To investigate whether lactate dehydrogenase A (LDHA), an important component of the LDH tetramer crucial for aerobic glycolysis, is associated with patient outcome and constitutes a therapeutic target in neuroblastoma (NB). Expression of LDHA mRNA and protein was determined in 709 and 110 NB patient samples, respectively, and correlated with survival and risk factors. LDHA and LDHB were depleted in human NB cell lines by CRISPR/Cas9 and shRNA, respectively, and aerobic glycolysis, clonogenicity, and tumorigenicity were determined. Expression of LDHA in relation to MYCN was measured in NB cell lines and in the TH-MYCN NB mouse model. Expression of LDHA, both on the mRNA and the protein level, was significantly and independently associated with decreased patient survival. Predominant cytoplasmic localization of LDHA protein was associated with poor outcome. Amplification and expression of did not correlate with expression of LDHA in NB cell lines or TH-MYCN mice, respectively. Knockout of LDHA inhibited clonogenicity, tumorigenicity, and tumor growth without abolishing LDH activity or significantly decreasing aerobic glycolysis. Concomitant depletion of LDHA and the isoform LDHB ablated clonogenicity while not abrogating LDH activity or decreasing aerobic glycolysis. The isoform LDHC was not expressed. High expression of LDHA is independently associated with outcome of NB, and NB cells can be inhibited by depletion of LDHA or LDHB. This inhibition appears to be unrelated to LDH activity and aerobic glycolysis. Thus, investigations of inhibitory mechanisms beyond attenuation of aerobic glycolysis are warranted, both in NB and normal cells. .

BackgroundNeuroblastoma is thought to originate from neural crest-derived cells. CD57 defines migratory neural crest cells in normal development and is expressed in neuroblastoma.Methodology and Principal FindingsWe investigated the role of CD57 expression in neuroblastoma cells ex situ and in situ. Compared to CD57low U-NB1 neuroblastoma cells, CD57high cells developed tumors with decreased latency after orthotopic transplantation into adrenal glands of mice. In addition, CD57high U-NB1 and SK-N-BE(2)-C neuroblastoma cells were also more clonogenic, induced more spheres and were less lineage-restricted. CD57high cells attached better to endothelial cells and showed enhanced invasiveness. While invasion of U-NB1 cells was inhibited by blocking antibodies against CD57, neither invasion of SK-N-BE(2)-C cells nor adhesion of U-NB1 and SK-N-BE(2)-C cells was attenuated. After tail vein injection only CD57high cells generated liver metastases, while overall metastatic rate was not increased as compared to CD57low cells. In stroma-poor neuroblastoma of patients CD57high cells were associated with undifferentiated tumor cells across all stages and tended to be more frequent after chemotherapy.ConclusionStrong expression of CD57 correlates with aggressive attributes of U-NB1 and SK-N-BE(2)-C neuroblastoma cells and is linked with undifferentiated neuroblastoma cells in patients.

CHD5, a tumor suppressor at 1p36, is frequently lost or silenced in poor prognosis neuroblastoma (NB) and many adult cancers. The role of CHD5 in metastasis is unknown. We confirm that low expression of CHD5 is associated with stage 4 NB. Forced expression of CHD5 in NB cell lines with 1p loss inhibited key aspects of the metastatic cascade in vitro: anchorage-independent growth, migration, and invasion. In vivo, formation of bone marrow and liver metastases developing from intravenously injected NB cells was delayed and decreased by forced CHD5 expression. Genome-wide mRNA sequencing revealed reduction of genes and gene sets associated with metastasis when CHD5 was overexpressed. Known metastasis-suppressing genes preferentially upregulated in CHD5-overexpressing NB cells included PLCL1. In patient NB, low expression of PLCL1was associated with metastatic disease and poor survival. Knockdown of PLCL1 and of p53 in IMR5 NB cells overexpressing CHD5 reversed CHD5-induced inhibition of invasion and migration in vitro. In summary, CHD5 is a metastasis suppressor in NB.

© F e r r a t a S t o r t i F o u n d a t i o nproducts inhibit replication and spread of MV. Deficiency in suppression of the type I IFN response in normal cells is part of the attenuated phenotype of vaccine strains of MV. 40,41 Many solid cancer cells are known to have a decreased IFN response (reviewed by Pitha 42 ), which can be exploited for oncolytic virotherapy. 43,44 It is unknown whether ALL cells have a deficient type I IFN response impacting on their response to viral infection, whether they harbor defects in the RIG-I/MDA-5 pathway or whether other mechanisms of increased susceptibility to attenuated MV are operative in ALL cells.In this study, we set out to investigate the hitherto unknown susceptibility of pediatric ALL to attenuated MV. Using our large collection of primary pediatric ALL propagated in immunodeficient mice 45 we show that MVEdm is remarkably effective against acute B-lineage ALL in the pre-clinical setting. Methods ALL cell lines, xenografts and patient samplesThe ALL cells lines Jurkat, CCRF-CEM, MOLT-4, REH, RS4;11 and NALM-6 were purchased. ALL cells from patients propagated in non-obese diabetic/severe combined immunodeficient mice (NOD/SCID) mice were procured from spleen tissue at a purity of above 90%. Primary patient ALL samples were obtained at diagnosis from pediatric patients with de novo ALL. Most patients were enrolled in the ALL-BFM study protocols. Patients' characteristics for the xenografts are listed in the Online Supplementary Table S1. The human study protocol was approved by the Ethical Review Board of the University Medical Center Ulm in accordance with the Declaration of Helsinki. Cell lines, xenografts and primary ALL cells were cultured in RPMI 1640 with 10% fetal calf serum, L-glutamine, penicillin and streptomycin. Human ALL NOD/SCID mouse modelNOD/SCID mice at a median age of 8-10 weeks (w) were used. Housing and treatment of animals were in accordance with state guidelines. ALL cells were injected into a lateral tail vein. After grafting, blood samples were evaluated at 1-w or 2-w intervals for human leukemia cells by determining CD45 + Ly5 -cells using flow cytometry. At necropsy cell suspensions from spleen, bone marrow and meninges were prepared and brains were procured. The presence of leukemic cells in the suspensions was determined by FACS analysis. Virus infectionEx vivo cells were infected with MV-Edm at a MOI of 1. ALL cell lines, xenografts, patient samples, peripheral blood mononuclear cells (PBMC), B and T cells were infected in serum-free RPMI 1640 medium at 37°C for 3 hours (h). For hematopoietic stem cells (HSC) serum-free IMDM medium was used. Medium was changed depending on the experiment. Replication of MV-Edm in Jurkat cells and PBMCTo quantitate viral replication in Jurkat cells, lysates were harvested 3, 24, 48 and 72 h after infection and added to Vero indicator cells. Syncytia were determined 72 h later. To compare replication in Jurkat cells to PBMC, lysates of PBMC were collected 72 h after infection and added to Vero ...

As high-risk neuroblastoma (NB) has a poor prognosis, new therapeutic modalities are needed. We therefore investigated the susceptibility of NB cells to γ-secretase inhibitor I (GSI-I). NOTCH signaling activity, the cellular effects of GSI-I and its mechanisms of cytotoxicity were evaluated in NB cells in vitro and in vivo. The results show that NOTCH signaling is relevant for human NB cells. Of the GSIs screened in vitro GSI-I was the most effective inhibitor of NB cells. Both MYCN-amplified and non-amplified NB cells were susceptible to GSI-I. Among the targets of GSI-I in NB cells were NOTCH and the proteasome. GSI-I caused G2/M arrest that was enhanced by acute activation of MYCN and led to mitotic dysfunction. GSI-I also induced proapoptotic NOXA. Survival of mice bearing an MYCN non-amplified orthotopic patient-derived NB xenograft was significantly prolonged by systemic GSI-I, associated with mitotic catastrophe and reduced angiogenesis, and without evidence of intestinal toxicity. In conclusion, the activity of GSI-I on multiple targets in NB cells and the lack of gastrointestinal toxicity in mice are advantageous and merit further investigations of GSI-I in NB.