal leukemia; a Kaplan-Meier overall survival plot is shown. This result was replicated in 2 independent experiments. (D) Serial flow cytometric analysis of peripheral blood shows a massive expansion of GFP⁺/Gr-1⁺ cells in the mCG-PR/JAK1 V658F cohort compared with that in the other experimental cohorts and controls. Error bars are mean ± SEM. (E) Flow cytometric analysis of peripheral blood, bone marrow, and spleen at the end of 1 JAK1 functional validation experiment showed no significant expansion of GFP⁺/Gr-1⁺ cells in cohorts other than mCG-PR/JAK1 V658F mice. Each symbol represents an individual mouse. Error bars are mean ± SEM.

Figure 4. Structural variant analysis of the sequenced mouse APL genome, with discovery and validation of a somatic 150-kb deletion on chromosome X that included nearly all of the Kdm6a gene also somatically deleted in 1 human AML sample.

(A) Copy number depth plot by sequence coverage for the tumor 9500 and 129/SvJ genomes in the same region on chromosome X that includes exons 3–29 of the Kdm6a locus. The 129/SvJ strain has no deletion in this region. (B) PCR validation of the tumor 9500 deletion. The PCR product was cloned and sequenced to confirm the breakpoints of the deletion. (C) High-resolution copy number depth plot of a human AML sample with a KDM6A deletion (exons 1–3 are deleted). The copy number plot compares the tumor to skin DNA array data from the same patient using the Affymetrix v6.0 SNP array platform. (A and C) The green lines represent the average copy number for the affected intervals, and the black lines represent the average copy number for the unaffected regions. Red dots represent individual copy number data points within the affected intervals, and blue dots represent individual copy number data points for the unaffected regions.

Figure 5. Heat map of Kdm6a deletions in 4 mouse APL tumors based on targeted NimbleGen custom 12 × 135 K CGH array data.

Kdm6a deletions are not residual germline CNVs (there are 2 copies of this region in the 129/SvJ strain). Deletions of Kdm6a in the 4 tumors have unique breakpoints and are not consistent with a founder effect or strain variant. On the right side of the heat map, B6 and 129 represent the B6 and 129/SvJ WT samples, respectively, that were used as controls, and 9500, 9520, 26, and 9495 represent individual mouse tumor numbers.

Figure 6. Schematic representation of the 4 Kdm6a deletions found in mouse APL tumors (tumors 9500, 9520, 26, and 9495) and a KDM6A deletion found in a human AML patient (sample 750152).

Mouse APL tumor 26 has 2 deletions involving Kdm6a, a 41.4-kb deletion and a smaller 8-kb deletion. Chr X, chromosome X.