Loss Of 53BP1 Research Articles

53BP1 deficiency leads to hyperrecombination using break-induced replication (BIR)

Break-induced replication (BIR) is mutagenic, and thus its use requires tight regulation, yet the underlying mechanisms remain elusive. Here we uncover an important role of 53BP1 in suppressing BIR after end resection at double strand breaks (DSBs), distinct from its end protection activity, providing insight into the mechanisms governing BIR regulation and DSB repair pathway selection. We demonstrate that loss of 53BP1 induces BIR-like hyperrecombination, in a manner dependent on Polα-primase-mediated end fill-in DNA synthesis on single-stranded DNA (ssDNA) overhangs at DSBs, leading to PCNA ubiquitination and PIF1 recruitment to activate BIR. On broken replication forks, where BIR is required for repairing single-ended DSBs (seDSBs), SMARCAD1 displaces 53BP1 to facilitate the localization of ubiquitinated PCNA and PIF1 to DSBs for BIR activation. Hyper BIR associated with 53BP1 deficiency manifests template switching and large deletions, underscoring another aspect of 53BP1 in suppressing genome instability. The synthetic lethal interaction between the 53BP1 and BIR pathways provides opportunities for targeted cancer treatment.

53BP1-the ‘Pandora’s box’ of genome integrity

53BP1 has several functions in the maintenance of genome integrity. It functions as a key mediator involved in double-strand break (DSB) repair, which functions to maintain a balance in the repair pathway choices and in preserving genomic stability. While its DSB repair functions are relatively well-characterized, its role in DNA replication and replication fork protection is less understood. In response to replication stress, 53BP1 contributes to fork protection by regulating fork reversal and restart. It helps maintain replication fork stability and speed, with 53BP1 loss leading to defective fork progression and increased sensitivity to replication stress agents. However, 53BP1's precise role in fork protection remains debated, as some studies have not observed protective effects. Therefore, it is critical to determine the role of 53BP1 in replication to better understand when it promotes replication fork protection, and the underlying mechanisms involved. Moreover, 53BP1's function in replication stress extends beyond its activity at active replication forks; it also forms specialized nuclear bodies (NBs) which protect stretches of under-replicated DNA (UR-DNA) transmitted from a previous cell cycle to daughter cells through mitosis. The mechanism of 53BP1 NBs in the coordination of replication and repair events at UR-DNA loci is not fully understood and warrants further investigation. The present review article focuses on elucidating 53BP1’s functions in replication stress (RS), its role in replication fork protection, and the significance of 53BP1 NBs in this context to provide a more comprehensive understanding of its less well-established role in DNA replication.

53BP1 deficiency leads to hyperrecombination using break-induced replication (BIR).

Break-induced replication (BIR) is mutagenic, and thus its use requires tight regulation, yet the underlying mechanisms remain elusive. Here we uncover an important role of 53BP1 in suppressing BIR after end resection at double strand breaks (DSBs), distinct from its end protection activity, providing insight into the mechanisms governing BIR regulation and DSB repair pathway selection. We demonstrate that loss of 53BP1 induces BIR-like hyperrecombination, in a manner dependent on Polα-primase-mediated end fill-in DNA synthesis on single-stranded DNA (ssDNA) overhangs at DSBs, leading to PCNA ubiquitination and PIF1 recruitment to activate BIR. On broken replication forks, where BIR is required for repairing single-ended DSBs (seDSBs), SMARCAD1 displaces 53BP1 to facilitate the localization of ubiquitinated PCNA and PIF1 to DSBs for BIR activation. Hyper BIR associated with 53BP1 deficiency manifests template switching and large deletions, underscoring another aspect of 53BP1 in suppressing genome instability. The synthetic lethal interaction between the 53BP1 and BIR pathways provides opportunities for targeted cancer treatment.

RAD18- and BRCA1-dependent pathways promote cellular tolerance to the nucleoside analog ganciclovir.

Ganciclovir (GCV) is a clinically important drug as it is used to treat viral infections. GCV is incorporated into the DNA during replication, where it interferes with subsequent replication on GCV-incorporated templates. However, the effects of GCV on the host genome and the mechanisms underlying cellular tolerance to GCV remain unclear. In this study, we explored these mechanisms using a collection of mutant DT40 cells. We identified RAD17/-, BRCA1-/-, and RAD18-/- cells as highly GCV-sensitive. RAD17, a component of the alternative checkpoint-clamp loader RAD17-RFC, was required for the activation of the intra-S checkpoint following GCV treatment. BRCA1, a critical factor for promoting homologous recombination (HR), was required for suppressing DNA double-strand breaks (DSBs). Moreover, RAD18, an E3-ligase involved in DNA repair, was critical in suppressing the aberrant ligation of broken chromosomes caused by GCV. We found that BRCA1 suppresses DSBs through HR-mediated repair and template switching (TS)-mediated damage bypass. Moreover, the strong GCV sensitivity of BRCA1-/- cells was rescued by the loss of 53BP1, despite the only partial restoration in the sister chromatid exchange events which are hallmarks of HR. These results indicate that BRCA1 promotes cellular tolerance to GCV through two mechanisms, TS and HR-mediated repair.

53BP1 loss elicits cGAS-STING-dependent antitumor immunity in ovarian and pancreatic cancer

53BP1 nucleates the anti-end resection machinery at DNA double-strand breaks, thereby countering BRCA1 activity. Loss of 53BP1 leads to DNA end processing and homologous recombination in BRCA1-deficient cells. Consequently, BRCA1-mutant tumors, typically sensitive to PARP inhibitors (PARPi), become resistant in the absence of 53BP1. Here, we demonstrate that the ‘leaky’ DNA end resection in the absence of 53BP1 results in increased micronuclei and cytoplasmic double-stranded DNA, leading to activation of the cGAS-STING pathway and pro-inflammatory signaling. This enhances CD8+ T cell infiltration, activates macrophages and natural killer cells, and impedes tumor growth. Loss of 53BP1 correlates with a response to immune checkpoint blockade (ICB) and improved overall survival. Immunohistochemical assessment of 53BP1 in two malignancies, high grade serous ovarian cancer and pancreatic ductal adenocarcinoma, which are refractory to ICBs, reveals that lower 53BP1 levels correlate with an increased adaptive and innate immune response. Finally, BRCA1-deficient tumors that develop resistance to PARPi due to the loss of 53BP1 are susceptible to ICB. Therefore, we conclude that 53BP1 is critical for tumor immunogenicity and underpins the response to ICB. Our results support including 53BP1 expression as an exploratory biomarker in ICB trials for malignancies typically refractory to immunotherapy.

BRCA1 and 53BP1 regulate reprogramming efficiency by mediating DNA repair pathway choice at replication-associated double-strand breaks

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.

Abstract A015: 53BP1 loss mediated PARP inhibitor resistance in BRCA1-deficient pancreatic cancer is overcome with immune checkpoint blockade

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer characterized by the presence of mutations in homologous recombination (HR) repair genes, including BRCA1 and BRCA2, in approximately 20% of diagnosed cases. 53BP1 (tumor protein p53 binding protein 1) nucleates the anti-end resection machinery at DNA double strand break (DSB)s thereby countering BRCA1 activity. Importantly, BRCA1-deficient PDACs that are typically sensitive to poly-ADP ribose polymerase inhibitors (PARPi) become resistant in the absence of 53BP1. Here, we demonstrate that loss of 53BP1 results in enhanced DNA end-resection, increased micronuclei, and cytoplasmic dsDNA, leading to activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, activating pro-inflammatory signaling. This not only enhances CD8+ T cell infiltration but also activates macrophages and natural killer (NK) cells culminating in tumor growth inhibition. Loss of 53BP1 correlates with response to immune checkpoint blockade (ICB) and improved overall survival. Finally, BRCA1-deficient tumors that develop resistance to PARPi due to the loss of 53BP1 are susceptible to ICB. Taken together, our findings provide a rationale for combining PARPi with ICB to overcome PARPi-resistance in BRCA1-deficient pancreatic cancers. Citation Format: Jeffrey Patterson-Fortin, Yajie Sun, Sen Han, Zhe Li, Zuzanna Nowicka, Yuna Hirohashi, Susan Kilgas, Jae Kyo Yi, Alexander Spektor, Wojciech Fendler, Panagiotis Konstantinopoulos, Alan D. D'Andrea, Dipanjan Chowdhury. 53BP1 loss mediated PARP inhibitor resistance in BRCA1-deficient pancreatic cancer is overcome with immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A015.

Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting

A synthetic lethal relationship exists between disruption of polymerase theta (Polθ), and loss of either 53BP1 or homologous recombination (HR) proteins, including BRCA1; however, the mechanistic basis of these observations are unclear. Here we reveal two distinct mechanisms of Polθ synthetic lethality, identifying dual influences of 1) whether Polθ is lost or inhibited, and 2) the underlying susceptible genotype. Firstly, we find that the sensitivity of BRCA1/2- and 53BP1-deficient cells to Polθ loss, and 53BP1-deficient cells to Polθ inhibition (ART558) requires RAD52, and appropriate reduction of RAD52 can ameliorate these phenotypes. We show that in the absence of Polθ, RAD52 accumulations suppress ssDNA gap-filling in G2/M and encourage MRE11 nuclease accumulation. In contrast, the survival of BRCA1-deficient cells treated with Polθ inhibitor are not restored by RAD52 suppression, and ssDNA gap-filling is prevented by the chemically inhibited polymerase itself. These data define an additional role for Polθ, reveal the mechanism underlying synthetic lethality between 53BP1, BRCA1/2 and Polθ loss, and indicate genotype-dependent Polθ inhibitor mechanisms.

YCH1899, a Highly Effective Phthalazin-1(2H)-one Derivative That Overcomes Resistance to Prior PARP Inhibitors.

Poly(ADP-ribose) polymerase inhibitors (PARPi) have significant efficacy in treating BRCA-deficient cancers, although resistance development remains an unsolved challenge. Herein, a series of phthalazin-1(2H)-one derivatives with excellent enzymatic inhibitory activity were designed and synthesized, and the structure-activity relationship was explored. Compared with olaparib and talazoparib, compound YCH1899 exhibited distinct antiproliferation activity against olaparib- and talazoparib-resistant cells, with IC50 values of 0.89 and 1.13 nM, respectively. Studies of the cellular mechanism revealed that YCH1899 retained sensitivity in drug-resistant cells with BRCA1/2 restoration or 53BP1 loss. Furthermore, YCH1899 had acceptable pharmacokinetic properties in rats and showed prominent dose-dependent antitumor activity in olaparib- and talazoparib-resistant cell-derived xenograft models. Overall, this study suggests that YCH1899 is a new-generation antiresistant PARPi that could provide a valuable direction for addressing drug resistance to existing PARPi drugs.

Multi-omics analysis reveals distinct non-reversion mechanisms of PARPi resistance in BRCA1- versus BRCA2-deficient mammary tumors

BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis), but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanismsthat do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance invivo, we combine molecular profiling with functional analysis of HR of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent reactivation of BRCA1/2. We observe restoration of HR in 62% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we find that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by PARG loss. Furthermore, combined multi-omics analysis identifies additional genes and pathways potentially involved in modulating PARPi response.

Abstract 6094: Longitudinal analysis of PARP inhibitor and platinum resistance in BRCA1/2m breast cancer using liquid biopsy

Abstract Background: Assessing how clinical resistance to PARP inhibitors develops has been challenging, due to the lack of coverage of potential resistance genes in sequencing panels and biopsies being subject to spatial heterogeneity. We studied the development of PARPi and/or platinum resistant disease using both tissue and a novel liquid biopsy assay, in patients treated for BRCA1/2-mutated metastatic breast cancer (BRCA1/2m mBC). Approach: A cohort of 35 mBC patients with germline or somatic BRCA1/2 mutations were identified as having developed PARPi or platinum resistant disease. Tumour biopsies were analysed by exome and transcriptome sequencing whereas ctDNA isolated from plasma sampled across the pre-treatment, response and eventual progression journey were analysed using the Guardant INFINITY platform equipped to detect mutations in >800 genes, and genome wide methylation, including promoter methylation in 398 cancer-related genes. Somatic mutations or regions of methylation that were associated with resistance were identified, including BRCA1/2 reversion mutations. Somatic mutations with the potential to cause PARPi resistance were annotated using CRISPR screen data and other functional analyses describing genes that alter PARPi synthetic lethality. Results: The most common resistance mechanism was BRCA1/2 reversion mutation (51%; n=8 BRCA1m patients, n=10 BRCA2m patients). Most reversions (77%) occurred via deletions (BRCA1, 70%; BRCA2, 79%) and exhibited microhomology use at junctions (> 80% for both BRCA1 and BRCA2). In 14 patients, multiple concurrent reversion mutations were detected with VAFs ranging from 0.1-40% for BRCA1 and 0.05-18% for BRCA2. Changes in VAF over time indicated that different reversions in the same patient may impart different fitness advantages in the face of treatment. Loss-of-function mutations in the 53BP1-Shieldin pathway were identified in two BRCA1m patients, and VUS mutations in the pathway were seen in two further patients. In some patients, reversion mutations co-occurred with either 53BP1-Shieldin pathway mutations or with replication fork stability mutations (PAXIP1 in BRCA2m patients) - indicating concurrent but mechanistically distinct forms of resistance develop in the same patient. We isolated a PDX model from one patient that exhibits resistance via both BRCA1 reversion and loss of 53BP1, allowing this phenomenon to be modelled in more detail. We also detected BRCA1 methylation in ctDNA in a patient with a somatic rearrangement in BRCA1. Conclusions: Liquid biopsy profiling of PARPi-resistance breast cancer patients indicates that many patients develop multiple reversion mutations, and that alterations in the 53BP1-Shieldin pathway are present but less frequent. The co-occurrence of 53BP1-Shieldin pathway mutations and reversion mutations suggests parallel mechanisms of resistance can operate in the same patient. Citation Format: Elizabeth Harvey-Jones, Maya Raghunandan, Luisa Robbez-Masson, Alaguthurai Thanussuyah, Roberta Liccardo, Arielle Yablonovitch, Mingyang Cai, Leylah Drusbosky, Michael Dorschner, Lorena Magraner Pardo, Rebecca Marlow, Asha Konde, Jennifer Trendell, John Alexander, Syed Haider, Chris Starling, Ioannis Roxanis, Jennifer Yen, Stephen J. Pettitt, Christopher J. Lord, Andrew N. Tutt. Longitudinal analysis of PARP inhibitor and platinum resistance in BRCA1/2m breast cancer using liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6094.

Abstract CT201: Ceralasertib and olaparib in the treatment of homologous recombination repair (HRR)-deficient platinum-sensitive ovarian cancer after progression on PARP inhibitors

Abstract Background: The majority of BRCA-mutant or HRR-deficient ovarian cancers are initially sensitive to PARP inhibitors (PARPis) but eventually develop resistance. The most commonly reported PARPi resistance mechanisms are (1) reversal of HRR deficiency through (a) reversion mutations in BRCA or other HRR genes (eg, RAD51C/D) or (b) DNA end resection rewiring with loss of 53BP1, REV7, or Shieldin complex components; or (2) stabilization of stalled replication forks through loss of PTIP or EZH2, preventing nuclease recruitment. The only confirmed PARPi resistance mechanism in the clinic to date is reversion mutations. Combined ATR and PARP inhibition is an attractive strategy that may overcome PARPi resistance, as has been shown in PARPi-resistant cell lines and patient-derived xenograft models (Yazinski et al Genes Dev 2017; Murai et al Oncotarget 2016; Kim et al Nat Commun 2020; data on file, AstraZeneca). The PARPi olaparib 300 mg twice daily (BID) continuously in combination with the ATR inhibitor ceralasertib 160 mg daily on days 1-7 of a 28-day cycle has demonstrated promising activity in the CAPRI study in patients with ovarian cancer who had progressed on PARPis (Wethington et al J Clin Oncol 2021). However, preclinical models of PARPi resistance suggest that a higher dose or duration of ceralasertib with a lower dose of olaparib may be more beneficial (data on file, AstraZeneca). Methods: D5330C00004 (NCT02264678) is a multicenter, modular, phase 1 study assessing ceralasertib in combination with other anti-cancer agents in adults with advanced solid malignancies. In Module 2, a lower dose of olaparib (150 mg BID continuous) and a longer duration of ceralasertib (80 mg BID, days 1-14 of a 28-day cycle) is now being explored in 2 expansion cohorts in patients with platinum-sensitive, high-grade, serous or endometrioid ovarian, fallopian tube, or primary peritoneal cancer with deleterious or suspected deleterious germline or somatic BRCA or RAD51C/D mutations, or HRR deficiency by Myriad MyChoice® or FoundationOne® CDx (F1CDx) assays. Patients will have progressed on prior PARPis after ≥6 months in the maintenance or treatment setting (≥12 months after first-line maintenance). Cohort 1 is enrolling ~30 patients immediately after progression on a PARPi; Cohort 2 is recruiting ~30 PARPi-treated patients after intervening platinum-based chemotherapy. Study endpoints include safety, tumor response by RECIST 1.1, and duration of response. Exploratory analyses include genomic and functional analysis of HRR restoration in tumor and circulating tumor DNA, and analysis of replication fork stability in organoid cultures established from mandatory fresh tumor biopsy samples. Recruitment began in July 2021 with the first patient dosed in August 2021. Citation Format: Geoffrey I. Shapiro, Bristi Basu, Anthony El-Khoueiry, Sophie Postel-Vinay, Seock-Ah Im, Sun Young Rha, Claire F. Friedman, Antoine Italiano, Yong Man Kim, Myong Cheol Lim, Rene Roux, Joyce F. Liu, Elhan Sanai, Simon A. Smith, Claire Smith, Sarah El Farhi, Alan Lau, Natalia Lukashchuk, Emma Dean, Matthew G. Krebs. Ceralasertib and olaparib in the treatment of homologous recombination repair (HRR)-deficient platinum-sensitive ovarian cancer after progression on PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT201.

Abstract P068: Different treatment schemes cause distinct PARP inhibitor resistance mechanisms in BRCA2-mutant ovarian cancer cells

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPis) represent a major advance in ovarian cancer management, now as treatment for recurrent ovarian cancer and as a maintenance therapy following platinum-based chemotherapy in the upfront and platinum-sensitive recurrent settings. Yet, a growing number of patients progress through PARPis, creating a need for further understanding on resistance mechanisms. We hypothesized that different treatment dosing/timing schemes would cause distinct mechanisms of acquired resistance to PARPis. To test this hypothesis, we exposed BRCA2-mutant PEO1 ovarian cancer cells to 0.5 μM olaparib, doubling its concentration up to 40 μM over 3–4 months (PEO1/OlaJR). The second PARPi-resistant PEO1 cell line (PEO1/OlaR) was developed using a relatively low initial dose of 5 nM olaparib, and gradually increasing its concentration up to 20 μM over 6 months. To confirm PARPi resistance, cell proliferation and viability were measured by MTT and colony formation assay, respectively. Both cell lines were resistant to two different PARPis beyond clinically achievable concentrations (olaparib IC50 141.7–372.8 μM and rucaparib IC50 37.1–51.2 μM) and demonstrated restored homologous recombination (HR) repair as evidenced by increased HR-reporter activity (1.32 to 1.45-fold compared to PEO1, p < 0.05) and RAD51 foci formation (5.2 to 6.1-fold compared to PEO1, p < 0.001). For PEO1/OlaJR, distinct resistance mechanisms to PARPi were noted: 1) promoting drug efflux confirmed by increased MDR efflux activity (1.92-fold compared to PEO1, p < 0.001) and increased protein levels of an ABCB1 transporter (7.2-fold compared to PEO1); and 2) enhancing HR repair restoration, likely via reduced REV7 protein expression (0.43-fold compared to PEO1). On the other hand, PEO1/OlaR exhibited a mesenchymal cell phenotype with greater invasion ability as measured by transwell invasion assay (2.25-fold compared to PEO1, p < 0.001) and restored HR repair with upregulation of TRIP13 protein (1.85-fold compared to PEO1). PEO1/OlaR also showed increased cell survival, possibly via activation of PI3K/AKT signaling, as evidenced by increased p-AKT (2.12-fold compared to PEO1). No loss of 53BP1 was observed in either PEO1/OlaJR or PEO1/OlaR based on whole exome sequencing and immunoblotting of 53BP1. BRCA2 reversion mutation status is currently under investigation. Altogether, our findings suggest that mechanisms of resistance to PARPis arise differently based on the duration and concentrations of drug treatment, highlighting the importance of appreciating interplay between specific treatment schemes and the downstream consequences. Thus, developing tailored therapeutic strategies should consider various mechanisms of PARPi resistance. Citation Format: Tzu-Ting Huang, Jayakumar R. Nair, Nitasha Gupta, Tomomi M. Yamamoto, Benjamin G. Bitler, Jung-Min Lee. Different treatment schemes cause distinct PARP inhibitor resistance mechanisms in BRCA2-mutant ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P068.

Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

The RNF8 and RNF168 Ubiquitin Ligases Regulate Pro- and Anti-Resection Activities at Broken DNA Ends During Non-Homologous End Joining

The RING-type E3 ubiquitin ligases RNF8 and RNF168 recruit DNA damage response (DDR) factors to chromatin flanking DNA double strand breaks (DSBs) including 53BP1, which protects DNA ends from resection during DNA DSB repair by non-homologous end joining (NHEJ). Deficiency of RNF8 or RNF168 does not lead to demonstrable NHEJ defects, but like deficiency of 53BP1, the combined deficiency of XLF and RNF8 or RNF168 leads to diminished NHEJ in lymphocytes arrested in G0/G1 phase. The function of RNF8 in NHEJ depends on its E3 ubiquitin ligase activity. Loss of RNF8 or RNF168 in G0/G1-phase lymphocytes leads to the resection of broken DNA ends, demonstrating that RNF8 and RNF168 function to protect DNA ends from nucleases, pos sibly through the recruitment of 53BP1. However, the loss of 53BP1 leads to more severe resection than the loss of RNF8 or RNF168. Moreover, in 53BP1-deficient cells, the loss of RNF8 or RNF168 leads to diminished DNA end resection. We conclude that RNF8 and RNF168 regulate pathways that both prevent and promote DNA end resection in cells arrested in G0/G1 phase.

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.

Abstract 2049: Cell-cycle dependent regulation of Sp1 at DSBs promotes repair via NHEJ and sensitizes cells to PARP inhibition

Abstract DNA double-strand breaks (DSBs) are life-threatening lesions that must be repaired to preserve chromosomal integrity. These lesions are repaired by two primary pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). The factors that mediate pathway choice are critical to maintain genomic integrity. While many of these factors, epigenetic changes and cell cycle stages are known, with HR predominating in S and G2, the underlying mechanisms that determine pathway choice are not well understood. Previously, we found that the transcription factor Sp1 is rapidly recruited to DSBs and is necessary for repair. Here, we demonstrate that Sp1 integrates repair pathway choice with cell cycle by promoting NHEJ in G1. Sp1 localizes to DSBs in G1 to recruit the NHEJ repair factors 53BP1 and Ku70. Sp1 is phosphorylated by Cyclin A/cdk2 upon entry into S phase, evicting both Sp1 and 53BP1 from the break site, thereby permitting BRCA1 binding and HR. Mutation of this phosphorylation site (Sp1-S59A) results in persistence of Sp1 and 53BP1 at DSBs in S phase cells, precluding BRCA1 binding and HR. We also demonstrate that Sp1 is sumoylated in S phase in response to damage. This sumoylation in recognized by the E3-ubiquitin ligase, RNF4, and therefore polyubiquitylates Sp1 and targets it for proteasomal degradation. Expression of a sumoylation deficient Sp1 (Sp1-K16R) results in Sp1 and 53BP1 occupancy at DSBs in S phase, inhibiting BRCA1 binding to DSBs and HR. HR proteins are commonly mutated in cancers, resulting in cellular “addiction” to alternative repair pathways. Exploiting this addiction is the basis of synthetic lethality and is applicable to the treatment of many types of malignancies. Similar to BRCA1 deficiency, expression of Sp1-S59A or Sp1-K16R also increases cell sensitivity to PARP inhibition in BRCA1+/+ cells because of the inability to recruit BRCA1. Alternatively, synthetic lethality in BRCA1-/- cells requires 53BP1, whereas loss of 53BP1 confers PARPi resistance in a BRCA1 deficient cell. Consistent with this, depletion of Sp1 also results in PARPi resistance in BRCA1-/- cells. Interestingly, Sp1 is overexpressed in many tumors and is associated with poor prognosis. These data corroborate our finding that Sp1 is required for NHEJ and provide preliminary evidence that Sp1 may be a biomarker for PARPi sensitivity. Citation Format: Michelle Swift, Jane Azizkhan-Clifford. Cell-cycle dependent regulation of Sp1 at DSBs promotes repair via NHEJ and sensitizes cells to PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2049.

Abstract IA-010: Deciphering DNA end resection at DSBs

Abstract Double stranded DNA break (DSB)s are repaired by two major mechanistically distinct pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). A decisive factor in the choice between DSB repair pathways is in the competition between DNA end protection (necessary for NHEJ) and DNA end resection (necessary for HR). As the ‘master regulator’ of DSB repair pathway choice, 53BP1, a chromatin-associated reader of epigenetic marks at DSBs, helps channel DSBs into the NHEJ pathway by restricting BRCA1-dependent end resection. 53BP1 nucleates the assembly of a higher order ensemble that includes the Shieldin complex, CST complex, and DNA polymerase (Pol) a-primase complex. Loss of BRCA1 provides a therapeutic opportunity as these tumors are exquisitely sensitive to inhibitors of poly (ADP-ribose) polymerase (PARP), and are also susceptible to platinum-based drugs. Loss of 53BP1 or any of the 53BP1-interacting proteins restores end resection and causes PARPi resistance in BRCA1-mutant tumors. We recently identified Dynein light chain like protein 1 (DYNLL1) as as anti-resection factor. Loss of DYNLL1 allowed DNA end resection and restored HR, thereby inducing resistance to platinum drugs and PARPi in BRCA1-mutant tumors. At the molecular level, DYNLL1 limited the nucleolytic resection of DNA ends by interacting with the resection machinery. Importantly, purified DYNLL1 interacted with MRE11 and inhibited its exonuclease activity in vitro. Now we observe that 53BP1 loss prevents DYNLL1 recruitment to DSBs and more broadly to chromatin. We hypothesize that 53BP1 inhibits MRE11-mediated DNA end resection by recruiting DYNLL1 and that DYNLL1 functions in parallel of Shieldin-CST to regulate DNA end resection and DSB repair pathway choice. We are investigating the crosstalk between the Shieldin-CST and DYNLL1-MRE11 arms of the 53BP1-dependent anti-DNA end resection machinery. Citation Format: Dipanjan Chowdhury. Deciphering DNA end resection at DSBs [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-010.

Centrosome defects cause microcephaly by activating the 53BP1-USP28-TP53 mitotic surveillance pathway.

Mutations in centrosome genes deplete neural progenitor cells (NPCs) during brain development, causing microcephaly. While NPC attrition is linked to TP53-mediated cell death in several microcephaly models, how TP53 is activated remains unclear. In cultured cells, mitotic delays resulting from centrosome loss prevent the growth of unfit daughter cells by activating a pathway involving 53BP1, USP28, and TP53, termed the mitotic surveillance pathway. Whether this pathway is active in the developing brain is unknown. Here, we show that the depletion of centrosome proteins in NPCs prolongs mitosis and increases TP53-mediated apoptosis. Cell death after a delayed mitosis was rescued by inactivation of the mitotic surveillance pathway. Moreover, 53BP1 or USP28 deletion restored NPC proliferation and brain size without correcting the upstream centrosome defects or extended mitosis. By contrast, microcephaly caused by the loss of the non-centrosomal protein SMC5 is also TP53-dependent but is not rescued by loss of 53BP1 or USP28. Thus, we propose that mutations in centrosome genes cause microcephaly by delaying mitosis and pathologically activating the mitotic surveillance pathway in the developing brain.

Loss of Nuclear DNA Ligase III Reverts PARP Inhibitor Resistance in BRCA1-Deficient Cells by Increasing DNA Replication Stress

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1, identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 is required for PARPi-induced fork acceleration in BRCA1-deficient cells and that LIG3 loss increases fork asymmetry. Furthermore, LIG3 depletion in BRCA1-deficient cells results in an increase in ssDNA gaps behind the replication forks, resulting in accumulation of chromosomal abnormalities. We also report that high expression of LIG3 in patients with invasive breast cancer correlates in with poorer overall survival, rendering LIG3 as a potential therapeutic target for enhancing PARPi sensitivity.