Sequential apoptotic and multiplexed proteomic evaluation of single cancer cells.

A potential cause of cancer relapse is pretreatment chemoresistant subpopulations. Identifying targetable features of subpopulations that are poorly primed for therapy-induced cell death may improve cancer therapy. Here, we develop and validate real-time BH3 profiling, a live and functional single-cell measurement of pretreatment apoptotic sensitivity that occurs upstream of apoptotic protease activation. On the same single cells, we perform cyclic immunofluorescence, which enables multiplexed immunofluorescence of more than 30 proteins on the same cell. Using cultured cells and rapid ex vivo cultures of colon cancer patient-derived xenograft (PDX) models, we identify Bak as a univariate correlate of apoptotic priming, find that poorly primed subpopulations can correspond to specific stages of the cell cycle, and, in some PDX models, identify increased expression of Bcl-XL, Mcl-1, or Her2 in subpopulations that are poorly primed for apoptosis. Last, we generate and validate mathematical models of single-cell priming that describe how targetable proteins contribute to apoptotic priming.

Metabolic perturbations sensitize triple-negative breast cancers to apoptosis induced by BH3 mimetics.

Cancer cells have differential metabolic dependencies compared to their nonmalignant counterparts. However, few metabolism-targeting compounds have been successful in clinical trials. Here, we investigated the metabolic vulnerabilities of triple-negative breast cancer (TNBC), particularly those metabolic perturbations that increased mitochondrial apoptotic priming and sensitivity to BH3 mimetics (drugs that antagonize antiapoptotic proteins). We used high-throughput dynamic BH3 profiling (HT-DBP) to screen a library of metabolism-perturbing small molecules, which revealed inhibitors of the enzyme nicotinamide phosphoribosyltransferase (NAMPT) as top candidates. In some TNBC cells but not in nonmalignant cells, NAMPT inhibitors increased overall apoptotic priming and induced dependencies on specific antiapoptotic BCL-2 family members. Treatment of TNBC cells with NAMPT inhibitors sensitized them to subsequent treatment with BH3 mimetics. The combination of a NAMPT inhibitor (FK866) and an MCL-1 antagonist (S63845) reduced tumor growth in a TNBC patient-derived xenograft model in vivo. We found that NAMPT inhibition reduced NAD+ concentrations below a critical threshold that resulted in depletion of adenine, which was the metabolic trigger that primed TNBC cells for apoptosis. These findings demonstrate a close interaction between metabolic and mitochondrial apoptotic signaling pathways and reveal that exploitation of a tumor-specific metabolic vulnerability can sensitize some TNBC to BH3 mimetics.

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.

A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion.

Dysregulation of the tightly controlled protein phosphorylation networks that govern cellular behavior causes cancer. The membrane-associated, intracellular protein tyrosine phosphatase PTP4A3 is overexpressed in human colorectal cancer and contributes to cell migration and invasion. To interrogate further the role of PTP4A3 in colorectal cancer cell migration and invasion, we deleted the Ptp4a3 gene from murine colorectal tumor cells. The resulting PTP4A3-/- cells exhibited impaired colony formation, spheroid formation, migration, and adherence compared with the paired PTP4A3fl/fl cells. We replicated these phenotypic changes using the new small-molecule, allosteric PTP4A3 inhibitor JMS-053. A related structure, JMS-038, which lacked phosphatase inhibition, displayed no cellular activity. Reduction in cell viability and colony formation by JMS-053 occurred in both mouse and human colorectal cell lines and required PTP4A3 expression. Ptp4a3 deletion increased the expression of extracellular matrix (ECM) and adhesion genes, including the tumor suppressor Emilin 1. JMS-053 also increased Emilin 1 gene expression. Moreover, The Cancer Genome Atlas genomic database revealed human colorectal tumors with high Ptp4a3 expression had low Emilin 1 expression. These chemical and biologic reagents reveal a previously unknown communication between the intracellular PTP4A3 phosphatase and the ECM and support efforts to pharmacologically target PTP4A3.-McQueeney, K. E., Salamoun, J. M., Ahn J. G., Pekic, P., Blanco, I. K., Struckman, H. L., Sharlow, E. R., Wipf, P., Lazo, J. S. A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion.

Targeting ovarian cancer and endothelium with an allosteric PTP4A3 phosphatase inhibitor.

Overexpression of protein tyrosine phosphatase PTP4A oncoproteins is common in many human cancers and is associated with poor patient prognosis and survival. We observed elevated levels of PTP4A3 phosphatase in 79% of human ovarian tumor samples, with significant overexpression in tumor endothelium and pericytes. Furthermore, PTP4A phosphatases appear to regulate several key malignant processes, such as invasion, migration, and angiogenesis, suggesting a pivotal regulatory role in cancer and endothelial signaling pathways. While phosphatases are attractive therapeutic targets, they have been poorly investigated because of a lack of potent and selective chemical probes. In this study, we disclose that a potent, selective, reversible, and noncompetitive PTP4A inhibitor, JMS-053, markedly enhanced microvascular barrier function after exposure of endothelial cells to vascular endothelial growth factor or lipopolysaccharide. JMS-053 also blocked the concomitant increase in RhoA activation and loss of Rac1. In human ovarian cancer cells, JMS-053 impeded migration, disrupted spheroid growth, and decreased RhoA activity. Importantly, JMS-053 displayed anticancer activity in a murine xenograft model of drug resistant human ovarian cancer. These data demonstrate that PTP4A phosphatases can be targeted in both endothelial and ovarian cancer cells, and confirm that RhoA signaling cascades are regulated by the PTP4A family.

Small molecule targeting of PTPs in cancer.

Protein tyrosine phosphatases (PTPs) undeniably have a central role in the development and progression of human cancers. Historically, however, PTPs have not been viewed as privileged drug targets, and progress on identifying potent, selective, and cell-active small molecule PTP inhibitors has suffered accordingly. This situation is rapidly changing, however, due to biochemical advances in the study of PTPs and recent small molecule screening campaigns, which have identified potent and mechanistically diverse lead structures. These compounds are facilitating the exploration of the fundamental cellular processes controlled by PTPs in cancers, and could form the inflection point for new therapeutic paradigms for the treatment of a range of cancers. Herein, we review recent advances in the discovery and biological annotation of cancer-relevant small molecule PTP inhibitors.

New Approaches to Difficult Drug Targets: The Phosphatase Story.

The drug discovery landscape is littered with promising therapeutic targets that have been abandoned because of insufficient validation, historical screening failures, and inferior chemotypes. Molecular targets once labeled as "undruggable" or "intractable" are now being more carefully interrogated, and while they remain challenging, many target classes are appearing more approachable. Protein tyrosine phosphatases represent an excellent example of a category of molecular targets that have emerged as druggable, with several small molecules and antibodies recently becoming available for further development. In this review, we examine some of the diseases that are associated with protein tyrosine phosphatase dysfunction and use some prototype contemporary strategies to illustrate approaches that are being used to identify small molecules targeting this enzyme class.

Localization of a Trypanosome Peroxin to the Endoplasmic Reticulum.

Trypanosoma brucei is the causative agent of diseases that affect 30,000-50,000 people annually. Trypanosoma brucei harbors unique organelles named glycosomes that are essential to parasite survival, which requires growth under fluctuating environmental conditions. The mechanisms that govern the biogenesis of these organelles are poorly understood. Glycosomes are evolutionarily related to peroxisomes, which can proliferate de novo from the endoplasmic reticulum or through the growth and division of existing organelles depending on the organism and environmental conditions. The effect of environment on glycosome biogenesis is unknown. Here, we demonstrate that the glycosome membrane protein, TbPex13.1, is localized to glycosomes when cells are cultured under high glucose conditions and to the endoplasmic reticulum in low glucose conditions. This localization in low glucose was dependent on the presence of a C-terminal tripeptide sequence. Our findings suggest that glycosome biogenesis is influenced by extracellular glucose levels and adds to the growing body of evidence that de novo glycosome biogenesis occurs in trypanosomes. Because the movement of peroxisomal membrane proteins is a hallmark of ER-dependent peroxisome biogenesis, TbPex13.1 may be a useful marker for the study such processes in trypanosomes.

Photooxygenation of an amino-thienopyridone yields a more potent PTP4A3 inhibitor.

The phosphatase PTP4A3 is an attractive anticancer target, but knowledge of its exact role in cells remains incomplete. A potent, structurally novel inhibitor of the PTP4A family was obtained by photooxygenation of a less active, electron-rich thienopyridone (1). Iminothienopyridinedione 13 displays increased solution stability and is readily obtained by two new synthetic routes that converge in the preparation of 1. The late-stage photooxygenation of 1 to give 13 in high yield highlights the potential of this reaction to modify the structure and properties of a biological lead compound and generate value for expanding the scope of an SAR investigation. Analog 13 should become a valuable tool for further exploration of the role of PTP4A3 in tumor progression.

Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.

Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1-3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins.

Investigational inhibitors of PTP4A3 phosphatase as antineoplastic agents.

INTRODUCTION: Protein tyrosine (Tyr) phosphatases have been implicated in many diseases, most notably in cancer. While there are a significant number of clinically approved inhibitors of protein Tyr kinases, there are no drugs specifically targeting protein Tyr phosphatases in clinical use despite the attractiveness of the molecular target. AREAS COVERED: This review examines the investigational challenges in identifying Tyr phosphatase inhibitors using the oncogenic phosphatase PTP4A3 as a prototype. The article includes a review of the structure, functionality and validation of PTP4A3 as a cancer target. It also provides an evaluation of existing small molecule and antibody inhibitors and provides new computational guidance for potentially more potent small molecule inhibitors. EXPERT OPINION: Tyr phosphatases, like PTP4A3, represent high value but ignored molecular targets for the treatment of cancer and other diseases. Although phosphatases are challenging targets, it seems likely that drug-like inhibitors of this important enzyme family would complement the growing number of protein Tyr kinase inhibitors. Animal models are beginning to provide validation for PTP4A3 as a molecular target for cancer progression and metastasis. The authors posit that greater efforts should be directed towards identifying Tyr phosphatase inhibitors for lead optimization and tool compounds to assist in interrogating and validating phosphatase involvement in physiological and pathological processes.

Protein-tyrosine phosphatase 4A3 (PTP4A3) promotes vascular endothelial growth factor signaling and enables endothelial cell motility.

Protein-tyrosine phosphatase 4A3 (PTP4A3) is highly expressed in multiple human cancers and is hypothesized to have a critical, albeit poorly defined, role in the formation of experimental tumors in mice. PTP4A3 is broadly expressed in many tissues so the cellular basis of its etiological contributions to carcinogenesis may involve both tumor and stromal cells. In particular, PTP4A3 is expressed in the tumor vasculature and has been proposed to be a direct target of vascular endothelial growth factor (VEGF) signaling in endothelial cells. We now provide the first in vivo experimental evidence that PTP4A3 participates in VEGF signaling and contributes to the process of pathological angiogenesis. Colon tumor tissue isolated from Ptp4a3-null mice revealed reduced tumor microvessel density compared with wild type controls. Additionally, vascular cells derived from Ptp4a3-null tissues exhibited decreased invasiveness in an ex vivo wound healing assay. When primary endothelial cells were isolated and cultured in vitro, Ptp4a3-null cells displayed greatly reduced migration compared with wild type cells. Exposure to VEGF led to an increase in Src phosphorylation in wild type endothelial cells, a response that was completely ablated in Ptp4a3-null cells. In loss-of-function studies, reduced VEGF-mediated migration was also observed when human endothelial cells were treated with a small molecule inhibitor of PTP4A3. VEGF-mediated in vivo vascular permeability was significantly attenuated in PTP4A3-deficient mice. These findings strongly support a role for PTP4A3 as an important contributor to endothelial cell function and as a multimodal target for cancer therapy and mitigating VEGF-regulated angiogenesis.