ENPP1 in Blood and Bone: Skeletal and Soft Tissue Diseases Induced by ENPP1 Deficiency.

The enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein that hydrolyzes extracellular ATP to generate pyrophosphate (PPi) and adenosine monophosphate, thereby contributing to downstream purinergic signaling pathways. The clinical phenotypes induced by ENPP1 deficiency are seemingly contradictory and include early-onset osteoporosis in middle-aged adults and life-threatening vascular calcifications in the large arteries of infants with generalized arterial calcification of infancy. The progressive overmineralization of soft tissue and concurrent undermineralization of skeleton also occur in the general medical population, where it is referred to as paradoxical mineralization to highlight the confusing pathophysiology. This review summarizes the clinical presentation and pathophysiology of paradoxical mineralization unveiled by ENPP1 deficiency and the bench-to-bedside development of a novel ENPP1 biologics designed to treat mineralization disorders in the rare disease and general medical population.

Catalysis-Independent ENPP1 Protein Signaling Regulates Mammalian Bone Mass.

Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency induces vascular/soft tissue calcifications in generalized arterial calcification of infancy (GACI), and low bone mass with phosphate-wasting rickets in GACI survivors (autosomal hypophosphatemic rickets type-2). ENPP1 haploinsufficiency induces early-onset osteoporosis and mild phosphate wasting in adults. Both conditions demonstrate the unusual combination of reduced accrual of skeletal mineral, yet excess and progressive heterotopic mineralization. ENPP1 is the only enzyme that generates extracellular pyrophosphate (PPi), a potent inhibitor of both bone and heterotopic mineralization. Life-threatening vascular calcification in ENPP1 deficiency is due to decreased plasma PPi; however, the mechanism by which osteopenia results is not apparent from an understanding of the enzyme's catalytic activity. To probe for catalysis-independent ENPP1 pathways regulating bone, we developed a murine model uncoupling ENPP1 protein signaling from ENPP1 catalysis, Enpp1T238A mice. In contrast to Enpp1asj mice, which lack ENPP1, Enpp1T238A mice have normal trabecular bone microarchitecture and favorable biomechanical properties. However, both models demonstrate low plasma Pi and PPi, increased fibroblast growth factor 23 (FGF23), and by 23 weeks, osteomalacia demonstrating equivalent phosphate wasting in both models. Reflecting findings in whole bone, calvarial cell cultures from Enpp1asj mice demonstrated markedly decreased calcification, elevated transcription of Sfrp1, and decreased nuclear ß-catenin signaling compared to wild-type (WT) and Enpp1T238A cultures. Finally, the decreased calcification and nuclear ß-catenin signaling observed in Enpp1asj cultures was restored to WT levels by knockout of Sfrp1. Collectively, our findings demonstrate that catalysis-independent ENPP1 signaling pathways regulate bone mass via the expression of soluble Wnt inhibitors such as secreted frizzled-related protein 1 (SFRP1), whereas catalysis dependent pathways regulate phosphate homeostasis through the regulation of plasma FGF23. © 2022 American Society for Bone and Mineral Research (ASBMR).

Direct targeting of amplified gene loci for proapoptotic anticancer therapy.

Gene amplification drives oncogenesis in a broad spectrum of cancers. A number of drugs have been developed to inhibit the protein products of amplified driver genes, but their clinical efficacy is often hampered by drug resistance. Here, we introduce a therapeutic strategy for targeting cancer-associated gene amplifications by activating the DNA damage response with triplex-forming oligonucleotides (TFOs), which drive the induction of apoptosis in tumors, whereas cells without amplifications process lower levels of DNA damage. Focusing on cancers driven by HER2 amplification, we find that TFOs targeting HER2 induce copy number-dependent DNA double-strand breaks (DSBs) and activate p53-independent apoptosis in HER2-positive cancer cells and human tumor xenografts via a mechanism that is independent of HER2 cellular function. This strategy has demonstrated in vivo efficacy comparable to that of current precision medicines and provided a feasible alternative to combat drug resistance in HER2-positive breast and ovarian cancer models. These findings offer a general strategy for targeting tumors with amplified genomic loci.

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Musculoskeletal Comorbidities and Quality of Life in ENPP1-Deficient Adults and the Response of Enthesopathy to Enzyme Replacement Therapy in Murine Models.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency leads to cardiovascular calcification in infancy, fibroblast growth factor 23 (FGF23)-mediated hypophosphatemic rickets in childhood, and osteomalacia in adulthood. Excessive enthesis mineralization and cervical spine fusion have been previously reported in patients with biallelic ENPP1 deficiency, but their effect on quality of life is unknown. We describe additional musculoskeletal complications in patients with ENPP1 deficiency, namely osteoarthritis and interosseous membrane ossification, and for the first time evaluate health-related quality of life (HRQoL) in patients with this disease, both subjectively via narrative report, and objectively via the Brief Pain Inventory-Short Form, and a Patient Reported Outcome Measurement Information System Physical Function (PROMIS PF) short form. Residual pain, similar in magnitude to that identified in adult patients with X-linked hypophosphatemia, was experienced by the majority of patients despite use of analgesic medications. Impairment in physical function varied from mild to severe. To assess murine ENPP1 deficiency for the presence of enthesopathy, and for the potential response to enzyme replacement therapy, we maintained Enpp1asj/asj mice on regular chow for 23 weeks and treated cohorts with either vehicle or a long-acting form of recombinant ENPP1. Enpp1asj/asj mice treated with vehicle exhibited robust calcification throughout their Achilles tendons, whereas two-thirds of those treated with ENPP1 enzyme replacement exhibited complete or partial suppression of the Achilles tendon calcification. Our combined results document that musculoskeletal complications are a significant source of morbidity in biallelic ENPP1 deficiency, a phenotype which is closely recapitulated in Enpp1asj/asj mice. Finally, we show that a long-acting form of recombinant ENPP1 prevents the development of enthesis calcification at the relatively modest dose of 0.3 mg/kg per week, suggesting that suppression of enthesopathy may be attainable upon dose escalation. © 2021 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Genetic pathways disrupted by ENPP1 deficiency provide insight into mechanisms of osteoporosis, osteomalacia, and paradoxical mineralization.

Ectonucleotide phosphatase/phosphodiesterase 1 (ENPP1) deficiency results in either lethal arterial calcifications ('Generalized Arterial Calcification of Infancy' - GACI), phosphate wasting rickets ('Autosomal Recessive Hypophosphatemic Rickets type 2' - ARHR2), early onset osteoporosis, or progressive spinal rigidity ('Ossification of the Posterior Longitudinal Ligament' - OPLL). As ENPP1 generates a strong endogenous mineralization inhibitor - extracellular pyrophosphate (PPi) - ENPP1 deficiency should not result in reduced bone volume, and therefore the mechanism ENPP1 associated osteoporosis is not apparent given current understanding of the enzyme's function. To investigate genetic pathways driving the skeletal phenotype of ENPP1 deficiency we compared gene expression in Enpp1asj/asj mice and WT sibling pairs by RNAseq and qPCR in whole bones, and in the liver and kidney by qPCR, directly correlating gene expression with measures of bone microarchitectural and biomechanical phenotypes. Unbiased analysis of the differentially expressed genes compared to relevant human disease phenotypes revealed that Enpp1asj/asj mice exhibit strong signatures of osteoporosis, ARHR2 and OPLL. We found that ENPP1 deficient mice exhibited reduced gene transcription of Wnt ligands in whole bone and increased transcription of soluble Wnt inhibitors in the liver and kidney, suggestive of multiorgan inhibition of Wnt activity. Consistent with Wnt suppression in bone, Collagen gene pathways in bone were significantly decreased and Fgf23 was significantly increased, all of which directly correlated with bone microarchitectural defects and fracture risk in Enpp1asj/asj mice. Moreover, the bone findings in 10-week old mice correlated with Enpp1 transcript counts but not plasma [PPi], suggesting that the skeletal phenotype at 10 weeks is driven by catalytically independent ENPP1 function. In contrast, the bone findings in 23-week Enpp1asj/asj mice strongly correlated with plasma PPi, suggesting that chronically low PPi drives the skeletal phenotype in older mice. Finally, correlation between Enpp1 and Fgf23 transcription suggested ENPP1 regulation of Fgf23, which we confirmed by dosing Enpp1asj/asj mice with soluble ENPP1-Fc and observing suppression of intact plasma FGF23 and ALP. In summary, our findings suggest that osteoporosis associated with ENPP1 deficiency involves the suppression of Wnt via catalytically independent Enpp1 pathways, and validates Enpp1asj/asj mice as tools to better understand OPLL and Paradoxical Mineralization Disorders.

Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites.

Next generation miRNA inhibition using short anti-seed PNAs encapsulated in PLGA nanoparticles.

Selective inhibition of microRNAs (miRNAs) offers a new avenue for cancer therapeutics. While most of the current anti-miRNA (antimiR) reagents target full length miRNAs, here we investigate novel nanoparticle-delivered short PNA probes containing cationic domains targeting the seed region of the miRNA for effective antimiR therapy. For proof of concept, we tested PNAs targeting miRNA-155 and employed poly(lactic-co-glycolic acid) (PLGA)-based nanoparticle formulation for delivery. A comprehensive evaluation of PLGA nanoparticles (NPs) containing short PNA probes showed significantly superior loading, release profile, and uniform size distribution, compared to conventional non-cationic PNA probes. Confocal microscopy and flow cytometry analyses showed efficient transfection efficiency and uniform distribution of PLGA NPs containing short PNA probes in the cytoplasm. Functional analysis also confirmed efficient miRNA-155 inhibition including an effect on its downstream target proteins. Further, reduced tumor growth was observed after systemic delivery of PLGA nanoparticles containing short PNA probes in vivo in a xenograft mouse model following inhibition of miR-155. There was no evidence of acute or chronic toxicity associated with systemic delivery of PLGA NPs containing short PNA probes in the mice. Overall, in this paper we present a novel antimiR strategy based on PLGA nanoparticle delivered short PNA probes for potential cancer therapy.

Human Heterozygous ENPP1 Deficiency Is Associated With Early Onset Osteoporosis, a Phenotype Recapitulated in a Mouse Model of Enpp1 Deficiency.

Biallelic ENPP1 deficiency in humans induces generalized arterial calcification of infancy (GACI) and/or autosomal recessive hypophosphatemic rickets type 2 (ARHR2). The latter is characterized by markedly increased circulating FGF23 levels and renal phosphate wasting, but aberrant skeletal manifestations associated with heterozygous ENPP1 deficiency are unknown. Here, we report three adult men with early onset osteoporosis who presented with fractures in the thoracic spine and/or left radius, mildly elevated circulating FGF23, and hypophosphatemia. Total hip bone mineral density scans demonstrated osteoporosis (Z-score < -2.5) and HRpQCT demonstrated microarchitectural defects in trabecular and cortical bone. Next-generation sequencing revealed heterozygous loss-of-function mutations in ENPP1 previously observed as biallelic mutations in infants with GACI. In addition, we present bone mass and structure data as well as plasma pyrophosphate (PPi) data of two siblings suffering from ARHR2 in comparison to their heterozygous and wild-type family members indicative of an ENPP1 gene dose effect. The skeletal phenotype in murine Enpp1 deficiency yielded nearly identical findings. Ten-week-old male Enpp1 asj/asj mice exhibited mild elevations in plasma FGF23 and hypophosphatemia, and micro-CT analysis revealed microarchitectural defects in trabecular and cortical bone of similar magnitude to HRpQCT defects observed in humans. Histomorphometry revealed mild osteomalacia and osteopenia at both 10 and 23 weeks. The biomechanical relevance of these findings was demonstrated by increased bone fragility and ductility in Enpp1 asj/asj mice. In summary, ENPP1 exerts a gene dose effect such that humans with heterozygous ENPP1 deficiency exhibit intermediate levels of plasma analytes associated with bone mineralization disturbance resulting in early onset osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

In vivo correction of anaemia in ß-thalassemic mice by γPNA-mediated gene editing with nanoparticle delivery.

The blood disorder, ß-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human ß-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% ß-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration.

MicroRNA silencing for cancer therapy targeted to the tumour microenvironment.

MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, microRNAs are critical cogs in numerous biological processes, and dysregulated microRNA expression is correlated with many human diseases. Certain microRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumours that depend on these microRNAs are said to display oncomiR addiction. Some of the most effective anticancer therapies target oncogenes such as EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (that is, antimiRs) is an evolving therapeutic strategy. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells. Here we introduce a novel antimiR delivery platform that targets the acidic tumour microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We find that the attachment of peptide nucleic acid antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produces a novel construct that could target the tumour microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumours (pH approximately 6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new model for using antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.

Molecular basis of purinergic signal metabolism by ectonucleotide pyrophosphatase/phosphodiesterases 4 and 1 and implications in stroke.

NPP4 is a type I extracellular membrane protein on brain vascular endothelium inducing platelet aggregation via the hydrolysis of Ap3A, whereas NPP1 is a type II extracellular membrane protein principally present on the surface of chondrocytes that regulates tissue mineralization. To understand the metabolism of purinergic signals resulting in the physiologic activities of the two enzymes, we report the high resolution crystal structure of human NPP4 and explore the molecular basis of its substrate specificity with NPP1. Both enzymes cleave Ap3A, but only NPP1 can hydrolyze ATP. Comparative structural analysis reveals a tripartite lysine claw in NPP1 that stabilizes the terminal phosphate of ATP, whereas the corresponding region of NPP4 contains features that hinder this binding orientation, thereby inhibiting ATP hydrolysis. Furthermore, we show that NPP1 is unable to induce platelet aggregation at physiologic concentrations reported in human blood, but it could stimulate platelet aggregation if localized at low nanomolar concentrations on vascular endothelium. The combined studies expand our understanding of NPP1 and NPP4 substrate specificity and range and provide a rational mechanism by which polymorphisms in NPP1 confer stroke resistance.

NPP4 is a procoagulant enzyme on the surface of vascular endothelium.

Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosphodiesterase-4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADP-mediated platelet aggregation at the site of vascular injury.

Kinetic analysis of autotaxin reveals substrate-specific catalytic pathways and a mechanism for lysophosphatidic acid distribution.

Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k(1) ≥500 µm(-1) s(-1)) and is hydrolyzed slowly (k(2) = 0.024 s(-1)). Release of the first hydrolysis product is random and rapid (≥1 s(-1)), whereas release of the second is slow and rate-limiting (0.005-0.007 s(-1)). Substrate binding and hydrolysis are slow and rate-limiting with LPC. Product release is sequential with choline preceding LPA. The catalytic pathway and kinetics depend strongly on the substrate, suggesting that ATX kinetics could vary for the various in vivo substrates. Slow catalysis with LPC reveals the potential for LPA signaling to spread to cells distal to the site of LPC substrate binding by ATX. An ATX mutant in which catalytic threonine at position 210 is replaced with alanine binds substrate weakly, favoring a role for Thr-210 in binding as well as catalysis. FTY720P, the bioactive form of a drug currently used to treat multiple sclerosis, inhibits ATX in an uncompetitive manner and slows the hydrolysis reaction, suggesting that ATX inhibition plays a significant role in lymphocyte immobilization in FTY720P-based therapeutics.

Autotaxin and lipid signaling pathways as anticancer targets.

Tumor progression and metastasis depend on signals in the tumor microenvironment acting on both the malignant cells and benign stroma to create an environment favorable to tumor expansion. A factor of emerging importance that acts in the tumor microenvironment is lysophosphatidic acid (LPA), a small signaling molecule that not only induces the transformation of benign cells into malignant invasive tumors, but also increases tumor growth, invasion, metastasis and angiogenesis. This review examines the LPA signaling pathway, its role in selected human malignancies, and the current state of development of inhibitors targeting molecules in this pathway.

Quantitative characterization of the interactions among c-myc transcriptional regulators FUSE, FBP, and FIR.

Human c-myc is critical for cell homeostasis and growth but is a potent oncogenic factor if improperly regulated. The c-myc far-upstream element (FUSE) melts into single-stranded DNA upon active transcription, and the noncoding strand FUSE recruits an activator [the FUSE-binding protein (FBP)] and a repressor [the FBP-interacting repressor (FIR)] to fine-tune c-myc transcription in a real-time manner. Despite detailed biological experiments describing this unique mode of transcriptional regulation, quantitative measurements of the physical constants regulating the protein-DNA interactions remain lacking. Here, we first demonstrate that the two FUSE strands adopt different conformations upon melting, with the noncoding strand DNA in an extended, linear form. FBP binds to the linear noncoding FUSE with a dissociation constant in the nanomolar range. FIR binds to FUSE more weakly, having its modest dissociation constants in the low micromolar range. FIR is monomeric under near-physiological conditions but upon binding of FUSE dimerizes into a 2:1 FIR(2)-FUSE complex mediated by the RRMs. In the tripartite interaction, our analysis suggests a stepwise addition of FIR onto an activating FBP-FUSE complex to form a quaternary FIR(2)-FBP-FUSE inhibitory complex. Our quantitative characterization enhances understanding of DNA strand preference and the mechanism of the stepwise complex formation in the FUSE-FBP-FIR regulatory system.

Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion.

Autotaxin (ATX) is a prometastatic enzyme initially isolated from the conditioned medium of human melanoma cells that stimulates a myriad of biological activities, including angiogenesis and the promotion of cell growth, survival, and differentiation through the production of lysophosphatidic acid (LPA). ATX increases the aggressiveness and invasiveness of transformed cells, and ATX levels directly correlate with tumor stage and grade in several human malignancies. To study the role of ATX in the pathogenesis of malignant melanoma, we developed antibodies and small-molecule inhibitors against recombinant human protein. Immunohistochemistry of paraffin-embedded human tissue shows that ATX levels are markedly increased in human primary and metastatic melanoma relative to benign nevi. Chemical screens identified several small-molecule inhibitors with binding constants ranging from nanomolar to low micromolar. Cell migration and invasion assays with melanoma cell lines show that ATX markedly stimulates melanoma cell migration and invasion, an effect suppressed by ATX inhibitors. The migratory phenotype can be rescued by the addition of the enzymatic product of ATX, LPA, confirming that the observed inhibition is linked to suppression of LPA production by ATX. Chemical analogues of the inhibitors show structure-activity relationships important for ATX inhibition and indicate pathways for their optimization. These studies suggest that ATX is an approachable molecular target for the rational design of chemotherapeutic agents directed against malignant melanoma.

Histopathologic and molecular aspects of CD56+ natural killer/ T-cell lymphoma of the testis.

Primary nasal-type natural killer/T-cell lymphoma of the testis is a rare malignancy. Although dissemination to the testis from other sites occurs somewhat more frequently than a primary presentation, even secondary testicular involvement is uncommon. In this article, the authors report on the comprehensive histopathologic, immunohistochemical, and molecular analysis of a case of primary testicular nasal-type natural killer/T-cell lymphoma, and review the features of 16 previously reported patients. The investigation carried out in this study indicates that the testicular nasal-type natural killer/T-cell lymphomas occur at a younger age than their B-cell counterparts, express cytoplasmic CD3 and surface CD56, and consistently show an infection by Epstein-Barr virus. These tumors have variable expression of T-cell antigens other than cytoplasmic CD3 and may show monoclonal rearrangement of T-cell receptor genes. Testicular natural killer/T-cell lymphomas of nasal type invariably follow an aggressive clinical course.

NMR-driven discovery of benzoylanthranilic acid inhibitors of far upstream element binding protein binding to the human oncogene c-myc promoter.

Reversal of aberrant gene expression that is induced by the proto-oncogene c-myc is likely to be effective for treating a variety of tumors that rely on this pathway for growth. One strategy to down-regulate the c-myc pathway is to target transcription factors that regulate its own expression. A host of proteins act in coordination to regulate c-myc expression and any one of them are theoretical targets for small-molecule therapy. Experimentally, it has been shown that the far upstream element (FUSE) binding protein (FBP) is essential for c-myc expression, and reductions in FBP levels both reduce c-myc expression and correlate with slower cell growth. FBP binds to ssDNA by capturing exposed DNA bases in a hydrophobic pocket. This suggests that a small molecule could be designed to occupy this pocket and inhibit FBP function. Using a variety of screening methodologies, we have identified ligands that bind to the DNA binding pockets of the KH domains of FBP. Gel shift analyses using full length FBP and a related transcription factor confirm that a small-molecule lead compound inhibits DNA binding in a specific manner. The benzoylanthranilic acid compounds described here represent leads in the design of FBP inhibitors that can serve as useful tools in the study of c-myc regulation and in the development of therapeutics that target the c-myc pathway.

Structure and dynamics of KH domains from FBP bound to single-stranded DNA.

Gene regulation can be tightly controlled by recognition of DNA deformations that are induced by stress generated during transcription. The KH domains of the FUSE-binding protein (FBP), a regulator of c-myc expression, bind in vivo and in vitro to the single-stranded far-upstream element (FUSE), 1,500 base pairs upstream from the c-myc promoter. FBP bound to FUSE acts through TFIIH at the promoter. Here we report the solution structure of a complex between the KH3 and KH4 domains of FBP and a 29-base single-stranded DNA from FUSE. The KH domains recognize two sites, 9-10 bases in length, separated by 5 bases, with KH4 bound to the 5' site and KH3 to the 3' site. The central portion of each site comprises a tetrad of sequence 5'd-ATTC for KH4 and 5'd-TTTT for KH3. Dynamics measurements show that the two KH domains bind as articulated modules to single-stranded DNA, providing a flexible framework with which to recognize transient, moving targets.