DNA polymerase theta (POLθ or POLQ) is synthetic lethal with homologous recombination (HR) deficiency and is thus a candidate target for HR-deficient cancers. Through high-throughput small-molecule screens, we identified the antibiotic novobiocin (NVB) as a specific POLθ inhibitor that selectively kills HR-deficient tumor cells in vitro and in vivo. NVB directly binds to the POLθ ATPase domain, inhibits its ATPase activity and phenocopies POLθ depletion. NVB kills HR-deficient breast and ovarian tumors in genetically engineered mouse models and xenograft and patient-derived xenograft models. Increased POLθ levels predict NVB sensitivity, and HR-deficient tumor cells with acquired resistance to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are sensitive to NVB in vitro and in vivo. Mechanistically, NVB-mediated cell death in PARPi-resistant cells arises from increased double-strand break end resection, leading to accumulation of single-stranded DNA intermediates and nonfunctional foci of the recombinase RAD51. Our results demonstrate that NVB may be useful alone or in combination with PARPi for treating HR-deficient tumors, including those with acquired PARPi resistance.
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Lord, C. J. & Ashworth, A. PARP inhibitors: synthetic lethality in the clinic. Science 355, 1152–1158 (2017).
Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921 (2005).
Bryant, H. E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).
Konstantinopoulos, P. A., Ceccaldi, R., Shapiro, G. I. & D’Andrea, A. D. Homologous recombination deficiency: exploiting the fundamental vulnerability of ovarian cancer. Cancer Discov. 5, 1137–1154 (2015).
Ledermann, J. et al. Olaparib maintenance therapy in platinum-sensitive relapsed ovarian cancer. N. Engl. J. Med. 366, 1382–1392 (2012).
Coleman, R. L. et al. Rucaparib maintenance treatment for recurrent ovarian carcinoma after response to platinum therapy (ARIEL3): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 390, 1949–1961 (2017).
Mirza, M. R. et al. Niraparib maintenance therapy in platinum-sensitive, recurrent ovarian cancer. N. Engl. J. Med. 375, 2154–2164 (2016).
Litton, J. K. et al. Talazoparib in patients with advanced breast cancer and a germline BRCA mutation. N. Engl. J. Med. 379, 753–763 (2018).
Lord, C. J. & Ashworth, A. Mechanisms of resistance to therapies targeting BRCA-mutant cancers. Nat. Med. 19, 1381–1388 (2013).
Sakai, W. et al. Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers. Nature 451, 1116–1120 (2008).
Ray Chaudhuri, A. et al. Replication fork stability confers chemoresistance in BRCA-deficient cells. Nature 535, 382–387 (2016).
Noordermeer, S. M. et al. The Shieldin complex mediates 53BP1-dependent DNA repair. Nature 560, 117–121 (2018).
Ghezraoui, H. et al. 53BP1 cooperation with the REV7–Shieldin complex underpins DNA structure-specific NHEJ. Nature 560, 122–127 (2018).
Ceccaldi, R. et al. Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair. Nature 518, 258–262 (2015).
Ceccaldi, R., Rondinelli, B. & D’Andrea, A. D. Repair pathway choices and consequences at the double-strand break. Trends Cell Biol. 26, 52–64 (2016).
Mateos-Gomez, P. A. et al. Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination. Nature 518, 254–257 (2015).
Wood, R. D. & Doublie, S. DNA polymerase θ (POLQ), double-strand break repair, and cancer. DNA Repair 44, 22–32 (2016).
Seki, M. et al. High-efficiency bypass of DNA damage by human DNA polymerase Q. EMBO J. 23, 4484–4494 (2004).
Yoon, J. H. et al. Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers. Cell 176, 1295–1309 (2019).
Chan, S. H., Yu, A. M. & McVey, M. Dual roles for DNA polymerase θ in alternative end-joining repair of double-strand breaks in Drosophila. PLoS Genet. 6, e1001005 (2010).
Goff, J. P. et al. Lack of DNA polymerase θ (POLQ) radiosensitizes bone marrow stromal cells in vitro and increases reticulocyte micronuclei after total-body irradiation. Radiat. Res. 172, 165–174 (2009).
Audebert, M., Salles, B. & Calsou, P. Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining. J. Biol. Chem. 279, 55117–55126 (2004).
Seki, M., Marini, F. & Wood, R. D. POLQ (Pol θ), a DNA polymerase and DNA-dependent ATPase in human cells. Nucleic Acids Res. 31, 6117–6126 (2003).
Ozdemir, A. Y., Rusanov, T., Kent, T., Siddique, L. A. & Pomerantz, R. T. Polymerase θ-helicase efficiently unwinds DNA and RNA–DNA hybrids. J. Biol. Chem. 293, 5259–5269 (2018).
Mateos-Gomez, P. A. et al. The helicase domain of Polθ counteracts RPA to promote alt-NHEJ. Nat. Struct. Mol. Biol. 24, 1116–1123 (2017).
Zahn, K. E., Jensen, R. B., Wood, R. D. & Doublie, S. Human DNA polymerase θ harbors DNA end-trimming activity critical for DNA repair. Mol. Cell 81, 1534–1547 (2021).
Beagan, K. et al. Drosophila DNA polymerase θ utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair. PLoS Genet. 13, e1006813 (2017).
Higgins, G. S. & Boulton, S. J. Beyond PARP–POLθ as an anticancer target. Science 359, 1217–1218 (2018).
Yusufzai, T. & Kadonaga, J. T. HARP is an ATP-driven annealing helicase. Science 322, 748–750 (2008).
Newman, J. A., Cooper, C. D. O., Aitkenhead, H. & Gileadi, O. Structure of the helicase domain of DNA polymerase θ reveals a possible role in the microhomology-mediated end-joining pathway. Structure 23, 2319–2330 (2015).
Higgins, G. S. et al. A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown. Cancer Res. 70, 2984–2993 (2010).
Eder, J. P., Wheeler, C. A., Teicher, B. A. & Schnipper, L. E. A phase I clinical trial of novobiocin, a modulator of alkylating agent cytotoxicity. Cancer Res. 51, 510–513 (1991).
Drusano, G. L. et al. Steady-state serum pharmacokinetics of novobiocin and rifampin alone and in combination. Antimicrob. Agents Chemother. 30, 42–45 (1986).
Kennedy, M. J. et al. Phase I and pharmacologic study of the alkylating agent modulator novobiocin in combination with high-dose chemotherapy for the treatment of metastatic breast cancer. J. Clin. Oncol. 13, 1136–1143 (1995).
Murren, J. R. et al. Phase I and pharmacokinetic study of novobiocin in combination with VP-16 in patients with refractory malignancies. Cancer J. 6, 256–265 (2000).
Liu, X. et al. Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer. Proc. Natl Acad. Sci. USA 104, 12111–12116 (2007).
Pantelidou, C. et al. PARP inhibitor efficacy depends on CD8+ T-cell recruitment via intratumoral STING pathway activation in BRCA-deficient models of triple-negative breast cancer. Cancer Discov. 9, 722–737 (2019).
Turner, N., Tutt, A. & Ashworth, A. Hallmarks of ‘BRCAness’ in sporadic cancers. Nat. Rev. Cancer 4, 814–819 (2004).
Taniguchi, T. et al. Disruption of the Fanconi anemia–BRCA pathway in cisplatin-sensitive ovarian tumors. Nat. Med. 9, 568–574 (2003).
Sugino, A., Higgins, N. P., Brown, P. O., Peebles, C. L. & Cozzarelli, N. R. Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc. Natl Acad. Sci. USA 75, 4838–4842 (1978).
Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M. & Neckers, L. M. The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone. J. Biol. Chem. 275, 37181–37186 (2000).
Hsieh, T. & Brutlag, D. ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings. Cell 21, 115–125 (1980).
Pocklington, M. J., Jenkins, J. R. & Orr, E. The effect of novobiocin on yeast topoisomerase type II. Mol. Gen. Genet. 220, 256–260 (1990).
Drean, A., Lord, C. J. & Ashworth, A. PARP inhibitor combination therapy. Crit. Rev. Oncol. Hematol. 108, 73–85 (2016).
Mirza, M. R., Pignata, S. & Ledermann, J. A. Latest clinical evidence and further development of PARP inhibitors in ovarian cancer. Ann. Oncol. 29, 1366–1376 (2018).
Parmar, K. et al. The CHK1 inhibitor prexasertib exhibits monotherapy activity in high-grade serous ovarian cancer models and sensitizes to PARP inhibition. Clin. Cancer Res. 25, 6127–6140 (2019).
Zhou, Y., Caron, P., Legube, G. & Paull, T. T. Quantitation of DNA double-strand break resection intermediates in human cells. Nucleic Acids Res. 42, e19 (2013).
Feng, W. et al. Genetic determinants of cellular addiction to DNA polymerase θ. Nat. Commun. 10, 4286 (2019).
Johnson, N. et al. Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance. Proc. Natl Acad. Sci. USA 110, 17041–17046 (2013).
Yazinski, S. A. et al. ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells. Genes Dev. 31, 318–332 (2017).
Lemee, F. et al. DNA polymerase θ up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability. Proc. Natl Acad. Sci. USA 107, 13390–13395 (2010).
Barber, L. J. et al. Comprehensive genomic analysis of a BRCA2 deficient human pancreatic cancer. PLoS ONE 6, e21639 (2011).
Eckelmann, B. J. et al. XRCC1 promotes replication restart, nascent fork degradation and mutagenic DNA repair in BRCA2-deficient cells. NAR Cancer 2, zcaa013 (2020).
Zhou, J. et al. Human CHD1 is required for early DNA-damage signaling and is uniquely regulated by its N terminus. Nucleic Acids Res. 46, 3891–3905 (2018).
Staudenbauer, W. L. & Orr, E. DNA gyrase: affinity chromatography on novobiocin–Sepharose and catalytic properties. Nucleic Acids Res. 9, 3589–3603 (1981).
Lim, K. S. et al. USP1 is required for replication fork protection in BRCA1-deficient tumors. Mol. Cell 72, 925–941 (2018).
Liu, J. F. et al. Establishment of patient-derived tumor xenograft models of epithelial ovarian cancer for preclinical evaluation of novel therapeutics. Clin. Cancer Res. 23, 1263–1273 (2017).
Harder, E. et al. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 12, 281–296 (2016).
Friesner, R. A. et al. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein–ligand complexes. J. Med. Chem. 49, 6177–6196 (2006).
Knight, J. L. et al. Leveraging data fusion strategies in multireceptor lead optimization MM/GBSA end-point methods. J. Chem. Theory Comput. 10, 3207–3220 (2014).
We thank the ICCB-Longwood Screening Facility at Harvard Medical School for their help with small-molecule screening. We thank P. Gokhale and Q. Zeng at the Belfer Center for Applied Cancer Science, as well as C. Yang and K. Parmar at the Center for DNA Damage and Repair, for their help with mouse studies. We thank C. Clairmont for providing RPE1 BRCA1−/− cells and purified TRIP13 protein and L. Moreau for her help with chromosome aberration assays. We thank H. Nguyen and S. Spisak for genomic analysis of POLQ-knockout clones. We thank C.-L. Tsai at the MD Anderson Cancer Center for providing the BLM-ATPase expression vector. We thank J. Stark for providing DR-GFP and EJ2 repair substrates, and G. Legube for providing DIvA cells for DSB end resection assays. This research was supported by a Stand Up To Cancer (SU2C)–Ovarian Cancer Research Fund Alliance–National Ovarian Cancer Coalition Dream Team Translational Research grant (SU2C-AACR-DT16-15) to A.D.D. SU2C is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. This work was also supported by the NIH (grants R37HL052725, P01HL048546, P50CA168504) and the US Department of Defense (grants BM110181 and BC151331P1), as well as grants from the Breast Cancer Research Foundation and the Fanconi Anemia Research Fund to A.D.D. This work was also supported by the Richard and Susan Smith Family Foundation, the Breast Cancer Research Foundation (BCRF-20-033) and the Basser Initiative at the Gray Foundation (167858/167856) (to A.D.D.). This work was also supported by the ERC starting grant (714162) and the Ville de Paris Emergences Program grant (DAE 137) to R.C. This work was also supported by an Ann Schreiber Mentored Investigator Award from the Ovarian Cancer Research Fund Alliance (457527), a Joint Center for Radiation Therapy Award from Harvard Medical School and a Breast & Gynecologic Cancer Innovation Award from the Susan F. Smith Center for Women’s Cancers at the DFCI to J.Z. J.A.T. and A.S. are supported by NIH grants P01 CA092548 and R35 CA220430, a Cancer Prevention Research Institute of Texas grant (RP180813) and a Robert A. Welch Chemistry Chair.
A.D.D. reports receiving commercial research grants from Eli Lilly and Company, Sierra Oncology and EMD Serono and is a consultant and/or advisory board member for Eli Lilly and Company, Sierra Oncology, Cedilla, Ideaya, Cyteir and EMD Serono. G.I.S. has received research funding from Eli Lilly, Merck KGaA/EMD Serono, Merck and Sierra Oncology, and he has served on advisory boards for Pfizer, Eli Lilly, G1 Therapeutics, Roche, Merck KGaA/EMD Serono, Sierra Oncology, Bicycle Therapeutics, Artios, Fusion Pharmaceuticals, Cybrexa Therapeutics, Astex, Almac, Ipsen, Bayer, Angiex, Daiichi Sankyo, Boehringer Ingelheim, ImmunoMet and Asana. The remaining authors declare no competing interests.
Peer review information Nature Cancer thanks Thomas Helleday and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Characterization of POLθ inhibitors obtained from the small-molecule screen, with biochemical assays and cell-based assays.
a, ADP-Glo assay for quantification of POLθ ATPase activity in various experimental conditions. N = 1 experiment. b, A secondary screen using the 72 initial hits in reducing POLθ ATPase activity (below 70 %, z-score < -4). The screen was done in the presence and absence of ssDNA. The four most promising hits advancing for further analysis are labeled (see Table S4 for analysis). Aurintricarboxylic acid (ATA), Reactive Blue 2 (RB), Suramin (SUR), and Novobiocin (NVB). Data shown are mean of n = 2 independent experiments. c, Quantification of POLθ and SMARCAL1 ATPase activity with indicated small molecules. The negative control is an inert small molecule (Vandetanib). Data shown are mean ± SD, n = 3 independent experiments. Statistics were performed using two tailed t-test with Welch’s correction. ***p < 0.001, **p < 0.01. d, Coomassie blue stained gels showing purified ATPase that were used in this study. e, Conjugation of NVB to epoxy-activated Sepharose-6B (Sigma). NVB conjugated beads showed light yellow color. The structure of NVB is shown. f, Pulldown experiments with NVB-conjugated beads and purified POLθ, SMARCAL1, CHD1, BLM, and RAD51. POLθ, SMARCAL1, CHD1, and BLM were detected by Western blot using anti-Flag antibody (Sigma #F1804) in this assay. RAD51 was detected by anti-RAD51 antibody (SantaCruz #398587). g, Pulldown experiments with NVB-conjugated beads and cell lysate from HEK293T cells expressing GFP-tagged full-length POLθ. Cell lysates were incubated with empty beads (EB), NVB-beads (NVB) or anti-GFP-beads (GFP) to assay for direct POLθ binding. GFP-POLθ bound to beads and input of each group were subjected to Western blot analysis, using an anti-GFP antibody. h, Competition assay between NVB-conjugated beads and free NVB when incubated with GFP-tagged full-length POLθ extracted from HEK293T cells. i, Thermal shift assay using GFP-POLQθ expressing HEK293T cell lysate incubated with NVB at the indicated temperatures. Supernatants after heat treatment were subjected to Western blot using anti-GFP and anti-Actin antibodies. j-l, Titration of indicated NVB (or DMSO) concentration with POLθ (j), BLM (k), or MRE11 (l) in the thermal shift assay to measure the effect of NVB on the protein stability. Average first derivative of the scattering profile is shown. Scattering peak max values are shown as mean ± SD, from n = 3 independent experiments.
a, Images and quantification of RAD51 foci in U2OS cells under increasing concentrations of NVB with or without gamma-irradiation (IR). b, Images and quantification of γH2AX foci in U2OS cells under increasing concentrations of NVB with or without IR. Data in (a) and (b) were pooled from two independent experiments. Each dot represents foci numbers in one cell. Scale bar = 10 μm.
Extended Data Fig. 3 Efficacy of NVB in BRCA1−/− GEMM and RAD51 pharmacodynamic study in TOV21G xenograft models.
a, NVB efficacy in the GEMM model (Tp53−/−Brca1−/− TNBC). Response of each tumor in each individual mouse is shown. Tumor chunks from GEMM mice were implanted in syngeneic FVB/129P2 mice, which were treated with PBS or 100 mg/kg NVB twice a day via IP for 5 weeks. b, Olaparib sensitivity of TOV21G (+ EV) or FANCF-complemented TOV21G (+ FANCF cDNA). c, Novobiocin sensitivity of TOV21G (+ EV) or FANCF-complemented TOV21G (+ FANCF cDNA). Mean ± SD of n = 6 biologically independent samples are shown in b and c. d-f, Immunohistochemical (IHC) study of the pharmacodynamic biomarker RAD51 after NVB and/or olaparib treatment in TOV21G tumors. d, Representative images of RAD51 IHC staining in xenografted TOV21G tumor cells. Tumor bearing NU(NCr)-Foxn1nu mice were treated with indicated drugs for 18 days before tumors were taken. FFPE tissue sections of the tumors were stained using an anti-RAD51 antibody and representative images (40X) are shown. e, Zoom-in of the IHC images in (d) to show RAD51 foci in detail. f, Quantification of RAD51 foci positive cells in TOV21G tumors. Cells with three or more RAD51 foci were counted as positive cells. Tumor samples from each group were processed and analyzed, and Mean±SD are shown, n = 4 for Olaparib and n = 6 for other groups. The total number of cells counted were shown in the graph (N). Statistical analysis was performed using one-way ANOVA in Prism, *, p < 0.05; **, p < 0.01; ***, p < 0.001.
a, Clonogenic survival of BRCA1−/− and WT RPE1 cells under increasing concentrations of the PARPi rucaparib. b, Clonogenic survival of BRCA2−/− and WT RPE1 cells under increasing concentrations of PARPi rucaparib. c, Clonogenic survival of BRCA2−/− and WT RPE1 cells under increasing concentrations of POLθ inhibitor. The survival fraction in a-c is normalized to the untreated samples. Data shown are Mean ± SEM, n = 3 independent experiments. d, Cell viability assay (CellTiter-Glo) in BRCA1−/− and WT RPE1 cells treated with indicated POLθ inhibitors (50 µM) or with the PARPi Olaparib as control. Cells were treated twice on days 1 and 4 and cell viability was measured on day 7. Data were mean ± SD, n = 3 biological replicates. Statistics analyses were two tailed t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001, ns, not significant (p > 0.05).
a, Quantification of apoptosis in BRCA1−/− and WT RPE1 cells under increasing concentrations of NVB. Mean ± SD, n = 3 independent experiments. b, Representative images of chromosomal aberrations (blue arrows) including radial figures (red arrows) in RPE1 (WT or BRCA1−/−) cells treated with NVB. c-d, Quantification of radial figures (c) and total chromosome aberration (d) in BRCA1−/− and WT RPE1 cells treated with NVB alone or in combination with mitomycin C (MMC). Data in c-d are mean ± SD of n = 3 independent experiments. Statistical significance was determined by one-way ANOVA multiple comparisons using uncorrected Fisher’s LSD test. ***, p < 0.001.
a–d, Verification of the U2OS-POLQ−/− cell line. a, Mapping the genetic alteration (deletions) in the U2OS POLQ knockout clone generated by CRISPR-Cas9. The targeted region was sequenced by Next Gen sequencing (number of reads are shown), and the deletions were mapped to POLQ locus of human genome on chromosome 3. b, A Western blot showing POLθ protein expression in WT and POLQ−/− U2OS cells. c, RAD51 foci assay in WT or POLQ−/− U2OS cells before and after (4 h) of 4 Gy IR. Mean of n = 2 independent experiments is shown. d, IR sensitivity of WT and POLQ−/− U2OS cells in clonogenic assays. Mean ± SD of n = 3 independent experiments are shown. e, NVB sensitivity of WT and POLQ−/− U2OS cells in clonogenic assays. Mean ± SD of n = 6 biologically independent samples are shown.
a, HSP90 client degradation assay in RPE1 and RPE1-BRCA1−/− cells. Cells were treated with a potent HSP90 inhibitor PU-H71 or NVB for 48 hours, and then cells were collected for Western blot analysis of the HSP90 client AKT1. b-c, HSP90 client degradation assay in MCF7 cells. Cells were treated with the potent HSP90 inhibitor PU-H71 or NVB for 24 hours, and then cells were collected for Western blot analysis of the HSP90 client proteins AKT1, CDK6 (b) and BRCA1 (c). Levels of HSP90 and HSP70 were also analyzed after NVB or PU-H71 treatment (c). d-e, Combination effect of etoposide and novobiocin in killing TOV21G cells (d) and CAPAN1 cells (e), showing their non-epistatic effects. Mean ± SD of n = 3 independent experiments are shown. f, CellTiter-Glo cell viability assay of empty vector (EV) and FANCF-complemented TOV21G cells, in the presence of olaparib, novobiocin or both. Mean ± SD, n = 4 biological replicates are shown. g, IC50 values of olaparib in TOV21G cells with or without NVB, derived from data in f. Mean ± SD of IC50 were shown, from n = 4 biological replicates.
Extended Data Fig. 8 Characterization of PARP inhibitor resistant clones derived from RPE1-BRCA1−/−.
a–c, Olaparib (a-b) and cisplatin (c) sensitivity of WT and BRCA1−/− RPE1 cells and the PARPi resistant clones of BRCA1−/− RPE1. Mean ± SD of biological replicates are shown, with n = 8 in A, n = 3 in B, and n = 8 in C. d, DNA fiber assay to measure the replication fork stability of the PARPi resistant clones. Mean±95%CI are shown from 3 independent experiments are shown, with n = number of fibers scored labeled. e, RAD51 foci analysis in WT and BRCA1−/− RPE1 cells and the PARPi resistant clones after irradiation (5Gy), to determine restoration of RAD51 foci in R clones. RAD51 was stained 4 hours after IR. Scale bar = 10 μm. f, A Western blot of WT and BRCA1−/− RPE cells and the PARPi resistant clones using a REV7 antibody. g, Immunofluorescence staining of 53BP1 in WT and BRCA1−/− RPE1 cells and the PARPi resistant clones after irradiation. Clone R4 showed much reduced 53BP1 foci after IR. h, Immunofluorescence staining of BRCA1 in WT and BRCA1−/− RPE1 cells and the PARPi resistant clones after irradiation. No BRCA1 foci was observed except in WT RPE1 cells. i, CellTiter-Glo assay to determine survival of PARPi-resistant and parental BRCA1−/− RPE1 cells upon CRISPR knockout of the POLQ gene. Data are mean ± SEM, with n = 4 biological replicates. Statistical analysis was t-test, *, p < 0.05; ns, not significant.
Extended Data Fig. 9 PARPi resistant MDA-MB-436 and UWB1.289 cells are sensitive to NVB, and NVB has synergy with PARPi in TOV21G cells.
a-b, Olaparib (a) and NVB (b) sensitivity of UWB1.289 and a PARPi resistant UWB1.289 clone (UWB1.289-YSR12) in CellTiter-Glo assays. Data shown are mean ± SD, n = 4 biological replicates. c, Western blot analysis of POLQ expression levels in in WT and BRCA1−/− RPE1 cells and the PARPi resistant BRCA1−/− clones. d, Olaparib sensitivity of PEO1 (BRCA2 mutated) and PEO4 cells (BRCA2 restored). e, NVB sensitivity of PEO1 and PEO4 cells. Data shown in d and e are Mean ± SD, n = 3 biological replicates. f, A Western blot shows POLQ expression in BRCA2-decient cells lines (PEO1) and their counterparts with BRCA2 reverted to wild type (PEO4).
Supplementary Tables 1–4 contain information and results from the small-molecule screen. Table 1, screen-related information. Table 2, complete results from the small-molecule screen. Table 3, top 72 initial hits from the small-molecule screen. Table 4, top ten hits that inhibited POLQ with or without ssDNA.
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Zhou, J., Gelot, C., Pantelidou, C. et al. A first-in-class polymerase theta inhibitor selectively targets homologous-recombination-deficient tumors. Nat Cancer 2, 598–610 (2021). https://doi.org/10.1038/s43018-021-00203-x
Nature Cancer (2021)