Promoter methylation in key DNA damage response genes shows a positive correlation with cumulative dose in chronically low-dose radiation-exposed individuals

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

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.

Evaluation of natural chronic low dose radiation exposure on telomere length and transcriptional response of shelterin complex in individuals residing in Kerala coast, India

Decision letter: Pan-cancer association of DNA repair deficiencies with whole-genome mutational patterns

Editor's evaluation: Pan-cancer association of DNA repair deficiencies with whole-genome mutational patterns

The high level natural radiation areas (HLNRA) of Kerala coast in south west India is unique for its wide variation in the background radiation dose (<1.0mGy to 45mGy/year) and vast population size. Several biological studies conducted in this area did not reveal any adverse effects of chronic low dose and low dose rate radiation on human population. In the present study, global transcriptome analysis was carried out in peripheral blood mono-nuclear cells of 36 individuals belonging to different background dose groups [NLNRA, (Group I, ≤1.50 mGy/year) and three groups of HLNRA; Group II, 1.51–5.0 mGy/year), Group III, 5.01-15mGy/year and Group IV, >15.0 mGy/year] to find out differentially expressed genes and their biological significance in response to chronic low dose radiation exposure. Our results revealed a dose dependent increase in the number of differentially expressed genes with respect to different background dose levels. Gene ontology analysis revealed majority of these differentially expressed genes are involved in DNA damage response (DDR) signaling, DNA repair, cell cycle arrest, apoptosis, histone/chromatin modification and immune response. In the present study, 64 background dose responsive genes have been identified as possible chronic low dose radiation signatures. Validation of 30 differentially expressed genes was carried out using fluorescent based universal probe library. Abundance of DDR and DNA repair genes along with pathways such as MAPK, p53 and JNK in higher background dose groups (> 5.0mGy/year) indicated a possible threshold dose for DDR signaling and are plausible reason of observing in vivo radio-adaptive response and non-carcinogenesis in HLNRA population. To our knowledge, this is the first study on molecular effect of chronic low dose radiation exposure on human population from high background radiation areas at transcriptome level using high throughput approach. These findings have tremendous implications in understanding low dose radiation biology especially, the effect of low dose radiation exposure in humans.

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.

Genetic effects due to long term exposure to low doses of ionizing radiation (LDIR) in humans are not well understood. Human population living in high level natural radiation areas (HLNRAs) of Kerala coast in India are continuously exposed to chronic LDIR emanating from monazite containing beach sand for many generations. The background radiation level in this area varies from < 1.0 to 45mGy/year. The people residing in HLNRAs sometimes receives background radiation dose which is approximately 10-40 times higher than the people living in adjacent normal level natural radiation areas (NLNRAs). This population provides a unique opportunity to identify, if present, a mutational signature due to chronic low-dose radiation exposure in humans. We have employed whole exome sequencing approach to determine germline mutational changes in the lymphocytes of healthy individuals from HLNRAs (mean background dose: 31.8 ± 5.4 mGy/year, mean age: 43.0 ± 5.9 years) and compared them with healthy individuals from NLNRAs (mean background dose: 0.9 ± 0.2 mGy/year, mean age: 43.0 ± 11.3 years). Our results revealed that the overall number of single nucleotide variants (SNVs) and insertions/deletions (indels) were not significantly different in HLNRA (7744 SNVs, 880 indels) and NLNRA (7951 SNVs, 856 indels) groups. A similar number of protein affecting mutations (PAMs) were observed in HLNRA (1925) and NLNRA (2082) individuals. Interestingly, several unique SNVs were identified in both the groups. In HLNRA, unique SNVs were overrepresented in genes involved in important biological pathways such as DNA repair (EXO1, PARP2, DDB1, POLQ, LIG1), epigenetic modification (KDM5D, SETDB2, KMT2B, BRD8, SIRT1), cell cycle progression (CDK14, CCND1) etc. Furthermore, significant predominance of C > T transitions which were unique to HLNRA group was observed preferentially at CpG dinucleotide regions. Analysis with REVEL and AloFT tools did not show any increase in potentially pathogenic mutations including those involved in carcinogenesis in HLNRA individuals exposed to chronic radiation. This study did not show any significant changes in genetic variants due to long term exposure to LDIR in human population living in HLNRAs of Kerala coast. However, presence of unique SNVs and C > T transitions in CpG islands of HLNRA individuals indicate the possible role of epigenetic mechanisms i.e. DNA methylation in response to chronic LDIR in this population. This study significantly enhances the current understanding of radiation induced genetic changes and associated cancer risk in human population.

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.

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

Objective To explore the effect of long-term low dose ionizing radiation on telomere length in adults. Methods Forty female residents aged more than 55 years old from high level natural background radiation area in Yangjiang city and forty age-matched female residents from control area in Enping city were selected by quota sampling as high background group and control group, respectively. Genomic DNA was isolated from their peripheral blood. Telomere length was determined using real time q-PCR. The t-test was used to compare the square roots of the means of two groups. The individuals were divided into four groups based on ages (55-, 60-, 65- and ≥70) and four groups based on BMI (<18.5, 18.5-23.99, 24.0-27.99 and ≥28.0). After adjusting age and BMI, multivariate linear regression analysis was performed to study the relationship between telomere length and cumulative exposure dose. The individuals were divided into longer telomere length group (≥2) and shorter telomere length group (<2). Logistic regression analysis was performed to study the relationship between telomere length and cumulative exposure dose. Results The average cumulative dose was(169.52±27.43)mSv for high background group and(47.52±6.50)mSv for control group. The telomere length of high background group was(1.98±1.25), shorter than that of control group (2.69±1.44) with statis-tically significant difference(t=2.24, P 0.05). Association between telomere length and cumulative dose was explored through Logistic regression, and odds ratio was taken as 0.992(95% CI, 0.985-0.999). There was a weak inverse association between telomere length and cumulative dose, because the odds ratio (OR) was very close to 1. Conclusions No obvious dose-effect relationship between telomere length of residents and cumulative radiation doses was found. But the long-term low dose ionizing radiation may lead to the shortening of the telomere length in adults. Key words: Telomere length; Low dose radiation; High background radiation area

Efficient repair of DNA double strand breaks in individuals from high level natural radiation areas of Kerala coast, south-west India

DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1 repeats and promoter methylation of DNA damage response (DDR) and DNA repair (DR)genes (PARP1, ATM, BRCA1, MLH1, XPC, RAD23B, APC, TNFα, DNMT3A, MRE11A, MGMT, CDKN2A, MTHFR) in human peripheral blood mononuclear cells (PBMCs) of healthy donors in response to γ-radiation. Methylation level was correlated with gene expression profile of selected DDR and DR genes (APC, MLH1, PARP1, MRE11A, TNFα, MGMT) to understand their role in gene regulation. Blood samples were collected from 15 random healthy donors, PBMCs were isolated, exposed to 0.1Gy (low) and 2.0Gy (high) doses of γ-radiation and proliferated for 48h and 72h. Genomic DNA and total RNA were isolated from irradiated PBMCs along with un-irradiated control. Methylation profile was determined from bisulphite converted DNA and amplified by methylation sensitive high resolution melting (MS-HRM)method. Total RNA was converted to cDNA and relative expression was analysed using real time quantitative-PCR. Our results revealed that at 0.1Gy, MRE11A and TNFα showed significant (P < 0.05) increase in methylation at 72h. At 2.0Gy, significant increase (P < 0.05) in methylation profile was observed at LINE1, MRE11A, PARP1, BRCA1, DNMT3A and RAD23B at 48h and 72h. PARP1 showed significant positive correlation of methylation status with gene expression. In conclusion, low and high doses of γ-radiation have significant influence on DNA methylation status of LINE1, DDR and DR genes suggesting their potential role as epigenetic signatures in human PBMCs, which can be further explored in human populations.

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.

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.