Project description:Neuroblastoma is an embryonal tumor which originates from neural crest progenitor cells that fail to differentiate along their predefined route to sympathetic neurons or sympatho-adrenergic adrenal cells. It is the most common extracranial tumor of childhood and accounts for 15% of all childhood cancer deaths. Especially patients suffering from high grade or relapsed neuroblastoma have poor outcome in spite of aggressive treatment regimens including autologous stem cell transplantation. Those patients are in urgent need of additional effective therapies which demands the development of targeted approaches. Dihydroorotatedehydrogenase (DHODH) is the fourth enzyme of the pyrimidine synthesis pathway which oxidizes dihydroorotate to orotate. In recent past it became a potential drug target for cancer treatment because of its keyrole in processing essential pyrimidine nucleotides. On the basis of the existing data, functional inhibition of DHODH is considered to be promising therapeutic option for several tumor entities like advanced colorectal, breast or lung-cancers. Leflunomide is an established drug in treatment of the autoimmune diseases rheumatoid arthritis and multiple sclerosis. In the liver Leflunomide becomes converted to its active metabolite called Teriflunomide, which inhibits the activity of DHODH directly. In recent times Leflunomide is also used for therapy against the Cytomegalovirus and the BK virus. Also for Melanoma was shown recently a decreased growth rate due to Leflunomide treatment in a zebrafish and a mouse model. As Melanoma is a malignant tumor of the skin, which derives also from neural crest progenitor cells, a coherent investigation of effictivity of Leflunomide in neuroblastoma celllines showed first promising results. The aim of our study was to reanalyse the effectivity of Leflunomide in Neuroblastoma and to shed further light in its biological mode of action. Three+B52 biological samples of the human neuroblastoma cell line IMR32 were treated with DMSO or Teriflunomide [118 ￂﾵM]

Project description:Neuroblastoma is the most common heterogeneous solid tumor in childhood, with limited treatments available for high-risk patients. Previous studies have shown that Dihydroorotate dehydrogenase (DHODH) plays an essential role in the de novo synthesis of pyrimidine and have identified it as a potential therapeutic target in cancer. Our previous studies have shown that the higher expression of DHODH is associated with the worst survival of neuroblastoma patients. Through knockdown experiments, we validated that reducing DHODH expression in neuroblastoma cells leads to a decrease in cell viability. Subsequently, we harnessed virtual screening software to repurpose drugs, targeting DHODH as potential neuroblastoma therapeutics. We conducted molecular docking simulations with a database of 2702 FDA-approved drugs on DHODH and identified a number of drugs that demonstrated high affinity for DHODH. Among these drugs, we found regorafenib, a multi-kinase inhibitor, effectively inhibited neuroblastoma cell proliferation and was more efficient than leflunomide, an FDA-approved DHODH inhibitor. To determine whether regorafenib is a novel DHODH inhibitor, we employed thermal shift and enzyme fluorescence assays. These assays revealed that regorafenib not only enhanced binding stability but also reduced the enzymatic activity of DHODH. Since regorafenib is a multi-kinase inhibitor, we confirm changes in downstream proteins by comparison with shDHODH to determine whether these alterations result from targeting DHODH. To further investigate the molecular mechanism of regorafenib in targeting DHODH, we conducted a proteome analysis using tandem mass tag (TMT) labeling. This involved comparing samples treated with regorafenib to those with DHODH knockdown. Using Liquid Chromatograph-mass spectrometry/ mass spectrometry (LC-MS/MS), a total of 4471 proteins and 31518 peptides were identified. Furthermore, we identified 343 differentially expressed proteins in the shDHODH sample and 225 in the regorafenib treatment sample. We performed gene ontology (GO) enrichment analysis on these two sets of samples. The results of the GO enrichment analysis, focusing on biological function, indicated that proteins were correlated with lipid metabolism. After a research search, the proteome data and validation through western blot revealed common differentially expressed proteins related to the mevalonate pathway and subsequently linked to ferroptosis. Additionally, previous studies suggest a connection between DHODH and ferroptosis, leading us to investigate whether regorafenib treatment influences ferroptosis as confirmation that our drug targets DHODH. Moreover, neuroblastoma cells could be rescued by ferroptosis inhibitor liproxstatin-1. In summary, our findings highlight the potential of using regorafenib to target DHODH, presenting a promising therapeutic strategy for high-risk neuroblastoma.

Project description:Neuroblastoma is the most common heterogeneous solid tumor in childhood, with limited treatments available for high-risk patients. Previous studies have shown that Dihydroorotate dehydrogenase (DHODH) plays an essential role in the de novo synthesis of pyrimidine and have identified it as a potential therapeutic target in cancer. Our previous studies have shown that the higher expression of DHODH is associated with the worst survival of neuroblastoma patients. Through knockdown experiments, we validated that reducing DHODH expression in neuroblastoma cells leads to a decrease in cell viability. Subsequently, we harnessed virtual screening software to repurpose drugs, targeting DHODH as potential neuroblastoma therapeutics. We conducted molecular docking simulations with a database of 2702 FDA-approved drugs on DHODH and identified a number of drugs that demonstrated high affinity for DHODH. Among these drugs, we found Regorafenib, a multi-kinase inhibitor, effectively inhibited neuroblastoma cell proliferation and was more efficient than leflunomide, an FDA-approved DHODH inhibitor. To determine whether Regorafenib is a novel DHODH inhibitor, we employed thermal shift and enzyme fluorescence assays. These assays revealed that Regorafenib not only enhanced binding stability but also reduced the enzymatic activity of DHODH. Since Regorafenib is a multi-kinase inhibitor, we confirm changes in downstream proteins by comparison with shDHODH to determine whether these alterations result from targeting DHODH. To further investigate the molecular mechanism of Regorafenib in targeting DHODH, we conducted a proteome analysis using tandem mass tag (TMT) labeling. This involved comparing samples treated with Regorafenib to those with DHODH knockdown. Using Liquid Chromatograph-mass spectrometry/ mass spectrometry (LC-MS/MS), a total of 4471 proteins and 31518 peptides were identified. Furthermore, we identified 377 differentially expressed proteins in the shDHODH sample and 225 in the Regorafenib treatment sample. We performed gene ontology (GO) enrichment analysis on these two sets of samples. The results of the GO enrichment analysis indicate that these proteins are associated with lipid metabolism in terms of biological functions. Through literature searches, we found that the results from proteomics analysis and Western blot validation both show that the commonly differentially expressed proteins are related to the mevalonate pathway. This pathway, when affected, influences ferroptosis. Additionally, we discovered a relationship between DHODH inhibition and lipid droplet production. In summary, previous studies have shown a connection between DHODH and ferroptosis. However, we identified a new mechanism where DHODH inhibition induces ferroptosis by affecting the mevalonate pathway. Therefore, our findings underscore the potential of using Regorafenib to target DHODH, proposing a novel and promising therapeutic strategy for high-risk neuroblastoma.

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