Neuroblastoma arises in early fetal development and its evolutionary duration predicts outcome.

Neuroblastoma, the most frequent solid tumor in infants, shows very diverse outcomes from spontaneous regression to fatal disease. When these different tumors originate and how they evolve are not known. Here we quantify the somatic evolution of neuroblastoma by deep whole-genome sequencing, molecular clock analysis and population-genetic modeling in a comprehensive cohort covering all subtypes. We find that tumors across the entire clinical spectrum begin to develop via aberrant mitoses as early as the first trimester of pregnancy. Neuroblastomas with favorable prognosis expand clonally after short evolution, whereas aggressive neuroblastomas show prolonged evolution during which they acquire telomere maintenance mechanisms. The initial aneuploidization events condition subsequent evolution, with aggressive neuroblastoma exhibiting early genomic instability. We find in the discovery cohort (n = 100), and validate in an independent cohort (n = 86), that the duration of evolution is an accurate predictor of outcome. Thus, insight into neuroblastoma evolution may prospectively guide treatment decisions.

MYCN mediates cysteine addiction and sensitizes neuroblastoma to ferroptosis.

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats.

Alternative lengthening of telomeres (ALT) occurs in ∼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.

Neuroblastoma-Telomere maintenance, deregulated signaling transduction and beyond.

The childhood malignancy neuroblastoma belongs to the group of embryonal tumors and originates from progenitor cells of the sympathoadrenal lineage. Treatment options for children with high-risk and relapsed disease are still very limited. In recent years, an ever-growing molecular diversity was identified using (epi)-genetic profiling of neuroblastoma tumors, indicating that molecularly targeted therapies could be a promising therapeutic option. In this review article, we summarize the various molecular subtypes and genetic events associated with neuroblastoma and describe recent advances in targeted therapies. We lay a strong emphasis on the importance of telomere maintenance mechanisms for understanding tumor progression and risk classification of neuroblastoma.

iTReX: Interactive exploration of mono- and combination therapy dose response profiling data.

High throughput screening methods, measuring the sensitivity and resistance of tumor cells to drug treatments have been rapidly evolving. Not only do these screens allow correlating response profiles to tumor genomic features for developing novel predictors of treatment response, but they can also add evidence for therapy decision making in precision oncology. Recent analysis methods developed for either assessing single agents or combination drug efficacies enable quantification of dose-response curves with restricted symmetric fit settings. Here, we introduce iTReX, a user-friendly and interactive Shiny/R application, for both the analysis of mono- and combination therapy responses. The application features an extended version of the drug sensitivity score (DSS) based on the integral of an advanced five-parameter dose-response curve model and a differential DSS for combination therapy profiling. Additionally, iTReX includes modules that visualize drug target interaction networks and support the detection of matches between top therapy hits and the sample omics features to enable the identification of druggable targets and biomarkers. iTReX enables the analysis of various quantitative drug or therapy response readouts (e.g. luminescence, fluorescence microscopy) and multiple treatment strategies (drug treatments, radiation). Using iTReX we validate a cost-effective drug combination screening approach and reveal the application's ability to identify potential sample-specific biomarkers based on drug target interaction networks. The iTReX web application is accessible at https://itrex.kitz-heidelberg.de.