Central memory phenotype drives success of checkpoint inhibition in combination with CAR T cells

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

Inhibiting phosphoglycerate dehydrogenase counteracts chemotherapeutic efficacy against MYCN‐amplified neuroblastoma

Here we sought metabolic alterations specifically associated with MYCN amplification as nodes to indirectly target the MYCN oncogene. Liquid chromatography-mass spectrometry-based proteomics identified seven proteins consistently correlated with MYCN in proteomes from 49 neuroblastoma biopsies and 13 cell lines. Among these was phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in de novo serine synthesis. MYCN associated with two regions in the PHGDH promoter, supporting transcriptional PHGDH regulation by MYCN. Pulsed stable isotope-resolved metabolomics utilizing C-13-glucose labeling demonstrated higher de novo serine synthesis in MYCN-amplified cells compared to cells with diploid MYCN. An independence of MYCN-amplified cells from exogenous serine and glycine was demonstrated by serine and glycine starvation, which attenuated nucleotide pools and proliferation only in cells with diploid MYCN but did not diminish these endpoints in MYCN-amplified cells. Proliferation was attenuated in MYCN-amplified cells by CRISPR/Cas9-mediated PHGDH knockout or treatment with PHGDH small molecule inhibitors without affecting cell viability. PHGDH inhibitors administered as single-agent therapy to NOG mice harboring patient-derived MYCN-amplified neuroblastoma xenografts slowed tumor growth. However, combining a PHGDH inhibitor with the standard-of-care chemotherapy drug, cisplatin, revealed antagonism of chemotherapy efficacy in vivo. Emergence of chemotherapy resistance was confirmed in the genetic PHGDH knockout model in vitro. Altogether, PHGDH knockout or inhibition by small molecules consistently slows proliferation, but stops short of killing the cells, which then establish resistance to classical chemotherapy. Although PHGDH inhibition with small molecules has produced encouraging results in other preclinical cancer models, this approach has limited attractiveness for patients with neuroblastoma

Defining the landscape of circular RNAs in neuroblastoma unveils a global suppressive function of MYCN

Circular RNAs (circRNAs) are a regulatory RNA class. While cancer-driving functions have been identified for single circRNAs, how they modulate gene expression in cancer is not well understood. We investigate circRNA expression in the pediatric malignancy, neuroblastoma, through deep whole-transcriptome sequencing in 104 primary neuroblastomas covering all risk groups. We demonstrate that MYCN amplification, which defines a subset of high-risk cases, causes globally suppressed circRNA biogenesis directly dependent on the DHX9 RNA helicase. We detect similar mechanisms in shaping circRNA expression in the pediatric cancer medulloblastoma implying a general MYCN effect. Comparisons to other cancers identify 25 circRNAs that are specifically upregulated in neuroblastoma, including circARID1A. Transcribed from the ARID1A tumor suppressor gene, circARID1A promotes cell growth and survival, mediated by direct interaction with the KHSRP RNA-binding protein. Our study highlights the importance of MYCN regulating circRNAs in cancer and identifies molecular mechanisms, which explain their contribution to neuroblastoma pathogenesis

Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond

Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond

Selective inhibition of HDAC8 decreases neuroblastoma growth in vitro and in vivo and enhances retinoic acid-mediated differentiation

For differentiation-defective malignancies, compounds that modulate transcription, such as retinoic acid and histone deacetylase (HDAC) inhibitors, are of particular interest. HDAC inhibitors are currently under investigation for the treatment of a broad spectrum of cancer diseases. However, one clinical drawback is class-specific toxicity of unselective inhibitors, limiting their full anticancer potential. Selective targeting of individual HDAC isozymes in defined tumor entities may therefore be an attractive alternative treatment approach. We have previously identified HDAC family member 8 (HDAC8) as a novel target in childhood neuroblastoma. Using small-molecule inhibitors, we now demonstrate that selective inhibition of HDAC8 exhibits antineuroblastoma activity without toxicity in two xenograft mouse models of MYCN oncogene-amplified neuroblastoma. In contrast, the unselective HDAC inhibitor vorinostat was more toxic in the same models. HDAC8-selective inhibition induced cell cycle arrest and differentiation in vitro and in vivo. Upon combination with retinoic acid, differentiation was significantly enhanced, as demonstrated by elongated neurofilament-positive neurites and upregulation of NTRK1. Additionally, MYCN oncogene expression was downregulated in vitro and tumor cell growth was markedly reduced in vivo. Mechanistic studies suggest that cAMP-response element-binding protein (CREB) links HDAC8- and retinoic acid-mediated gene transcription. In conclusion, HDAC-selective targeting can be effective in tumors exhibiting HDAC isozyme-dependent tumor growth in vivo and can be combined with differentiation-inducing agents

Clinical Presentation and Management of a Dinutuximab Beta Extravasation in a Patient with Neuroblastoma

Extravasation can present serious accidental complication of intravenous drug application. While monoclonal antibodies do not show the necrotic potential of cytotoxic chemotherapy drugs, considerable inflammatory toxicity can occur, necessitating standardized operating procedures for the management of their extravasation. Here, we report the clinical course and management of dinutuximab beta extravasation in a 3-year-old child. Dinutuximab beta is a chimeric monoclonal antibody targeting the GD2 disialoganglioside on the surface of neuroblastoma cells that has in recent years gained significant importance in the treatment of high-risk neuroblastoma, now contributing to both first- and second-line therapy protocols. The dinutuximab beta extravasation reported here occurred when the patient received the antibody cycle as a continuous infusion over a 10-day period after haploidentical stem cell transplantation for relapsed high-risk neuroblastoma. The extravasated dinutuximab beta caused local pain, swelling, and hyperemia accompanied by fever and an overall deterioration in the general condition. Laboratory diagnostics demonstrated an increase in C-reactive protein level and total white blood cell count. Clinical complication management consisted of intravenous fluid therapy, local dabbing with dimethyl sulfoxide (DMSO), analgesia with dipyrone, as well as application of intravenous antibiotics to prevent bacterial superinfection in the severely immunocompromised host. The patient considerably improved after six days with this treatment regimen and fully recovered by day 20

RITA displays anti-tumor activity in medulloblastomas independent of TP53 status

Current therapy of medulloblastoma, the most common malignant brain tumor of childhood, achieves 40-70% survival. Secondary chemotherapy resistance contributes to treatment failure, where TP53 pathway dysfunction plays a key role. MDM2 interaction with TP53 leads to its degradation. Reactivating TP53 functionality using small-molecule inhibitors, such as RITA, to disrupt TP53-MDM2 binding may have therapeutic potential. We show here that RITA decreased viability of all 4 analyzed medulloblastoma cell lines, regardless of TP53 functional status. The decrease in cell viability was accompanied in 3 of the 4 medulloblastoma cell lines by accumulation of TP53 protein in the cells and increased CDKN1A expression. RITA treatment in mouse models inhibited medulloblastoma xenograft tumor growth. These data demonstrate that RITA treatment reduces medulloblastoma cell viability in both in vitro and in vivo models, and acts independently of cellular TP53 status, identifying RITA as a potential therapeutic agent to treat medulloblastoma

Pharmacological activation of the p53 pathway by nutlin-3 exerts anti-tumoral effects in medulloblastomas

Medulloblastomas account for 20% of pediatric brain tumors. With an overall survival of 40%-70%, their treatment is still a challenge. The majority of medulloblastomas lack p53 mutations, but even in cancers retaining wild-type p53, the tumor surveillance function of p53 is inhibited by the oncoprotein MDM2. Deregulation of the MDM2/p53 balance leads to malignant transformation. Here, we analyzed MDM2 mRNA and protein expression in primary medulloblastomas and normal cerebellum and assessed the mutational status of p53 and MDM2 expression in 6 medulloblastoma cell lines. MDM2 expression was elevated in medulloblastomas, compared with cerebellum. Four of 6 medulloblastoma cell lines expressed wild-type p53 and high levels of MDM2. The tumor-promoting p53-MDM2 interaction can be inhibited by the small molecule, nutlin-3, restoring p53 function. Targeting the p53-MDM2 axis using nutlin-3 significantly reduced cell viability and induced either cell cycle arrest or apoptosis and expression of the p53 target gene p21 in these 4 cell lines. In contrast, DAOY and UW-228 cells harboring TP53 mutations were almost unaffected by nutlin-3 treatment. MDM2 knockdown in medulloblastoma cells by siRNA mimicked nutlin-3 treatment, whereas expression of dominant negative p53 abrogated nutlin-3 effects. Oral nutlin-3 treatment of mice with established medulloblastoma xenografts inhibited tumor growth and significantly increased survival. Thus, nutlin-3 reduced medulloblastoma cell viability in vitro and in vivo by re-activating p53 function. We suggest that inhibition of the MDM2-p53 interaction with nutlin-3 is a promising therapeutic option for medulloblastomas with functional p53 that should be further evaluated in clinical trials

Multiplexed Quantification of Four Neuroblastoma DNA Targets in a Single Droplet Digital PCR Reaction

The detection and characterization of cell-free DNA (cfDNA) in peripheral blood from neuroblastoma patients may serve as a minimally invasive approach to liquid biopsy. Major challenges in the analysis of cfDNA purified from blood samples are small sample volumes and low cfDNA concentrations. Droplet digital PCR (ddPCR) is a technology suitable for analyzing low levels of cfDNA. Reported here are two quadruplexed ddPCR assay protocols that reliably quantify MYCN and ALK copy numbers in a single reaction together with the two reference genes, NAGK and AFF3, and accurately estimate ALK(F1174L) (exon 23 position 3522, C>A) and ALK(R1275Q) (exon 25 position 3824, G>A) mutant allele fractions using cfDNA as input. The separation of positive and negative droplets was optimized for detecting two targets in each ddPCR fluorescence channel by the adjustment of the probe and primer concentrations of each target molecule. The quadruplexed assays were validated using a panel of 10 neuroblastoma cell lines and paired blood plasma and primary neuroblastoma samples from nine patients. Accuracy and sensitivity thresholds in quadruplexed assays corresponded well with those from the respective duplexed assays. Presented are two robust quadruplexed ddPCR protocols applicable in the routine clinical setting and that require only minimal plasma volumes for the assessment of MYCN and ALK oncogene status