Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH- MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.

Neuroblastoma is an aggressive pediatric cancer with a high rate of metastasis to the BM. Despite intensive treatments including high-dose chemotherapy, the overall survival rate for children with metastatic neuroblastoma remains dismal. Understanding the cellular and molecular mechanisms of the metastatic tumor microenvironment is crucial for developing new therapies and improving clinical outcomes. Here, we used single-cell RNA-Seq to characterize immune and tumor cell alterations in neuroblastoma BM metastases by comparative analysis with patients without metastases. Our results reveal remodeling of the immune cell populations and reprogramming of gene expression profiles in the metastatic niche. In particular, within the BM metastatic niche, we observed the enrichment of immune cells, including tumor-associated neutrophils, macrophages, and exhausted T cells, as well as an increased number of Tregs and a decreased number of B cells. Furthermore, we highlighted cell communication between tumor cells and immune cell populations, and we identified prognostic markers in malignant cells that are associated with worse clinical outcomes in 3 independent neuroblastoma cohorts. Our results provide insight into the cellular, compositional, and transcriptional shifts underlying neuroblastoma BM metastases that contribute to the development of new therapeutic strategies.

PPM1D is a negative regulator of p53 and genomic aberrations resulting in increased activity of PPM1D have been observed in cancers of different origins, indicating that PPM1D has oncogenic properties. We established a transgenic mouse model overexpressing PPM1D and showed that these mice developed a wide variety of cancers. PPM1D-expressing mice developed tumors phenotypically and genetically similar to tumors in mice with dysfunctional p53. T-cell lymphoblastic lymphoma was the most frequent cancer observed in these mice (55%) followed by adenocarcinomas (24%), leukemia (12%) and other solid tumors including neuroblastoma. Characterization of T-cell lymphomas in mice overexpressing PPM1D demonstrates Pten-deletion and p53-accumulation similar to mice with p53 loss-of-function. Also, Notch1 mutations which are recurrently observed in T-cell acute lymphoblastic lymphoma (T-ALL) were frequently detected in PPM1D-transgenic mice. Hence, PPM1D acts as an oncogenic driver in connection with cellular stress, suggesting that the PPM1D gene status and expression levels should be investigated in TP53 wild-type tumors.

Medulloblastoma is one of the most common malignant brain tumor types in children, with an overall survival of 70%. Mortality is associated with metastatic relapsed tumors. Rho-associated kinases (ROCKs), important for epithelial-mesenchymal transition (EMT) and proper nervous system development, have previously been identified as a promising drug target to inhibit cancer growth and metastatic spread. Here, we show that ROCKs are expressed in medulloblastoma, with higher ROCK2 mRNA expression in metastatic compared to non-metastatic tumors. By evaluating three ROCK inhibitors in a panel of medulloblastoma cell lines we demonstrated that medulloblastoma cells were sensitive for pharmacological ROCK inhibition. The specific ROCK inhibitor RKI-1447 inhibited the tumorigenicity in medulloblastoma cells as well as impeded cell migration and invasion. Differential gene expression analysis suggested that ROCK inhibition was associated with the downregulation of signaling pathways important in proliferation and metastasis e.g., TNFα via NFκβ, TGFβ, and EMT. Expression of key proteins in these pathways such as RHOA, RHOB, JUN, and vimentin was downregulated in ROCK inhibited cells. Finally, we showed that ROCK inhibition by RKI-1447 suppressed medulloblastoma growth and proliferation in vivo. Collectively, our results suggest that ROCK inhibition presents a potential new therapeutic option in medulloblastoma, especially for children with metastatic disease.

Neuroblastoma is a heterogeneous embryonal malignancy and the most deadly tumor of childhood, although a minor subset may show spontaneous differentiation. It arises from the multipotent neural crest lineage during development. Some of this multipotency is retained in neuroblastoma, which can give rise to both adrenergic and mesenchymal tumor cells. The mechanisms enabling such dual fates are unknown, but likely help neuroblastoma to evade existing therapies. To understand neuroblastoma plasticity, we analyzed patient tumors using single-cell transcriptomics. In addition to the heterogeneous adrenergic and mesenchymal populations, we identify a subpopulation of malignant cells resembling Schwann cell precursors (SCPs). This SCP-like population connects the adrenergic and mesenchymal compartments through transitions structurally reminiscent of the SCP cell-fate decision fork that occurs during normal development. While the directionality of such transitions in neuroblastoma remains to be established, this finding expands the potential reservoirs of malignant cells, and suggests intratumoral plasticity mechanisms relevant for therapeutic resistance and relapse.

Background/Objectives: Medulloblastoma (MB) is the most common high-grade paediatric brain tumour, with group 3 MB patients having the worst prognosis. A high prevalence of group 3 tumours shows overexpression of the MYC oncogene, making it a potential therapeutic target. However, attempts to directly inhibit MYC have so far demonstrated limited success. Dihydroorotate dehydrogenase (DHODH), a crucial enzyme of the pyrimidine biosynthesis process, has emerged as an up-and-coming target in oncology, as its inhibition has shown promise in several cancers. Methods: In this study, we investigated the efficacy of brequinar, a DHODH inhibitor, in MB, with a focus on group 3. In vitro, BRQ’s effects on cell viability and MYC expression were tested in seven MB cell lines. In vivo, a novel zebrafish xenograft model was used to evaluate BRQ’s impact on tumour growth and toxicity. Results: High DHODH expression was identified in group 3 and shh MB subgroups, correlating with poor survival and MYC expression. BRQ demonstrated nanomolar efficacy in inducing apoptosis and reducing MYC expression in group 3 MB cell lines. Finally, we established a novel zebrafish xenograft model and demonstrated that BRQ significantly inhibited tumour growth at non-toxic concentrations in vivo, particularly in the D458 metastatic MB cell line. Conclusions: Our findings indicate that DHODH is a promising therapeutic target in group 3 MBs. Furthermore, BRQ shows potential for clinical application, effectively reducing tumour growth and MYC expression in vitro and in vivo. Moreover, our newly established zebrafish xenograft model offers a promising avenue for rapid in vivo drug testing for use in MB.