Alternative End Joining Research Articles

The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks.

Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.

Establishment of a Transformation Coupled in vitro End Joining Assay to Estimate Radiosensitivity in Tumor Cells.

Here, we present a modified in vitro end-joining (EJ) assay to quantify EJ capacity, accuracy as well as pathway switch to alternative end-joining (Alt-EJ) or single strand annealing (SSA). A novel transformation assay was established to specifically measure circular repair products, which correlate with classical EJ efficiency. The EJ assay was validated using EJ-deficient mammalian cell lines (Ku80, DNA-PKcs, LigIV, or XRCC4 mutants). A pathway switch to Alt-EJ and SSA was seen exclusively in Ku-deficient cells. Circular EJ product formation correlated with cell survival and DSB repair capacity after X-irradiation. Investigation of 14 HNSCC cell lines revealed differences in the total EJ capacity but a broader variation in the amount of circular repair products. Sequencing of repair junctions in HNSCC cells demonstrated a predominance of high-fidelity EJ and an avoidance of both Alt-EJ and SSA. A significant correlation was observed between the amount of circular repair products, repair of IR-induced DSB and radiosensitivity. Collectively, these data indicate that the presented in vitro-EJ-assay can not only estimate the repair capacity of cancer cells to enable the stratification into radiosensitive or radioresistant, but can also identify repair pathway deregulation such as a switch to Alt-EJ or SSA, which enables tumor targeting.

Abstract 1569: TGFβ signaling suppresses alternative end joining associated with genomic mutations and PARP Inhibition sensitivity

Abstract Differential use of DNA repair pathways in cancer leads to distinct clinical outcomes following genotoxic therapies. We found that deficient TGFβ signaling due to human papilloma virus (HPV) in head and neck squamous cell carcinoma (HNSCC) results in impaired DNA repair proficiency, increased use of alternative end joining (alt-EJ) and that a 50-gene TGFβ signature is negatively correlated with prognosis (Clin Ca Res 2018, 24(23): 6001). To assess the molecular consequence of TGFβ signaling in the DNA damage response (DDR), we performed a Nanostring assay on SAS HNSCC and U251 glioblastoma cell lines using the 180 gene DNA damage and repair panel. The results showed that treatment with TGFβ type 1 receptor kinase inhibitor LY2157299 or TGFβ profoundly altered DDR gene expression profiles. TGFβ inhibition for 24 hr significantly (p<0.05) increased critical alt-EJ genes, POLQ (DNA polymerase theta) and PARP1 (poly(ADP-ribose) polymerase 1), which was confirmed by qRT-PCR. To interrogate the relationship between TGFβ and alt-EJ in The Cancer Genome Atlas (TCGA), we used the literature to curate a 36 gene alt-EJ signature. Single specimen gene set enrichment analyses (ssGSEA) showed that the alt-EJ signature was significantly anti-correlated with the TGFβ signature in 15 of 17 cancer types (PCC = -0.35; p =10-16). This anti-correlation was also observed in 875 cancer cell lines in Catalogue of Somatic Mutations in Cancer (COSMIC) database, suggesting a conserved, cancer cell intrinsic mechanism by which TGFβ regulates alt-EJ. Alt-EJ is an error prone DNA repair mechanism that generates gross genomic alterations. Consistent with this, low ssGSEA score for TGFβ and high ssGSEA score for alt-EJ are correlated with increased deletions or insertions in genome sequence of patient tumors in TCGA as well as cancer cell lines in COSMIC (p<0.05). PARP1 requirement prompted analyses of drug sensitivity data in the Sanger database, which showed that cell lines with high alt-EJ and low TGFβ ssGSEA score are significantly more sensitive to PARP1 inhibitors, olaparib and ABT-888, (p < 0.0001). These data lead to the novel hypothesis that TGFβ signaling actively suppresses alt-EJ. If true, the TGFβ/alt-EJ signature may be useful as a biomarker for precision cancer medicine with genotoxic agents, TGFβ or PARP inhibitors. Citation Format: Qi Liu, Luis Palomero, Jade Moore, Ines Guix, Miquel Angel Pujana, Mary Helen Barcellos-Hoff. TGFβ signaling suppresses alternative end joining associated with genomic mutations and PARP Inhibition sensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1569.

Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining.

To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70−/− and AtLig4−/−). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70−/− mutant was slightly more sensitive than the AtLig4−/− mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11–30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3–4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

XRCC1 promotes replication restart, nascent fork degradation and mutagenic DNA repair in BRCA2-deficient cells

Homologous recombination/end joining (HR/HEJ)-deficient cancers with BRCA mutations utilize alternative DNA double-strand break repair pathways, particularly alternative non-homologous end joining or microhomology-mediated end joining (alt-EJ/MMEJ) during S and G2 cell cycle phases. Depletion of alt-EJ factors, including XRCC1, PARP1 and POLQ, is synthetically lethal with BRCA2 deficiency; yet, XRCC1 roles in HR-deficient cancers and replication stress are enigmatic. Here, we show that after replication stress, XRCC1 forms an active repair complex with POLQ and MRE11 that supports alt-EJ activity in vitro. BRCA2 limits XRCC1 recruitment and repair complex formation to suppress alt-EJ at stalled forks. Without BRCA2 fork protection, XRCC1 enables cells to complete DNA replication at the expense of increased genome instability by promoting MRE11-dependent fork resection and restart. High XRCC1 and MRE11 gene expression negatively impacts Kaplan–Meier survival curves and hazard ratios for HR-deficient breast cancer patients in The Cancer Genome Atlas. The additive effects of depleting both BRCA2 and XRCC1 indicate distinct pathways for replication restart. Our collective data show that XRCC1-mediated processing contributes to replication fork degradation, replication restart and chromosome aberrations in BRCA2-deficient cells, uncovering new roles of XRCC1 and microhomology-mediated repair mechanisms in HR-deficient cancers, with implications for chemotherapeutic strategies targeting POLQ and PARP activities.

Epigenetic Regulation of DNA Repair Pathway Choice by MacroH2A1 Splice Variants Ensures Genome Stability

The inactive X chromosome (Xi) is inherently susceptible to genomic aberrations. Replication stress (RS) has been proposed as an underlying cause, but the mechanisms that protect from Xi instability remain unknown. Here, we show that macroH2A1.2, an RS-protective histone variant enriched on the Xi, is required for Xi integrity and female survival. Mechanistically, macroH2A1.2 counteracts its structurally distinct and equally Xi-enriched alternative splice variant, macroH2A1.1. Comparative proteomics identified a role for macroH2A1.1 in alternative end joining (alt-EJ), which accounts for Xi anaphase defects in the absence of macroH2A1.2. Genomic instability was rescued by simultaneous depletion of macroH2A1.1 or alt-EJ factors, and mice deficient for both macroH2A1 variants harbor no overt female defects. Notably, macroH2A1 splice variant imbalance affected alt-EJ capacity also in tumor cells. Together, these findings identify macroH2A1 splicing as a modulator of genome maintenance that ensures Xi integrity and may, more broadly, predict DNA repair outcome in malignant cells.

Regulation of Error-Prone DNA Double-Strand Break Repair and Its Impact on Genome Evolution.

Double-strand breaks are one of the most deleterious DNA lesions. Their repair via error-prone mechanisms can promote mutagenesis, loss of genetic information, and deregulation of the genome. These detrimental outcomes are significant drivers of human diseases, including many cancers. Mutagenic double-strand break repair also facilitates heritable genetic changes that drive organismal adaptation and evolution. In this review, we discuss the mechanisms of various error-prone DNA double-strand break repair processes and the cellular conditions that regulate them, with a focus on alternative end joining. We provide examples that illustrate how mutagenic double-strand break repair drives genome diversity and evolution. Finally, we discuss how error-prone break repair can be crucial to the induction and progression of diseases such as cancer.

SCFSKP2 regulates APC/CCDH1-mediated degradation of CTIP to adjust DNA-end resection in G2-phase

The cell cycle-dependent engagement of DNA-end resection at DSBs is regulated by phosphorylation of CTIP by CDKs, the central regulators of cell cycle transitions. Cell cycle transitions are also intimately regulated by protein degradation via two E3 ubiquitin ligases: SCFSKP2 and APC/CCDH1 complex. Although APC/CCDH1 regulates CTIP in G1– and G2-phase, contributions by SCFSKP2 have not been reported. We demonstrate that SCFSKP2 is a strong positive regulator of resection. Knockdown of SKP2, fully suppresses resection in several cell lines. Notably, this suppression is G2-phase specific and is not observed in S-phase or G1–phase cells. Knockdown of SKP2 inactivates SCFSKP2 causing APC/CCDH1 activation, which degrades CTIP. The stabilizing function of SCFSKP2 on CTIP promotes resection and supports gene conversion (GC), alternative end joining (alt-EJ) and cell survival. We propose that CDKs and SCFSKP2-APC/CCDH1 cooperate to regulate resection and repair pathway choice at DSBs in G2-phase.

Desiccation does not drastically increase the accessibility of exogenous DNA to nuclear genomes: evidence from the frequency of endosymbiotic DNA transfer

BackgroundAlthough horizontal gene transfer (HGT) is a widely accepted force in the evolution of prokaryotic genomes, its role in the evolution of eukaryotic genomes remains hotly debated. Some bdelloid rotifers that are resistant to extreme desiccation and radiation undergo a very high level of HGT, whereas in another desiccation-resistant invertebrate, the tardigrade, the pattern does not exist. Overall, the DNA double-strand breaks (DSBs) induced by prolonged desiccation have been postulated to open a gateway to the nuclear genome for exogenous DNA integration and thus to facilitate the HGT process, thereby enhancing the rate of endosymbiotic DNA transfer (EDT).ResultsWe first surveyed the abundance of nuclear mitochondrial DNAs (NUMTs) and nuclear plastid DNAs (NUPTs) in five eukaryotes that are highly resistant to desiccation: the bdelloid rotifers Adineta vaga and Adineta ricciae, the tardigrade Ramazzottius varieornatus, and the resurrection plants Dorcoceras hygrometricum and Selaginella tamariscina. Excessive NUMTs or NUPTs were not detected. Furthermore, we compared 24 groups of desiccation-tolerant organisms with their relatively less desiccation-tolerant relatives but did not find a significant difference in NUMT/NUPT contents.ConclusionsDesiccation may induce DSBs, but it is unlikely to dramatically increase the frequency of exogenous sequence integration in most eukaryotes. The capture of exogenous DNA sequences is possible only when DSBs are repaired through a subtype of non-homologous end joining, named alternative end joining (alt-EJ). Due to the deleterious effects of the resulting insertion mutations, alt-EJ is less frequently initiated than other mechanisms.

CRISPR with a Happy Ending: Non‐Templated DNA Repair for Prokaryotic Genome Engineering

The exploration of microbial metabolism is expected to support the development of a sustainable economy and tackle several problems related to the burdens of human consumption. Microorganisms have the potential to catalyze processes that are currently unavailable, unsustainable and/or inefficient. Their metabolism can be optimized and further expanded using tools like the clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) systems. These tools have revolutionized the field of biotechnology, as they greatly streamline the genetic engineering of organisms from all domains of life. CRISPR-Cas and other nucleases mediate double-strand DNA breaks, which must be repaired to prevent cell death. In prokaryotes, these breaks can be repaired through either homologous recombination, when a DNA repair template is available, or through template-independent end joining, of which two major pathways are known. These end joining pathways depend on different sets of proteins and mediate DNA repair with different outcomes. Understanding these DNA repair pathways can be advantageous to steer the results of genome engineering experiments. In this review, we discuss different strategies for the genetic engineering of prokaryotes through either non-homologous end joining (NHEJ) or alternative end joining (AEJ), both of which are independent of exogenous DNA repair templates.

The recent advances in non-homologous end-joining through the lens of lymphocyte development

Lymphocyte development requires ordered assembly and subsequent modifications of the antigen receptor genes through V(D)J recombination and Immunoglobulin class switch recombination (CSR), respectively. While the programmed DNA cleavage events are initiated by lymphocyte-specific factors, the resulting DNA double-strand break (DSB) intermediates activate the ATM kinase-mediated DNA damage response (DDR) and rely on the ubiquitously expressed classical non-homologous end-joining (cNHEJ) pathway including the DNA-dependent protein kinase (DNA-PK), and, in the case of CSR, also the alternative end-joining (Alt-EJ) pathway, for repair. Correspondingly, patients and animal models with cNHEJ or DDR defects develop distinct types of immunodeficiency reflecting their specific DNA repair deficiency. The unique end-structure, sequence context, and cell cycle regulation of V(D)J recombination and CSR also provide a valuable platform to study the mechanisms of, and the interplay between, cNHEJ and DDR. Here, we compare and contrast the genetic consequences of DNA repair defects in V(D)J recombination and CSR with a focus on the newly discovered cNHEJ factors and the kinase-dependent structural roles of ATM and DNA-PK in animal models. Throughout, we try to highlight the pending questions and emerging differences that will extend our understanding of cNHEJ and DDR in the context of primary immunodeficiency and lymphoid malignancies.

Increasing CRISPR/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors.

Histone deacetylase inhibitors (HDACis) affect DNA repair pathways by modulating multiple cellular machineries, including chromatin state, DNA repair factor modification, and the cell cycle. These machineries can differentially affect DNA repair outcomes. With the aim to investigate the impacts of HDACis on DNA repair following CRISPR/Cas9 cleavage from the mixed actions, we used two pan-HDACis, trichostatin A (TSA) and PCI-24781, to treat animal immortalized and primary cells, and studied CRISPR/Cas9-mediated genome editing results by nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. We first found that TSA and PCI-24781 increased NHEJ efficiency. However, further analysis of the total NHEJ events demonstrated that alternative end joining (alt-EJ) mainly contributed to the enhanced total NHEJ by HDACis. We then analyzed HDR efficiency with HDACi treatment and found that multiple HDR pathways, including homologous recombination, single strand annealing and single-stranded oligonucleotide (ssODN)-mediated HDR, were all increased with HDACi treatment. TSA also increased CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. Analyzing acetylation status of DNA repair factors showed that acetylation levels of classical NHEJ (c-NHEJ) factors KU70 and KU80 and alt-EJ factor PARP1 were significantly enhanced, but alt-EJ factor LIG3 and HDR factors Rad51 and Rad52 were not affected greatly, implying a differential impact on these repair pathways by HDACis. In addition, TSA and PCI-24781 can enrich cells in G2/M phase of the cell cycle which is beneficial for occurrence of HDR. These findings show that HDACis can effectively promote CRISPR-mediated homology-involved DNA repair, including HDR and alt-EJ pathways, through concerted action of multiple cellular machineries.

COM1, a factor of alternative non-homologous end joining, lagging behind the classic non-homologous end joining pathway in rice somatic cells.

The role of rice (Oryza sativa) COM1 in meiotic homologous recombination (HR) is well understood, but its part in somatic double-stranded break (DSB) repair remains unclear. Here, we show that for rice plants COM1 conferred tolerance against DNA damage caused by the chemicals bleomycin and mitomycin C, while the COM1 mutation did not compromise HR efficiencies and HR factor (RAD51 and RAD51 paralogues) localization to irradiation-induced DSBs. Similar retarded growth at the post-germination stage was observed in the com1-2 mre11 double mutant and the mre11 single mutant, while combined mutations in COM1 with the HR pathway gene (RAD51C) or classic non-homologous end joining (NHEJ) pathway genes (KU70, KU80, and LIG4) caused more phenotypic defects. In response to γ-irradiation, COM1 was loaded normally onto DSBs in the ku70 mutant, but could not be properly loaded in the MRE11RNAi plant and in the wortmannin-treated wild-type plant. Under non-irradiated conditions, more DSB sites were occupied by factors (MRE11, COM1, and LIG4) than RAD51 paralogues (RAD51B, RAD51C, and XRCC3) in the nucleus of wild-type; protein loading of COM1 and XRCC3 was increased in the ku70 mutant. Therefore, quite differently to its role for HR in meiocytes, rice COM1 specifically acts in an alternative NHEJ pathway in somatic cells, based on the Mre11-Rad50-Nbs1 (MRN) complex and facilitated by PI3K-like kinases. NHEJ factors, not HR factors, preferentially load onto endogenous DSBs, with KU70 restricting DSB localization of COM1 and XRCC3 in plant somatic cells.

HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells

(Molecular Cell 77, 384–394.e1–e4; January 16, 2020) The authors report two minor errors in Figure 3G of the originally published article. In this panel, there appeared a letter “E” that was not relevant to the figure and may have been confusing to readers. Also, in the same panel, the authors report having inadvertently copied an older version of the bar graph, with fewer replicates, which is different from the one provided to reviewers in the revised version of the manuscript. These errors were made during the generation of high-resolution images toward the end of the production process and have since been corrected online. The errors do not change the message of the figure, nor do they impact the overall conclusions of the article. The authors apologize for any inconvenience.Figure 3GHMCES Deficiency Leads to a Defect in CSR in the CH12 B Cell Line (original)View Large Image Figure ViewerDownload Hi-res image Download (PPT) HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B CellsShukla et al.Molecular CellDecember 2, 2019In BriefHMCES (5hmC binding, embryonic stem cell-specific protein), originally found to bind 5-hydroxymethylcytosine (5hmC), was recently reported to covalently crosslink to DNA at abasic sites. Shukla et al. show that HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells. Full-Text PDF Open Archive

Exploiting MYC-induced PARPness to target genomic instability in multiple myeloma.

Multiple myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability, which promotes disease progression and drug resistance. Since we previously demonstrated that LIG3-dependent repair is involved in the genomic instability, drug resistance and survival of MM cells, we here investigated the biological relevance of PARP1, a driver component of the alternative nonhomologous end joining pathway, in MM. We found a significant correlation between higher PARP1 mRNA expression and poor prognosis of MM patients. PARP1 knockdown or its pharmacological inhibition by olaparib impaired MM cell viability in vitro and was effective against in vivo xenografts of human MM. Anti-proliferative effects induced by PARP1 inhibition were correlated with an increase of DNA double-strand breaks, activation of the DNA damage response and apoptosis. Importantly, by comparing a gene expression signature of PARP-inhibitor sensitivity to our plasma cell dyscrasia gene expression profiling, we identified a subset of MM patients who could benefit from PARP inhibitors. In particular, gene set enrichment analysis suggested that high MYC expression correlates with sensitivity to PARP inhibitors in MM. Indeed, we identified MYC as a promoter of PARP1-mediated repair in MM and, consistently, we demonstrated that cytotoxic effects induced by PARP inhibition are mostly detectable in MYC-proficient MM cells. Taken together, our findings indicate that MYC-driven MM cells are addicted to PARP1 alternative non-homologous end joining repair, which therefore represents a druggable target in this still incurable disease.

DSBR-Seq Reveals Differential Roles of BRCA1 and BRCA2 in Alternative End Joining and Single Strand Templated Repair

Double strand break (DSB) repair mainly occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). We present an assay system that enables simultaneous measurement of all three pathways using Cas9-generated DSBs and next generation sequencing to profile and quantify pathway choice. The assay system has provided several insights. First, absence of the key Alt-EJ factor Pol q only abrogates ~50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. These fundamental differences between BRCA1 and BRCA2 deficiency have implications for therapeutic targeting of HR-deficient cancers. This assay can be used in any system which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

XRCC1 deficient triple negative breast cancers are sensitive to ATR, ATM and Wee1 inhibitor either alone or in combination with olaparib.

Background:PARP inhibitor (PARPi) monotherapy is a new strategy in BRCA germ-line deficient triple negative breast cancer (TNBC). However, not all patients respond, and the development of resistance limits the use of PARPi monotherapy. Therefore, the development of alternative synthetic lethality strategy, including in sporadic TNBC, is a priority. XRCC1, a key player in base excision repair, single strand break repair, nucleotide excision repair and alternative non-homologous end joining, interacts with PARP1 and coordinates DNA repair. ATR, ATM and Wee1 have essential roles in DNA repair and cell cycle regulation.Methods:Highly selective inhibitors of ATR (AZD6738), ATM (AZ31) and Wee1 (AZD1775) either alone or in combination with olaparib were tested for synthetic lethality in XRCC1 deficient TNBC or HeLa cells. Clinicopathological significance of ATR, ATM or Wee1 co-expression in XRCC1 proficient or deficient tumours was evaluated in a large cohort of 1650 human breast cancers.Results:ATR (AZD6738), ATM (AZ31) or Wee1 (AZD1775) monotherapy was selectively toxic in XRCC1 deficient cells. Selective synergistic toxicity was evident when olaparib was combined with AZD6738, AZ31 or AZD1775. The most potent synergistic interaction was evident with the AZD6738 and olaparib combination therapy. In clinical cohorts, ATR, ATM or Wee1 overexpression in XRCC1 deficient breast cancer was associated with poor outcomes.Conclusion:XRCC1 stratified DNA repair targeted combinatorial approach is feasible and warrants further clinical evaluation in breast cancer.

Abstract I14: Polymerase theta synthetic lethal interaction in homologous recombination-deficient pancreatic ductal adenocarcinoma

Abstract Recent genomic characterization of PDA reveals that between 20-25 % of PDA harbor recurrent mutations in genes, including BRCA1/2, PALB2, and ATM, which are critical for homologous recombination (HR), an important form of DNA repair. In many patients, these may be germline mutations. This subgroup of PDAs, termed HR-deficient PDA, has emerged as a defined biological entity associated with increased chemoresistance and a more aggressive disease course. The defects in HR observed in these tumors impart cells with a specific vulnerability to PARP inhibitors and platinum-containing therapy. Still, as observed in the case of many other targeted therapies, only a fraction of HR-defective patient tumors respond to PARP inhibition. More so, many patients that initially respond eventually often develop resistance and progress. Therefore, novel therapies which can be effective against HR-defective PDA, alone or in combination with PARP inhibitors or other combinatorial regimens, are urgently needed. We have recently determined that inactivation of the HR pathway is associated with overexpression of polymerase theta (PolO–, also known as POLQ) in PDA. POLQ is a key enzyme that regulates an alternative pathway of DNA repair, known as the alternative non-homologous end-joining (Alt-NHEJ) pathway. In the setting of defective HR, Alt-NHEJ becomes a critical pathway responsible for the repair of DNA breaks and POLQ inhibition in HR-defective tumor cells demonstrates a synthetic lethality phenotype, not observed in cells with intact HR. Furthermore, POLQ knockdown significantly upregulated the cGAS-STING pathway in HR-deficient PDAs linking the DNA damage response to the immune response. Overall, targeting POLQ may represent a novel and in a valuable therapeutic strategy in HR-defective pancreatic cancer, as POLQ inhibitors are currently in development for clinical use. Citation Format: Annie Wang, Andrea Zamperone, Mohammad Sohail, Lidong Wang, Fiyinfolu Balogun, Jiufeng Li, Ende Zhao, Daniel Diolaiti, Agnel Sfeir, Diane M. Simeone. Polymerase theta synthetic lethal interaction in homologous recombination-deficient pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I14.

Nonspecific Binding of RNA to PARP1 and PARP2 Does Not Lead to Catalytic Activation.

Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2, respectively), upon binding damaged DNA, become activated to add long chains of poly(ADP-ribose) (PAR) to themselves and other nuclear proteins. This activation is an essential part of the DNA damage response. The PAR modifications recruit the DNA repair machinery to sites of DNA damage and result in base excision and single-strand break repair, homologous recombination, nucleotide excision repair, and alternative nonhomologous end joining. More recently, both PARP1 and PARP2 have been shown to bind to or be activated by RNA, a property that could interfere with the function of PARP1 and PARP2 in the response to DNA damage or lead to necrosis by depletion of cellular NAD+. We have quantitatively evaluated the in vitro binding of a variety of RNAs to PARP1 and PARP2 and queried the ability of these RNAs to switch on enzymatic activity. We find that while both proteins bind RNAs without specificity toward sequence or structure, their interaction with RNA does not lead to auto-PARylation. Thus, although PARP1 and PARP2 are promiscuous with respect to activation by DNA, they both demonstrate exquisite selectivity against activation by RNA.

HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells.

HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.