Phosphatase PRL-3 Research Articles

Super-enhancer-driven TOX2 mediates oncogenesis in Natural Killer/T Cell Lymphoma

BackgroundExtranodal natural killer/T-cell lymphoma (NKTL) is an aggressive type of non-Hodgkin lymphoma with dismal outcome. A better understanding of disease biology and key oncogenic process is necessary for the development of targeted therapy. Super-enhancers (SEs) have been shown to drive pivotal oncogenes in various malignancies. However, the landscape of SEs and SE-associated oncogenes remain elusive in NKTL.MethodsWe used Nano-ChIP-seq of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs NKTL primary tumor samples. Integrative analysis of RNA-seq and survival data further pinned down high value, novel SE oncogenes. We utilized shRNA knockdown, CRISPR-dCas9, luciferase reporter assay, ChIP-PCR to investigate the regulation of transcription factor (TF) on SE oncogenes. Multi-color immunofluorescence (mIF) staining was performed on an independent cohort of clinical samples. Various function experiments were performed to evaluate the effects of TOX2 on the malignancy of NKTL in vitro and in vivo.ResultsSE landscape was substantially different in NKTL samples in comparison with normal tonsils. Several SEs at key transcriptional factor (TF) genes, including TOX2, TBX21(T-bet), EOMES, RUNX2, and ID2, were identified. We confirmed that TOX2 was aberrantly overexpressed in NKTL relative to normal NK cells and high expression of TOX2 was associated with worse survival. Modulation of TOX2 expression by shRNA, CRISPR-dCas9 interference of SE function impacted on cell proliferation, survival and colony formation ability of NKTL cells. Mechanistically, we found that RUNX3 regulates TOX2 transcription by binding to the active elements of its SE. Silencing TOX2 also impaired tumor formation of NKTL cells in vivo. Metastasis-associated phosphatase PRL-3 has been identified and validated as a key downstream effector of TOX2-mediated oncogenesis.ConclusionsOur integrative SE profiling strategy revealed the landscape of SEs, novel targets and insights into molecular pathogenesis of NKTL. The RUNX3-TOX2-SE-TOX2-PRL-3 regulatory pathway may represent a hallmark of NKTL biology. Targeting TOX2 could be a valuable therapeutic intervene for NKTL patients and warrants further study in clinic.

Abstract 672: Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3)

Abstract Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual-specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and is correlated with poor patient prognosis in a variety of solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. The development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is shallow and hydrophobic. Currently available small molecules do not exhibit binding specificity for PRL-3 over PRL family members, and the only research antibody specific for PRL-3 can only recognize denatured protein. To address the lack of tools available to study PRL-3, we have developed alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3. Nanobodies have emerged as a valuable research tool and show promise as cancer therapeutics as they are ~15kD and lack light chains, allowing them to reach cavities within active sites that conventional antibodies cannot normally reach. Nanobodies also maintain high specificity and affinity for their antigens. We identified seven unique nanobodies that bind to PRL-3 with no activity towards PRL-1 and PRL-2, making our nanobodies one of the first tools to selectively target PRL-3 in its native state. We used biolayer interferometry and found the nanobody binding affinity for PRL-3 to be within a KD of 30 - 300 nM, similar to that of antibodies currently on the market. We identified PRL-3:nanobody interactions with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and showed binding outside the active site. These data were confirmed by analyzing the effects of nanobodies on PRL-3 phosphatase activity and substrate binding. Our anti-PRL-3 nanobodies specifically pulled down PRL-3 over PRL-1/-2 in immunoprecipitation experiments. Finally, we used these nanobodies to analyze PRL-3 localization in fixed immunofluorescence experiments in human cancer cells. We found that a C-terminal tag on PRL-3, such as FLAG or GFP, enhanced PRL-3 localization to the membrane, compared to untagged protein, which may have confounded previous PRL-3 functional studies. We are currently utilizing these nanobodies in two ways to understand PRL-3’s role in cancer. First, we will use the nanobody to stabilize PRL-3 for X-ray crystallography to develop higher resolution structures that could contribute to substrate identification and drug design. Secondly, we will examine PRL-3 function and trafficking during various cancer processes, such as proliferation, invasion, and stress, to determine how PRL-3 localization contributes to cancer progression. Citation Format: Caroline Noel Smith, Kyle Kihn, Zachary A. Williamson, K. Martin Chow, Louis B. Hersh, Konstantin Korotkov, Daniel Deredge, Jessica S. Blackburn. Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 672.

PRL-3 promotes a positive feedback loop between STAT1/2-induced gene expression and glycolysis in multiple myeloma.

Over 34 000 patients are diagnosed yearly with multiple myeloma (MM), which remains a fatal malignancy. Expression of the phosphatase PRL-3 is associated with poor prognosis in MM patients, and Vandsemb etal. have demonstrated that PRL-3 contributes to enhanced MM cell fitness through activation of a glycolysis-associated feedback loop. PRL-3 resulted in increased expression of signal transducer and activator of transcription 1 (STAT1) and 2 (STAT2) and increased glycolysis. Increased glucose metabolism in turn activated STAT1/2 and interferon 1-related genes. This discovery advances the MM field by providing a new potential treatment avenue. Comment on: https://doi.org/10.1111/febs.16058.

The dual inhibition against the activity and expression of tyrosine phosphatase PRL-3 from a rhodanine derivative

Increasing evidences demonstrated that PRL-3 was associated with metastatic potential in a variety of cancers including CRC, gastric cancer, ovarian cancer and so on. PRL-3 knock down inhibited the development of metastasis by reducing the size of primary tumors and inhibiting the invasion and growth of cancer cells. Therefore, PRL-3 is a promising diagnostic marker and therapeutic target in tumors. So far, only several PRL-3 inhibitors have been reported. In this study, six rhodanine derivatives were synthesized and characterized. The compounds were evaluated against tyrosine phosphatase PRL-3. Among these compounds, 5-(5-chloro-2-(trifluoromethyl)benzylidene)-2-thioxothiazolidin-4-one (4) could effectively inhibit PRL-3 with IC50 value of 15.22 μM. Fluorescent assays suggested compound 4 tightly bound to tyrosine phosphatase PRL-3 with the molar ratio of 1:1, and the binding constant of 1.74 × 106 M−1. Compound 4 entered into SW-480 cells, selectively inhibited the expression of PRL-3 and increased the phosphorylation of PRL-3 substrates, and decreased the survival rate of SW-480 cells with IC50 of 6.64 μM and induced apoptosis. The results revealed that compound 4 is a dual functional inhibitor against the activity and expression of PRL-3 and a promising anti-cancer candidate targeting PRL-3.

Actin-Membrane Release Initiates Cell Protrusions.

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.

Abstract 3752: Identification of nanobodies specific for the oncogenic protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) that modulate its binding, stability, and phosphatase activity

Abstract PRL-3 is an oncogenic phosphatase across multiple cancer types, including colon, ovarian, melanoma, breast, and leukemia. While there is increasing interest in developing PRL-3 inhibitors for use in cancer research and treatment, there have been several issues in designing small molecule inhibitors for PRL-3, such as a shallow, negative charged active site, homology between the PRL-3 active site and other protein tyrosine phosphatases, and a high degree of overall homology between PRL-3 and family members PRL-1 and PRL-2. Nanobodies have recently emerged as an immensely useful research tool and show promise as a cancer therapeutic. Nanobodies are small, at ~15kD and lack light chains, allowing them to fit into spaces on target proteins that conventional antibodies cannot normally reach. Other advantages of nanobodies include their stability under stringent conditions, lack of immunogenicity, ability to permeate the cell, and a high specificity and affinity for their antigens. Using full-length PRL-3 protein as an immunogen in alpaca, I used phage display technology to identify alpaca nanobodies that had high affinity for PRL-3 through subtractive panning. I identified 18 unique nanobodies through sequencing; 14 of these were able to be expressed in bacteria and purified. The binding specificity of the nanobodies to PRL-3 over PRL-1 and PRL-2 was determined through an indirect ELISA assay, which showed that that 12 out of 14 anti-PRL-3 nanobodies bound PRL-3 significantly better than PRL-1 or PRL-2 (~25X times higher binding affinity to PRL-3, p<0.0001). I also identified several nanobodies that stabilize PRL-3 structure using a Differential Scanning Fluorescence (DSF) assays to analyze shifts in the melting temperature of PRL-3 following binding, with the ultimate goal of developing PRL-3/nanobody crystal structures to identify nanobody binding sites and find PRL-3 active site binders. Additionally, I have found the 6X-Histidine-tagged nanobodies are useful for PRL-3 western blot, immunoprecipitation, and immunofluorescence, and mCherry-tagged nanobodies (chromobodies) allow for analysis of PRL-3 trafficking. Nanobodies were also tested for their ability to inhibit the phosphatase activity of PRL-3, using both purified protein and functional in vitro assays. Preliminary results showed that several nanobodies decreased PRL-3 phosphatase activity, and cell-based assays to assess how nanobody expression affects PRL-3 function and cellular phenotype are ongoing. Overall, we have developed. both a novel research tool that can be used to gain insight into the structure and function of PRL-3 in normal and cancer cells, and a potentially new biologic inhibitor of PRL-3 that functions with high specificity and potency. Citation Format: Caroline N. Smith, K. Martin Chow, Louis B. Hersh, Jessica S. Blackburn. Identification of nanobodies specific for the oncogenic protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) that modulate its binding, stability, and phosphatase activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3752.

PRL3 enhances T-cell acute lymphoblastic leukemia growth through suppressing T-cell signaling pathways and apoptosis.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes and is largely driven by the NOTCH/MYC pathway. Yet, additional oncogenic drivers are required for transformation. Here, we identify protein tyrosine phosphatase type 4 A3 (PRL3) as a collaborating oncogenic driver in T-ALL. PRL3 is expressed in a large fraction of primary human T-ALLs and is commonly co-amplified with MYC. PRL3 also synergized with MYC to initiate early-onset ALL in transgenic zebrafish and was required for human T-ALL growth and maintenance. Mass spectrometry phosphoproteomic analysis and mechanistic studies uncovered that PRL3 suppresses downstream T cell phosphorylation signaling pathways, including those modulated by VAV1, and subsequently suppresses apoptosis in leukemia cells. Taken together, our studies have identified new roles for PRL3 as a collaborating oncogenic driver in human T-ALL and suggest that therapeutic targeting of the PRL3 phosphatase will likely be a useful treatment strategy for T-ALL.

Abstract 1789: The importance of the phosphatase PRL family sub-cellular localization during the cellular cycle

Abstract Acute lymphoblastic leukemia (ALL) is one of the most prevalent cancers diagnosed in adolescents. The survival rate for ALL is good during the initial diagnosis but is much less optimistic for patients who have relapsed ALL, due to limited treatment options. Additionally, the high dose cytotoxic chemotherapies given to ALL patients have long term adverse consequences. New and less toxic targeted therapies are needed for the treatment of ALL. We previously performed a large scale transplantation screen in zebrafish to identify genes associated with high relapse potential in ALL, and a top hit from this screen was the protein tyrosine phosphatase 4A3, PTP4A3 or PRL3. PRL3 overexpression in zebrafish enhances ALL progression and penetrance, and small molecule targeting of PRL3 in human cells rapidly induces apoptosis. The mechanisms through which PRL3 drive ALL progression and survival are unknown. PRL3 has a predicted nuclear localization site as well as a predicted prenylation motif, causing its localization to the plasma membrane. My preliminary data suggest that PRL3 shifts its localization between the plasma membrane and nucleus during the cell cycle. I am currently examining the localization of PRL3 and other PRL family members during different phases of the cell cycle and identifying signaling pathways that they may be involved with at these distinct sites to promote ALL progression, using CLIP-tag and SNAP-tag technologies that will allow me to image directly PRL localization in vitro. Biotin ligase approaches (Turbo-ID) will be used to determine the substrates of PRL activity and PRL interacting partners at these distinct locations. My project will provide scientific data that supports our hypothesis that PRL3 can transition between the membrane and nucleus, and plays a role in cell cycle. Identification of substrates of PRL3 that promote ALL progression may provide novel drug targets for this disease. Citation Format: Anna K. O'Leary, Jessica Blackburn. The importance of the phosphatase PRL family sub-cellular localization during the cellular cycle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1789.

Abstract 3695: A protein tyrosine phosphatase 4A3 (PRL-3)/Wnt signaling axis as a novel therapeutic target in acute lymphoblastic leukemia (ALL) relapse

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common pediatric malignancy and 15-20% of patients experience disease relapse, which is frequently more aggressive and treatment resistant than the primary disease, and often has unfavorable outcome. Relapse occurs because conventional chemotherapies are unable to reliably and completely eliminate leukemia stem cells (LSCs), which are the only cells within the leukemia with the ability to self-renew and remake or replenish a leukemia from a single cell. Eliminating LSCs can greatly improve patient outcome; for example, differentiation therapy, which blocks LSC self-renewal, has improved the survival of Acute Pro-myelocytic Leukemia to >95%. We have previously completed a >8,000 animal screen in a zebrafish Myc-induced ALL model and identified a panel of zebrafish ALL, in which the leukemia stem cell frequency is ~1 in every 10 cells, and identified a unique leukemia stem cell signature. The Wnt pathway was highly expressed by LSCs, and has also emerged in mouse studies as having an important role in LSC self-renewal in T-ALL. Current Wnt inhibitors have unacceptably toxicity in the clinic. I have found that Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL3) is highly expressed by ALL cells that also express Wnt pathways genes, and is not expressed by normal cell types. In a zebrafish Myc-induce ALL model, PRL3 expression significantly enhanced leukemia stem cell frequency, while inhibition of PRL3 reduced LSC numbers in vivo. In human cells, I found that PRL3 regulates the expression of downstream Wnt pathway target genes, and we are currently testing the effects of small molecule inhibition of PRL3 on the phosphorylation status of proteins involved in Wnt signaling, to define the mechanism of PRL3 action in this pathway. Apart from identifying novel interacting partners in the Wnt pathway, I have also developed a novel zebrafish reporter line that can be used to identify other small molecules that block Wnt signaling. In these fish, Wnt expressing leukemia stem cells are fluorescently tagged. These leukemias will be used for in vivo drug screens to identify FDA-approved compounds that block Wnt signaling and/or target leukemia stem cells without adverse effects on developing zebrafish larvae. In total, my research will define a novel role for the phosphatase PRL3 in self-renewal of leukemia stem cells via activation of Wnt signaling. These data will demonstrate that “untargetable” oncogenes like Wnt can be targeted by inhibiting easily druggable critical interacting proteins, such as phosphatases like PRL3. This work is also likely to have a positive translational impact by identifying FDA-approved drugs that block LSC self-renewal in ALL and other types of Wnt and/or PRL3 dependent cancers. Citation Format: Meghan G. Haney, Kristin O'Leary, Jessica S. Blackburn. A protein tyrosine phosphatase 4A3 (PRL-3)/Wnt signaling axis as a novel therapeutic target in acute lymphoblastic leukemia (ALL) relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3695.

PRL3-zumab as an immunotherapy to inhibit tumors expressing PRL3 oncoprotein

Tumor-specific antibody drugs can serve as cancer therapy with minimal side effects. A humanized antibody, PRL3-zumab, specifically binds to an intracellular oncogenic phosphatase PRL3, which is frequently expressed in several cancers. Here we show that PRL3-zumab specifically inhibits PRL3+ cancer cells in vivo, but not in vitro. PRL3 antigens are detected on the cell surface and outer exosomal membranes, implying an ‘inside-out’ externalization of PRL3. PRL3-zumab binds to surface PRL3 in a manner consistent with that in classical antibody-dependent cell-mediated cytotoxicity or antibody-dependent cellular phagocytosis tumor elimination pathways, as PRL3-zumab requires an intact Fc region and host FcγII/III receptor engagement to recruit B cells, NK cells and macrophages to PRL3+ tumor microenvironments. PRL3 is overexpressed in 80.6% of 151 fresh-frozen tumor samples across 11 common cancers examined, but not in patient-matched normal tissues, thereby implicating PRL3 as a tumor-associated antigen. Targeting externalized PRL3 antigens with PRL3-zumab may represent a feasible approach for anti-tumor immunotherapy.

PRL-3 Promotes Ubiquitination and Degradation of AURKA and Colorectal Cancer Progression via Dephosphorylation of FZR1.

The oncogenic phosphatase PRL-3 is highly expressed in metastatic colorectal cancer but not in nonmetastatic colorectal cancer or noncolorectal cancer metastatic cancers. Although the proinvasive capacity of PRL-3 has been validated in multiple types of cancer, its impact on colorectal cancer progression and the underlying mechanisms remain poorly understood. Here, we report that overexpressed PRL-3 stimulates G2-M arrest, chromosomal instability (CIN), self-renewal, and growth of colorectal cancer cells in xenograft models, while colorectal cancer cell proliferation is decreased. PRL-3-induced G2-M arrest was associated with decreased expression of Aurora kinase A (AURKA). PRL-3-promoted slow proliferation, CIN, self-renewal, and growth in xenografts were counteracted by ectopic expression of AURKA. Conversely, knockdown of PRL-3 resulted in low proliferation, S-phase arrest, impaired self-renewal, increased apoptosis, and diminished xenograft growth independently of AURKA. Analysis of colorectal cancer specimens showed that expression of PRL-3 was associated with high status of CIN and poor prognosis, which were antagonized by expression of AURKA. PRL-3 enhanced AURKA ubiquitination and degradation in a phosphatase-dependent fashion. PRL-3 interacted with AURKA and FZR1, a regulatory component of the APC/CFZR1 complex. Destabilization of AURKA by PRL-3 required PRL-3-mediated dephosphorylation of FZR1 and assembly of the APC/CFZR1 complex. Our study suggests that PRL-3-regulated colorectal cancer progression is collectively determined by distinct malignant phenotypes and further reveals PRL-3 as an essential regulator of APC/CFZR1 in controlling the stability of AURKA. SIGNIFICANCE: Dephosphorylation of FZR1 by PRL-3 facilitates the activity of APC/CFZR1 by destabilizing AURKA, thus influencing aggressive characteristics and overall progression of colorectal cancer.

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.

Abstract 2368: The phosphatase PRL3 as a novel therapeutic target in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is one of the most prevalent cancers that is diagnosed in adolescents. The survival rate for ALL is good during the initial diagnosis but is much less optimistic for patients who have relapsed ALL, due to limited treatment options. Additionally, the high-dose cytotoxic chemotherapies that ALL patients are given have long-term adverse consequences. New and less toxic targeted therapies are needed for the treatment of ALL. We previously performed a large-scale transplantation screen in zebrafish to identify genes associated with high relapse potential in ALL, and a top hit from this screen was the protein tyrosine phosphatase 4A3, PTP4A3 or PRL3. Upregulation of PRL3 is correlated progression of colorectal, breast, and gastric cancers, and is associated with poor patient prognosis. We have found that PRL3 is highly expressed in a subset of patient ALL samples, and I am examining the role of PRL3 in ALL progression. We have found that PRL3 expression enhances ALL proliferation, and promotes leukemia survival after cytotoxic chemotherapy treatment. Small-molecule inhibition of PRL3 induces apoptosis in ALL, and synergizes with common ALL chemotherapies. I am currently examining the localization of PRL3 and identifying signaling pathways that it may be involved with to promote ALL progression. If my project is successful, it will provide scientific data that support our hypothesis that PRL3 is related to acute lymphoblastic leukemia. If there is a correlation between PRL3 and ALL, PRL3 could be used as a more effective and less toxic treatment for patients with ALL. Citation Format: Anna K. O'Leary, Min Wei, Jessica Blackburn. The phosphatase PRL3 as a novel therapeutic target in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2368.

Abstract 2364: The phosphatase PRL-3 is a pro-survival oncogene in acute lymphoblastic leukemia

Abstract PRL-3, phosphatase of regenerating liver 3, is one member of a subfamily of protein tyrosine phosphatases that belongs to the dual-specificity phosphatases (DSPs). Accumulating evidence shows that PRL-3 is a biomarker of tumor progression and metastasis and high PRL-3 expression is associated with reduced overall and disease-free survival in a variety of solid tumors, suggesting it's importance in tumor recurrence. However, the role for PRL-3 in leukemia relapse has not yet been described. We have found that PRL-3 contributes to Acute Lymphoblastic Leukemia (ALL) malignancy by acting as a pro-survival gene, both in vitro and in vivo. Expression of PRL-3 in a MYC-induced zebrafish T-cell ALL model showed that PRL-3 expressing ALL had shorter latency and higher penetrance, compared with the fish injected with rag2:Myc; rag2:GFP alone. Growth after transplantation at low cell number was also significantly faster in the PRL3 expressing T-ALL, suggesting that PRL-3 plays an important role in ALL development and progression. We found PRL-3 to be highly expressed in a majority of human ALL compared to normal peripheral blood mononuclear cells (PBMNC), which suggests clinical relevance. Analysis of gene expression microarray data of 119 patient samples reveals that PRL-3 was highly expressed in 58% of the patients. In addition, PRL-3 had the highest expression in the most aggressive and treatment resistant subgroups of T-ALL, the TAL/LMO and Immature subtypes. PRL-3 is also expressed by human T-ALL cell lines using quantitative RT- PCR and immunoblotting. PRL-3 knockdown leads to significant apoptosis in T-ALL cells. The treatment of T-ALL cells using small molecule drugs including 2-cyano-2-ene-ester compound (Analog 3) and thieno [3,2-b] pyridine 1-oxide (thienopyridone) , also resulted in significant growth inhibition of T-ALL cells in part through >4-fold increase in apoptosis, measured by annexin V staining. In summary, our study provides evidence for a role of PRL-3 in T- ALL malignancy, and suggests that PRL3 may be a useful drug target in the treatment of this disease. Our future studies are focused on identifying the mechanisms by which PRL3 promotes T-ALL progression and survival. Citation Format: Min Wei, Kristin O'Leary, Jinpeng Liu, Chi Wang, Jessica Blackburn. The phosphatase PRL-3 is a pro-survival oncogene in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2364.

Abstract 4374: Non-canonical activation of β-catenin by PRL-3 phosphatase in acute myeloid leukemia

Abstract Aberrant activation of Wnt/β-catenin signaling pathway is essential for the development of AML; however, the mechanistic basis for the 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 subset 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. Citation Format: Phyllis SY Chong, Jianbiao Zhou, Jing Yuan Chooi, Wee Joo Chng, Jayantha Gunaratne, Qi Zeng. Non-canonical activation of β-catenin by PRL-3 phosphatase in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4374.

Mutational Analysis of a Conserved Glutamate Reveals Unique Mechanistic and Structural Features of the Phosphatase PRL-3.

Phosphatase of regenerating liver (PRL)-3 (PTP4A3) has gained much attention in cancer research due to its involvement in tumor promoting and metastatic processes. It belongs to the protein tyrosine phosphatase (PTP) superfamily and is thought to follow the catalytic mechanism shared by this family, which aside from the conserved active-site amino acids includes a conserved glutamic acid residue that is usually required for the integrity of the active site in PTPs. We noted that in structures of PRL-3, PRL-1, and PTEN these residues do not clearly align and therefore we sought to investigate if the glutamic acid residue fulfills its usual function in these proteins. Although this residue was essential for PTEN’s catalytic activity, it was nonessential for PRL-1 and PRL-3. Surprisingly, the mutation E50R increased PRL-3 activity against all tested in vitro substrates and also enhanced PRL-3-promoted cell adhesion and migration. We show that the introduction of Arg50 leads to an enhancement of substrate turnover for both PRL-3 and, to a lesser extent, PRL-1, and that the stronger gain in activity correlates with a higher structural flexibility of PRL-3, likely allowing for conformational adaptation during catalysis. Thus, in contrast to its crucial functions in other PTPs, this conserved glutamic acid can be replaced in PRL-3 without impairing the structural integrity. The variant with enhanced activity might serve as a tool to study PRL-3 in the future.

Expression of PRL-3 regulates proliferation and invasion of breast cancer cells in vitro.

The protein tyrosine phosphatase PRL-3 plays an important role in cancer cell migration, invasion and metastasis. In breast cancer, PRL-3 is overexpressed in 70-75% of tumors and even more frequently in lymph node metastases. Moreover, PRL-3 overexpression in breast cancer is associated with an adverse disease outcome. Aim of this study was to determine the role of PRL-3 in breast cancer cell proliferation, migration and invasion in vitro. PRL-3 mRNA expression was evaluated in 6 breast cancer cell lines by quantitative real-time PCR. To investigate the effect of PRL-3 expression in breast cancer cells in vitro we both up- and downregulated PRL-3 expression in breast cancer cells and performed in vitro wound repair cell motility assays and invasion assays. The influence of PRL-3 knockdown in MCF-7 cells on the expression of several gene products involved in cell invasion and cytoskeletal function was evaluated with real-time PCR. PRL-3 mRNA expression was demonstrated in all breast cancer cell lines evaluated. Knockdown of PRL-3 in MCF-7 cells resulted in decreased proliferation, wound healing and invasion. PRL-3 knockdown in MCF-7 cells resulted in a significant reduction of heparanase, MMP-9, actin gamma-2 and Myosin 9 expression, and significant elevation of E-cadherin. We conclude that PRL-3 is an important regulatory factor for breast cancer cell proliferation and invasion. Loss of PRL-3 function induces an antimetastatic gene expression profile in breast cancer cells. Due to its role in tumor growth and metastasis, PRL-3 emerges as a new therapeutic target in breast cancer therapy.

The Tyrosine Phosphatase PRL3 Drives T‐Cell Acute Lymphoblastic Leukemia Progression

T‐cell acute lymphoblastic leukemia (T‐ALL) is a major clinical challenge, representing 15–25% of all acute lymphoblastic leukemias in children and adults. The survival rate for pediatric patients with relapsed T‐ALL is a disheartening <30% in pediatric patients and <10% in adults. Current drugs against T‐ALL include highly cytotoxic chemotherapies, which are generally ineffective against relapse and are particularly hard for young children to receive. These harsh therapies can result in a spectrum of developmental issues and potent side‐effects. Therefore, it is imperative to develop molecularly targeted therapies that can effectively inhibit T‐ALL progression without contributing to the adverse side‐effects of general chemotherapy.We have found that zebrafish T‐ALL cells acquired amplification and high expression of the Protein Tyrosine Phosphatase 4A3 (PRL3) concurrent with evolution of enhanced relapse potential in a transplantation screen involving over 6,000 animals. Further, approximately 50% of human T‐ALL patients have high PRL3 expression. PRL3 has not been associated with leukemia onset or progression, but here we show overexpression of PRL3 leads to increased T‐ALL onset and aggressiveness in an in vivo transgenic zebrafish T‐ALL model. Small molecule inhibitors of PRL3 significantly reduced viability of established cultures of human T‐ALL cells in vitro. Based on these data, we hypothesize that PRL3 plays an important role in T‐ALL cell survival at all stages of leukemia progression. We are currently using fluorescent and confocal microscopy in zebrafish over‐expressing PRL3 to determine the cellular mechanisms by which PRL3 enhances leukemia onset. We are also generating PRL3 knockout lines using a CRISPR‐Cas9 for similar studies. Finally, we are treating zebrafish bearing T‐ALL with two specific, research‐grade PRL inhibitors to determine if blocking PRL3 phosphatase activity prevents leukemia onset and relapse. We have also identified several FDA‐approved, general phosphatase inhibitors that have potent anti‐PRL activity and are capable of killing ALL cells in vitro. Current work is focused on screening these inhibitors in vivo in our zebrafish models.We conclude that PRL3 is a novel gene that may be important for T‐ALL onset and progression. As a phosphatase, it is a unique druggable target. Future directions will focus on identifying the molecular mechanisms by which PRL3 drives leukemia progression, and moving top drug hits from our zebrafish screens into pre‐clinical testing in patient derived xenografts models in mice. We hope that our findings may lead to eventual clinical trials as alternatives to the traditional cytotoxic chemotherapy offered to pediatric T‐ALL patients now.Support or Funding InformationNCI 4R00CA181500NIGMS P30GM110787The V Foundation for Cancer Research

PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3

The phosphatases of regenerating liver (PRLs) are involved in tumorigenesis and metastatic cancer yet their cellular function remains unclear. Recent reports have shown PRL phosphatases bind tightly to the CNNM family of membrane proteins to regulate magnesium efflux. Here, we characterize the interactions between the CBS-pair (Bateman) domain of CNNM3 and either PRL2 or PRL3 using X-ray crystallography, isothermal titration calorimetry, and activity assays. We report four new crystal structures of PRL proteins bound to the CNNM3 CBS-pair domain that reveal the effects of cysteine disulphide formation and nucleotide binding on complex formation. We use comprehensive mutagenesis of the PRL3 catalytic site to quantify the importance of different PRL amino acids, including cysteine 104, leucine 108, and arginine 110, for CNNM binding and phosphatase activity. We show the PRL3 R138E mutant is selectively deficient in CNNM3 binding with the potential to distinguish between the downstream effects of phosphatase and CNNM-binding activities in vivo. Through a novel activity assay, we show that PRL3 has magnesium-sensitive phosphatase activity with ATP and other nucleotides. Our results identify a strong correlation between phosphatase activity and CNNM binding and support the contention that PRL function as pseudophosphatases regulated by chemical modifications of their catalytic cysteine.

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.