Publications

Supplementary Data from A Phase I Trial of TB-403 in Relapsed Medulloblastoma, Neuroblastoma, Ewing Sarcoma, and Alveolar Rhabdomyosarcoma

<p>Effects of VEGFR-1 signaling ablation on glucose/insulin tolerance and insulin production by the pancreas during obesity.</p>

Objective: Endothelial cells exposed to laminar shear stress express a thick glycocalyx on their surface that plays an important role in reducing vascular permeability and endothelial anti-inflammatory, antithrombotic, and antiangiogenic properties. Production and maintenance of this glycocalyx layer is dependent on cellular carbohydrate synthesis, but its regulation is still unknown. Approach and Results: Here, we show that biosynthesis of the major structural component of the endothelial glycocalyx, hyaluronan, is regulated by shear. Both in vitro as well as in in vivo, hyaluronan expression on the endothelial surface is increased on laminar shear and reduced when exposed to oscillatory flow, which is regulated by KLF2 (Krüppel-like Factor 2). Using a CRISPR-CAS9 edited small tetracysteine tag to endogenous HAS2 (hyaluronan synthase 2), we demonstrated increased translocation of HAS2 to the endothelial cell membrane during laminar shear. Hyaluronan production by HAS2 was shown to be further driven by availability of the hyaluronan substrates UDP-glucosamine and UDP-glucuronic acid. KLF2 inhibits endothelial glycolysis and allows for glucose intermediates to shuttle into the hexosamine- and glucuronic acid biosynthesis pathways, as measured using nuclear magnetic resonance analysis in combination with 13 C-labeled glucose. Conclusions: These data demonstrate how endothelial glycocalyx function and functional adaptation to shear is coupled to KLF2-mediated regulation of endothelial glycolysis.

Summary The pathogenesis of vascular tumors such as angiosarcomas is poorly understood. Cadherin expression inversely correlates with tumor malignancy and the endothelial specific VE-cadherin is low or absent in angiosarcomas, suggesting an inhibitory role for this protein in tumor progression. In this paper we report that Pmy T VE-cadherin null (VEC null) endothelial cells form larger vascular tumors in nude mice when injected subcutaneously as compared to isogenic VE-cadherin positive (VEC pos) cells. This effect requires the association of β-catenin to VE-cadherin, since a VE-cadherin mutant lacking the domain responsible for β-catenin binding (Δβcat) cannot rescue the phenotype. In VEC null cells β-catenin is phosphorylated and partly degraded. N-cadherin is increased and detected at junctions. VEC null cells also present an altered fibrinolytic activity with increases in tPA, uPA, uPAR and a strong reduction in PAI-1, which may be correlated to the high incidence of abrupt hemorrhages in VEC null tumors. Overall, these data strongly suggest that downregulation of VE-cadherin in endothelial tumors may have important consequences for tumor growth and bleeding complications.

Liver cirrhosis is a very complex disease in which several pathological processes such as inflammation, fibrosis, and pathological angiogenesis are closely integrated. We hypothesized that treatment with pharmacological agents with multiple mechanisms of action will produce superior results to those achieved by only targeting individual mechanisms. This study thus evaluates the therapeutic use of the multitargeted receptor tyrosine kinase inhibitor Sunitinib (SU11248). The in vitro effects of SU11248 were evaluated in the human hepatic stellate cell line LX-2 by measuring cell viability. The in vivo effects of SU11248 treatment were monitored in the livers of cirrhotic rats by measuring angiogenesis, inflammatory infiltrate, fibrosis, α-smooth muscle actin (α-SMA) accumulation, differential gene expression by microarrays, and portal pressure. Cirrhosis progression was associated with a significant enhancement of vascular density and expression of vascular endothelial growth factor-A, angiopoietin-1, angiopoietin-2, and placental growth factor in cirrhotic livers. The newly formed hepatic vasculature expressed vascular cellular adhesion molecule 1 and intercellular adhesion molecule 1. Interestingly, the expression of these adhesion molecules was adjacent to areas of local inflammatory infiltration. SU11248 treatment resulted in a significant decrease in hepatic vascular density, inflammatory infiltrate, α-SMA abundance, LX-2 viability, collagen expression, and portal pressure. Conclusion: These results suggest that multitargeted therapies against angiogenesis, inflammation, and fibrosis merit consideration in the treatment of cirrhosis. (Hepatology 2007.)

Objective— To study whether Notch signaling, which regulates cell fate decisions and vessel morphogenesis, controls lymphatic development. Methods and Results— In zebrafish embryos, sprouts from the axial vein have lymphangiogenic potential because they give rise to the first lymphatics. Knockdown of delta-like-4 (Dll4) or its receptors Notch-1b or Notch-6 in zebrafish impaired lymphangiogenesis. Dll4/Notch silencing reduced the number of sprouts producing the string of parchordal lymphangioblasts; instead, sprouts connecting to the intersomitic vessels were formed. At a later phase, Notch silencing impaired navigation of lymphatic intersomitic vessels along their arterial templates. Conclusion— These studies imply critical roles for Notch signaling in the formation and wiring of the lymphatic network.

Duchenne muscular dystrophy (DMD) is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of DMD. The expression of uPA is induced in mdx dystrophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular function. Bone marrow (BM) transplantation experiments revealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy.

Abstract Ovarian cancer accounts for the highest number of gynecologic-associated deaths in the developed world, and resistance to platinum-based therapy represents a major clinical and societal challenge in patients’ management. Since metabolism is intertwined with signaling pathways controlling cell death, we aimed to investigate to what extent metabolic adaptations could contribute to the development of the resistant phenotype. By performing isotope-labeled 13C-glucose tracer analysis in vitro, we found that, when they become resistant to platinum, ovarian cancer cells stop to synthetize serine and are characterized by significantly lower intracellular levels of this amino acid compared to sensitive cells. However, serine is required for cellular growth and survival, and resistant cells increase its uptake from the medium, becoming exquisitely vulnerable to serine starvation. We showed that, although resistant cells accumulate DNA damage upon platinum treatment, they have a higher capacity of repairing it because of increased poly(ADP)-ribose polymerase (PARP) activity, compared to the sensitive ones. Since PARP enzymes are major oxidized nicotinamide adenine dinucleotide (NAD+)-consuming enzymes, we collected evidence that serine synthesis downregulation, as a consequence of central carbon metabolic reshuffling, provides resistant cells with the advantage of sparing NAD+, thus sustaining PARP activation and allowing a more efficient DNA repair. We confirmed that downregulation of serine synthesis is a peculiar trait of resistant tumors also in vivo, using patient-derived xenografts (PDX) ovarian cancer models subjected to serine/glycine free diet. Moreover, analysis of The Cancer Genome Atlas Consortium (TCGA) ovarian cancer dataset revealed that tumors from platinum-resistant patients are characterized by a downregulation of serine biosynthetic enzymes, suggesting that serine auxotrophy could represent a novel and exploitable vulnerability of platinum-resistant ovarian cancers. Citation Format: Tom Van Nyen, Joao A.G. Duarte, Matteo Rossi, Mélanie Planque, Esther Zaal, Ali Talebi, Stijn Moens, Guy Eelen, Hugo Horlings, Johan Swinnen, Celia Berkers, Peter Carmeliet, Reuven Agami, Sarah-Maria Fendt, Diether Lambrechts, Daniela Annibali, Frederic Amant. Serine auxotrophy: A novel metabolic vulnerability of platinum-resistant ovarian cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A75.