Biology subjects

Eggert, A.

Publications and source records attributed to Eggert, A..

16 recordsLinked to original sources

bioRxiv2026

Quantitative and sensitive neuroblastoma minimal residual disease detection using extrachromosomal DNA (ecDNA) breakpoints

Sensitive detection of minimal residual disease (MRD) remains a major unmet need in high-risk neuroblastoma. MYCN amplification, a hallmark of high-risk disease, typically occurs on extrachromosomal DNA (ecDNA), but the potential of ecDNA-associated genomic rearrangements for individualized MRD monitoring has not been fully exploited. Here, we applied neuroblastoma-specific hybrid capture-based panel sequencing to identify patient-unique breakpoints within MYCN amplicons, and used Circle-seq and Nanopore sequencing to resolve the extrachromosomal amplicon structure in representative samples. Analysis of 8 neuroblastoma cell lines and 22 primary tumors identified 69 tumor-specific breakpoints. Those selected for assay development were validated by breakpoint-specific PCR and Sanger sequencing. Breakpoints detected in primary tumors remained detectable at relapse, supporting their stability as MRD markers. Breakpoint-specific real-time quantitative PCR and droplet digital PCR detected these junctions in bone marrow aspirates with high specificity and reached sensitivities down to a tumor DNA fraction of 10^-6. We applied this approach to 53 serial bone marrow aspirates from 14 patients with high-risk neuroblastoma to monitor MRD dynamics, resolving treatment response and molecular persistence. In six samples, breakpoint-positive DNA was detected in bone marrow that was negative by conventional cytology and immunocytology, highlighting the added value of molecular monitoring. Together, these findings establish ecDNA breakpoint-based detection as a strategy for MRD assessment in neuroblastoma, that is, in principle, applicable to any ecDNA-amplified oncogene.

cancer biology↗

bioRxiv2026

Sensitivity profiling reveals consistent drug responses across preclinical neuroblastoma models

Despite intensive treatment, overall survival for high-risk and relapse neuroblastoma patients remains below 50%. Even though comprehensive molecular profiling enables treatment stratification, druggable alterations have been identified for only a subset of patients. In vitro drug screening offers a complementary approach. Here, we compare the translational potential of three preclinical drug screening methods: ex vivo short-term, in vitro patient-derived organoid and in vivo patient-derived xenograft (PDX) drug testing. In total, 55 screens were performed from 38 neuroblastoma samples and five pediatric non-malignant samples, testing 77-224 drugs per screen. Ex vivo short-term drug screens achieved higher success rates than organoid screens (65% versus 23%) and shorter turnaround times (14 days versus 3-12 months). Matched samples showed consistent drug sensitivities across sample origin (patient versus PDX-derived; mean r = 0.84) and method (ex vivo short-term versus organoids; mean r = 0.87), demonstrating that ex vivo short-term screens recapitulate drug sensitivities found in long-term organoid models. In parallel, as part of the ITCC-P4 consortium, ten compounds were tested in vivo in eight PDX models, with samples matching the ex vivo screens. For seven out of ten clinically available compounds, ex vivo drug responses were comparable with in vivo responses in matched PDX models. These results demonstrate that, while organoids and PDX models remain essential for drug discovery, ex vivo short-term drug screening provides a rapid alternative for functional precision oncology in neuroblastoma.

cell biology↗

bioRxiv2026

The ITCC-P4 PDX platform of pediatric cancers for preclinical testing

Cancer is the leading cause of disease-related deaths among children in high-income countries. Tumor heterogeneity and lack of mechanism-of-action-based therapeutic options are key challenges to overcome in order to improve pediatric cancer patients survival. Here, we report the EU-IMI-2 funded public-private partnership "ITCC-Pediatric Preclinical Proof-of-Concept Platform" (ITCC-P4), which has built a large repertoire of patient-derived xenograft (PDX) models, representing all major solid pediatric cancer types, for in vivo drug testing. Three-hundred-fifty-three PDX models from diagnostic and relapsed pediatric cancers have been established and molecularly characterized, together with matched germline/tumor samples. As proof-of-concept, we present in vivo drug screening data in neuroblastoma and rhabdomyosarcoma models. PDX data, accessible at http://r2platform.com/itcc-p4, allow the selection of models based on oncogenic drivers and/or potential biomarkers for preclinical testing. Operated by a non-profit entity (www.itccp4.com), this sustainable platform aids academic and industrial researchers in developing and prioritizing innovative therapies for pediatric cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/703023v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@195ba30org.highwire.dtl.DTLVardef@f2c2d9org.highwire.dtl.DTLVardef@1d63f4dorg.highwire.dtl.DTLVardef@d60027_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Goats who stare at wolves - identifying natural response stimuli for an affect-driven attention bias test in small ruminants

Reliable and non-invasive assessment of affective states is crucial in animal welfare research. Linking affective states to cognitive processes, such as cognitive biases, provides a promising approach. An affect-driven attention bias - the tendency to focus more on threatening than on neutral or positive stimuli when in a negative affective state - has been observed in humans and non-human animal species. However, implementation is often hampered because ecologically relevant test setups need to be adapted to species-specific differences in the perception and processing of information from different modalities. As an initial step towards developing and validating an affect-driven attention bias test for goats, we aimed to identify visual stimuli perceived as potentially threatening using a looking-time paradigm. In a within-subject design, 30 adult female dwarf goats (Capra hircus) were presented with photographs of 12 animal species (six natural predators and six non-predators) from three taxa (mammals, reptiles, birds). Each image was shown for 10 seconds on one of two video screens, while the opposite screen remained white. Each goat completed four sessions (one per day) of six trials each: two sessions showing full-body and two sessions showing face/head-only images, resulting in 24 trials per subject. Analysis of relative looking duration revealed a significant interaction between animal taxon and predator category. Goats looked longer at predatory than non-predatory reptiles, while the opposite pattern was found for mammals. No difference was observed for birds. The presented body part (full body vs. face/head only) did not influence looking behaviour. These results suggest that photographs of predatory reptiles, particularly snakes, might be perceived as potentially threatening by goats, indicating their suitability as negative stimuli in future affect-driven attention bias tests. Further validation is needed to confirm their negative valence.

animal behavior and cognition↗

bioRxiv2025

circVDJ-seq for T cell clonotype detection in single-cell and spatial multi-omics

Monitoring T cell repertoires in human tissues provides important insights into immune response mechanisms in cancer, infectious diseases, and autoimmunity. However, retrieving VDJ information from single-cell and spatial transcriptomics workflows with 3-barcoding of cDNA remains resource-intensive or requires specialized sequencing equipment. Here, we introduce circVDJ-seq for simplified and cost-efficient T cell receptor (TCR) profiling from 3-directed workflows like single-cell or single-nucleus RNA sequencing, ATAC+RNA multi-omics, and spatial transcriptomics. Application of circVDJ-seq to freshly resected neuroblastomas and postmortem lymph nodes affected by pneumonia or COVID-19 reveals distinct immune microenvironments and T cell clonality patterns, highlighting broad utility across diverse clinical contexts.

genomics↗

bioRxiv2025

Personalized CRISPR Knock-In Cytokine Gene Therapy to Remodel the Tumor Microenvironment and Enhance CAR T Cell Therapy in Solid Tumors

The immunosuppressive tumor microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumors. To address this, we developed a novel gene therapeutic strategy that enables localized remodeling of the TME via tumor-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumor-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma--a pediatric solid tumor with a suppressive TME--as a model, we applied CancerPAM to sequencing data from cell lines and patients to identify optimal integration sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG). CRISPR-mediated CXCL10 knock-in into tumor cells significantly enhanced CAR T cell infiltration and antitumor efficacy both in vitro and in vivo. In vivo, CXCL10-expressing tumors showed significantly increased early CAR T cell infiltration and prolonged survival compared to controls. CancerPAM rankings correlated strongly with target-site specificity and knock-in efficiency, validating its predictive performance. Our findings establish CancerPAM as a powerful tool for safe and effective CRISPR-based interventions and provide a conceptual framework for integrating cytokine-driven TME remodeling with cellular immunotherapies. This personalized strategy holds promise for enhancing CAR T cells and other immunotherapies across immune-refractory solid tumors.

bioengineering↗

bioRxiv2024

Targeting MYCN upregulates L1CAM tumor antigen in MYCN-dysregulated neuroblastoma to increase CAR T cell efficacy

BackgroundCurrent treatment protocols have only limited success in pediatric patients with neuroblastomas harboring amplifications of the central oncogene, MYCN. Adoptive T cell therapy presents an innovative strategy to improve cure rates. However, L1CAM-targeting CAR T cells achieved only limited response against refractory/relapsed neuroblastoma in an ongoing phase I trial to date. Here, we investigate how oncogenic MYCN levels influence tumor cell response to CAR T cells, as one possible factor limiting success in trials. MethodsHigh MYCN levels were induced in SK-N-AS cells harboring the normal diploid MYCN complement using a tetracycline-inducible system. The inducible MYCN cell model or MYCN-amplified neuroblastoma cell lines were cocultured with L1CAM-CAR T cells. CAR T cell effector function was assessed via activation marker expression (flow cytometry), cytokine release and tumor cytotoxicity (biophotonic signal assessment). The cell model was characterized using RNA sequencing, and our data compared to publicly available RNA and proteomic data sets from neuroblastomas. ChIP-sequencing data was used to determine transcriptional L1CAM regulation by MYCN using public data sets. Synergism between CAR T cells and the MLN8237 AURKA inhibitor, which indirectly inhibits MYCN activity, was assessed in vitro using the Bliss model and in vivo in an immunocompromised mouse model. ResultsInducing high MYCN levels in the neuroblastoma cell model reduced L1CAM expression and, consequently, L1CAM-CAR T cell effector function (activation, cytokine release and cytotoxicity) in vitro. Primary neuroblastomas possessing high MYCN levels expressed lower levels of both the L1CAM transcript and L1CAM tumor antigen. Indirectly inhibiting MYCN via AURKA using MLN8237 treatment restored L1CAM expression on tumor cells in vitro and restored L1CAM-CAR T cell effector function. Combining MLN8237 and L1CAM-CAR T cell treatment synergistically increased neuroblastoma-directed killing in MYCN-overexpressing cells in vitro and in vivo concomitant with severe in vivo toxicity. ConclusionWe shed new light on a primary resistance mechanism in MYCN-driven neuroblastoma against L1CAM-CAR T cells via target antigen downregulation. These data suggest that combining L1CAM-CAR T cell therapy with pharmacological MYCN inhibition may benefit patients with high-risk neuroblastomas harboring MYCN amplifications.

immunology↗

bioRxiv2023

Spontaneous helping in pigs is mediated by helper's social attention and distress signals of individuals in need

Helping behaviour is of special interest for prosociality because it appears to be motivated by the needs of others. We developed a novel paradigm to investigate helping in pigs (Sus scrofa domesticus) and tested 75 individuals in eight groups in their home pens. Two identical compartments were attached to the pen, equipped with a window, and a door that could be opened from the outside by lifting a handle. Pigs in all groups spontaneously opened doors during a five-day familiarization. During testing, each pig was isolated once from its group and placed in one of the two compartments, in a counter-balanced order. In 85% of cases, pigs released a trapped group member from the test compartment within 20 minutes (median latency = 2.2 minutes). Pigs were more likely and quicker to open a door to free the trapped pig than to open a door to an empty compartment. Pigs who spent more time looking at the window of the test compartment were more likely to help. Distress signals by the trapped pig increased its probability of being helped. Responses are consistent with several criteria for identifying targeted helping, but results can also be explained by selfish motivations.

animal behavior and cognition↗

bioRxiv2023

Intercellular extrachromosomal DNA copy number heterogeneity drives cancer cell state diversity

Neuroblastoma is characterised by extensive inter- and intra-tumour genetic heterogeneity and varying clinical outcomes. One possible driver for this heterogeneity are extrachromosomal DNAs (ecDNA), which segregate independently to the daughter cells during cell division and can lead to rapid amplification of oncogenes. While ecDNA-mediated oncogene amplification has been shown to be associated with poor prognosis in many cancer entities, the effects of ecDNA copy number heterogeneity on intermediate phenotypes are still poorly understood. Here, we leverage DNA and RNA sequencing data from the same single cells in cell lines and neuroblastoma patients to investigate these effects. We utilise ecDNA amplicon structures to determine precise ecDNA copy numbers and reveal extensive intercellular ecDNA copy number heterogeneity. We further provide direct evidence for the effects of this heterogeneity on gene expression of cargo genes, including MYCN and its downstream targets, and the overall transcriptional state of neuroblastoma cells. These results highlight the potential for rapid adaptability of cellular states within a tumour cell population mediated by ecDNA copy number, emphasising the need for ecDNA-specific treatment strategies to tackle tumour formation and adaptation.

cancer biology↗

bioRxiv2022

Elimusertib outperforms standard of care chemotherapy in preclinical patient-derived pediatric solid tumor models

The small molecule inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), elimusertib, is currently being tested clinically in various cancer entities in adults and children. Its preclinical anti-tumor activity in pediatric malignancies, however, is largely unknown. We here assessed the preclinical activity of elimusertib in >40 cell lines and >30 patient-derived xenograft (PDX) models derived from common pediatric solid tumor entities. Detailed in vitro and in vivo molecular characterization of the treated models enabled the evaluation of response biomarkers. Pronounced objective response rates were observed for elimusertib monotherapy in PDX, when treated with a regimen currently used in clinical trials. Strikingly, elimusertib outperformed standard of care chemotherapies, particularly in alveolar rhabdomysarcoma PDX. Thus, elimusertib has strong preclinical anti-tumor activity in pediatric solid tumor models, which may translate to clinically meaningful responses in patients. Statement of translational relevanceElimusertib is a small molecule inhibitor of ATR. ATR inhibitors have shown promising results as anticancer agents in adult cancers, but there is limited information on their effectiveness in pediatric solid tumors. Using a cohort of 32 patient-derived xenografts from pediatric solid tumors, we here evaluated the therapeutic potential of elimusertib in vivo. Elimusertib reduced tumor volume growth in all samples. Elimusertib had very limited toxicity and was potent even in tumors with preexisting chemoresistance. Our preclinical data indicates that elimusertib is a safe and potent therapeutic option for pediatric solid tumors. This data may serve as a rationale for the development of pediatric clinical trials for ATR inhibitors.

cancer biology↗

bioRxiv2022

Amplicon structure creates collateral therapeutic vulnerability in cancer

Although DNA amplifications in cancers frequently harbor passenger genes alongside oncogenes, the functional consequence of such co-amplifications and their impact for therapy remains ill-defined. We discovered that passenger co-amplifications can create amplicon structure-specific collateral vulnerabilities. We present the DEAD-box helicase 1 (DDX1) gene as a bona fide passenger co-amplified with MYCN in cancers. Survival of cancer cells with DDX1 co-amplifications strongly depends on the mammalian target of rapamycin complex 1 (mTORC1). Mechanistically, aberrant DDX1 expression inhibits the tricarboxylic acid cycle through a previously unrecognized interaction with dihydrolipoamide S-succinyltransferase, a component of the alpha-ketoglutarate dehydrogenase complex. Cells expressing aberrant DDX1 levels compensate for the metabolic shift by enhancing mTORC1 activity. Consequently, pharmacological mTORC1 inhibition triggered cell death specifically in cells harboring the DDX1 co-amplification. This work highlights a significant contribution of passenger gene alterations to the therapeutic susceptibility of cancers. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/506647v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@58b27dorg.highwire.dtl.DTLVardef@12e2fccorg.highwire.dtl.DTLVardef@1440f33org.highwire.dtl.DTLVardef@141e933_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Copy-number dosage regulates telomere maintenance and disease-associated pathways in neuroblastoma

Telomere maintenance in neuroblastoma is linked to poor outcome and caused by either TERT activation or through alternative lengthening of telomeres (ALT). In contrast to TERT activation, commonly caused by genomic rearrangements or MYCN amplification, ALT is less well understood. Alterations at the ATRX locus are key drivers of ALT but only present in [~]50% of ALT tumors. To identify potential new pathways to telomere maintenance, we investigate allele-specific gene dosage effects from whole genomes and transcriptomes in 115 primary neuroblastomas. We show that copy-number dosage deregulates telomere maintenance, genomic stability, and neuronal pathways and identify upregulation of variants of histone H3 and H2A as a potential alternative pathway to ALT. We investigate the interplay between TERT activation, overexpression and copy-number dosage and reveal loss of imprinting at the RTL1 gene associated with poor clinical outcome. These results highlight the importance of gene dosage in key oncogenic mechanisms in neuroblastoma.

genomics↗

bioRxiv2021

Neuroblastoma signalling models unveil combination therapies targeting feedback-mediated resistance

Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and to the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospo-proteomics profiles confirm the cell-specific feedback effects and synergy of MEK and IGFR targeted treatements. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies, and our findings should be considered for the planning of future clinical trials introducing MEKi in the treatment of neuroblastoma.

cancer biology↗

bioRxiv2020

Exploiting a PAX3-FOXO1-induced synthetic lethal ATR dependency for rhabdomyosarcoma therapy

Pathognomonic PAX3-FOXO1 fusion oncogene expression is associated with poor outcome in rhabdomyosarcoma. Combining genome-wide CRISPR screening with cell- based functional genetic approaches, we here provide evidence that PAX3-FOXO1 induces replication stress, resulting in a synthetic lethal dependency to ATR-mediated DNA damage-response signaling in rhabdomyosarcoma. Expression of PAX3-FOXO1 in muscle progenitor cells was not only sufficient to induce hypersensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibited increased sensitivity to structurally diverse inhibitors of ATR, a dependency that could be validated genetically. Mechanistically, ATR inhibition led to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increased sensitivity of rhabdomyosarcoma cells to PARP inhibition in vitro, and combined ATR and PARP inhibition induced regression of primary patient-derived alveolar rhabdomyosarcoma xenografts in vivo. Moreover, a genome-wide CRISPR activation screen (CRISPRa) identified FOS gene family members as inducers of resistance against ATR inhibitors. Mechanistically, FOS gene family members reduced replication stress in rhabdomyosarcoma cells. Lastly, compassionate use of ATR inhibitors in two pediatric patients suffering from relapsed PAX3-FOXO1-expressing alveolar rhabdomyosarcoma showed signs of tolerability, paving the way to clinically exploit this novel synthetic lethal dependency in rhabdomyosarcoma.

cancer biology↗

bioRxiv2020

MYB5a/NEGAN activates petal anthocyanin pigmentation and shapes the MBW regulatory network in Mimulus luteusvar. variegatus

Much of the visual diversity of angiosperms is due to the frequent evolution of novel pigmentation patterns in flowers. The gene network responsible for anthocyanin pigmentation, in particular, has become a model for investigating how genetic changes give rise to phenotypic innovation. In the monkeyflower genus Mimulus, an evolutionarily recent gain of petal lobe anthocyanin pigmentation in M. luteus var. variegatus was previously mapped to genomic region pla2. Here, we use DNA sequence analysis and spatiotemporal patterns of gene expression to identify MYB5a - homologous to the NEGAN transcriptional activator from M. lewisii - as a likely candidate gene within the pla2 region. Transgenic manipulation of gene expression confirms that MYB5a is both necessary and sufficient for petal lobe anthocyanin pigmentation. The deployment of MYB5a/NEGAN to the petal lobe stands in contrast to its more restricted role as a nectar guide anthocyanin activator in other Mimulus species. Transcriptome sequencing of a MYB5a RNAi line reveals the degree to which other regulators of the anthocyanin pathway - including R3 MYB repressors and bHLH and WD40 co-activators - are responsive to the level of expression of MYB5a. Overall, this work reveals that a genetically simple change, which we hypothesize to be a regulatory mutation in cis to MYB5a, has cascading effects on gene expression, not only on the genes downstream of MYB5a but also on all of its known partners in the anthocyanin regulatory network. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/030536v3_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@120d538org.highwire.dtl.DTLVardef@8e6085org.highwire.dtl.DTLVardef@162f684org.highwire.dtl.DTLVardef@26860f_HPS_FORMAT_FIGEXP M_FIG Graphical abstract.Solid black arrows indicate the direction (though not magnitude) of gene expression change, following RNAi knockdown of MYB5a/NEGAN in M. l. variegatus. The number of black arrows corresponds to the number of gene copies identified in the transcriptome. Grey symbols denote positive and negative regulatory interactions. RTO is an R3 MYB protein that inhibits anthocyanin biosynthesis by sequestering bHLH proteins away from the MBW complex. C_FIG

genetics↗

bioRxiv2019

Enhancer hijacking determines intra- and extrachromosomal circular MYCN amplicon architecture in neuroblastoma

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA. The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyzed the MYCN amplicon structure and its chromatin landscape. This revealed two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplified a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons was characterized by high structural complexity, lacked key local enhancers, and instead contained distal chromosomal fragments, which harbored CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.

cancer biology↗