Suppress DNA Damage Research Articles

6686 Age-Associated Local GH Adversely Impacts the Microenvironmental Epithelial Landscape

Abstract Disclosure: V.M. Chesnokova: None. S. Zonis: None. T. Apaydin: None. C. Wang Valencia: None. R. Ainsworth: None. R. Barrett: None. A. Kettenbach: None. S. Melmed: None. The tissue microenvironmental homeostasis is disrupted with age-related changes. Knowledge of mechanisms enabling the age-associated tissue landscape is limited. Senescent cells increase with age, and the secretome released from senescence cells (SASP) affects neighboring cells, often creating a milieu favoring neoplasia. We previously showed that local non-pituitary epithelial growth hormone (GH) is induced and secreted from senescent cells, accumulates with age, and suppresses DNA repair, leading to abundant DNA damage. To examine SASP-derived autocrine GH actions, we analyzed normal human colon cells (hNCC) infected with lentivirus expressing hGH (lentiGH) or vector (lentiV). Mass spectrometry revealed significant changes in the Rho signaling pathway reflecting cell cytoskeletal rearrangements. To examine paracrine GH action, we co-cultured intact human 3D intestinal organoids with organoids infected with lentiGH for 5 weeks. Using whole-exome sequencing we found that local paracrine GH emanating from lentiGH organoids increases neighboring intact organoid cell chromosomal instability, inducing somatic mutations, including deletions, breakends, duplications, and insertions. Mechanisms for observed chromosomal instability include sequelae of GH-mediated suppressed DNA damage repair proteins including pATM, pATR, and pBRCA2, leading to DNA damage accumulation and favoring cell transformation over the long term. Furthermore, enriched gene ontology and KEGG pathway analysis of intact organoids exposed to paracrine GH identified distorted extracellular matrix (ECM) gene expression and focal adhesion pathways. We confirmed these genomic changes with Western blotting showing altered proteins associated with ECM and those responsible for cell structural rearrangements. Moreover, phospho-omics of GH-exposed cells revealed significant phosphorylation changes in proteins associated with cell skeletal rearrangement and cell migration pathways. We found that GH triggers these changes by EMT activation as shown by suppressing E-cadherin and inducing Twist2 and Snai1 transcription factors. Together, all these changes are consistent with observed increased migration, invasion, and anchorage-independent growth of hNCC infected with lentiGH or co-cultured with GH-secreting hNCC or with GH-secreting normal colon fibroblasts. As paracrine GH also facilitates metastases after intra-splenic injection of senescent cells in nude mice carrying xenografts secreting GH, our results indicate that accumulated and locally secreted GH in aging tissue enables a microenvironmental landscape favoring epithelial cell transformation. Presentation: 6/1/2024

Roflumilast Attenuates Microglial Senescence and Retinal Inflammatory Neurodegeneration Post Retinal Ischemia Reperfusion Injury Through Inhibiting NLRP3 Inflammasome.

Retinal ischemia-reperfusion (RIR) injury is implicated in various retinal diseases, leading to retinal ganglion cells (RGCs) degeneration. Microglial senescence exacerbates inflammation, contributing to neurodegeneration. This study aimed to investigate the potential therapeutic role of Roflumilast (Roflu) in ameliorating microglial senescence and neuroinflammation following RIR injury. C57BL/6J mice underwent RIR surgery, and Roflu treatment was administered intraperitoneally. BV2 microglial cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) to simulate ischemic conditions in vitro. SA-β-gal staining was used to detect cellular senescence. Quantitative PCR and ELISA were used to examine the levels of senescence-associated secretory phenotype (SASP) factors. Hematoxylin and eosin (H&E) staining was performed on retinal sections to assess retinal morphology and thickness. Surviving RGCs were labeled and quantified in retinal whole-mounts using immunofluorescence (IF). Furthermore, Western blot and IF staining were used to quantify the proteins associated with the cell cycle and NLRP3 inflammasomes. Roflu treatment reduced microglial senescence, ROS production, and secretion of pro-inflammatory cytokines in OGD/R-exposed BV2 cells. It also restored cell proliferation capacity and reversed OGD/R-induced cell cycle arrest. In vivo, Roflu alleviated retinal senescence, preserved retinal thickness, and protected against RGCs death in the RIR mouse model. Mechanistically, Roflu inhibited the NLRP3 inflammasome activation and suppressed DNA damage signaling pathway in microglia. Roflu exerts neuroprotective effects by mitigating microglial senescence and inflammation via inhibition of the NLRP3 inflammasome in RIR injury. These findings suggest that Roflu may serve as a promising therapeutic strategy for retinal diseases associated with ischemic injury by targeting microglial senescence.

Dimethyl Sulfoxide Attenuates Ionizing Radiation-induced Centrosome Overduplication and Multipolar Cell Division in Human Induced Pluripotent Stem Cells.

Centrosomes are important organelles for cell division and genome stability. Ionizing radiation exposure efficiently induces centrosome overduplication via the disconnection of the cell and centrosome duplication cycles. Over duplicated centrosomes cause mitotic catastrophe or chromosome aberrations, leading to cell death or tumorigenesis. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into all organs. To maintain pluripotency, PSCs show specific cellular dynamics, such as a short G1 phase and silenced cell-cycle checkpoints for high cellular proliferation. However, how exogenous DNA damage affects cell cycle-dependent centrosome number regulation in PSCs remains unknown. This study used human iPSCs (hiPSCs) derived from primary skin fibroblasts as a PSC model to address this question. hiPSCs derived from somatic cells could be a useful tool for addressing the radiation response in cell lineage differentiation. After radiation exposure, the hiPSCs showed a higher frequency of centrosome overduplication and multipolar cell division than the differentiated cells. To suppress the indirect effect of radiation exposure, we used the radical scavenger dimethyl sulfoxide (DMSO). Combined treatment with radiation and DMSO efficiently suppressed DNA damage and centrosome overduplication in hiPSCs. Our results will contribute to the understanding of the dynamics of stem cells and the assessment of the risk of genome instability for regenerative medicine.

FACT inhibitor CBL0137, administered in an optimized schedule, potentiates radiation therapy for glioblastoma by suppressing DNA damage repair.

Standard-of-care for glioblastoma remains surgical debulking followed by temozolomide and radiation. However, many tumors become radio-resistant while radiation damages surrounding brain tissue. Novel therapies are needed to increase the effectiveness of radiation and reduce the required radiation dose. Drug candidate CBL0137 is efficacious against glioblastoma by inhibiting histone chaperone FACT, known to be involved in DNA damage repair. We investigated the combination of CBL0137 and radiation on glioblastoma. In vitro, we combined CBL0137 with radiation on U87MG and A1207 glioblastoma cells using the clonogenic assay to evaluate the response to several treatment regimens, and the Fast Halo Assay to examine DNA repair. In vivo, we used the optimum combination treatment regimen to evaluate the response of orthotopic tumors in nude mice. In vitro, the combination of CBL0137 and radiation is superior to either alone and administering CBL0137 two hours prior to radiation, having the drug present during and for a prolonged period post-radiation, is an optimal schedule. CBL0137 inhibits DNA damage repair following radiation and affects the subcellular distribution of histone chaperone ATRX, a molecule involved in DNA repair. In vivo, one dose of CBL0137 is efficacious and the combination of CBL0137 with radiation increases median survival over either monotherapy. CBL0137 is most effective with radiation for glioblastoma when present at the time of radiation, immediately after and for a prolonged period post-radiation, by inhibiting DNA repair caused by radiation. The combination leads to increased survival making it attractive as a dual therapy.

FACT inhibitor CBL0137, administered in an optimized schedule, potentiates radiation therapy for glioblastoma by suppressing DNA damage repair.

Standard-of-care for glioblastoma remains surgical debulking followed by temozolomide and radiation. However, many tumors become radio-resistant while radiation damages surrounding brain tissue. Novel therapies are needed to increase the effectiveness of radiation and reduce the required radiation dose. Drug candidate CBL0137 is efficacious against glioblastoma by inhibiting histone chaperone FACT, known to be involved in DNA damage repair. We investigated the combination of CBL0137 and radiation on glioblastoma. In vitro, we combined CBL0137 with radiation on U87MG and A1207 glioblastoma cells using the clonogenic assay to evaluate the response to several treatment regimens, and the Fast Halo Assay to examine DNA repair. In vivo, we used the optimum combination treatment regimen to evaluate the response of orthotopic tumors in nude mice. In vitro, the combination of CBL0137 and radiation is superior to either alone and administering CBL0137 two hours prior to radiation, having the drug present during and for a prolonged period post-radiation, is an optimal schedule. CBL0137 inhibits DNA damage repair following radiation and affects the subcellular distribution of histone chaperone ATRX, a molecule involved in DNA repair. In vivo, one dose of CBL0137 is efficacious and the combination of CBL0137 with radiation increases median survival over either monotherapy. CBL0137 is most effective with radiation for glioblastoma when present at the time of radiation, immediately after and for a prolonged period post-radiation, by inhibiting DNA repair caused by radiation. The combination leads to increased survival making it attractive as a dual therapy.

COMMD10 inhibited DNA damage to promote the progression of gastric cancer

PurposeThe copper metabolism MURR1 domain 10 (COMMD10) plays a role in a variety of tumors. Here, we investigated its role in gastric cancer (GC).MethodsOnline prediction tools, quantitative real-time PCR, western blotting and immunohistochemistry were used to evaluate the expression of COMMD10 in GC. The effect of COMMD10 knockdown was investigated in the GC cell lines and in in vivo xenograft tumor experiments. Western blotting and immunofluorescence were used to explore the relationships between COMMD10 and DNA damage.ResultsThe expression of COMMD10 was upregulated in GC compared to that in para-cancerous tissue and correlated with a higher clinical TNM stage (P = 0.044) and tumor size (P = 0.0366). High COMMD10 expression predicted poor prognosis in GC. Knockdown of COMMD10 resulted in the suppression of cell proliferation, migration, and invasion, accompanied by cell cycle arrest and an elevation in apoptosis rate. Moreover, the protein expression of COMMD10 was decreased in cisplatin-induced DNA-damaged GC cells. Suppression of COMMD10 impeded DNA damage repair, intensified DNA damage, and activated ATM–p53 signaling pathway in GC. Conversely, restoration of COMMD10 levels suppressed DNA damage and activation of the ATM-p53 signaling cascade. Additionally, knockdown of COMMD10 significantly restrained the growth of GC xenograft tumors while inhibiting DNA repair, augmenting DNA damage, and activating the ATM–p53 signaling pathway in xenograft tumor tissue.ConclusionCOMMD10 is involved in DNA damage repair and maintains genomic stability in GC; knockdown of COMMD10 impedes the development of GC by exacerbating DNA damage, suggesting that COMMD10 may be new target for GC therapy.

Targeting mTOR and survivin concurrently potentiates radiation therapy in renal cell carcinoma by suppressing DNA damage repair and amplifying mitotic catastrophe

BackgroundRenal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation.Experimental designWe slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization.ResultsEY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy.ConclusionTaken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

Nicotinamide Mononucleotide improves oocyte maturation of mice with type 1 diabetes

BackgroundThe number of patients with type 1 diabetes rises rapidly around the world in recent years. Maternal diabetes has a detrimental effect on reproductive outcomes due to decreased oocyte quality. However, the strategies to improve the oocyte quality and artificial reproductive technology (ART) efficiency of infertile females suffering from diabetes have not been fully studied. In this study, we aimed to examine the effects of nicotinamide mononucleotide (NMN) on oocyte maturation of mouse with type 1 diabetes mouse and explore the underlying mechanisms of NMN’s effect.MethodsStreptozotocin (STZ) was used to establish the mouse models with type 1 diabetes. The successful establishment of the models was confirmed by the results of body weight test, fasting blood glucose test and haematoxylin and eosin (H&E) staining. The in vitro maturation (IVM) rate of oocytes from diabetic mice was examined. Immunofluorescence staining (IF) was performed to examine the reactive oxygen species (ROS) level, spindle/chromosome structure, mitochondrial function, actin dynamics, DNA damage and histone modification of oocytes, which are potential factors affecting the oocyte quality. The quantitative reverse transcription PCR (RT-qPCR) was used to detect the mRNA levels of Sod1, Opa1, Mfn2, Drp1, Sirt1 and Sirt3 in oocytes.ResultsThe NMN supplementation increased the oocyte maturation rate of the mice with diabetes. Furthermore, NMN supplementation improved the oocyte quality by rescuing the actin dynamics, reversing meiotic defects, improving the mitochondrial function, reducing ROS level, suppressing DNA damage and restoring changes in histone modifications of oocytes collected from the mice with diabetes.ConclusionNMN could improve the maturation rate and quality of oocytes in STZ-induced diabetic mice, which provides a significant clue for the treatment of infertility of the patients with diabetes.

Abstract P22: C16orf72/HAPSTR1/TAPR1 Interacts with BRCA1 and Senataxin to Regulate R-loops at Stalled Replication Forks and Confer Resistance to PARP Disruption

Abstract Poly (ADP-ribose) polymerase (PARP) proteins play a crucial role in DNA repair, and their inhibition is toxic in cells with defects in homologous recombination (HR). Whilst this interaction is well-established, other synthetic lethal interactions with PARP1/PARP2 disruption are poorly explored. Hence, we performed a genome-wide CRISPR screen to identify genes which are synthetic lethal with PARP1/PARP2 disruption. We discovered that the uncharacterized C16orf72/HAPSTR1/TAPR1 gene is essential for the survival of PARP1/PARP2 double knock-out cells but not the parental wildtype cells. We further show that C16orf72 is a novel modulator of R-loop and is crucial in facilitating replication fork restart and suppressing DNA damage in response to replication stress. We show that C16orf72 functions in a parallel pathway to PARP1/PARP2 to suppress DNA:RNA hybrids that accumulate at stalled replication forks, and it does this by interacting with BRCA1 and the DNA/RNA helicase Senataxin. Citation Format: Abhishek Bharadwaj Sharma, Muhammad Khairul Ramlee, Joel Kosmin, Martin R. Higgs, Amy Wolstenholme, George E. Ronson, Dylan Jones, Daniel Ebner, Noor Shamkhi, David Sims, Paul W. G. Wijnhoven, Josep Forment, Ian Gibbs-Seymour, Nicholas D. Lakin. C16orf72/HAPSTR1/TAPR1 Interacts with BRCA1 and Senataxin to Regulate R-loops at Stalled Replication Forks and Confer Resistance to PARP Disruption [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P22.

Identification of different classes of genome instability suppressor genes through analysis of DNA damage response markers.

Cellular pathways that detect DNA damage are useful for identifying genes that suppress DNA damage, which can cause genome instability and cancer predisposition syndromes when mutated. We identified 199 high-confidence and 530 low-confidence DNA damage-suppressing (DDS) genes in Saccharomyces cerevisiae through a whole-genome screen for mutations inducing Hug1 expression, a focused screen for mutations inducing Ddc2 foci, and data from previous screens for mutations causing Rad52 foci accumulation and Rnr3 induction. We also identified 286 high-confidence and 394 low-confidence diverse genome instability-suppressing (DGIS) genes through a whole-genome screen for mutations resulting in increased gross chromosomal rearrangements and data from previous screens for mutations causing increased genome instability as assessed in a diversity of genome instability assays. Genes that suppress both pathways (DDS+ DGIS+) prevent or repair DNA replication damage and likely include genes preventing collisions between the replication and transcription machineries. DDS+ DGIS- genes, including many transcription-related genes, likely suppress damage that is normally repaired properly or prevent inappropriate signaling, whereas DDS- DGIS+ genes, like PIF1, do not suppress damage but likely promote its proper, nonmutagenic repair. Thus, induction of DNA damage markers is not a reliable indicator of increased genome instability, and the DDS and DGIS categories define mechanistically distinct groups of genes.

High expression of BCAT1 sensitizes AML cells to PARP inhibitor by suppressing DNA damage response.

Previous evidence has confirmed that branched-chain aminotransferase-1 (BCAT1), a key enzyme governing branched-chain amino acid (BCAA) metabolism, has a role in cancer aggression partly by restricting αKG levels and inhibiting the activities of the αKG-dependent enzyme family. The oncogenic role of BCAT1, however, was not fully elucidated in acute myeloid leukemia (AML). In this study, we investigated the clinical significance and biological insight of BCAT1 in AML. Using q-PCR, we analyzed BCAT1 mRNAs in bone marrow samples from 332 patients with newly diagnosed AML. High BCAT1 expression independently predicts poor prognosis in patients with AML. We also established BCAT1 knockout (KO)/over-expressing (OE) AML cell lines to explore the underlying mechanisms. We found that BCAT1 affects cell proliferation and modulates cell cycle, cell apoptosis, and DNA damage/repair process. Additionally, we demonstrated that BCAT1 regulates histone methylation by reducing intracellular αKG levels in AML cells. Moreover, high expression of BCAT1 enhances the sensitivity of AML cells to the Poly (ADP-ribose) polymerase (PARP) inhibitor both in vivo and in vitro. Our study has demonstrated that BCAT1 expression can serve as a reliable predictor for AML patients, and PARP inhibitor BMN673 can be used as an effective treatment strategy for patients with high BCAT1 expression. KEY MESSAGES: High expression of BCAT1 is an independent risk factor for poor prognosis in patients with CN-AML. High BCAT1 expression in AML limits intracellular αKG levels, impairs αKG-dependent histone demethylase activity, and upregulates H3K9me3 levels. H3K9me3 inhibits ATM expression and blocks cellular DNA damage repair process. Increased sensitivity of BCAT1 high expression AML to PARP inhibitors may be used as an effective treatment strategy in AML patients.

Abstract B023: SMARCAL1 is a novel candidate therapeutic target for ALT-positive osteosarcoma

Abstract The DNA translocase SMARCAL1 has been characterized by our laboratory and others as a DNA replication-associated factor that promotes the remodeling of stalled replication forks to facilitate the repair of DNA damage and the restart of DNA synthesis. Recent genome-wide analyses of CRISPR-Cas9 loss-of-function screens in pediatric cancer cell lines have suggested an essential function for SMARCAL1 in a subset of pediatric tumors (Dharia NV et al., Nature Genetics, 2021). In agreement with these observations, pan-cancer analysis of dependency scores in over 1,000 cell lines from the DepMap project revealed an important role for SMARCAL1 in tumors of mesenchymal origin and especially in osteosarcoma. We found that SMARCAL1 dependency in osteosarcoma is selectively associated with lack of telomerase expression. In the absence of telomerase, cancer cells mostly rely on a recombination-based mechanism, known as ALT (Alternative Lengthening of Telomeres), for the maintenance of their telomere length. Surprisingly, by comparing genome-wide CRISPR screening data of known ALT-positive vs ALT-negative osteosarcoma cell lines, we found that SMARCAL1 is among the genes that are most selectively essential in ALT-positive tumors. We validated in multiple cellular models a strict correlation and functional relationship between ALT status and SMARCAL1 dependency. In line with previous findings (Cox KE et al., Cell Reports, 2016), we observed that SMARCAL1 localizes to a subset of telomeres in ALT-positive cells and loss of SMARCAL1 induces accumulation of telomere damage. SMARCAL1 enzymatic activity and its RPA-binding function contribute to suppress DNA damage in ALT-positive cells. Furthermore, our studies suggest that SMARCAL1 protects telomere stability in ALT-positive cancer cells by limiting PRIMPOL-mediated restart of stalled replication forks. In addition, SMARCAL1 deficiency enhances cell-intrinsic immune response and anti-tumor immunity. Collectively, our studies have uncovered SMARCAL1 as a novel candidate therapeutic target for the treatment of ALT-positive cancer patients. In these patients, SMARCAL1 inhibition may have a dual beneficial effect by promoting selective cancer cell toxicity combined with stimulation of anti-tumor immunity and enhancement of immunotherapy response. Citation Format: Angelo Taglialatela, Xiao Chen, Zahra Fatema Khan, Filemon Dela Cruz, Dua Shivi, Dillon Oswald, Azeroglu Benura, Eros Lazzerini Denchi, Min Jaewon, Andrew L. Kung, Alberto Ciccia. SMARCAL1 is a novel candidate therapeutic target for ALT-positive osteosarcoma [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 B023.

Targeting of RRM2 suppresses DNA damage response and activates apoptosis in atypical teratoid rhabdoid tumor

BackgroundAtypical teratoid rhabdoid tumors (ATRT) is a rare but aggressive malignancy in the central nervous system, predominantly occurring in early childhood. Despite aggressive treatment, the prognosis of ATRT patients remains poor. RRM2, a subunit of ribonucleotide reductase, has been reported as a biomarker for aggressiveness and poor prognostic conditions in several cancers. However, little is known about the role of RRM2 in ATRT. Uncovering the role of RRM2 in ATRT will further promote the development of feasible strategies and effective drugs to treat ATRT.MethodsExpression of RRM2 was evaluated by molecular profiling analysis and was confirmed by IHC in both ATRT patients and PDX tissues. Follow-up in vitro studies used shRNA knockdown RRM2 in three different ATRT cells to elucidate the oncogenic role of RRM2. The efficacy of COH29, an RRM2 inhibitor, was assessed in vitro and in vivo. Western blot and RNA-sequencing were used to determine the mechanisms of RRM2 transcriptional activation in ATRT.ResultsRRM2 was found to be significantly overexpressed in multiple independent ATRT clinical cohorts through comprehensive bioinformatics and clinical data analysis in this study. The expression level of RRM2 was strongly correlated with poor survival rates in patients. In addition, we employed shRNAs to silence RRM2, which led to significantly decrease in ATRT colony formation, cell proliferation, and migration. In vitro experiments showed that treatment with COH29 resulted in similar but more pronounced inhibitory effect. Therefore, ATRT orthotopic mouse model was utilized to validate this finding, and COH29 treatment showed significant tumor growth suppression and prolong overall survival. Moreover, we provide evidence that COH29 treatment led to genomic instability, suppressed homologous recombinant DNA damage repair, and subsequently induced ATRT cell death through apoptosis in ATRT cells.ConclusionsCollectively, our study uncovers the oncogenic functions of RRM2 in ATRT cell lines, and highlights the therapeutic potential of targeting RRM2 in ATRT. The promising effect of COH29 on ATRT suggests its potential suitability for clinical trials as a novel therapeutic approach for ATRT.

Epigenetically Downregulated Breast Cancer Gene 2 through Acetyltransferase Lysine Acetyltransferase 2B Increases the Sensitivity of Colorectal Cancer to Olaparib

Olaparib suppresses DNA damage repair by inhibiting the poly ADP ribose polymerase (PARP), especially in cancers with BRCA1/2 mutations or the BRCA-ness phenotype. However, the first trials showed that some patients with defective DNA damage repair are still resistant to olaparib. The recovery of the wildtype BRCA is a prominent mechanism of PARP inhibitor (PARPi) resistance in BRCA-deficient tumors, but additional molecular features of olaparib resistance remain poorly understood. The objective of our study was to find molecular parameters that contribute to olaparib response or resistance in CRC. We report that histone acetyltransferase KAT2B decreases BRCA2 expression by reducing the acetylation of the 27th amino acid in histone H3 (H3K27) at the promoter of the BRCA2 gene in colorectal cancer (CRC). This increases the sensitivity of CRC cells toward olaparib treatment. The H3K27ac binding domain of BRCA2 may be required for its transcription. Low endogenous KAT2B expression, which we identify in a subset of cultured BRCA2-expressing CRC cells, leads to an accumulation of γH2AX (more DNA damage), resulting in low PARPi resistance in BRCA-expressing cells. Our results reveal KAT2B and histone acetylation as regulators of BRCA2 expression and PARPi responses in BRCA2-expressing CRC cells, providing further insights into molecular prerequisites for targeting BRCA-functional tumors.

The entanglement of DNA damage and pattern recognition receptor signaling

Cells are under constant pressure to suppress DNA damage originating from both exogenous and endogenous sources. Cellular responses to DNA damage help to prevent mutagenesis and cell death that arises when DNA damage is either left unrepaired or repaired inaccurately. During the “acute phase” of DNA damage signaling, lesions are recognized, processed, and repaired to restore the primary DNA sequence whilst cell cycle checkpoints delay mitotic progression, cell death and the propagation of errors to daughter cells. Increasingly, there is recognition of a “chronic phase” of DNA damage signaling, exemplified by the secretion of dozens of cytokines days after the inciting damage event. In this review, we focus on the cellular origin of these chronic responses, the molecular pathways that control them and the increasing appreciation for the interconnection between acute and chronic DNA damage responses.

The Chromatin Remodeler CHD4 Sustains Ewing Sarcoma Cell Survival by Controlling Global Chromatin Architecture.

CRISPR/Cas9 screening in Ewing sarcoma identifies a dependency on CHD4, which is crucial for the maintenance of chromatin architecture to suppress DNA damage and a promising therapeutic target for DNA damage repair-deficient malignancies.

Venetoclax Synergizes with IMGN632, a Novel CD123-Targeting Antibody Conjugated to a DNA Alkylating Payload, By Suppressing DNA Damage Response and Potentiating Apoptosis in Acute Myeloid Leukemia in Vitro Models

CD123, a component of interleukin-3 receptor, is overexpressed on the surface of acute myeloid leukemia (AML) blasts and leukemic stem cells compared with normal hematopoietic progenitors, which makes this antigen an attractive therapeutic target. IMGN632 is a novel antibody-drug-conjugate comprising a high affinity CD123-targeting antibody and DNA alkylating cytotoxic payload. In preclinical studies, IMGN632 demonstrated high potency and selective cytotoxicity against AML leukemic cells vs normal myeloid progenitors, suggesting reduced likelihood of myelosuppression (Kovtun, 2018). Importantly, we previously showed high synergy of IMGN632 combination with BCL-2 inhibitor venetoclax (VEN) and DNA hypomethylating azacytidine (AZA) in AML cell lines and xenograft models (ASH 2020, #617). IMGN632 demonstrated encouraging single agent activity and favorable tolerability in relapsed/refractory AML patients (ASH 2019 #734, ASCO 2020 TPS7563, NCT03386513) and is currently tested in combination with VEN and/ or AZA in relapsed and frontline CD123-positive AML patients (NCT04086264). In this study, we provide new insight into molecular mechanisms underlying the synergistic efficacy of these combinations using AML models in vitro. AML cells with FLT3-ITD mutations expressed higher levels of CD123, as previously reported (Han 2013). FLT3-ITD cell lines were particularly sensitive to IMGN632 with IC 50 values at low ng/ml range. Correspondingly, as evidenced by large scale drug combination screen using BLISS independence model, triple IMGN632/VEN/AZA treatment was synergistic at multiple dosage levels and ratios, suggesting a favorable potency of triple combination in FLT3-mutated subtypes of AML associated with poor prognosis. The efficacy of IMGN632/VEN/AZA combination was however decreased in cells lacking functional TP53. shRNA-mediated knockdown of TP53 in FLT3-ITD MOLM13 cells resulted in increased cell viability, reduced apoptosis and downregulation of cleaved caspase-3 and PARP. While both treated wild-type and TP53 KD cells showed robust overexpression of phosphorylated histone H2AX (p-H2AX), TP53 KD cells expressed higher levels of anti-apoptotic MCL-1 and lower levels of pro-apoptotic BAX, indicative of their intrinsic inability to fully execute apoptosis and resulting in suppressed response to triple combination. Importantly, in sensitive AML cells, IMGN632 induced profound DNA damage, G 1 (MOLM13 and MOLM14 cells) or G 2/M (MV4-11 cells) phase cell cycle arrest and decline in S-phase population, all above augmented by VEN/AZA co-treatment. Observed increase in p-H2AX was accompanied by apoptotic caspase-3 and PARP cleavage and was fully prevented by QVD-OPh caspase inhibitor, suggesting that double-stranded DNA damage and p-H2AX triggered by triple combination were secondary events associated with apoptosis but not the initial cause of cell cycle arrest. Thus, given that cytotoxic payload of IMGN632 is a DNA alkylator, we next tested its impact on mediators of the DNA damage response (DDR). IMGN632 alone induced phosphorylation of p53 at Ser15, a step required for p53 transcriptional activity, and triggered activating phosphorylation of checkpoint kinase Chk1 (Ser345), known to facilitate cell cycle arrest and DNA damage repair, thus preventing treated cells from progressing through S-phase but also increasing the potential of repairing IMGN632-induced DNA damage (Fig. 1A). Upon combined IMGN632+VEN treatment, p53 phosphorylation was sustained, but p-ChK1 decreased, suggesting that VEN may impair protective DDR triggered by IMGN632, resulting in greater cell death. To support this hypothesis, we showed that IMGN632 treatment prior to VEN produced significant synergistic activity (Fig. 1B). Together, these results suggest that VEN, apart from its canonical inhibitory effect on anti-apoptotic BCL-2, exerts previously unrecognized ability to suppress DDR program in AML and augments activity of DNA damaging IMGN632. Failure of cells to sustain DDR in the presence of VEN constitutes a key aspect of high efficacy of IMGN/VEN/AZA combination in AML.

Study on the Multimodal Anticancer Mechanism of Ru(II)/Ir(III) Complexes Bearing a Poly(ADP-ribose) Polymerase 1 Inhibitor.

A series of novel ruthenium(II) and iridium(III) complexes (Ru1-Ru3 and Ir1-Ir3) with different ancillary ligands and a PARP-1-inhibitory chelating ligand 2-(2,3-dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (L1) were designed and prepared. The target complexes were structurally characterized by NMR and ESI-MS techniques. Among them, the crystal and molecular structures of Ir1 and Ir2 were also determined by X-ray crystallography. These complexes retained the PARP-1 enzyme inhibitory effect of L1 and showed potent antiproliferative activity on the tested cancer cell lines. The ruthenium(II) complexes Ru1-Ru3 were found to be more cytotoxic than the iridium(III) complexes Ir1-Ir3. Further investigations revealed that the most active complex Ru3 induced apoptosis in MCF-7 cells by multiple modes, inclusive of inducing DNA damage, suppressing DNA damage repair, disturbing cell cycle distribution, decreasing the mitochondrial membrane potential, and increasing the intracellular reactive oxygen species levels.

Cytosine–phosphate–guanine oligodeoxynucleotides alleviate radiation-induced kidney injury in cervical cancer by inhibiting DNA damage and oxidative stress through blockade of PARP1/XRCC1 axis

BackgroundRadiotherapy can cause kidney injury in patients with cervical cancer. This study aims to investigate the possible molecular mechanisms by which CpG-ODNs (Cytosine phosphate guanine-oligodeoxynucleotides) regulate the PARP1 (poly (ADP-ribose) polymerase 1)/XRCC1 (X-ray repair cross-complementing 1) signaling axis and its impact on radiation kidney injury (RKI) in cervical cancer radiotherapy.MethodsThe GSE90627 dataset related to cervical cancer RKI was obtained from the Gene Expression Omnibus (GEO) database. Bioinformatics databases and R software packages were used to analyze the target genes regulated by CpG-ODNs. A mouse model of RKI was established by subjecting C57BL/6JNifdc mice to X-ray irradiation. Serum blood urea nitrogen (BUN) and creatinine levels were measured using an automated biochemical analyzer. Renal tissue morphology was observed through HE staining, while TUNEL staining was performed to detect apoptosis in renal tubular cells. ELISA was conducted to measure levels of oxidative stress-related factors in mouse serum and cell supernatant. An in vitro cell model of RKI was established using X-ray irradiation on HK-2 cells for mechanism validation. RT-qPCR was performed to determine the relative expression of PARP1 mRNA. Cell proliferation activity was assessed using the CCK-8 assay, and Caspase 3 activity was measured in HK-2 cells. Immunofluorescence was used to determine γH2AX expression.ResultsBioinformatics analysis revealed that the downstream targets regulated by CpG-ODNs in cervical cancer RKI were primarily PARP1 and XRCC1. CpG-ODNs may alleviate RKI by inhibiting DNA damage and oxidative stress levels. This resulted in significantly decreased levels of BUN and creatinine in RKI mice, as well as reduced renal tubular and glomerular damage, lower apoptosis rate, decreased DNA damage index (8-OHdG), and increased levels of antioxidant factors associated with oxidative stress (SOD, CAT, GSH, GPx). Among the CpG-ODNs, CpG-ODN2006 had a more pronounced effect. CpG-ODNs mediated the inhibition of PARP1, thereby suppressing DNA damage and oxidative stress response in vitro in HK-2 cells. Additionally, PARP1 promoted the formation of the PARP1 and XRCC1 complex by recruiting XRCC1, which in turn facilitated DNA damage and oxidative stress response in renal tubular cells. Overexpression of either PARP1 or XRCC1 reversed the inhibitory effects of CpG-ODN2006 on DNA damage and oxidative stress in the HK-2 cell model and RKI mouse model.ConclusionCpG-ODNs may mitigate cervical cancer RKI by blocking the activation of the PARP1/XRCC1 signaling axis, inhibiting DNA damage and oxidative stress response in renal tubule epithelial cells.

DHX36 maintains genomic integrity by unwinding G-quadruplexes.

The guanine-rich stretch of single-stranded DNA (ssDNA) forms a G-quadruplex (G4) in a fraction of genic and intergenic chromosomal regions. The probability of G4 formation increases during events causing ssDNA generation, such as transcription and replication. In turn, G4 abrogates these events, leading to DNA damage. DHX36 unwinds G4-DNA in vitro and in human cells. However, its spatial correlation with G4-DNA in vivo and its role in genome maintenance remain unclear. Here, we demonstrate a connection between DHX36 and G4-DNA and its implications for genomic integrity. The nuclear localization of DHX36 overlapped with that of G4-DNA, RNA polymerase II, and a splicing-related factor. Depletion of DHX36 resulted in accumulated DNA damage, slower cell growth, and enhanced cell growth inhibition upon treatment with a G4-stabilizing compound; DHX36 expression reversed these defects. In contrast, the reversal upon expression of DHX36 mutants that could not bind G4 was imperfect. Thus, DHX36 may suppress DNA damage by promoting the clearance of G4-DNA for cell growth and survival. Our findings deepen the understanding of G4 resolution in the maintenance of genomic integrity.