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.

Fig. 1. Loss of DNPH1 sensitizes HR-deficient cells to PARP inhibitors.

(A) Volcano plot showing sgRNA scores from MAGeCK analysis of a genomewide CRISPR-Cas9 dropout/enrichment screen in eHAP MUS81^-/- cells (olaparib vs mock). Each point represents limit fold change (sensitizing sgRNAs to the left and resistance to the right) with corresponding MAGeCK score. BER and nucleotide metabolism factors are highlighted. (B) eHAP WT or KO cell lines were treated for 6 days with the indicated doses of olaparib, and viability was determined using CellTiter-Glo (mean with s.e.m; n=3). Data were analyzed using ANOVA for multiple comparisons. MUS81^-/- vs MUS81^-/- DNPH1 ^-/-, p = 0.0013; MUS81 ^-/- vs MUS81 ^-/- ITPA ^-/-, p = 0.0144. (C) DLD1 WT or KO cell lines were seeded for colony formation and treated continuously for 10 days with olaparib. Colonies were fixed and stained with crystal violet. Well diameter is 22 mm. (D) DLD1 WT or KO cell lines were treated continuously for 10 days with olaparib. Cell viability was determined, and data analyzed as in (B). BRCA2 ^-/- vs BRCA2 ^-/- DNPH1 ^-/-, p < 0.0001. (E) SUM149 WT (revertant) or KO cell lines were treated for 10 days with olaparib. Cell viability was determined, and data analyzed as in (B). BRCA1^mut vs BRCA1^mut DNPH1 ^-/-, p = 0.0021.

Fig. 2. DNPH1 is a nucleotide sanitizer that hydrolyses hmdUMP to prevent genomic DNA incorporation.

(A) Genomic DNA was extracted from eHAP WT or DNPH1^-/- cells, digested, and analyzed for its nucleoside composition by LC-MS. The graph depicts the ratio of the indicated nucleosides in DNPH1^-/- vs WT genomic DNA (mean with s.e.m; n=3). (B) Nucleotide pools were extracted from eHAP WT or DNPH1^-/- cells treated with 0.5 μM hmdU for 4 h and analyzed for hmdUMP by LC-MS. (C) SDS-PAGE of purified HIS-tagged DNPH1 and catalytic site mutant DNPH1^E104Q visualized by Instant Blue stain. (D) Schematic indicating the hydrolysis of hmdUMP by DNPH1 to form deoxyribose phosphate (dRP) and hydroxymethyl uracil (hmU). (E) Rates of dNMP hydrolysis by DNPH1 were determined by linear regression of data from Figure S3E. Each dNMP was analyzed independently. (F) Analysis of DNPH1 activity with deoxynucleoside monophosphates (dNMPs). DNPH1 or DNPH1^E104Q (4 μM) were incubated individually with the indicated substrates (1 mM) for 45 min. Products were analyzed by RP-HPLC and visualized as chromatograms. Untreated dNMPs and the nucleobase hmU are indicated. (G) ¹H NMR time-resolved spectroscopy of an equimolar mixture of dAMP, dCMP, dGMP, TMP and hmdUMP (0.5 mM each) following incubation with 1 μM DNPH1 for the indicated times. Time-dependent disappearance of hmdUMP (red box) and appearance of hydroxymethyl uracil are highlighted (blue box). The resonances of non-labile base protons from each nucleotide are shown.

Fig. 3. Sensitization of HR-deficient cells to PARPi by hmdU.

(A) eHAP MUS81^-/- cells were either untreated or treated with the indicated nucleosides (200 nM) in the presence or absence of olaparib (25 nM) for 6 days. Cell viability was determined using CellTiter-Glo (mean with s.e.m; n=3). (B) Patient-derived SUM149 BRCA1^mut (parental) and revertant (WT) cells were treated for 8 days with olaparib in the presence or absence of hmdU (1 μM). Cell viability was determined using CellTiter-Glo. BRCA1^mut vs BRCA1^mut + hmdU, p = 0.0004. (C) DLD1 WT and BRCA2 ^-/- cells were treated for 10 days with olaparib in the presence or absence of hmdU (1 μM). Cell viability was determined as in (B) BRCA2^-/- vs BRCA2^-/- + hmdU, p < 0.0001. (D) eHAP MUS81^-/- and MUS81^-/- DNPH1^-/- cells were either left untreated or treated with the indicated nucleosides (200 nM) for 6 days. Cell viability was determined as in (A). Bar chart shows the ratio of cell viability between MUS81^-/- DNPH1^-/- vs MUS81^-/- (mean with s.e.m; n=3). (E) DLD1 WT or KO cell lines were treated for 10 days with olaparib in the absence or presence of hmdU (50 nM). Cell viability was determined as in (B). BRCA2^-/- DNPH1^-/- vs BRCA2^-/- DNPH1^-/- + hmdU, p < 0.0001. DLD1 WT and BRCA2^-/- cells treated with olaparib only are from (C).

Fig. 4. hmdU is produced by metabolism of epigenetically modified nucleotides.

(A) Volcano plot showing sgRNA scores from MAGeCK analysis of CRISPR-Cas9 hmdC dropout/enrichment screen. Data are represented as in Fig. 1A (n=3). (B) eHAP KO cell lines were continuously treated with olaparib (50 nM) for 6 days and cell viabilities were determined using CellTiter-Glo. (C) Genomic DNA was extracted from the indicated eHAP cell lines, digested, and analyzed for hmdU by LC-MS. The ratio of hmdU levels to WT is indicated (mean with s.e.m; n=3). (D) eHAP cell lines were treated with hmdC (0.2 μM) for 24 hours. Genomic DNA was extracted, digested, and analyzed for hmdU by LC-MS. The relative genomic hmdU levels are indicated (mean with s.e.m; n=3) (E) eHAP MUS81^-/-, MUS81^-/- DNPH1^-/- or MUS81^-/- DNPH1^-/- cells transduced with lentiCRISPR-sgTET1 and sgTET2 were treated with olaparib (25 nM) in the absence or presence of hmdU (50 nM) for 6 days. Cell viability was determined as in (B). (F) Schematic showing the metabolism of hmdC originating from TET-mediated hydroxymethylated cytosine and other extracellular sources. Breakdown of DNA (for example, during DNA repair) releases epigenetically marked nucleotides, such as hmdCMP. To prevent their re-incorporation, they are degraded in a two-step process; (i) hmdCMP is deaminated to cytotoxic hmdUMP by DCTD, and (ii) DNPH1 hydrolyzes hmdUMP into hmU and dRP. DNPH1 deficiency leads to excess hmdUMP that becomes phosphorylated to hmdUTP by DTYMK and incorporated in DNA.

Fig. 5. hmdU promotes PARP trapping, DSB formation, and cell death through the actions of SMUG1.

(A) eHAP WT and KO cell lines were treated with olaparib (25 nM) and the indicated doses of hmdU for 6 days. Cell viability was determined using CellTiter-Glo (n=3). MUS81^-/- vs MUS81^-/- SMUG1^-/-, p = 0.0014. (B) eHAP WT and SMUG1^-/- cells were either untreated or pre-treated for 24 hours with hmdU (0.35 μM) or 2 hours with MMS (0.01%) followed by olaparib (10 μM) for 4 hours. Following subcellular fractionation the nuclear soluble or chromatin fractions were analyzed by immunoblotting with the indicated antibodies. (C) eHAP MUS81^-/- or MUS81^-/- DNPH1^-/- cells were transduced with lentiCRISPR-sgPARP1 or control (sgCTRL) and treated with olaparib for 6 days. Cell viability was determined as in (A). MUS81^-/- DNPH1^-/-/sgCTRL vs MUS81^-/- DNPH1^-/-/sgPARP1, p < 0.0001. (D) Top: PFGE analysis of DNA break formation in DLD1 WT or KO cell lines either untreated or treated with hmdU (2 μM) or co-treated with olaparib (0.1 μM) and hmdU (100 nM) for 72 hours. DNA breaks were visualized by ethidium bromide staining. Bottom: the same samples were analyzed by immunoblotting. (E) DLD1 WT and KO cell lines were either untreated or treated with olaparib (10 nM) and hmdU (0.1 μM) for 48 hours and subjected to immunofluorescence staining with a RAD51 antibody (green). DNA was stained by DAPI (blue). Scale bar is 10 μm. (F) Schematic overview of DNA fiber analysis. DLD1 WT or KO cells were untreated or co-treated with olaparib and hmdU for 48 hrs. Cells were labeled with CldU (20 mins; 20 μM) followed by IdU (20 mins; 150 μM). DNA fibers were spread on glass slides and subjected to immunofluorescence staining with CldU (red) and IdU (green) antibodies. Fork symmetry was assessed by measuring the lengths of the two bidirectional forks. (G) Plots showing the lengths of the bidirectional forks in DLD1 cell lines treated with olaparib (0.1 μM) and hmdU (1 μM) for 48 hrs. Bidirectional forks with >30% difference in lengths (outside red lines) were scored as asymmetrical and shown as percentage. (H) Plots showing the asymmetry factor from forks in (G). Horizontal line represents the median. BRCA2^-/- vs BRCA2^-/- DNPH1^-/-, p = 0.0028.

Fig. 6. Killing of PARPi-resistant BRCA1 cells by targeting DNPH1.

(A) Left panel: SUM149 BRCA1^mut (parental) and WT (revertant) cell lines were either untreated or treated with olaparib in the absence (black lines) or presence of hmdU (2 μM; red lines) for 8 days. Cell viability was determined using CellTiter-Glo (mean with s.e.m; n=3). Blue dotted line represents EC50 values. Center panel: As (left) but SUM149 BRCA1^mut (parental) and WT cell lines were either untreated or treated with the indicated doses of hmdU in the absence (black lines) or presence of DNPH1 KO (red lines) for 8 days. Right panel: Therapeutic index of the indicated treatments calculated from the ratio of EC50 values from (left) and (center) in SUM149 WT vs. BRCA1^mut cells. (B) Left: SUM149 BRCA1^mut/sg53BP1 or WT (revertant) cell lines were treated as in (A, left). For direct comparison, curves of WT and WT + hmdU from (A, left) are shown (solid black and red lines, respectively). Center: SUM149 BRCA1^mut/sg53BP1 or WT cell lines were treated as in (A, center). For direct comparison, curves of WT and DNPH1^-/- from (A, center) are shown (solid black and red lines, respectively). Right: Therapeutic index of the indicated treatments calculated from the ratio of EC50 values from (left) and (center) in SUM149 BRCA1^mut/sg53BP1 cells. (C) Left: SUM149 BRCA1^mut PARP1^-/- or WT cell lines were treated as in (A, left). For direct comparison, curves of WT and WT + hmdU from (A, left) are shown (solid black and red lines, respectively). Center: SUM149 BRCA1^mut PARP1^-/- or WT cell lines were treated as in (A, center). For direct comparison, curves of WT and DNPH1^-/- from (A, center) are shown (solid black and red lines, respectively). Right: Therapeutic index of the indicated treatments calculated from the ratio of EC50 values from (left) and (center) in BRCA1^mut PARP1^-/- cells. (D) Inhibition of DNPH1 activity towards hmdUMP by N6-benzyl-AMP (DNPH1i). DNPH1 (4 μM) was incubated with hmdUMP (1 mM) in the absence or presence of DNPH1i for 45 min. Products were analyzed by RP-HPLC and visualized as chromatograms. (E) SUM149 BRCA1^mut (parental) and WT (revertant) cell lines were either untreated or treated with hmdU in the absence or presence of DNPH1i (0.3 μM) for 8 days. Viability was determined using CellTiter-Glo (mean with s.e.m; n=3). BRCA1^mut vs BRCA1^mut + DNPH1i, p < 0.0001. (F) SUM149 BRCA1^mut PARP1^-/- or WT (revertant) cell lines were treated as in (E) (mean with s.e.m; n=3). BRCA1^mut PARP1^-/- vs BRCA1^mut PARP1^-/- + DNPH1i, p < 0.0001.