Checkpoint kinase 1 inhibition sensitises transformed cells to dihydroorotate dehydrogenase inhibition.

Stéphanie Arnould,Gisèle Matar,Charles Theillet,Florence Bernex,Jean Yohan Noël,Yoan Buscail,Yves L Janin,Charles Vincent,Hélène Delpech,Marta Jarlier,Charlène Berthet,Yoann Delorme,Pierre Olivier Vidalain,Nelly Pirot,Geneviève Rodier,Simon Lachambre,Claude Sardet

doi:10.18632/oncotarget.19199

Abstract

Reduction in nucleotide pools through the inhibition of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) has been demonstrated to effectively reduce cancer cell proliferation and tumour growth. The current study sought to investigate whether this antiproliferative effect could be enhanced by combining Chk1 kinase inhibition. The pharmacological activity of DHODH inhibitor teriflunomide was more selective towards transformed mouse embryonic fibroblasts than their primary or immortalised counterparts, and this effect was amplified when cells were subsequently exposed to PF477736 Chk1 inhibitor. Flow cytometry analyses revealed substantial accumulations of cells in S and G2/M phases, followed by increased cytotoxicity which was characterised by caspase 3-dependent induction of cell death. Associating PF477736 with teriflunomide also significantly sensitised SUM159 and HCC1937 human triple negative breast cancer cell lines to dihydroorotate dehydrogenase inhibition. The main characteristic of this effect was the sustained accumulation of teriflunomide-induced DNA damage as cells displayed increased phospho serine 139 H2AX (γH2AX) levels and concentration-dependent phosphorylation of Chk1 on serine 345 upon exposure to the combination as compared with either inhibitor alone. Importantly a similar significant increase in cell death was observed upon dual siRNA mediated depletion of Chk1 and DHODH in both murine and human cancer cell models. Altogether these results suggest that combining DHODH and Chk1 inhibitions may be a strategy worth considering as a potential alternative to conventional chemotherapies.

Highlights

Dihydroorotate dehydrogenase (EC 1.3.5.2; dihydroorotate dehydrogenase (DHODH)) is the one mitochondrial enzyme that is located on the outer surface of the inner membrane and takes part in the fourth and rate-limiting step of de novo pyrimidine biosynthesis [1]
While a 24-hour exposure to TFN had a limited effect on primary and immortalised cells, it strongly reduced proliferation of transformed cells in a concentrationdependent manner (p < 0.01). This differential effect was observed when these cell populations were exposed to another DHODH inhibitor, IPP-A017-A04 (Supplementary Figure 1; [10]), and the antiproliferative effect of both compounds was partly reversed by concomitant exposure to 50 μg/ml uridine (Supplementary Figure 2)
Conventional chemotherapies have been combined with Chk1 inhibitors in order to increase their cytotoxicity through the abrogation of S and G2/M Chk1-dependent DNA damage checkpoints

Summary

Introduction

Dihydroorotate dehydrogenase (EC 1.3.5.2; DHODH) is the one mitochondrial enzyme that is located on the outer surface of the inner membrane and takes part in the fourth and rate-limiting step of de novo pyrimidine biosynthesis [1]. It converts dihydroorotic acid to orotic acid whilst reducing ubiquinone to ubiquinol which makes DHODH a link between pyrimidine synthesis and respiratory electron transport chain. DHODH has emerged as a new therapeutic target in a wide spectrum of pathologies as de novo pyrimidine synthesis is extensively used in rapidly proliferating human or parasitic cells. DHODH inhibition effectively slowed down cancer cell and tumour growth of diverse tissue origins [20,21,22,23,24,25]

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