Strand Break Repair Research Articles

Abstract 3305: WSD-0628, a novel brain penetrant ATM inhibitor, radiosensitizes GBM and melanoma patient derived xenografts

Abstract Glioblastoma (GBM) are inherently resistant to radiation therapy (RT), and development of radiosensitizers is one strategy to overcome this limitation. Repair of DNA double strand breaks induced by RT are mediated by the protein kinase Ataxia Telangiectasia mutated (ATM). In this study, the novel ATM inhibitor WSD-0628 was evaluated in combination with RT using GBM and melanoma models. In vitro evaluation of 10uM WSD-0628 binding to a panel of potential drug targets, including receptors, ion channels, enzymes, and transporters, indicated a satisfactory safety profile with low risk for off-target liability. WSD-0628 potently inhibits ATM-mediated phosphorylation of the DNA damage response protein KAP1 in MCF-7 cells at sub-nanomolar (nM) concentrations (IC50 0.42nM) in comparison to much less potent inhibition of the related kinases ATR (phosphorylation of CHK1, IC50 742nM) or DNA-PKcs (auto-phosphorylation of DNA-PK, IC50 169nM) in HT29 cells assessed by ELISA. In U251 GBM cells, 30 nM WSD-0628 potently inhibited RT-induced phospho-KAP1 and robustly reduced clonogenic survival by 5-fold when combined with 5 Gy irradiation (combination vs RT alone, p<0.01). Similar potent radiosensitizing effects were seen in a melanoma brain metastasis PDX line M12 (10nM WSD-0628+IR-5Gy 1% survival vs 5% survival with IR-5Gy alone. p<0.01), and the SV-40 transformed astrocyte line SVG-A (10nM WSD-0628 + IR-2.5Gy survival 0.2% vs 15% with IR-2.5Gy alone. p<0.01). Evaluation of the pharmacokinetic profile of WSD-0628 in mice 2h after a single 5 mg/kg oral dose reveals a high level of free drug availability in the brain (34nM) and in the CSF (50nM) with little to no Pgp/BCRP substrate liability. An initial in vivo dose finding study in orthotopic GBM43 PDX yielded significant benefit with WSD-0628 at either 5 or 10 mg/kg PO daily when combined with radiation (2Gy QD for 5 days); Median survival for sham RT (29d) or RT alone (34d) were significantly different from RT combinations with 5 mg/kg (54d) and 10 mg/kg (73d; p<0.01 for both dose levels), although the higher dose combination was poorly tolerated with body weight loss between 15-20% one week after RT completion. Dosing of WSD-0628 (7.5 mg/kg PO, QD) given just before and 24h after a single dose of RT (12.5Gy) in mice with orthotopic M12 was well tolerated and provided robust radiosensitizing effects with median survival for combination treatment of over 180d vs 17d for control and 49d with RT alone groups (combination vs RT alone, p=0.04). Collectively, these results suggest a promising role for WSD-0628 in combination with RT in GBM and melanoma metastatic to the brain. Citation Format: Ann Mladek Tuma, Wei Zhong, Lily Liu, Danielle M. Burgenske, Brett L. Carlson, Katrina K. Bakken, Zeng Hu, Margaret A. Connors, Jann N. Sarkaria. WSD-0628, a novel brain penetrant ATM inhibitor, radiosensitizes GBM and melanoma patient derived xenografts [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 3305.

Abstract 6307: The botanical drug PBI-05204, a supercritical CO₂ extract of Nerium oleander, augments the antitumor efficacy of radiotherapy in treatment of human glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common as well as one of the most malignant types of brain cancer. Despite progress in development of novel therapies for the treatment of GBM, it remains largely incurable with a poor prognosis and a very low life expectancy. Recent studies have shown that oleandrin, a unique cardiac glycoside from Nerium oleander, as well as defined extract (PBI-05204) that contains this molecule, inhibit growth of human glioblastoma, and modulate glioblastoma patient-derived stem cell-renewal properties. The present study aimed to investigate the radiosensitization of PBI-05204 in glioblastoma using both in vitro and in vivo cancer models as well as to explore the potential mechanism of actions in GBM. The radiosensitizing effect of PBI-05204 was assessed against human GBM U87MG, U251, T98G and A172 cell lines as well as their relevant xenograft and orthotopic models. The induction of apoptosis, DNA damage and repair of DNA double strand breaks were assessed with determination of caspase 3 and 9 protein expression, DNA laddering, protein expression of rH2AX, Ku70, DNA-PKcs, and RAD51 as well as a Comet Assay. PBI-05204 treatment leads to an increased in vitro sensitivity of GBM cells, including U87MG, U251, T98G and A172 cells, to radiotherapy (RT) in which the main mechanisms are the transition from autophagy to apoptosis and enhanced DNA damage evidenced by increased expression of γH2AX. Additionally, relative increased expression of Ku70, DNA-PKcs and RAD51 due to RT were reduced by PBI-05204 in U87MG and U251 cells, suggesting PBI-05204 lessened RT mediated DNA repair. PBI-05204 significantly enhanced the RT mediated inhibition of tumor growth by 4.7-, 2.1- and 2.2-fold in U87MG, U251 and T98G xenograft models, respectively. The combination of RT and PBI-05204 showed a significantly enhancement of disease-free survival to 103.0 ± 63.2 days compared to the control group (p < 0.001) which was 3-fold longer than that of RT only group. Collectively, these results reveal that PBI-05204 enhances antitumor activity of RT in preclinical/murine models of human GBM. Given the fact that PBI-05204 has already been examined in Phase I and II clinical trials for cancer patients, its efficacy when combined with standard-of-care radiotherapy regimens in GBM should be explored in future clinical trials of this difficult to treat brain cancer. Citation Format: Alessandro Colapietro, Peiying Yang, Alessandra Rosetti, Andrea Mancini, Flora Vital, Stefano Martellucci, Francesco Marampon, Vincenzo Mattei, Giovanni Luca Gravina, Robert Newman, Claudio Festuccia. The botanical drug PBI-05204, a supercritical CO₂ extract of Nerium oleander, augments the antitumor efficacy of radiotherapy in treatment of human glioblastoma [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 6307.

Abstract 1406: Design and synthesis of novel cardiac glycosides by targeting the DNA damage response for cancer therapy

Abstract Cardiac Glycosides (CGs) are used to treat congestive heart disease. Their application has since been extended to cancer treatment. While the mechanism of action is still unknown, it was previously believed that Na+/K+ ATPase might play a crucial role in cancer mitigation. However, we recently reported that cardiac glycosides could specifically enhance the anti-cancer effect of chemotherapy in KRAS mutant lung cancer by inhibiting the DNA Damage Response (DDR), and these effects were independent of the Na+/K+ ATPase. Mechanistically, we showed that cardiac glycosides induced the degradation of UHRF1, an important player in promoting DNA double strand break (DSB) repair through homologous recombination. These results suggest the promise of derivatizing cardiac glycosides into potential chemo sensitizing agents. Yet, one of the major challenges with these compounds is their cardiotoxicity. This has created a need for developing a strategy to generate less cardiotoxic drugs that are effective in inhibiting the DNA damage response to safe and effective anticancer activity. Herein we report a multi-step synthetic route utilizing k-strophanthidin as the initial building block. A systematic structural design was applied that included modification of the sugar moiety, glycoside linker, stereochemistry, and lactone ring to create a library of O-glycosides and MeON-neoglycosides. These molecules were screened for anti-cancer properties by exploring their influence on the signaling/expression of various downstream kinases in the DDR pathway. We discovered the inhibitory activity of O-glycosides to be more potent than the MeON-neoglycosides or benzylidene-lactone modified compounds. These results demonstrate the ability to chemically synthesize CG derivatives to optimize for anticancer activity with the goal of identifying less cardiotoxic CGs. Citation Format: Diana Ainembabazi, Xinran Geng, Navnath Gavande, YouWei Zhang, John Turchi. Design and synthesis of novel cardiac glycosides by targeting the DNA damage response for cancer therapy [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 1406.

Abstract 811: Pharmacologic ascorbate opens a therapeutic window for ATM inhibition and radiotherapy in colorectal cancer

Abstract Purpose/Objective(s): The ATM protein is key to DNA double strand break (DSB) repair and ATM inhibitors are potent radiosensitizers. Clinical translation of these agents in combination with radiotherapy (RT) has been limited due to concerns for increased normal tissue toxicity. Pharmacologic Ascorbate (P-AscH-) radiosensitizes cancer cells via generation of a H2O2 flux while acting as a radioprotector in normal tissue via free radicle scavenging. We hypothesized that P-AscH- could open a therapeutic window for the combination of RT and ATM inhibition in colorectal cancer (CRC). Materials/Methods: Multiple human and murine CRC cell lines were used in vitro. Clonogenic survival was assessed after combinations of RT +/- P-AscH and DNA Repair Inhibitors (DRIs). Catalase expression was induced using HCT116 cells expressing a doxycycline-inducible catalase transgene. DSBs were quantified using neutral comet assays. Cell cycle distribution were assessed using flow cytometry. ATM localization/activation were assessed using IF. Normal tissue toxicity in vivo was assessed using IHC following whole-abdominal RT. Survival and tumor growth delay was assessed following 5Gyx3 +/- drug treatment to unilateral flank tumors in syngeneic/xenograft models. Results: DRIs were potent radiosensitizers in most CRC cell lines and the addition of P-AscH- further reduced clonogenic survival. In contrast, P-AscH- did not radiosensitize HUVEC or FHs-74int normal cell lines. P-AscH- significantly increased the number of DSBs in tumors after RT in vitro. P-AscH- simultaneously decreased nuclear localization and activation of pATM after RT and perturbed G2+M phase progression. In vivo, the addition of P-AscH- to RT + KU60019 significantly increased survival delayed tumor growth in syngeneic/xenograft models while ameliorating increased normal bowel toxicity as measured by jejunal crypt density, acute weight loss, rectal injury, and markers of oxidative stress following whole-abdominal RT. The effects of P-AscH were reversed by inducing the overexpression of catalase. Conclusion: P-AscH- improves both aspects of the therapeutic window of RT+ATM inhibition in CRC by simultaneously enhancing tumor efficacy while decreasing RT-mediated bowel toxicity. The effects of P-AscH- on clonogenic survival, initial and persistent DSBs, G2+M phase perturbations, ATM activation/localization, and in vivo survival and tumor growth delay were dependent on H202 flux. Normal tissue protection appears to be related to decreased oxidative stress. Citation Format: Cameron M. Callaghan, Ibrahim M. Abukhiran, Richard V. Van Rheeden, Amanda L. Kalen, Samuel N. Rodman, Michael S. Petronek, Kranti A. Mapuskar, Sarah L. Mott, Mitchell C. Coleman, Prabhat C. Goswami, John M. Buatti, Bryan G. Allen, Douglas R. Spitz, Joseph M. Caster. Pharmacologic ascorbate opens a therapeutic window for ATM inhibition and radiotherapy in colorectal cancer [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 811.

Watching right and wrong nucleotide insertion captures hidden polymerase fidelity checkpoints

Efficient and accurate DNA synthesis is enabled by DNA polymerase fidelity checkpoints that promote insertion of the right instead of wrong nucleotide. Erroneous X-family polymerase (pol) λ nucleotide insertion leads to genomic instability in double strand break and base-excision repair. Here, time-lapse crystallography captures intermediate catalytic states of pol λ undergoing right and wrong natural nucleotide insertion. The revealed nucleotide sensing mechanism responds to base pair geometry through active site deformation to regulate global polymerase-substrate complex alignment in support of distinct optimal (right) or suboptimal (wrong) reaction pathways. An induced fit during wrong but not right insertion, and associated metal, substrate, side chain and pyrophosphate reaction dynamics modulated nucleotide insertion. A third active site metal hastened right but not wrong insertion and was not essential for DNA synthesis. The previously hidden fidelity checkpoints uncovered reveal fundamental strategies of polymerase DNA repair synthesis in genomic instability.

APOBEC3 mutational signatures are associated with extensive and diverse genomic instability across multiple tumour types

BackgroundThe APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide 3) family of cytidine deaminases is responsible for two mutational signatures (SBS2 and SBS13) found in cancer genomes. APOBEC3 enzymes are activated in response to viral infection, and have been associated with increased mutation burden and TP53 mutation. In addition to this, it has been suggested that APOBEC3 activity may be responsible for mutations that do not fall into the classical APOBEC3 signatures (SBS2 and SBS13), through generation of double strand breaks.Previous work has mainly focused on the effects of APOBEC3 within individual tumour types using exome sequencing data. Here, we use whole genome sequencing data from 2451 primary tumours from 39 different tumour types in the Pan-Cancer Analysis of Whole Genomes (PCAWG) data set to investigate the relationship between APOBEC3 and genomic instability (GI).Results and conclusionsWe found that the number of classical APOBEC3 signature mutations correlates with increased mutation burden across different tumour types. In addition, the number of APOBEC3 mutations is a significant predictor for six different measures of GI. Two GI measures (INDELs attributed to INDEL signatures ID6 and ID8) strongly suggest the occurrence and error prone repair of double strand breaks, and the relationship between APOBEC3 mutations and GI remains when SNVs attributed to kataegis are excluded.We provide evidence that supports a model of cancer genome evolution in which APOBEC3 acts as a causative factor in the development of diverse and widespread genomic instability through the generation of double strand breaks. This has important implications for treatment approaches for cancers that carry APOBEC3 mutations, and challenges the view that APOBECs only act opportunistically at sites of single stranded DNA.

Modulating DNA Repair Pathways to Diversify Genomic Alterations in Saccharomyces cerevisiae.

ABSTRACTNuclease based genome editing systems have emerged as powerful tools to drive genomic alterations and enhance genome evolution via precise engineering in the various human and microbial cells. However, error-prone DNA repair has not been well studied previously to generate diverse genomic alterations and novel phenotypes. Here, we systematically investigated the potential interplay between DNA double strand break (DSB) repair and genome editing tools, and found that modulating the DSB end resection proteins could significantly improve mutational efficiency and diversity without exogenous DNA template in yeast. Deleting SAE2, EXO1, or FUN30, or overexpressing MRE11-H125N (nuclease-dead allele of MRE11), for DSB end resection markedly increased the efficiency of CRISPR/SpCas9 (more than 22-fold) and CRISPR/AsCpf1 (more than 30-fold)-induced mutagenesis. Deleting SAE2 or overexpressing MRE11-H125N substantially diversified CRISPR/SpCas9 or AsCpf1-induced mutation 2–3-fold at URA3 locus, and 3–5-fold at ADE2 locus. Thus, the error-prone DNA repair protein was employed to develop a novel mutagenic genome editing (mGE) strategy, which can increase the mutation numbers and effectively improve the ethanol/glycerol ratio of Saccharomyces cerevisiae through modulating the expression of FPS1 and GPD1. This study highlighted the feasibility of potentially reshaping the capability of genome editing by regulating the different DSB repair proteins and can thus expand the application of genome editing in diversifying gene expression and enhancing genome evolution.IMPORTANCE Most of the published papers about nuclease-assisted genome editing focused on precision engineering in human cells. However, the topic of inducing mutagenesis via error-prone repair has often been ignored in yeast. In this study, we reported that perturbing DNA repair, especially modifications of the various DSB end resection-related proteins, could greatly improve the mutational efficiency and diversity, and thus functionally reshape the capability of the different genome editing tools without requiring an exogenous DNA template in yeast. Specifically, mutagenic genome editing (mGE) was developed based on CRISPR/AsCpf1 and MRE11-H125N overexpression, and used to generate promoters of different strengths more efficiently. Thus, this work provides a novel method to diversify gene expression and enhance genome evolution.

Oxidative damage and DNA repair in desiccated recalcitrant embryonic axes of Acer pseudoplatanus L.

BackgroundMost plants encounter water stress at one or more different stages of their life cycle. The maintenance of genetic stability is the integral component of desiccation tolerance that defines the storage ability and long-term survival of seeds. Embryonic axes of desiccation-sensitive recalcitrant seeds of Acer pseudoplatnus L. were used to investigate the genotoxic effect of desiccation. Alkaline single-cell gel electrophoresis (comet assay) methodology was optimized and used to provide unique insights into the onset and repair of DNA strand breaks and 8-oxo-7,8-dihydroguanine (8-oxoG) formation during progressive steps of desiccation and rehydration.ResultsThe loss of DNA integrity and impairment of damage repair were significant predictors of the viability of embryonic axes. In contrast to the comet assay, automated electrophoresis failed to detect changes in DNA integrity resulting from desiccation. Notably, no significant correlation was observed between hydroxyl radical (٠OH) production and 8-oxoG formation, although the former is regarded to play a major role in guanine oxidation.ConclusionsThe high-throughput comet assay represents a sensitive tool for monitoring discrete changes in DNA integrity and assessing the viability status in plant germplasm processed for long-term storage.

Deciphering the mechanism of processive ssDNA digestion by the Dna2-RPA ensemble

Single-stranded DNA (ssDNA) commonly occurs as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors outcompete RPA to access ssDNA. Using the budding yeast Saccharomyces cerevisiae as a model system, we find that Dna2 — a key nuclease in DNA replication and repair — employs a bimodal interface to act with RPA both in cis and in trans. The cis-activity makes RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA delivers its bound ssDNA to Dna2. On the other hand, activity in trans is mediated by an acidic patch on Dna2, which enables it to function with a sub-optimal amount of RPA, or to overcome DNA secondary structures. The trans-activity mode is not required for cell viability, but is necessary for effective double strand break (DSB) repair.

DNA repair protein DNA-PK protects PC12 cells from oxidative stress-induced apoptosis involving AKT phosphorylation

BackgroundEmerging evidence suggest that DNA-PK complex plays a role in the cellular response to oxidative stress, in addition to its function of double strand break (DSB) repair. In this study we evaluated whether DNA-PK participates in oxidative stress response and whether this role is independent of its function in DNA repair.Methods and resultsWe used a model of H2O2-induced DNA damage in PC12 cells (rat pheochromocytoma), a well-known neuronal tumor cell line. We found that H2O2 treatment of PC12 cells induces an increase in DNA-PK protein complex levels, along with an elevation of DNA damage, measured both by the formation of γΗ2ΑX foci, detected by immunofluorescence, and γH2AX levels detected by western blot analysis. After 24 h of cell recovery, γΗ2ΑX foci are repaired both in the absence and presence of DNA-PK kinase inhibitor NU7026, while an increase of apoptotic cells is observed when DNA-PK activity is inhibited, as revealed by counting pycnotic nuclei and confirmed by FACS analysis. Our results suggest a role of DNA-PK as an anti-apoptotic factor in proliferating PC12 cells under oxidative stress conditions. The anti-apoptotic role of DNA-PK is associated with AKT phosphorylation in Ser473. On the contrary, in differentiated PC12 cells, were the main pathway to repair DSBs is DNA-PK-mediated, the inhibition of DNA-PK activity causes an accumulation of DNA damage.ConclusionsTaken together, our results show that DNA-PK can protect cells from oxidative stress induced-apoptosis independently from its function of DSB repair enzyme.Graphical

RNF19A-mediated ubiquitination of BARD1 prevents BRCA1/BARD1-dependent homologous recombination

BRCA1-BARD1 heterodimers act in multiple steps during homologous recombination (HR) to ensure the prompt repair of DNA double strand breaks. Dysfunction of the BRCA1 pathway enhances the therapeutic efficiency of poly-(ADP-ribose) polymerase inhibitors (PARPi) in cancers, but the molecular mechanisms underlying this sensitization to PARPi are not fully understood. Here, we show that cancer cell sensitivity to PARPi is promoted by the ring between ring fingers (RBR) protein RNF19A. We demonstrate that RNF19A suppresses HR by ubiquitinating BARD1, which leads to dissociation of BRCA1-BARD1 complex and exposure of a nuclear export sequence in BARD1 that is otherwise masked by BRCA1, resulting in the export of BARD1 to the cytoplasm. We provide evidence that high RNF19A expression in breast cancer compromises HR and increases sensitivity to PARPi. We propose that RNF19A modulates the cancer cell response to PARPi by negatively regulating the BRCA1-BARD1 complex and inhibiting HR-mediated DNA repair.

DNA damage-induced degradation of Sp1 promotes cellular senescence

Persistent DNA damage (genotoxic stress) triggers signaling cascades that drive cells into apoptosis or senescence to avoid replicating a damaged genome. Sp1 has been found to play a role in double strand break (DSB) repair, and a link between Sp1 and aging has also been established, where Sp1 protein, but not RNA, levels decrease with age. Interestingly, inhibition ATM reverses the age-related degradation of Sp1, suggesting that DNA damage signaling is involved in senescence-related degradation of Sp1. Proteasomal degradation of Sp1 in senescent cells is mediated via sumoylation, where sumoylation of Sp1 on lysine 16 is increased in senescent cells. Taking into consideration our previous findings that Sp1 is phosphorylated by ATM in response to DNA damage and that proteasomal degradation of Sp1 at DSBs is also mediated by its sumoylation and subsequent interaction with RNF4, we investigated the potential contribution of Sp1’s role as a DSB repair factor in mediating cellular senescence. We report here that Sp1 expression is decreased with a concomitant increase in senescence markers in response to DNA damage. Mutation of Sp1 at serine 101 to create an ATM phospho-null mutant, or mutation of lysine 16 to create a sumo-null mutant, prevents the sumoylation and subsequent proteasomal degradation of Sp1 and results in a decrease in senescence. Conversely, depletion of Sp1 or mutation of Sp1 to create an ATM phosphomimetic results in premature degradation of Sp1 and an increase in senescence markers. These data link a loss of genomic stability with senescence through the action of a DNA damage repair factor.

Nuclear Transglutaminase 2 interacts with topoisomerase II\u237a to promote DNA damage repair in lung cancer cells

BackgroundTo block repairs of DNA damages, especially the DNA double strand break (DSB) repair, can be used to induce cancer cell death. DSB repair depends on a sequential activation of DNA repair factors that may be potentially targeted for clinical cancer therapy. Up to now, many protein components of DSB repair complex remain unclear or poorly characterized. In this study, we discovered that Transglutaminase 2 (TG2) acted as a new component of DSB repair complex.MethodsA bioinformatic analysis was performed to identify DNA damage relative genes from dataset from The Cancer Genome Atlas. Immunofluorescence and confocal microscopy were used to monitor the protein localization and recruitment kinetics. Furthermore, immunoprecipitation and mass spectrometry analysis were performed to determine protein interaction of both full-length and fragments or mutants in distinct domain. In situ lung cancer model was used to study the effects cancer therapy in vivo.ResultsAfter DSB induction, cytoplasmic TG2 was extensively mobilized and translocated into nucleus after phosphorylated at T162 site by DNA-PKcs. Nuclear TG2 quickly accumulated at DSB sites and directly interacting with Topoisomerase IIα (TOPOIIα) with its TGase domain to promote DSB repair. TG2 deficient cells lost capacity of DSB repair and become susceptible to ionizing radiation. Specific inhibition of TG2-TOPOIIα interaction by glucosamine also significantly inhibited DSB repair, which increased sensitivity in lung cancer cells and engrafted lung cancers.ConclusionsThese findings elucidate new mechanism of TG2 in DSB repair trough directly interacting with TOPOIIα, inhibition of which provided potential target for overcoming cancer resistance.

Abstract 618: Spatio-temporal multiomics analysis reveals distinct molecular features in recurrent stage I non-small cell lung cancers after R0 tumor resection

Abstract A portion of stage I non-small cell lung cancer (NSCLC) patients who have received R0 resection experience unexpected tumor recurrence during follow up. Some of them even recurred within 12 months after surgery, which is unable to predict by any clinical characteristics. Few study investigates the molecular features of such short-term recurrent tumors. Multiomics analysis were performed on 81 stage I NSCLC patients who had received R0 resection, based on deep whole-exome sequencing (average depth >500x) on multi-region 239 tumor tissue samples and 81 matched-adjacent normal specimens, as well as RNA sequencing and plasma targeted circulating tumor DNA detection. Dynamic dimension comparison of genomic alteration features between tumor recurrence and non-recurrence patients was made according to 60-months median time of follow up. Published WES datasets of Stage I NSCLCs of 131 East Asian (EAS LUADs), 62 European (TRACERx) and 277 American patients (TCGA) were extracted as validation. 60.5% patients were non-smokers. Lower chromosomal instability was found in our cohort characterized by less trunk CNVs and lower CNV coverage on genome as compared to the European cohort. Age-related signature mutations significantly accumulated in recurrence-free tumors. Somatic mutations occurred in DNA damage repair pathways were more likely to cause tumor recurrence (p=0.04), particularly in homologous recombination (HRR) (p=0.017) and translesion synthesis (TLS) (p=0.009) pathways. Dynamic analysis discovered the essential genes in Fanconi anemia (FA), TLS and HRR pathways that coordinate together for DNA interstrand crosslink (ICL) recognition and faithful repair of DNA double strand breaks (DSBs) during DNA replication stress frequently mutated in early tumor recurrence patients (n=7/11, 63%). Twenty-two focal CNV-driver genes, including cell proliferation-related RECQL4 (8q24.3), PIK3CA (3q26.32), BCL9 (1q21.2), TBL1XR1 (3q26.32) frequently amplified in recurrent tumors, as well as BCL3 and CBLC (19q13.31-32) in the early recurrent tumors. RNA-seq data demonstrated cell cycle pathway significantly up-regulated in recurrent tumors. More TMB, CNV fraction and genome doubling events in FA-TLS-HRR alteration patients verified genomic instability caused by dysfunction of cell cycle control and ICL-DSB repair contributing to early tumor recurrence. Integrative model cross clinical and molecular features stratified stage I patient prognosis in both our cohort and European cohort, in which and high risk patients were more detected by circulating tumor DNA. This is the first study to dynamically investigate the genomic features specifically in stage I lung NSCLC according to the precise tumor recurrence time, which provides important clues to postoperative treatment strategy and drug target research. Citation Format: Kezhong Chen, Airong Yang, Shuangxiu Wu, Yuntao Nie, Haifeng Shen, Jian Bai, Lin Wu, Fan Yang, Jun Wang. Spatio-temporal multiomics analysis reveals distinct molecular features in recurrent stage I non-small cell lung cancers after R0 tumor resection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 618.

Abstract PO-018: Hypoxia activated prodrugs of DNA-dependent protein kinase inhibitors as radiosensitisers of head and neck squamous cell carcinomas

Abstract Inhibition of the repair of radiation-induced DNA double strand breaks offers the potential to sensitize tumors to radiation therapy. DNA-dependent protein kinase (DNA-PK) is a well validated target for radiosensitisation, but inhibition also has the potential to radiosensitize normal tissues within the radiation field, and to target normal tissue functions outside of DNA-PK’s canonical role in DNA repair. Exploiting hypoxia to selectively activate hypoxia-activated prodrugs of DNA-PK inhibitors (DNA-PKi) in tumors may enhance tumor selectivity, with the advantage of sensitizing the most radioresistant (hypoxic) subpopulation, as well as sensitizing adjacent oxic cells through a local bystander effect. We recently conducted a drug discovery campaign, scaffold-hopping from the pan-PIKK inhibitor dactolisib to identify new DNA-PKi. We screened compounds against three signal targets DNA-PK, PI3Kα and mTOR, and determined in vitro sensitization of UT-SCC-54C head and neck squamous cell carcinoma (HNSCC) cells using a growth inhibition endpoint. We identified a highly selective lead DNA-PKi, SN39536, and a corresponding hypoxia-activated prodrug, SN39884. SN39536 is an effective inhibitor of DNA-PK activity, both biochemically and in cells, with substantial selectivity compared to phosphoinositide-3-kinase (PI3K) enzymes and other PI3K-related kinase enzymes. SN39536 provided radiosensitization of DNA-PK proficient HAP1 cells, but was inactive against isogenic cells with knockout of the DNA-PK catalytic subunit (PRKDC), demonstrating on-mechanism activity. SN39536 also provided robust radiosensitization of oxic UT-SCC-54C HNSCC cells using a clonogenic endpoint. Prodrug SN39884 was deactivated as a DNA-PKi and released SN39536 under reductive conditions. Incubation of the prodrug with HCT116 cells overexpressing cytochrome P450 oxidoreductase or UT-SCC-54C cells led to hypoxia-dependent metabolism of the prodrug and efficient release of SN39536. SN39536 effectively sensitized UT-SCC-54C cells to radiation under either aerobic or anoxic conditions, while prodrug SN39884 only sensitized cells under anoxia. SN39536 and its prodrug also demonstrated radiosensitization of hypoxic cells in UT-SCC-54C tumor xenografts using an ex vivo clonogenic survival endpoint. We have identified a new class of potent and selective DNA-PK inhibitors and demonstrated the ability of a hypoxia-activated prodrug to selectively release the inhibitor under hypoxia in vitro and in vivo. Citation Format: Michael P. Hay, Way W. Wong, Benjamin D. Dickson, Gary J. Cheng, Liew P. Liew, Cho R. Hong, William R. Wilson, Stephen M. Jamieson. Hypoxia activated prodrugs of DNA-dependent protein kinase inhibitors as radiosensitisers of head and neck squamous cell carcinomas [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-018.

Abstract PO-020: Telomerase reverse transcriptase inhibitor NU-1 enhances radiation and chemotherapy sensitivity by disrupting DNA repair and accelerating cellular senescence

Abstract Upon reactivation in cancer, telomerase adds TTAGGG DNA repeats to the ends of chromosomes to prevent telomere erosion and senescence despite deregulated cell proliferation. The telomerase catalytic core consists of telomerase reverse transcriptase (TERT) and the template RNA, telomerase RNA (TR). While TR is broadly expressed, TERT is silenced in somatic cells, leading to progressive telomere shortening with each cell division leading to replicative senescence or apoptosis. That most cancers can maintain telomere integrity despite continued cell division has made TERT an attractive target for cancer therapy. We recently reported NU-1, a novel covalent telomerase inhibitor derived from the natural product chrolactomycin, that irreversibly blocks the telomerase activity of TERT. In examining mechanisms of action, our RNA-seq analysis found that NU-1 affects not only telomere maintenance but also DNA damage response (DDR) pathways. TERT has previously been implicated in multiple cancer-related pathways, including cell survival and repair of DNA double strand breaks (DSBs). To further explore the effects of TERT inhibition on the DDR, we used the human ER+ breast cancer cell line MCF7 to examine radiation response in the presence or absence of TERT inhibitors, including NU-1, chrolactomycin, BIBR1532 and MST-312. TERT inhibition was sufficient to delay both the resolution of γH2AX foci, a marker for DDR signaling, and DSB repair based on neutral comet assay. Surviving cells displayed increased cellular senescence after TERT inhibition. Importantly, only a minor fraction of the persistent γH2AX foci induced by TERT inhibitors colocalized with telomeres, based on binding of PNA probes. Examining mechanisms of DSB repair, NU-1 or BIBR1532 appeared to primarily inhibit the non-homologous end joining (NHEJ) DSB repair pathway versus less effect on homologous recombination (HR) repair, using Traffic Light Reporter assays. Similarly, NU-1 enhanced loss of cell proliferation after treatment with irinotecan, doxorubicin, etoposide, or paclitaxel in multiple TERT-expressing cancer cell lines. This effect was absent in the TERT negative Saos2 cell line. These results establish NU-1 as a chemical probe to explore TERT catalytic activity as a determinant of DNA damage tolerance and suggest telomerase inhibitors as tumor-specific radio/chemo-sensitizers. Citation Format: Yue Liu, Stephen J. Kron, Rick C. Betori, Karl A. Scheidt, Grant Frost, Ding Wu, Don J. Wolfgeher, Elena V. Efimova, Scott B. Cohen. Telomerase reverse transcriptase inhibitor NU-1 enhances radiation and chemotherapy sensitivity by disrupting DNA repair and accelerating cellular senescence [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-020.

DNA Repair Biosensor-Identified DNA Damage Activities of Endophyte Extracts from Garcinia cowa.

Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.

Parp3 promotes astrocytic differentiation through a tight regulation of Nox4-induced ROS and mTorc2 activation

Parp3 is a member of the Poly(ADP-ribose) polymerase (Parp) family that has been characterized for its functions in strand break repair, chromosomal rearrangements, mitotic segregation and tumor aggressiveness. Yet its physiological implications remain unknown. Here we report a central function of Parp3 in the regulation of redox homeostasis in continuous neurogenesis in mice. We show that the absence of Parp3 provokes Nox4-induced oxidative stress and defective mTorc2 activation leading to inefficient differentiation of post-natal neural stem/progenitor cells to astrocytes. The accumulation of ROS contributes to the decreased activity of mTorc2 as a result of an oxidation-induced and Fbxw7-mediated ubiquitination and degradation of Rictor. In vivo, mTorc2 signaling is compromised in the striatum of naïve post-natal Parp3-deficient mice and 6 h after acute hypoxia-ischemia. These findings reveal a physiological function of Parp3 in the tight regulation of striatal oxidative stress and mTorc2 during astrocytic differentiation and in the acute phase of hypoxia-ischemia.

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos

CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F0 generation.

XRN2 interactome reveals its synthetic lethal relationship with PARP1 inhibition

Persistent R-loops (RNA–DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5′-3′-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2’s association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition.