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Abstract

Neuroblastoma (NB) represents a malignant neoplasm that arises from precursor cells of the sympathetic nervous system. This disease entity is responsible for a considerable proportion of childhood cancer-related mortalities, emphasizing the pressing need for effective therapeutic interventions. Despite the availability of several therapeutic options for the treatment of high-risk NB, patients frequently experience relapse even after exhibiting an initial positive response to treatment. Moreover, the relapsed tumors in the majority of cases demonstrate a high degree of resistance to alternative therapeutic interventions. Amplified MYCN is a defining characteristic of a high-risk subtype of NB, which is often accompanied by advanced metastatic disease and an unfavorable prognosis in terms of overall survival. MYCN is an oncoprotein that broadly reprograms tumor cells, induces proliferation, and blocks neuronal differentiation. Additionally, MYCN plays a crucial role in maintaining redox balance by activating antioxidant responses and significantly affecting cellular metabolism (Alborzinia et al., 2022; Floros et al., 2021; Lu et al., 2021). Although amplified MYCN is a well-known risk factor for NB, direct targeting of MYCN remains challenging. Therefore, researchers are exploring alternative therapeutic strategies that exploit metabolic vulnerabilities related to MYCN. According to recent studies conducted by Alborzinia et al. (2022), a functional association has been established between oncogenic MYCN and ferroptosis, a form of iron-dependent, regulated cell death. The research suggests that NB cells that have amplified MYCN are highly dependent on cysteine, and deprivation of this amino acid from the growth medium resulted in significant lipid peroxidation. However, co-administration of ferrostatin-1, a ferroptosis inhibitor, was found to prevent this effect. To further understand NB cell lines' sensitivity to ferroptosis induction, this study focused on an extensive characterization of NB cell lines to identify the genetic and molecular mechanisms underlying their addiction to cystine and sensitivity to ferroptotic cell death. Based on the acquired data, it is apparent that the sensitivity to ferroptosis is influenced by the cellular lineage state. Specifically, cell lines with greater mesenchymal characteristics exhibit heightened resistance to the induction of ferroptosis. Additionally, by correlating baseline gene expression data to sensitivity to ferroptosis induction, the Q10-related pathways were identified as an important resistance mechanism against ferroptosis. Notably, these pathways were found to be downregulated in MYCN-amplified tumors, which suggests their possible sensitivity to ferroptosis. Moreover, the response of NB cell lines to various ferroptotic stress stimuli was investigated. Transcriptome profiling of NB cells subjected to cystine depletion revealed an upregulation of the ATF4-related stress response, which was more prominent in MYCN-amplified cell lines. In contrast, MYCN-non-amplified cells exhibited increased cystine uptake, likely due to sufficient levels of glutamate available for exchange with cystine. In addition, decreased glutamate levels were observed in MYCN-amplified NB tumors, emphasizing the role of glutamate in thiol metabolism. Furthermore, the role of the transulfuration pathway in NB sensitivity to ferroptotic cell death was explored. Downregulation of one of the key enzymes in de novo cysteine synthesis pathway (CTH) was linked to increased sensitivity to ferroptosis inducers and accumulation of toxic lipid peroxides. On the transcriptome level, CTH downregulation led to the upregulation of cysteine uptake / restoration mechanisms and adrenergic-to-mesenchymal transition. In addition, stable isotope tracing experiments confirmed the involvement of methionine-derived cysteine in glutathione synthesis. In this study, the significance of glutathione peroxidase 4 (GPX4), a crucial factor in ferroptosis, has also been investigated. Treatment with ferroptosis inducers has demonstrated that the downregulation of GPX4 increases the sensitivity of NB cells to ferroptosis induction. Furthermore, transcriptomic profiling has revealed that GPX4 knockdown results in upregulation of genes implicated in the TNFA signaling pathway and adrenergic-to-mesenchymal transition. These findings suggest that the loss of cellular identity is a potential adaptive response mechanism to the stress imposed by ferroptosis. To extend the in vitro results to clinically relevant scenarios, a regression model was developed using the data from the cell lines and subsequently applied to a dataset of 600 NB tumors. The results of this model suggest that MYCN-amplified NB tumors are more likely to be vulnerable to ferroptosis induction. Interestingly, the expression of MYCN and its associated targets were found to be the most differentially expressed genes between tumors predicted to be sensitive or resistant to the ferroptosis-inducing agents. Taken together, the study's findings suggest that targeting ferroptosis may represent a promising therapeutic strategy for MYCN or c-MYC-driven NB tumors. The study's comprehensive profiling of NB cell lines has contributed significantly to the understanding of ferroptosis regulation, and it allowed to identify specific mechanisms that can serve as vulnerabilities of MYC-driven tumors.