Fig 2: USP1 inhibitor treatment results in accumulation of ssDNA gaps in a RAD18-dependent manner. A and B, DNA fiber assays with or without S1 nuclease treatment for analysis of ssDNA gaps. A, Representative images of the DNA fibers showing presence or absence of ssDNA gaps after S1 nuclease treatment are shown. Top, schematics of nascent DNA labeling with two nucleotide analogs CldU and IdU in presence or absence of drugs (olaparib or I-138), followed by incubation with or without S1 nuclease. Reduction in IdU length after S1 nuclease treatment indicates presence of ssDNA gaps in UWB1.289 cells. B, Bottom, quantification of the IdU tract lengths evaluating the presence or absence of ssDNA gaps. Each dot indicates one fiber; at least 200 fibers were analyzed per condition. The horizontal bars indicate the median of the IdU tract lengths in each experimental condition. C, Volcano plot showing genes targeted by sgRNAs that differentially dropped out (blue) or became enriched (red) in USP1 inhibitor (TNG0869) versus DMSO-treated UWB1.289 cells in a CRISPR screen. D, aniPOND analysis of MDA-MB-436 cells after treatment with I-138 (1 µmol/L) or DMSO for 4 hours. Top, schematic of the drug treatment and nascent DNA labeling with EdU in MDA-MB-436 cells. Bottom, Western blots of input or captured proteins showing that RAD18, USP1, and Ub-PCNA proteins are enriched at the replication fork after treatment with I-138. E, Western blots of the soluble and chromatin fractions from MDA-MB-436 cells treated with USP1 inhibitor I-138 for 4 hours. F, Top, representative immunofluorescence images of the proximity ligation–based assay (PLA) foci in MDA-MB-436 cells depicting increased localization of RAD18 at the EdU-labeled nascent DNA after I-138 treatment. Bottom, quantification of proximity ligation–based assay foci per cell nucleus in DMSO- or I-138–treated cells. Between 100 and 300 cells were analyzed per condition. G, Top, schematic of PCNA regulation through ubiquitination by RAD18 and deubiquitination by USP1. Bottom, Western blots of the lysates from MDA-MB-436 cells after transfection with siCtrl or siRAD18, followed by treatment with the USP1 inhibitor I-138 for 24 hours. H, Clonogenic survival assays of siCtrl or siRAD18 transfected UWB1.289 cells in presence of a USP1 inhibitor I-138. Graphical quantitation of the proportion of colonies from cells treated with increasing concentrations of the USP1 inhibitor I-138 compared with control cells treated with DMSO is shown. UWB1.289 cells were transfected with siCtrl or siRAD18 and treated in six-well plates for 10 days with I-138. I, DNA fiber assays with or without S1 nuclease treatment for analysis of ssDNA gaps. Top, schematics of nascent DNA labeling with two nucleotide analogs CldU and IdU in presence or absence of I-138, followed by incubation with or without S1 nuclease in UWB1.289 cells after transfection with siCtrl or siRAD18. Bottom, quantification of the IdU tract lengths evaluating the presence or absence of ssDNA gaps. Each dot indicates one fiber; at least 200 fibers were analyzed per condition. The horizontal bars indicate the median of the lengths in each experimental condition as described on the bottom. P values were calculated using the Mann–Whitney test. *, P > 0.05; ***, P < 0.001.

Fig 3: USP1 inhibition results in ssDNA gap accumulation and killing of BRCA1-deficient ovarian cancer patient-derived organoids. A, Representative images of the morphology of ovarian cancer patient-derived organoids. B, Quantitation of RAD51 foci and γH2AX foci in DF4104 (BRCA1-WT) and DF3888 (BRCA1-mutant) patient-derived organoids after IR treatment. C, USP1 mRNA levels in patient-derived organoids. The gene expression levels were normalized to the internal standard gene, GAPDH. The means and SE were determined from triplicate measurements in two biologic replicates. P values were calculated using the t test. Internal normalization for the baseline expression of USP1 mRNA was made with DF4732 (BRCA1/2 WT PDO) for comparison. D, Survival plots of BRCA1-WT and BRCA1-mutant patient-derived organoids exposed to a graded concentration of USP1 inhibitor TNG6132. Cells were plated to 96-well U-bottom ultralow attachment plates and incubated for 48 hours prior to drug treatment. Viability was assessed at 7 days using CellTiter-Glo 3D reagent. E, DNA fiber assays with and without S1 nuclease treatment in patient-derived organoids. Top, schematics of nascent DNA labeling with two nucleotide analogs CldU and IdU in presence or absence of drugs (niraparib or TNG6132), followed by treatment with or without S1 nuclease. Bottom, quantification of the IdU tract lengths of the fibers evaluating the presence or absence of ssDNA gaps. Each dot indicates one fiber; at least 100 fibers were analyzed per condition. The horizontal bars indicate the median of the lengths in each experimental condition as described at the bottom. P values were calculated using the Mann–Whitney test. F, A table summarizing the drug sensitivity and ssDNA gap assay results of BRCA1-WT and BRCA1-mutant patient-derived organoids. ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Fig 4: USP1 inhibition synergizes with ssDNA gap–inducing agents such as PARP inhibitors and POLQ inhibitors. A, Schematic of the model depicting accumulation of ssDNA gaps after inhibition of PARP, POLQ, or USP1. B and D, Synergy between the USP1 inhibitor I-138 and olaparib (B) or I-138 and novobiocin (D) in indicated cell lines. Left, Bliss synergy/antagonism levels on the experimental combination dose–response surface for cells treated with graded concentrations of I-138 and olaparib or I-138 and novobiocin. Bliss scores greater than zero (green/blue shading) indicate synergy. Cells were grown for 24 hours in triplicate before exposure to graded concentrations of I-138 and olaparib or I-138 and novobiocin. Viability was assessed at 7 days using CellTiter-Glo reagent. Right, IC50 plots for olaparib sensitivity (B) or novobiocin sensitivity (D) of the indicated cells in presence or absence of I-138. C and E, DNA fiber assays with or without S1 nuclease treatment for analysis of ssDNA gaps. Top, schematics of nascent DNA labeling with two nucleotide analogs CldU and IdU in presence or absence of drugs (olaparib, I-138, or novobiocin), followed by incubation with or without S1 nuclease in indicated cell lines. Bottom, quantification of the IdU tract lengths of the fibers evaluating the presence or absence of ssDNA gaps in indicated cell lines. Each dot indicates one fiber; at least 200 fibers were analyzed per condition. The horizontal bars indicate the median of the lengths in each experimental condition as described on the bottom. P values were calculated using the Mann–Whitney test. *, P > 0.05; ***, P < 0.001.

Fig 5: Inhibition of DNA polymerase α induces SLFN11-dependent cell death in multiple cell lines.a, An experimental scheme depicting how cell viability was measured in indicated cell lines after transient knockout of SLFN11 and challenge with DNA polymerase α inhibitor (ST1926). b, A western blot depicting SLFN11 protein levels after transient knockout of SLFN11 in the indicated cell lines. c–g, Dose–response curves of HeLa Kyoto iCas9 (c), HT-1080 iCas9 (d), TOV112D iCas9 (e), A673 iCas9 (f) and eHAP iCas9 (g) cells challenged with the indicated doses of DNA polymerase α inhibitor (ST1926). Experiments were performed in n = 3 biological replicates. Data are presented as mean ± s.d. h, In WT cells expressing SLFN11 (blue panel), PrimPol repriming activity restricts accumulation of ssDNA at cisplatin-stalled replication forks caused by uncoupling of the helicase and DNA polymerase, thereby preventing SLFN11 activation and cell death. Similarly, inhibition of DNA polymerase α uncouples DNA replication on each strand to drive ssDNA accumulation at forks in a PrimPol-independent manner. In PrimPol-deficient cells (green panel), cisplatin-induced DNA damage drives accumulation of ssDNA at replication forks in a USP1-dependent manner, leading to RPA exhaustion and SLFN11 activation. USP1-dependent downregulation of the FA pathway allows for full SLFN11 activation and subsequent ribosome stalling, ISR activation and cell death, as previously described38. In SLFN11-deficient cells (red panel), cisplatin-induced DNA damage or inhibition of DNA polymerase α causes ssDNA accumulation at forks and RPA exhaustion. Loss of SLFN11 prevents signal transduction through ribosome stalling and GCN2, preventing SLFN11-dependent cell death. Instead, prolonged RPA exhaustion leads to fork breakage and replication catastrophe as previously described9, leading to DSBs that are repaired by the NHEJ pathway, leading to cell death. Source numerical data and unprocessed blots are available in the source data. PolAi, Pol A inhibitor.Source data