Metabolic reprogramming contributes to radioprotection by protein kinase Cδ

Angela M Ohm; Trisiani Affandi; Julie A Reisz; M Cecilia Caino; Angelo D'Alessandro; Mary E Reyland

doi:10.1016/j.jbc.2023.105186

Metabolic reprogramming contributes to radioprotection by protein kinase Cδ

J Biol Chem. 2023 Oct;299(10):105186. doi: 10.1016/j.jbc.2023.105186. Epub 2023 Aug 21.

Authors

Angela M Ohm¹, Trisiani Affandi¹, Julie A Reisz², M Cecilia Caino³, Angelo D'Alessandro², Mary E Reyland⁴

Affiliations

¹ Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
² Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
³ Department of Pharmacology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
⁴ Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA. Electronic address: Mary.Reyland@cuanschutz.edu.

Abstract

Loss of protein kinase Cδ (PKCδ) activity renders cells resistant to DNA damaging agents, including irradiation; however, the mechanism(s) underlying resistance is poorly understood. Here, we have asked if metabolic reprogramming by PKCδ contributes to radioprotection. Analysis of global metabolomics showed that depletion of PKCδ affects metabolic pathways that control energy production and antioxidant, nucleotide, and amino acid biosynthesis. Increased NADPH and nucleotide production in PKCδ-depleted cells is associated with upregulation of the pentose phosphate pathway (PPP) as evidenced by increased activation of G6PD and an increase in the nucleotide precursor, 5-phosphoribosyl-1-pyrophosphate. Stable isotope tracing with U-[¹³C₆] glucose showed reduced utilization of glucose for glycolysis in PKCδ-depleted cells and no increase in U-[¹³C₆] glucose incorporation into purines or pyrimidines. In contrast, isotope tracing with [¹³C₅, ¹⁵N₂] glutamine showed increased utilization of glutamine for synthesis of nucleotides, glutathione, and tricarboxylic acid intermediates and increased incorporation of labeled glutamine into pyruvate and lactate. Using a glycolytic rate assay, we confirmed that anaerobic glycolysis is increased in PKCδ-depleted cells; this was accompanied by a reduction in oxidative phosphorylation, as assayed using a mitochondrial stress assay. Importantly, pretreatment of cells with specific inhibitors of the PPP or glutaminase prior to irradiation reversed radioprotection in PKCδ-depleted cells, indicating that these cells have acquired codependency on the PPP and glutamine for survival. Our studies demonstrate that metabolic reprogramming to increase utilization of glutamine and nucleotide synthesis contributes to radioprotection in the context of PKCδ inhibition.

Keywords: apoptosis; metabolism; protein kinase Cδ; radioprotection; salivary gland.