PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia

Abstract

Alterations of genes encoding transcriptional regulators of lymphoid development are a hallmark of B-progenitor acute lymphoblastic leukemia (B-ALL) and most commonly involve PAX5, encoding the DNA-binding transcription factor paired-box 5. The majority of PAX5 alterations in ALL are heterozygous, and key PAX5 target genes are expressed in leukemic cells, suggesting that PAX5 may be a haploinsufficient tumor suppressor. To examine the role of PAX5 alterations in leukemogenesis, we performed mutagenesis screens of mice heterozygous for a loss-of-function Pax5 allele. Both chemical and retroviral mutagenesis resulted in a significantly increased penetrance and reduced latency of leukemia, with a shift to B-lymphoid lineage. Genomic profiling identified a high frequency of secondary genomic mutations, deletions, and retroviral insertions targeting B-lymphoid development, including Pax5, and additional genes and pathways mutated in ALL, including tumor suppressors, Ras, and Janus kinase-signal transducer and activator of transcription signaling. These results show that in contrast to simple Pax5 haploinsufficiency, multiple sequential alterations targeting lymphoid development are central to leukemogenesis and contribute to the arrest in lymphoid maturation characteristic of ALL. This cross-species analysis also validates the importance of concomitant alterations of multiple cellular growth, signaling, and tumor suppression pathways in the pathogenesis of B-ALL.

Figure 1

Kaplan-Meier survival curves of ENU- or MMLV-treated Pax5 wild-type or heterozygous mice. (A-B) Data for ENU-treated mice. (C-D) MMLV data. (A,C) Data for all tumors (ie, B, T, myeloid, or mixed) show increased penetrance and reduced latency in Pax5 heterozygous mice treated with either ENU or MMLV. (B,D) Data for B-lineage ALL tumors only, showing the markedly increased penetrance of B-ALL in the context of Pax5 haploinsufficiency. P values were determined using the log-rank (Mantel-Cox) test.

Figure 2

Representative immunophenotype data for ENU- and MMLV-induced tumors. (A-B) Each tumor was stained with a panel of markers to determine T, B, or myeloid lineage, with B220 expression indicative of likely B-cell lineage. (C-D) Two representative ENU- and MMLV-induced tumors that exhibited variation in the degree of immunophenotypic maturation, with Hardy fraction A tumors show on the left (B220⁺CD19⁻) and Hardy fraction B tumors (B220⁺CD19⁺) shown on the right.

Figure 3

Secondary Pax5 alterations in ENU- and MMLV-induced B-cell tumors. (A) (Left) Representative log₂ ratio array-CGH data at the Pax5 locus at 4qB1, showing intragenic deletions for 3 cases. (Right) Probe level data for the same cases relative to the diploid (log₂ ratio = 0) state. The deletions involve exon 6 (DW5552), exon 7-8 (DW5582), and exon 7 (DW5588), each of which results in a frameshift premature truncation of translation and loss of PAX5 activity. All Pax5 deletions identified in the study are depicted in supplemental Figure 5. (B) Location of PAX5 sequence mutations identified by exome and Sanger sequencing. Mutations were largely restricted to the paired domain and are predicted to disrupt DNA binding and PAX5 transcriptional activation. (C-D) Structural modeling of the PAX5 DNA-binding domain (yellow) complexed with ETS1 (data not shown) and DNA (purple/blue) showing mutated amino acid residues (magenta) juxtaposed to the major or minor grooves of the DNA double helix (for details, see supplemental Figure 8 and supplemental Table 7).

Figure 4

Structural modeling and functional consequences of JAK mutations identified in ENU- and MMLV-induced tumors. Modeling of (A) JAK1 and (B) JAK3 mutations was performed using the crystal structure of the JAK2 pseudokinase domain (PDB ID code 4FVP). Each mutation was predicted to disrupt the active site of the JAK1/3 pseudokinase domain and result in constitutive activation of JAK-STAT signaling. (C) JAK1/3 mutations identified in this study and prior studies of human ALL were expressed in IL-3-dependent Ba/F3 cells and conferred cytokine-independent proliferation. MIG, MSCV-IRES-GFP retroviral vector; EV, empty vector. (D) IL-3-independent Ba/F3 cells were treated with increasing concentrations of the JAK inhibitor AZD1480 showing submicromolar inhibition of proliferation, which was accompanied by inhibition of JAK-STAT activation (representative phosphoflow cytometry and IC₅₀ data are provided in supplemental Figure 9). Data are normalized to untreated cells.