Extracorporeal membrane oxygenation in long-term COVID-19 with severe neutropenia and thrombocytopenia after allogeneic hematopoietic stem cell transplantation: a case report.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) was associated with potentially life-threatening complications. Among patients supported by extracorporeal membrane oxygenation (ECMO), those who underwent HSCT had a worse prognosis than those who did not. Advances in HSCT and critical care management have improved the prognosis of ECMO-supported HSCT patients. CASE: The patient in the remission stage of lymphoma after 22 months of allogeneic hematopoietic stem cell transplantation, suffered from ARDS, severe neutropenia, thrombocytopenia, and long-term COVID-19. We evaluated the benefits and risks of ECMO for the patient, including the possibility of being free from ECMO, the status of malignancy, the interval from HSCT to ARDS, the function of the graft, the amount of organ failure, and the comorbidities. ECMO was ultimately used to save his life. CONCLUSIONS: We did not advocate for the general use of ECMO in HSCT patients and we believed that highly selected patients, with well-controlled tumors, few comorbidities, and fewer risk factors for death, tended to benefit from ECMO with well ICU management.

Plasma biomarker panel for major depressive disorder by quantitative proteomics using ensemble learning algorithm: A preliminary study.

Major depressive disorder (MDD) is a major international public health issue; thus, investigating its underlying mechanisms and identifying suitable biomarkers to enable its early detection are imperative. Using data-independent acquisition-mass spectrometry-based proteomics, the plasma of 44 patients with MDD and 25 healthy controls was studied to detect differentially expressed proteins. Bioinformatics analyses, such as Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, Protein-Protein Interaction network, and weighted gene co-expression network analysis were employed. Moreover, an ensemble learning technique was used to build a prediction model. A panel of two biomarkers, L-selectin and an isoform of the Ras oncogene family was identified. With an area under the receiver operating characteristic curve of 0.925 and 0.901 for the training and test sets, respectively, the panel was able to distinguish MDD from the controls. Our investigation revealed numerous potential biomarkers and a diagnostic panel based on several algorithms, which may contribute to the future development of a plasma-based diagnostic approach and better understanding of the molecular mechanisms of MDD.

Value of endometrial thickness for the detection of endometrial cancer and atypical hyperplasia in asymptomatic postmenopausal women.

BACKGROUND: The role of transvaginal sonography (TVS) in screening endometrial cancer and hyperplasia is significant in postmenopausal women. The objective of this study is to determine the endometrium thickness (ET) cut-off to distinguish premalignancy and malignancy in asymptomatic postmenopausal women. METHODS: We retrospectively evaluated data of 968 eligible patients among 2537 asymptomatic postmenopausal women with ET ≥ 5 mm examined by TVS who were subjected to hysteroscopy and endometrial biopsy between January 1, 2017, and June 30, 2020 in an urban tertiary specialized hospital in China. The patients were divided into two groups according to the pathology outcomes: benign, and atypical hyperplasia (AH) and endometrial carcinoma (EC). The risk factors and the optimal cut-off of ET for detecting AH and EC were determined by logistic regression analysis and receiver operating characteristic curve. RESULTS: 2537 patients were offered hysteroscopy during a 42-month period. Finally, 968 patients were included for further analysis. Of these, 8 (0.8%) women were diagnosed with EC and 5 (0.5%) women with AH. The mean ET of AH and EC group was substantially higher than that in benign group (10.4 mm vs. 7.7 mm, P < 0.05). ET was significantly correlated with AH and EC shown by logistic regression analysis with an odds ratio (OR) of 1.252 (95% confidence interval [CI] 1.107-1.416, P < 0.001). The optimal cut-off value for AH and EC was found to be 8 mm with the maximum AUC of 0.715 (95% CI 0.686-0.743, P < 0.001), with a sensitivity of 0.846, a specificity of 0.609, positive likelihood ratio (LR+) of 2.164 and negative likelihood ratio (LR-) of 0.253. CONCLUSION: An ET cut-off of ≥ 8 mm shows a reasonable performance to detect AH and EC in asymptomatic postmenopausal women, thereby avoiding more invasive endometrial biopsy.

Cyclic di-AMP as endogenous adjuvant enhanced BCG-induced trained immunity and protection against Mycobacterium tuberculosis in mice.

Bacillus Calmette-Guérin (BCG) is a licensed prophylactic vaccine against tuberculosis (TB). Current TB vaccine efforts focus on improving BCG effects through recombination or genetic attenuation and/or boost with different vaccines. Recent years, it was revealed that BCG could elicit non-specific heterogeneous protection against other pathogens such as viruses through a process termed trained immunity. Previously, we constructed a recombinant BCG (rBCG-DisA) with elevated c-di-AMP as endogenous adjuvant by overexpressing di-adenylate cyclase of Mycobacterium tuberculosis DisA, and found that rBCG-DisA induced enhanced immune responses by subcutaneous route in mice after M. tuberculosis infection. In this study, splenocytes from rBCG-DisA immunized mice by intravenous route (i.v) elicited greater proinflammatory cytokine responses to homologous and heterologous re-stimulations than BCG. After M. tuberculosis infection, rBCG-DisA immunized mice showed hallmark responses of trained immunity including potent proinflammatory cytokine responses, enhanced epigenetic changes, altered lncRNA expressions and metabolic rewiring in bone marrow cells and other tissues. Moreover, rBCG-DisA immunization induced higher levels of antibodies and T cells responses in the lung and spleen of mice after M. tuberculosis infection. It was found that rBCG-DisA resided longer than BCG in the lung of M. tuberculosis infected mice implying prolonged duration of vaccine efficacy. Then, we found that rBCG-DisA boosting could prolong survival of BCG-primed mice over 90 weeks against M. tuberculosis infection. Our findings provided in vivo experimental evidence that rBCG-DisA with c-di-AMP as endogenous adjuvant induced enhanced trained immunity and adaptive immunity. What's more, rBCG-DisA showed promising potential in prime-boost strategy against M. tuberculosis infection in adults.

One step preparation of multifunctional poly (ether sulfone) thin films with potential for wound dressing.

The increasing demand for higher-quality medical care has resulted in the obsolescence of traditional biomaterials. Medical care is currently transitioning from an era depending on single-functional biomaterials to one that is supported by multifunctional and stable biomaterials. Herein, long-lasting multifunctional poly(ether sulfone) thin films (MPFs) containing heparin-mimic groups and a quaternary ammonium compound (QAC) were prepared via semi-interpenetrating polymer network (SIPN) strategy. The MPFs, with rough surface and inner finger-like macrovoid, had better hydrophilicity and anti-protein fouling ability, as revealed by scanning electron microscopy (SEM), atomic force microscope (AFM) and water contact angle (WCA) and protein adsorption tests. The results of platelet adhesion and activation, and clotting time confirmed that the hemocompatibility of the MPFs was significantly improved. From cell culture and germ-culture test, it was noted that the overall trend of human umbilical vein endothelial cell (HUVEC) proliferation was enhanced by a combination of heparin-mimic groups and QAC, whereas the growth of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was significantly prohibited. In addition, the MPFs were capable of modulating the expression level of basic fibroblast growth factor (bFGF) and transforming growth factor-beta1 (TGF-ß1) in fibroblast, which was beneficial to controlling the formation of hypertrophic scar. In summary, the MPFs had potential to be used in the field of wound management and the study might help guide the design of surface structure of wound dressing.

ANO1: More Than Just Calcium-Activated Chloride Channel in Cancer.

ANO1, a calcium-activated chloride channel (CACC), is also known as transmembrane protein 16A (TMEM16A). It plays a vital role in the occurrence, development, metastasis, proliferation, and apoptosis of various malignant tumors. This article reviews the mechanism of ANO1 involved in the replication, proliferation, invasion and apoptosis of various malignant tumors. Various molecules and Stimuli control the expression of ANO1, and the regulatory mechanism of ANO1 is different in tumor cells. To explore the mechanism of ANO1 overexpression and activation of tumor cells by studying the different effects of ANO1. Current studies have shown that ANO1 expression is controlled by 11q13 gene amplification and may also exert cell-specific effects through its interconnected protein network, phosphorylation of different kinases, and signaling pathways. At the same time, ANO1 also resists tumor apoptosis and promotes tumor immune escape. ANO1 can be used as a promising biomarker for detecting certain malignant tumors. Further studies on the channels and the mechanism of protein activity of ANO1 are needed. Finally, the latest inhibitors of ANO1 are summarized, which provides the research direction for the tumor-promoting mechanism of ANO1.

Uncovering the Gene Regulatory Network of Maize Hybrid ZD309 under Heat Stress by Transcriptomic and Metabolomic Analysis.

Maize is an important cereal crop but is sensitive to heat stress, which significantly restricts its grain yield. To explore the molecular mechanism of maize heat tolerance, a heat-tolerant hybrid ZD309 and its parental lines (H39_1 and M189) were subjected to heat stress, followed by transcriptomic and metabolomic analyses. After six-day-heat treatment, the growth of ZD309 and its parental lines were suppressed, showing dwarf stature and rolled leaf compared with the control plants. ZD309 exhibited vigorous growth; however, M189 displayed superior heat tolerance. By transcriptomic and metabolomic analysis, hundreds to thousands of differentially expressed genes (DEGs) and metabolites (DEMs) were identified. Notably, the female parent H39 shares more DEGs and DEMs with the hybrid ZD309, indicating more genetic gain derived from the female instead of the male. A total of 299 heat shock genes detected among three genotypes were greatly aggregated in sugar transmembrane transporter activity, plasma membrane, photosynthesis, protein processing in the endoplasmic reticulum, cysteine, and methionine metabolism. A total of 150 heat-responsive metabolites detected among three genotypes were highly accumulated, including jasmonic acid, amino acids, sugar, flavonoids, coumarin, and organic acids. Integrating transcriptomic and metabolomic assays revealed that plant hormone signal transduction, cysteine, and methionine metabolism, and α-linolenic acid metabolism play crucial roles in heat tolerance in maize. Our research will be facilitated to identify essential heat tolerance genes in maize, thereby contributing to breeding heat resistance maize varieties.

The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma.

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin-RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136-400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.

Effect of the interference with DRP1 expression on the biological characteristics of glioma stem cells.

In the present study, a model of glioma stem cells (GSCs) was established and combined with molecular targeting drugs in order to observe its inhibitory effect on the proliferation and biological characteristics of GSCs, with the aim of providing a potential target for the treatment of glioma. On the basis of a relatively classical induction strategy with neuron induction medium, a large number of GSC-like cells in good condition and globular growth were amplified in vitro, which had the potential to differentiate into neurons, oligodendrocytes and astrocytes/glioma cells. It was observed that the interference with dynamin-related protein 1 expression using Mdivi-1, a mitochondrial mitotic inhibitor, at the optimal concentration, decreased the expression level of stem cell-associated genes, inhibited proliferation and promoted apoptosis in GSCs. The present study provided an experimental basis for a novel strategy of cancer treatment with tumor stem cells as the target.

Feasibility Analysis of Cell-Free DNA Derived from Plasma of Lung Cancer Patients for Next-Generation Sequencing.

Purpose: The quality of specimens directly affects the experimental results. The stability and structural integrity of nucleic acids in samples have a decisive influence on high-throughput sequencing results. Next-generation sequencing (NGS) provides the most comprehensive criteria for evaluating the specimen quality. To test the quality of cell-free DNA (cfDNA) from lung cancer plasma samples stored in our biobank, we conducted a study to evaluate the quality in terms of the genetic level. Methods: A total of 189 peripheral blood samples were collected from patients from patients with EGFR-positive nonsmall cell lung cancer who were seen and treated in Jilin Provincial Cancer Hospital from August 2012 to March 2018. Twelve milliliters of peripheral blood samples were collected and centrifuged at 4°C, 2000 rpm for 15 minutes. Plasma samples were dispensed into cryotubes and stored at -80°C. Plasma cfDNA was extracted by a DNA extraction kit (Qiagen) and the DNA concentration was detected by a Qubit 3.0 fluorometer. Results: The total volume of cfDNA extraction at baseline was 50 µL, the median concentration according to Qubit was 0.633 ng/µL, the range was 0.331-6.09 ng/µL, and the median total DNA was 34.25 ng, ranging from 20.35 to 304.5 ng. The median value of the Qubit concentration in advanced plasma samples was 0.838 ng/µL, ranging from 0.24 to 21.9 ng/µL, and median total DNA was 41.9 ng, ranging from 12.0 to 1095.0 ng. Based on the aforementioned quality assessment factors, 4 of 189 frozen lung cancer baseline plasma samples were not included in further analyses, and for the remaining 185 cases of cfDNA >20 ng, the pass rate was 97.9%. In 143 frozen lung cancer advanced stage plasma samples, 133 cases of cfDNA >20 ng, the pass rate was 93%. Conclusion: Frozen lung cancer plasma samples stored in the biobank for 1-6 years at -80°C under certain conditions still retain a high level of cfDNA, which is suitable for NGS detection.

De novo variants in the Helicase-C domain of CHD8 are associated with severe phenotypes including autism, language disability and overgrowth.

CHD8, which encodes Chromodomain helicase DNA-binding protein 8, is one of a few well-established Autism Spectrum Disorder (ASD) genes. Over 60 mutations have been reported in subjects with variable phenotypes, but little is known concerning genotype-phenotype correlations. We have identified four novel de novo mutations in Chinese subjects: two nonsense variants (c.3562C>T/p.Arg1188X, c.2065C>A/p.Glu689X), a splice site variant (c.4818-1G>A) and a missense variant (c.3502T>A/p.Tyr1168Asn). Three of these were identified from a 445-member ASD cohort by ASD gene panel sequencing of the 96 subjects who remained negative after molecular testing for copy number variation, Rett syndrome, FragileX and tuberous sclerosis complex (TSC). The fourth (p.Glu689X) was detected separately by diagnostic trio exome sequencing. We used diagnostic instruments and a comprehensive review of phenotypes, including prenatal and postnatal growth parameters, developmental milestones, and dysmorphic features to compare these four subjects. In addition to autism, they also presented with prenatal onset macrocephaly, intellectual disability, overgrowth during puberty, sleep disorder, and dysmorphic features, including broad forehead with prominent supraorbital ridges, flat nasal bridge, telecanthus and large ears. For further comparison, we compiled a comprehensive list of CHD8 variants from the literature and databases, which revealed constitutive and somatic truncating variants in the HELIC (Helicase-C) domain in ASD and in cancer patients, respectively, but not in the general population. Furthermore, HELIC domain mutations were associated with a severe phenotype defined by a greater number of clinical features, lower verbal IQ, and a prominent, consistent pattern of overgrowth as measured by weight, height and head circumference. Overall, this study adds to the ASD-associated loss-of-function mutations in CHD8 and highlights the clinical importance of the HELIC domain of CHD8.

Cesarean scar endometriosis: presentation of 198 cases and literature review.

BACKGROUND: Cesarean scar endometriosis (CSE) is the most common type of abdominal wall endometriosis (AWE). The aim of this study was to systematically identify the clinical features of CSE and recommend precautionary measures. METHODS: A large, retrospective study was undertaken with CSE patients treated surgically at our hospital between January 2005 and December 2017. RESULTS: A total of 198 CSE patients were enrolled, with a mean age of 32.0 ± 4.0 years. The main complaint of the patients was abdominal mass (98.5%), followed by cyclic pain (86.9%). The latency period of CSE was 31.6 ± 23.9 months, and the duration between the onset of symptoms and this surgery was 28.3 ± 25.0 months. A majority (80.8%, n = 160) of the patients had undergone a Pfannenstiel incision, and a minority (19.2%, n = 38) a vertical midline incision. The latency period of CSE in the case of a Pfannenstiel incision was significantly shorter than that in the case of a vertical midline incision (24.0 vs 33.0 months, P = 0.006). A total of 187 (94.4%) patients had a single endometrioma, 11 (5.6%) patients had multiple endometriomas, and the 11 multiple-endometrioma patients had all undergone a Pfannenstiel incision. Lesions of endometrioma were common in corner sites, after either incision: 142/171 (83.0%) in Pfannenstiel incision scars and 32/38 (84.2%) in vertical incision scars. CONCLUSIONS: The findings of this study indicate that the Pfannenstiel incision carries a higher risk of CSE than the vertical midline incision. Thorough cleaning at the conclusion of CS, particularly of both corner sites of the adipose layer and the fascia layer, is strongly recommended for CSE prevention. Further studies might provide additional recommendations.

Overexpression of PRDM13 inhibits glioma cells via Rho and GTP enzyme activation protein.

PR (PRDI­BFI and RIZ) domain containing (PRDM) proteins have been shown to be important in several types of human cancer. PRDM13, a member of the PRDM family, contains transcriptional regulators involved in modulating several cellular processes. However, the function of PRDM13 in glioma remains to be elucidated. The purpose of the present study was to evaluate the expression and effect of PRDM13 on glioma cells. It was found that the expression of PRDM13 was reduced in glioma cells, and the overexpression of PRDM13 significantly decreased the proliferation, migration and invasion of U87 glioma cells. Through validation of RNA­sequencing analysis, genes regulating cell proliferation and migration were classified from Gene Ontology sources. In addition, PRDM13 was shown to be associated with Rho protein and GTP enzyme activation protein. The over-expression of PRDM13 upregulated deleted in liver cancer 1 (DLC1) to inhibit the proliferation and invasion of U87 cells. In conclusion, PRDM13 decreased the proliferation and invasion of U87 cells, and may be of potential value for glioma therapy.

Concurrent Inhibition of Neurosphere and Monolayer Cells of Pediatric Glioblastoma by Aurora A Inhibitor MLN8237 Predicted Survival Extension in PDOX Models.

Purpose: Pediatric glioblastoma multiforme (pGBM) is a highly aggressive tumor in need of novel therapies. Our objective was to demonstrate the therapeutic efficacy of MLN8237 (alisertib), an orally available selective inhibitor of Aurora A kinase (AURKA), and to evaluate which in vitro model system (monolayer or neurosphere) can predict therapeutic efficacy in vivoExperimental Design: AURKA mRNA expressions were screened with qRT-PCR. In vitro antitumor effects were examined in three matching pairs of monolayer and neurosphere lines established from patient-derived orthotopic xenograft (PDOX) models of the untreated (IC-4687GBM), recurrent (IC-3752GBM), and terminal (IC-R0315GBM) tumors, and in vivo therapeutic efficacy through log rank analysis of survival times in two models (IC-4687GBM and IC-R0315GBM) following MLN8237 treatment (30 mg/kg/day, orally, 12 days). Drug concentrations in vivo and mechanism of action and resistance were also investigated.Results: AURKA mRNA overexpression was detected in 14 pGBM tumors, 10 PDOX models, and 6 cultured pGBM lines as compared with 11 low-grade gliomas and normal brains. MLN8237 penetrated into pGBM xenografts in mouse brains. Significant extension of survival times were achieved in IC-4687GBM of which both neurosphere and monolayer were inhibited in vitro, but not in IC-R0315GBM of which only neurosphere cells responded (similar to IC-3752GBM). Apoptosis-mediated MLN8237 induced cell death, and the presence of AURKA-negative and CD133+ cells appears to have contributed to in vivo therapy resistance.Conclusions: MLN8237 successfully targeted AURKA in a subset of pGBMs. Our data suggest that combination therapy should aim at AURKA-negative and/or CD133+ pGBM cells to prevent tumor recurrence. Clin Cancer Res; 24(9); 2159-70. ©2018 AACR.

Dimethylation of Histone 3 Lysine 9 is sensitive to the epileptic activity, and affects the transcriptional regulation of the potassium channel Kcnj10 gene in epileptic rats.

Potassium channels can be affected by epileptic seizures and serve a crucial role in the pathophysiology of epilepsy. Dimethylation of histone 3 lysine 9 (H3K9me2) and its enzyme euchromatic histone­lysine N­methyltransferase 2 (G9a) are the major epigenetic modulators and are associated with gene silencing. Insight into whether H3K9me2 and G9a can respond to epileptic seizures and regulate expression of genes encoding potassium channels is the main purpose of the present study. A total of 16 subtypes of potassium channel genes in pilocarpine­modelled epileptic rats were screened by reverse transcription­quantitative polymerase chain reaction, and it was determined that the expression ATP­sensitive inward rectifier potassium channel 10 (Kcnj10) increased in hippocampus and insular cortex, while the expression of most of the other subtypes decreased. The total level of H3K9me2 decreased in the model group compared with the control. The Kcnj10 gene encoding the Kir4.1 channel was selected to analyse changes in H3K9me2 in the promoter region by the chromatin immuno­precipitation method. Anti­H3K9me2 and anti­G9a antibodies were used to identify the modified DNAs. Five primers were designed across the promoter region of the Kcnj10 gene. In epileptic hippocampi, the relative abundance of H3K9me2 and G9a in the promoter region of Kcnj10 decreased markedly. Removal of the H3K9me2 repressive mark resulted in decreased transcriptional inhibition of the Kcnj10 gene and therefore increased its expression. In the cultured C6 cells, specific inhibition of the enzymatic activity of G9a by 2­(Hexahydro­4­methyl­1H­1,4­diazepin­1­yl)­6,7­di­ methoxy­N­(1­(phenyl­methyl)­4­piperidinyl)­4­quinazolinamine tri­hydrochloride hydrate (bix01294) resulted in upregulation of the expression of Kir4.1 proteins. The present study demonstrated that H3K9me2 and G9a are sensitive to epileptic seizure activity during the acute phase of epilepsy and can affect the transcriptional regulation of the Kcnj10 channel.

The novel kinase inhibitor ponatinib is an effective anti-angiogenic agent against neuroblastoma.

Background High-risk neuroblastoma has poor outcomes with high rates of relapse despite aggressive treatment, and novel therapies are needed to improve these outcomes. Ponatinib is a multi-tyrosine kinase inhibitor that targets many pathways implicated in neuroblastoma pathogenesis. We hypothesized that ponatinib would be effective against neuroblastoma in preclinical models. Methods We evaluated the effects of ponatinib on survival and migration of human neuroblastoma cells in vitro. Using orthotopic xenograft mouse models of human neuroblastoma, we analyzed tumors treated with ponatinib for growth, gross and histologic appearance, and vascularity. Results Ponatinib treatment of neuroblastoma cells resulted in decreased cell viability and migration in vitro. In mice with orthotopic xenograft neuroblastoma tumors, treatment with ponatinib resulted in decreased growth and vascularity. Conclusions Ponatinib reduces neuroblastoma cell viability in vitro and reduces tumor growth and vascularity in vivo. The antitumor effects of ponatinib suggest its potential as a novel therapeutic agent for neuroblastoma, and further preclinical testing is warranted.

Nifurtimox Is Effective Against Neural Tumor Cells and Is Synergistic with Buthionine Sulfoximine.

Children with aggressive neural tumors have poor survival rates and novel therapies are needed. Previous studies have identified nifurtimox and buthionine sulfoximine (BSO) as effective agents in children with neuroblastoma and medulloblastoma. We hypothesized that nifurtimox would be effective against other neural tumor cells and would be synergistic with BSO. We determined neural tumor cell viability before and after treatment with nifurtimox using MTT assays. Assays for DNA ladder formation and poly-ADP ribose polymerase (PARP) cleavage were performed to measure the induction of apoptosis after nifurtimox treatment. Inhibition of intracellular signaling was measured by Western blot analysis of treated and untreated cells. Tumor cells were then treated with combinations of nifurtimox and BSO and evaluated for viability using MTT assays. All neural tumor cell lines were sensitive to nifurtimox, and IC50 values ranged from approximately 20 to 210 µM. Nifurtimox treatment inhibited ERK phosphorylation and induced apoptosis in tumor cells. Furthermore, the combination of nifurtimox and BSO demonstrated significant synergistic efficacy in all tested cell lines. Additional preclinical and clinical studies of the combination of nifurtimox and BSO in patients with neural tumors are warranted.

Binimetinib inhibits MEK and is effective against neuroblastoma tumor cells with low NF1 expression.

BACKGROUND: Novel therapies are needed for children with high-risk and relapsed neuroblastoma. We hypothesized that MAPK/ERK kinase (MEK) inhibition with the novel MEK1/2 inhibitor binimetinib would be effective in neuroblastoma preclinical models. METHODS: Levels of total and phosphorylated MEK and extracellular signal-regulated kinase (ERK) were examined in primary neuroblastoma tumor samples and in neuroblastoma cell lines by Western blot. A panel of established neuroblastoma tumor cell lines was treated with increasing concentrations of binimetinib, and their viability was determined using MTT assays. Western blot analyses were performed to examine changes in total and phosphorylated MEK and ERK and to measure apoptosis in neuroblastoma tumor cells after binimetinib treatment. NF1 protein levels in neuroblastoma cell lines were determined using Western blot assays. Gene expression of NF1 and MEK1 was examined in relationship to neuroblastoma patient outcomes. RESULTS: Both primary neuroblastoma tumor samples and cell lines showed detectable levels of total and phosphorylated MEK and ERK. IC50 values for cells sensitive to binimetinib ranged from 8 nM to 1.16 µM, while resistant cells did not demonstrate any significant reduction in cell viability with doses exceeding 15 µM. Sensitive cells showed higher endogenous expression of phosphorylated MEK and ERK. Gene expression of NF1, but not MEK1, correlated with patient outcomes in neuroblastoma, and NF1 protein expression also correlated with responses to binimetinib. CONCLUSIONS: Neuroblastoma tumor cells show a range of sensitivities to the novel MEK inhibitor binimetinib. In response to binimetinib, sensitive cells demonstrated complete loss of phosphorylated ERK, while resistant cells demonstrated either incomplete loss of ERK phosphorylation or minimal effects on MEK phosphorylation, suggesting alternative mechanisms of resistance. NF1 protein expression correlated with responses to binimetinib, supporting the use of NF1 as a biomarker to identify patients that may respond to MEK inhibition. MEK inhibition therefore represents a potential new therapeutic strategy for neuroblastoma.

A Polymorphism in the FGFR4 Gene Is Associated With Risk of Neuroblastoma and Altered Receptor Degradation.

BACKGROUND: Outcomes for children with high-risk neuroblastoma are poor, and improved understanding of the mechanisms underlying neuroblastoma pathogenesis, recurrence, and treatment resistance will lead to improved outcomes. Aberrant growth factor receptor expression and receptor tyrosine kinase signaling are associated with the pathogenesis of many malignancies. A germline polymorphism in the FGFR4 gene is associated with increased receptor expression and activity and with decreased survival, treatment resistance, and aggressive disease for many malignancies. We therefore investigated the role of this FGFR4 polymorphism in neuroblastoma pathogenesis. MATERIALS AND METHODS: Germline DNA from neuroblastoma patients and matched controls was assessed for the FGFR4 Gly/Arg388 polymorphism by RT-PCR. Allele frequencies were assessed for association with neuroblastoma patient outcomes and prognostic features. Degradation rates of the FGFR4 Arg388 and Gly388 receptors and rates of receptor internalization into the late endosomal compartment were measured. RESULTS: Frequency of the FGFR4 AA genotype and the prevalence of the A allele were significantly higher in patients with neuroblastoma than in matched controls. The Arg388 receptor demonstrated slower degradation than the Gly388 receptor in neuroblastoma cells and reduced internalization into multivesicular bodies. CONCLUSIONS: The FGFR4 Arg388 polymorphism is associated with an increased prevalence of neuroblastoma in children, and this association may be linked to differences in FGFR4 degradation rates. Our study provides the first evidence of a role for FGFR4 in neuroblastoma, suggesting that FGFR4 genotype and the pathways regulating FGFR4 trafficking and degradation may be relevant for neuroblastoma pathogenesis.