Oncogenic Role of PAX5 in B Cell Malignancies

Tumors of hematopoietic origin are often associated with specific chromosomal translocations, which result in the activation of oncogenes controlling differentiation, proliferation or cell survival. The most frequent targets of chromosomal translocations are transcription factor genes, which regulate important aspects of hematopoietic development. One of these genes, PAX5, has been implicated in the formation of two distinct B cell tumors. Under normal physiological conditions, the B-cell-specific transcription factor Pax5 plays an essential role in controlling the commitment, development, function and identity of B cells throughout B lymphopoiesis, which was shown by previous work of our group. B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood cancer. A subset of these leukemias contain translocations that generate novel transcription factors by fusing the N-terminal DNA-binding domain of Pax5 with C-terminal sequences of different partner proteins. These novel transcription factors may contribute to tumorigenesis by interfering with the normal function of Pax5, which is expressed from the second intact PAX5 allele. A subfamily of B-cell non-Hodgin lympoma (B-NHL) is characterized by IGH-PAX5 translocations, which lead to Pax5 overexpression in mature B cells by bringing the PAX5 gene under the control of potent regulatory elements of the immunoglobulin heavy-chain (IGH) locus. The goal of this grant application is to elucidate the oncogenic role of Pax5 in transgenic mouse models of BCP-ALL and B-NHL by reconstructing the corresponding human PAX5 translocations by conditional knock-in in the mouse Pax5 and Igh genes, respectively. By molecular, cellular and histological analyses of these mouse models, we will address the following questions: (1) Do the Pax5 translocation alleles upon short-term activation interfere with B cell differentiation? (2) Which pathways and molecular functions of the wild-type Pax5 protein are affected by the Pax5 translocations? (3) Do leukemias or lymphomas develop upon long-term activation of the Pax5 translocations in the mouse? (4) Which oncogenes or tumor suppressor genes cooperate with the Pax5 translocations to accelerate tumor development? Experimental answers to these questions will provide important molecular insight into how the deregulation of Pax5 expression or function affects normal B lymphopoiesis and contributes to tumor development.

B-cell precursor acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, and the transcription factor gene PAX5 is one of the most frequently mutated genes (> 30%) in B-ALL patients. About 2.5% of all B-ALL cases contain PAX5 translocations that generate PAX5 fusion proteins by linking the N-terminal part of PAX5 with the C-terminal sequences of different partner proteins, such as the transcriptional regulators ETV6 and FOXP1. All PAX5 fusion proteins contain the N-terminal DNA-binding paired domain, but lack the potent C-terminal transactivation region of PAX5. As all leukemia cells still contain a wild-type PAX5 allele in addition to the PAX5 translocation, the PAX5 fusion proteins are thought to contribute to B-ALL formation by interfering with the normal function of PAX5. In this FWF project, we have investigated the oncogenic role of two PAX5 translocations in B-ALL by generating knock-in mice expressing the Pax5-Etv6 or Pax5-Foxp1 fusion protein from the endogenous Pax5 locus. Similar to B-ALL tumors, B cell development was arrested at an early stage in Pax5Etv6/+ and Pax5Foxp1/+ mice. This developmental block resulted from the deregulation of many genes in early B lymphocytes of these knock-in mice, which was caused by an altered and broader DNA-binding specificity of the Pax5 fusion proteins as shown for Pax5-Etv6. One allele or both alleles of the closely linked CDKN2A and CDKN2B genes, which code for potent tumor suppressors controlling cell proliferation, are known to be deleted in human B-ALLs with PAX5 translocations, suggesting that the CDKN2A/B deletion cooperates with PAX5 translocations in tumor development. Indeed, Pax5Etv6/+ Cdkna2ab+/ mice constitute a faithful mouse model for the corresponding human disease, as these compound mutant mice rapidly develop B-ALL in contrast to Pax5Etv6/+ or Cdkna2ab+/ mice. By comparing the Pax5-Etv6-specific gene expression signatures between mouse Pax5Etv6/+ Cdkna2ab+/ B-ALLs and human PAX5-ETV6-positive B-ALLs in ongoing RNA-sequencing experiments, we will identify common target genes in a genome-wide manner that could explain the oncogenic role of the PAX5-ETV6 protein. In summary, this FWF project has provided novel important insight into the oncogenic role of PAX5 translocations in B-ALL and has generated the basis for ongoing experiments to identify critical target genes that may offer possible intervention strategies for interfering with B-ALL development.