Abstract 2172: Functional studies on leukemic PAX5 fusion proteins

Abstract

Abstract Recent analyses of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have revealed that the gene encoding the transcription factor PAX5, which is essential for B-lymphocyte commitment and maintenance, is frequently affected by genetic alterations including mutations, deletions, and translocations. Chromosome rearrangements resulting in the expression of chimeric PAX5 fusion proteins account for about 2% of all BCP-ALL cases. In these chimeric proteins the N-terminus of PAX5 comprising the DNA-binding paired domain is always retained, but intriguingly the C-terminal fusion partners are substantially heterogeneous. A thorough characterization of PAX5 fusion proteins is, thus, required to determine their oncogenic properties and to unravel whether they affect common or distinct pathways. Analysis of the subcellular localization of tagged versions of PAX5 fusion proteins including PAX5-BRD1, PAX5-HIPK1, PAX5-POM121, and PAX5-DACH1 in HeLa cells using indirect immunofluorescence showed that all fusions were mainly nuclear. These data are in line with the presence of the DNA-binding paired domain and at least one nuclear localization signal, and provide further evidence that PAX5 fusion proteins potentially act as aberrant transcription factors. However, PAX5-HIPK1, which lacks the kinase domain of HIPK1, displayed a diffuse nuclear and cytoplasmic distribution pattern. Intriguingly, co-transfection of PAX5-HIPK1 and wild-type HIPK1 into HeLa cells, which normally express only low levels of HIPK1, resulted in localization of both proteins to nuclear speckles indicating that (auto-)phosphorylation by HIPK1 may target the two proteins to these PML-body-like structures. Further studies are ongoing to verify whether this is indeed the case. In addition, assessment of the self-interaction properties of the mentioned PAX5 chimeric proteins by co-immunoprecipitation (Co-IP) determined that PAX5-DACH1 was able to oligomerize. Bioinformatic analyses identified coiled-coil regions in the C-terminal Dachshund boxes of DACH1 and DACH2 as putative oligomerization motifs. Despite the differences in their self-interaction properties, chromatin-immunoprecipitation (ChIP) revealed that all examined fusion proteins were able to occupy PAX5 target loci (e.g. CD79A) suggesting that the paired domain was sufficient to recruit the proteins to the specific DNA-sequences. Together, on the one hand, our data support the current concept that all PAX5 fusion proteins share the common feature to act as dominant negative forms of PAX5. On the other hand, as well we demonstrate that the diverse fusion proteins possess distinct properties, which may be responsible for differences in the pathogenesis of the respective leukemia. 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 2172. doi:10.1158/1538-7445.AM2011-2172