Impaired DNA Damage Repair Research Articles

Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro

Stromal antigen 1 and 2 (STAG1 and STAG2) are two mutually exclusive components of the cohesin complex that is crucial for centromeric and telomeric cohesion. Beyond its structural role, STAG2 also plays a pivotal role in homologous recombination (HR) repair and has emerged as a promising therapeutic target in cancer treatment. Here, we employed a fluorescence polarization (FP)-based high-throughput screening and identified KPT-6566 as a dual inhibitor of STAG1 and STAG2. Biochemical and biophysical analyses demonstrated that KPT-6566 directly binds to STAG1 and STAG2, disrupting their interactions with SCC1 and double-stranded DNA. A metaphase chromosome spread assay showed that KPT-6566 causes premature chromosome separation and induces chromosome damages in HeLa cells. Furthermore, KPT-6566 also impairs DNA damage repair, leading to the accumulation of double-strand breaks and cell apoptosis. Finally, KPT-6566 can sensitize HeLa and HepG2 cells to PARP inhibitor Olaparib and the NHEJ inhibitor UMI-77, exhibiting a synergistic effect in suppressing cell proliferation. Our findings highlight the potential of STAG1/2 as promising therapeutic targets in cancer treatment, particularly when they are targeted in combination with other DNA damage response inhibitors.

Coordinated protein modules define DNA damage responses to carboplatin at single cell resolution in human ovarian carcinoma models.

Tubo-ovarian high-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy and frequently responds to platinum-based chemotherapy because of common genetic and somatic impairment of DNA damage repair (DDR) pathways. The mechanisms of clinical platinum resistance are diverse and poorly molecularly defined. Consequently, there are no biomarkers or medicines that improve patient outcomes. Herein we use single cell mass cytometry (CyTOF) to systematically evaluate the phosphorylation and abundance of proteins known to participate in the DNA damage response (DDR). Single cell analyses of highly characterized HGSC cell lines that phenocopy human patients show that cells with comparable levels of intranuclear platinum, a proxy for carboplatin uptake, undergo different cell fates. Unsupervised analyses revealed a continuum of DDR responses. Decompositional methods were used to identify eight distinct protein modules of carboplatin resistance and sensitivity at single cell resolution. CyTOF profiling of primary and secondary platinum-resistance patient models shows that a complex DDR sensitivity module is strongly associated with response, suggesting it as a potential tool to clinically characterize complex drug resistance phenotypes.

Mitochondrial dysfunction and impaired DNA damage repair through PICT1 dysregulation in alveolar type II cells in emphysema

BackgroundAlveolar type II (ATII) cells have a stem cell potential in the adult lung and repair the epithelium after injury induced by harmful factors. Their damage contributes to emphysema development, characterized by alveolar wall destruction. Cigarette smoke is the main risk factor for this disease development.MethodsATII cells were obtained from control non-smoker and smoker organ donors and emphysema patients. Isolated cells were used to study the role of PICT1 in this disease. Also, a cigarette smoke-induced murine model of emphysema was applied to define its function in disease progression further.ResultsDecreased PICT1 expression was observed in human and murine ATII cells in emphysema. PICT1 was immunoprecipitated, followed by mass spectrometry analysis. We identified MRE11, which is involved in DNA damage repair, as its novel interactor. PICT1 and MRE11 protein levels were decreased in ATII cells in this disease. Moreover, cells with PICT1 deletion were exposed to cigarette smoke extract. This treatment induced cellular and mitochondrial ROS, cell cycle arrest, nuclear and mitochondrial DNA damage, decreased mitochondrial respiration, and impaired DNA damage repair.ConclusionsThis study indicates that PICT1 dysfunction can negatively affect genome stability and mitochondrial activity in ATII cells, contributing to emphysema development. Targeting PICT1 can lead to novel therapeutic approaches for this disease.

The radiosensitivity of niraparib tosylate to esophageal squamous cell carcinoma is stronger under hypoxia and higher-pressure conditions

Background: Radiotherapy is an effective method for esophageal squamous cell carcinoma (ESCC) treatment show reduced effectiveness in long-term due to reduced sensitivity of cancer cells to radiotherapy. Niraparib tosylate, a poly ADP-ribose polymerase (PARP) inhibitor may enhance the radiosensitivity of these cells. Hence, the present study was aimed to study the effects of niraparib tosylate on ESCC proliferation, apoptosis and cell cycle along with radiosensitivity enhancement efficiency. Materials and methods: ESCC cell lines, KYSE-30 were subjected to niraparib tosylate or irradiation treatments or combination of both. CCK8 and colony formation assays were performed to monitor cell growth status. Cell cycle and apoptosis level were investigated through flow cytometry. The expressions of targeted genes were detected at protein levels by western blot. Besides, xenografts were successfully established in immunocompromised mice. Student’s t-test was used to compare data and P < 0.05 was considered statistically significant. Results: Treatment with niraparib tosylate showed G2/M cycle arrest and apoptosis in KYSE-30 cells and the rate was observed to be more significant with combination therapy. These effects were further strengthened when cells were cultured under hypoxia and high atmospheric pressure. An enhanced susceptibility to radiotherapy was observed with niraparib tosylate combination. The homologous recombination related regulator, Rad51, showed remarkable upregulation in cancer cells exposed to combination of drug-radiation treatment. The tumor burden was also relieved in xenograft mouse models with combination of drug-radiation treatment, indicating the potential of niraparib tosylate use clinical cancer therapy. Conclusion: From the results, a novel function of niraparib tosylate can be established, impairing DNA damage repair efficiency to increase the susceptibility of ESCC to radiotherapy, resulting in cell apoptosis and G2/M cycle arrest in vitro and in vivo.

Functional Analysis of BARD1 Missense Variants on Homology-Directed Repair in Ovarian and Breast Cancers.

Women with germline BRCA1 mutations face an increased risk of developing breast and ovarian cancers. BARD1 (BRCA1 associated RING domain 1) is an essential heterodimeric partner of BRCA1, and mutations in BARD1 are also associated with these cancers. While BARD1 mutations are recognized for their cancer susceptibility, the exact roles of numerous BARD1 missense mutations remain unclear. In this study, we conducted functional assays to assess the homology-directed DNA repair (HDR) activity of all BARD1 missense substitutions identified in 55 breast and ovarian cancer samples, using the real-world data from the COSMIC and cBioPortal databases. Seven BARD1 variants (V85M, P187A, G491R, R565C, P669L, T719R, and Q730L) were confirmed to impair DNA damage repair. Furthermore, cells harboring these BARD1 variants exhibited increased sensitivity to the chemotherapeutic drugs, cisplatin, and olaparib, compared to cells expressing wild-type BARD1. These findings collectively suggest that these seven missense BARD1 variants are likely pathogenic and may respond well to cisplatin-olaparib combination therapy. This study not only enhances our understanding of BARD1's role in DNA damage repair but also offers valuable insights into predicting therapy responses in patients with specific BARD1 missense mutations.

Contribution of Germline TET2 Variants to the Pathogenesis of Kidney Diseases through Impaired DNA Damage Repair and Activation of Cytosolic Nucleotide Sensors

Contribution of Germline TET2 Variants to the Pathogenesis of Kidney Diseases through Impaired DNA Damage Repair and Activation of Cytosolic Nucleotide Sensors

Bisphenol AP inhibits mouse oocyte maturation in vitro by disrupting cytoskeleton architecture and cell cycle processes

Bisphenol A (BPA) is among the extensively researched environmental endocrine-disrupting chemicals (EDCs), and its utilization is restricted owing to the detrimental impacts it has on human health. Bisphenol AP (BPAP) is one of the alternatives to BPA, but the influence of BPAP on human health has not been elucidated. The objective of the current research was to determine the influence of BPAP exposure on the in vitro maturation of mouse oocytes and to explore its potential reproductive toxicity. BPAP exposure was found to inhibit polar body extrusion during mouse oocyte maturation, resulting in an arrest at the metaphase I stage of meiosis. Exposure to BPAP led to sustained activation of BubR1, preventing the degradation of both Securin and Cyclin B1. Mechanistically, BPAP exposure disrupts spindle assembly and chromosome alignment. Levels of acetylated α-tubulin were significantly elevated in BPAP-treated oocytes, reflecting decreased spindle stability. Exposure to BPAP also induced DNA damage and impaired DNA damage repair. In addition, BPAP exposure altered histone modification levels. In summary, this investigation suggests that exposure to BPAP can influence cytoskeletal assembly, interfere with cell cycle progression, induce DNA damage, alter histone modifications, and ultimately impede oocyte meiotic maturation. This investigation enhances understanding of the impact of bisphenol analogs on female gametes, underscoring that BPAP cannot be considered a reliable replacement for BPA.

USP14 inhibition promotes DNA damage repair and represses ovarian granulosa cell senescence in premature ovarian insufficiency

BackgroundPremature ovarian insufficiency (POI) is a condition characterized by a substantial decline or loss of ovarian function in women before the age of 40. However, the pathogenesis of POI remains to be further elucidated, and specific targeted drugs which could delay or reverse ovarian reserve decline are urgently needed. Abnormal DNA damage repair (DDR) and cell senescence in granulosa cells are pathogenic mechanisms of POI. Ubiquitin-specific protease 14 (USP14) is a key enzyme that regulates the deubiquitylation of DDR-related proteins, but whether USP14 participates in the pathogenesis of POI remains unclear.MethodsWe measured USP14 mRNA expression in granulosa cells from biochemical POI (bPOI) patients. In KGN cells, we used IU1 and siRNA-USP14 to specifically inhibit USP14 and constructed a cell line stably overexpressing USP14 to examine its effects on DDR function and cellular senescence in granulosa cells. Next, we explored the therapeutic potential of IU1 in POI mouse models induced by D-galactose.ResultsUSP14 expression in the granulosa cells of bPOI patients was significantly upregulated. In KGN cells, IU1 treatment and siUSP14 transfection decreased etoposide-induced DNA damage levels, promoted DDR function, and inhibited cell senescence. USP14 overexpression increased DNA damage, impaired DDR function, and promoted cell senescence. Moreover, IU1 treatment and siUSP14 transfection increased nonhomologous end joining (NHEJ), upregulated RNF168, Ku70, and DDB1, and increased ubiquitinated DDB1 levels in KGN cells. Conversely, USP14 overexpression had the opposite effects. Intraperitoneal IU1 injection alleviated etoposide-induced DNA damage in granulosa cells, ameliorated the D-galactose-induced POI phenotype, promoted DDR, and inhibited cell senescence in ovarian granulosa cells in vivo.ConclusionsUpregulated USP14 in ovarian granulosa cells may play a role in POI pathogenesis, and targeting USP14 may be a potential POI treatment strategy. Our study provides new insights into the pathogenesis of POI and a novel POI treatment strategy.

Fance deficiency impaired DNA damage repair of prospermatogonia and altered the repair dynamics of spermatocytes

BackgroundNon-obstructive azoospermia (NOA) is the most severe form of male infertility and affects approximately 1% of men worldwide. Fanconi anemia (FA) genes were known for their essential role in DNA repair and growing evidence showed the crucial role of FA pathway in NOA. However, the underlying mechanisms for Fance deficiency lead to a serious deficit and delayed maturation of male germ cells remain unclear.MethodsWe used Fance deficiency mouse model for experiments, and collected testes or epididymides from mice at 8 weeks (8W), 17.5 days post coitum (dpc), and postnatal 11 (P11) to P23. The mice referred to three genotypes: wildtype (Fance+/+), heterozygous (Fance+/-), and homozygous (Fance−/−). Hematoxylin and eosin staining, immunofluorescence staining, and surface spread of spermatocytes were performed to explore the mechanisms for NOA of Fance−/− mice. Each experiment was conducted with a minimum of three biological replicates and Kruskal-Wallis with Dunn’s correction was used for statistical analysis.ResultsIn the present study, we found that the adult male Fance−/− mice exhibited massive germ cell loss in seminiferous tubules and dramatically decreased sperms in epididymides. During the embryonic period, the number of Fance−/− prospermatogonia decreased significantly, without impacts on the proliferation (Ki-67, PCNA) and apoptosis (cleaved PARP, cleaved Caspase 3) status. The DNA double-strand breaks (γH2AX) increased at the cellular level of Fance−/− prospermatogonia, potentially associated with the increased nonhomologous end joining (53BP1) and decreased homologous recombination (RAD51) activity. Besides, Fance deficiency impeded the progression of meiotic prophase I of spermatocytes. The mechanisms entailed the reduced recruitment of the DNA end resection protein RPA2 at leptotene and recombinases RAD51 and DMC1 at zygotene. It also involved impaired removal of RPA2 at zygotene and FANCD2 foci at pachytene. And the accelerated initial formation of crossover at early pachytene, which is indicated by MLH1.ConclusionsFance deficiency caused massive male germ cell loss involved in the imbalance of DNA damage repair in prospermatogonia and altered dynamics of proteins in homologous recombination, DNA end resection, and crossover, providing new insights into the etiology and molecular basis of NOA.

CYP2E1 in 1,4-dioxane metabolism and liver toxicity: insights from CYP2E1 knockout mice study.

1,4-Dioxane (DX), an emerging water contaminant, is classified as a Group 2B liver carcinogen based on animal studies. Understanding of the mechanisms of action of DX liver carcinogenicity is important for the risk assessment and control of this environmental pollution. Previous studies demonstrate that high-dose DX exposure in mice through drinking water for up to 3 months caused liver mild cytotoxicity and oxidative DNA damage, a process correlating with hepatic CYP2E1 induction and elevated oxidative stress. To access the role of CYP2E1 in DX metabolism and liver toxicity, in the current study, male and female Cyp2e1-null mice were exposed to DX in drinking water (5000 ppm) for 1 week or 3 months. DX metabolism, redox and molecular investigations were subsequently performed on male Cyp2e1-null mice for cross-study comparisons to similarly treated male wildtype (WT) and glutathione (GSH)-deficient Gclm-null mice. Our results show that Cyp2e1-null mice of both genders were resistant to DX-induced hepatocellular cytotoxicity. In male Cyp2e1-null mice exposed to DX for 3 months, firstly, DX metabolism to β-hydroxyethoxyacetic acid was reduced to ~ 36% of WT levels; secondly, DX-induced hepatic redox dysregulation (lipid peroxidation, GSH oxidation, and activation of NRF2 antioxidant response) was substantially attenuated; thirdly, liver oxidative DNA damage was at a comparable level to DX-exposed WT mice, accompanied by suppression of DNA damage repair response; lastly, no aberrant proliferative or preneoplastic lesions were noted in DX-exposed livers. Overall, this study reveals, for the first time, that CYP2E1 is the main enzyme for DX metabolism at high dose and a primary contributor to DX-induced liver oxidative stress and associated cytotoxicity. High dose DX-induced genotoxicity may occur via CYP2E1-independent pathway(s), potentially involving impaired DNA damage repair.

KAT6A Condensates Impair PARP1 Trapping of PARP Inhibitors in Ovarian Cancer.

Most clinical PARP inhibitors (PARPis) trap PARP1 in a chromatin-bound state, leading to PARPi-mediated cytotoxicity. PARPi resistance impedes the treatment of ovarian cancer in clinical practice. However, the mechanism by which cancer cells overcome PARP1 trapping to develop PARPi resistance remains unclear. Here, it is shown that high levels of KAT6A promote PARPi resistance in ovarian cancer, regardless of its catalytic activity. Mechanistically, the liquid-liquid phase separation (LLPS) of KAT6A, facilitated by APEX1, inhibits the cytotoxic effects of PARP1 trapping during PARPi treatment. The stable KAT6A-PARP1-APEX1 complex reduces the amount of PARP1 trapped at the DNA break sites. In addition, inhibition of KAT6A LLPS, rather than its catalytic activity, impairs DNA damage repair and restores PARPi sensitivity in ovarian cancer both in vivo and in vitro. In conclusion, the findings demonstrate the role of KAT6A LLPS in fostering PARPi resistance and suggest that repressing KAT6A LLPS can be a potential therapeutic strategy for PARPi-resistant ovarian cancer.

Lower ratio of IMPDH1 to IMPDH2 sensitizes gliomas to chemotherapy.

Gliomas are the most common primary tumors of the central nervous system, with approximately half of patients presenting with the most aggressive form of glioblastoma. Although several molecular markers for glioma have been identified, they are not sufficient to predict the prognosis due to the extensive genetic heterogeneity within glioma. Our study reveals that the ratio of IMPDH1 to IMPDH2 expression levels serves as a molecular indicator for glioma treatment prognosis. Patients with a higher IMPDH1/IMPDH2 ratio exhibit a worse prognosis, while those with a lower ratio display a more favorable prognosis. We further demonstrate that IMPDH1 plays a crucial role in maintaining cellular GTP/GDP levels following DNA damage compared to IMPDH2. In the absence of IMPDH1, cells experience an imbalance in the GTP/GDP ratio, impairing DNA damage repair capabilities and rendering them more sensitive to TMZ. This study not only introduces a novel prognostic indicator for glioma clinical diagnosis but also offers innovative insights for precise and stratified glioma treatment.

Blocking lipid synthesis induces DNA damage in prostate cancer and increases cell death caused by PARP inhibition.

Androgen deprivation therapy (ADT) is the primary treatment for prostate cancer; however, resistance to ADT invariably develops, leading to castration-resistant prostate cancer (CRPC). Prostate cancer progression is marked by increased de novo synthesis of fatty acids due to overexpression of fatty acid synthase (FASN), making this enzyme a therapeutic target for prostate cancer. Inhibition of FASN results in increased intracellular amounts of ceramides and sphingomyelin, leading to DNA damage through the formation of DNA double-strand breaks and cell death. We found that combining a FASNi with the poly-ADP ribose polymerase (PARP) inhibitor olaparib, which induces cell death by blocking DNA damage repair, resulted in a more pronounced reduction in cell growth than that caused by either drug alone. Human CRPC organoids treated with a combination of PARP and FASNi were smaller, had decreased cell proliferation, and showed increased apoptosis and necrosis. Together, these data indicate that targeting FASN increases the therapeutic efficacy of PARP inhibitors by impairing DNA damage repair, suggesting that combination therapies should be explored for CRPC.

Abstract 1439: Elucidation of the pathogenesis of prostate cancer harboring mutations of epigenetic factor UTX using genetically engineered mice

Abstract Introduction and Objective: Histone modifications are important for tissue homeostasis, and their mutations are involved in carcinogenesis. Mutations in a histone modifier, UTX (KDM6A) are common in prostate cancer (PCa), which is frequently associated with loss of its counterpart UTY (KDM6C). However, mechanisms underlying the development of UTX-deficient PCa are not clear. We aimed to address this issue by generating and analyzing genetically-engineered mice. Methods: We generated mice with deletion of both Utx and Uty in the prostate tissue (UtxΔ, UtyΔ) and crossed with mice with a heterozygous deletion of p53 (p53+/−) to create UtxΔ, UtyΔ and p53+/− compound mice. High-fat diet (HFD) that is known to promote cancer growth was administered to mice. We found that UtxΔ, UtyΔ, p53+/− mice treated with HFD developed PCa with Gleason score (GS) = 3+3. Isolated prostate tissues were subjected to pathologic, molecular, and cellular analyses. In addition, RM-2 mouse PCa cells which do not express UTY were knocked down for Utx (siUtx) and control (siNC) by siRNAs to assess changes in the abilities of proliferation and migration. Results: RM-2 siUtx cells showed increased proliferation and migration compared with RM-2 siNC cells. By performing high-throughput RNA sequencing (RNA seq) and gene set enrichment analysis (GSEA) using mouse prostate tissues, we found that loss of UTX in the prostate activated inflammatory pathways, whereas inactivated DNA damage repair pathways. Immunostaining prostate tissues with an antibody to a macrophage marker, F4/80, exhibited significant accumulation of macrophages in UtxΔ, UtyΔ, p53+/+ mice compared to Utx+, UtyΔ, p53+/+ mice. In addition, immunofluorescent staining of irradiated prostate tissues with an antibody against a DNA repair marker, γH2AX, revealed that the time required for DNA damage repair was markedly prolonged in UtxΔ, UtyΔ, p53+/+ mice compared with Utx+, UtyΔ, p53+/+ mice. Conclusions: These results suggest that loss of UTX contributes to prostate cancer development through promoting inflammation and impairing DNA damage repair, which may serve as novel therapeutic targets in PCa patients with UTX mutations. Citation Format: NOBUHITO MURAMOTO, Masayuki Iwasaki, Yasuyuki Sera, Hisamitsu Ide, Shoichiro Mukai, Tsuyoshi Fukushima, Shigeo Horie, Hiroaki Honda. Elucidation of the pathogenesis of prostate cancer harboring mutations of epigenetic factor UTX using genetically engineered mice [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 1439.

Abstract 5941: Small molecule NSC59984 synergizes with PARP inhibitors in BRCA1 wild type ovarian cancer

Abstract PARP inhibitors (PARPi) targeting poly (ADP-ribose) polymerase are the first clinically approved drugs designed to apply synthetic lethality in BRCA1 mutant/deficient cancer. However, the treatment can cause reversion of BRCA mutation which results in drug resistance. To explore new strategies to improve antitumor efficacy of PARPi, we applied PARPi in combination with a small molecule NSC59984 for cancer therapy. NSC59984 is a small molecular targeting mutant p53 degradation and activating p73 via ROS-ERK2-MDM2 pathway. The treatments with radiation or hydrogen peroxide showed that NSC59984 enhanced DNA comet tails and gamma-H2AX, correlated to reduction of rad51 foci in cancer cells. These results suggest that NSC59984 impairs DNA damage repair via abrogation of homologous recombination (HR). We further applied the combinational treatment of NSC59984 and PARPi in BRCA1 wild-type ovarian cancer cells and found that NSC59984 synergized with PARPi to reduce cell viability, inhibit colony formation, and increase cell death. The combination treatment enhanced DNA damage and correlated with reduction of BRCA1 at the protein level. These results, taken together, suggest that the induction of BRCAness causes a synthetical lethality with PARPi in BRCA1 wild-type cancer cells. Cell cycle profiling showed that the cells were arrested at the G2/M phase in response to combinational treatment. At the G2 phase arrest, the high doses of the combinational drugs led to cell death via mitotic catastrophe, and the intermediate doses induced cellular senescence defined by senescence phenotypic hallmarks including senescence-associated beta-gal staining and a secretome consistent with senescence-associated secretory phenotype (SASP). Therapy-induced senescence (TIS) in cancer cells is considered as one of the mechanisms of tumor recurrence and drug resistance. To reduce the risk of TIS in treatment, we applied senolytic treatment to target senescent cells in the combinational treatment. The senolytic drug ABT263 treatment eliminated the senescent cancer cells from the combinational treatment. ABT263, NSC59984 + PARPi combinational treatment further reduced cell viability. Our study provides a rationale for small molecular compounds targeting HR deficiency in combination with PARPi to treat BRCA1wild-type ovarian cancer cells, and additional senolytic treatment may be required to limit resistance by removal of the TIS. Citation Format: Shengliang Zhang, Lanlan Zhou, Leiqing Zhang, Maximilian Pinho-Schwermann, Benedito Carneiro, John Sedivy, Wafik S. El-Deiry. Small molecule NSC59984 synergizes with PARP inhibitors in BRCA1 wild type ovarian cancer [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 5941.

Abstract 4762: Tumor treating fields (TTFields) increase cancer cell membrane permeability and improve sensitivity to doxorubicin in vitro and in vivo

Abstract Background: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. While the mechanism of action of TTFields has been associated with an antimitotic effect and impaired DNA damage repair mechanisms, the application of TTFields has also been shown to increase the membrane permeability of glioblastoma cells. The current study examined whether increased cell membrane permeability is relevant in other tumor types and may be leveraged to facilitate uptake of the anticancer agent doxorubicin (DOX). Methods: Breast mammary carcinoma (4T1), breast adenocarcinoma (MCF-7), uterine sarcoma (MES-SA), lung fibroblasts (MRC-5), and brain endothelial (HBMVEC) cells were treated with TTFields using the inovitroTM system. Intracellular 7-aminoactinomycin D (7-AAD) accumulation and cell count were measured using flow cytometry to determine membrane permeability and cytotoxicity, respectively. TTFields (300 kHz) together with DOX were applied to 4T1 cells DOX-sensitive (4T1-S) and DOX-resistant (4T1-R, generated by repeated DOX exposure of the 4T1-S cells), followed by flow cytometry analysis of DOX accumulation (24 h treatment) and cell survival using cell count (72 h treatment). For proof of concept, mice were orthotopically inoculated with 4T1-S cells, followed by TTFields treatment for 72 h and injection of DOX (5 mg/kg) 24 h before ending TTFields treatment. DOX fluorescence was measured by flow cytometry in single-cell tumor suspension and whole tumor in vivo imaging system (IVIS). For the efficacy studies, mice were orthotopically inoculated with 4T1-S or 4T1-R cells. Tumors were allowed to grow, and then the mice were treated with TTFields (300 kHz) or sham-heat continuously for 8 days, with DOX (1 mg/kg for 4T1-S cells; 5 mg/kg for 4T1-R cells) or vehicle 2- and 6-days following treatment initiation. Tumor volumes were measured using caliper. Results: TTFields increased 7-AAD accumulation in cancer but not normal cells. Maximal TTFields-induced cellular permeability was identified at 300 kHz for 4T1 cells, whereas TTFields-induced cytotoxicity was the highest at 150 kHz. The application of TTFields allowed DOX accumulation to the same extent in both DOX-resistant and DOX-sensitive cells and sensitized both cell types to DOX cytotoxicity. A 2- to 3-fold higher DOX accumulation was observed in tumors isolated from mice treated with TTFields relative to control mice. Significantly lower tumor volume was observed in mice treated with TTFields and DOX relative to control or mice treated with DOX alone. Conclusions: TTFields increased cancer cell permeability, resulting in enhanced intracellular accumulation of DOX. Concomitant treatment with TTFields and DOX can improve treatment efficacy in DOX-resistant tumors and sensitize DOX-sensitive tumors to lower DOX concentrations. Citation Format: Bella Koltun, Tali Voloshin, Tal Kan, Cfir David, Lilach Koren, Yaara Porat, Alexandra Volodin, Noa Kaynan, Anat Klein-Goldberg, Rom Paz, Boris Brant, Yiftah Barsheshet, Efrat Zemer-Tov, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor treating fields (TTFields) increase cancer cell membrane permeability and improve sensitivity to doxorubicin in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4762.

E2F8-CENPL pathway contributes to homologous recombination repair and chemoresistance in breast cancer

Chemoresistance poses a significant obstacle to the treatment of breast cancer patients. The increased capacity of DNA damage repair is one of the mechanisms underlying chemoresistance. Bioinformatic analyses showed that E2F8 was associated with cell cycle progression and homologous recombination (HR) repair of DNA double-strand breaks (DSBs) in breast cancer. E2F8 knockdown suppressed cell growth and attenuated HR repair. Accordingly, E2F8 knockdown sensitized cancer cells to Adriamycin and Cisplatin. Centromere protein L (CENPL) is a transcriptional target by E2F8. CENPL overexpression in E2F8-knockdowned cells recovered at least in part the effect of E2F8 on DNA damage repair and chemotherapy sensitivity. Consistently, CENPL knockdown impaired DNA damage repair and sensitized cancer cells to DNA-damaging drugs. These findings demonstrate that targeting E2F8-CENPL pathway is a potential approach to overcoming chemoresistance.

Cell-Type-Dependent Recruitment Dynamics of FUS Protein at Laser-Induced DNA Damage Sites.

Increased signs of DNA damage have been associated to aging and neurodegenerative diseases. DNA damage repair mechanisms are tightly regulated and involve different pathways depending on cell types and proliferative vs. postmitotic states. Amongst them, fused in sarcoma (FUS) was reported to be involved in different pathways of single- and double-strand break repair, including an early recruitment to DNA damage. FUS is a ubiquitously expressed protein, but if mutated, leads to a more or less selective motor neurodegeneration, causing amyotrophic lateral sclerosis (ALS). Of note, ALS-causing mutation leads to impaired DNA damage repair. We thus asked whether FUS recruitment dynamics differ across different cell types putatively contributing to such cell-type-specific vulnerability. For this, we generated engineered human induced pluripotent stem cells carrying wild-type FUS-eGFP and analyzed different derivatives from these, combining a laser micro-irradiation technique and a workflow to analyze the real-time process of FUS at DNA damage sites. All cells showed FUS recruitment to DNA damage sites except for hiPSC, with only 70% of cells recruiting FUS. In-depth analysis of the kinetics of FUS recruitment at DNA damage sites revealed differences among cellular types in response to laser-irradiation-induced DNA damage. Our work suggests a cell-type-dependent recruitment behavior of FUS during the DNA damage response and repair procedure. The presented workflow might be a valuable tool for studying the proteins recruited at the DNA damage site in a real-time course.

Abstract B007: NAD+ metabolism drives PARPi resistance in ovarian cancer cells

Abstract NAD+ is a coenzyme for redox reactions and a cofactor for enzymes such as PARPs and Sirtuins. The nuclear enzyme PARP1 is a major consumer of NAD+, which is cleaved during the catalytic activation of PARP1 in response to DNA damage. PARP1 inhibitors compete with NAD+ for the catalytic site of PARP1 and reduce the catalytic activity of PARP1 and impair DNA damage repair. Recently PARP inhibitors (PARPi) have shown efficacy in the treatment of ovarian cancers and most patients with defects in homologous repair of DNA damage are now on PARPi maintenance. But, heterogeneous responses to PARPi is limiting their clinical efficacy. To identify the mechanisms of PARPi resistance, we generated a panel of ovarian cancer cells with acquired resistance to niraparib (PARPiR), an FDA approved PARP inhibitor. We demonstrated that the PARPiR cells are also resistant to other FDA approved PARP inhibitors rucaparib and olaparib. This data suggested that a common mechanism of resistance underlies poor response to the FDA approved PARP inhibitors. Since PARP1 activity is tightly controlled by the cellular NAD+ levels, we next investigated the role of NAD+ biosynthesis in PARPi resistance. Metabolomic profiling of the parental and PARPiR cells demonstrated that PARPiR cells have elevated levels of NAD+ metabolism. Indeed, treating the PARPiR cells with inhibitors of NAD+ synthesis restored the sensitivity to PARP inhibitors. Moreover, culturing the previously sensitive parental ovarian cancer cells in NAD+ precursors decreased their sensitivity to PARP inhibitors. These data collectively demonstrated that high levels of NAD+ metabolism causes PARP inhibitor resistance in ovarian cancer cells. Complementary to the metabolomic analysis, results from phenotypic assays in PARPiR cells revealed that high levels of NAD+ maintain cellular quiescence in PARPiR cells. In summary, our results demonstrated that high levels of NAD+ biosynthesis causes PARPi resistance by maintaining cellular quiescence. There is an urgent need to identify the mechanisms of resistance to PARP inhibitors to increase their clinical efficacy. Our study identified that maintenance of quiescence is a fundamental mechanism of PARPi resistance. We also demonstrated that inhibiting NAD+ biosynthesis reverses PARP inhibitor resistance. Citation Format: Subin Myong, Hardik Shah, Benedikt Nagele, Reshma Rajesh, Sridevi Challa. NAD+ metabolism drives PARPi resistance in ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B007.

Abstract 1918 Elevated dNTP Pools Synergize with Ionizing Radiation to Impair DNA Damage Repair and Promote Apoptosis in Therapy Resistant Cancers

Abstract 1918 Elevated dNTP Pools Synergize with Ionizing Radiation to Impair DNA Damage Repair and Promote Apoptosis in Therapy Resistant Cancers