Systematic analysis of RNA-binding proteins identifies targetable therapeutic vulnerabilities in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor with a strong tendency to metastasize, limiting the prognosis of affected patients. Genomic, epigenomic and transcriptomic analyses have demonstrated the exquisite molecular complexity of this tumor, but have not sufficiently defined the underlying mechanisms or identified promising therapeutic targets. To systematically explore RNA-protein interactions relevant to OS, we define the RNA interactomes together with the full proteome and the transcriptome of cells from five malignant bone tumors (four osteosarcomata and one malignant giant cell tumor of the bone) and from normal mesenchymal stem cells and osteoblasts. These analyses uncover both systematic changes of the RNA-binding activities of defined RNA-binding proteins common to all osteosarcomata and individual alterations that are observed in only a subset of tumors. Functional analyses reveal a particular vulnerability of these tumors to translation inhibition and a positive feedback loop involving the RBP IGF2BP3 and the transcription factor Myc which affects cellular translation and OS cell viability. Our results thus provide insight into potentially clinically relevant RNA-binding protein-dependent mechanisms of osteosarcoma.

Loss of LAT1 sex-dependently delays recovery after caerulein-induced acute pancreatitis.

BACKGROUND: The expression of amino acid transporters is known to vary during acute pancreatitis (AP) except for LAT1 (slc7a5), the expression of which remains stable. LAT1 supports cell growth by importing leucine and thereby stimulates mammalian target of rapamycin (mTOR) activity, a phenomenon often observed in cancer cells. The mechanisms by which LAT1 influences physiological and pathophysiological processes and affects disease progression in the pancreas are not yet known. AIM: To evaluate the role of LAT1 in the development of and recovery from AP. METHODS: AP was induced with caerulein (cae) injections in female and male mice expressing LAT1 or after its knockout (LAT1 Cre/LoxP). The development of the initial AP injury and its recovery were followed for seven days after cae injections by daily measuring body weight, assessing microscopical tissue architecture, mRNA and protein expression, protein synthesis, and enzyme activity levels, as well as by testing the recruitment of immune cells by FACS and ELISA. RESULTS: The initial injury, evaluated by measurements of plasma amylase, lipase, and trypsin activity, as well as the gene expression of dedifferentiation markers, did not differ between the groups. However, early metabolic adaptations that support regeneration at later stages were blunted in LAT1 knockout mice. Especially in females, we observed less mTOR reactivation and dysfunctional autophagy. The later regeneration phase was clearly delayed in female LAT1 knockout mice, which did not regain normal expression of the pancreas-specific differentiation markers recombining binding protein suppressor of hairless-like protein (rbpjl) and basic helix-loop-helix family member A15 (mist1). Amylase mRNA and protein levels remained lower, and, strikingly, female LAT1 knockout mice presented signs of fibrosis lasting until day seven. In contrast, pancreas morphology had returned to normal in wild-type littermates. CONCLUSION: LAT1 supports the regeneration of acinar cells after AP. Female mice lacking LAT1 exhibited more pronounced alterations than male mice, indicating a sexual dimorphism of amino acid metabolism.

Germline RET variants underlie a subset of paediatric osteosarcoma.

BACKGROUND: Although considerable effort has been put into decoding of the osteosarcoma genome, very little is known about germline mutations that underlie this primary malignant tumour of bone. METHODS AND RESULTS: We followed here a coincidental finding in a multiple endocrine neoplasia family in which a 32-year-old patient carrying a germline pathogenic RET mutation developed an osteosarcoma 2 years after the resection of a medullary thyroid carcinoma. Sequencing analysis of additional 336 patients with osteosarcoma led to the identification of germline activating mutations in the RET proto-oncogene in three cases and somatic amplifications of the gene locus in five matched tumours (4%, n=5/124 tumours). Functional analysis of the pathogenic variants together with an integrative analysis of osteosarcoma genomes confirmed that the mutant RET proteins couple functional kinase activity to dysfunctional ligand binding. RET mutations further co-operated with alterations in TP53 and RB1, suggesting that osteosarcoma pathogenesis bears reminiscence to the stepwise model of medullary thyroid carcinoma. CONCLUSIONS: After Li-Fraumeni-predisposing mutations in TP53, RET becomes the second most mutated cancer-predisposing gene in the germline of patients with osteosarcoma. Hence, early identification of RET mutation carriers can help to identify at-risk family members and carry out preventive measures.

Choroid plexus LAT2 and SNAT3 as partners in CSF amino acid homeostasis maintenance.

BACKGROUND: Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Although derived from blood plasma, it is nearly protein-free (~ 250-fold less) and contains about 2-20-fold less free amino acids, with the exception of glutamine (Gln) which is nearly equal. The aim of this study was to determine which amino acid transporters are expressed in mouse CP epithelium in order to gain understanding about how this barrier maintains the observed amino acid concentration gradient. METHODS: Expression of amino acid transporters was assessed in isolated choroid plexuses (CPs) by qRT-PCR followed by localization studies using immunofluorescence with specific antibodies. The impact of LAT2 (Slc7a8) antiporter deletion on CSF amino acids was determined. RESULTS: The purity of isolated choroid plexuses was tested on the mRNA level using specific markers, in particular transthyretin (Ttr) that was enriched 330-fold in CP compared to cerebral tissue. In a first experimental round, 14 out of 32 Slc amino acid transporters tested on the mRNA level by qPCR were selected for further investigation. Out of these, five were considered highly expressed, SNAT1 (Slc38a1), SNAT3 (Slc38a3), LAT2 (Slc7a8), ASC1 (Slc7a10) and SIT1 (Slc6a20b). Three of them were visualized by immunofluorescence: SNAT1 (Slc38a1), a neutral amino acid-Na+ symporter, found at the blood side basolateral membrane of CP epithelium, while SNAT3 (Slc38a3), an amino acid-Na+ symporter and H+ antiporter, as well as LAT2 (Slc7a8), a neutral amino acid antiporter, were localized at the CSF-facing luminal membrane. In a LAT2 knock-out mouse model, CSF Gln was unchanged, whereas other amino acids normally 2-20-fold lower than in plasma, were increased, in particular the LAT2 uptake substrates leucine (Leu), valine (Val) and tryptophan (Trp) and some other amino acids such as glutamate (Glu), glycine (Gly) and proline (Pro). CONCLUSION: These results suggest that Gln is actively transported by SNAT1 from the blood into CP epithelial cells and then released luminally into CSF via SNAT3 and LAT2. Its efflux via LAT2 may drive the reuptake from the CSF of essential amino acid substrates of this antiporter and thereby participates to maintaining the amino acid gradient between plasma and CSF.

Prexasertib (LY2606368) reduces clonogenic survival by inducing apoptosis in primary patient-derived osteosarcoma cells and synergizes with cisplatin and talazoparib.

Progress in the systemic control of osteosarcoma has been limited over the past decades thus indicating the urgent clinical need for the development of novel treatment strategies. Therefore, we have recently developed new preclinical models to study promising novel agents for the treatment of pediatric osteosarcoma. The checkpoint kinase (chk) inhibitor prexasertib (LY2606368) and its salt form (LSN2940930) have recently been shown to be active in adult and pediatric malignancies, including sarcoma. We have now tested the potency of prexasertib in clonogenic survival assays in two new lines of primary patient-derived osteosarcoma cells and in two established osteosarcoma cell lines as a single agent and in combination with cisplatin and the poly ADP-ribose polymerase (PARP) inhibitor talazoparib. Prexasertib alone results in strongly reduced clonogenic survival at low nanomolar concentrations and acts by affecting cell cycle progression, induction of apoptosis and induction of double-stranded DNA breakage at concentrations that are well below clinically tolerable and safe plasma concentrations. In combination with cisplatin and talazoparib, prexasertib acts in a synergistic fashion. Chk1 inhibition by prexasertib and its combination with the DNA damaging agent cisplatin and the PARP-inhibitor talazoparib thus emerges as a potential new treatment option for pediatric osteosarcoma which will now have to be tested in preclinical primary patient derived in vivo models and clinical studies.

Differential Impact of Dietary Branched Chain and Aromatic Amino Acids on Chronic Kidney Disease Progression in Rats.

The metabolism of dietary proteins generates waste products that are excreted by the kidney, in particular nitrogen-containing urea, uric acid, ammonia, creatinine, and other metabolites such as phosphates, sulfates, and protons. Kidney adaptation includes an increase in renal plasma flow (RPF) and glomerular filtration rate (GFR) and represents a burden for diseased kidneys increasing the progression rate of CKD. The present study aimed at identifying potential differences between amino acid (AA) groups constituting dietary proteins regarding their impact on RPF, GFR, and CKD progression. We utilized the well-established 5/6 nephrectomy (5/6 Nx) CKD model in rats and submitted the animals for 5 weeks to either the control diet (18% casein protein) or to diets containing 8% casein supplemented with 10% of a mix of free amino acids, representing all or only a subset of the amino acids contained in casein. Whereas the RPF and GFR measured in free moving animals remained stable during the course of the diet in rats receiving the control mix, these parameters decreased in animals receiving the branched chain amino acid (BCAA) supplementation and increased in the ones receiving the aromatic amino acids (AAAs). In animals receiving essential amino acids (EAAs) containing both BCAAs and AAAs, there was only a small increase in RPF. The kidneys of the 5/6 Nx rats receiving the BCAA diet showed the strongest increase in smooth muscle actin and collagen mRNA expression as a result of higher level of inflammation and fibrosis. These animals receiving BCAAs also showed an increase in plasma free fatty acids pointing to a problem at the level of energy metabolism. In contrast, the animals under AAA diet showed an activation of AMPK and STAT3. Taken together, our results demonstrate that subsets of EAAs contained in dietary proteins, specifically BCAAs and AAAs, exert contrasting effects on kidney functional parameters and CKD progression.

Histone 3.3 hotspot mutations in conventional osteosarcomas: a comprehensive clinical and molecular characterization of six H3F3A mutated cases.

BACKGROUND: Histone 3.3 (H3.3) hotspot mutations in bone tumors occur in the vast majority of giant cell tumors of bone (GCTBs; 96%), chondroblastomas (95%) and in a few cases of osteosarcomas. However, clinical presentation, histopathological features, and additional molecular characteristics of H3.3 mutant osteosarcomas are largely unknown. METHODS: In this multicentre, retrospective study, a total of 106 conventional high-grade osteosarcomas, across all age groups were re-examined for hotspot mutations in the H3.3 coding genes H3F3A and H3F3B. H3.3 mutant osteosarcomas were re-evaluated in a multidisciplinary manner and analyzed for genome-wide DNA-methylation patterns and DNA copy number aberrations alongside H3.3 wild-type osteosarcomas and H3F3A G34W/L mutant GCTBs. RESULTS: Six osteosarcomas (6/106) carried H3F3A hotspot mutations. No mutations were found in H3F3B. All patients with H3F3A mutant osteosarcoma were older than 30 years with a median age of 65 years. Copy number aberrations that are commonly encountered in high-grade osteosarcomas also occurred in H3F3A mutant osteosarcomas. Unlike a single osteosarcoma with a H3F3A K27M mutation, the DNA methylation profiles of H3F3A G34W/R mutant osteosarcomas were clearly different from H3.3 wild-type osteosarcomas, but more closely related to GCTBs. The most differentially methylated promoters between H3F3A G34W/R mutant and H3.3 wild-type osteosarcomas were in KLLN/PTEN (p < 0.00005) and HIST1H2BB (p < 0.0005). CONCLUSIONS: H3.3 mutations in osteosarcomas may occur in H3F3A at mutational hotspots. They are overall rare, but become more frequent in osteosarcoma patients older than 30 years. Osteosarcomas carrying H3F3A G34W/R mutations are associated with epigenetic dysregulation of KLLN/PTEN and HIST1H2BB.

Histone deacetylase inhibition sensitizes osteosarcoma to heavy ion radiotherapy.

BACKGROUND: Minimal improvements in treatment or survival of patients with osteosarcoma have been achieved during the last three decades. Especially in the case of incomplete tumor resection, prognosis remains poor. Heavy ion radiotherapy (HIT) and modern anticancer drugs like histone deacetylase inhibitors (HDACi) have shown promising effects in osteosarcoma in vitro. In this study, we tested the effect of HIT and the combination of HIT and the HDACi suberoylanilide hydroxamic acid (SAHA) in a xenograft mouse model. METHODS: Osteosarcoma xenografts were established by subcutaneous injection of KHOS-24OS cells and treated with either vehicle (DMSO), SAHA, HIT or HIT and SAHA. Tumor growth was determined and tumor necrosis, proliferation rate, apoptotic rate as well as vessel density were evaluated. RESULTS: Here, we show that the combination of HIT and SAHA induced a significant delay of tumor growth through increased rate of apoptosis, increased expression of p53 and p21(Waf1/Cip1), inhibition of proliferation and angiogenesis compared to tumors treated with HIT only. CONCLUSION: HIT and in particular the combination of HIT and histone deacetylase inhibition is a promising treatment strategy in OS and may be tested in clinical trials.

Establishment of a patient-derived orthotopic osteosarcoma mouse model.

BACKGROUND: Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. METHODS: Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. RESULTS: A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. CONCLUSION: We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.

Cardiac safety of Modified Vaccinia Ankara for vaccination against smallpox in a young, healthy study population.

BACKGROUND: Conventional smallpox vaccines based on replicating vaccinia virus (VV) strains (e.g. Lister Elstree, NYCBOH) are associated with a high incidence of myo-/pericarditis, a severe inflammatory cardiac complication. A new smallpox vaccine candidate based on a non-replicating Modified Vaccinia Ankara (MVA) poxvirus has been assessed for cardiac safety in a large placebo-controlled clinical trial. METHODS: Cardiac safety of one and two doses of MVA compared to placebo was assessed in 745 healthy subjects. Vaccinia-naïve subjects received either one dose of MVA and one dose of placebo, two doses of MVA, or two doses of placebo by subcutaneous injection four weeks apart; vaccinia-experienced subjects received a single dose of MVA. Solicited and unsolicited adverse events (AE) and cardiac safety parameters (recorded as Adverse Events of Special Interest, AESI) were monitored after each injection. RESULTS: A total of 5 possibly related AESI (3 cases of palpitations, 2 of tachycardia) were reported during the study. No case of myo- or pericarditis occurred. One possibly related serious AE (SAE) was reported during the 6-month follow-up period (sarcoidosis). The most frequently observed AEs were injection site reactions. CONCLUSIONS: Vaccination with MVA was safe and well tolerated and did not increase the risk for development of myo-/pericarditis. TRIAL REGISTRATION: ClinicalTrials.gov NCT00316524.

CCN1 mutation is associated with atrial septal defect.

The genetic basis of congenital heart disease remains unknown in most of the cases. Recently, a novel mouse model shed new light on the role of CCN1/CYR61, a matricellular regulatory factor, in cardiac morphogenesis. In a candidate gene approach, we analyzed a cohort of 143 patients with atrial septal defects (ASD) by sequencing the coding exons of CCN1. In addition to three frequent polymorphisms, we identified an extremely rare novel heterozygous missense mutation (c.139C > T; p.R47W) in one patient with severe ASD. The mutation leads to an exchange of residues with quite different properties in a highly conserved position of the N-terminal insulin-like growth factor binding protein module. Further bioinformatic analysis, exclusion of known ASD disease genes as well as the exclusion of the mutation in a very high number of ethnically matched controls (more than 1,000 individuals) and in public genetic databases, indicates that the p.R47W variant is a probable disease-associated mutation. The report about ASD in mice in heterozygous Ccn 1 +/- animals strongly supports this notion. Our study is the first to suggest a relationship between a probable CCN1 mutation and ASD. Our purpose here was to draw attention to CCN1, a gene that we believe may be important for genetic analysis in patients with congenital heart disease.

The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients.

BACKGROUND: The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K⁺ channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl⁻ secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. METHODS: We studied the effects of 1-EBIO on CFTR-mediated Cl⁻ secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl⁻ secretion. RESULTS: Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl⁻ secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl⁻ secretion by 39.2±6.7% (P<0.001) via activation of basolateral Ca²âº-activated and clotrimazole-sensitive KCNN4 K⁺ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl⁻ secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl⁻ conductance. CONCLUSIONS: We conclude that 1-EBIO potentiates Cl⁻secretion in native CF tissues expressing CFTR mutants with residual Cl⁻ channel function by activation of basolateral KCNN4 K⁺ channels that increase the driving force for luminal Cl⁻ exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

Fisheries and aquaculture industries involvement to control product health and quality safety to satisfy consumer-driven objectives on retail markets in Europe.

Over the past years the export of agricultural and fishery products from developing countries has substantially increased to markets within the OECD. Retailers and importers are expanding their international operations to meet consumer demands for year-round delivery of products. Moreover, consumers have become increasingly concerned about the safety of food, including those derived from aquatic resources [FAO/NACA/WHO Joint Study Group, 1999. Report food safety issues associated with products from aquaculture. WHO Technical Report Series No 883: VII, pp. 1-55]. Governments and leading businesses are responding by imposing new safety regulations and standards to the international food system (e.g. HACCP, EUREP-GAP), product liability and labeling [Reilly, A., Howgate, P., Käferstein, F., 1997. Safety hazards and the application of HACCP in aquaculture. In: Proceedings of the Second International Conference on Fish Inspection and Quality Control: A Global Focus, Arlington, VA, 19-24 May 1996. Technomic Publishing, Lancaster, PA, pp. 353-373]. Initial concerns for imports of aquacultural products from developing to industrialized countries focussed on bacterial contamination [Buras, N. 1993. Microbial safety of produce from wastewater-fed aquaculture. In: Pullin, R.V.C., Rosenthal, H., MacLean, J.L.(Eds.), Proceedings of ICLARM Conferences, vol. 31, pp. 285-295]. Today, if trade opportunities are to be maintained, these countries must adapt to a full array of regulations and standards. This paper describes four scenarios in aquaculture and fishing product trade between developing countries and countries in the European Union.

The growth rate of Mycobacterium smegmatis depends on sufficient porin-mediated influx of nutrients.

Mycobacteria have a unique outer membrane (OM) that is thicker than any other known biological membrane. Nutrients cross this permeability barrier by diffusion through porins. MspA is the major porin of Mycobacterium smegmatis. In this study we showed that three paralogues of MspA, namely MspB, MspC and MspD are also porins. However, only the mspA and mspC genes were expressed in the wild-type strain. None of the single deletion mutants displayed a significant OM permeability defect except for the mspA mutant. Deletion of the mspA gene caused activation of transcription of mspB and/or mspD in three independent strains by unknown chromosomal mutations. It is concluded that mspB and mspD provide backup porins for M. smegmatis. This also indicated that a minimal porin-mediated OM permeability is essential for survival of M. smegmatis. Electron microscopy in combination with quantitative image analysis of protein gels revealed that the number of pores per cell dropped from 2400 to 800 and 150 for the DeltamspA and DeltamspA DeltamspC mutant (ML10) respectively. The very low number of pores correlated well with the at least 20-fold lower channel activity of detergent extracts of the ML10 strain and its 15- and 75-fold lower permeability to nutrient molecules such as serine and glucose respectively. The amount of Msp porin and the OM permeability of the triple porin mutant lacking mspA, mspC and mspD was not altered. The growth rate of M. smegmatis dropped drastically with its porin-mediated OM permeability in contrast to porin mutants of Escherichia coli. These results show that porin-mediated influx of nutrients is a major determinant of the growth rate of M. smegmatis.