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Liquid biopsy strategies in pediatric patients are challenging due to low body weight. This study investigated cfDNA size distribution and concentration in blood, bone marrow, cerebrospinal fluid, and urine from 84 patients with neuroblastoma classified as low (n = 28), intermediate (n = 6), or high risk (n = 50) to provide key data for liquid biopsy biobanking strategies. The average volume of blood and bone marrow plasma provided ranged between 1 and 2 mL. Analysis of 637 DNA electropherograms obtained by Agilent TapeStation measurement revealed five different major profiles and characteristic DNA size distribution patterns for each of the biofluids. The proportion of samples containing primarily cfDNA was, at 85.5%, the highest for blood plasma. The median cfDNA concentration amounted to 6.28 ng/mL (blood plasma), 58.2 ng/mL (bone marrow plasma), 0.08 ng/mL (cerebrospinal fluid), and 0.49 ng/mL (urine) in samples. Meta-analysis of the dataset demonstrated that multiple cfDNA-based assays employing the same biofluid sample optimally require sampling volumes of 1 mL for blood and bone marrow plasma, 2 mL for cerebrospinal fluid, and as large as possible for urine samples. A favorable response to treatment was associated with a rapid decrease in blood-based cfDNA concentration in patients with high-risk neuroblastoma. Blood-based cfDNA concentration was not sufficient as a single parameter to indicate high-risk disease recurrence. We provide proof of concept that monitoring neuroblastoma-specific markers in very small blood volumes from infants is feasible.

Simple Summary: Solid tumor cells can lose or heterogeneously express antigens to become resistant to chimeric antigen receptor (CAR) T cell therapy. Here, we explore whether epigenetic manipulation to unleash antigen-independent killing mechanisms can overcome this hurdle. KDM1A is overexpressed in many cancers and removes lysine methylation on histones that keeps the DNA firmly packed to selectively activate or repress gene activity, depending on the specific lysine target. KDM1A also regulates the expression of nonhistone proteins. We inhibited KDM1A in the childhood tumor, neuroblastoma, to increase FAS expression on tumor cells. The FAS receptor can be triggered to induce cell death when bound by the FAS ligand on CAR and other activated T cells present in the tumor environment, even if the tumor cells lack the target antigen. FAS upregulation via KDM1A inhibition sensitized neuroblastoma cells to FAS-FASL-mediated killing and augmented CAR T cell therapy against antigen-poor or even antigen-negative neuroblastoma. Abstract: Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising treatment strategy, however, therapeutic success against solid tumors such as neuroblastoma remains modest. Recurrence of antigen-poor tumor variants often ultimately results in treatment failure. Using antigen-independent killing mechanisms such as the FAS receptor (FAS)-FAS ligand (FASL) axis through epigenetic manipulation may be a way to counteract the escape achieved by antigen downregulation. Analysis of public RNA-sequencing data from primary neuroblastomas revealed that a particular epigenetic modifier, the histone lysine demethylase 1A (KDM1A), correlated negatively with FAS expression. KDM1A is known to interact with TP53 to repress TP53-mediated transcriptional activation of genes, including FAS. We showed that pharmacologically blocking KDM1A activity in neuroblastoma cells with the small molecule inhibitor, SP-2509, increased FAS cell-surface expression in a strictly TP53-dependent manner. FAS upregulation sensitized neuroblastoma cells to FAS-FASL-dependent killing and augmented L1CAM-directed CAR T cell therapy against antigen-poor or even antigen-negative tumor cells in vitro. The improved therapeutic response was abrogated when the FAS-FASL interaction was abolished with an antagonistic FAS antibody. Our results show that KDM1A inhibition unleashes an antigen-independent killing mechanism via the FAS-FASL axis to make tumor cell variants that partially or totally suppress antigen expression susceptible to CAR T cell therapy.

The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.

Simple Summary The childhood tumor, neuroblastoma, has a broad clinical presentation. Risk assessment at diagnosis is particularly difficult in molecularly heterogeneous high-risk cases. Here we investigate the potential of imaging mass spectrometry to directly detect intratumor heterogeneity on the protein level in tissue sections. We show that this approach can produce discriminatory peptide signatures separating high- from low- and intermediate-risk tumors, identify 8 proteins aassociated with these signatures and validate two marker proteins using tissue immunostaining that have promise for further basic and translational research in neuroblastoma. We provide proof-of-concept that mass spectrometry-based technology could assist early risk assessment in neuroblastoma and provide insights into peptide signature-based detection of intratumor heterogeneity. Abstract Risk classification plays a crucial role in clinical management and therapy decisions in children with neuroblastoma. Risk assessment is currently based on patient criteria and molecular factors in single tumor biopsies at diagnosis. Growing evidence of extensive neuroblastoma intratumor heterogeneity drives the need for novel diagnostics to assess molecular profiles more comprehensively in spatial resolution to better predict risk for tumor progression and therapy resistance. We present a pilot study investigating the feasibility and potential of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to identify spatial peptide heterogeneity in neuroblastoma tissues of divergent current risk classification: high versus low/intermediate risk. Univariate (receiver operating characteristic analysis) and multivariate (segmentation, principal component analysis) statistical strategies identified spatially discriminative risk-associated MALDI-based peptide signatures. The AHNAK nucleoprotein and collapsin response mediator protein 1 (CRMP1) were identified as proteins associated with these peptide signatures, and their differential expression in the neuroblastomas of divergent risk was immunohistochemically validated. This proof-of-concept study demonstrates that MALDI-MSI combined with univariate and multivariate analysis strategies can identify spatially discriminative risk-associated peptide signatures in neuroblastoma tissues. These results suggest a promising new analytical strategy improving risk classification and providing new biological insights into neuroblastoma intratumor heterogeneity.

The immunosuppressive microenvironment in solid tumors is thought to form a barrier to the entry and efficacy of cell-based therapies such as chimeric antigen receptor (CAR) T cells. Combining CAR T cell therapy with checkpoint inhibitors has been demonstrated to oppose immune escape mechanisms in solid tumors and augment antitumor efficacy. We evaluated PD-1/PD-L1 signaling capacity and the impact of an inhibitor of this checkpoint axis in an in vitro system for cancer cell challenge, the coculture of L1CAM-specific CAR T cells with neuroblastoma cell lines. Fluorescence-activated cell sorting-based analyses and luciferase reporter assays were used to assess PD-1/PD-L1 expression on CAR T and tumor cells as well as CAR T cell ability to kill neuroblastoma cells. Coculturing neuroblastoma cell lines with L1CAM-CAR T cells upregulated PD-L1 expression on neuroblastoma cells, confirming adaptive immune resistance. Exposure to neuroblastoma cells also upregulated the expression of the PD-1/PD-L1 axis in CAR T cells. The checkpoint inhibitor, nivolumab, enhanced L1CAM-CAR T cell-directed killing. However, nivolumab-enhanced L1CAM-CAR T cell killing did not strictly correlate with PD-L1 expression on neuroblastoma cells. In fact, checkpoint inhibitor success relied on strong PD-1/PD-L1 axis expression in the CAR T cells, which in turn depended on costimulatory domains within the CAR construct, and more importantly, on the subset of T cells selected for CAR T cell generation. Thus, T cell subset selection for CAR T cell generation and CAR T cell prescreening for PD-1/PD-L1 expression could help determine when combination therapy with checkpoint inhibitors could improve treatment efficacy.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase that promotes G2/M-phase transition, is expressed in elevated levels in high-risk neuroblastomas and correlates with unfavorable patient outcome. Recently, we and others have presented PLK1 as a potential drug target for neuroblastoma, and reported that the BI2536 PLK1 inhibitor showed antitumoral actvity in preclinical neuroblastoma models. Here we analyzed the effects of GSK461364, a competitive inhibitor for ATP binding to PLK1, on typical tumorigenic properties of preclinical in vitro and in vivo neuroblastoma models. GSK461364 treatment of neuroblastoma cell lines reduced cell viability and proliferative capacity, caused cell cycle arrest and massively induced apoptosis. These phenotypic consequences were induced by treatment in the low-dose nanomolar range, and were independent of MYCN copy number status. GSK461364 treatment strongly delayed established xenograft tumor growth in nude mice, and significantly increased survival time in the treatment group. These preclinical findings indicate PLK1 inhibitors may be effective for patients with high-risk or relapsed neuroblastomas with upregulated PLK1 and might be considered for entry into early phase clinical trials in pediatric patients. OA gold

OA gold The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.

Treatment for neuroblastoma, the most common extracranial childhood tumor, spans a broad range of aggressiveness that mirrors the risk profiles of disease subtypes, with high-risk neuroblastoma still presenting a clinical challenge. Currently, most patients with relapsed neuro-blastoma die of disease and present a major challenge for treatment. New therapeutic options are urgently needed to improve patient survival. Activating mutations in the gene encoding the anaplastic lymphoma kinase (ALK) remain the most frequent druggable mutations identified in neuroblastomas to date. Preclinical data support an oncogene addiction of neuroblastoma cells to mutated ALK and demonstrate that ALK inhibitory therapy strongly combats tumor models. Most recently, pediatric phase I testing has been completed for the first approved ALK inhibitor, Crizotinib, showing very encouraging antitumoral results in neuroblastoma patients. Subsequently, an international phase I study with the second generation ALK inhibitor, LDK-378, will be launched that makes ALK inhibitory therapy also available to pediatric patients in Germany.

MYCN amplification is a common feature of aggressive tumour biology in neuroblastoma. The MYCN transcription factor has been demonstrated to induce or repress expression of numerous genes. MicroRNAs (miRNA) are a recently discovered class of short RNAs that repress translation and promote mRNA degradation by sequence-specific interaction with mRNA. Here, we sought to analyse the role of MYCN in regulation of miRNA expression. Using a miRNA microarray containing 384 different miRNAs and a set of 160 miRNA real-time PCR assays to validate the microarray results, 7 miRNAs were identified that are induced by MYCN in vitro and are upregulated in primary neuroblastomas with MYCN amplification. Three of the seven miRNAs belong to the miR-106a and miR-17 clusters, which have previously been shown to be regulated by c-Myc. The miR-17–92 polycistron also acts as an oncogene in haematopoietic progenitor cells. We show here that miR-221 is also induced by MYCN in neuroblastoma. Previous studies have reported miR-221 to be overexpressed in several other cancer entities, but its regulation has never before been associated with Myc. We present evidence of miRNA dysregulation in neuroblastoma. Additionally, we report miRNA induction to be a new mechanism of gene expression downregulation by MYCN. © 2007 Wiley-Liss, Inc.

The Trk family consists of three receptor tyrosine kinases, each of which can be activated by one or more of four neurotrophins-NGF, BDNF, NT3 and NT4. Neurotrophins mediate their multiple effects through a number of distinct intracellular signaling cascades regulating such diverse biological responses as cell survival, proliferation and differentiation in normal and neoplastic neuronal cells. Expression of Trk receptors also plays an important role in the biology and clinical behavior of neuroblastomas. High expression of TrkA is present in neuroblastomas with favorable biological features and highly correlated with patient survival, whereas TrkB is mainly expressed on unfavorable, aggressive neuroblastomas. This short review discusses recent data on the biological roles of TrkA and TrkB signaling in neuroblastoma. (C) 2005 Elsevier Ireland Ltd. All rights reserved

Notch proteins are the receptors for an evolutionarily highly conserved signalling pathway that regulates numerous cell fate decisions during development. Signal transduction involves the presenilin-dependent intracellular processing of Notch and nuclear translocation of the intracellular domain of Notch, Notch-IC. Notch-IC associates with the DNA-binding protein RBP-Jkappa/CBF-1 to activate transcription of Notch target genes. In the absence of Notch signalling, RBP-Jkappa/CBF-1 acts as a transcriptional repressor through the recruitment of histone deacetylase (HDAC) corepressor complexes. We identified SHARP as an RBP-Jkappa/CBF-1-interacting corepressor in a yeast two-hybrid screen. In cotransfection experiments, SHARP-mediated repression was sensitive to the HDAC inhibitor TSA and facilitated by SKIP, a highly conserved SMRT and RBP-Jkappa-interacting protein. SHARP repressed Hairy/Enhancer of split (HES)-1 promoter activity, inhibited Notch-1-mediated transactivation and rescued Notch-1-induced inhibition of primary neurogenesis in Xenopus laevis embryos. Based on our data, we propose a model in which SHARP is a novel component of the HDAC corepressor complex, recruited by RBP-Jkappa to repress transcription of target genes in the absence of activated Notch.

MYCN transcriptionally represses CD9 to trigger an invasion-metastasis cascade in neuroblastoma Johannes Fabian, Desiree Opitz, Kristina Althoff, Marco Lodrini, Kathy Astrahantseff, Barbara Hero, Ruth Volland, Anneleen Beckers, Katleen de Preter, Nitin S Patil, Mohammed L Abba, Theresa M Thole, Jasmin Wunschel, Annette Kunkele, Jamie Hu, Leonille Schweizer, Gunhild Mechtersheimer, Daniel R Carter, Belamy B Cheung, Odilia Popanda, Andreas von Deimling, Kai-Oliver Henrich, Frank Westermann, Manfred Schwab, Jan Koster, Rogier Versteeg, Glenn M Marshall, Frank Speleman, Margot Zoeller, Heike Allgayer, Matthias Fischer, Frank Berthold, Andreas E Kulozik, Olaf Witt, Angelika Eggert, Johannes H Schulte, Hedwig E Deubzer