Insights into Pathomechanisms and Treatment Development in Heritable Ectopic Mineralization Disorders: Summary of the PXE International Biennial Research Symposium—2016

MEETING REPORT

Insights into Pathomechanisms and Treatment Development in Heritable Ectopic Mineralization Disorders: Summary of the PXE International Biennial Research Symposium—2016 Jouni Uitto1, Qiaoli Li1, Koen van de Wetering1, Andra´s Va´radi2 and Sharon F. Terry3 Pseudoxanthoma elasticum is a prototype of heritable ectopic mineralization disorders, with phenotypic overlap with generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Recent observations have suggested that the reduced inorganic pyrophosphate/phosphate ratio is the cause of soft connective tissue mineralization in these disorders. PXE International, a patient advocacy organization, supports research in part by sponsoring biennial research symposia on these disorders; the latest meeting was held in September 2016 at Thomas Jefferson University, Philadelphia. This report summarizes the progress in pseudoxanthoma elasticum and other ectopic mineralization disorders, as presented in the symposium, with focus on translational aspects of precision medicine toward improved diagnostics and treatment development for these currently intractable disorders. Journal of Investigative Dermatology (2017) 137, 790e795; doi:10.1016/j.jid.2016.12.014

INTRODUCTION

Heritable ectopic mineralization disorders comprise a group of diseases with a wide spectrum of clinical manifestations due to deposition of calcium hydroxyapatite minerals on soft connective tissues. The prototype of these conditions is pseudoxanthoma elasticum (PXE), a multisystem disorder with primary clinical findings in the skin, the eyes, and the cardiovascular system (Li et al., 2016a; Neldner, 1988) (Figure 1). Although PXE is usually a late-onset, slowly progressing disease, the clinical manifestations particularly in the eyes and the arterial blood vessels cause considerable morbidity and occasional early mortality. In the spectrum of heritable ectopic mineralization disorders are other conditions, including generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC), with overlapping phenotypic features with PXE (Li and Uitto, 2013; Nitschke and Rutsch, 2012). However,

GACI is characterized by severe, early onset mineralization of the cardiovascular system, and the diagnosis is often made by prenatal ultrasound. Without treatment, as many as 60% of the children die from cardiovascular complications within the first 6 months of life (Rutsch et al., 2008). In contrast, ACDC is a lateonset mineralization disorder of elderly individuals, calcium deposition noted primarily in the arteries of the lower extremities and in periarticular ligaments (St Hilaire et al., 2011). The classic form of PXE is caused by mutations in the ABCC6 gene, while GACI harbors mutations in ENPP1 in most cases, and ACDC is caused by mutations in NT5E (Figure 2). Although there is an overlap in clinical phenotypes, some patients with GACI have also been shown to harbor mutations in ABCC6, and some patients with clinical manifestations consistent with PXE have mutations in ENPP1 (Li et al., 2014a; Nitschke et al., 2012). These observations have suggested the

possibility of shared pathomechanistic pathways for these conditions. PXE International, a lay organization established in 1995, advocates for patients and families with PXE and supports research on ectopic mineralization disorders. PXE International organizes a biennial research symposium in the United States. The latest PXE International Research Symposium, “Progress in Diagnostics, Research, and Treatment,” was held on 15e17 September 2016 in the Department of Dermatology and Cutaneous Biology at Thomas Jefferson University, Philadelphia, PA. The symposium also served as the venue of inauguration of the PXE International Center of Excellence in Research and Clinical Care at Jefferson. This center will serve as an umbrella bridging the laboratory studies to clinical trials and patient care. The symposium was attended by the international cadre of premiere scientists on ectopic mineralization disorders. The

1 Department of Dermatology and Cutaneous Biology, Sidney Kimmel Medical College, and PXE International Center of Excellence in Research and Clinical Care, Thomas Jefferson University, Philadelphia, Pennsylvania, USA; 2Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Budapest, Hungary; and 3PXE International, Washington, District of Columbia, USA

Correspondence: Jouni Uitto, Department of Dermatology and Cutaneous Biology, Sidney Kimmel Medical College at Thomas Jefferson University, 233 S. 10th Street, Suite 450 BLSB, Philadelphia, Pennsylvania 19107, USA. E-mail: [email protected] Abbreviations: ACDC, arterial calcification due to CD73 deficiency; AMP, adenosine monophosphate; ATP, adenosine triphosphate; GACI, generalized arterial calcification of infancy; PPi, inorganic pyrophosphate; PTC, premature termination codon; PXE, pseudoxanthoma elasticum; TNAP, tissue nonspecific alkaline phosphatase Received 12 October 2016; revised 7 December 2016; accepted 11 December 2016

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ª 2017 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.

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Figure 1. Clinical features and histopathology of ectopic mineralization in pseudoxanthoma elasticum. (a, b) Cutaneous findings consist of discrete yellowish papules that coalesce into inelastic, leathery plaques on the predilection sites, such as the side of the neck. (c, d) Histopathology of the affected skin reveals accumulation of pleomorphic elastotic material that becomes progressively calcified (left panel, Verhoeff-van Giesson elastic stain; right panel, von Kossa calcium stain). (e) Eye involvement is signified by angioid streaks reflecting breakage in the calcified Bruch’s membrane (arrows). These breaks allow neovascularization from the underlying capillaries to the retina (arrowheads) leading to loss of visual acuity and blindness unless treated. (f) Extensive mineralization of arterial vasculature, as illustrated by left renal artery, can result in nephrogenic hypertension, intermittent claudication and occasionally early myocardial infarcts and strokes (adapted from Li et al., 2014a).

meeting presentations developed a number of innovative concepts and provided significant insights into pathomechanism of these disorders, with translational implications. ANIMAL MODELS OF ECTOPIC MINERALIZATION DISORDERS

After the discovery of ABCC6 mutations being responsible for PXE, two mouse

models were created by targeted ablation of Abcc6 (Gorgels et al., 2005; Klement et al., 2005). These models have served as a platform to explore pathomechanisms of PXE and to test various pharmacological interventions. Subsequently, a hypomorphic Abcc6 mutant allele was found in four different mouse strains with variations in disease severity, indicating that PXE

is not just a simple Mendelian trait, but the phenotype can be modulated by the contribution of genetic modifiers, including epigenetics (Berndt et al., 2013; Li et al., 2012). In fact, quantitative trait locus analyses have tentatively identified a number of genetic loci involved directly or indirectly in the pathogenesis of PXE (Li et al., unpublished). These studies have been complemented by identification of rare modifier variants that alter the severity of cardiovascular disease in patients with PXE, as revealed by whole exome sequencing in patients with mild versus severe cardiovascular manifestations (De Vilder et al., unpublished). Furthermore, systematic evaluation of a large library of ABCC6 mutations has identified mutations leading to altered protein folding and altered protein function. Preliminary data have also demonstrated that stabilization of mutant ABCC6, via specific domaindomain interactions, can facilitate proper trafficking and function of the mutant proteins, providing a mechanism for stabilization of the protein and rescue of the phenotype due to diseasecausing mutations (Ran and Thibodeau, 2017). In addition to PXE, several spontaneous and engineered mouse models have been shown to recapitulate features of GACI due to genetic alterations in the Enpp1 gene. Some of these mouse strains, such as Enpp1tm1Gdg, Enpp1asj-2J, and ttw (tiptoe walking) mice, have complete inactivation of ENPP1 resulting in severe phenotypes, whereas a hypomorphic variant, Enpp1asj, has approximately 15% of residual ENPP1 activity resulting in a milder disease (Li et al., 2014b). Finally, an Nt5e null mouse (Nt5etm1jgsc) has recently been characterized with respect to ectopic mineralization, demonstrating extensive calcium deposition in the juxta-articular ligaments, mimicking features of ACDC (Li et al., 2014c). In addition to mouse models, an Abcc6 null rat model devoid of ABCC6 was recently developed, and complete necropsy revealed ectopic mineralization in the skin, eyes, and arteries in a similar manner as in Abcc6 / mice and in patients with PXE (Li et al., 2017). The advantages of the rat over mouse models of PXE include its bigger size that allows www.jidonline.org

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Figure 2. Schematic illustration of the generation of PPi and Pi, and heritable diseases featuring perturbations in the promineralization/antimineralization networks. Loss-of-function mutations in the ABCC6, ENPP1, NT5E, and ALPL genes cause autosomal recessive pseudoxanthoma elasticum (PXE), generalized arterial calcification of infancy (GACI), arterial calcification due to CD73 deficiency (ACDC), and infantile hypophosphatasia (HOPS), respectively. ABCC6, a putative transmembrane transporter, mediates ATP release from hepatocytes to extracellular space where ATP is converted to PPi and AMP by ENPP1, a membrane-bound pyrophosphatase/phosphodiesterase; CD73 converts AMP to Pi and adenosine, the latter one being an inhibitor of tissue nonspecific alkaline phosphatase (TNAP), an extracellular, yet membrane-associated protein, that hydrolyzes PPi to Pi. Deficiencies in ABCC6, ENPP1, and CD73 proteins lead to reduced plasma PPi levels and PPi/Pi ratio, thereby promoting hydroxyapatite mineralization in peripheral tissues. TNAP deficiency is associated with severe hypophosphatasia and defective bone mineralization due to significantly increased plasma pyrophosphate levels. ANKH is a transmembrane protein that transports PPi across the plasma membrane and stimulates the elaboration of extracellular PPi from intracellular stores. Loss of ANKH function in patients and in progressive ankylosis mice leads to decreased extracellular PPi and increased hydroxyapatite deposition in articular cartilage (ank phenotype). Gain-of-function mutations in ANKH gene can cause calcium pyrophosphate deposition disease (CPPD) due to excess extracellular PPi. The PPi/Pi balance is, therefore, critical for prevention of ectopic mineralization and maintenance of normal skeletal mineralization. Please note that these reactions do not necessarily take place in the same cell. ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; EC, extracellular; IC, intracellular; PPi, inorganic pyrophosphate.

physiological experiments, such as liver and kidney perfusions (see below). PATHOMECHANISMS OF PXE—THE CRITICAL ROLE OF INORGANIC PYROPHOSPHATE

The classic form of PXE is caused by mutations in ABCC6, a putative efflux transporter of the ABC family of proteins, primarily localized in the basolateral plasma membrane of hepatocytes in the liver and in proximal tubules of the kidneys (Scheffer et al., 2002). Early on, a hypothesis was advanced that PXE is a metabolic disease caused by lack of circulating antimineralization factor(s), but the precise nature of the molecule transported from the intracellular milieu 792

of the cells to extracellular space by ABCC6 remained unresolved (Uitto et al., 2001). It was recently shown, however, that cells expressing ABCC6 excrete large amounts of nucleoside triphosphates, particularly adenosine triphosphate (ATP) (Jansen et al., 2013). In the extracellular space, ectonucleotidases hydrolyze the secreted ATP into adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi), a potent inhibitor of mineralization (Figure 2). This observation suggested that the release of ATP in an ABCC6-dependent manner is the critical pathogenic feature of PXE, and PPi may indeed be the circulating factor whose deficiency allows ectopic mineralization of the

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peripheral tissues, such as skin, retina of the eyes and the arterial blood vessels, to take place. In support of this hypothesis is the observation that plasma PPi levels in Abcc6 knockout mice and rats are only approximately 30% of that in control animals, and patients with established ABCC6 mutations similarly have low plasma PPi concentrations (Jansen et al., 2014; Li et al., 2017). In situ liver and kidney perfusions performed in the rat model of PXE, coupled with quantitation of the PPi/Pi ratio in the perfusate, demonstrated that the liver plays a central role in contributing to the plasma PPi levels under physiologic conditions (Li et al., 2017). At the same time, PPi levels in Enpp1 null mice are very low, often undetectable, further supporting the hypothesis that reduced PPi levels, and in particular a low PPi/Pi ratio, are the critical determinant of ectopic mineralization in these disorders (Li et al., 2014b; Lomashvili et al., 2014). Parallel studies on fibroblasts from patients with PXE and conditionally targeted mouse models of PXE have suggested complementary pathomechanisms potentially contributing to the low PPi levels (Ziegler et al., unpublished). First, under osteogenic culture conditions, cells from patients with PXE have been shown to calcify, suggesting a provoked cell-autonomous defect, with the notion that deficiency of ABCC6 in both local and distant cells may be necessary to drive the pathologic calcification (Boraldi et al., 2014; Dabisch-Ruthe et al., 2014; Ziegler et al., 2014, 2015). It has also been suggested that cells from patients with PXE show increased expression and activity of tissue nonspecific alkaline phosphatase (TNAP), an enzyme that degrades PPi to Pi, thus altering the PPi/Pi ratio (Boraldi et al., 2014; Dabisch-Ruthe et al., 2014). Collectively, these studies suggest that perturbations in the pathways resulting in the synthesis of PPi by hydrolysis of ATP, and its further processing by TNAP to Pi can result in the altered PPi/Pi ratio (Figure 2). At the same time, CD73 hydrolyzes AMP to Pi and adenosine, the latter one being an inhibitor of TNAP (Markello et al., 2011). Thus, in the absence of CD73 in ACDC, TNAP is activated resulting in increased levels of Pi and creating a promineralization

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environment. Collectively, there is a critical pathway controlling the PPi/Pi ratio, and its aberrations explain the development of ectopic mineralization in PXE, GACI, and ACDC (Figure 2). TREATMENT PROSPECTS PPi administration

With the assumption that reduced PPi levels are the critical feature of PXE and other ectopic mineralization disorders, one straightforward approach would be to replenish plasma PPi levels (O’Neill et al., 2011). This concept has been tested by subcutaneous administration of PPi to Abcc6 / mice that develop ectopic mineralization in the heart as a result of freeze injury, known as dystrophic cardiac calcification (Brampton et al., 2014). Even though PPi has been thought to be unstable and have a short half-life in circulation, there is a possibility that administration of PPi can be used for treatment of patients with PXE. Bisphosphonates

Although PPi was considered to be unstable and quickly metabolized, stable PPi analogs, with considerably longer half-lives in blood, have been tested in mouse models of ectopic mineralization. A number of studies utilizing either Abcc6 or Enpp1 mutant mice have demonstrated that bisphosphonates, particularly etidronate, are absorbed from the intestine and prevent ectopic mineralization in these mouse models (Li et al., 2015, 2016c, 2016b). Bisphosphonates have two independent modes of action on mineralization: (i) inhibition of the calcium hydroxyapatite deposition by binding to the surface of the crystals preventing their growth, and (ii) inhibition of osteoclastic activity, the basis for their use in treatment of osteoporosis (Drake et al., 2008). Etidronate, one of the first generation bisphosphonates, has a profile favoring antimineralization over anti-osteoclastic activity, and indeed, it was shown to prevent ectopic mineralization in soft connective tissues (Li et al., 2017). At the same time, etidronate administration increased the bone mineral density and corrected the mineralization defect noted in Enpp1asj mice. Thus, bisphosphonates, perhaps with further development of compounds with more

powerful antimineralization properties, would provide dual benefits to the patients with PXE. In this context, it should be noted that etidronate has been used for prenatal therapy by administration to pregnant mothers with a history of GACI or in whom a diagnosis of fetal GACI was suspected by ultrasound during the second trimester. It has been suggested that etidronate therapy lowers the perinatal mortality, suggesting that administration of etidronate during pregnancy may be a promising prenatal rescue therapy for GACI (Rutsch et al., 2008). ENPP1 protein replacement

GACI is caused by reduced ENPP1 activity, and consequently, this enzyme replacement would be expected to counteract the clinical phenotype. Indeed, a recent study demonstrated that recombinant ENPP1, when administered into Enpp1asj mice as a model of GACI, resulted in elevated plasma PPi levels, reduced the extent of ectopic mineralization, and prevented mortality in this mouse model (Albright et al., 2015). It has also been suggested that similar ENPP1 replacement therapy might be helpful for PXE. This suggestion is based, at least in part, on the observation that in ABCC6-deficient mice and in patients with PXE, even in the complete absence of ABCC6 transporter activity, there is approximately 30e40% residual plasma level of PPi, suggesting alternate sources of ATP (Jansen et al., 2014; Li et al., 2017). In this context, another potential treatment strategy would be to increase the release of ATP from tissues and cells other than hepatocytes in an ABCC6 independent manner (Lohman et al., 2012). Such an increase in ATP pool, particularly when combined with recombinant ENPP1, would be expected to result in increased plasma PPi levels. It should be noted that abnormally high levels of PPi can result in deposition in calcium pyrophosphate crystals, as has been shown in patients with gain-of-function mutations in ANKH resulting in increased pyrophosphate transport from cartilage and causing pyrophosphate arthropathy (Rosenthal and Ryan, 2016). TNAP inhibition

Assuming the critical role of the PPi/Pi ratio in determining the extent of

hydroxyapatite deposition and the severity of the associated clinical phenotype, all factors contributing to the PPi levels should be considered as potential targets for pharmacologic intervention. One of such molecules is TNAP which hydrolyzes PPi to Pi (Figure 2). In fact, genetic deficiency of TNAP results in hypophosphatasia that in TNAP null mice results in embryonic lethality due to lack of bone formation (Millan and Whyte, 2016). However, controlled inhibition of TNAP by orally available TNAP inhibitors has been suggested to prevent calcification in ABCC6 mutant cells in vitro, and they attenuated the phenotype in Abcc6 / mice in vivo; however, their effects on bone mineralization have not been examined (Ziegler et al., unpublished). On the basis of these preclinical observations, TNAP inhibitors could serve as a potential avenue to prevent ectopic mineralization in a controlled manner. Premature termination codon read through

Approximately 300 distinct mutations in ABCC6 have been identified so far in patients with PXE. Identification of specific mutations, with elucidation of the consequences at the mRNA and protein levels, has paved the way for the application of precision medicine for this disorder. For example, the notion that approximately 35% of all mutant alleles in ABCC6 consist of premature termination codon (PTC) mutations has formed the basis to use PTC readthrough molecules for therapy in PXE. Initial studies utilizing PTC124 as the prototypic molecule demonstrated in cell culture systems synthesis of full length protein, although at low levels (Zhou et al., 2013). More recent studies have utilized amlexanox, a molecule that in addition to PTC read-through capability counteracts nonsensemediated mRNA decay, suggesting its potential usefulness for a subset of patients with PXE caused by PTC mutations (Uitto et al., in press). Chaperone therapy

Another example of the mutation-based application of precision medicine for PXE revolves around chaperone-based correction of mistargeting of mutant ABCC6 protein. It has been demonstrated that a number of ABCC6 missense www.jidonline.org

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mutations cause conformational alterations that result in altered intracellular trafficking and subcellular mislocalization of the mutant protein (Le Saux et al., 2011; Pomozi et al., 2014). A chemical chaperone, 4-phenylbutyrate, has been shown in in vitro culture systems and in wild-type mice to be capable of correcting the trafficking defect, allowing the mutant ABCC6 protein to be targeted to the proper localization at the basolateral surface of hepatocytes (Jin et al., 2015; Le Saux et al., 2011; Pomozi et al., 2014). These observations have been extended to in vivo with demonstrations that 4phenylbutyrate attenuated the dystrophic cardiac mineralization phenotype in an Abcc6 knockout mouse (Pomozi et al., 2016). Thus, these examples of precision medicine, based on the knowledge of specific mutations in ABCC6, serve as examples of potential development of innovative treatment approaches to counteract ectopic mineralization disorders.

Challenges of clinical trials on PXE

CLINICAL TRIALS Magnesium trial

Significant progress, as summarized by this PXE International Biennial Research Symposium, has been made in understanding the pathomechanisms of ectopic mineralization disorders, with potential for significant translation in the near future. In fact, in addition to the existing clinical trials, further innovative approaches, including PPi and enzyme replacement therapies, are being contemplated. Finally, understanding of the pathomechanisms of ectopic mineralization disorders in these rare heritable diseases will provide insights that may also be applicable to common conditions with mineral deposition in soft connective tissues, such as vascular calcification in the elderly populations.

A clinical trial has tested the potential of elevated dietary magnesium to counteract ectopic mineralization in PXE (NCT01525875). This trial was based on previous demonstrations that an increase in dietary magnesium by 5-fold over that in control diet completely abolished mineralization in Abcc6 / mice (LaRusso et al., 2009). Conversely, lowering the magnesium content to approximately 20% of the control level accelerated the mineralization process (Jiang and Uitto, 2012). A 2-year double-blinded, placebocontrolled trial on 40 patients with PXE has been recently completed, and the results are currently being analyzed.

The two ongoing studies cited above highlight some of the difficulties encountered in designing clinical trials for PXE. First, the disease is progressing slowly and the interval progression is somewhat unpredictable, making quantification of the clinical findings difficult. Although the imaging techniques used in both studies provide more precise quantifiable data, the fact that these treatments are unlikely to reverse the existing mineral deposits, as suggested by the previous mouse studies, necessitates a long treatment period for any significant changes to be observed. Nevertheless, improved imaging technologies to quantitate the calcification and utilization of PPi as a potential biomarker of short-term efficacy of treatment modalities may provide tools to quantitate such changes in future clinical trials with increased precision. CONCLUSIONS

CONFLICT OF INTEREST The authors state no conflicts of interest.

Etidronate trial

Another clinical trial currently in progress is designed to study the effect of etidronate on vascular calcification in patients with PXE. This randomized placebo-controlled 1-year trial has enrolled more than 70 patients in the Netherlands, and the study is in progress (NTR5180). This study will attempt to assess the degree of mineralization specifically in the leg arteries by 18F-NaF positron emission tomography-computed tomography imaging in patients with PXE. 794

ACKNOWLEDGMENTS This research symposium was partially supported by the NIH/NIAMS grant R13AR070643 as well as by PXE International and The Jefferson Institute of Molecular Medicine. Carol Kelly assisted in manuscript preparation.

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