Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA -deficient cells to PARP inhibitors

Abstract

Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.

XRCC1 Prevents Replication Fork Instability during Misincorporation of the DNA Demethylation Bases 5-Hydroxymethyl-2′-Deoxycytidine and 5-Hydroxymethyl-2′-Deoxyuridine

“…Our data shows that the mechanism behind the aforementioned cytotoxicity is related to increased PARP1 chromatin retention at 5hmC/5hmU removal intermediates associated to replication fork collisions. In agreement with our data, a recently published work shows that contamination of the nucleotide pool by 5hmU-derived from 5hmC leads to 5hmU misincorporation in the genome, requiring some components of the BER pathway (SMUG1, PARP1, PARG) to promote cell survival [27]. The mechanism for this cytotoxicity seems to be related to olaparib trapped-PARP1 at 5hmU-processing sites persisting long enough as to be head-on collided by the replisome during the following cell cycle.…”

“…The mechanism for this cytotoxicity seems to be related to olaparib trapped-PARP1 at 5hmU-processing sites persisting long enough as to be head-on collided by the replisome during the following cell cycle. However, our work uncovers that 5hmU processing at replication forks directly impact in fork dynamics, and diverges from the model proposed by Fugger et al [27]. Our model would imply that BER processing of 5hmC or 5hmU substrates occurred straight after misincorporation, probably in the context of reversed replication forks.…”

“…We next examined if trapped PARP1-DNA complex by olaparib at the 5hmU DNA lesions incurred in increased genomic stability. In agreement with the role of PARP1 at signaling 5hmU DNA breaks [27], olaparib treatment largely exacerbated 5hmU-mediated γ-H2AX foci formation (Figure 5A), at a larger extent compared to the same dose of 5hmC. Consistent with the role of 5hmC at blocking ongoing replication fork progression (Figure 5B), 5hmU also hindered replication fork progression at larger extent than 5hmC exposure, which was exacerbated by olaparib treatment, thus trapping PARP1 at 5hmU sites.…”

“…A recent report shows that 5hmC can be taken up by cells and deaminated to 5hmU in the cytoplasm, contaminating the free nucleotide pool [27]. 5hmU is readily incorporated during DNA synthesis and removed from DNA by an active BER mechanism involving SMUG1, PARG and PARP1, suggesting that 5hmC-derived 5hmU is a novel source of genomic instability.…”

An enzyme-mediated bioorthogonal labeling method for genome-wide mapping of 5-hydroxymethyluracil

“… 18 It is worth noting that the loss of nucleotide salvage factor DNPH1 can give rise to aberrant incorporation of 5-hmdU into human cells. 19 The post-replicative formation of 5hmU occurs via hydroxylation of thymine, which can be mediated by ten-eleven translocation (TET) dioxygenases in mammals 20 and J-binding proteins (JBPs) in protozoan genomes, 21,22 respectively. Another proposed mechanism for the formation of 5hmU is through the deamination of 5hmC by activation-induced cytidine deaminase (AID) or apolipoprotein B mRNA-editing catalytic polypeptide-like (APOBEC) family enzymes.…”