USP7 controls NGN3 stability and pancreatic endocrine lineage development.

Understanding the factors and mechanisms involved in beta-cell development will guide therapeutic efforts to generate fully functional beta cells for diabetes. Neurogenin 3 (NGN3) is the key transcription factor that marks endocrine progenitors and drives beta-cell differentiation. Here we screen for binding partners of NGN3 and identify the deubiquitylating enzyme USP7 as a key regulator of NGN3 stability. Mechanistically, USP7 interacts with, deubiquitinates and stabilizes NGN3. In vivo, conditional knockout of Usp7 in the mouse embryonic pancreas causes a dramatic reduction in islet formation and hyperglycemia in adult mice, due to impaired NGN3-mediated endocrine specification during pancreatic development. Furthermore, pharmacological inhibition of USP7 during endocrine specification in human iPSC models of beta-cell differentiation decreases NGN3 expressing progenitor cell numbers and impairs beta cell differentiation. Thus, the USP7-NGN3 axis is an essential mechanism for driving endocrine development and beta-cell differentiation, which can be therapeutically exploited.

Efficacy of mecobalamin (vitamin B12) in the treatment of long-term pain in women diagnosed with fibromyalgia: protocol for a randomised, placebo-controlled trial.

INTRODUCTION: Fibromyalgia causes long-term pain. It affects at least 2% of the population, the majority being women. In addition, extended symptoms corresponding to vitamin B12 deficiency occur. Findings from several studies have indicated that vitamin B12 may be a possible treatment for pain in fibromyalgia. The aim of the proposed study is to evaluate whether vitamin B12 decreases pain sensitivity and the experience of pain (ie, hyperalgesia and allodynia) in women with fibromyalgia. METHODS AND ANALYSIS: The study is a randomised, placebo-controlled, single-blind, clinical trial with two parallel groups which are administered mecobalamin (vitamin B12) or placebo over 12 weeks. 40 Swedish women aged 20-70 years with an earlier recorded diagnosis of fibromyalgia are randomised into the placebo group or the treatment group, each consisting of 20 participants. Outcomes consist of questionnaires measured at baseline and after 12 weeks of treatment. A final re-evaluation will then follow 12 weeks after treatment ends. The primary outcome is tolerance time, maximised to 3 min, which is assessed using the cold pressor test. In order to broaden the understanding of the lived experience of participants, qualitative interviews will be conducted using a phenomenological approach on a lifeworld theoretical basis (reflective lifeworld research approach). ETHICS AND DISSEMINATION: The protocol for the study is approved by the local ethical committee at Linkoping (EPM; 2018/294-31, appendices 2019-00347 and 2020-04482). The principles of the Helsinki Declaration are followed regarding oral and written consent to participate, confidentiality and the possibility to withdraw participation from the study at any time. The results will primarily be communicated through peer-reviewed journals and conferences. TRIAL REGISTRATION NUMBER: NCT05008042.

Clinically relevant treatment of PDX models reveals patterns of neuroblastoma chemoresistance.

Chemotherapy resistance and relapses are common in high-risk neuroblastoma (NB). Here, we developed a clinically relevant in vivo treatment protocol mimicking the first-line five-chemotherapy treatment regimen of high-risk NB and applied this protocol to mice with MYCN-amplified NB patient-derived xenografts (PDXs). Genomic and transcriptomic analyses were used to reveal NB chemoresistance mechanisms. Intrinsic resistance was associated with high genetic diversity and an embryonic phenotype. Relapsed NB with acquired resistance showed a decreased adrenergic phenotype and an enhanced immature mesenchymal-like phenotype, resembling multipotent Schwann cell precursors. NBs with a favorable treatment response presented a lineage-committed adrenergic phenotype similar to normal neuroblasts. Novel integrated phenotypic gene signatures reflected treatment response and patient prognosis. NB organoids established from relapsed PDX tumors retained drug resistance, tumorigenicity, and transcriptional cell states. This work sheds light on the mechanisms of NB chemotherapy response and emphasizes the importance of transcriptional cell states in chemoresistance.

Simultaneous determination of folate and methotrexate metabolites in serum by LC-MS/MS during high-dose methotrexate therapy.

High-dose methotrexate (HDMTX) is a central component in the treatment of acute lymphoblastic leukemia, osteosarcoma, and some lymphomas and brain tumors. MTX is given at lethal doses and then is followed by rescue treatment with folinic acid (FA). Despite FA rescue, many patients suffer severe toxicity. The pharmacokinetics of FA rescue have not been sufficiently studied. However, optimization of FA rescue could potentially increase anti-tumor effects, whilst decreasing organ toxicity. Here, we describe our efforts to establish and optimize a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the simultaneous determination of five essential components of the folate cycle, as well as MTX and its two metabolites. The method was applied to 6 individual patients receiving HDMTX, with 3 or 4 measurements for each patient. The method allows analysis of samples that were initially frozen. This notion, together with the test results in the 6 pilot patients, shows the feasibility of this method to study MTX and FA pharmacokinetics during HDMTX treatment. The method has the potential to optimize HDMTX and FA rescue treatment in individual patients.

Anti-tumor effects of rigosertib in high-risk neuroblastoma.

High-risk neuroblastoma has a poor prognosis despite intense treatment, demonstrating the need for new therapeutic strategies. Here we evaluated the effects of rigosertib (ON-01910.Na) in preclinical models of high-risk neuroblastoma. Among several hundred cancer cell lines representing 24 tumor types, neuroblastoma was the most sensitive to rigosertib. Treatment of MYCN-amplified neuroblastoma organoids resulted in organoid disintegration, decreased cell viability, and increased apoptotic cell death. Neuroblastoma response to rigosertib involved G2M cell cycle arrest and decreased phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204). Rigosertib delayed tumor growth and prolonged survival of mice carrying neuroblastoma MYCN-amplified PDX tumors (median survival: 31 days, treated; 22 days, vehicle) accompanied with increased apoptosis in treated tumors. We further identified vincristine and rigosertib as a potential promising drug combination treatment. Our results show that rigosertib might be a useful therapeutic agent for MYCN-amplified neuroblastomas, especially in combination with existing agents.

Identification of the major rabbit and guinea pig semen coagulum proteins and description of the diversity of the REST gene locus in the mammalian clade Glires.

The seminal vesicle secretions of guinea pig and rabbit were analyzed for semen coagulum proteins. Using SDS-PAGE we discovered a previously not fully recognized semen coagulum protein, Svp5, in the guinea pig and a single predominant component, SVP200, in the rabbit. Potential genes of these proteins were identified in genome databases by their homology with human and murine genes. The structure of their fullength transcripts was determined using seminal vesicle cDNA and sequencing primers based on genomic sequences. Homology searching indicated that both Svp5 and SVP200 were synthesized from composite genes that were the result of merger between two genes showing homology with human SEMG2 and PI3. For a deeper understanding of the evolution of the genes, we retrieved and analyzed genome sequences from the REST gene loci, encompassing genes of semen coagulum proteins and related rapidly evolving seminal vesicle-transcribed genes, of 14 rodents and 2 lagomorphs. The analysis showed that rodents of the suborders myomorpha, hystricomorpha, and castorimorpha had unique sets of REST genes, whereas sciuromorpha seemed to be lacking such genes. It also indicated a closer relationship between myomorpha and castorimorpha than to rodents of the two other analyzed suborders. In the lagomorph species, the pika appeared to be devoid of REST genes, whereas the rabbit had a single expressed REST gene, SVP200, and two pseudogenes. The structural similarity of semen coagulum proteins in rabbit and hystricomph species suggests that they are closely related. This was also supported by other similarities at their REST gene loci, e.g. the finding of a PI3-like gene in the rabbit that also had features in common with caltrin2 of hystricomorph rodents. The homologies indicate that hystricomorpha may have separated from myomorpha and castorimorpha before the separation of hystricomorpha from lagomorpha.

Therapeutic targeting of KSP in preclinical models of high-risk neuroblastoma.

Neuroblastoma is a childhood malignancy with often dismal prognosis; relapse is common despite intense treatment. Here, we used human tumor organoids representing multiple MYCN-amplified high-risk neuroblastomas to perform a high-throughput drug screen with approved or emerging oncology drugs. Tumor-selective effects were calculated using drug sensitivity scores. Several drugs with previously unreported anti-neuroblastoma effects were identified by stringent selection criteria. ARRY-520, an inhibitor of kinesin spindle protein (KSP), was among those causing reduced viability. High expression of the KSP-encoding gene KIF11 was associated with poor outcome in neuroblastoma. Genome-scale loss-of-function screens in hundreds of human cancer cell lines across 22 tumor types revealed that KIF11 is particularly important for neuroblastoma cell viability. KSP inhibition in neuroblastoma patient-derived xenograft (PDX) cells resulted in the formation of abnormal monoastral spindles, mitotic arrest, up-regulation of mitosis-associated genes, and apoptosis. In vivo, KSP inhibition caused regression of MYCN-amplified neuroblastoma PDX tumors. Furthermore, treatment of mice harboring orthotopic neuroblastoma PDX tumors resulted in increased survival. Our results suggested that KSP inhibition could be a promising treatment strategy in children with high-risk neuroblastoma.

Discovery of epi-Enprioline as a Novel Drug for the Treatment of Vincristine Resistant Neuroblastoma.

Neuroblastoma is a childhood solid tumour originating from undifferentiated neural progenitor cells of the sympathetic nervous system. Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. In the present study, we aimed to identify novel drugs that can inhibit the growth and survival of chemoresistant neuroblastoma. High-throughput screening identified a small molecule, epi-enprioline that was able to induce apoptosis of vincristine-resistant neuroblastoma cells via the mitochondrial apoptotic pathway. Epi-enprioline reduced tumour growth in multiple preclinical models, including an orthotopic neuroblastoma patient-derived xenograft model in vivo. In summary, our data suggest that epi-enprioline can be considered as a lead compound for the treatment of vincristine-resistant neuroblastoma uncovering a novel strategy, which can be further explored as a treatment for drug-resistant neuroblastoma.

Tick-borne Encephalitis Vaccine Failures: A 10-year Retrospective Study Supporting the Rationale for Adding an Extra Priming Dose in Individuals Starting at Age 50 Years.

BACKGROUND: Southern Sweden is endemic for tick-borne encephalitis (TBE), with Stockholm County as one of the high-risk areas. Our aim in this study was to describe cases of vaccine failures and to optimize future vaccination recommendations. METHODS: Patients with TBE were identified in the notification database at the Department of Communicable Disease Control and Prevention in Stockholm County during 2006-2015. Vaccine failure was defined as TBE despite adherence to the recommended vaccination schedule with at least 2 doses. Clinical data were extracted from medical records. RESULTS: A total of 1004 TBE cases were identified, 53 (5%) were defined as vaccine failures. In this latter group, the median age was 62 years (6-83). Forty-three (81%) patients were aged >50 years and 2 were children. Approximately half of the patients had comorbidities, with diseases affecting the immune system accounting for 26% of all cases. Vaccine failures following the third or fourth vaccine dose accounted for 36 (68%) of the patients. Severe and moderate TBE disease affected 81% of the cases. CONCLUSIONS: To our knowledge, this is the largest documented cohort of TBE vaccine failures. Vaccine failure after 5 TBE vaccine doses is rare. Our data provide rationale for adding an extra priming dose to those aged ≥50 years.

Anti-tumor effects of PIM/PI3K/mTOR triple kinase inhibitor IBL-302 in neuroblastoma.

The PI3K pathway is a major driver of cancer progression. However, clinical resistance to PI3K inhibition is common. IBL-302 is a novel highly specific triple PIM, PI3K, and mTOR inhibitor. Screening IBL-302 in over 700 cell lines representing 47 tumor types identified neuroblastoma as a strong candidate for PIM/PI3K/mTOR inhibition. IBL-302 was more effective than single PI3K inhibition in vitro, and IBL-302 treatment of neuroblastoma patient-derived xenograft (PDX) cells induced apoptosis, differentiated tumor cells, and decreased N-Myc protein levels. IBL-302 further enhanced the effect of the common cytotoxic chemotherapies cisplatin, doxorubicin, and etoposide. Global genome, proteome, and phospho-proteome analyses identified crucial biological processes, including cell motility and apoptosis, targeted by IBL-302 treatment. While IBL-302 treatment alone reduced tumor growth in vivo, combination therapy with low-dose cisplatin inhibited neuroblastoma PDX growth. Complementing conventional chemotherapy treatment with PIM/PI3K/mTOR inhibition has the potential to improve clinical outcomes and reduce severe late effects in children with high-risk neuroblastoma.

The Interactome of Palmitoyl-Protein Thioesterase 1 (PPT1) Affects Neuronal Morphology and Function.

Palmitoyl-protein thioesterase 1 (PPT1) is a depalmitoylation enzyme that is mutated in cases of neuronal ceroid lipofuscinosis (NCL). The hallmarks of the disease include progressive neurodegeneration and blindness, as well as seizures. In the current study, we identified 62 high-confident PPT1-binding proteins. These proteins included a self-interaction of PPT1, two V-type ATPases, calcium voltage-gated channels, cytoskeletal proteins and others. Pathway analysis suggested their involvement in seizures and neuronal morphology. We then proceeded to demonstrate that hippocampal neurons from Ppt1-/- mice exhibit structural deficits, and further investigated electrophysiology parameters in the hippocampi of mutant mice, both in brain slices and dissociated postnatal primary cultures. Our studies reveal new mechanistic features involved in the pathophysiology of this devastating neurodegenerative disease.

Patient-Derived Xenograft Models Reveal Intratumor Heterogeneity and Temporal Stability in Neuroblastoma.

Patient-derived xenografts (PDX) and the Avatar, a single PDX mirroring an individual patient, are emerging tools in preclinical cancer research. However, the consequences of intratumor heterogeneity for PDX modeling of biomarkers, target identification, and treatment decisions remain underexplored. In this study, we undertook serial passaging and comprehensive molecular analysis of neuroblastoma orthotopic PDXs, which revealed strong intrinsic genetic, transcriptional, and phenotypic stability for more than 2 years. The PDXs showed preserved neuroblastoma-associated gene signatures that correlated with poor clinical outcome in a large cohort of patients with neuroblastoma. Furthermore, we captured spatial intratumor heterogeneity using ten PDXs from a single high-risk patient tumor. We observed diverse growth rates, transcriptional, proteomic, and phosphoproteomic profiles. PDX-derived transcriptional profiles were associated with diverse clinical characteristics in patients with high-risk neuroblastoma. These data suggest that high-risk neuroblastoma contains elements of both temporal stability and spatial intratumor heterogeneity, the latter of which complicates clinical translation of personalized PDX-Avatar studies into preclinical cancer research.Significance: These findings underpin the complexity of PDX modeling as a means to advance translational applications against neuroblastoma. Cancer Res; 78(20); 5958-69. ©2018 AACR.

Proteomics insights into infantile neuronal ceroid lipofuscinosis (CLN1) point to the involvement of cilia pathology in the disease.

Mutations in the depalmitoylation enzyme, palmitoyl protein thioesterase (PPT1), result in the early onset neurodegenerative disease known as Infantile Neuronal Ceroid Lipofuscinosis. Here, we provide proteomic evidence suggesting that PPT1 deficiency could be considered as a ciliopathy. Analysis of membrane proteins from brain enriched for acylated proteins from neonate Ppt1 knock out and control mice revealed a list of 88 proteins with differential expression levels. Amongst them, we identified Rab3IP, which regulates ciliogenesis in concert with Rab8 and Rab11. Immunostaining analysis revealed that PPT1 is localized in the cilia. Indeed, an unbiased proteomics analysis on isolated cilia revealed 660 proteins, which differed in their abundance levels between wild type and Ppt1 knock out. We demonstrate here that Rab3IP, Rab8 and Rab11 are palmitoylated, and that palmitoylation of Rab11 is required for correct intracellular localization. Cells and brain preparations from Ppt1-/- mice exhibited fewer cells with cilia and abnormally longer cilia, with both acetylated tubulin and Rab3IP wrongly distributed along the length of cilia. Most importantly, the analysis revealed a difference in the distribution and levels of the modified proteins in cilia in the retina of mutant mice versus the wildtype, which may be important in the early neurodegenerative phenotype. Overall, our results suggest a novel link between palmitoylated proteins, cilial organization and the pathophysiology of Neuronal Ceroid Lipofuscinosis.

Outbreak of enterovirus D68 of the new B3 lineage in Stockholm, Sweden, August to September 2016.

We report an enterovirus D68 (EV-D68) outbreak in Stockholm Sweden in 2016. Between 22 August and 25 September EV-D68 was detected in 74/495 respiratory samples analysed at the Karolinska University Hospital. During the peak week, 30/91 (33%) samples were EV-D68 positive. Viral protein (VP)P4/VP2 sequencing revealed that cases were caused by B3 lineage strains. Forty-four (59%) EV-D68-positive patients were children aged ≤ 5 years. Ten patients had severe respiratory or neurological symptoms and one died.

Changes in glycoprotein expression between primary breast tumour and synchronous lymph node metastases or asynchronous distant metastases.

BACKGROUND: Breast cancer is a very heterogeneous disease and some patients are cured by the surgical removal of the primary tumour whilst other patients suffer from metastasis and spreading of the disease, despite adjuvant therapy. A number of prognostic and treatment predictive factors have been identified such as tumour size, oestrogen (ER) and progesterone (PgR) receptor status, human epidermal growth factor receptor type 2 (HER2) status, histological grade, Ki67 and age. Lymph node involvement is also assessed during surgery to determine if the tumour has spread which requires dissection of the axilla and adjuvant treatment. The prognostic and treatment predictive factors assessing the nature of the tumour are all routinely based on the status of the primary tumour. RESULTS: We have analysed a unique tumour set of fourteen primary breast cancer tumours with matched synchronous axillary lymph node metastases and a set of nine primary tumours with, later developed, matched distant metastases from different sites in the body. We used a pairwise tumour analysis (from the same individual) since the difference between the same tumour-type in different patients was greater. Glycopeptide capture was used in this study to selectively isolate and quantify N-linked glycopeptides from tumours mixtures and the captured glycopeptides were subjected to label-free quantitative tandem mass spectrometry analysis. Differentially expressed proteins between primary tumours and matched lymph node metastasis and distant metastasis were identified. Two of the top hits, ATPIF1 and tubulin ß-chain were validated by immunohistochemistry to be differentially regulated. CONCLUSIONS: We show that the expression of a large number of glycosylated proteins change between primary tumours and matched lymph node metastases and distant metastases, confirming that cancer cells undergo a molecular transformation during the spread to a secondary site. The proteins are part of important pathways such as cell adhesion, migration pathways and immune response giving insight into molecular changes needed for the tumour to spread. The large difference between primary tumours and lymph node and distant metastases also suggest that treatment should be based on the phenotype of the lymph node and distant metastases.

Global genomic and transcriptomic analysis of human pancreatic islets reveals novel genes influencing glucose metabolism.

Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5'-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.

Discovery-based protein expression profiling identifies distinct subgroups and pathways in leiomyosarcomas.

UNLABELLED: Soft tissue sarcomas (STS) are malignant tumors of mesenchymal origin. A substantial portion of these tumors exhibits complex karyotypes and lack characterized chromosomal aberrations. Owing to such properties, both histopathologic and molecular classification of these tumors has been a significant challenge. This study examines the protein expression of a large number of human STS, including subtype heterogeneity, using two-dimensional gel proteomics. In addition, detailed proteome profiles of a subset of pleomorphic STS specimens using an in-depth mass-spectrometry approach identified subgroups within the leiomyosarcomas with distinct protein expression patterns. Pathways analysis indicates that key biologic nodes like apoptosis, cytoskeleton remodeling, and telomere regulation are differentially regulated among these subgroups. Finally, investigating the similarities between protein expression of leiomyosarcomas and undifferentiated pleomorphic sarcomas (UPS) revealed similar protein expression profiles for these tumors, in comparison with pleomorphic leiomyosarcomas. IMPLICATIONS: These results suggest that UPS tumors share a similar lineage as leiomyosarcomas and are likely to originate from different stages of differentiation from mesenchymal stem cells to smooth muscle cells.

A central role for GRB10 in regulation of islet function in man.

Variants in the growth factor receptor-bound protein 10 (GRB10) gene were in a GWAS meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type 2 diabetes (T2D) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. GRB10 is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. GRB10 knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. Together, these findings suggest that tissue-specific methylation and possibly imprinting of GRB10 can influence glucose metabolism and contribute to T2D pathogenesis. The data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father.

WNT5A-mediated ß-catenin-independent signalling is a novel regulator of cancer cell metabolism.

WNT5A has been identified as an important ligand in the malignant progression of a number of tumours. Although WNT5A signalling is often altered in cancer, the ligand's role as either a tumour suppressor or oncogene varies between tumour types and is a contemporary issue for investigators of ß-catenin-independent WNT signalling in oncology. Here, we report that one of the initial effects of active WNT5A signalling in malignant melanoma cells is an alteration in cellular energy metabolism and specifically an increase in aerobic glycolysis. This was found to be at least in part due to an increase in active Akt signalling and lactate dehydrogenase (LDH) activity. The clinical relevance of these findings was strengthened by a strong correlation (P < 0.001) between the expression of WNT5A and LDH isoform V in a cohort of melanocytic neoplasms. We also found effects of WNT5A on energy metabolism in breast cancer cells, but rather than promoting aerobic glycolysis as it does in melanoma, WNT5A signalling increased oxidative phosphorylation rates in breast cancer cells. These findings support a new role for WNT5A in the metabolic reprogramming of cancer cells that is a context- dependent event.