John W. M. Creemers

Purpose of Review An aneurysm is a frequently occurring and potentially life-threatening cardiovascular disease. However, limited clinical therapeutic drugs are available. Here, we aimed to provide a framework for aneurysm treatment by targeting Furin. Recent Findings Our recent study reported, for the first time, that several Furin variants are found in patients with aneurysm, indicating the important role of Furin in aneurysm. Given that Furin is an important endoproteolytic enzyme that modulates the biochemical activity of proproteins involved in aneurysm formation and progression, interfering with Furin could be a promising strategy for aneurysm treatment. Summary Since several Furin substrates like TGF-β1, NOTCH, IGF1R/IR and MMPs/ADAMs are closely associated with the hallmarks of aneurysm, Furin is a potential target for aneurysm treatment. However, significant gaps remain in our understanding of Furin in the different biological the hallmarks of aneurysms, due to the different role of Furin substrates in regulating the hallmarks of aneurysm. Further research is needed to clarify how the summarized interfering strategies targeting Furin impact on the aneurysm formation and progression in vivo.

KRAS and BRAF mutations drive colorectal cancer (CRC) progression by sustaining aberrant signaling and promoting therapeutic resistance. Here, we identify TGF-β1-COX-2 axis as a critical regulatory pathway mediated by Furin in CRC harboring KRAS or BRAF mutation. Genetic silencing or pharmacological inhibition of Furin in KRAS-mutant (KPN) and BRAF-mutant (BPN) tumor-derived cells suppressed tumor growth, reduced angiogenesis, and enhanced CD8⁺ T cell infiltration in mouse tumor models. KRAS- and BRAF-mutant organoids with impaired Furin activity exhibited increased sensitivity to 5-FU and irinotecan, whereas only KPN organoids were sensitive to oxaliplatin. Mechanistically, Furin inhibition via shRNA or the Furin inhibitor MI1148 blocked IGF-1 receptor and TGF-β1 precursor maturation and signaling, which was associated with repressed COX-2 expression. Conversely, COX-2 overexpression elevated TGF-β1 levels, which in turn enhanced Furin expression, establishing a feed-forward loop that promoted tumor progression and angiogenesis. Moreover, Furin inhibition largely disrupted the activity of multiple kinases linked to KRAS and BRAF oncogenic signaling. In CRC patient samples, Furin expression positively correlated with KRAS, BRAF, TGF-β1, and COX-2. Collectively, these findings identify Furin as a pivotal regulator of oncogenic signaling in KRAS- and BRAF-mutant CRC, and highlight the therapeutic potential of targeting the Furin-TGF-β1-COX-2 axis.

Fibroblast growth factor 23 (FGF23) is a phosphate-regulating hormone produced by osteocytes. In iron deficiency anemia (IDA) and in chronic kidney disease (CKD), FGF23 is also produced by erythroid cells. Recent studies have suggested that rising circulating FGF23 is negatively associated with erythropoiesis in IDA and CKD. However, the distinct contributions of bone- and erythroid-produced FGF23 to anemia in IDA remain unclear. Using the conditional deletion of Fgf23 in osteocytes (Fgf23Dmp1-cKO) and in erythroid cells (Fgf23HbB-cKO) in mice fed a control (Ctr) or an iron deficient (ID) diet, we first determined that in ID, osteocytes and erythroid cells are distinct sources of circulating intact FGF23 (iFGF23) and FGF23 cleaved peptides, respectively. We further show that erythroid-specific deletion of Fgf23 corrected anemia in ID mice, and overexpression induced anemia in Ctr mice unlike osteocyte-specific deletion or overexpression of Fgf23. Importantly, erythroid-specific deletion of Furin (FurinHbB-cKO), the enzyme responsible for FGF23 cleavage, led to increased production of iFGF23 from erythroid cells and aggravated ID-induced anemia. iFGF23 also dose-dependently blocked the differentiation of erythroid progenitors in culture triggering mitochondrial dysfunction leading to impaired erythropoiesis. These effects were fully suppressed by co-treatment with an FGFR1 inhibitor. Finally, erythroid-specific deletion of Fgf23 in an animal model of progressive CKD prevented the development of anemia of CKD. In aggregate, our results show that erythroid-expressed FGF23 is a negative regulator of erythropoiesis that contributes to anemia via direct paracrine FGFR1 activation in erythroid precursors.

Loss of the prolyl endopeptidase-like (PREPL) protein causes congenital myasthenic syndrome-22 (CMS22), a rare neuromuscular and metabolic disorder. PREPL belongs to the serine hydrolase superfamily, but its physiological substrates remain unknown. Based on the predicted lipid binding pocket in its crystal structure and its in vitro esterase activity, we hypothesized that PREPL might act as a lipase in vivo and directly regulate lipid metabolism. To test this, we performed unbiased lipidomics in Prepl knockout (KO) mouse brains and CRISPR-Cas9-generated KO cell lines. Across tissue and cell types, global phospholipid composition was largely unchanged, with only modest, non-significant increases in lysophospholipids, arguing against a direct role of PREPL in (lyso)phospholipid turnover. In contrast, PREPL KO HEK293T cells exhibited a significant accumulation of triacylglycerols (TAGs) and an increased number of lipid droplets, indicating a selective shift toward lipid storage. Given the central role of peroxisomes in lipid metabolism, we assessed PREPL localization and examined peroxisome number, morphology, and levels of key peroxisomal proteins. PREPL did not localize to peroxisomes, and peroxisome number and proteins levels were largely unchanged. However, KO cells displayed elongated peroxisomes, a phenotype possibly linked to mitochondrial dysfunction. Indeed, previous studies have shown that PREPL localizes to mitochondria and is required for respiratory chain activity and oxidative phosphorylation. These mitochondrial defects are predicted to impair fatty acid β-oxidation and disrupt redox balance, thereby promoting TAG synthesis and lipid droplet biogenesis as adaptive responses. Overall, our findings indicate that PREPL does not act as a canonical lipase but indirectly alters lipid homeostasis through its critical role in mitochondrial function. Elevated TAG levels and altered peroxisome morphology likely represent secondary consequences of impaired mitochondrial fatty acid metabolism in PREPL-deficient cells. These results establish a mechanistic link between mitochondrial dysfunction and lipid remodeling in PREPL deficiency, providing novel insights into the metabolic pathology of CMS22.

KRAS and BRAF mutations drive colorectal cancer (CRC) progression by sustaining aberrant signaling and promoting therapeutic resistance. Here, we identify TGF-β1-COX-2 axis as a critical regulatory pathway mediated by Furin in CRC harboring KRAS or BRAF mutation. Genetic silencing or pharmacological inhibition of Furin in KRAS-mutant (KPN) and BRAF-mutant (BPN) tumor-derived cells suppressed tumor growth, reduced angiogenesis, and enhanced CD8⁺ T cell infiltration in mouse tumor models. KRAS- and BRAF-mutant organoids with impaired Furin activity exhibited increased sensitivity to 5-FU, oxaliplatin, and irinotecan. Mechanistically, Furin inhibition via shRNA or the Furin inhibitor MI1148 blocked IGF-1 receptor and TGF-β1 precursor maturation and signaling, which was associated with repressed COX-2 expression. Conversely, COX-2 over-expression elevated TGF-β1 levels, which in turn enhanced Furin expression, establishing a feed-forward loop that promoted tumor progression and angiogenesis. Moreover, Furin inhibition largely disrupted the activity of multiple kinases linked to KRAS and BRAF oncogenic signaling. In CRC patient samples, Furin expression positively correlated with KRAS, BRAF, TGF-β1, and COX-2. Collectively, these findings identify Furin as a pivotal regulator of oncogenic signaling in KRAS- and BRAF-mutant CRC, and highlight the therapeutic potential of targeting the Furin-TGF-β1-COX-2 axis.

Congenital myasthenic syndrome-22 (CMS22, OMIM 616224) is a rare genetic disorder caused by deleterious genetic variation in the prolyl endopeptidase-like ( PREPL ) gene. Previous reports have described patients with deletions and nonsense variants in PREPL , but nothing is known about the effect of missense variants in the pathology of CMS22. In this study, we have functionally characterized missense variants in PREPL from 3 patients with CMS22, all with hallmark phenotypes. Biochemical evaluation revealed that these missense variants do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria and disease mechanism. Structural analysis showed that the variants affect regions most likely involved in intraprotein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the 3 variants. The importance of nonhydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines, which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the transgolgi network. In conclusion, these studies showed that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense variants that do not necessarily result in a loss of hydrolytic activity of PREPL.

Aortic aneurysms (AA) frequently involve dysregulation of transforming growth factor β (TGF-β)-signalling in the aorta. Here, FURIN was tested as aneurysm predisposition gene given its role as proprotein convertase in pro-TGF-β maturation. Rare FURIN variants were detected by whole-exome sequencing of 781 unrelated aortic aneurysm patients and affected relatives. Thirteen rare heterozygous FURIN variants occurred in 3.7% (29) unrelated index AA patients, of which 72% had multiple aneurysms or a dissection. FURIN maturation and activity of these variants were decreased in vitro. Patient-derived fibroblasts showed decreased pro-TGF-β processing, phosphorylation of downstream effector SMAD2 and kinases ERK1/2, and steady-state mRNA levels of the TGF-β-responsive ACTA2 gene. In aortic tissue, collagen and fibrillin fibres were affected. One variant (R745Q), observed in 10 unrelated cases, affected TGF-β signalling variably, indicating effect modification by individual genetic backgrounds. FURIN is a novel, frequent genetic predisposition for abdominal-, thoracic-, and multiple aortic or middle sized artery aneurysms in older patients, by affecting intracellular TGF-β signalling, depending on individual genetic backgrounds.

Congenital myasthenic syndrome-22 (CMS22) is a rare genetic disorder caused by mutations in the prolyl endopeptidase-like ( PREPL ) gene. Since previous reports only described patients with deletions and nonsense mutations in PREPL , nothing is known about the effect of missense mutations in the pathology of CMS22. In this study, we have characterized missense mutations in PREPL in three CMS22 patients, all with hallmark phenotypes. Biochemical evaluation revealed that these missense mutations do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria. Structural analysis shows that the mutations affect regions most likely involved in intra-protein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the three mutants. The importance of non-hydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the trans-Golgi network. In conclusion, these studies show that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense mutations that do not necessarily result in a loss of hydrolytic activity of PREPL. Graphical

Loss of prolyl endopeptidase-like ( PREPL ) encoding a serine hydrolase with (thio)esterase activity leads to the recessive metabolic disorder Congenital Myasthenic Syndrome-22 (CMS22). It is characterized by severe neonatal hypotonia, feeding problems, growth retardation, and hyperphagia leading to rapid weight gain later in childhood. The phenotypic similarities with Prader-Willi syndrome (PWS) are striking, suggesting that similar pathways are affected. The aim of this study was to identify changes in the hypothalamic-pituitary axis in mouse models for both disorders and to examine mitochondrial function in skin fibroblasts of patients and knockout cell lines. We have demonstrated that Prepl is downregulated in the brains of neonatal PWS-IC ^-p/+m mice. In addition, the hypothalamic-pituitary axis is similarly affected in both Prepl ^-/- and PWS-IC ^-p/+m mice resulting in defective orexigenic signaling and growth retardation. Furthermore, we demonstrated that mitochondrial function is altered in PREPL knockout HEK293T cells and can be rescued with the supplementation of coenzyme Q10. Finally, PREPL-deficient and PWS patient skin fibroblasts display defective mitochondrial bioenergetics. The mitochondrial dysfunction in PWS fibroblasts can be rescued by overexpression of PREPL. In conclusion, we provide the first molecular links between CMS22 and PWS, raising the possibility that PREPL substrates might become therapeutic targets for treating both disorders. Graphical