Fork Stability Research Articles

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.

BRCA2 C-terminal clamp restructures RAD51 dimers to bind B-DNA for replication fork stability.

Tumor suppressor protein BRCA2 acts with RAD51 in replication-fork protection (FP) and homology-directed DNA break repair (HDR). Critical for cancer etiology and therapy resistance, BRCA2 C-terminus was thought to stabilize RAD51-filaments after they assemble on single-stranded (ss)DNA. Here we determined the detailed crystal structure for BRCA2 C-terminal interaction-domain (TR2i) with ATP-bound RAD51 prior to DNA binding. In contrast to recombinogenic RAD51-filaments comprising extended ATP-bound RAD51 dimers, TR2i unexpectedly reshapes ATP-RAD51 into a unique dimer conformation accommodating double-stranded B-DNA binding unsuited for HDR initiation. Structural, biochemical, and molecular results with interface-guided mutations uncover TR2i's FP mechanism. Proline-driven secondary-structure stabilizes residue triads and spans the RAD51 dimer engaging pivotal interactions of RAD51 M210 and BRCA2 S3291/P3292, the cyclin-dependent kinase (CDK) phosphorylation site that toggles between FP during S-phase and HDR in G2. TR2i evidently acts as an allosteric clamp switching RAD51 from ssDNA to double-stranded and B-DNA binding enforcing FP over HDR.

The effect of replication protein A inhibition and post-translational modification on ATR kinase signaling

The ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair to ensure genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions (RPA-DBi) and RPA protein–protein interactions (RPA-PPIi); both activities are required for ATR activation. Here, we biochemically reconstitute the ATR kinase signaling pathway and demonstrate that RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of target proteins with selectivity advantages over active site ATR inhibitors. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation does not affect ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway-targeted cancer therapeutics.

The recent advance and prospect of poly(ADP-ribose) polymerase inhibitors for the treatment of cancer.

Chemotherapies are commonly used in cancer therapy, their applications are limited to low specificity, severe adverse reactions, and long-term medication-induced drug resistance. Poly(ADP-ribose)polymerase (PARP)inhibitors are a novel class of antitumor drugs developed to solve these intractable problems based on the mechanism of DNA damage repair, which have been widely applied in the treatment of ovarian cancer, breast cancer, and other cancers through inducing synthetic lethal effect and trapping PARP-DNAcomplex in BRCA gene mutated cancer cells. In recent years, PARP inhibitors have been widely used in combination with various first-line chemotherapy drugs, targeted drugs and immune checkpoint inhibitors to expand the scope of clinical application. However, the intricate mechanisms underlying the drug resistance to PARP inhibitors, including the restoration of homologous recombination, stabilization of DNA replication forks, overexpression of drug efflux protein, and epigenetic modifications pose great challenges and desirability in the development of novel PARP inhibitors. In this review, we will focus on the mechanism, structure-activity relationship, and multidrug resistance associated with the representative PARP inhibitors. Furthermore, we aim to provide insights into the development prospects and emerging trends to offer guidance for the clinical application and inspiration for the development of novel PARP inhibitors and degraders.

Profound T Lymphocyte and DNA Repair Defect Characterizes Schimke Immuno-Osseous Dysplasia

Schimke immuno-osseous dysplasia is a rare multisystemic disorder caused by biallelic loss of function of the SMARCAL1 gene that plays a pivotal role in replication fork stabilization and thus DNA repair. Individuals affected from this disease suffer from disproportionate growth failure, steroid resistant nephrotic syndrome leading to renal failure and primary immunodeficiency mediated by T cell lymphopenia. With infectious complications being the leading cause of death in this disease, researching the nature of the immunodeficiency is crucial, particularly as the state is exacerbated by loss of antibodies due to nephrotic syndrome or immunosuppressive treatment. Building on previous findings that identified the loss of IL-7 receptor expression as a possible cause of the immunodeficiency and increased sensitivity to radiation-induced damage, we have employed spectral cytometry and multiplex RNA-sequencing to assess the phenotype and function of T cells ex-vivo and to study changes induced by in-vitro UV irradiation and reaction of cells to the presence of IL-7. Our findings highlight the mature phenotype of T cells with proinflammatory Th1 skew and signs of exhaustion and lack of response to IL-7. UV light irradiation caused a severe increase in the apoptosis of T cells, however the expression of the genes related to immune response and regulation remained surprisingly similar to healthy cells. Due to the disease’s rarity, more studies will be necessary for complete understanding of this unique immunodeficiency.

The multifaceted roles of the Ctf4 replisome hub in the maintenance of genome integrity

At the core of cellular life lies a carefully orchestrated interplay of DNA replication, recombination, chromatin assembly, sister-chromatid cohesion and transcription. These fundamental processes, while seemingly discrete, are inextricably linked during genome replication. A set of replisome factors integrate various DNA transactions and contribute to the transient formation of sister chromatid junctions involving either the cohesin complex or DNA four-way junctions. The latter structures serve DNA damage bypass and may have additional roles in replication fork stabilization or in marking regions of replication fork blockage. Here, we will discuss these concepts based on the ability of one replisome component, Ctf4, to act as a hub and functionally link these processes during DNA replication to ensure genome maintenance.

Harnessing DNA replication stress to target RBM10 deficiency in lung adenocarcinoma

The splicing factor RNA-binding motif protein 10 (RBM10) is frequently mutated in lung adenocarcinoma (LUAD) (9-25%). Most RBM10 cancer mutations are loss-of-function, correlating with increased tumorigenesis and limiting the efficacy of current LUAD targeted therapies. Remarkably, therapeutic strategies leveraging RBM10 deficiency remain unexplored. Here, we conduct a CRISPR-Cas9 synthetic lethality (SL) screen and identify ~60 RBM10 SL genes, including WEE1 kinase. WEE1 inhibition sensitizes RBM10-deficient LUAD cells in-vitro and in-vivo. Mechanistically, we identify a splicing-independent role of RBM10 in regulating DNA replication fork progression and replication stress response, which underpins RBM10-WEE1 SL. Additionally, RBM10 interacts with active DNA replication forks, relying on DNA Primase Subunit 1 (PRIM1) that synthesizes Okazaki RNA primers. Functionally, we demonstrate that RBM10 serves as an anchor for recruiting Histone Deacetylase 1 (HDAC1) to facilitate H4K16 deacetylation and R-loop homeostasis to maintain replication fork stability. Collectively, our data reveal a role of RBM10 in fine-tuning DNA replication and provide therapeutic arsenal for targeting RBM10-deficient tumors.

The Effect of Replication Protein A Inhibition and Post-Translational Modification on ATR Kinase Signaling.

The ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair towards ensuring genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions, termed RPA-DBi, and RPA protein-protein interactions, termed RPA-PPIi; both activities are required for ATR activation. Here, we employ a biochemically reconstituted ATR kinase signaling pathway and demonstrate that both RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of downstream target proteins. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation has no effect on ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway targeted cancer therapeutics.

Farm to Fork Stability of Phytochemicals and Micronutrients in Brassica oleracea and Allium Vegetables.

Brassica oleracea and Allium vegetables are known for their unique, family specific, water-soluble phytochemicals, glucosinolates, and S-alk(en)yl-l-cysteine sulfoxides, respectively. However, they are also important delivery systems of several other health-related compounds, such as carotenoids (lipid-soluble phytochemicals), vitamin C (water-soluble micronutrient), and vitamin K1 (lipid-soluble micronutrient). When all-year-round availability or transport over long distances is targeted for these often seasonal, locally grown vegetables, processing becomes indispensable. However, the vegetable processing chain, which consists of multiple steps (e.g., pretreatment, preservation, storage, preparation), can impact the nutritional quality of these vegetables corresponding to the nature of the health-related compounds and their susceptibility to (bio)chemical conversions. Since information about the impact of the vegetable processing chain is scattered per compound or processing step, this review targets an integration of the state of the art and discusses needs for future research. Starting with a discussion on substrate-enzyme location within the vegetable matrix, an overview is provided of the impact and potential of processing, encompassing a wide range of (nonenzymatic) conversions.

Structure and repair of replication-coupled DNA breaks.

Using CRISPR-Cas9 nicking enzymes, we examined the interaction between the replication machinery and single-strand breaks, one of the most common forms of endogenous DNA damage. We show that replication fork collapse at leading-strand nicks generates resected single-ended double-strand breaks (seDSBs) that are repaired by homologous recombination (HR). If these seDSBs are not promptly repaired, arrival of adjacent forks creates double-ended DSBs (deDSBs), which could drive genomic scarring in HR-deficient cancers. deDSBs can also be generated directly when the replication fork bypasses lagging-strand nicks. Unlike deDSBs produced independently of replication, end resection at nick-induced seDSBs and deDSBs is BRCA1-independent. Nevertheless, BRCA1 antagonizes 53BP1 suppression of RAD51 filament formation. These results highlight distinctive mechanisms that maintain replication fork stability.

Abstract IA009: Single strand DNA GAP accumulation as a functional biomarker for DNA Repair Inhibitors

Abstract Deficiency of homologous recombination (HR)-mediated DNA repair occurs through genetic or epigenetic inactivation of the BRCA1 and BRCA2 (BRCA1/2) genes. HR-deficiency also provides unique opportunities for targeted therapy, through the mechanism of synthetic lethality, as exemplified by the hypersensitivity of BRCA1/2-mutated tumors to PARP inhibitors (PARPi). To date, several PARP inhibitors have been approved for clinical use. The promising clinical response of patients with germline BRCA1/2-mutations prompted the use of PARPi for patients with somatic BRCA1/2 mutations as well. In addition, these results extended the use of PARPi for various types of ovarian, breast, pancreatic, and prostate tumors with HR defects. PARPi resistance is emerging as the major obstacle to clinical effectiveness in patients with HR-deficient tumors. PARPi resistance results from several independent mechanisms, leading to the restoration of Homologous Recombination and/or Replication Fork stabilization. The absence of alternative options for patients with tumors with innate or acquired resistance underlines the urgency to develop additional therapeutics. More recent studies have identified new synthetic lethal opportunities for BRCA1/2 deficient tumors. Knockout of the genes for POLQ (DNA polymerase theta) or for USP1 (Ubiquitin Specific Protease 1) results in killing of these tumors, and inhibitors of these enzymes are emerging as promising agents for HR-deficient tumors. POLQ and USP1 expression is particularly high in subtypes of breast and ovarian tumors with defects in HR. As a result, POLQi or USP1i in HR-deficient tumors induces cell death. POLQi or USP1i can synergize with PARPi in killing HR-deficient tumors or PARPi-resistant tumors. BRCA1/2 deficient tumors have an increase in single strand DNA gaps (ssGAPs) near replication forks. Treatment with PARPi results in an increase in the size and number for these ssGAPs, ultimately leading to DSBs and cell death. Interestingly, PARPi resistance results, at least in part, from the ability of resistant cells to close the ssGAPs through enhanced POLQ and USP1 activity, thereby providing a rationale for the combination of PARP1, POLQi, and USP1 in HR-deficient cancers. USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. Moreover, this synergy was observed in a set of patient-derived ovarian cancer organoids (PDOs), thus confirming the sensitivity of BRCA1-deficient cells to inhibition by these agents. The accumulation of ssDNA gaps after treatment with a USP1i, PARP, or POLQi correlated with the sensitivity to these drugs in all models tested. Ovarian cancer PDOs provide a powerful tool for detecting drug synergy and for rapid in vitro sensitivity testing. The detection of ssDNA gap accumulation may be a useful predictive biomarker for response to DNA Repair inhibitors as monotherapy or in combination in ongoing clinical trials. Citation Format: Alan D. D'Andrea. Single strand DNA GAP accumulation as a functional biomarker for DNA Repair Inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA009.

Abstract IA008: Exploiting tumor selective vulnerabilities with structure based drug design

Abstract It is well appreciated that the integrity of both pluripotent and terminally differentiated cellular genomes is under constant pressure from damage provoked by cell-intrinsic and environmental factors. As such, defects in the cellular machinery preserving genomic integrity lead to the accumulation of variants that can promote cancer, but they also confer tumor-cell specific vulnerabilities that present synthetic-lethal therapeutic opportunities. The deepening mechanistic appreciation of tumor-selective pressure on DNA replication-fork fidelity, namely oncogene-induced replication stress, has provoked great interest in the prosecution of drug targets within the cellular machinery that otherwise supports replication fork stability. Poly (ADP-Ribose) glycohydrolase (PARG) is an enzyme with a unique role in the resolution of DNA damage repair and DNA replication fork restart through hydrolysis of Poly (ADP-ribose) (PAR) chains. IDE161 is a potent, selective small molecule inhibitor of PARG that is being developed as an anti-cancer therapeutic for patients with advanced or metastatic cancer harboring defects in homologous recombination repair. In culture, the accumulation of PAR chains upon PARG inhibition causes delayed repair of DNA breaks, replication stress and mitotic catastrophe in settings with collateral liabilities in DNA damage repair and/or replication fork stability. Genome-wide CRISPR-mediated fitness screens across multiple genetic backgrounds revealed strong synthetic lethal interactions of IDE161 with otherwise non-essential components of cellular machinery supporting base excision repair, homologous recombination repair and replication fork stability. In addition, evaluation of IDE161 antiproliferative activity across a panel of 350 molecularly characterized cancer cell lines returned a response profile that was only partially overlapping with that observed with PARPi and was enriched for models harboring vulnerabilities associated with replication stress and/or defects in DNA repair. Notably, many homologous recombination deficient (HRD) cell models display differential dependency on PARG activity vs PARP1/2 activity, suggesting PARG inhibition as a potential therapeutic strategy distinct from PARP inhibitors. In vivo assessment of IDE161 anti-tumor activity in CDX and PDX models mirrored these mechanistic relationships. A number of HRD PDX models displayed a superior response to IDE161 versus PARPi, including robust regressions selectively observed with IDE161 versus niraparib in BRCA2 altered ER+, HER2-breast cancers. Thus, IDE161 has potential as novel targeted therapy that exploits the synthetic lethal relationship between PARG and genomic instability, leading to selective anti-proliferative activity in tumors harboring defects in DNA repair and replication fidelity. Citation Format: Reeja Maskey, Diana Munoz, Megan Conway, Arjun Rao, Vidhya Nagarajan, Ivan Shabalin, Kelly Trego, Jonathan Ryan, Peter Teriete, Christian Frey, Josh Taygerly, Paul Barsanti, Claire Neilan, Jasgit Sachdev, Michael White. Exploiting tumor selective vulnerabilities with structure based drug design [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA008.

Dual roles of UFMylation on stalling fork stability

Dual roles of UFMylation on stalling fork stability

H2AX promotes replication fork degradation and chemosensitivity in BRCA-deficient tumours

Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.

Abstract RF01-05: Functional assessment of RAD51 foci and replication fork dynamics in PARPi resistant BRCA1/2 mutated breast cancer

Abstract Background: Although platinum salts (Pt) or Poly (ADP-Ribose) Polymerase inhibitors (PARPi) are effective in treating homologous recombination defective (HRD) breast cancer, resistance often emerges, especially in advanced disease. Predicting response and relapse is complex, even in patients with germline BRCA1/2 mutations (gBRCA1/2m). Clinically approved HRD detection methods are limited to identification of pathogenic mutations in HR genes or mutational signatures in the genome of tumors caused by HRD. In PDX models derived from HRD breast cancer, the restoration of nuclear RAD51 foci formation, a key feature of functional HR, can predict resistance to HRD-targeted treatment.1 In addition, the restoration of replication fork stability, despite PARPi or induction of Pt adducts that induce replication fork arrest and collapse, confers resistance in pre-clinical models2; to date this resistance mechanism has not been clinically validated. Here we analyse RAD51 foci in FFPE samples and DNA replication fork dynamics, using DNA fibre combing assays, in patient derived organoids (PDO) relating these to clinical response to illustrate how such assays might predict clinical HRD-targeted therapy resistance in metastatic breast cancer (MBC). Patients and Methods: We used immunofluorescent detection of RAD51 foci as a marker for HR proficiency (HRP) in tumors from 29 patients with gBRCA1/2m with MBC treated with HRD-targeted treatment (n=6 PARPi, n=2 Pt and n=21 both agents in sequence). All patients developed resistance that was either de novo or acquired. RAD51 and BRCA1 foci were scored in a minimum of 50 geminin (a marker of S/G2 phase) positive tumor cells; cells with ≥5 foci were classified as positive, and tumours where ≤10% or >10% of cells were positive were considered HRD or HRP, respectively. DNA replication fork dynamics were assessed in with or without PARPi using thymidine analogue labelling. DNA fibre analysis was performed in PDOs developed from fresh tumor sampling of HRD-targeted treatment sensitive or resistant tumors, to determine the relationship between replication fork dynamics, stability and PARPi sensitivity. Results: RAD51 analysis was performed on 9 treatment naïve samples (n=9 patients), 8 samples obtained after HRD-targeted treatment resistance (n=8 patients) and 27 samples obtained pre and post HRD-targeted treatment resistance (n=12 patients). Functional HRD by RAD51 in treatment naive samples was seen in 100% (n=17) of patients with acquired resistance and 66% of patients with de novo resistance. All patients, whether with de novo or acquired resistance, exhibited high RAD51 scores in post-resistance tumour samples, suggesting restoration of HR function is the dominant mechanism of PARPi resistance. As such, RAD51 analysis shows potential as a biomarker of clinical PARPi resistance. Replication fork stability fibre was analysed after exposure to potent PARP1 trapping inhibitors or an ATRi control in 3 PDOs (2 gBRCA1m, 1 BRCAwt). As controls we used an isogenic 2D cell line with and without a CRISPR engineered BRCA1 reversion mutation (SUM149 parental BRCA1m and SUM149BS*1 revertant) or with a PARP1 mutation (SUM149 TR2 clone) that prevents PARP1 being trapped on DNA. These experiments indicate that replication fork dynamics can be assessed in “patient derived” models of breast cancer and that PARPi sensitivity was associated with PARPi induced replication fork instability (median ratio IdU:CldU in resistant lines: 0.98, sensitive lines: 0.60). Conclusion: We show that HRP restoration and RAD51 foci in advanced BRCA1/2m breast cancers is the dominant form resistance to HRD-targeted treatment. We also demonstrate for the first time that analysis of DNA replication fork dynamics can be carried out in breast cancer PDOs and could be further explored as a functional predictive biomarker of PARPi resistance. 1.Pellegrino et al, 2022 (PMID: 35425960) 2.Chaudhuri et al, 2016 (PMID: 27443740) Citation Format: Elizabeth Harvey-Jones, Maya Raghunandan, Luisa Robbez-Masson, Thanussuyah Alaguthurai, Alba Llop-Guevara, Jennifer Trendell, Olga Rodriguez, Marta Guzman, Daniel Weekes, Christelle deRenty, Eleanor Knight, Rebecca Marlow, Christopher Starling, Ruifang Liu, Nivedita Ravindran, Ioannis Roxanis, Natalia Lukashchuk, Violeta Serra, Stephen Pettitt, Christopher Lord, Andrew NJ Tutt. Functional assessment of RAD51 foci and replication fork dynamics in PARPi resistant BRCA1/2 mutated breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF01-05.

Exploration of poly (ADP-ribose) polymerase inhibitor resistance in the treatment of BRCA1/2-mutated cancer.

Breast cancer susceptibility 1/2 (BRCA1/2) genes play a crucial role in DNA damage repair, yet mutations in these genes increase the susceptibility to tumorigenesis. Exploiting the synthetic lethality mechanism between BRCA1/2 mutations and poly(ADP-ribose) polymerase (PARP) inhibition has led to the development and clinical approval of PARP inhibitor (PARPi), representing a milestone in targeted therapy for BRCA1/2 mutant tumors. This approach has paved the way for leveraging synthetic lethality in tumor treatment strategies. Despite the initial success of PARPis, resistance to these agents diminishes their efficacy in BRCA1/2-mutant tumors. Investigations into PARPi resistance have identified replication fork stability and homologous recombination repair as key factors sensitive to PARPis. Additionally, studies suggest that replication gaps may also confer sensitivity to PARPis. Moreover, emerging evidence indicates a correlation between PARPi resistance and cisplatin resistance, suggesting a potential overlap in the mechanisms underlying resistance to both agents. Given these findings, it is imperative to explore the interplay between replication gaps and PARPi resistance, particularly in the context of platinum resistance. Understanding the impact of replication gaps on PARPi resistance may offer insights into novel therapeutic strategies to overcome resistance mechanisms and enhance the efficacy of targeted therapies in BRCA1/2-mutant tumors.

PARP1 UFMylation ensures the stability of stalled replication forks

The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1 activation. However, it is unclear how the activity of PARP1 is regulated. Here, we found that UFMylation is required for the efficient activation of CHK1 by UFMylating PARP1 at K548 during replication stress. Inactivation of UFL1, the E3 enzyme essential for UFMylation, delayed CHK1 activation and inhibits nascent DNA degradation during replication blockage as seen in PARP1-deficient cells. An in vitro study indicated that PARP1 is UFMylated at K548, which enhances its catalytic activity. Correspondingly, a PARP1 UFMylation-deficient mutant (K548R) and pathogenic mutant (F553L) compromised CHK1 activation, the restart of stalled replication forks following replication blockage, and chromosome stability. Defective PARP1 UFMylation also resulted in excessive nascent DNA degradation at stalled replication forks. Finally, we observed that PARP1 UFMylation-deficient knock-in mice exhibited increased sensitivity to replication stress caused by anticancer treatments. Thus, we demonstrate that PARP1 UFMylation promotes CHK1 activation and replication fork stability during replication stress, thus safeguarding genome integrity.

PTIP UFMylation promotes replication fork degradation in BRCA1-deficient cells

“BRCAness” defines cancers that are homologous recombination (HR)-deficient due to BRCA1 or BRCA2 mutations. These mutations confer synthetic lethality with PARP1/2 inhibitors. The chromatin regulator PTIP promotes stalled replication fork degradation in “BRCAness” cells, but the underlying mechanism by which PTIP regulates stalled replication fork stability is unclear. Here, we performed a series of in vitro analyses to dissect the function of UFMylation in regulating fork stabilization in BRCA1-deficient cells. By denaturing co-immunoprecipitation, we first found that replication stress can induce PTIP UFMylation. Interestingly, this post-translational modification promotes end resection and degradation of nascent DNA at stalled replication forks in BRCA1-deficient cells. By cell viability assay, we found that PTIP-depleted and UFL1-depleted BRCA1 knockdown cells are less sensitive to PARP inhibitors than the siRNA targeting negative control BRCA1-deficient cells. These results identify a new mechanism by which PTIP UFMylation confers chemoresistance in BRCA1-deficient cells.

UFL1 triggers replication fork degradation by MRE11 in BRCA1/2-deficient cells.

The stabilization of stalled forks has emerged as a crucial mechanism driving resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient tumors. Here, we identify UFL1, a UFM1-specific E3 ligase, as a pivotal regulator of fork stability and the response to PARP inhibitors in BRCA1/2-deficient cells. On replication stress, UFL1 localizes to stalled forks and catalyzes the UFMylation of PTIP, a component of the MLL3/4 methyltransferase complex, specifically at lysine 148. This modification facilitates the assembly of the PTIP-MLL3/4 complex, resulting in the enrichment of H3K4me1 and H3K4me3 at stalled forks and subsequent recruitment of the MRE11 nuclease. Consequently, loss of UFL1, disruption of PTIP UFMylation or overexpression of the UFM1 protease UFSP2 protects nascent DNA strands from extensive degradation and confers resistance to PARP inhibitors in BRCA1/2-deficient cells. These findings provide mechanistic insights into the processes underlying fork instability in BRCA1/2-deficient cells and offer potential therapeutic avenues for the treatment of BRCA1/2-deficient tumors.

RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication.

The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.