SUMMARY Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. Here we report that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduces NMG expression and substantially depletes nucleotide pools, which attenuates GTP dependent protein (GTPase) activity and cell invasion. Addition of exogenous guanosine or GTP restores the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. Additionally, mtp53 creates a dependency on the nucleoside salvage pathway enzyme deoxycytidine kinase (dCK) for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53 harboring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleoside salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53’s control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.

Co-targeting of both de novo and salvage pathways for dCTP biosynthesis shows efficacy in T-ALL and B-ALL.

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.

BackgroundDNA methylation is important for the maintenance of the silent state of genes on the inactive X chromosome (Xi). Here, we screened for siRNAs and chemicals that reactivate an Xi-linked reporter in the presence of 5-aza-2′-deoxycytidine (5-aza-2′-dC), an inhibitor of DNA methyltransferase 1, at a concentration that, on its own, is not sufficient for Xi-reactivation.ResultsWe found that inhibition of ribonucleotide reductase (RNR) induced expression of the reporter. RNR inhibition potentiated the effect of 5-aza-2′-dC by enhancing its DNA incorporation, thereby decreasing DNA methylation levels genome-wide. Since both 5-aza-2′-dC and RNR-inhibitors are used in the treatment of hematological malignancies, we treated myeloid leukemia cell lines with 5-aza-2′-dC and the RNR-inhibitor hydroxyurea, and observed synergistic inhibition of cell growth and a decrease in genome-wide DNA methylation.ConclusionsTaken together, our study identifies a drug combination that enhances DNA demethylation by altering nucleotide metabolism. This demonstrates that Xi-reactivation assays can be used to optimize the epigenetic activity of drug combinations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0034-4) contains supplementary material, which is available to authorized users.