Abstract 2171: The fusion protein PAX5-JAK2 constitutively activates JAK-STAT signaling

Abstract

Abstract In B-cell precursor acute lymphoblastic leukemia the transcription factor PAX5, a master regulator of B-cell commitment and maintenance, is recurrently fused to several different partner proteins including other transcription factors, structural proteins, and the tyrosine kinase (TK) JAK2. Mutations and translocations affecting JAK2 are found in a variety of hematopoietic malignancies and lead to its constitutive activation and cytokine-independent JAK-STAT signaling. The chimeric PAX5-JAK2 protein contains the DNA binding domain of PAX5 and the JH1 kinase domain of JAK2. Reciprocal JAK2-PAX5 transcripts are also expressed, and the respective JAK2-PAX5 fusion protein consists of the JAK2 JH2-JH7 domains fused to the PAX5 partial homeodomain and the transactivation and inhibitory domains. In a first step towards the understanding of the function of these chimeric proteins, their subcellular localization was determined. Transfection of tagged fusion proteins into HEK293 and HeLa cells displayed nuclear localization of PAX5-JAK2. In contrast, JAK2-PAX5 fusion proteins were mainly localized in the cytoplasm. In order to determine whether PAX5-JAK2 is tyrosine phosphorylated and whether PAX5-JAK2 or JAK2-PAX5 is capable of activating STAT proteins, intracellular phosphoprotein analyses using flow cytometry were performed. Transiently transfected unstimulated JAK2-deficient human gamma2A cells exhibited a high degree of phosphorylation of PAX5-JAK2 compared to JAK2 wild-type protein, strongly suggesting cytokine stimulation-independent TK activity of the fusion protein. Additionally, expression of PAX5-JAK2 but not JAK2-PAX5 resulted in constitutive phosphorylation of STATs 1, 3, and 5. The capability of PAX5-JAK2 to induce STAT5 was confirmed by reporter gene assays. Mutation of the respective tyrosine residues in PAX5-JAK2 completely abolished the phosphorylation of STATs demonstrating that their activation is phospho-PAX5-JAK2-dependent. As it has been shown that PAX5 fusion proteins can act as competitive inhibitors of wild-type PAX5, we also performed a CD19 luciferase assay which showed that PAX5-JAK2 has a negative effect on the activation capacity of wild-type protein. Additional analysis including gene expression profiling of patient material will be performed to further evaluate the function of the chimeric proteins. Together, PAX5-JAK2 appears to affect JAK-STAT signaling and may at the same time interfere with normal B-cell development. In contrast to ETV6-JAK2 – the only other JAK2 chimeric protein analyzed in detail to date – which resides in the cytoplasm and is tyrosine phosphorylated upon dimerization, PAX5-JAK2 localizes to the nucleus and lacks any putative self-association motif. Hence, PAX5-JAK2 represents the first nuclear JAK2-fusion protein, which constitutively activates the JAK-STAT signaling pathway. This project is funded by the Austrian Science Fund FWF (P21554). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2171. doi:10.1158/1538-7445.AM2011-2171