Mediated DNA Damage Response Research Articles

AXL Inhibition Induces DNA Damage and Replication Stress in Non-Small Cell Lung Cancer Cells and Promotes Sensitivity to ATR Inhibitors.

AXL, a TAM (TYRO3, AXL, and MERTK) family receptor tyrosine kinase, is increasingly being recognized as a key determinant of resistance to targeted therapies, as well as chemotherapy and radiation in non-small cell lung cancer (NSCLC) and other cancers. We further show here that high levels of AXL and epithelial-to-mesenchymal transition were frequently expressed in subsets of both treatment-naïve and treatment-relapsed NSCLC. Previously, we and others have demonstrated a role for AXL in mediating DNA damage response (DDR), as well as resistance to inhibition of WEE1, a replication stress response kinase. Here, we show that BGB324 (bemcentinib), a selective small-molecule AXL inhibitor, caused DNA damage and induced replication stress, indicated by ATR/CHK1 phosphorylation, more significantly in TP53-deficient NSCLC cell lines. Similar effects were also observed in large-cell neuroendocrine carcinoma (LCNEC) cell lines. High AXL protein levels were also associated with resistance to ATR inhibition. Combined inhibition of AXL and ATR significantly decreased cell proliferation of NSCLC and LCNEC cell lines. Mechanistically, combined inhibition of AXL and ATR significantly increased RPA32 hyperphosphorylation and DNA double-strand breaks and induced markers of mitotic catastrophe. Notably, NSCLC cell lines with low levels of SLFN11, a known predictive biomarker for platinum and PARP inhibitor sensitivity, were more sensitive to AXL/ATR cotargeting. These findings demonstrate a novel and unexpected role for AXL in replication stress tolerance, with potential therapeutic implications. IMPLICATIONS: These findings demonstrate that the combination of AXL and ATR inhibitors could be a promising therapeutic combination for NSCLC, LCNEC, and other cancers.

Contemporary Perspective on a Foundational Paper in Role of Structural Maintenance of Chromosome Complex in Mediating DNA Damage Response Checkpoint

Contemporary Perspective on a Foundational Paper in Role of Structural Maintenance of Chromosome Complex in Mediating DNA Damage Response Checkpoint

Bortezomib synergizes with nocodazole in p53 mediated DNA damage response signalling in A549 lung cancer cells

Bortezomib synergizes with nocodazole in p53 mediated DNA damage response signalling in A549 lung cancer cells

SLX4-XPF mediates DNA damage responses to replication stress induced by DNA-protein interactions.

The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA-protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4-XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4-ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.

Acute Myeloid Leukaemia in Elderly: Expression Analysis of DNA Damage Response (DDR) and Anti-Apoptosis Genes Identify Novel Targets for Potential “Synergistic Lethality”

Background: Genetic instability and heterogeneity are fundamental to AML biology and pivotal to its prognosis. Acute myeloid leukaemia (AML) is most frequent in older adults (>60 years) and a significant majority are considered "treatment-naïve" given medical comorbidities, high-risk disease biology, and poor tolerance to chemotherapy. These patients receive low-intensity regimens, mostly hypomethylating agents due to impaired tolerance to induction chemotherapy, resulting in low overall survival. Thus, there is a critical need to develop targeted therapies capable of rapidly inducing a high rate of clinical response, with better tolerability and durable responses for these elderly AML patients. DNA damage response (DDR) is a specialized and highly orchestrated signalling cascade to maintain genetic stability and is closely linked to the cell cycle. The cell cycle checkpoints result in arrest and the resumption of cell cycle progression when DNA damage has been repaired. However, the DNA repair failure will direct cells towards cellular senescence or apoptosis. Hence, DDR and apoptosis are intricately related physiological processes. In AML, mutations in key regulators of gene expression and/or chromatin structure, such as p53, K-RAS, and isocitrate dehydrogenase 1 and 2 (IDH1/2) results in defective DDR. Defects in DDR are also age-related; therefore, resulting in an exponential increase in the incidence of cancer with age. The emerging treatment regimens for elderly AML are focusing on synergistic lethality using a combination of DDR inhibitors (PARP1) and/ or anti-apoptotic inhibitors (BCL2) in combination with low dose anthracycline and additional targeted therapies like (IDH2/DNTM etc.). However, the wider implication and influence of DDR genes either alone or in combination with anti-apoptotic genes remains uncharacterized in elderly AML. In this pilot study, we screened a large cohort of AML samples for mRNA expression for a series of DDR and apoptotic genes to investigate the age-related variation in expression of these molecules. We observed distinct differences in DDR and anti-apoptotic gene expression between elderly AML compared to paediatric patients. We anticipate, that these preliminary observations will pave the pathway for future comprehensive studies to expand the employment of synergistic lethality strategy for elderly AML patients. Methods: We employed RNA extracted from formalin-fixed paraffin-embedded (FFPE) diagnostic tissuesamples in 100 AML patients. Age defined the categorization of patients into three groups; a, <18 years (n=34); b,18-60 years (n=32) and c, >60 years (n=34). nCounter (NanoString Technologies) platform was used for the quantification of mRNA. Qlucore Omics Explorer software was employed with defined criteria (fold change >2.0; p<0.01 and q <0.05) for statistical analysis. Results:We noted that several DDR mediators (BRIP1, POLD1, XRCC4) are differentially up-regulated in the AML samples linked with elderly patient group, alongside anti-apoptotic genes (IDH2, IKBKB) when compared with samples from patient <18 years (Figure 1). Moreover, DDR effectors (BRCA1& ATM) were up-regulated in the elderly cohort relative to paediatric AML samples, suggesting potential targets for treating elderly AML (Figure2). Conclusions:This pilot study identifies several additional and novel DDR targets; which can be exploited to enhance chemotherapeutic efficacy in "treatment fit" as well as "treatment naïve" elderly AML patients. Besides, we also report numerous novel anti-apoptotic molecules up-regulated in elderly AML. Taken together the preliminary data, presented here, expand the repertoire for targeted therapy in elderly AML with a specific focus on synergistic lethality. Disclosures No relevant conflicts of interest to declare.

Global H3K79 di-methylation mediates DNA damage response to PAH exposure in Chinese coke oven workers.

Polycyclic aromatic hydrocarbons (PAHs) are the main contaminants of coke oven emissions which can induce serious genetic damage in coke oven workers. Epigenetic alternations play essential roles in the regulation of DNA damage effect of PAHs. Previous studies indicate that H3K79 di-methylation (H3K79me2) is integral in DNA damage repair. However, the potential role of H3K79me2 in DNA damage response (DDR) following PAHs exposure is still unclear. In this study, we recruited 256 male coke oven workers and control workers, and examined H3K79me2 and DNA damage in their peripheral blood lymphocytes (PBLCs). The results showed that global H3K79me2 of coke oven workers was 29.3% less than that of the controls (P<0.001). The H3K79me2 was negatively correlated with the concentration of urinary 1-hydroxypyrene (1-OHP) (β=-0.235, P<0.001) and level of genetic damage evaluated by comet assay (βTail DNA %=-0.313, P<0.001; βOTM=-0.251, P=0.008). Consistently, we found that benzo(a)pyrene (BaP) inhibited H3K79me2 in immortalized human bronchial epithelial (HBE) cells in a time-dependent manner. In order to explore the function of H3K79me2 in PAHs DDR, we established histone 3.1/3.3 K79A mutant cells (H3K79A) to suppress H3K79me2. H3K79A cells showed more serious DNA damage and decreased cell viability than control cells after BaP treatment. In addition, we also found that the expression of DOT1L, the only methyltransferase in H3K79, was repressed by BaP dose-dependently. DOT1L knockdown resulted in decreased H3K79me2 level and aggravated DNA damage after BaP exposure. This suggests that BaP induces H3K79me2 repression via inhibiting DOT1L expression. In conclusion, these findings indicate that PAH exposure decreases the level of global H3K79me2, which is integral for DNA damage response regulation of PAHs.

Targeting of the E3 ubiquitin-protein ligase HUWE1 impairs DNA repair capacity and tumor growth in preclinical multiple myeloma models

Experimental evidence suggests that ubiquitin-protein ligases regulate a number of cellular processes involved in tumorigenesis. We analysed the role of the E3 ubiquitin-protein ligase HUWE1 for pathobiology of multiple myeloma (MM), a still incurable blood cancer. mRNA expression analysis indicates an increase in HUWE1 expression levels correlated with advanced stages of myeloma. Pharmacologic as well as RNAi-mediated HUWE1 inhibition caused anti-proliferative effects in MM cell lines in vitro and in an MM1.S xenotransplantation mouse model. Cell cycle analysis upon HUWE1 inhibition revealed decreased S phase cell fractions. Analyses of potential HUWE1-dependent molecular functions did not show involvement in MYC-dependent gene regulation. However, HUWE1 depleted MM cells displayed increased DNA tail length by comet assay, as well as changes in the levels of DNA damage response mediators such as pBRCA1, DNA-polymerase β, γH2AX and Mcl-1. Our finding that HUWE1 might thus be involved in endogenous DNA repair is further supported by strongly enhanced apoptotic effects of the DNA-damaging agent melphalan in HUWE1 depleted cells in vitro and in vivo. These data suggest that HUWE1 might contribute to tumour growth by endogenous repair of DNA, and could therefore potentially be exploitable in future treatment developments.

Usnic Acid Inhibits Proliferation and Migration through ATM Mediated DNA Damage Response in RKO Colorectal Cancer Cell.

Usnic Acid (UA), also known as lichenol, has been reported to have inhibitory effects on a variety of cancer cells, but its specific mechanism remained to be elucidated. Tumor chemotherapy drugs, especially DNA damage chemotherapeutic drugs, target Chromosomal DNA, but their spontaneous and acquired drug resistance are also an urgent problem to be solved. Therefore, drug combination research has become the focus of researchers. Here, we evaluated the tumor-suppressing molecular mechanism of UA in colorectal cancer cells RKO from the perspective of the ATM-mediated DNA damage signaling pathway through H2O2 simulating DNA damage chemotherapeutic drugs. CCK8 cell proliferation assay was used to determine the inhibition of RKO cells by hydrogen peroxide and UA alone or in combination, and wound healing assay was applied to determine the effect of the drug on cell migration. Transfected cells with miRNA18a-5p mimics and inhibitors, MDC and DCFH-DA staining for the measurement of autophagy and ROS, cell cycle and apoptosis were detected by flow cytometry, expressions of microRNA and mRNA were determined by fluorescence quantitative PCR, and protein by Western blot. We found that UA can upregulate ATM via miR-18a to activate the DNA damage signaling pathway and inhibit the proliferation and migration of RKO cells in a concentration-dependent manner. At the same time, DNA damage responses, including cell cycle, autophagy, apoptosis and ROS levels, are also regulated by UA. Therefore, UA combined with DNA damage chemotherapeutic drugs may be an effective treatment for cancer.

Abstract 4198: Role of receptor tyrosine kinase AXL in mediating DNA damage response

Abstract AXL is a receptor tyrosine kinase that is often overexpressed in many cancers. It contributes to tumor progression, metastasis and drug resistance through activating downstream signaling cascades, making it an emerging therapeutic target. Moreover, the first-in-class AXL inhibitor R428 (BGB321) was lately approved by the FDA for the treatment of relapsed or refractory acute myeloid leukemia. Previously, R428 (BGB321) was also reported to show selective sensitivity towards ovarian cancers with a Mesenchymal molecular subtype. Recently, a novel role of AXL in the regulation of DNA damage responses has been brought to light. Here, we further explored AXL's role in mediating DNA damage responses by using ovarian cancer as a disease model. AXL inhibition using R428 resulted in the increase DNA damage foci in ovarian cancer cells SKOV3 and HeyA8. This occurred concurrently with the upregulation of classic DNA damage response signaling molecules such as γH2Ax, ATM and ATR. RNA sequencing results further validated this observation, that R428-treated cells showed downregulation of genes involved in cell cycle control and DNA replication, suggesting the decrease in proliferation. Through SILAC co-immunoprecipitation mass spectrometry, we uncovered a novel binding partner of AXL that could potentially regulate the DNA damage repair upon the inhibition of AXL, giving us insights to how AXL inhibition might contribute to DNA damage. Furthermore, our results showed that AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress, paving ways to the rationale for potential combinatory use of AXL and DNA damage repair inhibitors. Citation Format: Xun Hui Yeo, Wai Leong Tam, Ruby Yun-Ju Huang. Role of receptor tyrosine kinase AXL in mediating DNA damage response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4198.

Abstract 4707: Long noncoding RNA FER1L4 is a novel p73 transcriptional target and inhibits colon cancer cell migration and invasion in a p73-dependent manner

Abstract p73 is a member of the p53 tumor suppressor family, which transactivates p53-responsive genes and mediates DNA damage response. Recent evidences suggest that p73 exerts its tumor suppressor functions by suppressing metastasis, but the exact mechanism remains unknown. It is increasingly evident that long non-coding RNAs (lncRNAs) play a significant role in tumor suppression. However, we still have a limited knowledge of the clinical significance of lncRNAs in colorectal cancer. The present study is aimed to identify novel lncRNAs that play a role in p73-mediated suppression of metastasis in colorectal cancer cells. p73 was knocked down in HCT116p53−/−p73+/+ cells and transcriptome analysis was performed to detect the differentially expressed lncRNAs in presence and absence of p73. The top two up-regulated and down-regulated lncRNAs were selected for further analysis based on the false discovery rate (FDR), fold-change (FC) and P-values. Out of these, FER1L4 lncRNA was found to be significantly induced by DNA damage in a p73-dependent manner. Through bioinformatics analysis, we identified two p73-binding sites in FER1L4 promoter. Consistent with this, p73 binding to FER1L4 promoter was confirmed through luciferase reporter assays and Chromatin Immunoprecipitation (ChIP) assays. Site-directed mutagenesis of both the binding sites totally abrogated p73 responsiveness, indicating that both the sites are equally responsible and essential for p73 binding. In addition, Real-time quantitative PCR demonstrated a rapid increase in endogenous FER1L4 mRNA upon induction of p73. Furthermore, knockdown of FER1L4 and p73 significantly increased the invasion and migration rate of colorectal cancer cells as confirmed by wound-healing assay. Also, knockdown of FER1L4 decreased the expression of E-cadherin, a cancer metastasis suppressor, and increased the expression of other prominent EMT markers such as N-cadherin, Snail, Vimentin and Fibronectin. Cell functional assays further revealed that FER1L4 causes a G2/M cell cycle arrest in a p73-dependent manner in HCT116p53−/−p73+/+ colon cancer cells and upon FER1L4kd, normal cell cycle progression was observed. Annexin V/PI and TUNEL apoptosis assays revealed that FER1L4 induced apoptosis in HCT116p53−/−p73+/+ colon cancer cells with increase in time-dependent treatment of etoposide and FER1L4kd inhibited apoptosis even in the presence of p73. The protein expression level of several pro-apoptotic genes such as Bad, Bax, Bik, Bim, BID, Bak and PUMA decreased upon FER1L4kd and p73kd, confirming that FER1L4 plays a role in p73-mediated apoptosis and cell cycle arrest. Taken together, we provide evidence that FER1L4 is a direct transcriptional target of p73 and p73 exerts its anti-metastatic function by inducing the expression of FER1L4 in response to genotoxic stress. Citation Format: Apoorva Uboveja, Yatendra Kumar Satija, Chanchal Bareja, Daman Saluja. Long noncoding RNA FER1L4 is a novel p73 transcriptional target and inhibits colon cancer cell migration and invasion in a p73-dependent manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4707.

Abstract 2380: A novel role for PACS-1 in suppression of oncogenic replication stress in cancer cells

Abstract PACS-1 is a protein involved in trafficking, especially transport of proteins like furin and Mannose-6-phosphate into the trans-golgi network. Intriguingly, our studies identified dynamic translocation of PACS-1 during cell division and its association with chromatin in tumor cells. Downregulation of PACS-1 in cancer cell lines attenuated cell growth, induced replication stress mediated DNA damage response (DDR), such as ATM/ATR-mediated checkpoint activation, and formation RPA, FANCD2 and pH2AX foci. Consistent with these observations, live-cell imaging revealed increased nuclear translocation of PACS-1 during S-phase and until the complete cell division. These results point to a novel role for PACS-1 during normal replication or in the suppression of oncogenic replication in tumor cells. However, a direct role for PACS-1 in cancer development or progression is not clear. Analysis of the cancer genome atlas (TCGA) data revealed occurrence of PACS-1 amplifications, mutations and deletions in cancers of different tissues. PACS-1 depletion leads to proteasome-mediated degradation of HDAC2 and HDAC3, compromised chromatin maturation, leads to replication stress and genomic instability. Intriguingly, stable overexpression of PACS-1 in MDA-MB-231 cells enhanced malignant behavior of tumor cells in their ability to form soft agar colonies and matrigel invasion compared to the cells that are expressing vector control. PACS-1 downregulation promotes replication stress in tumor cells suggesting that its upregulation in tumors may be important for replication stress recovery in cancer cells and for an efficient response to chemotherapy induced DNA damage. Furthermore, Prognoscan analysis indicated that PACS-1 expression levels correlates with poor survival in multiple studies, including breast, ovarian and lung cancers, suggesting a role for PACS-1 in tumorigenesis and a biomarker for poor prognosis. Citation Format: Chinnadurai Mani, Kaushlendra Tripathi, David W. Clark, Gary Thomas, Komaraiah Palle. A novel role for PACS-1 in suppression of oncogenic replication stress in cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2380.

Abstract 2961: Olaparib mediated DNA damage response and its resistant mechanism in cervical cancer cells

Abstract Cervical cancer is still the most common gynecologic cancer in developing countries. Olaparib is a poly-(ADR-ribose)-polymerase 1 (PARP1) inhibitor targeting at DNA strand break repair. However its efficacy in cervical cancer is unclear. This study aimed to evaluate the therapeutic potential of olaparib in cervical cancer. Olaparib remarkably reduced the PARylation activity and arrested cervical cancer cells at G2/M phase, and was associated with reduced level of a mitosis marker, phosphor-histone H3 as indicated by propidium iodide DNA staining and immunofluorescence. Cytotoxicity of olaparib against cervical cancer cells was determined by XTT assay with IC50 ranged from 35.49µM in C4I to 87.17µM in SiHa. The difference in olaparib sensitivity between C4I and SiHa was consistently demonstrated in annexin V based apoptotic assay, where a higher level of apoptotic response was found in C4I (21.12%) compared to SiHa (6.37%). Immunofluorescent staining and western blot for gH2AX clearly showed DNA double-strand break accumulation in both cell lines upon PARP1 blockade. Lower expression level of RAD51, a homologous recombination (HR) repair factor, was also observed in C4I, when compared to SiHa. Besides, gH2AX/RAD51 foci co-immunofluorescent line scan image analysis showed that there was reduced level of DNA damage recognition by RAD51 (26.80%), compared to SiHa (65.31%). These findings suggested that there was a reduced HR activity in C4I and might explain the higher sensitivity to olaparib compared to SiHa. We also demonstrated the therapeutic potential of olaparib in cervical cancer, and the rationale of using RAD51 as a predictive biomarker for olaparib sensitivity. Citation Format: Ka Yu Tse, Stephanie Si Liu, Horace Hok Yeung Lee, Philip Pun Ching Ip, Kin Tak Chan. Olaparib mediated DNA damage response and its resistant mechanism in cervical cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2961.

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

Abstract Purpose: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors like palbociclib, ribociclib, and abemaciclib have improved progression free survival in patients with metastatic, estrogen receptor positive (ER+) breast cancer, acquired resistance to these drugs limits their efficacy. Despite promising new studies defining the utility of CDK4/6 inhibitors in the upfront, non-metastatic setting, there is limited data available on the effects of concurrent CDK4/6 inhibition and radiation (RT). Methods: Transcriptomic and proteomic expression data was used to quantify changes in RNA and protein expression in ER+ breast cancer cell lines (MCF-7, T47D) after short term (16 hour) CDK4/6 inhibition. Proliferation assays were used to determine the half-maximal inhibitory concentration (IC50) of palbociclib, ribociclib, and abemaciclib. Clonogenic survival assays were performed to calculate the radiation enhancement ratio (rER) and the surviving fraction at 2 Gy for each treatment. Homologous recombination (HR) proficiency was assessed using RAD51 and γH2AX foci formation and a pYFP reporter was used to assess non-homologous end joining (NHEJ) efficiency. Western blots were used to quantify protein expression. MCF-7 xenografts were used to study the efficacy of combination (palbociclib + RT) therapy in vivo. MCF-7 and T47D cell lines with acquired resistance to CDK4/6 inhibition (IC50 &amp;gt;1uM) were used for comparison in all assays. Results: Transcriptomic and proteomic analyses identified changes in expression of DNA damage response mediators and cell cycle machinery with short term CDK4/6 inhibition. Palbociclib significantly radiosensitized ER+ cell lines at concentrations at or below the IC50 value in clonogenic survival assays (MCF-7 rER: 1.22-1.52, T47D rER: 1.23-1.50) and led to a decrease in the surviving fraction of cells at 2 Gy (p &amp;lt; 0.001). Similar results were observed in ribociclib- (rER: 1.08 - 1.68) and abemaciclib-treated (rER: 1.19 - 2.05) cells. MCF-7 and T47D cells treated with CDK4/6 inhibition and RT showed a decrease in RAD51 foci formation, suggesting a decrease in HR efficiency (p &amp;lt; 0.001). However, CDK4/6 inhibition did not affect NHEJ efficiency (p &amp;gt; 0.05). CDK4/6 inhibition + RT led to a decrease in expression of protein expression of HR meditators like p-CHK1 but did not affect phosphorylation of NHEJ proteins like pKu80/pKu70. Cells with acquired resistance to CDK4/6 inhibition did not demonstrate radiosensitization (MCF-7 rER: 0.93 - 1.03, T47D rER: 0.96 - 1.11) or changes in RAD51 foci formation with combination treatment. Conclusions: Our data suggests that CDK4/6 inhibitor-mediated radiosensitization may be effective in ER+ breast cancers prior to the development of CDK4/6 inhibitor resistance. These studies provide preclinical rationale to test CDK4/6 inhibition + RT in women with locally-advanced ER+ breast cancer at high risk for locoregional recurrence. Citation Format: Andrea M. Pesch, Nicole Hirsh, Benjamin C. Chandler, Anna R. Michmerhuizen, Cassandra L. Ritter, Marlie Androsiglio, Kari Wilder-Romans, Meilan Liu, Christina L. Gersch, Jose M. Larios, James M. Rae, Corey W. Speers. CDK4/6 inhibitor-mediatated radiosensitization of estrogen receptor positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6280.

Single-Cell Transcriptome Analysis of Colon Cancer Cell Response to 5-Fluorouracil-Induced DNA Damage

DNA damage often induces heterogeneous cell-fate responses, such as cell-cycle arrest and apoptosis. Through single-cell RNA sequencing (scRNA-seq), we characterize the transcriptome response of cultured colon cancer cell lines to 5-fluorouracil (5FU)-induced DNA damage. After 5FU treatment, a single population of colon cancer cells adopts three distinct transcriptome phenotypes, which correspond to diversified cell-fate responses: apoptosis, cell-cycle checkpoint, and stress resistance. Although some genes are regulated uniformly across all groups of cells, many genes showed group-specific expression patterns mediating DNA damage responses specific to the corresponding cell fate. Some of these observations are reproduced at the protein level by flow cytometry and are replicated in cells treated with other 5FU-unrelated genotoxic drugs, camptothecin and etoposide. This work provides a resource for understanding heterogeneous DNA damage responses involving fractional killing and chemoresistance, which are among the major challenges in current cancer chemotherapy.

Effect of inhibition of receptor tyrosine kinase AXL by a selective small molecular inhibitor R428 (BGB321) on DNA damage repair response in ovarian cancer cells.

e15640 Background: AXL is a receptor tyrosine kinase that is often overexpressed in many cancers. It contributes to tumor progression, metastasis and drug resistance through activating downstream signaling cascades, making it an emerging therapeutic target. The first-in-class AXL inhibitor R428 (BGB321) was approved by the FDA for the treatment of relapsed or refractory acute myeloid leukemia. R428 (BGB321) was also reported to show selective sensitivity towards ovarian cancers (OC) with a Mesenchymal (Mes) molecular subtype. Recently, a novel role of AXL in the regulation of DNA damage responses has been described. In this study, we explored further the role of AXL in mediating DNA damage responses by using OC as a disease model. Methods: OC cell lines were treated with R428. Accumulation of γH2AX positive foci was assessed for DNA damage response. Western blotting for γH2AX, ATM and ATR levels were performed. Dose response curves of ATR inhibitors were generated by treating OC cells with the fixed dose of R428 (IC20 concentration of each cell line). Results: AXL inhibition by using R428 resulted in the increase of DNA damage foci in Mes OC cells SKOV3 and HeyA8. This occurred concurrently with the up-regulation of classic DNA damage response signaling molecules such as γH2AX, ATM and ATR. The IC50 of the ATR inhibitor significantly decreased for 2-3 folds in all OC cell lines tested. AXL inhibitor R428 sensitized both BRCA-mutated and non-BRCA-mutated OC cells to a potent and highly selective ATR inhibitor. Conclusions: Our results showed that AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress, paving ways to the rationale for potential combinatory use of AXL and DNA damage repair inhibitors.

SMCHD1 promotes ATM-dependent DNA damage signaling and repair of uncapped telomeres.

Structural maintenance of chromosomes flexible hinge domain-containing protein 1 (SMCHD1) has been implicated in X-chromosome inactivation, imprinting, and DNA damage repair, and mutations in SMCHD1 can cause facioscapulohumeral muscular dystrophy. More recently, SMCHD1 has also been identified as a component of telomeric chromatin. Here, we report that SMCHD1 is required for DNA damage signaling and non-homologous end joining (NHEJ) at unprotected telomeres. Co-depletion of SMCHD1 and the shelterin subunit TRF2 reduced telomeric 3'-overhang removal in time-course experiments, as well as the number of chromosome end fusions. SMCHD1-deficient cells displayed reduced ATM S1981 phosphorylation and diminished formation of γH2AX foci and of 53BP1-containing telomere dysfunction-induced foci (TIFs), indicating defects in DNA damage checkpoint signaling. Removal of TPP1 and subsequent activation of ATR signaling rescued telomere fusion events in TRF2-depleted SMCHD1 knockout cells. Together, these data indicate that SMCHD1 depletion reduces telomere fusions in TRF2-depleted cells due to defects in ATM-dependent checkpoint signaling and that SMCHD1 mediates DNA damage response activation upstream of ATM phosphorylation at uncapped telomeres.

P73 \u2013 NAV3 axis plays a critical role in suppression of colon cancer metastasis

p73 is a member of the p53 tumor suppressor family, which transactivates p53-responsive genes and mediates DNA damage response. Recent evidences suggest that p73 exerts its tumor suppressor functions by suppressing metastasis, but the exact mechanism remains unknown. Here, we identify Navigator-3 (NAV3), a microtubule-binding protein, as a novel transcriptional target of p73, which gets upregulated by DNA damage in a p73-dependent manner and plays a vital role in p73-mediated inhibition of cancer cell invasion, migration, and metastasis. Induction of p73 in response to DNA damage leads to rapid increase in endogenous NAV3 mRNA and protein levels. Through bioinformatic analysis, we identified two p73-binding sites in NAV3 promoter. Consistent with this, p73 binding to NAV3 promoter was confirmed through luciferase, Chromatin Immunoprecipitation, and site-directed mutagenesis assays. Abrogation of NAV3 and p73 expression significantly increased the invasion and migration rate of colorectal cancer cells as confirmed by wound-healing, cell invasion, and cell migration assays. Also, knockdown of NAV3 decreased the expression of E-cadherin and increased the expression of other prominent mesenchymal markers such as N-cadherin, Snail, Vimentin, and Fibronectin. Immunohistochemistry analysis revealed the downregulation of both NAV3 and p73 expression in metastatic colon cancer tissues as compared to non-metastatic cancer tissues. Additionally, the expression pattern of NAV3 and p73 showed extensively significant correlation in both non-metastatic and metastatic human colon cancer tissue samples. Taken together, our study provide conclusive evidence that Navigator-3 is a direct transcriptional target of p73 and plays crucial role in response to genotoxic stress in p73-mediated inhibition of cancer cell invasion, migration, and metastasis.

Assessment of cytotoxic and genotoxic potentials of a mononuclear Fe(II) Schiff base complex with photocatalytic activity in Trigonella

BackgroundIn recent times, coordination complexes of iron in various oxidation states along with variety of ligand systems have been designed and developed for effective treatment of cancer cells without adversely affecting the normal cell and tissues of various organs. MethodsIn this study, we have evaluated the mechanism of action of a Fe(II) Schiff base complex in the crop plant Trigonella foenum-graecum L. (Fenugreek) as the screening system by using morphological, cytological, biochemical and molecular approaches. Further functional characterization was performed using MCF-7 cell line and solid tumour model for the assessment of anti-tumour activity of the complex. ResultsOur results indicate efficiency of the Fe(II) Schiff base complex in the induction of double strand breaks in DNA. Complex treatment clearly induced cytotoxic and genotoxic damage in Trigonella seedlings. The Fe-complex treatment caused cell cycle arrest via the activation of ATM-ATR kinase mediated DNA damage response pathway with the compromised expression of CDK1, CDK2 and CyclinB1 protein in Trigonella seedlings. In cultured MCF-7 cells, the complex induces cytotoxicity and DNA fragmentation through intracellular ROS generation. Fe-complex treatment inhibited tumour growth in solid tumour model with no additional side effects. ConclusionThe growth inhibitory and cytotoxic effects of the complex result from activation of DNA damage response along with oxidative stress and cell cycle arrest. General significanceOverall, our results have provided comprehensive information on the mechanism of action and efficacy of a Fe(II) Schiff base complex in higher eukaryotic genomes and indicated its future implications as potential therapeutic agent.

Targeting bromodomain protein ANCCA/ATAD2 enhances the efficacy of DNA‑damaging chemotherapy agents and radiation.

Bromodomain proteins such as BRD4 chromatin regulator are attractive cancer therapeutic targets. ANCCA (AAA+ nuclear coregulatory cancer‑associated protein, also known as ATPase family AAA domain containing 2 or ATAD2) is a novel oncology drug target and contains a bromodomain and an ATPase domain. Our research group as well as others previously identified ANCCA/ATAD2 as a putative oncogene and a poor prognosis factor in many types of cancer including triple‑negative breast cancer (TNBC). In the present study, it is reported for the first time that the expression of ANCCA was highly induced by DNA‑damaging chemotherapy agents such as carboplatin, doxorubicin and mitomycin C, as well as ionizing radiation. Notably, ANCCA is required for efficient dissolution of DNA damage foci and homologous recombination. Further studies revealed that ANCCA mediates the optimal expression and activation of DNA damage response and repair factors including Chk1, Chk2 and BRCA1, and that ANCCA is recruited to the promoter of BRCA1 in response to DNA damage. Moreover, ANCCA knockdown sensitizes TNBC cells to carboplatin. Collectively, these data provide the first evidence indicating that ANCCA is a novel mediator of DNA damage response and repair and that targeting ANCCA can enhance the efficacy of radiation and chemotherapies.

High Fidelity Deep Sequencing Reveals No Effect of ATM, ATR, and DNA-PK Cellular DNA Damage Response Pathways on Adenovirus Mutation Rate.

Most DNA viruses exhibit relatively low rates of spontaneous mutation. However, the molecular mechanisms underlying DNA virus genetic stability remain unclear. In principle, mutation rates should not depend solely on polymerase fidelity, but also on factors such as DNA damage and repair efficiency. Most eukaryotic DNA viruses interact with the cellular DNA damage response (DDR), but the role of DDR pathways in preventing mutations in the virus has not been tested empirically. To address this goal, we serially transferred human adenovirus type 5 in cells in which the telangiectasia-mutated PI3K-related protein kinase (ATM), the ATM/Rad3-related (ATR) kinase, and the DNA-dependent protein kinase (DNA-PK) were chemically inactivated, as well as in control cells displaying normal DDR pathway functioning. High-fidelity deep sequencing of these viral populations revealed mutation frequencies in the order of one-millionth, with no detectable effect of the inactivation of DDR mediators ATM, ATR, and DNA-PK on adenovirus sequence variability. This suggests that these DDR pathways do not play a major role in determining adenovirus genetic diversity.