Abstract 1108: Global discovery of dysregulated protein expression and phosphorylation networks identifies Leo1 as a key substrate of PRL-3 phosphatase in leukemogenesis.

Abstract

Abstract Cytogenetically normal acute myeloid leukemia (AML) constitutes about 40-50% of human AML cases, is a highly heterogeneous and poorly understood. Identification of novel oncogenic alterations and pathways are the key to the better understanding and discovery of useful therapeutic targets in the improved management of AML. PRL-3 is a small 20kDa prenylated dual-specificity phosphatase that is over-expressed in about 50% of acute myeloid leukemia (AML) and associated with poorer survival, but the mechanisms regulating the associated malignancy remains poorly understood. Our in vitro data demonstrates that ectopic PRL-3 expression in the factor-dependent TF1 AML cells confers cytokine-independent growth, induces colony-forming ability in methylcellulose media and tumorigenesis in vivo. To systematically characterize novel substrates of PRL-3 in leukemia, unbiased large-scale proteomics and phosphoproteomics analyses were performed between the parental TF1 cells and their malignant PRL-3 transfectant counterparts to discover critical differences in signaling networks. We obtained quantitative measurements on 803 proteins, where 331 were significantly up-regulated (>1.5-fold) and 67 were under-expressed (<0.6-fold). More importantly, PRL-3 alters the phosphorylation status of 192 proteins. Our proteomics profiling and in vitro validation data revealed that Leo1, component of the Polymerase II-associating factor 1 (PAF) complex, is upregulated and dephosphorylated by PRL-3. Mechanistically, PRL-3 regulates Leo1 by relieving the repressive H3K9me3 mark on Leo1 promoter through direct binding of JMJD2C histone demethylase, promoting transcriptionally permissive chromatin state leading to efficient Leo1 gene expression. Conversely, abrogation of PRL-3 using RNAi reduced Leo1 levels and destabilizes the PAF complex, consequently leading to the downregulation of PAF-regulated pluripotency target genes like Sox2, Sox4 and Tbx3. Our functional analyses corroborated the contribution of Leo1 towards PRL-3 oncogenicity, whereby inhibition of Leo1 in PRL-3 cells impeded cell proliferation, induced significant apoptosis and abolished colony formation. Finally, we relate these data to clinical relevance where 46% of the human AML patient samples and 45.5% of the AML cell lines showed a positive correlation between PRL-3 expression and abnormal Leo1 activation. In conclusion, our work allows the identification of bona fide PRL-3 targets and signaling networks, which established starting points for a complete enumeration of PRL-3 function in AML signaling. The improved understanding of this network provides valuable leads for designing future therapies targeting PRL-3 in leukemia and carcinomas. Citation Format: Phyllis SY Chong, Jian Biao Zhou, Tiannan Guo, Siu Kwan Sze, Qi Zeng, Wee Joo Chng. Global discovery of dysregulated protein expression and phosphorylation networks identifies Leo1 as a key substrate of PRL-3 phosphatase in leukemogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1108. doi:10.1158/1538-7445.AM2013-1108