Here we sought metabolic alterations specifically associated with MYCN amplification as nodes to indirectly target the MYCN oncogene. Liquid chromatography‐mass spectrometry‐based proteomics identified seven proteins consistently correlated with MYCN in proteomes from 49 neuroblastoma biopsies and 13 cell lines. Among these was phosphoglycerate dehydrogenase (PHGDH), the rate‐limiting enzyme in de novo serine synthesis. MYCN associated with two regions in the PHGDH promoter, supporting transcriptional PHGDH regulation by MYCN. Pulsed stable isotope‐resolved metabolomics utilizing 13C‐glucose labeling demonstrated higher de novo serine synthesis in MYCN‐amplified cells compared to cells with diploid MYCN. An independence of MYCN‐amplified cells from exogenous serine and glycine was demonstrated by serine and glycine starvation, which attenuated nucleotide pools and proliferation only in cells with diploid MYCN but did not diminish these endpoints in MYCN‐amplified cells. Proliferation was attenuated in MYCN‐amplified cells by CRISPR/Cas9‐mediated PHGDH knockout or treatment with PHGDH small molecule inhibitors without affecting cell viability. PHGDH inhibitors administered as single‐agent therapy to NOG mice harboring patient‐derived MYCN‐amplified neuroblastoma xenografts slowed tumor growth. However, combining a PHGDH inhibitor with the standard‐of‐care chemotherapy drug, cisplatin, revealed antagonism of chemotherapy efficacy in vivo. Emergence of chemotherapy resistance was confirmed in the genetic PHGDH knockout model in vitro. Altogether, PHGDH knockout or inhibition by small molecules consistently slows proliferation, but stops short of killing the cells, which then establish resistance to classical chemotherapy. Although PHGDH inhibition with small molecules has produced encouraging results in other preclinical cancer models, this approach has limited attractiveness for patients with neuroblastoma.
Development of new anticancer drugs with currently available animal models is hampered by the fact that human cancer cells are embedded in an animal-derived environment. Neuroblastoma is the most common extracranial solid malignancy of childhood. Major obstacles include managing chemotherapy-resistant relapses and resistance to induction therapy, leading to early death in very-high-risk patients. Here, we present a three-dimensional (3D) model for neuroblastoma composed of IMR-32 cells with amplified genes of the myelocytomatosis viral related oncogene MYCN and the anaplastic lymphoma kinase (ALK) in a renal environment of exclusively human origin, made of human embryonic kidney 293 cells and primary human kidney fibroblasts. The model was produced with two pneumatic extrusion printheads using a commercially available bioprinter. Two drugs were exemplarily tested in this model: While the histone deacetylase inhibitor panobinostat selectively killed the cancer cells by apoptosis induction but did not affect renal cells in the therapeutically effective concentration range, the peptidyl nucleoside antibiotic blasticidin induced cell death in both cell types. Importantly, differences in sensitivity between two-dimensional (2D) and 3D cultures were cell-type specific, making the therapeutic window broader in the bioprinted model and demonstrating the value of studying anticancer drugs in human 3D models. Altogether, this cancer model allows testing cytotoxicity and tumor selectivity of new anticancer drugs, and the open scaffold design enables the free exchange of tumor and microenvironment by any cell type.
The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.
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