Purpose: Targeting of KITand platelet-derived growth factor receptor (PDGFR) tyrosine kinases by imatinib is an effective anticancer strategy. However, mutations of the gatekeeper residue (T670 in KITand T681in PDGFRh) render the two kinases resistant to imatinib. The aim of this study was to evaluate whether sorafenib , a multitargeted ATP-competitive inhibitor of KITand PDGFR, was active against imatinib-resistant KITand PDGFRh kinases. Experimental Design: We used in vitro kinase assays and immunoblot with phosphospecific antibodies to determine the activity of sorafenib on KIT and PDGFRh kinases. We also exploited reporter luciferase assays to measure the effects of sorafenib on KIT and PDGFRh downstream signaling events. The activity of sorafenib on interleukin-3^independent proliferation of Ba/F3 cells expressing oncogenic KITor its imatinib-resistant T670I mutant was also tested. Results: Sorafenib efficiently inhibited gatekeeper mutants of KIT and PDGFRh (IC 50 for KIT T670I, 60 nmol/L; IC 50 for PDGFRh T681I, 110 nmol/L). Instead, it was less active against activation loop mutants of the two receptors (IC 50 for KIT D816V, 3.8 Amol/L; IC 50 for PDGFRh D850V, 1.17 Amol/L) that are also imatinib-resistant. Sorafenib blocked receptor autophosphorylation and signaling of KIT and PDGFRh gatekeeper mutants in intact cells as well as activation of AP1-responsive and cyclin D1 gene promoters, respectively. Finally, the compound inhibited KITdependent proliferation of Ba/F3 cells expressing the oncogenic KIT mutant carrying the T670I mutation. Conclusions: Sorafenib might be a promising anticancer agent for patients carrying KIT and PDGFRh gatekeeper mutations.The KIT and platelet-derived growth factor receptors (PDGFR) are members of the type III subclass of receptor tyrosine kinases. KIT is the receptor for stem cell factors (SCF), whereas PDGFRa and PDGFRh are the receptors for platelet-derived growth factors (PDGF; ref. 1). The structure of these receptors includes an extracellular domain with five immunoglobulinlike motifs, a single membrane-spanning domain, and a cytoplasmic tyrosine kinase domain. The kinase domain is split by a kinase insert sequence into an ATP-binding region and a phosphotransferase region (1).KIT, PDGFRa, and PDGFRh are frequently activated in neoplastic diseases. More than 30 gain-of-function mutations in KIT, either single amino acid changes or small deletions/ insertions, have been identified in such highly malignant human neoplastic diseases as gastrointestinal stromal tumors (GIST) and mastocytosis. GISTs are the most common type of sarcoma arising in the digestive tract and are generally distinguished from other abdominal sarcomas by the expression of KIT. Approximately 80% of these tumors show activating mutations in KIT (2). GIST mutations cluster in the KIT juxtamembrane region, whereas most mutations associated with mastocytosis target a specific aspartate residue (D816) in the kinase activation loop (3). Fusion of PDGFRa with different genes has been found in ...

MCM proteins are overexpressed in ATC and sustain the high proliferative capacity of ATC cells.

ZD6474 (vandetanib, Zactima, Astra Zeneca) is an anilinoquinazoline used to target the receptor tyrosine kinase RET in familial and sporadic thyroid carcinoma (IC 50 : 100 nM). The aim of this study was to identify molecular determinants of RET sensitivity to ZD6474. Here, we show that mutation of RET tyrosine 806 to cysteine (Y806C) induced RET kinase resistance to ZD6474 (IC 50 : 933 nM). Y806 maps close to the gate-keeper position at the RET kinase nucleotide-binding pocket. Although tyrosine 806 is a RET auto-phosphorylation site, its substitution to phenylalanine (Y806F) did not markedly affect RET susceptibility to ZD6474 (IC 50 : 87 nM), suggesting that phosphorylation of Y806 is not required for compound binding. Accordingly, the introduction of a phosphomimetic residue (Y806E) also caused resistance to ZD6474, albeit of a lesser degree (IC 50 : 512 nM) than the cysteine mutation. Y806C/E RET mutants were also resistant to ZD6474 with respect to intracellular signalling and activation of an AP1-responsive promoter. We conclude that Y806 is a molecular determinant of RET sensitivity to ZD6474. Y806C is a natural RET mutation identified in a patient affected by multiple endocrine neoplasia type 2B. Based on its rare occurrence, it is unlikely that Y806C will be a frequent cause of refractoriness to ZD6474; however, it may be envisaged that mutations at this site can mediate secondary resistance formation in patients treated with the compound.

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Background Preterm birth affects almost 9–11% of newborns and is one of the leading causes of childhood neurodevelopmental disabilities; the underlying molecular networks are poorly defined. In neurons, retrotransposons LINE-1 (L1) are an active source of genomic mosaicism that is deregulated in several neurological disorders; early life experience has been shown to regulate L1 activity in mice. Methods Very preterm infants were randomized to receive standard care or early intervention. L1 methylation was measured at birth and at hospital discharge. At 12 and 36 months, infants’ neurodevelopment was evaluated with the Griffiths Scales. L1 methylation and CNVs were measured in mouse brain areas at embryonic and postnatal stages. Results Here we report that L1 promoter is hypomethylated in preterm infants at birth and that an early intervention program, based on enhanced maternal care and positive multisensory stimulation, restores L1 methylation levels comparable to healthy newborns and ameliorates neurodevelopment in childhood. We further show that L1 activity is fine-tuned in the perinatal mouse brain, suggesting a sensitive and vulnerable window for the L1 epigenetic setting. Conclusions Our results open the field on the inspection of L1 activity as a novel molecular and predictive approach to infants’ prematurity-related neurodevelopmental outcomes. Trial registration ClinicalTrial.gov (NCT02983513). Registered on 6 December 2016, retrospectively registered.

Necrotizing enterocolitis (NEC) is a devastating gut disease in preterm neonates. In NEC animal models, mesenchymal stromal cells (MSCs) administration has reduced the incidence and severity of NEC. We developed and characterized a novel mouse model of NEC to evaluate the effect of human bone marrow-derived MSCs (hBM-MSCs) in tissue regeneration and epithelial gut repair. NEC was induced in C57BL/6 mouse pups at postnatal days (PND) 3–6 by (A) gavage feeding term infant formula, (B) hypoxia/hypothermia, and (C) lipopolysaccharide. Intraperitoneal injections of PBS or two hBM-MSCs doses (0.5 × 106 or 1 × 106) were given on PND2. At PND 6, we harvested intestine samples from all groups. The NEC group showed an incidence of NEC of 50% compared with controls (p < 0.001). Severity of bowel damage was reduced by hBM-MSCs compared to the PBS-treated NEC group in a concentration-dependent manner, with hBM-MSCs (1 × 106) inducing a NEC incidence reduction of up to 0% (p < 0.001). We showed that hBM-MSCs enhanced intestinal cell survival, preserving intestinal barrier integrity and decreasing mucosal inflammation and apoptosis. In conclusion, we established a novel NEC animal model and demonstrated that hBM-MSCs administration reduced the NEC incidence and severity in a concentration-dependent manner, enhancing intestinal barrier integrity.

Very preterm infants are exposed to prenatal inflammatory processes and early postnatal hemodynamic and respiratory complications, but limited data are available about the endothelial effect of these conditions. The present pilot study investigates the perinatal endothelial phenotype in very preterm infants (VPIs) and explores its predictive value on neonatal mortality and hemodynamic and respiratory complications. Angiopoietin 1 (Ang-1), Ang-2, E-selectin, vascular adhesion molecule 1 (VCAM-1), tissue factor (TF), and endothelin 1 (ET-1) concentrations were tested in first (T1), 3rd (T2), and 7–10th (T3) day of life in 20 VPIs using Luminex technology and compared with 14 healthy full-term infants (FTIs). Compared to FTIs, VPIs had lower Ang-1 at T1 and T2; higher Ang-2 at T1, T2, and T3; higher Ang-2/Ang-1 ratio at T1, T2, and T3; lower E-selectin at T1, T2, and T3; higher VCAM-1 at T1; higher TF at T2. No differences in concentrations were found in neonatal deaths. VPIs with hemodynamic or respiratory complications had higher Ang-2 at T3. Perinatal low Ang-1 and high Ang-2 associated with high VCAM-1 and TF in VPIs suggest a proinflammatory endothelial phenotype, resulting from the synergy of a pathological prenatal inheritance and a premature extrauterine transition.

Millions of infants are born prematurely every year worldwide. Prematurity, particularly at lower gestational ages, is associated with high mortality and morbidity and is a significant global health burden. Pregnancy complications and preterm birth syndrome strongly impact neonatal clinical phenotypes and outcomes. The vascular endothelium is a pivotal regulator of fetal growth and development. In recent years, the key role of uteroplacental pathologies impairing endothelial homeostasis is emerging. Conditions leading to very and extremely preterm birth can be classified into two main pathophysiological patterns or endotypes: infection/inflammation and dysfunctional placentation. The first is frequently related to chorioamnionitis, whereas the second is commonly associated with hypertensive disorders of pregnancy and fetal growth restriction. The nature, timing, and extent of prenatal noxa may alter fetal and neonatal endothelial phenotype and functions. Changes in the luminal surface, oxidative stress, growth factors imbalance, and dysregulation of permeability and vascular tone are the leading causes of endothelial dysfunction in preterm infants. However, the available evidence regarding endothelial physiology and damage is limited in neonates compared to adults. Herein, we discuss the current knowledge on endothelial dysfunction in the infectious/inflammatory and dysfunctional placentation endotypes of prematurity, summarizing their molecular features, available biomarkers, and clinical impact. Furthermore, knowledge gaps, shadows, and future research perspectives are highlighted.