Key DNA Damage Response Genes Research Articles

Identification of the key DNA damage response genes for predicting immunotherapy and chemotherapy efficacy in lung adenocarcinoma based on bulk, single-cell RNA sequencing, and spatial transcriptomics

BackgroundImmune checkpoint inhibitors (ICI) plus chemotherapy is the preferred first-line treatment for advanced driver-negative lung adenocarcinoma (LUAD). The DNA damage response (DDR) is the main mechanism underlying chemotherapy resistance, and EGLN3 is a key DDR component. MethodWe conducted an analysis utilizing TCGA and GEO databases employing multiple labels-WGCNA, DEGs, and prognostic assessments. Using bulk RNA-seq and scRNA-seq data, we isolated EGLN3 as the single crucial DDR gene. Spatial transcriptome analysis revealed the spatial differential distribution of EGLN3. TIDE/IPS scores and pRRophetic/oncoPredict R packages were used to predict resistance to ICI and chemotherapy drugs, respectively. ResultsEGLN3 was overexpressed in LUAD tissues (p < 0.001), with the high EGLN3 expression group exhibiting a poor prognosis (p = 0.00086, HR: 1.126 [1.039−1.22]). Spatial transcriptome analysis revealed EGLN3 overexpression in cancerous and hypoxic regions, positively correlating with DDR-related and TGF-β pathways. Drug response predictions indicated EGLN3's resistance to the common chemotherapy drugs, including cisplatin (p = 6.1e−14), docetaxel (p = 1.1e−07), and paclitaxel (p = 4.2e−07). Furthermore, on analyzing the resistance mechanism, we found that EGLN3 regulated DDR-related pathways and induced chemotherapy resistance. Additionally, EGLN3 influenced TGF-β signaling, Treg cells, and cancer-associated fibroblast cells, culminating in immunotherapy resistance. Moreover, validation using real-world data, such as GSE126044, GSE135222, and, IMvigor210, substantiated the response trends to immunotherapy and chemotherapy. ConclusionsEGLN3 emerges as a potential biomarker predicting lower response to both immunotherapy and chemotherapy, suggesting its promise as a therapeutic target in advanced LUAD.

RNA Expression of DNA Damage Response Genes in Muscle-Invasive Bladder Cancer: Influence on Outcome and Response to Adjuvant Cisplatin-Based Chemotherapy.

Background: Perioperative cisplatin-based chemotherapy (CBC) can improve the outcome of patients with muscle-invasive bladder cancer (MIBC), but it is still to be defined which patients benefit. Mutations in DNA damage response genes (DDRG) can predict the response to CBC. The value of DDRG expression as a marker of CBC treatment effect remains unclear. Material and methods: RNA expression of the nine key DDRG (BCL2, BRCA1, BRCA2, ERCC2, ERCC6, FOXM1, RAD50, RAD51, and RAD52) was assessed by qRT-PCR in a cohort of 61 MICB patients (median age 66 y, 48 males, 13 females) who underwent radical cystectomy in a tertiary care center. The results were validated in the The Cancer Genome Atlas (TCGA) cohort of MIBC (n = 383). Gene expression was correlated with disease-free survival (DFS) and overall survival (OS). Subgroup analyses were performed in patients who received adjuvant cisplatin-based chemotherapy (ACBC) (Mannheim n = 20 and TCGA n = 75). Results: Low expression of RAD52 was associated with low DFS in both the Mannheim and the TCGA cohorts (Mannheim: p = 0.039; TCGA: p = 0.017). This was especially apparent in subgroups treated with ACBC (Mannheim: p = 0.0059; TCGA: p = 0.012). Several other genes showed an influence on DFS in the Mannheim cohort (BRCA2, ERCC2, FOXM1) where low expression was associated with poor DFS (p < 0.05 for all). This finding was not fully supported by the data in the TCGA cohort, where high expression of FOXM1 and BRCA2 correlated with poor DFS. Conclusion: Low expression of RAD52 correlated with decreased DFS in the Mannheim and the TCGA cohort. This effect was especially pronounced in the subset of patients who received ACBC, making it a promising indicator for response to ACBC on the level of gene expression.

Abstract 1374: Preclinical investigation of ATR inhibition alone and in combination with PARP inhibition in high risk neuroblastoma

Abstract Background:Neuroblastoma (NB) is the commonest extra-cranial malignant solid tumour of childhood and one of the most difficult to cure. DNA damage response (DDR) defects are frequently observed in high risk NB including allelic loss and loss of function mutations in key DDR genes, oncogene induced replication stress (RS) and cell cycle checkpoint dysfunction. Cancer cells with defective cell cycle checkpoint signalling and/or increased oncogene-driven RS are acutely dependent on the DNA damage sensor kinase ATR. This study aims to identify features of NB cell lines leading to ATR inhibitor sensitivity. As PARP inhibition causes RS through unrepaired single strand DNA breaks progressing to replication, we hypothesise that ATR inhibition will increase PARP inhibitor cytotoxicity. Aims: 1) To determine which molecular features lead to sensitivity to VE-821 (ATR inhibitor) in cell lines derived from high-risk NB tumours. 2) To assess synergism between PARP inhibition with olaparib and ATR inhibition in high risk NB cell lines and to measure RS. Materials and Methods: Cell proliferation in response to 72 hours treatment with VE-821 was assessed by XTT assay (Roche) in a panel of 11 NB cell lines and the effect of ATR inhibition on olaparib growth inhibition in 4 cell lines: SHSY5Y, SKNAS, NGP and N20_R1. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS respectively. RS was also measured by γH2AX foci formation using immunofluorescent microscopy. Results: VE-821 caused significantly more growth inhibition in MYCN amplified cell lines and cell lines with low ATM protein expression by XTT assay (p&amp;lt;0.05 Mann-Whitney U test). Olaparib (5 µM) treatment increased CHK1S345 and H2AXS129 phosphorylation after 24 hours treatment in all cell lines. H2AXS129 phosphorylation and foci number was further increased with the addition of VE-821 (1 µM). ATR inhibition prevented CHK1S345 phosphorylation. In cell proliferation assays, combination index analysis (Calcusyn) showed that ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (&amp;lt;1 µM) (CI value 0.04-0.89). Conclusion: MCYN amplification and low ATM protein expression are determinants of ATRi sensitivity in NB cell lines. ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (&amp;lt;1 µM) and further increases the replication stress caused by PARP inhibition. Citation Format: Harriet E. Southgate, Lindi Chen, Nicola J. Curtin, Deborah A. Tweddle. Preclinical investigation of ATR inhibition alone and in combination with PARP inhibition in high risk neuroblastoma [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 1374.

ATR Inhibition Potentiates PARP Inhibitor Cytotoxicity in High Risk Neuroblastoma Cell Lines by Multiple Mechanisms.

Background: High risk neuroblastoma (HR-NB) is one the most difficult childhood cancers to cure. These tumours frequently present with DNA damage response (DDR) defects including loss or mutation of key DDR genes, oncogene-induced replication stress (RS) and cell cycle checkpoint dysfunction. Aim: To identify biomarkers of sensitivity to inhibition of Ataxia telangiectasia and Rad3 related (ATR), a DNA damage sensor, and poly (ADP-ribose) polymerase (PARP), which is required for single strand break repair. We also hypothesise that combining ATR and PARP inhibition is synergistic. Methods: Single agent sensitivity to VE-821 (ATR inhibitor) and olaparib (PARP inhibitor), and the combination, was determined using cell proliferation and clonogenic assays, in HR-NB cell lines. Basal expression of DDR proteins, including ataxia telangiectasia mutated (ATM) and ATR, was assessed using Western blotting. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS, respectively. RS and homologous recombination repair (HRR) activity was also measured by γH2AX and Rad51 foci formation using immunofluorescence. Results: MYCN amplification and/or low ATM protein expression were associated with sensitivity to VE-821 (p < 0.05). VE-821 was synergistic with olaparib (CI value 0.04–0.89) independent of MYCN or ATM status. Olaparib increased H2AXS129 phosphorylation which was further increased by VE-821. Olaparib-induced Rad51 foci formation was reduced by VE-821 suggesting inhibition of HRR. Conclusion: RS associated with MYCN amplification, ATR loss or PARP inhibition increases sensitivity to the ATR inhibitor VE-821. These findings suggest a potential therapeutic strategy for the treatment of HR-NB.

Targeting the DNA Damage Response for the Treatment of High Risk Neuroblastoma.

Despite intensive multimodal therapy, the survival rate for high risk neuroblastoma (HR-NB) remains <50%. Most cases initially respond to treatment but almost half will subsequently relapse with aggressive treatment resistant disease. Novel treatments exploiting the molecular pathology of NB and/or overcoming resistance to current genotoxic therapies are needed before survival rates can significantly improve. DNA damage response (DDR) defects are frequently observed in HR-NB including allelic deletion and loss of function mutations in key DDR genes, oncogene induced replication stress and cell cycle checkpoint dysfunction. Exploiting defects in the DDR has been a successful treatment strategy in some adult cancers. Here we review the genetic features of HR-NB which lead to DDR defects and the emerging molecular targeting agents to exploit them.

Pan-Cancer Analysis of Potential Synthetic Lethal Drug Targets Specific to Alterations in DNA Damage Response.

Alterations in DNA damage response (DDR) is one of the several hallmarks of cancer. Genomic instability resulting from a disrupted DDR mechanism is known to contribute to cancer progression, and are subjected to radiation, cytotoxic, or more recently targeted therapies with limited success. Synthetic lethality (SL), which is a condition where simultaneous loss-of-function of the genes from complementary pathways result in loss of viability of cancer cells have been exploited to treat malignancies resulting from defects in certain DDR pathways. Albeit being a promising therapeutic strategy, number of SL based drugs currently in clinical trial is limited. In this work we performed a comprehensive pan-cancer analysis of alterations in 10 DDR pathways with different components of DNA repair. Using unsupervised clustering of single sample enrichment of these pathways in 7,272 tumor samples from 17 tumor types from TCGA, we identified three prominent clusters, each associated with specific DDR mechanisms. Somatic mutations in key DDR genes were found to be dominant in each of these three clusters with distinct DDR component. Using a machine-learning based algorithm we predicted SL partners specific to somatic mutations in key genes representing each of the three DDR clusters and identified potential druggable targets. We explored the potential FDA-approved drugs for targeting the predicted SL genes and tested the sensitivity using the drug screening data in cell lines with mutation in the primary DDR genes. We have shown clinical relevance, for selected targetable SL interactions using Kaplan-Meier analysis in terms of improved disease-free survival. Thus, our computational framework provides a basis for clinically relevant and actionable SL based drug targets specific to alterations in DDR pathways.

DNA damage response genes mark the early transition from colitis to neoplasia in colitis-associated colon cancer

Chronic intestinal inflammation predisposes patients with Inflammatory Bowel Disease (IBD) to Colitis-Associated Cancer (CAC). In the setting of chronic inflammation, microsatellite instability (MSI) results from early loss of DNA damage response (DDR) genes, ultimately leading to tumor formation. Despite continued efforts to improve early detection of high risk, pre-dysplastic regions in IBD patients, current macroscopic and genetic surveillance modalities remain limited. Therefore, understanding the regulation of key DDR genes in the progression from colitis to cancer may improve molecular surveillance of CAC. To evaluate DDR gene regulation in the transition from colitis to tumorigenesis, we utilized the well-established Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) pre-clinical murine model of CAC in C57BL/6 mice. In order to assess colonic tumor burden in the setting of mutagen and intestinal irritation, tumors were visualized and graded in real time through high-resolution murine colonoscopy. Upon sacrifice, colons were opened and assessed for macroscopic tumor via high magnification surgical lenses (HMSL). Tissues were then sectioned and separated into groups based on the presence or absence of macroscopically visible tumor. Critical DDR genes were evaluated by semi-quantitative RT-PCR. Interestingly, colon tissue with macroscopically visible tumor (MVT) and colon tissue prior to observable tumor (the non-macroscopically visible tumor-developing group, NMVT) were identical in reduced mRNA expression of mlh1, anapc1, and ercc4 relative to colitic mice without mutagen, or those receiving mutagen alone. Colitis alone was sufficient to reduce colonic ercc4 expression when compared to NMVT mice. Therefore, reduced ercc4 expression may mark the early transition to CAC in a pre-clinical model, with expression reduced prior to the onset of observable tumor. Moreover, the expression of select DDR genes inversely correlated with chronicity of inflammatory disease. These data suggest ercc4 expression may define early stages in the progression to CAC.

Abstract 4146: Selinexor synergizes with DNA damaging agents through down-regulation of key DNA damage response genes

Abstract Background: Selinexor is a first-in-class, orally bioavailable SINE (Selective Inhibitor of Nuclear Export) compound currently in phase 1/2 clinical trials of patients with solid and hematological malignancies. SINE compounds bind exportin 1 (XPO1/CRM1) and inhibit nuclear export, resulting in nuclear retention of major tumor suppressor proteins (TSPs). We previously found through Reverse Phase Protein Array analysis that selinexor treatment alters the level of key DNA damage response (DDR) proteins. We also observed enhanced anti-tumor activity when combining selinexor with standard DNA damaging agents (DDA) in pre-clinical models and clinical studies. We hypothesize that a reduction in DDR protein expression by selinexor drives the synergy observed in combination with DDAs. Methods: Solid and hematological cancer cell lysates treated with selinexor +/- DDAs (cisplatin, doxorubicin, docetaxel and gemcitabine) were analyzed by immunoblotting and qPCR. Comet assay and γH2A.X staining were analyzed by immunofluorescence to evaluate DNA damage. Mice bearing MDA-MB-231 (triple negative breast cancer) tumors were treated with sub-toxic doses of selinexor (2.5 mg/kg), cisplatin (4 mg/kg) or docetaxel (4 mg/kg) alone or in combination. Tumor growth and mouse weight were followed for 25 days. Results: Selinexor reduced the expression of DDR mRNA and proteins involved in single-stranded (SSB) and double-stranded break (DSB) repair as early as 2 hours post treatment. We observed a dose-dependent correlation between the degree of DDR protein reduction and selinexor cytotoxicity in vitro. Selinexor prevented DDR activation following exposure to DDAs and led to synergistic cell death. Treatment of cells with selinexor plus DDA showed enhanced activity in comet assays and an increase in γH2A.X staining when compared to treatment with the single agents. Exposure to selinexor 6 - 24 hours after DDA treatment inhibited the DDR mechanism and was more synergistic than the opposite order (Sel → DDA) or simultaneous treatment. In a MDA-MB-231 xenograft, treatment with selinexor, docetaxel (SSB) or cisplatin (DSB) alone resulted in 66.7%, 51.5% or 26.6% TGI, respectively when compared to vehicle control whereas sequential treatment of docetaxel or cisplatin followed by selinexor resulted in 93.9% TGI or 9.6% tumor regression, respectively. No significant weight loss was seen in any of the groups by the end of the study. IHC staining and immunoblot analysis of tumor tissue and sections showed a reduction of DDR proteins. Conclusion: Selinexor synergizes with DDAs by increasing both SSB and DSB through inhibition of relevant DDR proteins. This data supports clinical development of selinexor in combination with standard chemotherapeutic agents in patients with solid and hematological malignancies. The results also suggest that sequential treatment of patients with DDAs followed by selinexor might be more beneficial. Citation Format: Trinayan Kashyap, Christian Argueta, Boris Klebanov, Oscar Gonzalez, Erkan Baloglu, Yosef Landesman, Margaret Lee, Humphrey Gardner, Sharon Shacham, William Senapedis. Selinexor synergizes with DNA damaging agents through down-regulation of key DNA damage response genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4146. doi:10.1158/1538-7445.AM2017-4146

Abstract MIP-043: OVARIAN CARCINOMA CDK12 MUTATIONS IMPAIR GENE-SPECIFIC POL II ELONGATION AND FAITHFUL REPAIR OF DNA DOUBLE STRAND BREAKS

Abstract The Cdk12/CycK complex is a novel transcription elongation-associated kinase that promotes expression of a subset of RNA polymerase II (Pol II) genes, including those of the DNA damage response (DDR) network. CDK12 is mutated in many cancers, particularly in high-grade serous ovarian carcinoma (HGS-OvCa), where it is one of only nine genes with statistically recurrent somatic mutations. However, the influence of these mutations on the kinase and their link to cancerogenesis remain ill-defined. Here, we show that most CDK12 mutations interfere with formation of the complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations identifies coordinated down-regulation of key DDR genes, particularly those of the homologous recombination (HR) repair pathway. Moreover, we note that production of nascent HR gene transcripts is compromised significantly upon CDK12 and CYCK knockdown. Indeed, Cdk12 occupies these genes and promotes phosphorylation of the Pol II's C-terminal domain (CTD) at Serine 2 (Ser2-P), the hallmark of effective transcription elongation, indicating that the down-regulated genes are directly controlled by Cdk12/CycK. Functionally, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful repair of DNA double strand breaks via HR. In summary, we provide molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations could elicit defects in HR-mediated and possibly other DNA repair pathways, leading to genomic instability underlying the genesis of HGS-OvCa. Citation Format: Kingsley M. Ekumi, Hana Paculova, Tina Lenasi, Vendula Pospichalova, Christian A. Bösken, Jana Rybarikova, Vitezslav Bryja, Matthias Geyer, Dalibor Blazek and Matjaz Barboric. OVARIAN CARCINOMA CDK12 MUTATIONS IMPAIR GENE-SPECIFIC POL II ELONGATION AND FAITHFUL REPAIR OF DNA DOUBLE STRAND BREAKS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-043.

The retinoblastoma homolog RBR1 mediates localization of the repair protein RAD51 to DNA lesions in Arabidopsis

The retinoblastoma protein (Rb), which typically functions as a transcriptional repressor of E2F‐regulated genes, represents a major control hub of the cell cycle. Here, we show that loss of the Arabidopsis Rb homolog RETINOBLASTOMA‐RELATED 1 (RBR1) leads to cell death, especially upon exposure to genotoxic drugs such as the environmental toxin aluminum. While cell death can be suppressed by reduced cell‐proliferation rates, rbr1 mutant cells exhibit elevated levels of DNA lesions, indicating a direct role of RBR1 in the DNA‐damage response (DDR). Consistent with its role as a transcriptional repressor, we find that RBR1 directly binds to and represses key DDR genes such as RADIATION SENSITIVE 51 (RAD51), leaving it unclear why rbr1 mutants are hypersensitive to DNA damage. However, we find that RBR1 is also required for RAD51 localization to DNA lesions. We further show that RBR1 is itself targeted to DNA break sites in a CDKB1 activity‐dependent manner and partially co‐localizes with RAD51 at damage sites. Taken together, these results implicate RBR1 in the assembly of DNA‐bound repair complexes, in addition to its canonical function as a transcriptional regulator.

Abstract 1271: The transcription factor BCL6 is a rational target in non-small cell lung cancer (NSCLC)

Abstract BCL6 is a transcription factor that promotes lymphomagenesis by repressing target genes involved in DNA damage sensing and response. In NSCLC patients, BCL6 is amplified in 40% of Squamous Cell Carcinomas (197/501, TCGA) and in 2.2% of Adenocarcinomas (5/230, TCGA), suggesting a potential oncogenic role for BCL6 in lung cancer. To determine whether BCL6 plays a role in sustaining NSCLC, we first measured the expression of BCL6 in a panel of 15 NSCLC cell lines and found BCL6 expression in 15/15 cell lines. Furthermore, in 7/15 cells lines (46%) we found BCL6 gene amplification. To gain insight into BCL6 function, we engineered two NSCLC cell lines with BCL6 amplification (H1299 and H838) to stably express BCL6-shRNA. BCL6 silencing resulted in 3-5 fold mRNA increase of key DNA damage response genes such as ATR, Chek1, p21 and p53. BCL6-mediated repression of DNA damage response genes has functional effects as BCL6 silencing resulted into i) G1 cell-cycle arrest as determine by flow cytometry analysis, ii) 1.5-2 fold increase in cell doubling time, iii) 40-60% reduction in colony formation. These results suggest that a major role of BCL6 in lung cancer could be to enable proliferation under genotoxic stress, a function that BCL6 exerts physiologically in B-cells during antibody generation. Under these premises, BCL6 inhibition should preferentially affect the proliferation of cells with elevated genomic instability. Thus, we measured the amount of chromosomal aberration harbored by the proliferating fractions of BCL6-shRNA cells compared to the isogenic parental cells using micronuclei assay. BCL6 silencing resulted in 30-40% reduction of micronuclei amount in the proliferating fraction of cells, supporting our hypothesis. Moreover, exposing NSCLC cells to DNA damaging agents leads to 2-3 fold increase in BCL6 expression regardless the presence of BCL6 amplification. In lymphomas, BCL6 represses genes by recruiting co-repressors to form “repressosome” complexes in gene promoters and enhancers. In particular, repression of DNA damage response genes is achieved by the recruitment of three co-repressors (SMRT, N-CoR, and BCOR) to the BTB-domain of BCL6. To determine whether the BTB-domain of BCL6 is mediating the repression of DNA damage genes in NSCLC, we employed FX1085, a small molecule that specifically interacts with the BCL6 BTB domain and prevents the formation of the repressosome complex. Exposure to FX1085 resulted in 3-10 fold increase in ATR, Chek1, p21 and p53 mRNA levels and prevented the proliferation of NSCLC cells with elevated genomic instability. Moreover, FX1085 affected the survival of 6/15 NSCLC cell lines (40%) at GI50 doses comparable to those effective in lymphoma cells, indicating that BCL6 maybe a suitable therapeutic target in NSCLC. Overall, our data indicate a role for BCL6 in sustaining NSCLC genomic instability and suggest that this protein may be a potential therapeutic target in this disease. Citation Format: Rossella Marullo, Haelee Ahn, Mariano Cardenas, Ari Melnick, Fengtian Xue, Leandro Cerchietti. The transcription factor BCL6 is a rational target in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1271.

Abstract AS21: Ovarian carcinoma CDK12 mutations misregulate DNA repair genes via deficient formation and function of the Cdk12/CycK complex

Abstract The Cdk12/CycK complex promotes gene expression by phosphorylating the C-terminal domain of RNA polymerase II. In previous work we found that CDK12/CycK complex maintains genomic stability via regulation of expression of several key DNA damage response genes such as BRCA1, ATR, FANCD2 or FANCI. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations interfere with the Cdk12/CycK complex formation, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations identifies coordinated down-regulation of key DNA damage response genes. Accordingly, we observed that the mutant Cdk12 proteins fail to promote the repair of DNA double strand breaks via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability that underlies the genesis of the cancer.. Citation Format: Kingsley M. Ekumi, Hana Paculova,Vendula Pospichalova, Christian A. Bosken,Vitezslav Bryja, Matthias Geyer, Dalibor Blazek, Matjaz Barboric. Ovarian carcinoma CDK12 mutations misregulate DNA repair genes via deficient formation and function of the Cdk12/CycK complex [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS21.

Abstract C233: Activation of p53 by the NEDD8 activating enzyme inhibitor MLN4924 is independent of Cdt1 and involves the cullin-RING ligase CRL4-DTL.

Abstract NEDD8-activating enzyme (NAE) is an E1 involved in the activation of a large family of E3 ubiquitin ligases termed the cullin-RING ligases (CRLs) through conjugation of cullin proteins with the ubiquitin-like modifier NEDD8. Polyubiquitination of CRL substrates targets them for degradation by the 26S proteasome. NAE thereby regulates the stability of many proteins including those required for cancer cell growth and survival. MLN4924 is an investigational small molecule inhibitor of NAE with 2-3 orders of magnitude selectivity over the related E1’s ubiquitin-activating enzyme (UAE) and sumo-activating enzymes (SAE; Ref. 1). Recently, a genome-wide siRNA screen in the A375 melanoma cell line identified key DNA damage response genes affecting sensitivity of cells to MLN4924, many of which were associated with the p53 pathway which activates a p21-dependent intra-S-phase checkpoint (2). A comparison of the effects of TP53 RNAi in A375 cells to genetic deletion of TP53 in HCT116 cells (HD 104-001 cells, Horizon Discovery Ltd) suggests that p21 induces a cell cycle arrest within 24 h, whereas analysis of the p53-regulated transcriptional signature indicates that proapoptotic proteins are up-regulated at later times. Surprisingly, stabilization of p53 by MLN4924 appears independent of the replication licensing factor Cdt1, ATR activation, or p53 neddylation, but rather involves the E3 ligase complex Cul4A/B-Ddb1-DTL. Furthermore, Nutlin-3induced ubiquitination of p53 could be blocked by MLN4924, suggesting that Mdm2 and DTL may cooperate in the ubiquitination of p53. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C233. Citation Format: Jonathan L. Blank, Xiaozhen J. Liu, Hugues Bernard, Hua Liao, Katherine Cosmopoulos, Eric S. Lightcap. Activation of p53 by the NEDD8 activating enzyme inhibitor MLN4924 is independent of Cdt1 and involves the cullin-RING ligase CRL4-DTL. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C233.

Pleiotropic Effects of the Trichloroethylene-Associated P81S VHL Mutation on Metabolism, Apoptosis, and ATM-Mediated DNA Damage Response

BackgroundThe risk relevance of the P81S von Hippel-Lindau (VHL) gene hotspot mutation identified in clear cell renal cell carcinoma from individuals exposed occupationally to trichloroethylene (TCE) is not known. VHL mutations in hereditary VHL syndrome strongly correlate with phenotypic associations, but specific sporadic mutations in VHL that uniquely alter its protein function may provide a selective growth advantage for somatic cells harboring these mutations.MethodsVHL deficient (Vhl -/-) mouse embryonic stem cells were generated that stably express wild-type, P81S, or R167Q human VHL protein. Under hypoxic conditions, cell lines were examined for hypoxia-inducible transcription factor family (HIF) stabilization and E3-ubiquitin ligase complex interactions. In vivo, teratomas were examined for tumor size, proliferation, apoptosis, and immunohistochemistry and subjected to gene expression analysis. Wild-type, R167Q, and P81S VHL-expressing teratomas were also exposed to 5 Gy ionizing radiation to quantify apoptotic response. Proliferation and apoptosis and teratoma growth were analyzed by either Student t test or analysis of variance with Bonferroni correction. All statistical tests were two-sided.ResultsThe P81S VHL mutation produces deregulation of HIF factors in cell culture but exhibits a growth advantage in the tumor microenvironment, in part because of suppression of apoptosis (P81S mean = 0.9%, 95% confidence interval = 0.6 to 1.2%; WT mean = 7.6%; 95% confidence interval = 6.4 to 8.8%; P < .001) coupled with sustained proliferation. Transcriptional analysis of P81S teratomas revealed the induction of metabolic pathways, antiapoptotic genes, and global suppression of key DNA damage response genes not observed in VHL wild-type or R167Q mutants. In vivo irradiation exposure showed that P81S mutant is resistant to ionizing radiation–induced apoptosis.ConclusionsThe TCE-associated P81S VHL mutation can initiate a unique adaptive response required for selective tumor growth through pleiotropic effects on metabolic diversification, apoptosis suppression, and alteration of the DNA damage response.

DNA Damage in Mammalian Neural Stem Cells Leads to Astrocytic Differentiation Mediated by BMP2 Signaling through JAK-STAT

SummaryThe consequences of DNA damage generation in mammalian somatic stem cells, including neural stem cells (NSCs), are poorly understood despite their potential relevance for tissue homeostasis. Here, we show that, following ionizing radiation-induced DNA damage, NSCs enter irreversible proliferative arrest with features of cellular senescence. This is characterized by increased cytokine secretion, loss of stem cell markers, and astrocytic differentiation. We demonstrate that BMP2 is necessary to induce expression of the astrocyte marker GFAP in irradiated NSCs via a noncanonical signaling pathway engaging JAK-STAT. This is promoted by ATM and antagonized by p53. Using a SOX2-Cre reporter mouse model for cell-lineage tracing, we demonstrate irradiation-induced NSC differentiation in vivo. Furthermore, glioblastoma assays reveal that irradiation therapy affects the tumorigenic potential of cancer stem cells by ablating self-renewal and inducing astroglial differentiation.

Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency

The impact and consequences of damage generation into genomic DNA, especially in the form of DNA double-strand breaks, and of the DNA-damage response (DDR) pathways that are promptly activated, have been elucidated in great detail. Most of this research, however, has been performed on proliferating, often cancerous, cell lines. In a mammalian body, the majority of cells are terminally differentiated (TD), and derives from a small pool of self-renewing somatic stem cells. Here, we comparatively studied DDR signaling and radiosensitivity in neural stem cells (NSC) and their TD-descendants, astrocytes - the predominant cells in the mammalian brain. Astrocytes have important roles in brain physiology, development and plasticity. We discovered that NSC activate canonical DDR upon exposure to ionizing radiation. Strikingly, astrocytes proved radioresistant, lacked functional DDR signaling, with key DDR genes such as ATM being repressed at the transcriptional level. Nevertheless, astrocytes retain the expression of non-homologous end-joining (NHEJ) genes and indeed they are DNA repair proficient. Unlike in NSC, in astrocytes DNA-PK seems to be the PI3K-like protein kinase responsible for γH2AX signal generation upon DNA damage. We also demonstrate the lack of functional DDR signaling activation in vivo in astrocytes of irradiated adult mouse brains, although adjacent neurons activate the DDR.