Mutations in the B-Cell Transcription Factor PAX5 Found in B-Progenitor Acute Lymphoblastic Leukemia Impair Normal PAX5 Activity.
Authors: Miller Christopher B. , Mullighan Charles G. , Downing James R. 
Published: April 2020

Abstract
Using genome-wide profiling of DNA copy number abnormalities using high-resolution single nucleotide polymorphism arrays, we recently identified a high frequency of genomic aberrations involving the PAX5 gene in pediatric B-progenitor ALL. PAX5 is a critical transcriptional regulator of B lymphocyte commitment and differentiation. Mutations, including partial tandem duplication, complete and focal deletions, point mutations in the DNA-binding or transactivation domain, and three translocations that encode PAX5 fusion proteins were observed in 31.7% of B-ALL. The PAX5 deletions were mono-allelic and resulted in either loss of the entire gene, or the deletion of only a subset of the exons leading to the production of PAX5 proteins that lacked the DNA-binding paired domain (exons 2–4) and/or the transcriptional activation domain (exons 7–10). In murine systems, the complete absence of PAX5 results in the arrest of B-cell development at the pro-B-cell stage prior to immunoglobulin heavy chain rearrangement, whereas haploinsufficiency leads to a partial block in B-cell development. Importantly, in the primary leukemia samples, the mono-allelic loss of PAX5 was associated with reduced expression of PAX5 by flow cytometry and quantitative RT-PCR, suggesting that haploinsufficiency contributes to the block in differentiation characteristic of B-progenitor ALL. To determine if the other identified PAX5 mutations result in hypomorphic alleles, we analyzed the DNA-binding and transcriptional activity of the encoded proteins. DNA-binding activity was assessed by electrophoretic mobility gel-shift assays using a labeled oligonucleotide probes from the promoters of the PAX5 target genes CD19 and CD79A (mb-1), and transcriptional activity was assessed by a luciferase-based reporter assays using the PAX5-dependent reporter plasmid, luc-CD19. Analysis was performed on the paired-domain mutants P80R and P34Q, the focal deletions Δe2-5, Δe2-6, Δe2-7, Δe2-8, and Δe6-8, and the PAX5-ETV6 and PAX5-FOXP1 translocation-encoded fusion proteins. As expected, DNA-binding was abrogated in deletion mutants that lacked the paired domain (Δe2-5, Δe2-6, Δe2-7, Δe2-8). In contrast, the PAX5 Δe6-8, which retains the paired DNA binding domain but lacks a significant portion of the transcriptional regulatory domain, had normal DNA binding activity. Importantly, the paired domain point mutants impaired DNA-binding in a promoter specific manner, with P80R having a marked reduction in binding to both the CD19 and mb-1 promoters, whereas P34Q showed reduced binding only to the mb-1 promoter. Surprisingly, the PAX5-ETV6 and the PAX5-FOXP1 translocations had markedly reduced DNA-binding activity despite retention of the PAX5 paired domain. As expected each of the mutants with impaired or absent DNA-binding activity were found to have markedly reduced transcriptional activity when compared to wild type PAX5. Similarly, those mutants with altered or deleted transcriptional activation domains had reduced transcriptional activity, as did the two PAX5 translocation-encoded fusion proteins (PAX5-ETV6 and PAX5-FOXP1). Moreover, transfection of increasing amounts of PAX5-ETV6 or PAX5-FOXP1 together with a fixed amount of wild type PAX5 revealed that the fusion proteins competitively inhibit the transcriptional activation of wild type PAX5. Taken together, these data indicate that the identified PAX5 mutations impair DNA-binding and/or transcriptional activity. This loss of normal PAX5 function in turn would contribute to the observed arrest in B-cell development seen in ALL.