End Resection Research Articles

TREX2 enables efficient genome disruption mediated by paired CRISPR-Cas9 nickases that generate 3′-overhanging ends

Paired SpCas9 nickases (SpCas9n) are an effective strategy to reduce off-target effect in genome editing. However, this approach is not efficient with 3'-overhanging ends, limiting its applications. In order to expand the utility of paired SpCas9n in genome editing, we tested the effect of the TREX2 3'-5' exonuclease on repair of 3'-overhanging ends. We found ectopic overexpression of Trex2 stimulates the efficiency of paired SpCas9n in genome disruption with 3'-overhanging ends up to 400-fold with little stimulation of off-target editing. TREX2 overexpressed preferentially deletes entire 3' overhangs but has no significant effect on 5' overhangs. Trex2 overexpression also stimulates genome disruption by paired SpCas9n that potentially generate short 3'-overhanging ends at overlapping SpCas9n target sites, suggesting sequential nicking of overlapping target sites by SpCas9n. This approach is further simplified with improved efficiency and safety by fusion of TREX2 and particularly its DNA-binding-deficient mutant to SpCas9n. Junction analysis at overlapping targets revealed the different extent of end resection of 3' single-stranded DNA (ssDNA) by free TREX2 and TREX2 fused to SpCas9n. SpCas9n-TREX2 fusion is more convenient and safer than overexpression of free TREX2 to process 3'-overhanging ends for efficient genome disruption by paired SpCas9n, allowing practical use of this TREX2-based strategy in genome editing.

Human HELQ regulates DNA end resection at DNA double-strand breaks and stalled replication forks.

Following a DNA double strand break (DSB), several nucleases and helicases coordinate to generate single-stranded DNA (ssDNA) with 3' free ends, facilitating precise DNA repair by homologous recombination (HR). The same nucleases can act on stalled replication forks, promoting nascent DNA degradation and fork instability. Interestingly, some HR factors, such as CtIP and BRCA1, have opposite regulatory effects on the two processes, promoting end resection at DSB but inhibiting the degradation of nascent DNA on stalled forks. However, the reason why nuclease actions are regulated by different mechanisms in two DNA metabolism is poorly understood. We show that human HELQ acts as a DNA end resection regulator, with opposing activities on DNA end resection at DSBs and on stalled forks as seen for other regulators. Mechanistically, HELQ helicase activity is required for EXO1-mediated DSB end resection, while ssDNA-binding capacity of HELQ is required for its recruitment to stalled forks, facilitating fork protection and preventing chromosome aberrations caused by replication stress. Here, HELQ synergizes with CtIP but not BRCA1 or BRCA2 to protect stalled forks. These findings reveal an unanticipated role of HELQ in regulating DNA end resection at DSB and stalled forks, which is important for maintaining genome stability.

New Facets of DNA Double Strand Break Repair: Radiation Dose as Key Determinant of HR versus c-NHEJ Engagement.

Radiation therapy is an essential component of present-day cancer management, utilizing ionizing radiation (IR) of different modalities to mitigate cancer progression. IR functions by generating ionizations in cells that induce a plethora of DNA lesions. The most detrimental among them are the DNA double strand breaks (DSBs). In the course of evolution, cells of higher eukaryotes have evolved four major DSB repair pathways: classical non-homologous end joining (c-NHEJ), homologous recombination (HR), alternative end-joining (alt-EJ), and single strand annealing (SSA). These mechanistically distinct repair pathways have different cell cycle- and homology-dependencies but, surprisingly, they operate with widely different fidelity and kinetics and therefore contribute unequally to cell survival and genome maintenance. It is therefore reasonable to anticipate tight regulation and coordination in the engagement of these DSB repair pathway to achieve the maximum possible genomic stability. Here, we provide a state-of-the-art review of the accumulated knowledge on the molecular mechanisms underpinning these repair pathways, with emphasis on c-NHEJ and HR. We discuss factors and processes that have recently come to the fore. We outline mechanisms steering DSB repair pathway choice throughout the cell cycle, and highlight the critical role of DNA end resection in this process. Most importantly, however, we point out the strong preference for HR at low DSB loads, and thus low IR doses, for cells irradiated in the G2-phase of the cell cycle. We further explore the molecular underpinnings of transitions from high fidelity to low fidelity error-prone repair pathways and analyze the coordination and consequences of this transition on cell viability and genomic stability. Finally, we elaborate on how these advances may help in the development of improved cancer treatment protocols in radiation therapy.

ATR-binding lncRNA ScaRNA2 promotes cancer resistance through facilitating efficient DNA end resection during homologous recombination repair

BackgroundOur previous study first showed that ATR-binding long noncoding RNA (lncRNA) is necessary for ATR function and promotes cancer resistance. However, the specific lncRNAs instrumental in ATR activation remain largely unclear, which limits our comprehensive understanding of this critical biological process.MethodsRNA immunoprecipitation (RIP) followed by RNA sequencing was employed to identify ATR-binding lncRNAs, which were further validated using RIP-qPCR assays. Immunofluorescence staining and Western blotting were applied to detect the activation of DNA damage repair factors. After the effect of scaRNA2 on cellular sensitivity to DNA-damaging reagents was determined, the effects of scaRNA2 on radiotherapy were investigated in patient-derived organoids and xenograft preclinical models. The clinical relevance of scaRNA2 was also validated in tissues isolated from rectal cancer patients.ResultsScaRNA2 was identified as the most enriched ATR-binding lncRNA and was found to be essential for homologous recombination (HR) mediated DNA damage repair. Furthermore, scaRNA2 knockdown abrogated the recruitment of ATR and its substrates in response to DNA damage. Mechanistically, scaRNA2 was observed to be necessary for Exo1-mediated DNA end resection and bridged the MRN complex to ATR activation. Knockdown of scaRNA2 effectively increased the sensitivity of cancer cells to multiple kinds of DNA damage-related chemoradiotherapy. Preclinically, knockdown of scaRNA2 improved the effects of radiotherapy on patient-derived organoids and xenograft models. Finally, an increase in scaRNA2 colocalized with ATR was also found in clinical patients who were resistant to radiotherapy.ConclusionsScaRNA2 was identified as the most abundant lncRNA bound to ATR and was demonstrated to bridge DNA end resection to ATR activation; thus, it could be applied as a potent target for combined cancer treatments with chemoradiotherapy.

Robust Radiosensitization by Combined Treatment of Cancer Cells with Talazoparib and Polθ Inhibitors

The PARP inhibitor talazoparib is synthetically lethal with HR-defective tumors and functions as a potent radiosensitizer specifically of cancer cells. Talazoparib exerts this unique radiosensitizing property by shifting ionizing radiation (IR)-induced DNA double strand break (DSB) repair towards error-prone alternative end-joining (alt-EJ). DNA polymerase theta (Polθ, encoded by POLQ) is a key component of alt-EJ. Here, we tested the hypothesis that inhibition of alt-EJ with Polθ ablation or using specific small molecule inhibitors can further increase talazoparib-induced radiosensitization. U2OS, A549, and their corresponding POLQ ablated/mutant cell lines were treated with talazoparib and/or Polθ inhibitors ART558/novobiocin prior to irradiation. siRNAs against CtIP, MRE11, EXO1; and a specific inhibitor of DNA2 were employed to suppress DNA end resection. Radiosensitization was assessed by clonogenic survival. Olaparib, rucaparib, and veliparib were also tested under similar conditions. DSB repair and end resection were measured by scoring γH2AX and RPA nuclear foci, respectively. Chromosomal abnormalities were assessed using G2-specific cytogenetics analysis. Genetic ablation or pharmacological inhibition of Polθ robustly enhanced talazoparib mediated radiosensitization by ∼40-70%. Notably, Polθ inhibition had a much lower effect (by ∼7-17%) when combined with other clinically used PARP inhibitors, olaparib, rucaparib, and veliparib. Polθ inhibition significantly suppressed talazoparib-induced translocation formation in irradiated cells. In addition, combined treatment with Polθ inhibitor and talazoparib attenuated DSB repair, resulting in ∼60% unresolved γH2AX foci and ∼40% unresolved chromatid breaks at 5h post IR. Talazoparib promoted resection of DNA ends as demonstrated by an increase in RPA foci. The resection process requires the activities of CtIP and MRE11, but not of DNA2 or EXO1. Finally, CtIP and MRE11 knockdown impaired radiosensitization following a combined talazoparib/Polθ inhibition treatment. Talazoparib increases the reliance of irradiated cancer cells on Polθ-mediated alt-EJ owing to the increased CtIP/MRE11-dependent resection it produces. Combining talazoparib with Polθ inhibitors has therefore great potential in improving radiotherapy of human tumors.

Short-range end resection requires ATAD5-mediated PCNA unloading for faithful homologous recombination.

Homologous recombination (HR) requires bidirectional end resection initiated by a nick formed close to a DNA double-strand break (DSB), dysregulation favoring error-prone DNA end-joining pathways. Here we investigate the role of the ATAD5, a PCNA unloading protein, in short-range end resection, long-range resection not being affected by ATAD5 deficiency. Rapid PCNA loading onto DNA at DSB sites depends on the RFC PCNA loader complex and MRE11-RAD50-NBS1 nuclease complexes bound to CtIP. Based on our cytological analyses and on an in vitro system for short-range end resection, we propose that PCNA unloading by ATAD5 is required for the completion of short-range resection. Hampering PCNA unloading also leads to failure to remove the KU70/80 complex from the termini of DSBs hindering DNA repair synthesis and the completion of HR. In line with this model, ATAD5-depleted cells are defective for HR, show increased sensitivity to camptothecin, a drug forming protein-DNA adducts, and an augmented dependency on end-joining pathways. Our study highlights the importance of PCNA regulation at DSB for proper end resection and HR.

Dynamics of the DYNLL1-MRE11 complex regulate DNA end resection and recruitment of Shieldin to DSBs.

The extent and efficacy of DNA end resection at DNA double-strand breaks (DSB) determine the repair pathway choice. Here we describe how the 53BP1-associated protein DYNLL1 works in tandem with the Shieldin complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1, where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1, predominantly in G1 cells. Shieldin localization to DSBs depends on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be resensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.

Simulating Space Conditions Evokes Different DNA Damage Responses in Immature and Mature Cells of the Human Hematopoietic System.

The impact of space radiation and microgravity on DNA damage responses has been discussed controversially, largely due to the variety of model systems engaged. Here, we performed side-by-side analyses of human hematopoietic stem/progenitor cells (HSPC) and peripheral blood lymphocytes (PBL) cultivated in a 2D clinostat to simulate microgravity before, during and after photon and particle irradiation. We demonstrate that simulated microgravity (SMG) accelerates the early phase of non-homologous end joining (NHEJ)-mediated repair of simple, X-ray-induced DNA double-strand breaks (DSBs) in PBL, while repair kinetics in HSPC remained unaltered. Repair acceleration was lost with increasing LET of ion exposures, which increases the complexity of DSBs, precluding NHEJ and requiring end resection for successful repair. Such cell-type specific effect of SMG on DSB repair was dependent on the NF-кB pathway pre-activated in PBL but not HSPC. Already under unperturbed growth conditions HSPC and PBL suffered from SMG-induced replication stress associated with accumulation of single-stranded DNA and DSBs, respectively. We conclude that in PBL, SMG-induced DSBs promote repair of radiation-induced damage in an adaptive-like response. HSPC feature SMG-induced single-stranded DNA and FANCD2 foci, i.e., markers of persistent replication stress and senescence that may contribute to a premature decline of the immune system in space.

Centriolar subdistal appendages promote double-strand break repair through homologous recombination.

The centrosome is a cytoplasmic organelle with roles in microtubule organization that has also been proposed to act as a hub for cellular signaling. Some centrosomal components are required for full activation of the DNA damage response. However, whether the centrosome regulates specific DNA repair pathways is not known. Here, we show that centrosome presence is required to fully activate recombination, specifically to completely license its initial step, the so-called DNA end resection. Furthermore, we identify a centriolar structure, the subdistal appendages, and a specific factor, CEP170, as the critical centrosomal component involved in the regulation of recombination and resection. Cells lacking centrosomes or depleted for CEP170 are, consequently, hypersensitive to DNA damaging agents. Moreover, low levels of CEP170 in multiple cancer types correlate with an increase of the mutation burden associated with specific mutational signatures and a better prognosis, suggesting that changes in CEP170 can act as a mutation driver but could also be targeted to improve current oncological treatments.

RBM14 promotes DNA end resection during homologous recombination repair.

DNA double-strand break (DSB) repair by homologous recombination (HR) is crucial for the maintenance of genome stability and integrity. In this study, we aim to identify novel RNA binding proteins (RBPs) involved in HR repair because little is known about RBP function in HR. For this purpose, we carry out pulldown assays using a synthetic ssDNA/dsDNA structure coated with replication protein A (RPA) to mimic resected DNA, a crucial intermediate in HR-mediated DSB repair. Using this approach, we identify RNA-binding motif protein 14 (RBM14) as a potential binding partner. We further show that RBM14 interacts with an essential HR repair factor, CtIP. RBM14 is crucial for CtIP recruitment to DSB sites and for subsequent RPA coating and RAD51 replacement, facilitating efficient HR repair. Moreover, inhibition of RBM14 expression sensitizes cancer cells to X-ray irradiation. Together, our results demonstrate that RBM14 promotes DNA end resection to ensure HR repair and may serve as a potential target for cancer therapy.

Long-range DNA end resection supports homologous recombination by checkpoint activation rather than extensive homology generation.

Homologous recombination (HR), the high-fidelity mechanism for double-strand break (DSB) repair, relies on DNA end resection by nucleolytic degradation of the 5¢-terminated ends. However, the role of long-range resection mediated by Exo1 and/or Sgs1-Dna2 in HR is not fully understood. Here, we show that Exo1 and Sgs1 are dispensable for recombination between closely-linked repeats, but are required for interchromosomal repeat recombination in Saccharomyces cerevisiae. This context-specific requirement for long-range end resection is connected to its role in activating the DNA damage checkpoint. Consistent with this role, checkpoint mutants also show a defect specifically in interchromosomal recombination. Furthermore, artificial activation of the checkpoint partially restores interchromosomal recombination to exo1∆ sgs1∆ cells. However, cell cycle delay is insufficient to rescue the interchromosomal recombination defect of exo1∆ sgs1∆ cells, suggesting an additional role for the checkpoint. Given that the checkpoint is necessary for DNA damage-induced chromosome mobility, we propose that the importance of the checkpoint, and therefore long-range resection, in interchromosomal recombination is due to a need to increase chromosome mobility to facilitate pairing of distant sites. The need for long-range resection is circumvented when the DSB and its repair template are in close proximity.

The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment.

DNA double-strand breaks are the most toxic lesions repaired through the non-homologous and joining (NHEJ) or the homologous recombination (HR), which is dependent on the generation of single-strand tails, by the DNA end resection mechanism. The resolution of the HR intermediates leads to error-free repair (Gene Conversion) or the mutagenic pathways (Single Strand Annealing and Alternative End-Joining); the regulation of processes leading to the resolution of the HR intermediates is not fully understood. Here, we used a hydrophilic extract of a new tomato genotype (named DHO) in order to modulate the Camptothecin (CPT) DNA damage response. We demonstrated increased phosphorylation of Replication Protein A 32 Serine 4/8 (RPA32 S4/8) protein in HeLa cells treated with the CPT in combination with DHO extract with respect to CPT alone. Moreover, we pointed out a change in HR intermediates resolution from Gene Conversion to Single Strand Annealing through the modified DNA repair protein RAD52 homolog (RAD52), DNA excision repair protein ERCC-1 (ERCC1) chromatin loading in response to DHO extract, and CPT co-treatment, with respect to the vehicle. Finally, we showed an increased sensitivity of HeLa cell lines to DHO extract and CPT co-treatment suggesting a possible mechanism for increasing the efficiency of cancer therapy. We described the potential role of DHO extract in the modulation of DNA repair, in response to Camptothecin treatment (CPT), favoring an increased sensitivity of HeLa cell lines to topoisomerase inhibitor therapy.

MND1 enables homologous recombination in somatic cells primarily outside the context of replication.

Faithful and timely repair of DNA double-strand breaks (DSBs) is fundamental for the maintenance of genomic integrity. Here, we demonstrate that the meiotic recombination co-factor MND1 facilitates the repair of DSBs in somatic cells. We show that MND1 localizes to DSBs, where it stimulates DNA repair through homologous recombination (HR). Importantly, MND1 is not involved in the response to replication-associated DSBs, implying that it is dispensable for HR-mediated repair of one-ended DSBs. Instead, we find that MND1 specifically plays a role in the response to two-ended DSBs that are induced by irradiation (IR) or various chemotherapeutic drugs. Surprisingly, we find that MND1 is specifically active in G2 phase, whereas it only marginally affects repair during S phase. MND1 localization to DSBs is dependent on resection of the DNA ends and seemingly occurs through direct binding of MND1 to RAD51-coated ssDNA. Importantly, the lack of MND1-driven HR repair directly potentiates the toxicity of IR-induced damage, which could open new possibilities for therapeutic intervention, specifically in HR-proficient tumors.

UbcH5c-dependent activation of DNA-dependent protein kinase in response to replication-mediated DNA double-strand breaks

Camptothecin (CPT) exhibits strong cytotoxicity by inducing DNA double-strand breaks (DSBs) through DNA replication. Unlike radiation-induced DSBs, which have two DNA ends, CPT-induced DSBs are considered to have only one DNA end. However, the differences in cellular responses to one-ended and two-ended DSBs are not well understood. Our previous study showed that proteasome inhibitor treatment suppressed CPT-induced activation of DNA-PK, a factor required for non-homologous end-joining in DSB repair, suggesting that the ubiquitin-proteasome pathway is involved in DNA-PK activation in response to one-ended DSBs. To identify the ubiquitination factors required for DNA-PK activation, we screened an siRNA library against E2 ubiquitin-conjugating enzymes and identified UbcH5c. Knockdown of UbcH5c suppressed DNA-PK activation caused by CPT, but not by the radio-mimetic drug neocarzinostatin. UbcH5c-dependent DNA-PK activation occurred independent of DNA end resection. Furthermore, loss of UbcH5c reduced DNA-PK-dependent chromosomal aberrations and suppressed the activation of cell cycle checkpoint in response to CPT. These results suggest that UbcH5c regulates DNA-PK activation in response to one-ended DSBs caused by replication fork collapse. To our knowledge, this is the first report of a DSB repair-related factor that is differentially involved in the response to one- and two-ended DSBs.

The SETD2 Methyltransferase Supports Productive HPV31 Replication through the LEDGF/CtIP/Rad51 Pathway.

The human papillomavirus (HPV) life cycle takes place in the stratified epithelium, with the productive phase being activated by epithelial differentiation. The HPV genome is histone-associated, and the life cycle is epigenetically regulated, in part, by histone tail modifications that facilitate the recruitment of DNA repair factors that are required for viral replication. We previously showed that the SETD2 methyltransferase facilitates the productive replication of HPV31 through the trimethylation of H3K36 on viral chromatin. SETD2 regulates numerous cellular processes, including DNA repair via homologous recombination (HR) and alternative splicing, through the recruitment of various effectors to histone H3 lysine 36 trimethylation (H3K36me3). We previously demonstrated that the HR factor Rad51 is recruited to HPV31 genomes and is required for productive replication; however, the mechanism of Rad51 recruitment has not been defined. SET domain containing 2 (SETD2) promotes the HR repair of double-strand breaks (DSBs) in actively transcribed genes through the recruitment of carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) to lens epithelium-derived growth factor (LEDGF)-bound H3K36me3, which promotes DNA end resection and thereby allows for the recruitment of Rad51 to damaged sites. In this study, we found that reducing H3K36me3 through the depletion of SETD2 or the overexpression of an H3.3K36M mutant leads to an increase in γH2AX, which is a marker of damage, on viral DNA upon epithelial differentiation. This is coincident with decreased Rad51 binding. Additionally, LEDGF and CtIP are bound to HPV DNA in a SETD2-dependent and H3K36me3-dependent manner, and they are required for productive replication. Furthermore, CtIP depletion increases DNA damage on viral DNA and blocks Rad51 recruitment upon differentiation. Overall, these studies indicate that H3K36me3 enrichment on transcriptionally active viral genes promotes the rapid repair of viral DNA upon differentiation through the LEDGF-CtIP-Rad51 axis. IMPORTANCE The productive phase of the HPV life cycle is restricted to the differentiating cells of the stratified epithelium. The HPV genome is histone-associated and subject to epigenetic regulation, though the manner in which epigenetic modifications contribute to productive replication is largely undefined. In this study, we demonstrate that SETD2-mediated H3K36me3 on HPV31 chromatin promotes productive replication through the repair of damaged DNA. We show that SETD2 facilitates the recruitment of the homologous recombination repair factors CtIP and Rad51 to viral DNA through LEDGF binding to H3K36me3. CtIP is recruited to damaged viral DNA upon differentiation, and, in turn, recruits Rad51. This likely occurs through the end resection of double-strand breaks. SETD2 trimethylates H3K36me3 during transcription, and active transcription is necessary for Rad51 recruitment to viral DNA. We propose that the enrichment of SETD2-mediated H3K36me3 on transcriptionally active viral genes upon differentiation facilitates the repair of damaged viral DNA during the productive phase of the viral life cycle.

MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1.

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access ofPOLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role ofMSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.

Surgical removal of periapical pathology with root end resection

In children and young adults, traumatic dental injuries (TDIs) to permanent teeth are common. Of all dental injuries, crown fractures and luxations of these teeth are the most frequent. For a successful outcome, accurate diagnosis, treatment planning, and follow-up are critical. This article presents a case report series of surgical management of traumatic injury with periapical lesion with rooth end resection using root end filling materials.

Shieldin complex assembly kinetics and DNA binding by SHLD3

The Shieldin complex represses end resection at DNA double-strand breaks (DSBs) and thereby serves as a pro-non homologous end joining (NHEJ) factor. The molecular details of the assembly of Shieldin and its recruitment to DSBs are unclear. Shieldin contains two REV7 molecules, which have the rare ability to slowly switch between multiple distinct native states and thereby could dynamically control the assembly of Shieldin. Here, we report the identification of a promiscuous DNA binding domain in SHLD3. At the N-terminus, SHLD3 interacts with a dimer of REV7 molecules. We show that the interaction between SHLD3 and the first REV7 is remarkably slow, while in contrast the interaction between SHLD3 and SHLD2 with a second REV7 molecule is fast and does not require structural remodeling. Overall, these results provide insights into the rate-limiting step of the molecular assembly of the Shieldin complex and its recruitment at DNA DSBs.

Abstract 6190: Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers

Abstract Cancers deficient in homologous recombination (HR) repair secondary to mutations in genes such as BRCA1 or BRCA2, are dependent on alternative DNA damage response (DDR) pathways to maintain genomic integrity, rendering them susceptible to synthetic lethal targeting of these pathways. Recently, inhibitors of polymerase theta (POLθ, encoded by POLQ), the critical enzyme in microhomology-mediated end-joining (MMEJ), have been shown to be synthetic lethal with HR repair deficiency (Zhou et al. Nature Cancer 2021). Both HR and MMEJ require nucleolytic DNA end-resection to allow for DSB repair, and we have previously shown that MMEJ acts as a barrier to DNA end-resection at DSBs (Patterson-Fortin et al. Cancer Research 2022). Given the synthetic lethality between HR and MMEJ leads to unrestrained DNA end-resection generating chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, we hypothesized that POLθ inhibition in HR-deficient cancers would activate the cGAS/STING innate immune response pathway and facilitate immunotherapy. To investigate the interactions of POLθ inhibition with the immune microenvironment in HR-deficient cancers, we used human cell lines and genetically modified mouse models representative of BRCA1-deficient triple-negative breast cancer (TNBC) and BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). Genetic or pharmacological inhibition of POLθ using novobiocin, a first-in-class inhibitor of the POLθ ATPase domain, induced significantly increased cytosolic dsDNA contained in micronuclei. This free DNA was sensed by the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS), increasing synthesis of cGAMP, in HR-deficient tumor cells but not in HR-proficient tumor cells. Increased cGAMP bound to and activated stimulator of interferon genes (STING), triggering phosphorylation of TBK1 and ultimately of IRF3. Activation of the cGAS/STING pathway by POLθ inhibition drove the expression of type I interferon response elements, including PD-L1. Depletion of STING by siRNA or by CRISPR abrogated this pro-inflammatory signaling and abolished the anti-tumor efficacy of novobiocin-mediated POLθ inhibition. Pharmacologic inhibition of POLθ enhanced Granzyme B+ CD8+ T-cell tumor infiltration. Importantly, antibody-mediated depletion of CD8+ T-cell severely compromised the anti-tumor efficacy of novobiocin-mediated POLθ inhibition, whereas anti-tumor activity of POLθ inhibition was augmented with the addition of either anti-PD-1 or anti-CTLA-4 antibodies. These results demonstrate that POLθ inhibition in HR-deficient cancers mediates a pro-inflammatory response in HR-deficient TNBC or PDAC tumor microenvironments, and that immune checkpoint blockade inhibition enhances the therapeutic efficacy of POLθ inhibition. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Constantia Pantelidou, Tin Phan, Carter Grochala, Anita K. Mehta, Jennifer L. Guerriero, Gerburg M. Wulf, Brian M. Wolpin, Ben Z. Stanger, Andrew J. Aguirre, James M. Cleary, Alan D. D'Andrea, Geoffrey I. Shapiro. Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers [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 6190.

ERCC6L2 mitigates replication stress and promotes centromere stability

Structurally complex genomic regions, such as centromeres, are inherently difficult to duplicate. The mechanism behind centromere inheritance is not well understood, and one of the key questions relates to the reassembly of centromeric chromatin following DNA replication. Here, we define ERCC6L2 as a key regulator of this process. ERCC6L2 accumulates at centromeres and promotes deposition of core centromeric factors. Interestingly, ERCC6L2-/- cells show unrestrained replication of centromeric DNA, likely caused by the erosion of centromeric chromatin. Beyond centromeres, ERCC6L2 facilitates replication at genomic repeats and non-canonical DNA structures. Notably, ERCC6L2 interacts with the DNA-clamp PCNA through an atypical peptide, presented here in a co-crystal structure. Finally, ERCC6L2 also restricts DNA end resection, acting independently of the 53BP1-REV7-Shieldin complex. We propose a mechanistic model, which reconciles seemingly distinct functions of ERCC6L2 in DNA repair and DNA replication. These findings provide a molecular context for studies linking ERCC6L2 to human disease.