PAX5/TEL Has An Opposite Dominant Effect on Endogenous PAX5, Affecting Its Transcriptional Regulation, B Cell Adhesion and BCR Signaling In Murine Pre-BI Cells
Authors: Fazio Grazia , Cazzaniga Valeria , Palmi Chiara , Galbiati Marta , Giordan Marco , Kronnie Truus te , Rolink Antonius , Biondi Andrea , Cazzaniga Giovanni 
Published: October 2019

Abstract
Abstract 2482

Introduction.
PAX5 is a transcription factor with both activation and repression functions, essential for B-cell development. Recently, it has been found as frequent target of abnormalities in B-cell precursor ALL, showing point mutations, deletions or involvement in chromosomal translocations. The functional role of these lesions is still poorly understood. In previous experiments in mouse pre-BI cells, we showed that the PAX5/TEL protein acts as an aberrant transcription factor with repressor function, causing a block on B-cell differentiation, short-term IL-7 independence and resistance to the anti-proliferative and pro-apoptotic effects of TGFbeta1. Moreover, PAX5/TEL enhances cell migration towards CXCL12, with the over expression of CXCR4.

Aim.
The aim of the present study was to comprehensively understand how PAX5/TEL affects the transcription process and eventually interferes with PAX5 and TEL pathways and how these modulate cellular processes.

Methods.
We analyzed gene expression profiles in pre-BI cells transduced either by MIGR-PAX5/TEL-IRES-GFP or by MIGR-GFP (Affymetrix Gene Chip technology, Mouse array 430A 2.0). Validation of Differentially Expressed Genes has been performed by quantitative RQ-PCR and FACS analyses. In vitro adhesion assays have been performed on VCAM1-coated slides.

Results.
PAX5/TEL significantly modulated the transcription process: among 340 differentially expressed genes, 61% were down- and 39% up-regulated. Both up and down-regulated genes encompass numerous PAX5-target genes; in particular, PAX5/TEL represses genes which are normally activated by PAX5, and, vice versa, it activates genes physiologically repressed by PAX5. Moreover, gene function classification analyses suggested that PAX5/TEL modulates molecules which are related to fundamental cellular processes, such as phosphorylation, transcription, B cell receptor signaling, as well as adhesion. In particular, we demonstrated the modulation of surface antigens responsible of extra cellular binding as well as the modulation of intracellular molecules involved in the signaling of adhesion regulation, such as CD44, SDC4, EDG1, NEDD9, BCAR3, PLEKHA2, SPHK1. Moreover, in vitro adhesion assays on VCAM1-coated slides showed a significant reduction of adhesion capacities in PAX5/TEL positive cells. In agreement with our previous results, which showed down-regulation of CD19, BLNK/SLP-65 and MB-1/CD79a, both genes involved in BCR signaling, we demonstrated the additional repression of numerous key molecules fundamental for this pathway, such as SIGLECG/CD22, IRF4, LCP2/SLP-75, SLAMF6/LY108, PLEKHA2, PRKD2, IKZF2, IKZF3. Furthermore, we functionally validated the impairment of BCR-signaling and demonstrated that PAX/TEL transduced pre-BI cells are completely blocked in IgM protein expression, loosing the ability to complete the VDJ rearrangement and consequently express the m-chain on the cells surface, compared to the control cells.

Conclusions.
These analyses further sustain the role of PAX5/TEL as an aberrant transcription factor. Its effect on endogenous PAX5 does not represent a classical dominant negative role; indeed, we defined this effect as an ‘opposite dominance’, since PAX5/TEL caused the up-regulation of PAX5-repressed genes and, vice versa, the down-regulation of PAX5-activated targets. The biological consequences of this aberrant transcriptional activity are the impairment of B cell receptor signaling and the reduced adhesion capacity, both fundamental processes in B-cells, potentially involved in tumor transformation and in leukemia.

Disclosures:
No relevant conflicts of interest to declare.