Phyllis Sy Chong

The pro-metastasis tyrosine phosphatase, PRL-3 (PTP4A3), is a novel mediator of oncogenic function of BCR-ABL in human chronic myeloid leukemia.

BackgroundResistance to tyrosine kinase inhibitors (TKIs) remains a challenge in management of patients with chronic myeloid leukemia (CML). A better understanding of the BCR-ABL signalling network may lead to better therapy.FindingsHere we report the discovery of a novel downstream target of BCR-ABL signalling, PRL-3 (PTP4A3), an oncogenic tyrosine phosphatase. Analysis of CML cancer cell lines and CML patient samples reveals the upregulation of PRL-3. Inhibition of BCR-ABL signalling either by Imatinib or by RNAi silencing BCR-ABL reduces PRL-3 and increases cleavage of PARP. In contrast, the amount of PRL-3 protein remains constant or even increased in response to Imatinib treatment in drug resistant cells expressing P210 T315I. Finally, analysis with specific shRNA shows PRL-3 involvement in the proliferation and self-renewal of CML cells.ConclusionsThese data support a role for PRL-3 in BCR-ABL signalling and CML biology and may be a potential therapeutic target downstream of BCR-ABL in TKI resistant mutant cells.

LEO1 Is Regulated by PRL-3 and Mediates Its Oncogenic Properties in Acute Myelogenous Leukemia

PRL-3, an oncogenic dual-specificity phosphatase, is overexpressed in 50% of acute myelogenous leukemia (AML) and associated with poor survival. We found that stable expression of PRL-3 confers cytokine independence and growth advantage of AML cells. However, how PRL-3 mediates these functions in AML is not known. To comprehensively screen for PRL3-regulated proteins in AML, we performed SILAC-based quantitative proteomics analysis and discovered 398 significantly perturbed proteins after PRL-3 overexpression. We show that Leo1, a component of RNA polymerase II-associated factor (PAF) complex, is a novel and important mediator of PRL-3 oncogenic activities in AML. We described a novel mechanism where elevated PRL-3 protein increases JMJD2C histone demethylase occupancy on Leo1 promoter, thereby reducing the H3K9me3 repressive signals and promoting Leo1 gene expression. Furthermore, PRL-3 and Leo1 levels were positively associated in AML patient samples (N=24; P<0.01). On the other hand, inhibition of Leo1 reverses PRL-3 oncogenic phenotypes in AML. Loss of Leo1 leads to destabilization of the PAF complex and downregulation of SOX2 and SOX4, potent oncogenes in myeloid transformation. In conclusion, we identify an important and novel mechanism by which PRL-3 mediates its oncogenic function in AML.

Phosphatase of regenerating liver-3 is regulated by signal transducer and activator of transcription 3 in acute myeloid leukemia

Overexpression of protein-tyrosine phosphatase of regenerating liver 3 (PRL-3) has been identified in about 50% of patients with acute myeloid leukemia (AML). The mechanism of regulation of PRL-3 remains obscure. Signal transducer and activator of transcription 3 (STAT3), a latent transcriptional factor, has also been often found to be activated in AML. We first identified STAT3-consensus-binding sites in the promoter of PRL-3 genes. Then we experimentally validated the direct binding and transcriptional activation. We applied shRNA-mediated knockdown and overexpression approaches in STAT3(-/-) liver cells and leukemic cells to validate the functional regulation of PRL-3 by STAT3. A STAT3 core signature, derived through data mining from publicly available gene expression data, was employed to correlate PRL-3 expression in large AML patient samples. We discovered that STAT3 binds to the -201 to -210 region of PRL-3, which was conserved between human and mouse. Importantly, PRL-3 protein was significantly reduced in mouse STAT3-knockout liver cells compared with STAT3-wild type counterparts, and ectopic expression of STAT3 in these cells led to a pronounced increase in PRL-3 protein. We demonstrated that STAT3 functionally regulated PRL-3, and STAT3 core signature was enriched in AML with high PRL-3 expression. Targeting either STAT3 or PRL-3 reduced leukemic cell viability. Silencing PRL-3 impaired invasiveness and induced leukemic cell differentiation. In conclusion, PRL-3 was transcriptionally regulated by STAT3. The STAT3/PRL-3 regulatory loop contributes to the pathogenesis of AML, and it might represent an attractive therapeutic target for antileukemic therapy.

LIN28B Activation by PRL-3 Promotes Leukemogenesis and a Stem Cell-like Transcriptional Program in AML

PRL-3 (PTP4A3), a metastasis-associated phosphatase, is also upregulated in patients with acute myeloid leukemia (AML) and is associated with poor prognosis, but the underlying molecular mechanism is unknown. Here, constitutive expression of PRL-3 in human AML cells sustains leukemogenesis in vitro and in vivo. Furthermore, PRL-3 phosphatase activity dependently upregulates LIN28B, a stem cell reprogramming factor, which in turn represses the let-7 mRNA family, inducing a stem cell–like transcriptional program. Notably, elevated levels of LIN28B protein independently associate with worse survival in AML patients. Thus, these results establish a novel signaling axis involving PRL-3/LIN28B/let-7, which confers stem cell–like properties to leukemia cells that is important for leukemogenesis. Implications: The current study offers a rationale for targeting PRL-3 as a therapeutic approach for a subset of AML patients with poor prognosis. Mol Cancer Res; 15(3); 294–303. ©2016 AACR.

Non-canonical activation of β-catenin by PRL-3 phosphatase in acute myeloid leukemia

Aberrant activation of Wnt/β-catenin signaling pathway is essential for the development of AML; however, the mechanistic basis for this dysregulation is unclear. PRL-3 is an oncogenic phosphatase implicated in the development of LSCs. Here, we identified Leo1 as a direct and specific substrate of PRL-3. Serine-dephosphorylated form of Leo1 binds directly to β-catenin, promoting the nuclear accumulation of β-catenin and transactivation of TCF/LEF downstream target genes such as cyclin D1 and c-myc. Importantly, overexpression of PRL-3 in AML cells displayed enhanced sensitivity towards β-catenin inhibition in vitro and in vivo, suggesting that these cells are addicted to β-catenin signaling. Altogether, our study revealed a novel regulatory role of PRL-3 in the sustenance of aberrant β-catenin signaling in AML. PRL-3 may serve as a biomarker to select for the subset of AML patients who are likely to benefit from treatment with β-catenin inhibitors. Our study presents a new avenue of cancer inhibition driven by PRL-3 overexpression or β-catenin hyperactivation.

A loss-of-function genetic screening reveals synergistic targeting of AKT/mTOR and WTN/β-catenin pathways for treatment of AML with high PRL-3 phosphatase

BackgroundProtein tyrosine phosphatase of regenerating liver 3 (PRL-3) is overexpressed in a subset of AML patients with inferior prognosis, representing an attractive therapeutic target. However, due to relatively shallow pocket of the catalytic site of PRL-3, it is difficult to develop selective small molecule inhibitor.MethodsIn this study, we performed whole-genome lentiviral shRNA library screening to discover synthetic lethal target to PRL-3 in AML. We used specific small molecule inhibitors to validate the synthetic lethality in human PRL-3 high vs PRL-3 low human AML cell lines and primary bone marrow cells from AML patients. AML mouse xenograft model was used to examine the in vivo synergism.ResultsThe list of genes depleted in TF1-hPRL3 cells was particularly enriched for members involved in WNT/β-catenin pathway and AKT/mTOR signaling. These findings prompted us to explore the impact of AKT/mTOR signaling inhibition in PRL-3 high AML cells in combination with WNT/β-catenin inhibitor. VS-5584, a novel, highly selective dual PI3K/mTOR inhibitor, and ICG-001, a WNT inhibitor, were used as a combination therapy. A synthetic lethal interaction between mTOR/AKT pathway inhibition and WNT/β-catenin was validated by a variety of cellular assays. Notably, we found that treatment with these two drugs significantly reduced leukemic burden and prolonged survival of mice transplanted with human PRL-3 high AML cells, but not with PRL-3 low AML cells.ConclusionsIn summary, our results support the existence of cooperative signaling networks between AKT/mTOR and WNT/β-catenin pathways in PRL-3 high AML cells. Simultaneous inhibition of these two pathways could achieve robust clinical efficacy for this subtype of AML patient with high PRL-3 expression and warrant further clinical investigation.

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

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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