End Joining DNA Repair Pathway Research Articles

Negative feedback loop in the activation of non-homologous end joining DNA repair pathway in Helicobacter pylori infected patients with gastritis

This study aimed to investigate the activation of error-prone DNA repair pathway in response to Helicobacter pylori infection. Relative changes in the expression levels of genes involved in the non-homologous end-joining pathway (NHEJ) in H. pylori-infected (Cases) and non-infected patients (Controls) with chronic gastritis were measured. A significant increase in the relative expression level of TP53, and significant decrease in the relative transcription of lncRNA LINP1 and XRCC5 were detected in the case group. The transcription of Lig4 and XRCC6 was increased in the case group, which was not statistically significant. The Spearman’s Correlation Coefficient analysis showed a significant positive-correlation between the transcriptional levels of LINP1 and XRCC4/XRCC5/Lig4, and between XRCC5 and TP53/Lig4 both in the case and control groups. Moreover, a significant positive correlation between LinP1 and XRCC6 in the case, and a significant positive correlation between XRCC4 and Lig4, and a negative correlation between TP53 and LinP1/XRCC4/XRCC5 in the control group was detected. Although a relative difference was detected in transcriptional levels of the NHEJ gene mediators, downregulation of LinP1 in H. pylori-infected patients proposed the activation of a negative feedback loop, which may interfere with the NHEJ activity at the early stages of gastritis.

Characterization of a KU70-disrupted strain of the mannosylerythritol lipid-producing yeast Pseudozyma tsukubaensis constructed by a marker recycling system.

The basidiomycetous yeast Pseudozyma tsukubaensis is known as an industrial mannosylerythritol lipid producer. In this study, the PtURA5 marker gene was deleted by homologous recombination. Using the PtURA5-deleted mutant as a host strain, we obtained a derivative disrupted for the PtKU70 gene, a putative ortholog of the KU70 gene encoding a protein involved in the nonhomologous end-joining pathway of DNA repair. Subsequently, the introduced PtURA5 gene was re-deleted by marker recycling. These results demonstrated that the PtURA5 gene can be used as a recyclable marker gene. Although the frequency of homologous recombination has been shown to be increased by KU70 disruption in other fungi, the PtKU70-disrupted strain of P. tsukubaensis did not demonstrate an elevated frequency of homologous recombination. Furthermore, the PtKU70-disrupted strain did not show increased susceptibility to bleomycin. These results suggested that the function of this KU70 ortholog in P. tsukubaensis is distinct from that in other fungi.

Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks

BackgroundCRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks.ResultsUsing long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells.ConclusionsOur findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

Abstract 7128: Novel Ku-DNA binding inhibitors impact on the cellular and in vivo DNA damage response to radio- and radiomimetic-therapy

Abstract DNA-PK, the DNA-dependent protein kinase, is a validated target for cancer therapeutics that plays a central role in the non-homologous end joining (NHEJ) DNA repair pathway and the DNA damage response (DDR). NHEJ is a key mechanism of DNA double-strand breaks (DSB) repair, especially those induced by ionizing radiation (IR). Ku 70/80 heterodimer is required as initiator in this process, acting as an essential DNA-binding component of DNA-PK that senses DNA damage. Blocking the DNA-PK kinase activity in combination with DSB inducing agents has been widely used as a therapeutic strategy to drive clinical efficacy of radiation therapy for cancer treatment. We have reported a unique approach of DNA-PK inhibition by developing Ku-DNA binding inhibitors (Ku-DBi’s), small molecule inhibitors that target the interaction between Ku70/80 and DNA. Ku-DBi’s demonstrated a direct interaction with Ku70/80, a potent inhibition of Ku-DNA binding and DNA-PK catalytic activity, and in vitro and cellular NHEJ inhibitory activity. This results in the potentiation of non-small cell lung cancer (NSCLC) cellular sensitivity to DSB generating therapies, as a function of inhibiting DNA-PKcs autophosphorylation and dysregulation of signaling to the DDR. In this study, we have expanded our structure activity relationship analyses to focus on optimizing selectivity, cellular uptake and Ku inhibitory activity. The new series of Ku-DBi’s include oxindole derivatives of the X80 core structure that achieve these goals. These novel compounds display increased cellular uptake while retaining potent Ku inhibitory activity and enhanced cellular inhibition on DNA-DSB repair. Ku-DBi’s optimization has enabled in vivo analyses of Ku-DBi - ionizing radiation combination therapy in human xenograft models of non-small cell lung cancer. The impact of Ku-DBi treatment on therapeutic efficacy, toxicity, Ku/DNA-PK inhibition, and DDR signaling will be presented. These data represent a significant advance in the development of Ku-DNA binding inhibitors and their therapeutic intervention for cancer treatment. Citation Format: Pamela L. Mendoza-Munoz, Dineshsinha Chauhan, Navnath S. Gavande, Joseph R. Dynlacht, Joy E. Garrett, John J. Turchi. Novel Ku-DNA binding inhibitors impact on the cellular and in vivo DNA damage response to radio- and radiomimetic-therapy [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 7128.

The BAP1 nuclear deubiquitinase is involved in the nonhomologous end-joining pathway of double-strand DNA repair through interaction with DNA-PK.

BRCA1-associated protein 1 (BAP1) has emerged as a major tumor suppressor gene in diverse cancer types, notably in malignant pleural mesothelioma (DPM), and has also been identified as a germline cancer predisposition gene for DPM and other select cancers. However, its role in the response to DNA damage has remained unclear. Here, we show that BAP1 inactivation is associated with increased DNA damage both in Met-5A human mesothelial cells and human DPM cell lines. Through proteomic analyses, we identified PRKDC as an interaction partner of BAP1 protein complexes in DPM cells and 293 T human embryonic kidney cells. PRKDC encodes the catalytic subunit of DNA protein kinase (DNA-PKcs) which functions in the nonhomologous end-joining (NHEJ) pathway of DNA repair. Double-stranded DNA damage resulted in prominent nuclear expression of BAP1 in DPM cells and phosphorylation of BAP1 at serine 395. A plasmid-based NHEJ assay confirmed a significant effect of BAP1 knockdown on cellular NHEJ activity. Combination treatment with X-ray irradiation and gemcitabine (as a radiosensitizer) strongly suppressed the growth of BAP1-deficient cells. Our results suggest reciprocal positive interactions between BAP1 and DNA-PKcs, based on phosphorylation of BAP1 by the latter and deubiquitination of DNA-PKcs by BAP1. Thus, functional interaction of BAP1 with DNA-PKcs supports a role for BAP1 in NHEJ DNA repair and may provide the basis for new therapeutic strategies and new insights into its role as a tumor suppressor.

Abstract A027: Development of small molecule inhibitors of the Ku-DNA interaction: Impacts on NHEJ, DDR signaling, optimizing genome editing technologies, and therapeutic intervention for the treatment of cancer

Abstract DNA-PK, the DNA-dependent protein kinase is a validated target for cancer therapeutics that drives the DNA damage response (DDR) and plays a critical role in the non-homologous end joining (NHEJ) DNA repair pathway. NHEJ is responsible for the repair of DNA double strand breaks (DSB), particularly those induced by ionizing radiation (IR). The generation of DNA DSBs drives clinical efficacy of radiation therapy and numerous DNA damaging chemotherapeutic drugs used to treat cancer. Modulating the DSB repair pathways is an effective mechanism to increase clinical efficacy of IR and certain chemotherapeutic regimens. We have taken the novel approach to inhibit target the DDR and NHEJ by developing small molecule inhibitors of the DSB sensor Ku, and its interaction with DNA. The Ku heterodimer serves as initiator of the NHEJ pathway following DSB induction and is the key DNA-binding component of the DNA-PK. We have created an extensive library of Ku DNA binding inhibitors (Ku-DBis) and demonstrate a direct interaction with the Ku heterodimer and potent inhibition of Ku-DNA binding and DNA-PK catalytic activity. In vitro analysis of a series of Ku truncation mutants defines the necessary domains for Ku-DBi activity. Ku-DBi’s show potent inhibition of in vitro and cellular NHEJ and, as a result, potentiate cellular sensitivity to DSB-inducing agents. Enhancement of sensitivity is a function of inhibiting DNA-PKcs autophosphorylation and dysregulation of signaling to the DDR. Recent chemical optimization has identified independent pharmacophores that drive off-target cytotoxicity, direct Ku inhibitory potency, and enhance cellular uptake activity. These data served as the basis for creation of a novel series of Ku-DBi’s towards interrogating NHEJ and DDR signaling in vivo, optimizing genome editing technologies, and therapeutic intervention for the treatment of cancer. Citation Format: John J. Turchi, Pamela L. Mendoza-Munoz, Pamela S. VanderVere-Carozza, Navnath S. Gavande, Joseph R. Dynlacht, Joy E. Garrett, Dineshsinha Chauhan, Katherine S. Pawelczak. Development of small molecule inhibitors of the Ku-DNA interaction: Impacts on NHEJ, DDR signaling, optimizing genome editing technologies, and therapeutic intervention for the treatment of cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A027.

260 Insertion of the CFTR cDNA in the CFTR locus in human bronchial epithelial cells is improved by inhibiting the nonhomologous end joining DNA repair pathway

260 Insertion of the CFTR cDNA in the CFTR locus in human bronchial epithelial cells is improved by inhibiting the nonhomologous end joining DNA repair pathway

Trichoderma harzianum marker-free strain construction based on efficient CRISPR/Cas9 recyclable system: A helpful tool for the study of biological control agents

This article reports the first successful marker-free genetic engineering of Trichoderma harzianum, a widely studied biocontrol agent with great potential for use in sustainable agriculture. T. harzianum has been shown to control plant diseases, promote plant growth, and improve plant tolerance to abiotic stress. A specific CRISPR/Cas9 gene-editing system, based in self-replicative AMA1-plasmid, was used to generate T. harzianum mutant strains, targeting the albA (pks4) and ku70 genes, involved in melanin synthesis and non-homologous end joining DNA repair pathway, respectively. The resulting albA-deficient strain displayed a loss of pigmentation in their conidia, confirming phenotypically and genotypically the efficacy of the CRISPR/Cas9 system in generating targeted gene knockouts in T. harzianum. A marker-free Δku70 strain was also constructed, with the maintenance of its original fitness, providing a helpful tool for precise genetic engineering in T. harzianum, due to NHEJ-deficiency, able to be used in in vitro and in vivo (in planta) studies. The study highlights the potential of using CRISPR/Cas9 technology to facilitate the study of the molecular processes involved in the biocontrol and plant growth-promoting properties of T. harzianum.

Non-Homologous End-Joining Pathway Genotypes Significantly Associated with Nasopharyngeal Carcinoma Susceptibility.

Defects in the non-homologous end-joining (NHEJ) DNA repair pathway lead to genomic instability and carcinogenesis. However, the roles of individual NHEJ genes in nasopharyngeal carcinoma (NPC) etiology are not well-understood. The aim of this study was to assess the contribution of NHEJ genotypes, including XRCC4 (rs6869366, rs3734091, rs28360071, rs28360317, rs1805377), XRCC5 (rs828907, rs11685387, rs9288518), XRCC6 (rs5751129, rs2267437, rs132770, rs132774), XRCC7 rs7003908, and Ligase4 rs1805388, to NPC risk, with 208 NPC patients and 416 controls. Genotype-phenotype correlations were also investigated by measuring mRNA and protein expression in adjacent normal tissues and assessing the NHEJ repair capacity in blood lymphocytes from 43 NPC patients. The results showed significant differences in the distributions of variant genotypes at XRCC4 rs3734091, rs28360071, and XRCC6 rs2267437 between the cases and controls. The variant genotypes of these three polymorphisms were associated with significantly increased NPC risks. NPC patients with the risk genotypes at XRCC6 rs2267437 had significantly reduced expression levels of both mRNA and protein, as well as a lower NHEJ repair capacity, than those with the wild-type genotype. In conclusion, XRCC4 rs3734091, rs28360071, and XRCC6 rs2267437 in the NHEJ pathway were associated with NPC susceptibility. XRCC6 rs2267437 can modulate mRNA and protein expression and the NHEJ repair capacity.

Polymorphisms in the Nonhomologous End-joining DNA Repair Pathway are Associated with HPV Integration in Cervical Dysplasia.

HPV integration in premalignant lesions and is thought to be an important driver of carcinogenesis. However, it is unclear what factors promote integration. The use of targeted genotyping among women presenting with cervical dysplasia has the potential to be an effective tool in assessing the likelihood of progression to cancer.

DNA Ligase 4 Contributes to Cell Proliferation against DNA-PK Inhibition in MYCN-Amplified Neuroblastoma IMR32 Cells.

Identifying the vulnerability of altered DNA repair machinery that displays synthetic lethality with MYCN amplification is a therapeutic rationale in unfavourable neuroblastoma. However, none of the inhibitors for DNA repair proteins are established as standard therapy in neuroblastoma. Here, we investigated whether DNA-PK inhibitor (DNA-PKi) could inhibit the proliferation of spheroids derived from neuroblastomas of MYCN transgenic mice and MYCN-amplified neuroblastoma cell lines. DNA-PKi exhibited an inhibitory effect on the proliferation of MYCN-driven neuroblastoma spheroids, whereas variable sensitivity was observed in those cell lines. Among them, the accelerated proliferation of IMR32 cells was dependent on DNA ligase 4 (LIG4), which comprises the canonical non-homologous end-joining pathway of DNA repair. Notably, LIG4 was identified as one of the worst prognostic factors in patients with MYCN-amplified neuroblastomas. It may play complementary roles in DNA-PK deficiency, suggesting the therapeutic potential of LIG4 inhibition in combination with DNA-PKi for MYCN-amplified neuroblastomas to overcome resistance to multimodal therapy.

Abstract 875: Breast cancer bioinformatics: Untangling the roles of nucleolin and BRCA1 in dysregulated DNA repair

Abstract Proteins that enable cells to sense and repair DNA damage are essential for maintaining the stability of genomes across cell divisions; dysregulation of these processes is an established hallmark of tumorigenesis. Nucleolin (NCL), a major RNA binding protein (RBP) and a caretaker tumor suppressor protein BRCA1 (the breast & ovarian cancer susceptibility gene) colocalize in breast cancer. Both NCL and BRCA1 have defined roles in homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways which are often dysregulated in breast cancer. BRCA1, along with a complex molecular network of signaling proteins gets recruited to damaged replication forks to initiate the HR pathway where NCL function is also implicated. However, how NCL collaborates with BRCA1 to orchestrate the complex mechanism of DNA repair under stress conditions remains unknown. To address this gap in knowledge, we have applied in silico approaches to uncover the key players which drive the molecular interactions of NCL and BRCA1 at the damaged sites. We utilized the NIH PPI, IntAct, STRING, BioGRID, GeneMania, PrePPI, and Mentha databases to derive an overlapping interactome. In this study we present a comprehensive interactome analysis and 47 proteins identified that interact with both NCL and BRCA1. As expected, majority of these common interactors participate in DNA damage damage response along with several that are implicated in regulating the gene expression via chromatin remodeling/RNA binding or controlling cell proliferation. We classified this interactome into 5 major categories based on their GO functional annotations: chromatin remodeling, DNA damage, DNA repair, RNA-binding proteins, and cell cycle. Previously, we have successfully used computational approaches to model the RNA-binding domains (RBD) of NCL and delineate the binding interfaces between NCL-RBD and a subset of miRNA that are specifically dysregulated in breast cancer. Using the same strategy, we have modeled the full length NCL to provide a complete structural representation of the protein. Our model fills the gap in missing NCL structural data, especially the unexplored N- and C - termini that may play important roles in NCL protein-protein interactions. Our results provide predicted interaction scenarios between NCL and the subset of the overlapping interactome of NCL and BRCA1, focused on DNA repair mechanisms. These in silico models are critical to understand the protein complexity at the interface of damaged DNA to identify candidates that can be targeted in breast carcinoma. Citation Format: Nitu Farhin, Andy Lam, Anjana D. Saxena, Shaneen Singh. Breast cancer bioinformatics: Untangling the roles of nucleolin and BRCA1 in dysregulated DNA repair [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 875.

Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor kit). FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK (DNA-dependent protein kinase), is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G and D816V) or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared with empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited extracellular signal–regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase mTOR) activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers.

Incorporation of 53BP1 into phase-separated bodies in cancer cells during aberrant mitosis.

53BP1 (also known as TP53BP1) is a key mediator of the non-homologous end joining (NHEJ) DNA repair pathway, which is the primary repair pathway in interphase cells. However, the mitotic functions of 53BP1 are less well understood. Here, we describe 53BP1 mitotic stress bodies (MSBs) formed in cancer cell lines in response to delayed mitosis. These bodies displayed liquid-liquid phase separation characteristics, were close to centromeres, and included lamin A/C and the DNA repair protein RIF1. After release from mitotic arrest, 53BP1 MSBs decreased in number and moved away from the chromatin. Using GFP fusion constructs, we found that the 53BP1 oligomerization domain region was required for MSB formation, and that inclusion of the 53BP1 N terminus increased MSB size. Exogenous expression of 53BP1 did not increase MSB size or number but did increase levels of MSB-free 53BP1. This was associated with slower mitotic progression, elevated levels of DNA damage and increased apoptosis, which is consistent with MSBs suppressing a mitotic surveillance by 53BP1 through sequestration. The 53BP1 MSBs, which were also found spontaneously in a subset of normally dividing cancer cells but not in non-transformed cells (ARPE-19), might facilitate the survival of cancer cells following aberrant mitoses. This article has an associated First Person interview with the first author of the paper.

POLQ suppresses genome instability and alterations inDNA repeat tract lengths.

DNA polymerase theta (POLQ) is a principal component of the alternative non-homologous end-joining (ANHEJ) DNA repair pathway that ligates DNA double-strand breaks. Utilizing independent models of POLQ insufficiency during telomere-driven crisis, we found that POLQ–/– cells are resistant to crisis-induced growth deceleration despite sustaining inter-chromosomal telomere fusion frequencies equivalent to wild-type (WT) cells. We recorded longer telomeres in POLQ–/– than WT cells pre- and post-crisis, notwithstanding elevated total telomere erosion and fusion rates. POLQ–/– cells emerging from crisis exhibited reduced incidence of clonal gross chromosomal abnormalities in accordance with increased genetic heterogeneity. High-throughput sequencing of telomere fusion amplicons from POLQ-deficient cells revealed significantly raised frequencies of inter-chromosomal fusions with correspondingly depreciated intra-chromosomal recombinations. Long-range interactions culminating in telomere fusions with centromere alpha-satellite repeats, as well as expansions in HSAT2 and HSAT3 satellite and contractions in ribosomal DNA repeats, were detected in POLQ–/– cells. In conjunction with the expanded telomere lengths of POLQ–/– cells, these results indicate a hitherto unrealized capacity of POLQ for regulation of repeat arrays within the genome. Our findings uncover novel considerations for the efficacy of POLQ inhibitors in clinical cancer interventions, where potential genome destabilizing consequences could drive clonal evolution and resistant disease.

R-loop Mediated DNA Damage and Impaired DNA Repair in Spinal Muscular Atrophy.

Defects in DNA repair pathways are a major cause of DNA damage accumulation leading to genomic instability and neurodegeneration. Efficient DNA damage repair is critical to maintain genomicstability and support cell function and viability. DNA damage results in the activation of cell death pathways, causing neuronal death in an expanding spectrum of neurological disorders, such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), Alzheimer’s disease (AD), and spinal muscular atrophy (SMA). SMA is a neurodegenerative disorder caused by mutations in the Survival Motor Neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motor neurons due to low levels of the SMN protein. The molecular mechanism of selective motor neuron degeneration in SMA was unclear for about 20 years. However, several studies have identified biochemical and molecular mechanisms that may contribute to the predominant degeneration of motor neurons in SMA, including the RhoA/ROCK, the c-Jun NH2-terminal kinase (JNK), and p53-mediated pathways, which are involved in mediating DNA damage-dependent cell death. Recent studies provided insight into selective degeneration of motor neurons, which might be caused by accumulation of R-loop-mediated DNA damage and impaired non-homologous end joining (NHEJ) DNA repair pathway leading to genomic instability. Here, we review the latest findings involving R-loop-mediated DNA damage and defects in neuron-specific DNA repair mechanisms in SMA and discuss these findings in the context of other neurodegenerative disorders linked to DNA damage.

Abstract 796: Dual inhibition of NHEJ and MMEJ induces synthetic lethality in TP53 mutant cancers

Abstract DNA repair pathway inhibitors are a new class of anti-cancer drugs that are advancing in clinical trials. While inhibitors of targets in the Non-Homologous End Joining (NHEJ) DNA repair pathway, such as DNA-dependent protein kinase (DNA-PK), are available for clinical use, it remains unclear which cancers are vulnerable to these agents. In a genome-wide CRISPR knockout screen with the DNA-PK inhibitor M3814, we identify loss of POLQ, encoding polymerase theta, and other genes in the microhomology-mediated end-joining (MMEJ) pathway as key predictors of sensitivity to DNA-PK inhibition, whereas loss of TP53 conferred resistance to DNA-PK inhibition. Inhibition of DNA-PK led to increased DNA double strand break end-resection, increased expression of polymerase theta, and activation of MMEJ repair. Combined DNA-PK inhibition by M3814 and polymerase theta inhibition by novobiocin resulted in synthetic lethality mediated by the accumulation of resected DNA and apoptosis. Significantly, this drug combination efficiently killed TP53-deficient human patient-derived xenografts and the corresponding tumor organoids. Taken together, our results provide a rationale for the combination of an inhibitor of DNA-PK mediated NHEJ and an inhibitor of polymerase theta mediated MMEJ in an anti-cancer trial. If DNA-PK is inhibited, cancers develop a hyper-dependence on MMEJ and an upregulation of DNA double strand break end resection and polymerase theta expression. Similarly, P53-deficiency which confers resistance to DNA-PK inhibition, also leads to a hyper-dependence on MMEJ and an upregulation of polymerase theta expression. Thus, a combination of DNA-PK and polymerase theta inhibitors may provide a precision treatment strategy for TP53-mutant solid tumors, known to account for 50% of newly diagnosed cancers. Citation Format: Jeffrey Patterson-Fortin, Arindam Bose, Wei-Chih Tsai, Carter Grochala, Huy Nguyen, Jia Zhou, Kalindi Parmar, Jean-Bernard Lazaro, Joyce Liu, Kelsey McQueen, Geoffrey I. Shapiro, David Kozono, Alan D. D'Andrea. Dual inhibition of NHEJ and MMEJ induces synthetic lethality in TP53 mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 796.

Rational design, synthesis and biological evaluation of dual PARP-1/2 and TNKS1/2 inhibitors for cancer therapy

Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors are the first and most successful drugs designed to exploit the concept of synthetic lethality (SL) between PARP-1 and BRCA1/2, which provides a novel strategy for tumor treatment. However, narrowed indications and resistance to PARP-1 inhibitors have hampered their further clinical application. Inducing “BRCAness” by targeting other targets, which will directly or indirectly disturb the homologous recombination (HR) repair pathway of double-strand DNA breaks (DSBs), is a promising strategy for expanding the clinical application of PARP-1 inhibitors and overcoming resistance to these inhibitors. Tankyrase1/2 (TNKS1/2) are involved in the nonhomologous end-joining (NHEJ) DNA repair pathway by regulating Wnt/β-catenin signaling. TNKS1/2 can also induce a “BRCAness” phenotype by regulating Wnt signaling, which increases the sensitivity of tumor cells with BRCA proficiency to PARP-1 inhibitors. These results suggest that cotargeting PARP1/2 and TNKS1/2 not only exerts a synergistic effect in the treatment of tumors but also provides a novel strategy for expanding the clinical application of PARP-1 inhibitors and overcoming resistance to PARP-1 inhibitors. Therefore, a series of dual PARP-1/2 and TNKS1/2 inhibitors were rationally designed, synthesized, and evaluated for their pharmacological properties. Among these candidates, compound I-9 showed excellent inhibitory activity as it inhibited PARP-1/2 and TNKS1/2 with IC50 values of 0.25 nM, 1.2 nM, 13.5 nM and 4.15 nM, respectively. I-9 exhibited favorable synergistic antitumor efficacy in both BRCA-mutant and BRCA-wild-type cancer lines. Moreover, I-9 exerted prominent dose-dependent antitumor activity in an HCT116 cell-derived xenograft model and was significantly more efficacious than olaparib and E7449. Overall, the present study indicated that I-9, a dual PARP-1/2 and TNKS1/2 inhibitor, is a novel and promising agent for cancer therapy.

Senescence and impaired DNA damage responses in alpha-synucleinopathy models

α-Synuclein is a crucial element in the pathogenesis of Parkinson’s disease (PD) and related neurological diseases. Although numerous studies have presented potential mechanisms underlying its pathogenesis, the understanding of α-synuclein-mediated neurodegeneration remains far from complete. Here, we show that overexpression of α-synuclein leads to impaired DNA repair and cellular senescence. Transcriptome analysis showed that α-synuclein overexpression led to cellular senescence with activation of the p53 pathway and DNA damage responses (DDRs). Chromatin immunoprecipitation analyses using p53 and γH2AX, chromosomal markers of DNA damage, revealed that these proteins bind to promoters and regulate the expression of DDR and cellular senescence genes. Cellular marker analyses confirmed cellular senescence and the accumulation of DNA double-strand breaks. The non-homologous end joining (NHEJ) DNA repair pathway was activated in α-synuclein-overexpressing cells. However, the expression of MRE11, a key component of the DSB repair system, was reduced, suggesting that the repair pathway induction was incomplete. Neuropathological examination of α-synuclein transgenic mice showed increased levels of phospho-α-synuclein and DNA double-strand breaks, as well as markers of cellular senescence, at an early, presymptomatic stage. These results suggest that the accumulation of DNA double-strand breaks (DSBs) and cellular senescence are intermediaries of α-synuclein-induced pathogenesis in PD.

A Bioluminescence Resonance Energy Transfer-Based Reporter System: Characterization and Applications.

Genome editing strategies and DNA repair research need powerful analytical tools. We generated a bioluminescence resonance energy transfer (BRET)-based reporter for the quantification of indel frequencies induced by DNA repair. The BRET reporter, expressed as a single molecule, consists of a mutated Renilla reniformis luciferase domain and a GFP2 domain separated by a shuttle-cloning box for the integration of any given endonuclease target sequence. The luciferase activity acts both as energy donor and as the internal standard, while the loss of GFP2 fluorescence acts as a reporter for the out-of-frame sequence alterations that result from the DNA repair via the non-homologous end joining/microhomology-mediated end joining DNA repair pathways of the endonuclease-mediated DNA double-strand break. This results in a decrease of the fluorescence/luminescence ratio. Employing this reporter in different experimental scenarios, using different cell lines and diseases targeted, we quantified the influence of both protein knockdown of DNA repair pathways as well as guide RNA mismatches on CRISPR-mediated nuclease activity and subsequent repair based on mutagenic repair on the reporter. In conclusion, we demonstrated this BRET-based reporter to be a robust and sensitive analytical tool for assessment of variety of different genome editing-based approaches.