Protein Tyrosine Phosphatase 4A3 Research Articles

Abstract 60: Functional mapping of glioblastoma recurrence reveals targetable dependencies in an axonal guidance pathway in highly invasive brain cancers

Abstract Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Genetic perturbation or small molecule inhibition of PTP4A2 represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic effects of PTP4A2 inhibition. Since a pan-PTP4A inhibitor was limited by poor penetrance across the blood brain barrier (BBB) in vivo, a second-generation chimeric antigen receptor (CAR)-T cell therapy was engineered against ROBO1 that elicits specific and potent anti-tumor responses in vivo. A single dose of anti-ROBO1 CAR-T cells doubles median survival in patient-derived xenograft (PDX) models of recurrent glioblastoma, and also eradicates tumors in around 50% of mice engrafted with PDX models of lung-to-brain metastases and pediatric relapsed medulloblastoma. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A-ROBO1 signaling axis at GBM recurrence, with potential in other malignant brain tumors. Citation Format: Sheila Kumari Singh, Chirayu Chokshi, Vaseem Shaikh, Benjamin Brakel, Alisha Anand, Chitra Venugopal, Thomas Kislinger, Kevin Henry, Martin Rossotti, Kevin Brown, John Lazo, Jason Moffat. Functional mapping of glioblastoma recurrence reveals targetable dependencies in an axonal guidance pathway in highly invasive brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 60.

SYST-28 FUNCTIONAL MAPPING REVEALS WIDESPREAD REMODELLING AND A NOVEL TARGETABLE PTP4A2-ROBO1 SIGNALING AXIS AT GLIOBLASTOMA RECURRENCE

Abstract Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation or small molecule inhibition of PTP4A2 represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment and aberrant ROBO1 signaling axis is associated with higher tumorigenicity in GBM. ROBO1 was highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs) in rGBM, brain metastasis (BM) and medulloblastoma (MB). Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. We therefore developed and validated second-generation CAR-T cells against ROBO1, which demonstrated upregulation of activation markers, enhanced cytokine release, markedly increased proliferation, and induction of potent and specific tumor cell death compared to untransduced cells in all the three brain cancers. These findings were further validated in vivo in rGBM, MB and BM models, where ROBO1 CAR-T cells showed significant reduction in tumor burden and increase in survival of treated mice. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A2-ROBO1 signaling axis at GBM recurrence.

Prognostic Biomarkers in Pituitary Tumours: A Systematic Review.

Pituitary tumours (PTs) are the second most common intracranial tumour. Although the majority show benign behaviour, they may exert aggressive behaviour and can be resistant to treatment. The aim of this review is to report the recently identified biomarkers that might have possible prognostic value. Studies evaluating potentially prognostic biomarkers or a therapeutic target in invasive/recurrent PTs compared with either non-invasive or non-recurrent PTs or normal pituitaries are included in this review. In the 28 included studies, more than 911 PTs were evaluated. A systematic search identified the expression of a number of biomarkers that may be positively correlated with disease recurrence or invasion in PT, grouped according to role: (1) insensitivity to anti-growth signals: minichromosome maintenance protein 7; (2) evasion of the immune system: cyclooxygenase 2, arginase 1, programmed cell death protein 1 (PD-1)/programmed death ligand 2, cluster of differentiation (CD) 80/CD86; (3) sustained angiogenesis: endothelial cell-specific molecule, fibroblast growth factor receptor, matrix metalloproteinase 9, pituitary tumour transforming gene; (4) self-sufficiency in growth signals: epidermal growth factor receptor; and (5) tissue invasion: matrix metalloproteinase 9, fascin protein. Biomarkers with a negative correlation with disease recurrence or invasion include: (1) insensitivity to anti-growth signals: transforming growth factor β1, Smad proteins; (2) sustained angiogenesis: tissue inhibitor of metalloproteinase 1; (3) tissue invasion: Wnt inhibitory factor 1; and (4) miscellaneous: co-expression of glial fibrillary acidic protein and cytokeratin, and oestrogen receptors α36 and α66. PD-1/programmed cell death ligand 1 showed no clear association with invasion or recurrence, while cyclin A, cytotoxic T lymphocyte-associated protein 4, S100 protein, ephrin receptor, galectin-3 , neural cell adhesion molecule, protein tyrosine phosphatase 4A3 and steroidogenic factor 1 had no association with invasion or recurrence of PT. With the aim to develop a more personalized approach to the treatment of PT, and because of the limited number of molecular targets currently studied in the context of recurrent PT and invasion, a better understanding of the most relevant of these biomarkers by well-d esigned interventional studies will lead to a better understanding of the molecular profile of PT. This should also meet the increased need of treatable molecular targets.

Abstract 60: Functional mapping reveals widespread remodelling and unrecognized pathway dependencies in recurrent glioblastoma

Abstract Glioblastoma is a highly fatal brain cancer. The underlying functional drivers of treatment resistance and disease recurrence are unclear. By applying a genome-wide CRISPR-Cas9 library to patient-derived glioblastoma stem cell models, we systematically map genetic dependencies in patient-matched pre-treatment primary and post-treatment recurrent tumor cells. These insights reveal a large-scale remodelling of genetic dependency profiles at disease recurrence, arming recurrent tumor cells with newly-acquired genetic drivers and further loss of tumor suppressors. These analyses support parallel tumor-intrinsic mechanisms of treatment resistance which rely on acquisition of immunosuppressive capacity, including a defective mismatch repair pathway, ablation of PTEN activity, and a novel combination of de novo mutations in SWI/SNF components. We map a multilayered genetic and functional response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel driver of self-renewal, proliferation and tumorigenicity at glioblastoma recurrence. Mechanistically, genetic perturbation and a small molecule inhibitor of PTP4A2 results in greater survival and reduced tumor growth in patient-derived models of recurrent glioblastoma. Citation Format: Chirayu R. Chokshi, Kevin Brown, Chitra Venugopal, Jason Moffat, Sheila K. Singh. Functional mapping reveals widespread remodelling and unrecognized pathway dependencies in recurrent glioblastoma [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 60.

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.

A Protein Tyrosine Phosphatase 4A3 (PTP4A3) inhibitor prevents and repairs pulmonary endothelial barrier dysfunction and ameliorates acute lung injury provoked by the SARS‐CoV‐2 Spike protein subunit 1 in k18‐hACE2 transgenic mice

PTP4A3 regulates various inflammatory and cytoskeletal signaling pathways (e.g., STAT3, RhoA, ERK, AKT) that are involved in endothelial barrier dysfunction and acute lung injury (ALI). We have investigated the actions of JMS‐053, a small PTP4A3 inhibitor, in both in vitro and in vivo models of SARS‐CoV‐2 Spike protein subunit 1 (S1SP)‐induced endothelial inflammation and acute lung injury. S1SP (10nM) caused a time‐dependent endothelial barrier dysfunction in human lung microvascular endothelial cell monolayers that was ameliorated by JMS‐053 (12.5µM) administered either as pre‐treatment (4 hours prior to S1SP) or post‐treatment (5 hours after S1SP). In K18‐hACE2 transgenic mice, which express the human ACE2 receptor for SARS‐CoV‐2 S1SP, we instilled S1SP intratracheally, post‐treated with JMS‐053 (10mg/kg i.p. at 1, 24 and 48 hours after S1SP) or vehicle, and investigated molecular, functional and histological outcomes 72 hours later. JMS‐053 mice displayed lower alveolar cellularity, proteinosis and “cytokine storm” (i.e., IL‐6, IL‐1β, IL‐17), and lower levels of phosphorylated (activated) STAT3 and NF‐kB, compared to mice instilled with S1SP and treated with vehicle. Moreover, JMS‐053 greatly reduced quantifiable histological evidence of injury, compared to vehicle‐treated animals. This data suggests that inhibition of PTP4A3 could represent a critical target for novel therapeutic approaches towards endothelial dysfunction, inflammation and acute lung injury.

Downregulated Expression of miRNA-130a-5p Aggravates Hepatoma Progression via Targeting PTP4A2.

Background Hepatoma is a leading cause of death worldwide, with high metastasis and recurrence rates. The aberrant expression of miRNA-130a-5p is involved in the development and progression of various cancers. However, there are no studies investigating the role of miRNA-130a-5p in hepatoma. The present study is aimed at clarifying the functional role of miRNA-130a-5p in hepatoma progression. Methods The expression levels of miRNA-130a-5p in hepatoma tissues and cell lines were detected by qRT-PCR assays. Bioinformatic analysis, gain-/loss-of-function experiments, and luciferase activity assays were conducted to verify whether miRNA-130a-5p is targeted by protein tyrosine phosphatase 4A2 (PTP4A2). The functions of miRNA-130a-5p and PTP4A2 in hepatoma were determined by cell proliferation assays. Results The expression of miRNA-130a-5p was downregulated in hepatoma tissues and was related to poor prognosis. However, the expression level of PTP4A2 was contradictory to that of miRNA-130a-5p, and PTP4A2 upregulation could aggravate hepatoma progression. The ectopic overexpression of PTP4A2 promoted hepatoma cell proliferation in vitro, which could be reversed by miRNA-130a-5p. Conclusions Our study implies that miRNA-130a-5p, which is downregulated in hepatoma tissues, can suppress hepatoma cell proliferation via targeting PTP4A2.

STEM-05. FUNCTIONAL MAPPING REVEALS WIDESPREAD REMODELLING AND UNRECOGNIZED PATHWAY DEPENDENCIES IN RECURRENT GLIOBLASTOMA

Abstract Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses support parallel tumor-intrinsic mechanisms of treatment resistance which rely on acquisition of immunosuppressive capacity, including a defective mismatch repair pathway, ablation of PTEN activity, and a novel combination of de novo mutations in SWI/SNF components. We map a multilayered genetic and functional response to resist chemoradiotherapy and drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel driver of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation and a small molecule inhibitor of PTP4A2 repress axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and exploit a genetic dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. We conclude that functional reprogramming drives tumorigenicity and present a dependence on a PTP4A2-ROBO1 signaling axis at GBM recurrence.

Abstract 2305: 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 has been linked to poor patient prognosis in a variety of tumor types. The mechanisms by which PRL-3 promotes tumor progression are not well understood, which is in part due to lack of tools to study this protein. There is an intense need for research-grade antibodies in the PRL field. However, the development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is both shallow and hydrophobic. The most specific research antibody against PRL-3 only interacts with denatured protein, which limits its use experimentally. There is currently a humanized monoclonal antibody, PRL-3-zumab, that has good specificity for PRL-3 in cell culture and in vivo, and is in Phase 2 clinical trial for treating gastric and hepatocellular carcinomas; unfortunately, PRL-3-zumab is not currently available for the research community to use. To address this, we have designed, purified, and tested alpaca-derived PRL-3 single domain antibodies, or nanobodies. Nanobodies have emerged as a useful research tool and show promise as a cancer therapeutic. Nanobodies are only ~15kD, and lack light chains which allows them to fit into cavities in target proteins that conventional antibodies cannot normally reach. Other advantages of nanobodies include their stability under stringent conditions, lack of immunogenicity, and a high specificity and affinity for their antigens. We have identified 7 unique nanobodies that bind to PRL-3 in ELISA with no activity towards PRL-1 and PRL-2. Nanobodies were found to have variable binding affinity for PRL-3 using biolayer interferometry. Anti-PRL-3 nanobodies immunoprecipitated PRL-3 from HEK293T cell lysates both when overexpressed and using endogenous protein levels, with most nanobodies showing no cross-reactivity with PRL-1 and PRL-2. Each nanobody was also utilized to define PRL-3 localization in human colon cancer cells in immunofluorescence cell staining, which showed PRL-3 localized to the plasma membrane and the nucleus. Following validation of nanobody specificity for PRL-3, we began determining the binding site for each nanobody to PRL-3 as well as determining how nuclear PRL-3 contributes to cancer progression. These anti-PRL-3 nanobodies are the first tool that allow for the study of PRL-3 in multiple cell-based assays without the issue of cross-reactivity with other PRLs, a tool that has yet to exist in the PRL field. They can now be utilized to study the structure, function, and localization of PRL-3 in both normal and cancer cells to determine and define the oncogenic role that PRL-3 plays in multiple types of cancer. Finally, nanobodies against PRL-3 will also serve as a useful first step in the development of biologics to target PRL-3 in clinic. Citation Format: Caroline N. Smith, K. Martin Chow, Louis B. Hersh, 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 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2305.

A screen of FDA-approved drugs identifies inhibitors of protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3)

Protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) is highly expressed in a variety of cancers, where it promotes tumor cell migration and metastasis leading to poor prognosis. Despite its clinical significance, small molecule inhibitors of PRL-3 are lacking. Here, we screened 1443 FDA-approved drugs for their ability to inhibit the activity of the PRL phosphatase family. We identified five specific inhibitors for PRL-3 as well as one selective inhibitor of PRL-2. Additionally, we found nine drugs that broadly and significantly suppressed PRL activity. Two of these broad-spectrum PRL inhibitors, Salirasib and Candesartan, blocked PRL-3-induced migration in human embryonic kidney cells with no impact on cell viability. Both drugs prevented migration of human colorectal cancer cells in a PRL-3 dependent manner and were selective towards PRLs over other phosphatases. In silico modeling revealed that Salirasib binds a putative allosteric site near the WPD loop of PRL-3, while Candesartan binds a potentially novel targetable site adjacent to the CX5R motif. Inhibitor binding at either of these sites is predicted to trap PRL-3 in a closed conformation, preventing substrate binding and inhibiting function.

Abstract PR009: The functional genomic landscape of recurrent glioblastoma

Abstract Glioblastoma (GBM) is a highly fatal brain cancer where mortality is predominantly caused by disease recurrence and lack of second-line therapies. Here, utilizing genome-wide CRISPR-Cas9 screening, we uncover treatment-based conditional genetic interactions modulating sensitivity to combined radiation therapy (RT) and temozolomide (TMZ), the standard of care treatment administered to over 70% of glioblastoma patients. To investigate therapy-induced changes in the functional landscape of glioblastoma stem cells (GSCs) without confounding patient-to-patient differences, we systematically mapped genetic dependencies in patient-matched pre-treatment primary and post-treatment recurrent GSCs. This comparison highlights a remodelling of genes and pathways regulating tumor cell survival at disease relapse, arming recurrent GSCs with newly acquired genetic drivers and further loss of tumor suppressors. Among these genes, we identify PTP4A2, encoding protein tyrosine phosphatase 4A2, as a major regulator of stemness and tumorigenicity in recurrent GSCs. Genetic perturbation or pharmacological inhibition of PTP4A2 influences the phosphorylation status and expression of interacting proteins belonging to axonal guidance pathways, as phosphoproteomic studies showed specific enrichment in axon guidance regulator Roundabout Guidance Receptor 1 (ROBO1) in recurrent GSCs. We therefore designed and developed efficacious Camelid biotinylated single-domain antibody (bio-sdAb) and tetrameric complexes targeting human ROBO1 (MKRo-20). Subsequently, we orthotopically engrafted recurrent GSCs into immunocompromised mice and administered local treatments of tetrameric MKRo-20. All mice treated with tetrameric MKRo-20 were rendered tumor free, suggesting that modulation of ROBO1 with bio-sdAbs presents a new immunotherapeutic strategy to target recurrent glioblastoma. Our work reveals a context-specific dependence on axonal guidance through a PTP4A2-ROBO axis in recurrent glioblastoma, highlighting new avenues of therapeutic intervention for second-line therapy in GBM. This abstract is also being presented as PO025. Citation Format: Chirayu Chokshi, David Tieu, Kevin Brown, Chitra Venugopal, Lina Liu, Laura Kuhlman, Katherine Chan, Amy Tong, Neil Savage, Dillon McKenna, Nikoo Aghaei, Minomi Subapanditha, Joseph M Salamoun, Martin Rossotti, Peter Wipf, Elizabeth Sharlow, John S. Lazo, Thomas Kislinger, Kevin Henry, Yu Lu, Jason Moffat, Sheila K. Singh. The functional genomic landscape of recurrent glioblastoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR009.

Correction to "Structure of the Complex of an Iminopyridinedione Protein Tyrosine Phosphatase 4A3 Phosphatase Inhibitor with Human Serum Albumin".

Correction to "Structure of the Complex of an Iminopyridinedione Protein Tyrosine Phosphatase 4A3 Phosphatase Inhibitor with Human Serum Albumin".

Structure of the Complex of an Iminopyridinedione Protein Tyrosine Phosphatase 4A3 Phosphatase Inhibitor with Human Serum Albumin.

Protein tyrosine phosphatase (PTP) 4A3 is frequently overexpressed in human solid tumors and hematologic malignancies and is associated with tumor cell invasion, metastasis, and a poor patient prognosis. Several potent, selective, and allosteric small molecule inhibitors of PTP4A3 were recently identified. A lead compound in the series, JMS-053 (7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione), has a long plasma half-life (∼ 24 hours) in mice, suggesting possible binding to serum components. We confirmed by isothermal titration calorimetry that JMS-053 binds to human serum albumin. A single JMS-053 binding site was identified by X-ray crystallography in human serum albumin at drug site 3, which is also known as subdomain IB. The binding of JMS-053 to human serum albumin, however, did not markedly alter the overall albumin structure. In the presence of serum albumin, the potency of JMS-053 as an in vitro inhibitor of PTP4A3 and human A2780 ovarian cancer cell growth was reduced. The reversible binding of JMS-053 to serum albumin may serve to increase JMS-053's plasma half-life and thus extend the delivery of the compound to tumors. SIGNIFICANCE STATEMENT: X-ray crystallography revealed that a potent, reversible, first-in-class small molecule inhibitor of the oncogenic phosphatase protein tyrosine phosphatase 4A3 binds to at least one site on human serum albumin, which is likely to extend the compound's plasma half-life and thus assist in drug delivery into tumors.

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.

Endosulfan triggers epithelial-mesenchymal transition via PTP4A3-mediated TGF-β signaling pathway in prostate cancer cells

Endosulfan is a persistent organochlorine pesticide that bioaccumulates in human body through the food chain and thus represents a potential risk to public health. Despite epidemiological studies, the molecular mechanisms underlying the carcinogenic effects of endosulfan in the prostate remain poorly understood. In this study, we investigated the effect of endosulfan on epithelial-mesenchymal transition (EMT) in human prostate cancer PC3 and DU145 cells. Endosulfan induced alterations of EMT biomarkers, reflecting repression of E-cadherin expression and induction of fibronectin, snail2, ZEB2, Twist1 and Vimentin. The expression of Protein-tyrosine Phosphatase 4A3 (PTP4A3) at mRNA and protein levels was upregulated by endosulfan. PTP4A3 inhibitor reversed the changes of EMT biomarkers, PTP4A3 and p-Smad2/Smad2, but did not affect the upregulation of Cleaved-Notch1 and Jagged1 in endosulfan-exposed cells. Endosulfan promoted cell migration and invasion, which were rescued by specific inhibitors for PTP4A3, TGF-β signaling and Notch signaling, respectively. These findings suggest that endosulfan promoted cell migration and invasion with the induction of EMT through PTP4A3-mediated TGF-β signaling pathway in prostate cancer cells.

Endosulfan promotes cell migration via PTP4A3-mediated signaling pathways in HUVECs.

Endosulfan is a persistent organic pollutant and can cause endothelial dysfunction, closely related to cardiovascular diseases. Endothelial cell migration plays a critical role in atherosclerosis and angiogenesis. This study was aimed to investigate the effect of environmentally relevant doses of endosulfan and underlying molecular mechanism on endothelial cell migration. Human umbilical vein endothelial cells (HUVECs) were treated with DMSO (control) or endosulfan (0.1, 1, 10 and 20μM) in the presence or absence of inhibitors. Wound healing and Transwell assay were employed to explore the effect of endosulfan on endothelial cell migration. The expression of genes or proteins was assayed by real-time PCR or immunoblotting. The results showed that endosulfan at relative low concentration (0.1, 1, 10 and 20μM) increased cell migration ability horizontally and vertically at 12h after exposure. In line with this cellular effect, Protein-tyrosine Phosphatase 4A3 (PTP4A3) expression was significantly increased in endosulfan-exposed endothelial cells. Specific inhibitor of PTP4A3 significantly inhibited 20μM endosulfan-induced cell migration, the expression and phosphorylation of Src and phosphorylation of focal adhesion kinase (FAK). Exposure to endosulfan resulted in activation of various signaling pathways including phosphoinositide 3-kinase (PI3K)/AKT, mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB), which were suppressed by PTP4A3 inhibitor or specific inhibitor for each signaling pathway. Exposure to endosulfan significantly reduced nitric oxide production and caused oxidative stress in HUVECs. These findings suggest that endosulfan promoted cell migration through PTP4A3-mediated various signaling pathways in endothelial cells.

Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) drives migration and progression of T-cell acute lymphoblastic leukemia in vitro and in vivo

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer. There are no immunotherapies and few molecularly targeted therapeutics available for treatment of this malignancy. The identification and characterization of genes and pathways that drive T-ALL progression are critical for the development of new therapies for T-ALL. Here, we determined that the protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) plays a critical role in T-ALL initiation and progression by promoting leukemia cell migration. PRL-3 is highly expressed in patient T-ALL samples at both the mRNA and protein levels compared to normal lymphocytes. Knock-down of PRL-3 expression using short-hairpin RNA (shRNA) in human T-ALL cell lines significantly impeded T-ALL cell migration capacity in vitro and reduced their ability to engraft and proliferate in vivo in xenograft mouse models. Additionally, PRL-3 overexpression in a Myc-induced zebrafish T-ALL model significantly accelerated disease onset and shortened the time needed for cells to enter blood circulation. Reverse-phase protein array (RPPA) and gene set enrichment analysis (GSEA) revealed that the SRC signaling pathway is affected by PRL-3. Immunoblot analyses validated that manipulation of PRL-3 expression in T-ALL cells affected the SRC signaling pathway, which is directly involved in cell migration, although Src was not a direct substrate of PRL-3. More importantly, T-ALL cell growth and migration were inhibited by small molecule inhibition of PRL-3, suggesting that PRL-3 has potential as a therapeutic target in T-ALL. Taken together, our study identifies PRL-3 as an oncogenic driver in T-ALL both in vitro and in vivo and provides a strong rationale for targeted therapies that interfere with PRL-3 function.

Combination of ERK2 and STAT3 Inhibitors Promotes Anticancer Effects on Acute Lymphoblastic Leukemia Cells.

Deregulated activation of signaling through the RAS/RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/RAF/MEK/ERK) and signal transducer and activator of transcription (STAT) pathways is involved in numerous hematological malignancies, making it an attractive therapeutic target. This study aimed to assess the effect of the combination of ERK2 inhibitor VX-11e and STAT3 inhibitor STA-21 on acute lymphoblastic leukemia cell lines REH and MOLT-4. REH and MOLT-4 cell lines were cultured with each drug alone and in combination. Cell viability, ERK activity, cell cycle distribution, apoptosis and oxidative stress induction were assessed by flow cytometry. Protein levels of STAT3, phospho-STAT3, protein tyrosine phosphatase 4A3 (PTP4A3), survivin, p53 and p21 were determined by western blotting. VX-11e in combination with STA-21 significantly inhibited cell viability, induced G0/G1 cell-cycle arrest, enhanced production of reactive oxygen species, and induced apoptosis. These effects were associated with an increased level of p21 protein in REH cells and with reduced levels of phopho-STAT3, survivin and PTP4A3 proteins in MOLT-4 cells. Our findings provide a rationale for combined inhibition of RAS/RAF/MEK/ERK and STAT3 pathways in order to enhance anticancer effects against acute lymphoblastic leukemia cells.

STEM-27. LEVERAGING FUNCTIONAL GENETIC DEPENDENCIES IN TREATMENT-REFRACTORY GLIOBLASTOMA

Abstract As the most common primary brain tumor in adults causing death, Glioblastoma (GBM) remains a therapeutic challenge. Unchanged for almost two decades, standard therapy is ineffective at preventing disease relapse with a median patient survival of < 15 months. Stem cell-like subpopulations of tumor cells, known as brain tumor initiating cells (BTICs), evade standard therapy and lead to relapse. Whereas previous studies largely focus on pre-treatment primary GBM (pGBM), we conducted a panel of genome-wide CRISPR-Cas9 gene knockout screens to determine modulators of treatment resistance and de novo genetic vulnerabilities arising at disease recurrence. Using our in vitro model of conventional therapy, we identified genes modulating sensitivity and resistance to Temozolomide and/or radiation therapy in patient-derived pGBM BTICs. Genes modulating sensitivity belong to Fanconi anaemia nuclear complex, interstrand cross link repair, and regulation of stem cell maintenance and differentiation. Following in vitro validation of gene knockouts conferring treatment sensitization in multiple pGBM BTIC lines, we continued to conduct the first genome-wide CRISPR-Cas9 screens in patient-derived rGBM BTICs. Focusing on genetic vulnerabilities arising de novo at disease relapse, we introduce the context-specific role of protein tyrosine phosphatase 4A2 (PTP4A2) in rGBM. Genetic knockout or small molecule targeting of PTP4A2 leads to a context-specific vulnerability of rGBM self renewal capacity and in vivo tumorigenecity. To continue our analysis of treatment-refractory GBM and overcome intertumoral heterogeneity, we conducted genome-wide CRISPR-Cas9 gene knockout screens and whole cell proteomics on patient-matched pGBM and rGBM BTICs. With >1000 differentially essential genes, combined functional genetic and proteomic analyses implicates genes involved in mRNA splicing, nucleotide metabolism, and activation of gene expression by sterol regulatory element-binding protein. Together, our functional genetic approach elucidates novel genes regulating treatment resistance and disease recurrence in GBM.

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.