Karin Hansson

High-risk neuroblastoma is an aggressive pediatric malignancy characterized by a high proliferative capacity, intrinsic replication stress, and poor clinical outcomes. Although mitotic inhibitors show preclinical efficacy, their clinical use is limited by dose-dependent toxicity. In this study, we performed high-throughput combinatorial drug screening using MYCN-amplified (MNA) human neuroblastoma tumoroids to identify drugs with orthogonal mechanisms that synergize with low-dose mitotic inhibition. Promising drug combinations were prioritized using a multifactorial approach including dose–response matrices across multiple neuroblastoma tumoroids and CD34+ cord blood cells, synergy area scores, and machine learning–based response predictions. Several therapeutic classes were selected, including kinase inhibitors, differentiation agents, and hormone therapies. Notably, combined ataxia telangiectasia and Rad 3–related (ATR) inhibition by elimusertib and mitotic kinesin spindle protein (KSP) inhibition showed additive to mild synergy and induced neuroblastoma cell death. This combination reduced tumor growth and prolonged survival in neuroblastoma MNA, chemoresistant patient-derived xenografts. Furthermore, alternating dosing schedules were well tolerated and maintained stable disease control in vivo. Collectively, these findings establish combined ATR and low-dose mitotic inhibition as a promising therapeutic strategy for MNA neuroblastoma.

Relapse and treatment resistance are common in children with high-risk neuroblastoma, and novel therapies are needed. Conventional drug discovery is slow, expensive, often fails in practice, and consequently falls short in addressing pediatric and rare conditions. In such instances, drug repurposing is a promising strategy. Here, we used two independent in silico prediction tools including machine learning to identify approved drugs for repurposing against neuroblastoma. The combination of statins and phenothiazines showed strong synergistic effects in human neuroblastoma organoids, decreased tumor growth, and prolonged survival in MYCN -amplified neuroblastoma patient-derived xenografts. The drug combination altered cholesterol metabolism through two different mechanisms and induced a phenotypic change toward an adrenergic state in vitro, which was associated with enhanced chemosensitivity. Integration of the drug combination into standard-of-care chemotherapy regressed tumors and prolonged survival in chemoresistant patient-derived xenografts. Thus, a combination of safe and approved medications added to standard-of-care chemotherapy outperforms chemotherapy alone in chemoresistant neuroblastoma.

Neuroblastoma (NB) is one of the most lethal childhood cancers due to its propensity to become treatment resistant. By spatial mapping of subclone geographies before and after chemotherapy across 89 tumor regions from 12 NBs, we find that densely packed territories of closely related subclones present at diagnosis are replaced under effective treatment by islands of distantly related survivor subclones, originating from a different most recent ancestor compared to lineages dominating before treatment. Conversely, in tumors that progressed under treatment, ancestors of subclones dominating later in disease are present already at diagnosis. Chemotherapy treated xenografts and cell culture models replicate these two contrasting scenarios and show branching evolution to be a constant feature of proliferating NB cells. Phylogenies based on whole genome sequencing of 505 individual NB cells indicate that a rich repertoire of parallel subclones emerges already with the first oncogenic mutations and lays the foundation for clonal replacement under treatment.

Understanding the factors and mechanisms involved in beta-cell development will guide therapeutic efforts to generate fully functional beta cells for diabetes. Neurogenin 3 (NGN3) is the key transcription factor that marks endocrine progenitors and drives beta-cell differentiation. Here we screen for binding partners of NGN3 and identify the deubiquitylating enzyme USP7 as a key regulator of NGN3 stability. Mechanistically, USP7 interacts with, deubiquitinates and stabilizes NGN3. In vivo, conditional knockout of Usp7 in the mouse embryonic pancreas causes a dramatic reduction in islet formation and hyperglycemia in adult mice, due to impaired NGN3-mediated endocrine specification during pancreatic development. Furthermore, pharmacological inhibition of USP7 during endocrine specification in human iPSC models of beta-cell differentiation decreases NGN3 expressing progenitor cell numbers and impairs beta cell differentiation. Thus, the USP7-NGN3 axis is an essential mechanism for driving endocrine development and beta-cell differentiation, which can be therapeutically exploited.

Chemotherapy resistance and relapses are common in high-risk neuroblastoma (NB). Here, we developed a clinically relevant in vivo treatment protocol mimicking the first-line five-chemotherapy treatment regimen of high-risk NB and applied this protocol to mice with MYCN -amplified NB patient-derived xenografts (PDXs). Genomic and transcriptomic analyses were used to reveal NB chemoresistance mechanisms. Intrinsic resistance was associated with high genetic diversity and an embryonic phenotype. Relapsed NB with acquired resistance showed a decreased adrenergic phenotype and an enhanced immature mesenchymal–like phenotype, resembling multipotent Schwann cell precursors. NBs with a favorable treatment response presented a lineage-committed adrenergic phenotype similar to normal neuroblasts. Novel integrated phenotypic gene signatures reflected treatment response and patient prognosis. NB organoids established from relapsed PDX tumors retained drug resistance, tumorigenicity, and transcriptional cell states. This work sheds light on the mechanisms of NB chemotherapy response and emphasizes the importance of transcriptional cell states in chemoresistance.

Neuroblastoma (NB) is a pediatric cancer of the sympathetic nervous system that accounts for 15% of childhood cancer deaths. High-risk (HR) NB tumors are treated with an aggressive multimodal therapy, including the COJEC induction protocol which is a combination of five different chemotherapeutic agents. However, around 15% of primary tumors are refractory from the start and over 60% present treatment resistant relapses. In this project we aim to decipher the mechanisms involved in treatment resistance in HR-NB. Using various NB patient-derived xenograft (PDX) models we designed and tested a clinically-relevant protocol that mimics COJEC treatment and surgical resection. RNA-sequencing, SNP analysis, and histology, were used to explore the molecular mechanisms involved in treatment response. With our COJEC-like protocol we were able to replicate the original patient response in our PDX models. NB PDX1 presented upfront progression and a lack of morphological differentiation. At a molecular level it was characterized by genome duplication and high number of chromosomal aberrations, as well as a high expression of early development/mesenchymal and cell cycle genes. In contrast, NB PDX3 presented an overall good response, leading to a 22% relapse rate after surgery. Relapsed tumors displayed high expression of early development/mesenchymal genes, similar to those seen in refractory PDX1. Meanwhile, the PDX3 tumors collected during surgery that did not relapse (cured) showed low MYCN expression, downregulation of cell cycle and DNA repair genes, along with a strong upregulation of nervous system/nor-adrenergic genes. These genetic signatures derived from the cured PDX3 samples also correlated with good prognosis in large cohorts of HR-NB patients. Despite observing a convergent/parallel evolution of specific genetic aberrations across our PDXs, their presence or absence does not predict prognosis. Instead, the high RNA expression levels of those specific genes correlates with treatment response both in our PDX models and in patient cohorts. Finally, we established 3D tumor organoids derived from treated PDX tumors and confirmed they maintain the molecular characteristics of the parental tumor as well as the acquired COJEC resistance and the tumorigenic capacity, making them an excellent tool to test novel therapeutics against HR-NB. In conclusion, we designed a clinically-relevant COJEC-like protocol that can mimic patient response in PDX models. Early developmental gene signatures appear to correlate with treatment resistance in HR-NB, while the expression of nor-adrenergic signatures after chemotherapy are associated with good prognosis, independently of the accumulation of copy number aberrations. Based on our results, we propose that a combination of DNA and RNA analysis at the surgical resection time point after COJEC could be of clinical interest to evaluate COJEC treatment response and predict future relapses. Citation Format: Adriana Mañas, Kristina Aaltonen, Natalie Andersson, Karin Hansson, Alexandra Seger, Hiroaki Yasui, Katarzyna Radke, Javanshir Esfandyari, David Lindgren, David Gisselsson, Daniel Bexell. Modeling and deciphering COJEC resistance in high risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3901.

Neuroblastoma is a childhood solid tumor commonly located in the adrenal gland. High-risk neuroblastoma patients undergo multidrug and multi-interventional treatment but despite these efforts many patients develop resistance to chemotherapy and eventually relapse. Neuroblastoma patients face an urgent need of novel therapeutic strategies that could effectively combat the disease. In a recent study we identified kinesin spindle protein (KSP) as a highly effective target in high-risk neuroblastoma. Pharmacological inhibition of KSP resulted in complete regression of a subset of neuroblastoma subcutaneous PDX tumors and tumor growth delay in orthotopic PDXs (Hansson, Radke et al, Sci. Transl. Med 2020). The purpose of the present study is to identify novel synergistic drug combinations effective against neuroblastoma and investigate their translational potential. We designed, optimized, and performed a high-throughput combination screen using 3D tumor organoids that represent three MYCN-amplified neuroblastoma tumors. KSP inhibition was tested in combination with the FIMM drug set of 527 approved and investigational drugs. Screening was performed stepwise: • Step 1: High-throughput drug screening 527 drug combinations and machine learning prediction (DECREASE tool) of drug combination synergy based on minimal required input. • Step 2: Screening of 6x6 dose-response matrices of top 26 predicted hits in high-risk neuroblastoma models. • Step 3: Screening of 6x6 dose-response matrices of top 26 hits in CD34+ cord blood controls to identify compounds that have low toxicity towards proliferating fraction of hematopoietic cells. Overall, we identified seven drug candidates (P1-P7). They classify as: kinase inhibitors, differentiating modifiers, and one hormone therapy. Screened combinations were chosen based on selective efficacy (difference between anti-neuroblastoma efficacy and efficacy towards CD34+ cord blood controls). Identified drug combinations are common among three high-risk neuroblastoma models and have most synergistic area score (MSA) over five. We further plan to validate seven hits in neuroblastoma models in vitro, investigate MOA of the most promising combination and test it in PDX models in vivo. Citation Format: Katarzyna Radke, Karin Hansson, Jani Saarela, Aleksandr Ianevski, Philipp Ianevski, Aurélie Baudet, Mattias Magnusson, Daniel Bexell. High-throughput combination screen for identifying novel therapies against high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3892.

KSP inhibition results in cell death through mitotic arrest and causes regression of neuroblastoma PDX tumors in mice.

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