Autosomal Recessive Hypophosphatemic Rickets Type 2 Associated with a Novel ENPP1 Variant in a Taiwanese Girl.

Autosomal recessive hypophosphatemic rickets (HR) type 2 (ARHR2) is a rare form of HR caused by variant of the gene encoding ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Our patient presented with a history of unsteady gait and progressively bowing legs that had commenced at the age of 1 year. Laboratory tests revealed an elevated level of fibroblast growth factor 23 (FGF23), hypophosphatemia, and a high urine phosphate level. Radiography revealed the typical features of rickets. Next-generation sequencing identified a previously reported c.783C>G (p.Tyr261Ter) and a novel c.1092-42A>G variant in the ENPP1 gene. The patient was prescribed oral phosphates and active vitamin D and underwent guided growth of both distal femora and proximal tibiae commencing at the age of 3 years. No evidence of generalized arterial calcification was apparent during follow-up, and growth rate was satisfactory.

Synovial Membrane Is a Major Producer of Extracellular Inorganic Pyrophosphate in Response to Hypoxia.

Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In inflammatory conditions, the synovial membrane (SM) is subjected to transient hypoxia, especially during movement. CPPD formation is supported by an increase in extracellular inorganic pyrophosphate (ePPi) levels, which are mainly controlled by the transporter Ank and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We demonstrated previously that transforming growth factor (TGF)-ß1 increased ePPi production by inducing Ank and Enpp1 expression in chondrocytes. As the TGF-ß1 level raises in synovial fluid under hypoxic conditions, we investigated whether hypoxia may transform SM as a major source of ePPi production. Synovial fibroblasts and SM explants were exposed to 10 ng/mL of TGF-ß1 in normoxic or hypoxic (5% O2) culture conditions. Ank and Enpp1 expression were assessed by quantitative PCR, Western blot and immunohistochemistry. ePPi was quantified in culture supernatants. RNA silencing was used to define the respective roles of Ank and Enpp1 in TGF-ß1-induced ePPi generation. The molecular mechanisms involved in hypoxia were investigated using an Ank promoter reporter plasmid for transactivation studies, as well as gene overexpression and RNA silencing, the respective role of hypoxia-induced factor (HIF)-1 and HIF-2. Our results showed that TGF-ß1 increased Ank, Enpp1, and therefore ePPi production in synovial fibroblasts and SM explants. Ank was the major contributor in ePPi production compared to ENPP1. Hypoxia increased ePPi levels on its own and enhanced the stimulating effect of TGF-ß1. Hypoxic conditions enhanced Ank promoter transactivation in an HIF-1-dependent/HIF-2-independent fashion. We demonstrated that under hypoxia, SM is an important contributor to ePPi production in the joint through the induction of Enpp1 and Ank. These findings are of interest as a rationale for the beneficial effect of anti-inflammatory drugs on SM in crystal depositions.

Vascular calcification in chronic kidney disease associated with pathogenic variants in ABCC6.

Vascular calcification is prevalent in chronic kidney disease (CKD). Genetic causes of CKD account for 10-20% of adult-onset disease. Vascular calcification is thought to be one of the most important risk factors for increased cardiovascular morbidity and mortality in CKD patients and is detectable in 80% of patients with end stage kidney disease (ESKD). Despite the high prevalence of vascular calcification in CKD, no single gene cause has been described. We hypothesized that variants in vascular calcification genes may contribute to disease pathogenesis in CKD, particularly in families who exhibit a predominant vascular calcification phenotype. We developed a list of eight genes that are hypothesized to play a role in vascular calcification due to their involvement in the ectopic calcification pathway: ABCC6, ALPL, ANK1, ENPP1, NT5E, SLC29A1, SLC20A2, and S100A12. With this, we assessed exome data from 77 CKD patients, who remained unsolved following evaluation for all known monogenic causes of CKD. We also analyzed an independent cohort (Ontario Neurodegenerative Disease Research Initiative (ONDRI), n = 520) who were screened for variants in ABCC6 and compared this to a control cohort of healthy adults (n = 52). We identified two CKD families with heterozygous pathogenic variants (R1141X and A667fs) in ABCC6. We identified 10 participants from the ONDRI cohort with heterozygous pathogenic or likely pathogenic variant in ABCC6. Replication in a healthy control cohort did not reveal any variants. Our study provides preliminary data supporting the hypothesis that ABCC6 may play a role in vascular calcification in CKD. By screening CKD patients for genetic causes early in the diagnostic pathway, patients with genetic causes associated with vascular calcification can potentially be preventatively treated with new therapeutics with aims to decrease mortality.

[1,2,4]Triazolo[1,5-a]pyrimidine derivatives: Structure-activity relationship study leading to highly selective ENPP1 inhibitors.

The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as its dominant negative regulator. Since activation of the innate immune system is a promising strategy for the treatment of various infectious diseases and cancers, ENPP1 inhibitors have attracted great attention as candidate drugs. We have previously identified small-molecule ENPP1 inhibitors having a [1,2,4]triazolo[1,5-a]pyrimidine scaffold by means of chemical screening using a fluorescence probe, TG-mAMP. In this study, we evaluated the structure-activity relationships of the hit and lead compounds in detail, and succeeded in developing compounds that strongly and selectively inhibit ENPP1 not only in vitro, but also in cellular systems.

T Cell-Derived Apoptotic Extracellular Vesicles Hydrolyze cGAMP to Alleviate Radiation Enteritis via Surface Enzyme ENPP1.

Radiation enteritis is the most common complication of pelvic radiotherapy, but there is no effective prevention or treatment drug. Apoptotic T cells and their products play an important role in regulating inflammation and maintaining physiological immune homeostasis. Here it is shown that systemically infused T cell-derived apoptotic extracellular vesicles (ApoEVs) can target mice irradiated intestines and alleviate radiation enteritis. Mechanistically, radiation elevates the synthesis of intestinal 2'3' cyclic GMP-AMP (cGAMP) and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) proinflammatory pathway. After systemic infusion of ApoEVs, the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) enriches on the surface of ApoEVs hydrolyze extracellular cGAMP, resulting in inhibition of the cGAS-STING pathway activated by irradiation. Furthermore, after ApoEVs are phagocytosed by phagocytes, ENPP1 on ApoEVs hydrolyzed intracellular cGAMP, which serves as an intracellular cGAMP hydrolyzation mode, thereby alleviating radiation enteritis. The findings shed light on the intracellular and extracellular hydrolysis capacity of ApoEVs and their role in inflammation regulation.

Bioactivity of 2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-2,3-dihydrochromen-4-one from pistacia chinensis on ENPP1 and aldose reductase.

Pistacia chinensis is used as a decorative tree and currently studied as a source of biofuels. Besides, its parts and extracts are endowed with several therapeutic uses which have been widely explored in traditional medicine and that are related to its rich composition in phytochemicals. Molecular docking and enzymatic inhibition tests were used to study the activity of eriodictyol, a flavonoid extracted from the barks of P. chinensis, against ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and aldose reductase (ALR2). The compound was highlighted as a micromolar inhibitor in vitro (IC50 = 263.76 ± 1.32 µM and 4.21 ± 0.94 µM, respectively) and docking showed that eriodictyol efficiently targets the binding sites of the enzymes. In conclusion, this study unveils the potential of eriodictyol on enzymes that are involved in immunostimulation and in complications of diabetes mellitus.

Quantitative correlation of ENPP1 pathogenic variants with disease phenotype.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein which hydrolyzes extracellular phosphoanhydrides into bio-active molecules that regulate, inter alia, ectopic mineralization, bone formation, vascular endothelial proliferation, and the innate immune response. The clinical phenotypes produced by ENPP1 deficiency are disparate, ranging from life-threatening arterial calcifications to cutaneous hypopigmentation. To investigate associations between disease phenotype and enzyme activity we quantified the enzyme velocities of 29 unique ENPP1 pathogenic variants in 41 patients enrolled in an NIH study along with 33 other variants reported in literature. We correlated the relative enzyme velocities with the presenting clinical diagnoses, performing the catalytic velocity measurements simultaneously in triplicate using a high-throughput assay to reduce experimental variation. We found that ENPP1 variants associated with autosomal dominant phenotypes reduced enzyme velocities by 50 % or more, whereas variants associated with insulin resistance had non-significant effects on enzyme velocity. In Cole disease the catalytic velocities of ENPP1 variants associated with AD forms trended to lower values than those associated with autosomal recessive forms - 8-32 % vs. 33 % of WT, respectively. Additionally, ENPP1 variants leading to life-threatening vascular calcifications in GACI patients had widely variable enzyme activities, ranging from no significant differences compared to WT to the complete abolishment of enzyme velocity. Finally, disease severity in GACI did not correlate with the mean enzyme velocity of the variants present in affected compound heterozygotes but did correlate with the more severely damaging variant. In summary, correlation of ENPP1 enzyme velocity with disease phenotypes demonstrate that enzyme velocities below 50 % of WT levels are likely to occur in the context of autosomal dominant disease (due to a monoallelic variant), and that disease severity in GACI infants correlates with the more severely damaging ENPP1 variant in compound heterozygotes, not the mean velocity of the pathogenic variants present.

Identification of the extracellular membrane protein ENPP3 as a major cGAMP hydrolase and innate immune checkpoint.

2'3'-Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) is a second messenger synthesized upon detection of cytosolic double-stranded DNA (dsDNA) and passed between cells to facilitate downstream immune signaling. Ectonucleotide pyrophosphatase phosphodiesterase I (ENPP1), an extracellular enzyme, was the only metazoan hydrolase known to regulate cGAMP levels to dampen anti-cancer immunity. Here, we uncover ENPP3 as the second and likely the only other metazoan cGAMP hydrolase under homeostatic conditions. ENPP3 has a tissue expression pattern distinct from ENPP1's and accounts for all cGAMP hydrolysis activity in ENPP1-deficient mice. Importantly, we also show that, as with ENPP1, selectively abolishing ENPP3's cGAMP hydrolysis activity results in diminished cancer growth and metastasis of certain tumor types in a stimulator of interferon genes (STING)-dependent manner. Both ENPP1 and ENPP3 are extracellular enzymes, suggesting the dominant role that extracellular cGAMP must play as a mediator of cell-cell innate immune communication. Our work demonstrates that ENPP1 and ENPP3 non-redundantly dampen extracellular cGAMP-STING signaling, pointing to ENPP3 as a target for cancer immunotherapy.

TET3 boosts hepatocyte autophagy and impairs non-alcoholic fatty liver disease by increasing ENPP1 promoter hypomethylation.

BACKGROUND: Dysregulated ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family occurs in metabolic reprogramming pathological processes. Nonetheless, the epigenetic mechanisms by which ENPP family impacts NAFLD, also known as metabolic dysfunction-associated steatotic liver disease (MASLD), is poorly appreciated. METHODS: We investigated the causes and consequences of ENPP1 promoter hypomethylation may boost NAFLD using NAFLD clinical samples, as well as revealed the underlying mechanisms using high-fat diet (HFD) + carbon tetrachloride (CCl4) induced mouse model of NAFLD and FFA treatment of cultured hepatocyte. RESULTS: Herein, we report that the expression level of ENPP1 are increased in patients with NAFLD liver tissue and in mouse model of NAFLD. Hypomethylation of ENPP1, is associated with the perpetuation of hepatocyte autophagy and liver fibrosis in the NAFLD. ENPP1 hypomethylation is mediated by the DNA demethylase TET3 in NAFLD liver fibrosis and hepatocyte autophagy. Additionally, knockdown of TET3 methylated ENPP1 promoter, reduced the ENPP1 expression, ameliorated the experimental NAFLD. Mechanistically, TET3 epigenetically promoted ENPP1 expression via hypomethylation of the promoter. Knocking down TET3 can inhibit the hepatocyte autophagy but an overexpression of ENPP1 showing rescue effect. CONCLUSIONS: We describe a novel epigenetic mechanism wherein TET3 promoted ENPP1 expression through promoter hypomethylation is a critical mediator of NAFLD. Our findings provide new insight into the development of preventative measures for NAFLD.

Tumor Exosomal ENPP1 Hydrolyzes cGAMP to Inhibit cGAS-STING Signaling.

To evade immune surveillance, tumor cells express ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) on the surface of their membrane, which degrades extracellular cyclic GMP-AMP (cGAMP), thereby inhibiting the cyclic GMP-AMP synthase (cGAS) stimulator of interferon gene (STING) DNA-sensing pathway. To fully understand this tumor stealth mechanism, it is essential to determine whether other forms of ENPP1 with hydrolytic cGAMP activity also are present in the tumor microenvironment to regulate this innate immune pathway. Herein, it is reported that various tumor-derived exosomes carry ENPP1, and can hydrolyze synthetic 2'3'-cGAMP and endogenous 2'3'-cGAMP produced by cells to inhibit cGAS-STING pathway in immune cells. Moreover, tumor exosomal ENPP1 also can hydrolyze 2'3'-cGAMP bound to LL-37 (an effective transporter of 2'3'-cGAMP) to inhibit STING signaling. Furthermore, high expression of ENPP1 in exosomes is observed isolated from human breast and lung cancer tissue, and tumor exosomal ENPP1 inhibited the immune infiltration of CD8+ T cells and CD4+ T cells. The results elucidate the essential function of tumor exosomal ENPP1 in the cGAS-STING pathway, furthering understanding of the crosstalk between the tumor cells and immune system.

Insight into small-molecule inhibitors targeting extracellular nucleotide pyrophosphatase/phosphodiesterase1 for potential multiple human diseases.

Extracellular nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as a type II transmembrane glycoprotein. It plays a crucial role in various biological processes, such as bone mineralization, cancer cell proliferation, and immune regulation. Consequently, ENPP1 has garnered attention as a promising target for pharmacological interventions. Despite its potential, the development of clinical-stage ENPP1 inhibitors for solid tumors, diabetes, and silent rickets remains limited. However, there are encouraging findings from preclinical trials involving small molecules exhibiting favorable therapeutic effects and safety profiles. This perspective aims to shed light on the structural properties, biological functions and the relationship between ENPP1 and diseases. Additionally, it focuses on the structure-activity relationship of ENPP1 inhibitors, with the intention of guiding the future development of new and effective ENPP1 inhibitors.

Natural history of ENPP1 deficiency: Nationwide Turkish Cohort Study of autosomal-recessive hypophosphataemic rickets type 2.

OBJECTIVE: Autosomal-recessive hypophosphataemic rickets type 2 (ARHR2) is a rare disease that is reported in survivors of generalized arterial calcification of infancy (GACI). DESIGN, PATIENTS AND MEASUREMENT: The objective of this study was to characterize a multicenter paediatric cohort with ARHR2 due to ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) deficiency and with a diagnosis of GACI or GACI-related findings. The clinical, biochemical and genetic characteristics of the patients were retrospectively retrieved. RESULTS: We identified 18 patients from 13 families diagnosed with ARHR2. Fifteen of the patients had an ENPP1 variation confirmed with genetic analyses, and three were siblings of one of these patients, who had clinically diagnosed hypophosphataemic rickets (HRs) with the same presentation. From nine centres, 18 patients, of whom 12 (66.7%) were females, were included in the study. The mean age at diagnosis was 4.2 ± 2.2 (1.6-9) years. The most frequently reported clinical findings on admission were limb deformities (66.6%) and short stature (44.4%). At diagnosis, the mean height SD was -2.2 ± 1.3. Five of the patients were diagnosed with GACI in the neonatal period and treated with bisphosphonates. Other patients were initially diagnosed with ARHR2, but after the detection of a biallelic variant in the ENPP1 gene, it was understood that they previously had clinical findings associated with GACI. Three patients had hearing loss, and two had cervical fusion. After the treatment of HRs, one patient developed calcification, and one developed intimal proliferation. CONCLUSION: ARHR2 represents one manifestation of ENPP1 deficiency that usually manifests later in life than GACI. The history of calcifications or comorbidities that might be associated with GACI will facilitate the diagnosis in patients with ARHR2, and patients receiving calcitriol and phosphate medication should be carefully monitored for signs of calcification or intimal proliferation.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 inhibitors: Research progress and prospects.

The cancer immunotherapies involved in cGAS-STING pathway have been made great progress in recent years. STING agonists exhibit broad-spectrum anti-tumor effects with strong immune response. As a negative regulator of the cGAS-STING pathway, ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) can hydrolyze extracellular 2', 3'-cGAMP and reduce extracellular 2', 3'-cGAMP concentration. ENPP1 has been validated to play important roles in diabetes, cancers, and cardiovascular disease and now become a promising target for tumor immunotherapy. Several ENPP1 inhibitors under development have shown good anti-tumor effects alone or in combination with other agents in clinical and preclinical researches. In this review, the biological profiles of ENPP1 were described, and the structures and the structure-activity relationships (SAR) of the known ENPP1 inhibitors were summarized. This review also provided the prospects and challenges in the development of ENPP1 inhibitors.

Second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP): the cell autonomous and non-autonomous roles in cancer progression.

Cytosolic double-stranded DNA (dsDNA) is frequently accumulated in cancer cells due to chromosomal instability or exogenous stimulation. Cyclic GMP-AMP synthase (cGAS) acts as a cytosolic DNA sensor, which is activated upon binding to dsDNA to synthesize the crucial second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP) that in turn triggers stimulator of interferon genes (STING) signaling. The canonical role of cGAS-cGAMP-STING pathway is essential for innate immunity and viral defense. Recent emerging evidence indicates that 2'3'-cGAMP plays an important role in cancer progression via cell autonomous and non-autonomous mechanisms. Beyond its role as an intracellular messenger to activate STING signaling in tumor cells, 2'3'-cGAMP also serves as an immunotransmitter produced by cancer cells to modulate the functions of non-tumor cells especially immune cells in the tumor microenvironment by activating STING signaling. In this review, we summarize the synthesis, transmission, and degradation of 2'3'-cGAMP as well as the dual functions of 2'3'-cGAMP in a STING-dependent manner. Additionally, we discuss the potential therapeutic strategies that harness the cGAMP-mediated antitumor response for cancer therapy.

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.

Targeting ENPP1 for cancer immunotherapy: Killing two birds with one stone.

Cancer immunotherapy, particularly with immune checkpoint inhibitors, has revolutionized the paradigm of cancer treatment. Nevertheless, the efficacy of cancer immunotherapy remains limited in most clinical settings due to the lack of a preexisting antitumor T-cell response in tumors. Therefore, the clinical outcomes of cancer immunotherapy must be improved crucially. With increased awareness of the importance of the innate immune response in the recruitment of T cells, as well as the onset and maintenance of the T cell response, great interest has been shown in activating the cGAS-STING signaling pathway to awaken the innate immune response, thereby orchestrating both innate and adaptive immune responses to induce tumor clearance. However, tumor cells have evolved to overexpress ectonucleotide pyrophosphate phosphodiesterase 1 (ENPP1), which degrades the immunotransmitter 2',3'-cGAMP and promotes the production of immune-suppressing adenosine, resulting in inhibition of the anticancer immune response in the tumor microenvironment. Clinically, ENPP1 overexpression is closely associated with poor prognosis in patients with cancer. Conversely, depleting or inhibiting ENPP1 has been verified to elevate extracellular 2',3'-cGAMP levels and inhibit the generation of adenosine, thereby reinvigorating the anticancer immune response for tumor elimination. A variety of ENPP1 inhibitors have recently been developed and have demonstrated significant promise for cancer immunotherapy. In this review, we provide an overview of ENPP1, dissect its immunosuppressive mechanisms, and discuss the development of ENPP1 inhibitors with the potential to further improve the efficacy of cancer immunotherapy.

ENPP1 ameliorates vascular calcification via inhibiting the osteogenic transformation of VSMCs and generating PPi.

This study aims to investigate the impact of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) on vascular calcification in rats. The rationale behind studying ENPP1's role in vascular calcification lies in its potential to modulate calcification processes. Understanding this relationship can offer insights into novel therapeutic avenues for addressing vascular calcification-related disorders. In this experiment, vascular smooth muscle cell (VSMC) calcification was induced using ß-glycerophosphoric acid. Subsequently, recombinant AAV9-carrying ENPP1 was introduced into VSMCs to achieve both in vitro and in vivo overexpression of ENPP1. The findings indicate that ENPP1 overexpression significantly reduces calcium and phosphorus content in the aorta (P < 0.05). Alizarin red and von Kossa staining reveal notable reductions in calcium salt deposits in VSMCs and aorta, respectively. Notably, the expression levels of BMP-2, PINP, OC, and BALP were substantially decreased in VSMCs (P < 0.05), underscoring ENPP1's role in impeding osteoblast-like transdifferentiation of VSMCs. Additionally, ENPP1 overexpression led to a significant increase in pyrophosphate (PPi) levels compared to control rats (P < 0.05). In conclusion, this study suggests that ENPP1 contributes to alleviating vascular calcification by elevating PPi levels and inhibiting the phenotypic transformation of VSMCs. These findings shed light on the potential therapeutic role of ENPP1 in mitigating vascular calcification-related complications.

ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway in breast cancer.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.

Genetics of Diffuse Idiopathic Skeletal Hyperostosis and Ossification of the Spinal Ligaments.

PURPOSE OF REVIEW: The study aims to provide updated information on the genetic factors associated with the diagnoses 'Diffuse Idiopathic Skeletal Hyperostosis' (DISH), 'Ossification of the Posterior Longitudinal Ligament' (OPLL), and in patients with spinal ligament ossification. RECENT FINDINGS: Recent studies have advanced our knowledge of genetic factors associated with DISH, OPLL, and other spinal ossification (ossification of the anterior longitudinal ligament [OALL] and the yellow ligament [OYL]). Several case studies of individuals afflicted with monogenic disorders, such as X-linked hypophosphatemia (XLH), demonstrate the strong association of fibroblast growth factor 23-related hypophosphatemia with OPLL, suggesting that pathogenic variants in PHEX, ENPP1, and DMP1 are associated with FGF23-phosphate wasting phenotype and strong genetic factors placing patients at risk for OPLL. Moreover, emerging evidence demonstrates that heterozygous and compound heterozygous ENPP1 pathogenic variants inducing 'Autosomal Recessive Hypophosphatemic Rickets Type 2' (ARHR2) also place patients at risk for DISH and OPLL, possibly due to the loss of inhibitory plasma pyrophosphate (PPi) which suppresses ectopic calcification and enthesis mineralization. Our findings emphasize the importance of genetic and plasma biomarker screening in the clinical evaluation of DISH and OPLL patients, with plasma PPi constituting an important new biomarker for the identification of DISH and OPLL patients whose disease course may be responsive to ENPP1 enzyme therapy, now in clinical trials for rare calcification disorders.