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Abstract

Neuroblastoma is the most common extracranial solid tumor in children and shows variable clinical outcomes. Neuroblastoma comprises epigenetically regulated subtypes, sustained by super enhancer networks and characterized by subtype specific gene sets. The adrenergic subtype shows a more differentiated, adrenal linage committed identity while the mesenchymal subtype displays a more undifferentiated stem cell like phenotype. This diversity contributes to the clinical heterogeneity as mesenchymal cells show elevated resistance to conventional chemotherapy drugs and occur more often in relapses. However, comprehensive analysis of subtype-specific vulnerabilities and optimal therapeutic interventions remain an ongoing challenge. Drug sensitivity profiling based on metabolic activity for 75 clinically relevant drugs for a panel of 24 neuroblastoma cell lines showed varying drug sensitivity profiles across the subtypes. Elevated sensitivity to chemotherapeutic drugs was confirmed for the adrenergic subtype, whereas increased sensitivity to MEK inhibitors was revealed as novel vulnerability for the mesenchymal subtype. Neuroblastoma subtypes differ in their cellular phenotype, therefore I developed a high-content imaging pipeline to quantify distinct morphological characteristics. Mesenchymal neuroblastoma cells showed an enlarged cellular phenotype and increased lysosomal content. Using changes in lysosomal content as readout for an image-based drug screen, I highlighted differences in the lysosomal stress response among neuroblastoma subtypes and demonstrated a higher increase in lysosome amount in response to chemotherapy treatment for mesenchymal neuroblastoma. To test, if higher lysosomal content in combination with the previously identified drug hits such as the MEKi could serve as a targetable vulnerability in the mesenchymal subtype, I did synergy combination screens, including image-based and metabolic readouts. Here, the lysosome was targeted by lysomotropic drugs and inhibitors of lysosomal acid sphingomyelinase. Although the results revealed synergistic effects for some combinations, a general heightened sensitivity for the mesenchymal subtype could not be demonstrated. Gene expression analysis revealed an upregulation of senescence secretome-associated pathways in mesenchymal neuroblastoma cells. Moreover, senescence was induced upon treatment of mesenchymal cells with inhibitors of the MAPK pathway, suggesting a metabolic alteration, which is druggable by senolytic treatments. The combination of senescence inducing drugs and senolytic agents was indeed synergistic in a sequential synergy screen setting. In summary, metabolic and morphological drug screens identified subtype-specific differences in drug sensitivity and phenotypic adaptation to drug treatment. Targeting lysosomal acid sphingomyelinase or senolytic signaling in combination with inhibitors of the MAPK pathway may be a possible therapeutic approach for neuroblastoma and will have to be evaluated in further studies.