Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle, independently of its on-target effect

Birte Arlt,Guido Mastrobuoni,Jasmin Wuenschel,Kathy Astrahantseff,Angelika Eggert,Stefan Kempa,Hedwig E Deubzer

doi:10.1080/14756366.2021.1935917

Abstract

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.

Highlights

Neuroblastoma is the most common extracranial paediatric solid tumour that arises from precursor cells in the developing sympathetic nervous system
We extended proliferation monitoring to a panel of two neuroblastoma cell lines each expressing either high-level (BE(2)-C and Kelly) or low-level (SH-EP and SK-N-AS) phosphoglycerate dehydrogenase (PHGDH) (Figure 1(A))
These data indicate that NCT-503 is effective in a PHGDH-independent manner in neuroblastoma cells, raising the possibility that some part of NCT-503 action may be caused by off-target effects

Summary

Introduction

Neuroblastoma is the most common extracranial paediatric solid tumour that arises from precursor cells in the developing sympathetic nervous system. Indirect approaches targeting metabolic alterations driven by high-level MYCN amplification recently identified an enhanced dependency of these cells on the serine-glycine onecarbon metabolic pathway[3]. This network generates one-carbon units that are utilised in various metabolic reactions, including nucleotide biosynthesis, methylation reactions and maintaining redox balance. It is well appreciated that the small-molecule PHGDH inhibitor, NCT503, reduces production of glucose-derived serine and attenuates the growth of PHGDH-dependent cell lines and xenograft tumours[4]. We have shown that NCT-503 reduces proliferation in vitro and initial tumour growth in vivo in MYCNamplified neuroblastoma cells[5]. We assessed proliferation and applied pulsed stable isotope-resolved metabolomics utilising 13C-glucose labelling[6] in cellular models for neuroblastoma expressing varying levels of the NCT-503 target, PHGDH, with the aim to assess the impact of NCT-503 treatment on central carbon metabolism

Methods

Results

Conclusion