Nonhomologous End Joining Efficiency Research Articles

Exocyclic and Linker Editing of Lys63-linked Ubiquitin Chains Modulators Specifically Inhibits Non-homologous End-joining Repair.

Despite the great advances in discovering cyclic peptides against protein targets, their reduced aqueous solubility, cell permeability, and activity of the cyclic peptide restrict its utilization in advanced biological research and therapeutic applications. Here we report on a novel approach of structural alternation of the exocyclic and linker parts that led to a new derivative with significantly improved cell activity allowing us to dissect its mode of action in detail. We have identified an effective cyclic peptide (CP7) that induces approximately a 9-fold increase in DNA damage accumulation and a remarkable increase in apoptotic cancer cell death compared to the reported molecule. Notably, treating cells with CP7 leads to a dramatic decrease in the efficiency of non-homologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs), which is accompanied by an increase in homologous recombination (HR) repair. Interestingly, treating BRCA1-deficient cells with CP7 restores HR integrity, which is accompanied by increased resistance to CP7. Additionally, CP7 treatment increases the sensitivity of cancer cells to ionizing radiation. Collectively, our findings demonstrate that CP7 is a selective inhibitor of NHEJ, offering a potential strategy to enhance the effectiveness of radiation therapy.

Low-dose ionizing radiation-induced RET/PTC1 rearrangement via the non-homologous end joining pathway to drive thyroid cancer.

Thyroid cancer incidence increases worldwide annually, primarily due to factors such as ionizing radiation (IR), iodine intake, and genetics. Papillary carcinoma of the thyroid (PTC) accounts for about 80% of thyroid cancer cases. RET/PTC1 (coiled-coil domain containing 6 [CCDC6]-rearranged during transfection) rearrangement is a distinctive feature in over 70% of thyroid cancers who exposed to low doses of IR in Chernobyl and Hiroshima‒Nagasaki atomic bombings. This study aims to elucidate mechanism between RET/PTC1 rearrangement and IR in PTC. N-thy-ori-3-1 cells were subjected to varying doses of IR (2/1/0.5/0.2/0.1/0.05Gy) of IR at different days, and result showed low-dose IR-induced RET/PTC1 rearrangement in a dose-dependent manner. RET/PTC1 has been observed to promote PTC both in vivo and in vitro. To delineate the role of different DNA repair pathways, SCR7, RI-1, and Olaparib were employed to inhibit non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ), respectively. Notably, inhibiting NHEJ enhanced HR repair efficiency and reduced IR-induced RET/PTC1 rearrangement. Conversely, inhibiting HR increased NHEJ repair efficiency and subsequent RET/PTC1 rearrangement. The MMEJ did not show a markable role in this progress. Additionally, inhibiting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) decreased the efficiency of NHEJ and thus reduced IR-induced RET/PTC1 rearrangement. To conclude, the data suggest that NHEJ, rather than HR or MMEJ, is the critical cause of IR-induced RET/PTC1 rearrangement. Targeting DNA-PKcs to inhibit the NHEJ has emerged as a promising therapeutic strategy for addressing IR-induced RET/PTC1 rearrangement in PTC.

Coevolution of non-homologous end joining efficiency and encephalization.

Double-strand breaks (DSB), the most difficult to repair DNA damage, are mainly repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). Previous studies seem to indicate that primates, and particularly humans, have a better NHEJ system. A distinctive feature of the primate lineage (beside longevity) is encephalization, i.e., the expansion of the brain relative to body mass (BM). Using existing transcriptome data from 34 mammalian species, we investigated the possible correlations between the expression of genes involved in NHEJ and encephalization, BM, and longevity. The same was done also for genes involved in the HR pathway. We found that, while HR gene expression is better correlated with longevity, NHEJ gene expression is strongly (and better) correlated with encephalization. Since the brain is composed of postmitotic cells, DSB repair should be mainly performed by NHEJ in this organ. Therefore, we interpret the correlation we found as an indication that NHEJ efficiency coevolved with encephalization.

TRIM33 loss in multiple myeloma is associated with genomic instability and sensitivity to PARP inhibitors

Deletions of chromosome 1p (del(1p)) are a recurrent genomic aberration associated with poor outcome in Multiple myeloma (MM.) TRIM33, an E3 ligase and transcriptional co-repressor, is located within a commonly deleted region at 1p13.2. TRIM33 is reported to play a role in the regulation of mitosis and PARP-dependent DNA damage response (DDR), both of which are important for maintenance of genome stability. Here, we demonstrate that MM patients with loss of TRIM33 exhibit increased chromosomal instability and poor outcome. Through knockdown studies, we show that TRIM33 loss induces a DDR defect, leading to accumulation of DNA double strand breaks (DSBs) and slower DNA repair kinetics, along with reduced efficiency of non-homologous end joining (NHEJ). Furthermore, TRIM33 loss results in dysregulated ubiquitination of ALC1, an important regulator of response to PARP inhibition. We show that TRIM33 knockdown sensitizes MM cells to the PARP inhibitor Olaparib, and this is synergistic with the standard of care therapy bortezomib, even in co-culture with bone marrow stromal cells (BMSCs). These findings suggest that TRIM33 loss contributes to the pathogenesis of high-risk MM and that this may be therapeutically exploited through the use of PARP inhibitors.

Abstract 4540: SY-7021, a novel DNA-PK inhibitor, exhibits significant anti-tumor activity in vitro and in vivo

Abstract Cancer therapies such as ionizing radiation or topoisomerase II inhibitors (for example, doxorubicin) induce DNA double-strand breaks (DSBs), which can subsequently be repaired by homologous recombination or non-homologous end joining (NHEJ). DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine protein kinase complex, is a pivotal component of the NHEJ process that maintain genome integrity. The critical role of DNA-PK in DNA damage response (DDR) and the dysregulated DNA-PK expression in cancer make it an intriguing therapeutic target for cancer, especially when combined with DSBs agents. Here, we identified a highly potent and selective DNA-PK inhibitor, SY-7021, with an IC50 value of 0.242 nM on DNA-PK kinase and high selectivity on ATM and ATR (greater than 400-fold) in biochemical assays. In a reporter assay for NHEJ repair, SY-7021 was able to dose-dependently reduce cellular NHEJ efficiency. Functionally, SY-7021 inhibited cell proliferation in multiple cancer cell lines alone or combined with doxorubicin. Basically, combination of SY-7021 and doxorubicin induced significant G2/M phase arrest and cell apoptosis, as well as enhanced the phosphorylation on Ser139 of γH2AX, Tyrosine 68 of CHK2 and Serine15 of p53 in MDA-MB-468 cells. In a subcutaneous NCI-H1703 xenograft tumor model, SY-7021 administered orally twice daily achieved dose-dependent tumor growth inhibition in vivo, with a tumor growth inhibition (TGI) of 105.6% at 60 mg/kg and no significant weight loss was observed. In addition, SY-7021 demonstrates good PK properties and acceptable safety profiles in in vivo studies. Collectively, SY-7021, a potent and selective DNA-PK inhibitor, shows significant inhibition on tumor growth in in vitro and in vivo studies, providing a rationale treatment for multiple tumors in monotherapy or in combination with other agents. Citation Format: Kai Zhang, Zhihua Liu, Yuhao Gao, Chang Lu, Shikang Cheng, Xijie Liu, Hong Luo, Yinghui Sun. SY-7021, a novel DNA-PK inhibitor, exhibits significant anti-tumor activity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4540.

Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair.

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells using GFP Based Reporter Systems.

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.

Double-strand DNA break repair: molecular mechanisms and therapeutic targets.

Double-strand break (DSB), a significant DNA damage brought on by ionizing radiation, acts as an initiating signal in tumor radiotherapy, causing cancer cells death. The two primary pathways for DNA DSB repair in mammalian cells are nonhomologous end joining (NHEJ) and homologous recombination (HR), which cooperate and compete with one another to achieve effective repair. The DSB repair mechanism depends on numerous regulatory variables. DSB recognition and the recruitment of DNA repair components, for instance, depend on the MRE11-RAD50-NBS1 (MRN) complex and the Ku70/80 heterodimer/DNA-PKcs (DNA-PK) complex, whose control is crucial in determining the DSB repair pathway choice and efficiency of HR and NHEJ. In-depth elucidation on the DSB repair pathway's molecular mechanisms has greatly facilitated for creation of repair proteins or pathways-specific inhibitors to advance precise cancer therapy and boost the effectiveness of cancer radiotherapy. The architectures, roles, molecular processes, and inhibitors of significant target proteins in the DSB repair pathways are reviewed in this article. The strategy and application in cancer therapy are also discussed based on the advancement of inhibitors targeted DSB damage response and repair proteins.

Effect of Helicobacter pylori on non-homologous end joining-mediated repair of proximal DNA double-strand breaks in GCV6 cells

Infection with Helicobacter pylori is the strongest known risk factor for gastric cancer, which is one of the leading causes of cancer-related mortality worldwide. H. pylori can contribute to carcinogenesis by inducing the genomic instability of infected cells through increasing accumulation of DNA double-stranded breaks (DSBs) and deregulating DSB repair systems. However, the mechanism of this phenomenon is still being elucidated. This study aims to investigate the impact of H. pylori on the efficacy of non-homologous end joining (NHEJ)-mediated repair of DSB. In this study, we used a human fibroblast cell line bearing a single copy of an NHEJ-reporter substrate stably inserted into the genome, which provides a quantitative measurement of NHEJ. Our results indicated the potential abilities of H. pylori strains to alter the NHEJ-mediated repair of proximal DSB in infected cells. In addition, we found an association between the alteration in the NHEJ efficiency, and the inflammation responses of infected cells caused by H. pylori.

Chloroquine-Induced DNA Damage Synergizes with Nonhomologous End Joining Inhibition to Cause Ovarian Cancer Cell Cytotoxicity.

Ovarian cancer (OC) is the most lethal gynecological malignancy; therefore, more effective treatments are urgently needed. We recently reported that chloroquine (CQ) increased reactive oxygen species (ROS) in OC cell lines (OCCLs), causing DNA double-strand breaks (DSBs). Here, we analyzed whether these lesions are repaired by nonhomologous end joining (NHEJ), one of the main pathways involved in DSB repair, and if the combination of CQ with NHEJ inhibitors (NHEJi) could be effective against OC. We found that NHEJ inhibition increased the persistence of γH2AX foci after CQ-induced DNA damage, revealing an essential role of this pathway in the repair of the lesions. NHEJi decreased the proliferation of OCCLs and a strong in vitro synergistic effect on apoptosis induction was observed when combined with CQ. This effect was largely abolished by the antioxidant N-Acetyl-L-cysteine, revealing the critical role of ROS and DSB generation in CQ/NHEJi-induced lethality. We also found that the NHEJ efficiency in OCCLs was not affected by treatment with Panobinostat, a pan-histone deacetylase inhibitor that also synergizes with CQ in OCCLs by impairing homologous recombination. Accordingly, the triple combination of CQ-NHEJi-Panobinostat exerted a stronger in vitro synergistic effect. Altogether, our data suggest that the combination of these drugs could represent new therapeutic strategies against OC.

BRCA2 C-Terminal RAD51-Binding Domain Confers Resistance to DNA-Damaging Agents.

Breast cancer type 2 susceptibility (BRCA2) protein is crucial for initiating DNA damage repair after chemotherapy with DNA interstrand crosslinking agents or X-ray irradiation, which induces DNA double-strand breaks. BRCA2 contains a C-terminal RAD51-binding domain (CTRBD) that interacts with RAD51 oligomer-containing nucleofilaments. In this study, we investigated CTRBD expression in cells exposed to X-ray irradiation and mitomycin C treatment. Surprisingly, BRCA2 CTRBD expression in HeLa cells increased their resistance to X-ray irradiation and mitomycin C. Under endogenous BRCA2 depletion using shRNA, the sensitivities of the BRCA2-depleted cells with and without the CTRBD did not significantly differ. Thus, the resistance to X-ray irradiation conferred by an exogenous CTRBD required endogenous BRCA2 expression. BRCA2 CTRBD-expressing cells demonstrated effective RAD51 foci formation and increased homologous recombination efficiency, but not nonhomologous end-joining efficiency. To the best of our knowledge, our study is the first to report the ability of the BRCA2 functional domain to confer resistance to X-ray irradiation and mitomycin C treatment by increased homologous recombination efficiency. Thus, this peptide may be useful for protecting cells against X-ray irradiation or chemotherapeutic agents.

The Choice of a Donor Molecule in Genome Editing Experiments in Animal Cells

Genome editing is a powerful tool that allows study of the properties of genes or changes to be made to the genetic sequence. Programmable nucleases that can induce double-strand breaks in the genomic sequence of interest have been developed over the past few decades. After initiation of a double-strand break (DSB) in DNA, the DSB can be repaired by the NHEJ (non-homologous end joining), which leads to various errors and gene knockout. Other repair options, HDR (homology directed repair) or SSTR (single-strand template repair), allow researchers to make desired changes in the gene. HDR occurs in the presence of a donor template, in natural conditions the donor template is a sister chromatid. The efficiency of HDR and SSTR is significantly lower than the efficiency of NHEJ in genome editing. Double-stranded, single-stranded and long single-stranded DNAs are used to increase efficiency and to make desired changes in genomic DNA. In this review, we discuss donor molecules that are used for DSB repair using HDR or SSTR during genome editing, their application, and modifications to increase the efficiency of HDR and SSTR.

WASH interacts with Ku to regulate DNA double-stranded break repair

SummaryThe Wiskott-Aldrich syndrome protein and SCAR homolog (WASH), an actin nucleation-promoting factor, is present in the nucleus where it regulates gene transcription and maintains nuclear organization. Here, we show that WASH interacts with core non-homologous end-joining (NHEJ) factors including Ku70/Ku80 and DNA-PKcs, and Ku70/Ku80 is involved in the recruitment of WASH to the sites of DNA double-stranded break (DSB). WASH depletion leads to increased cell sensitivity and impaired DNA repair capacity in response to etoposide-induced DSBs and reduces NHEJ efficiency. Mechanistically, we show that loss of WASH inhibits the phosphorylation of DNA-PKcs, H2AX, and KAP1 after DSB induction and reduces chromatin relaxation and the recruitment of several downstream NHEJ factors to DSBs. Moreover, WASH role in DSB repair depends on its conserved C-terminal VCA domain and Arp2/3 activation. Our findings reveal a function and mechanistic insight for WASH in DNA DSB repair by the NHEJ pathway.

CDK4/6 Inhibition and Radiation as a Treatment Strategy to Improve Local Disease Control in Breast Cancers With Poor Prognoses

Locoregional control remains an issue in women with multi-node positive estrogen receptor-positive (ER+) breast cancer and many women with triple-negative breast cancer (TNBC). There is growing evidence that CDK4/6 inhibition (CDK4/6i) sensitizes breast cancer cells to ionizing radiation (RT) by suppressing the DNA damage response, but the role of RB is currently unclear. We sought to understand the role of RB and the implications of RB loss in ER+ and TNBC.Clonogenic survival assays were used to calculate radiosensitivity and calculate enhancement ratios (rER). Cellular viability was quantified 72 hours after drug addition to calculate half-maximal inhibitory concentrations (IC50s). Phospho- and total RB expression levels were assessed by immunoblotting. DNA repair was assessed with γH2AX and RAD51 immunofluorescence. GFP-based plasmid reporter systems were used to assess homologous recombination (HR) and non-homologous end joining (NHEJ) competency. Isogenic RB1 knockout cells were generated with CRISPR-Cas9, and siRNA was used for transient RB1 knockdown. G1 cell cycle arrest was quantified using propidium iodide-based flow cytometry. In vivo efficacy of CDK4/6 inhibition + RT was assessed using MDA-MB-231 xenografts.Four TNBC cell lines with intact RB expression were radiosensitized (rER 1.08 - 2.22) after CDK4/6i with palbociclib, ribociclib, or abemaciclib, but two RB null TNBC models were not radiosensitized with combination treatment (rER: 0.84 - 1.00). In ER+ and TNBC cell lines, response to CDK4/6i + RT was accurately predicted by the presence or absence of RB protein, and higher RB expression led to increased radiosensitization of breast cancer cell lines at lower doses. In addition, pRB expression decreased in wild type TNBC cell lines treated with CDK4/6i + RT. HR was suppressed with CDK4/6i in RB wild type - but not mutant - cell lines. NHEJ efficiency in RB wild type TNBC was unchanged with CDK4/6i. Genetic knockdown of RB1 led to the loss of radiosensitization in ER+ and TNBC cell lines (rER: 0.84 - 1.13) as well as a decrease in sensitivity to CDK4/6i monotherapy. Parental and Cas9 control TNBC cells arrested in G1 24 hours after CDK4/6i and remained arrested at 48 hours, but RB1 CRISPR TNBC cells did not arrest after drug treatment. In a TNBC xenograft model with wild type RB expression (MDA-MB-231), CDK4/6i + RT led to significant radiosensitization in vivo and delayed time to tumor doubling.RB loss mitigates CDK4/6 inhibitor-mediated radiosensitization of both ER+ and TNBC cells. Loss of RB expression prevents CDK4/6i-mediated radiosensitization, suggesting that RB expression may be a valuable clinical biomarker to predict efficacy of CDK4/6i + RT in future clinical trials. Our data also provide the preclinical rationale for CDK4/6i with RT not just in ER+ BC, but also in women with TNBC that express RB.

The conserved Tpk1 regulates non-homologous end joining double-strand break repair by phosphorylation of Nej1, a homolog of the human XLF.

The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine–threonine kinase, encompassing three catalytic (Tpk1–3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the tpk1 catalytic subunit reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ factor (nej1), co-function in the same pathway, and parallel to the NHEJ factor yku80. Chromatin immunoprecipitation and resection data suggest that tpk1 deletion influences repair protein recruitments and DNA resection. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair and nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.

Short-term CDK4/6 Inhibition Radiosensitizes Estrogen Receptor-Positive Breast Cancers.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have improved progression-free survival for metastatic, estrogen receptor-positive (ER+) breast cancers, but their role in the nonmetastatic setting remains unclear. We sought to understand the effects of CDK4/6 inhibition (CDK4/6i) and radiotherapy in multiple preclinical breast cancer models. Transcriptomic and proteomic analyses were used to identify significantly altered pathways after CDK4/6i. Clonogenic assays were used to quantify the radiotherapy enhancement ratio (rER). DNA damage was quantified using γH2AX staining and the neutral comet assay. DNA repair was assessed using RAD51 foci formation and nonhomologous end joining (NHEJ) reporter assays. Orthotopic xenografts were used to assess the efficacy of combination therapy. Palbociclib significantly radiosensitized multiple ER+ cell lines at low nanomolar, sub IC50 concentrations (rER: 1.21-1.52) and led to a decrease in the surviving fraction of cells at 2 Gy (P < 0.001). Similar results were observed in ribociclib-treated (rER: 1.08-1.68) and abemaciclib-treated (rER: 1.19-2.05) cells. Combination treatment decreased RAD51 foci formation (P < 0.001), leading to a suppression of homologous recombination activity, but did not affect NHEJ efficiency (P > 0.05). Immortalized breast epithelial cells and cells with acquired resistance to CDK4/6i did not demonstrate radiosensitization (rER: 0.94-1.11) or changes in RAD51 foci. In xenograft models, concurrent palbociclib and radiotherapy led to a significant decrease in tumor growth. These studies provide preclinical rationale to test CDK4/6i and radiotherapy in women with locally advanced ER+ breast cancer at high risk for locoregional recurrence.

INDEL detection, the 'Achilles heel' of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels.

Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

End Processing Factor APLF Promotes NHEJ Efficiency and Contributes to TMZ- and Ionizing Radiation-Resistance in Glioblastoma Cells.

PurposeGlioblastoma (GBM) is the most commonly diagnosed primary brain tumor in adults. Despite a variety of advances in the understanding of GBM cancer biology during recent decades, very few of them were applied into treatment, and the survival rate of GBM patients has not been improved majorly due to the low chemosensitivity to temozolomide (TMZ) or low radiosensitivity. Therefore, it is urgent to elucidate mechanisms of TMZ- and IR-resistance and develop novel therapeutic strategies to improve GBM treatment.MethodsTMZ- and IR-resistant cell lines were acquired by continuous exposing parental GBM cells to TMZ or IR for 3 months. Cell viability was determined by using Sulforhodamine B (SRB) assay. Protein and mRNA expression were examined by Western blotting assay and quantitative polymerase chain reaction (qPCR) assay, respectively. Homologous recombination (HR) and nonhomologous end joining (NHEJ) efficiency were measured by HR and NHEJ reporter assay. Cell apoptosis was determined by Caspase3/7 activity. Autophagy was analyzed using CYTO-ID® Autophagy detection kit. Tumor growth was examined by U87 xenograft mice model.ResultsDNA repair efficiency of non-homologous end joining (NHEJ) pathway is significantly increased in TMZ- and IR-resistant GBM cells. Importantly, APLF, which is one of the DNA end processing factors in NHEJ, is upregulated in TMZ- and IR-resistant GBM cells and patients. APLF deficiency significantly decreases NHEJ efficiency and improves cell sensitivity to TMZ and IR both in vitro and in vivo.ConclusionOur study provides evidence for APLF serving as a promising, novel target in GBM chemo- and radio-therapy.

USP14 Regulates DNA Damage Response and Is a Target for Radiosensitization in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) represents ~85% of the lung cancer cases. Despite recent advances in NSCLC treatment, the five-year survival rate is still around 23%. Radiotherapy is indicated in the treatment of both early and advanced stage NSCLC; however, treatment response in patients is heterogeneous. Thus, identification of new and more effective treatment combinations is warranted. We have identified Ubiquitin-specific protease 14 (USP14) s a regulator of major double-strand break (DSB) repair pathways in response to ionizing radiation (IR) by its impact on both non-homologous end joining (NHEJ) and homologous recombination (HR) in NSCLC. USP14 is a proteasomal deubiquitinase. IR treatment increases levels and DSB recruitment of USP14 in NSCLC cell lines. Genetic knockdown, using shUSP14 expression or pharmacological inhibition of USP14, using IU1, increases radiosensitization in NSCLC cell lines, as determined by a clonogenic survival assay. Moreover, shUSP14-expressing NSCLC cells show increased NHEJ efficiency, as indicated by chromatin recruitment of key NHEJ proteins, NHEJ reporter assay, and increased IR-induced foci formation by 53BP1 and pS2056-DNA-PKcs. Conversely, shUSP14-expressing NSCLC cells show decreased RPA32 and BRCA1 foci formation, suggesting HR-deficiency. These findings identify USP14 as an important determinant of DSB repair in response to radiotherapy and a promising target for NSCLC radiosensitization.

Abstract 6280: CDK4/6 inhibitor-mediatated radiosensitization of estrogen receptor positive breast cancer

Abstract Purpose: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors like palbociclib, ribociclib, and abemaciclib have improved progression free survival in patients with metastatic, estrogen receptor positive (ER+) breast cancer, acquired resistance to these drugs limits their efficacy. Despite promising new studies defining the utility of CDK4/6 inhibitors in the upfront, non-metastatic setting, there is limited data available on the effects of concurrent CDK4/6 inhibition and radiation (RT). Methods: Transcriptomic and proteomic expression data was used to quantify changes in RNA and protein expression in ER+ breast cancer cell lines (MCF-7, T47D) after short term (16 hour) CDK4/6 inhibition. Proliferation assays were used to determine the half-maximal inhibitory concentration (IC50) of palbociclib, ribociclib, and abemaciclib. Clonogenic survival assays were performed to calculate the radiation enhancement ratio (rER) and the surviving fraction at 2 Gy for each treatment. Homologous recombination (HR) proficiency was assessed using RAD51 and γH2AX foci formation and a pYFP reporter was used to assess non-homologous end joining (NHEJ) efficiency. Western blots were used to quantify protein expression. MCF-7 xenografts were used to study the efficacy of combination (palbociclib + RT) therapy in vivo. MCF-7 and T47D cell lines with acquired resistance to CDK4/6 inhibition (IC50 &amp;gt;1uM) were used for comparison in all assays. Results: Transcriptomic and proteomic analyses identified changes in expression of DNA damage response mediators and cell cycle machinery with short term CDK4/6 inhibition. Palbociclib significantly radiosensitized ER+ cell lines at concentrations at or below the IC50 value in clonogenic survival assays (MCF-7 rER: 1.22-1.52, T47D rER: 1.23-1.50) and led to a decrease in the surviving fraction of cells at 2 Gy (p &amp;lt; 0.001). Similar results were observed in ribociclib- (rER: 1.08 - 1.68) and abemaciclib-treated (rER: 1.19 - 2.05) cells. MCF-7 and T47D cells treated with CDK4/6 inhibition and RT showed a decrease in RAD51 foci formation, suggesting a decrease in HR efficiency (p &amp;lt; 0.001). However, CDK4/6 inhibition did not affect NHEJ efficiency (p &amp;gt; 0.05). CDK4/6 inhibition + RT led to a decrease in expression of protein expression of HR meditators like p-CHK1 but did not affect phosphorylation of NHEJ proteins like pKu80/pKu70. Cells with acquired resistance to CDK4/6 inhibition did not demonstrate radiosensitization (MCF-7 rER: 0.93 - 1.03, T47D rER: 0.96 - 1.11) or changes in RAD51 foci formation with combination treatment. Conclusions: Our data suggests that CDK4/6 inhibitor-mediated radiosensitization may be effective in ER+ breast cancers prior to the development of CDK4/6 inhibitor resistance. These studies provide preclinical rationale to test CDK4/6 inhibition + RT in women with locally-advanced ER+ breast cancer at high risk for locoregional recurrence. Citation Format: Andrea M. Pesch, Nicole Hirsh, Benjamin C. Chandler, Anna R. Michmerhuizen, Cassandra L. Ritter, Marlie Androsiglio, Kari Wilder-Romans, Meilan Liu, Christina L. Gersch, Jose M. Larios, James M. Rae, Corey W. Speers. CDK4/6 inhibitor-mediatated radiosensitization of estrogen receptor positive breast cancer [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 6280.