Global discovery of dysregulated protein expression and phosphorylation networks identifies leo1 as a novel target of PRL-3 phosphatase (PTP4A3) in leukemogenesis

Abstract

Identification of novel oncogenic alterations and pathways are the keys to the understanding and discovery of therapeutic targets in the improved management of AML. PRL-3 is a small 20kDa prenylated dual-specificity phosphatase that is over-expressed in 50% of AML and associated with poor survival, but the mechanisms regulating the associated malignancy remains unknown. Our data demonstrates that ectopic PRL-3 expression in the factor-dependent TF1 AML cells confers cytokine-independent growth, induces colony-forming ability and tumorigenesis in vivo. To characterize novel substrates of PRL-3, unbiased large-scale SILAC-based MS was performed between the parental TF1 and isogenic TF1-PRL3 cells 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). Importantly, PRL-3 alters the phosphorylation status of 192 proteins. Our proteomics profiling and in vitro validation revealed that Leo1, component of the Polymerase II-associating factor 1 (PAF) complex, is upregulated and dephosphorylated by PRL-3. Mechanistically, PRL-3 upregulates Leo1 by relieving the repressive H3K9me3 mark on Leo1 promoter through direct binding of JMJD2C histone demethylase, promoting a permissive chromatin state for Leo1 transcription. 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 (n=24) showed PRL3+Leo1+ expression, with a statistically significant association of P<0.01. In conclusion, our work allows the identification of bona fide PRL-3 targets, which established starting points for a complete enumeration of PRL-3 function in AML signaling and provides valuable leads for designing future therapies targeting PRL-3 in leukemia.