ENPP1 Research Articles

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (K121Q rs1044498) polymorphism is associated with diabetic nephropathy but not obesity among type-2 diabetes mellitus Egyptian patients

IntroductionGenetics contribute to the development of type-2 diabetes mellitus (T2DM), its complications, and phenotypes such as diabetic nephropathy (DN) and obesity. Although the likely associations among the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene, DN and obesity have been massively investigated, the results are still controversial. This study aimed to assess whether ENPP1 K121Q (A/C rs1044498) variant is associated with DN and obesity in T2DM Egyptian patients. We genotyped this variant in a total of 183 T2DM Egyptian patients who were classified into cases (91 participants with moderately increased albuminuria or severely increased albuminuria ≥ 30 mg/g) and controls (92 patients with normoalbuminuria <30 mg/g) using TaqMan technology.ResultsPatients, with the C (minor/risk) allele, had significantly higher moderately increased albuminuria/severely increased albuminuria levels (P < 0.001) and albumin-to-creatinine ratio (P = 0.001) than those with the wild A allele. AC and CC genotypes of ENPP1 K121Q (A/C, rs1044498) variant and its C-allele frequencies are significantly higher in cases than controls (P = 0.043 and 0.013), respectively, and in patients with estimated glomerular-filtration rate (eGFR) less than 60 than those with eGFR more than 60 (P = 0.014 and 0.004), respectively. AC and CC genotypes are associated with cases with a significant odds ratio in both dominant [odds ratio (OR): 2.003, 95% confidence interval (CI): 1.106–3.628, P = 0.022) and additive (OR: 1.865, 95% CI: 1.134–3.070, P = 0.014) models of inheritance and in patients with eGFR less than 60 in the dominant model of inheritance (OR: 2.398, 95% CI: 1.258–4.571, P = 0.008), but showed no association with obesity (P > 0.05).ConclusionENPP1 K121Q (A/C, rs1044498) variant is associated with DN but not obesity among Egyptian T2DM patients.

Abstract LBA009: Orally available ENPP1 inhibitor, TXN10128, restores STING activation in tumor microenvironment and confers anti-tumor responses in combination with immune checkpoint blockade

Abstract Background Recently, there are growing needs of immune modulators that can convert cold tumors into hot tumors, which can be utilized for combination treatment with existing immune related therapies. An orally available small molecule that is capable of activating innate immune response can be an ideal candidate to meet those needs. Upon binding to 2’3’-cGAMP, STING activates TBK1-IRF3 signaling cascade in cancer cells as well as host cells and promotes innate immune responses against cancer cells, leading to T cell mediated anti-tumor immunity by facilitating T cell priming and infiltration. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), a transmembrane protein highly expressed in subset of cancer cells, has been known to hydrolyze 2’3’-cGAMP and negatively regulates the STING activation. Inhibition of ENPP1 can prevent 2’3’-cGAMP degradation in tumor microenvironment (TME) and restore STING activation, leading to innate immune responses. It has been shown that ENPP1 inhibitor can enhance anti-tumor immunity by restoring 2’3’-cGAMP level in TME and can prevent tumor growth when co-treated with immune checkpoint blockade (ICB) as well as with radiation therapy. Methods The activity of ENPP1 inhibitors were measured in enzymatic assay using 2’3’-cGAMP as substrate and in cellular STING activation assay using IRF3-responsive reporter. Also, the effects on innate immune response were estimated in LIN (Lymphocyte INfiltration) assay measuring lymphocyte infiltration and TMED (TME in Dish) assay measuring tumor spheroid growth and immune cell activation. In vivo efficacy of TXN10128 (PO dosing) was evaluated in MC38 syngeneic model in combination with and anti-PD-L1 (IV dosing). Tumor growth was monitored and immune cells in tumor were analyzed. Results TXN10128 inhibited ENPP1 activity with single digit nanomolar potency in enzyme assay and further induced STING activation in cellular assay. In 3D spheroid co-culture condition, TXN10128 enhances lymphocyte infiltration and inhibits the spheroid growth. TXN10128 has a drug-like properties desirable for oral administration in terms of physicochemical properties and pharmacokinetics parameters. Systemic exposure of TXN10128 by PO dosing resulted in synergistic tumor growth inhibition with anti-PD-L1 antibody and improved tumor-infiltrating lymphocytes (TIL) profile as expected in MC38 syngeneic model. Additional in vivo studies are on-going to expand applicable cancer types to TXN10128 treatment. Conclusions TXN10128 is a potent and selective ENPP1 inhibitor that can exert immune response in 3D co-culture condition, which is consistent with tumor growth inhibition and favorable TIL profile in animal model. Together with promising drug-likeness, these studies demonstrate that TXN10128 is a suitable candidate for clinical investigation as a combination partner with existing immunotherapies. Citation Format: Sungjoon Kim, Imran Ali, Ahran Yu, Sun woo Lee, Sung young Park, Jung hwan Choi, Yong-yea Park, Chan sun Park. Orally available ENPP1 inhibitor, TXN10128, restores STING activation in tumor microenvironment and confers anti-tumor responses in combination with immune checkpoint blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA009.

Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) due to ENPP1-deficiency.

Awareness for hypophosphatemic rickets has increased in the last years, based on the availability of specific medical treatments. Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) is a rare form of hypophosphatemic rickets, which is known to develop in survivors of generalized arterial calcification of infancy (GACI). Both disorders are based on a deficiency of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and present with a high clinical variability and a lack of a phenotype-genotype association. ARHR2 is characterized by phosphate wasting due to elevated fibroblast growth factor 23 (FGF23) levels and might represent a response of the organism to minimize ectopic calcification in individuals with ENPP1-deficiency. This report reviews the recent clinical and preclinical data on this ultra-rare disease in childhood.

Abstract 16747: Rapidly Lethal Dilated Cardiomyopathy Secondary To Generalized Arterial Calcification Of Infancy

Case Presentation: A previously healthy 3-month-old infant presented with cardiorespiratory arrest, from which she was successfully resuscitated. A dilated cardiomyopathy with severely depressed systolic function was diagnosed. ECG showed high voltage QRS complexes and generalized alteration of repolarization. In the following 12 hours, she suffered 2 other cardiac arrests, recuperated with defibrillation. Hemodinamic stability was achieved but brain death was diagnosed 36 hours after the onset of symptoms. Autopsy showed striking calcification of the right and left coronary arteries, with narrowing of the arterial lumen, causing extensive subendocardial infarction. Calcification also affected the aorta, pulmonary arteries, thyroid, kidney and other splanchnic arteries. Generalized arterial calcification of infancy (GACI) was diagnosed and a genetic study found two biallelic variants in ABCC6 gene: p.Arg1114Cys and p.Trp38Ser, both previously described in elastic pseudoxanthoma (PXE), but not in GACI. Genotyping of the healthy parents confirmed genetic segregation with biallelic variants. Discussion: GACI is an extremely rare genetic disease characterized by widespread arterial calcification and narrowing of large and medium-sized vessels. The usual clinical presentation is heart failure in fetal life or in the first months of infancy. In most cases it is lethal, with death occurring within a few hours or days after the onset of symptoms, although clinical involvement is highly variable and cases with long survival have been described. GACI is an autosomal recessive disease secondary to biallelic variants in the ENPP1 gene (67% of cases) and in the ABCC6 gene (9%). The variants found in our patient had not been previously described in GACI, just in PXE, a much milder disease with usually normal lifespan. This case confirms that both entities reflect two extremes of a clinical spectrum of ectopic calcification instead of two different disorders. The aim of presenting this case is to remind clinicians of this rare etiology in neonates or infants with dilated cardiomyopathy. In case of death, autopsy should always be requested. When this condition is diagnosed, genetic study will be positive in 75% of cases, allowing prenatal counseling.

Hepatitis B Virus Promotes Hepatocellular Carcinoma Progression Synergistically With Hepatic Stellate Cells via Facilitating the Expression and Secretion of ENPP2.

Background: Hepatitis B virus (HBV) infection is a major risk factor causing hepatocellular carcinoma (HCC) development, but the molecular mechanisms are not fully elucidated. It has been reported that virus infection induces ectonucleotide pyrophosphatase-phosphodiesterase 2 (ENPP2) expression, the latter participates in tumor progression. Therefore, the aim of the present study was to investigate whether HBV induced HCC malignancy via ENPP2. Methods: HCC patient clinical data were collected and prognosis was analyzed. Transient transfection and stable ectopic expression of the HBV genome were established in hepatoma cell lines. Immunohistochemical staining, RT-qPCR, western blot, and ELISA assays were used to detect the expression and secretion of ENPP2. Finally, CCK-8, colony formation, and migration assays as well as a subcutaneous xenograft mouse model were used to investigate the influence of HBV infection, ENPP2 expression, and activated hepatic stellate cells (aHSCs) on HCC progression in vitro and in vivo. Results: The data from cancer databases indicated that the level of ENPP2 was significant higher in HCC compared within normal liver tissues. Clinical relevance analysis using 158 HCC patients displayed that ENPP2 expression was positively correlated with poor overall survival and disease-free survival. Statistical analysis revealed that compared to HBV-negative HCC tissues, HBV-positive tissues expressed a higher level of ENPP2. In vitro, HBV upregulated ENPP2 expression and secretion in hepatoma cells and promoted hepatoma cell proliferation, colony formation, and migration via enhancement of ENPP2; downregulation of ENPP2 expression or inhibition of its function suppressed HCC progression. In addition, aHSCs strengthened hepatoma cell proliferation, migration in vitro, and promoted tumorigenesis synergistically with HBV in vivo; a loss-function assay further verified that ENPP2 is essential for HBV/aHSC-induced HCC progression. Conclusion: HBV enhanced the expression and secretion of ENPP2 in hepatoma cells, combined with aHSCs to promote HCC progression via ENPP2.

Plasmacytoid dendritic cell activation is dependent on coordinated expression of distinct amino acid transporters

Human plasmacytoid dendritic cells (pDCs) are interleukin-3 (IL-3)-dependent cells implicated in autoimmunity, but the role of IL-3 in pDC biology is poorly understood. We found that IL-3-induced Janus kinase 2-dependent expression of SLC7A5 and SLC3A2, which comprise the large neutral amino acid transporter, was required for mammalian target of rapamycin complex 1 (mTORC1) nutrient sensor activation in response to toll-like receptor agonists. mTORC1 facilitated increased anabolic activity resulting in type I interferon, tumor necrosis factor, and chemokine production and the expression of the cystine transporter SLC7A11. Loss of function of these amino acid transporters synergistically blocked cytokine production by pDCs. Comparison of invitro-activated pDCs with those from lupus nephritis lesions identified not only SLC7A5, SLC3A2, and SLC7A11 but also ectonucleotide pyrophosphatase-phosphodiesterase 2 (ENPP2) as components of a shared transcriptional signature, and ENPP2 inhibition also blocked cytokine production. Our data identify additional therapeutic targets for autoimmune diseases in which pDCs are implicated.

A DOCK1 Gene-Derived Circular RNA Is Highly Expressed in Luminal Mammary Tumours and Is Involved in the Epithelial Differentiation, Growth, and Motility of Breast Cancer Cells.

Simple SummaryIn the present study, we define the expression of 15 selected circular RNAs, using a panel of 18 breast cancer cell lines recapitulating the heterogeneity of tumors and consisting of three distinct groups according to the mesenchymal/epithelial phenotype. We identify circDOCK1-1 generated from the DOCK1 gene as an important regulator of the epithelial differentiation of mammary tumour cells. In addition, our high-throughput transcriptomic studies provide information on the genes that are regulated by circDOCK1-1, casting new light on the gene networks involved in the processes underlying the development of mammary tumours. Our results further indicate that circDOCK1-1 controls cell–cell adhesion and consequently random as well as directional motility. Besides their interest from a basic point of view, the data generated may be of translational relevance, as they suggest that strategies aimed at increasing the amounts or at stimulating the activity of circDOCK1-1 may be of significance for the treatment of a subset of triple-negative breast cancer patients.Circular RNAs are regulatory molecules involved in numerous cellular processes and may be involved in tumour growth and diffusion. Here, we define the expression of 15 selected circular RNAs, which may control the process of epithelial-to-mesenchymal transition, using a panel of 18 breast cancer cell lines recapitulating the heterogeneity of these tumours and consisting of three groups according to the mesenchymal/epithelial phenotype. A circular RNA from the DOCK1 gene (hsa_circ_0020397) shows low/undetectable levels in triple-negative mesenchymal cell lines, while its content is high in epithelial cell lines, independent of estrogen receptor or HER2 positivity. RNA-sequencing experiments performed on the triple-negative/mesenchymal MDA-MB-231 and MDA-MB-157 cell lines engineered to overexpress hsa_circ_0020397 demonstrate that the circRNA influences the expression of 110 common genes. Pathway analysis of these genes indicates that overexpression of the circular RNA differentiates the two mesenchymal cell lines along the epithelial pathway and increases cell-to-cell adhesion. This is accompanied by growth inhibition and a reduction in the random/directional motility of the cell lines. The upregulated AGR2, ENPP1, and PPP1R9A genes as well as the downregulated APOE, AQP3, CD99L2, and IGFBP4 genes show an opposite regulation by hsa_circ_0020397 silencing in luminal CAMA1 cells. The results provide novel insights into the role played by specific circular RNAs in the generation/progression of breast cancer.

Comparative immunolocalization of tissue nonspecific alkaline phosphatase and ectonucleotide pyrophosphatase/phosphodiesterase 1 in murine bone

ObjectiveThis study aimed to demonstrate the immunolocalization and gene expression of tissue nonspecific alkaline phosphatase (TNALP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in osteoblasts, preosteoblasts, and osteocytes of murine bone to provide clues for a better understanding of the supply of phosphate ions (Pi) during bone mineralization. MethodsSix-week-old male C57BL/6J mice (n = 6) were fixed with a paraformaldehyde solution, and the right femora were extracted for immunodetection of TNALP and ENPP1, while the left tibiae were used for reverse transcription polymerase chain reaction to evaluate Tnalp and Enpp1 gene expression. ResultsTNALP was intensely localized on the basolateral cell membranes of mature osteoblasts and preosteoblastic cells. There was little immunoreactivity of TNALP on the secretory surface of the osteoblasts and no TNALP reactivity in the osteocytes. In contrast, ENPP1 was observed throughout the cytoplasm of mature osteoblasts and osteocytes embedded in bone but was not observed in preosteoblasts. Together, despite the fact that the osteoid is a site of matrix vesicle-mediated mineralization, ENPP1, which inhibits mineralization by providing pyrophosphates, was localized in close proximity of the osteoid, whereas TNALP, which facilitates mineralization by providing Pi, was relatively distant from the osteoid. ConclusionIt seems likely that the differential localization of TNALP and ENPP1 around the osteoid observed at the microscopic level may provide preferential micro-circumstance for a balanced concentration of Pi and pyrophosphate for bone mineralization.

Levels of serum ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) predicts severity of abdominal aortic calcification in end-stage renal disease patients receiving regular dialysis.

To investigate the correlation between serum ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) level and severity of abdominal vascular calcification in end-stage renal disease (ESRD) patients receiving dialysis. A total of 124 patients were consecutively enrolled into the study in our local institution. Based on the Kidney Disease Improving Global Outcomes (KDIGO) guidelines and recommendations, abdomen lateral X-ray was used to determine abdominal aortic calcification score (AACS) for each patient at enrollment. Patients were divided into three groups based on AACS: no or mild calcification group, moderate calcification group, and severe calcification group. The relationships between ENPP1 levels and AACS were assessed by Spearman analysis and the value of ENPP1 in predicting severity of abdominal aortic calcification was evaluated by receiver operating characteristic (ROC). The level of ENPP1 in dialysis patients was (7.68 ± 1.67) ng/ml. There was no significant difference in serum ENPP1 level between peritoneal dialysis patients and hemodialysis patients (p > 0.05). The AACS of dialysis patients was negatively correlated with ENPP1 value (r=-0.70). Compared to no/mild calcification patients, the levels of serum ENPP1 in patients with moderate/severe calcification were decreased significantly (p < 0.01). The severity of vascular calcification was correlated with serum ENPP1 value, the severer the vascular calcification, the lower the serum ENPP1 level, and the difference was statistically significant (all p < 0.05). The area under ROC curve of ENPP1 was 0.90, the corresponding sensitivity was 0.86, and the specificity was 0.87. Levels of serum ENPP1 in non-diabetic ESRD patients are negatively related to the severity of abdominal aortic vascular calcification.

Abstract 53: Discovery of novel potent and orally bioavailable small-molecule inhibitors of ENPP1 to stabilize cGAMP and ATP in the tumor microenvironment and boost anti-tumor immunity

Abstract Extracellular 2',3'-cyclic GMP-AMP (cGAMP) has a critical immune-transmitter role in the tumor microenvironment (TME). Tumor cells produce and secrete cGAMP, which primes immune cells for tumor rejection through STING (stimulator of interferon genes) signalling (Marcus et al., Immunity 2018; Carozza et al., Nature Cancer 2020). Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) is the only enzyme known to hydrolyze extracellular cGAMP. In concert with the ectoATPase CD39 and the 5'-nucleotidase CD73, ENPP1 also contributes to the generation of an immunosuppressive TME by converting extracellular ATP into adenosine. ENPP1 is overexpressed in a number of tumor types, including breast cancer, liver cancer, thyroid cancer and sarcomas, and has been associated with poor outcome. Hence, inhibition of ENPP1 is an emerging strategy to augment anti-tumor immunity by stabilizing extracellular cGAMP and ATP, thereby turning cold tumors into immunologically hot tumors. Here, we report on the identification and characterization of novel chemical series of ENPP1 inhibitors. A drug-like small-molecule library (~160,000 compounds) was screened to identify inhibitors of ENPP1-mediated cGAMP hydrolysis. Hits were confirmed to also inhibit ATP hydrolysis by ENPP1, but not nucleotide hydrolysis mediated by the closely related ENPP2 (autotaxin) enzyme. A selection of hits, chemically distinct from previously reported ENPP1 inhibitors (such as those based on QS1; Carroza et al, Cell Chem Biol, 2020), were subjected to hit-to-lead optimization supported by structure-based guidance. This led to the identification of ENPP1 inhibitors with sub-nM potency on ENPP1 in biochemical assays, which maintained a &amp;gt;1000x selectivity window with respect to ENPP2 and to other phosphodiesterases. These compounds translated well to stabilization of nucleotides in the presence of ENPP1-overexpressing cancer cell lines, with IC50 values in the nM range. A selection of compounds was profiled for DMPK (drug metabolism and pharmacokinetic) parameters, and compounds with a suitable profile were prioritized for in vivo evaluation. Orally bio-available, metabolically stable compounds were assessed in mouse syngeneic tumor models, selected on the basis of high ENPP1 and high cGAS (cGAMP synthase) expression. Stabilization of cGAMP, activation of a STING-mediated cytokine response and immune-cell infiltration/activation in the TME were used as pharmacodynamic endpoints. An update on in vivo efficacy data in a range of ENPP1-positive tumor models will be provided during the presentation. These novel orally bioavailable ENPP1 inhibitors unleash local, TME-restricted, innate immune activation, and hold the promise to overcome the current limitations of direct STING agonists. Citation Format: Matthias Versele, Javier del Pino Garcia, Ilse Vandecaetsbeek, Karolien Castermans, Ranie Kellens, Wanda Haeck, Hugo Klaassen, Arnaud Bourin, Dries De Clercq, Sara Allasia, Sandro Boland, Arnaud Marchand, Patrick Chaltin, Mathieu Bollen. Discovery of novel potent and orally bioavailable small-molecule inhibitors of ENPP1 to stabilize cGAMP and ATP in the tumor microenvironment and boost anti-tumor immunity [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 53.

Identification of Plasma Cell Antigen PC-1 as a Novel Cell Surface Marker of Germinal Center, Memory and Plasmacytoma B Cells.

The murine plasma cell (PC) alloantigen PC-1, also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), is expressed on PCs and by many non-lymphoid tissues. PC-1 is best known for its role in phosphate metabolism, hydrolyzing NTP to generate pyrophosphate. However, its expression and function in B lymphocytes remains unclear. Here we describe a previously unknown expression pattern of PC-1 in murine late stage B cells and B lymphomas. In normal mice, expression of PC-1, determined by flow cytometry, was very low on bone marrow pro-B, pre-B, immature B, and mature B cells, and on splenic follicular and marginal zone B cells. However, peritoneal B cells expressed significantly higher levels of PC-1 than splenic naïve B cells. CD5+ B1a cells expressed more PC-1 than B1b and B2 cells. Interestingly, PC-1 distinguishes two subsets of B1a: PC-1hi and PC-1lo. The PC-1hi B1a cells were IgMlo, B220lo, and larger in size while PC-1lo B1a cells were IgMhi, B220hi, and smaller in size. The PC-1hi B1a cells express higher levels of Blimp-1 transcripts than the PC-1lo B1a cells, indicating that the PC- 1hi B1a cells may exhibit features of pre-plasma cells. Furthermore, after immunization with NP-KLH, germinal center (GC) B cells, PCs and early memory B cells expressed 2–4-fold more PC-1 than naïve B cells. Naïve B cells stimulated with LPS or anti-IgM plus anti-CD40 antibodies also dramatically upregulated PC-1 expression. Finally, gene expression profiling studies of primary lymphomas in NFS.V(+) congenic mice revealed high expression levels of PC-1 in plasmacytomas, immunoblastic lymphomas, and centroblastic-follicular lymphoma types. These studies demonstrate that PC-1 can be a useful marker for identification of stages in GC and post-GC cell differentiation and of lymphomas with a GC or PC origin. The function of PC-1 in B cells is currently under investigation.

Influence of Single Nucleotide Polymorphism of ENPP1 and ADIPOQ on Insulin Resistance and Obesity: A Case-Control Study in a Javanese Population.

Single nucleotide polymorphisms (SNPs) in obesity-related genes, such as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) and adiponectin (ADIPOQ), potentially increase the risk of insulin resistance, the most common metabolic dysregulation related to obesity. We investigated the association of ENPP1 SNP K121Q (rs1044498) with insulin resistance and ADIPOQ SNP + 267G > T (rs1501299) with circulating adiponectin levels in a case–control study involving 55 obese and 55 lean Javanese people residing in Yogyakarta, Indonesia. Allele frequency was determined by a chi squared test or Fisher’s exact test with an expected value less than 0.05. Odds ratios and 95% confidence intervals were estimated by regression logistic analysis. The presence of the Q121 allele of ENPP1 resulted in significantly higher fasting glucose, fasting insulin levels, and HOMA-IR, as compared to homozygous K121 carriers. The risk of insulin resistance was elevated in obese individuals carrying Q121 instead of homozygous K121. Adiponectin level was significantly lower in the obese group as compared to the lean group. Obese individuals carrying homozygous protective alleles (TT) of ADIPOQ tended to have lower adiponectin levels as compared to GT and GG carriers, however, we did not find statistically significant effects of the +276G > T SNP of the ADIPOQ gene on the plasma adiponectin levels or on the development of obesity.

Generalized Arterial Calcification of Infancy Type 1 (GACI1): Identification of a Novel Pathogenic Variant (c.1715T>C (p.Leu572Ser)).

Generalized Arterial Calcification of Infancy (GACI) is a rare disease inherited in a recessive manner, with severe and diffuse early onset of calcifications along the internal elastic lamina in large and medium size arteries. The diagnosis results are from clinical manifestations, imaging, histopathologic exams, and genetic tests. GACI is predominantly caused by biallelic pathogenic variant in the ENPP1 gene (GACI1, OMIM#208000) and, to a lesser extent, by pathogenic variants in the ABCC6 gene (GACI2, OMIM#614473). We present a novel variation in the ENPP1 gene identified in a patient clinically diagnosed with GACI and confirmed by genetic investigation and autopsy as GACI type 1. The sequence analysis of the patient’s ENPP1 gene detected two heterozygous variants c.1412A>G (p.Tyr471Cys) and c.1715T>C (p.Leu572Ser). The variant c.1715T>C (p.Leu572Ser) has not been described yet in the literature and in mutation databases. A genetic analysis was also carried out for the parents of the newborn; the heterozygous pathogenic variant c.1412A>G (p.Tyr471Cys) was detected in the mother’s ENPP1 gene, and a sequence analysis of the father’s ENPP1 gene revealed the novel heterozygous variant c.1715T>C (p.Leu572Ser). Our results showed that the variant c.1715T>C (p.Leu572Ser) may have a pathogenic role in the development of GACI type1 (GACI1, OMIM#208000), at least when associated with the pathogenic c.1412A>G (p.Tyr471Cys) variant. The identification of novel mutations potentially enabled genotype/phenotype associations that will ultimately have an impact on clinical management and prognosis for the disease.

Abstract #1003877: The Lifelong Impact of ENPP1 Deficiency: Outcomes From a Patient and Caregiver Survey

Pyrophosphate (PPi) acts as a potent inhibitor of mineralization. Patients deficient in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), a protein involved in PPi metabolism, have low serum PPi and heterogeneous clinical manifestations, including arterial calcification; cardiac, neurological, and gastrointestinal (GI) dysfunction; skeletal abnormalities; and hearing loss. This study was intended to characterize the burden of disease from a patient or caregiver/parent perspective.

Type 2 diabetes is associated with the MTNR1B gene, a genetic bridge between circadian rhythm and glucose metabolism, in a Turkish population.

Type 2 diabetes (T2D) is a complicated public health problem in Turkey as well as worldwide. Genome-wide approaches have been guiding in very challenging situations, such as the elucidation of genetic variations underlying complex diseases such as T2D. Despite intensive studies worldwide, few studies have determined the genetic susceptibility to T2D in Turkish populations. In this study, we investigated the effect of genes that are strongly associated with T2D in genome-wide association (GWA) studies, including MTNR1B, CDKAL1, THADA, ADAMTS9 and ENPP1, on T2D and its characteristic traits in a Turkish population. In 824 nonobese individuals (454 T2D patients and 370 healthy individuals), prominent variants of these GWA genes were genotyped by real-time PCR using the LightSNiP Genotyping Assay System. The SNP rs1387153 C/T, which is located 28kb upstream of the MTNR1B gene, was significantly associated with T2D and fasting blood glucose levels (P < 0.05). The intronic SNP rs10830963 C/G in the MTNR1B gene was not associated with T2D, but it was associated with fasting blood glucose, HbA1C and LDL levels (P < 0.05). The other important GWA loci investigated in our study were not found to be associated with T2D or its traits. Only the SNP rs1044498 (A/C variation) in the ENPP1 gene was determined to be related to fasting blood glucose (P < 0.05). Our study suggests, consistent with the literature, that the MTNR1B locus, which has a prominent role in glucose regulation, is associated with T2D development by affecting blood glucose levels in our population.

Identification of cell surface targets for CAR-T cell therapies and antibody–drug conjugates in breast cancer

BackgroundTwo promising therapeutic strategies in oncology are chimeric antigen receptor-T cell (CAR-T) therapies and antibody–drug conjugates (ADCs). To be effective and safe, these immunotherapies require surface antigens to be sufficiently expressed in tumors and less or not expressed in normal tissues. To identify new targets for ADCs and CAR-T specifically targeting breast cancer (BC) molecular and pathology-based subtypes, we propose a novel in silico strategy based on multiple publicly available datasets and provide a comprehensive explanation of the workflow for a further implementation.MethodsWe carried out differential gene expression analyses on The Cancer Genome Atlas BC RNA-sequencing data to identify BC subtype-specific upregulated genes. To fully explain the proposed target-discovering methodology, as proof of concept, we selected the 200 most upregulated genes for each subtype and undertook a comprehensive analysis of their protein expression in BC and normal tissues through several publicly available databases to identify the potentially safest and viable targets.ResultsWe identified 36 potentially suitable and subtype-specific tumor surface antigens (TSAs), including fibroblast growth factor receptor-4 (FGFR4), carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), GDNF family receptor alpha 1 (GFRA1), integrin beta-6 (ITGB6) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We also identified 63 potential TSA pairs that might be appropriate for co-targeting strategies. Finally, we validated subtype specificity in a cohort of our patients, multiple BC cell lines and the METABRIC database.ConclusionsOverall, our in silico analysis provides a framework to identify novel and specific TSAs for the development of new CAR-T and antibody-based therapies in BC.

High expression of ENPP1 in high-grade serous ovarian carcinoma predicts poor prognosis and as a molecular therapy target.

Recent studies have shown that the expression of ENPP1 is related to differentiation, death, dissemination and chemosensitivity of tumor cells. So far, there is no research in ovarian carcinoma. This study aimed at exploring the role of ENPP1 gene in ovarian carcinoma, the relationship with prognostic indicators and chemotherapy resistance, and investigates the possibility of molecular targeted therapy. The expression of ENPP1 in 41 normal ovarian epithelial tissues, 97 ovarian serous cystadenoma and 103 HGSOC tissues was detected by IHC. In ovarian cancer tissues and ovarian cancer cell lines, mRNA and protein expression of ENPP1 was determined by qRT-PCR and Western blot. The ENPP1 expression was knockdowned by siRNA. Cell proliferation was measured with the BrdU Cell Proliferation ELISA. Cell migration and invasion were detected by Wound-Healing, Transwell migration and Matrigel invasion assay. Caspase 3 activity was determined by the CaspACE. The expression of EMT markers such as E-cadherin, N-cadherin, and Vimentin was measured, and the expression of PCNA and MMP9 was also be detected. The results showed that the expression of ENPP1 was significantly increased in high-grade ovarian serous carcinoma, the number of strong expression was 85.4% (22.3%+63.1%) and only 1.03% (1.03%+0.0%) in serous cystadenoma, but no in normal ovarian epithelium (P< 0.05). And the stronger the expression of ENPP1, the later the FIGO stage and the poorer differentiation of cells (P = 0.001 or <0.001, respectively). However, no correlation was found between the expression of ENPP1 and chemosensitivity. ENPP1 was also highly expressed in ovarian cancer tissues and in epithelial ovarian cancer cell lines (A2780, CaoV3, OVCAR3, SKOV3 and 3ao). After down-regulation of ENPP1 expression by RNA interference, the cell proliferation, migration and invasion of ovarian cancer cell decreased significantly, the expression of apoptosis related gene caspase 3 increased significantly, while the expression of PCNA and MMP9 was significantly down regulated. In addition, EMT biological characteristics of A2780 and SKOV3 cells were also inhibited. In summary, the increased expression of ENPP1 may be related to the occurrence of HGSOC, and indicate that the disease progresses rapidly and the prognosis is poor. ENPP1 may be considered as a potential molecular therapeutic target.

Development of a High-Throughput Assay to Identify Inhibitors of ENPP1.

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP2/GMP2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z' value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2'3'cGAMP compared with 2'3'c-diGMP and showed no activity with 3'3'c-diAMP. The Transcreener AMP2/GMP2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.

Ablation of Pyrophosphate Regulators Promotes Periodontal Regeneration

Biomineralization is regulated by inorganic pyrophosphate (PPi), a potent physiological inhibitor of hydroxyapatite crystal growth. Progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) act to increase local extracellular levels of PPi, inhibiting mineralization. The periodontal complex includes 2 mineralized tissues, cementum and alveolar bone (AB), both essential for tooth attachment. Previous studies demonstrated that loss of function of ANK or ENPP1 (reducing PPi) resulted in increased cementum formation, suggesting PPi metabolism may be a target for periodontal regenerative therapies. To compare the effects of genetic ablation of Ank, Enpp1, and both factors concurrently on cementum and AB regeneration, mandibular fenestration defects were created in Ank knockout (Ank KO), Enpp1 mutant (Enpp1asj/asj), and double KO (dKO) mice. Genetic ablation of Ank, Enpp1, or both factors increased cementum regeneration compared to controls at postoperative days (PODs) 15 and 30 (Ank KO: 8-fold, 3-fold; Enpp1asj/asj: 7-fold, 3-fold; dKO: 11-fold, 4-fold, respectively) associated with increased fluorochrome labeling and expression of mineralized tissue markers, dentin matrix protein 1 (Dmp1/DMP1), osteopontin (Spp1/OPN), and bone sialoprotein (Ibsp/BSP). Furthermore, dKO mice featured increased cementum thickness compared to single KOs at POD15 and Ank KO at POD30. No differences were noted in AB volume between genotypes, but osteoblast/osteocyte markers were increased in all KOs, partially mineralized osteoid volume was increased in dKO versus controls at POD15 (3-fold), and mineral density was decreased in Enpp1asj/asj and dKOs at POD30 (6% and 9%, respectively). Increased numbers of osteoclasts were present in regenerated AB of all KOs versus controls. These preclinical studies suggest PPi modulation as a potential and novel approach for cementum regeneration, particularly targeting ENPP1 and/or ANK. Differences in cementum and AB regeneration in response to reduced PPi conditions highlight the need to consider tissue-specific responses in strategies targeting regeneration of the entire periodontal complex.

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 β-catenin signaling compared to wild-type (WT) and Enpp1T238A cultures. Finally, the decreased calcification and nuclear β-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).