EPCO-04. RADIOTHERAPY IS ASSOCIATED WITH GLOBAL METHYLATION ALTERATIONS IN PATIENT DERIVED GLIOBLASTOMA CELL LINES

Abstract PURPOSE/OBJECTIVE(S) In glioblastoma, DNA methylation states are the most predictive marker of overall survival and response to therapy. Our understanding of how epigenetic states, such as DNA methylation, are “mis-repaired” after DNA damage repair is scant, hampering our ability to understand how treatment associated DNA methylation alterations may drive tumor resistance and growth. MATERIALS AND METHODS Three different patient derived IDH wild-type glioma stem cell (GSC) lines, in duplicates, were treated with radiation (20 Gray in 10 fractions vs. sham control) and allowed to recover prior to DNA methylation analysis with 850K methylation arrays. To analyze the methylation array data via bioinformatic methods we used RnBeads (version 2.4.0) and R (version 3.6.1) packages. We further focused our analysis to specific genomic regions, including CpG islands, promoters, gene bodies and CTCF motifs to understand how methylation alterations may differ between these and other genomic contexts following radiation. RESULTS There were widespread differential methylation (pre-treatment vs. radiation treatment) changes among the genomic regions examined. Interestingly, we found differential methylation changes at CTCF motifs, which play important DNA-methylation dependent roles in gene expression and chromatin architecture regulation. Hierarchical clustering, PCA and MDS analysis of DNA methylation status amongst CpG islands, promoters, gene bodies and CTCF domains revealed strong intra-sample differences, but not inter-sample differences (between GSC lines), suggesting radiation associated methylation alterations maybe loci and context dependent. CONCLUSION Radiation treatment is associated with wide-spread alterations of DNA methylation states in this patient derived glioblastoma model. Such alterations may drive gene expression changes or genomic architecture alterations that lead to treatment resistance, warranting further mechanistic investigation of the interplay between radiation induced DNA damage and local epigenetic state restoration following DNA damage repair.

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Genome-Wide DNA Methylation Analysis Shows Enrichment of Differential Methylation in “Open Seas” and Enhancers and Reveals Hypomethylation in DNMT3A Mutated Cytogenetically Normal AML (CN-AML)

Blood ◽

10.1182/blood.v120.21.653.653 ◽

2012 ◽

Vol 120 (21) ◽

pp. 653-653 ◽

Cited By ~ 2

Author(s):

Ying Qu ◽

Andreas Lennartsson ◽

Verena I. Gaidzik ◽

Stefan Deneberg ◽

Sofia Bengtzén ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Differential Methylation ◽

Methylation Analysis ◽

Cpg Sites ◽

Genome Wide ◽

Genomic Regions ◽

Methylation Patterns

Abstract Abstract 653 DNA methylation is involved in multiple biologic processes including normal cell differentiation and tumorigenesis. In AML, methylation patterns have been shown to differ significantly from normal hematopoietic cells. Most studies of DNA methylation in AML have previously focused on CpG islands within the promoter of genes, representing only a very small proportion of the DNA methylome. In this study, we performed genome-wide methylation analysis of 62 AML patients with CN-AML and CD34 positive cells from healthy controls by Illumina HumanMethylation450K Array covering 450.000 CpG sites in CpG islands as well as genomic regions far from CpG islands. Differentially methylated CpG sites (DMS) between CN-AML and normal hematopoietic cells were calculated and the most significant enrichment of DMS was found in regions more than 4kb from CpG Islands, in the so called open sea where hypomethylation was the dominant form of aberrant methylation. In contrast, CpG islands were not enriched for DMS and DMS in CpG islands were dominated by hypermethylation. DMS successively further away from CpG islands in CpG island shores (up to 2kb from CpG Island) and shelves (from 2kb to 4kb from Island) showed increasing degree of hypomethylation in AML cells. Among regions defined by their relation to gene structures, CpG dinucleotide located in theoretic enhancers were found to be the most enriched for DMS (Chi χ2<0.0001) with the majority of DMS showing decreased methylation compared to CD34 normal controls. To address the relation to gene expression, GEP (gene expression profiling) by microarray was carried out on 32 of the CN-AML patients. Totally, 339723 CpG sites covering 18879 genes were addressed on both platforms. CpG methylation in CpG islands showed the most pronounced anti-correlation (spearman ρ =-0.4145) with gene expression level, followed by CpG island shores (mean spearman rho for both sides' shore ρ=-0.2350). As transcription factors (TFs) have shown to be crucial for AML development, we especially studied differential methylation of an unbiased selection of 1638 TFs. The most enriched differential methylation between CN-AML and normal CD34 positive cells were found in TFs known to be involved in hematopoiesis and with Wilms tumor protein-1 (WT1), activator protein 1 (AP-1) and runt-related transcription factor 1 (RUNX1) being the most differentially methylated TFs. The differential methylation in WT 1 and RUNX1 was located in intragenic regions which were confirmed by pyro-sequencing. AML cases were characterized with respect to mutations in FLT3, NPM1, IDH1, IDH2 and DNMT3A. Correlation analysis between genome wide methylation patterns and mutational status showed statistically significant hypomethylation of CpG Island (p<0.0001) and to a lesser extent CpG island shores (p<0.001) and the presence of DNMT3A mutations. This links DNMT3A mutations for the first time to a hypomethylated phenotype. Further analyses correlating methylation patterns to other clinical data such as clinical outcome are ongoing. In conclusion, our study revealed that non-CpG island regions and in particular enhancers are the most aberrantly methylated genomic regions in AML and that WT 1 and RUNX1 are the most differentially methylated TFs. Furthermore, our data suggests a hypomethylated phenotype in DNMT3A mutated AML. Disclosures: No relevant conflicts of interest to declare.

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Abstract AP10: REAL-TIME ASSESSMENT OF HGSC DNA DAMAGE REPAIR DEFECTS AND DEFECT-INDUCED RESPONSE TO THERAPY IN OVARIAN CANCER ORGANOIDS

10.1158/1557-3265.ovcasymp18-ap10 ◽

2019 ◽

Author(s):

Sarah J. Hill ◽

Brennan Decker ◽

Emma A. Roberts ◽

Chunyu Yang ◽

Neil S. Horowitz ◽

...

Keyword(s):

Ovarian Cancer ◽

Dna Damage ◽

Real Time ◽

Dna Damage Repair ◽

Response To Therapy ◽

Induced Response ◽

Damage Repair ◽

Time Assessment

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A Novel Role for Cathepsin S as a Potential Biomarker in Triple Negative Breast Cancer

Journal of Oncology ◽

10.1155/2019/3980273 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Richard D. A. Wilkinson ◽

Roberta E. Burden ◽

Sara H. McDowell ◽

Darragh G. McArt ◽

Stephen McQuaid ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Dna Damage ◽

Adjuvant Therapy ◽

Dna Damage Repair ◽

Cathepsin S ◽

Damage Repair ◽

Potential Biomarker ◽

Repair Pathways ◽

Improved Outcome

Cathepsin S (CTSS) has previously been implicated in a number of cancer types, where it is associated with poor clinical features and outcome. To date, patient outcome in breast cancer has not been examined with respect to this protease. Here, we carried out immunohistochemical (IHC) staining of CTSS using a breast cancer tissue microarray in patients who received adjuvant therapy. We scored CTSS expression in the epithelial and stromal compartments and evaluated the association of CTSS expression with matched clinical outcome data. We observed differences in outcome based on CTSS expression, with stromal-derived CTSS expression correlating with a poor outcome and epithelial CTSS expression associated with an improved outcome. Further subtype characterisation revealed high epithelial CTSS expression in TNBC patients with improved outcome, which remained consistent across two independent TMA cohorts. Furtherin silicogene expression analysis, using both in-house and publicly available datasets, confirmed these observations and suggested high CTSS expression may also be beneficial to outcome in ER-/HER2+ cancer. Furthermore, high CTSS expression was associated with the BL1 Lehmann subgroup, which is characterised by defects in DNA damage repair pathways and correlates with improved outcome. Finally, analysis of matching IHC analysis reveals an increased M1 (tumour destructive) polarisation in macrophage in patients exhibiting high epithelial CTSS expression. In conclusion, our observations suggest epithelial CTSS expression may be prognostic of improved outcome in TNBC. Improved outcome observed with HER2+ at the gene expression level furthermore suggests CTSS may be prognostic of improved outcome in ER- cancers as a whole. Lastly, from the context of these patients receiving adjuvant therapy and as a result of its association with BL1 subgroup CTSS may be elevated in patients with defects in DNA damage repair pathways, indicating it may be predictive of tumour sensitivity to DNA damaging agents.

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Changes in DNA Damage Repair Gene Expression and Cell Cycle Gene Expression Do Not Explain Radioresistance in Tamoxifen-Resistant Breast Cancer

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504019x15555794826018 ◽

2020 ◽

Vol 28 (1) ◽

pp. 33-40 ◽

Cited By ~ 3

Author(s):

Annemarie E. M. Post ◽

Johan Bussink ◽

Fred C. G. J. Sweep ◽

Paul N. Span

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cell Cycle ◽

Dna Damage ◽

Dna Damage Repair ◽

Cross Resistance ◽

Damage Repair ◽

Breast Cancer Cohort ◽

Tamoxifen Resistant ◽

Repair Genes

Tamoxifen-induced radioresistance, reported in vitro, might pose a problem for patients who receive neoadjuvant tamoxifen treatment and subsequently receive radiotherapy after surgery. Previous studies suggested that DNA damage repair or cell cycle genes are involved, and could therefore be targeted to preclude the occurrence of cross-resistance. We aimed to characterize the observed cross-resistance by investigating gene expression of DNA damage repair genes and cell cycle genes in estrogen receptor-positive MCF-7 breast cancer cells that were cultured to tamoxifen resistance. RNA sequencing was performed, and expression of genes characteristic for several DNA damage repair pathways was investigated, as well as expression of genes involved in different phases of the cell cycle. The association of differentially expressed genes with outcome after radiotherapy was assessed in silico in a large breast cancer cohort. None of the DNA damage repair pathways showed differential gene expression in tamoxifen-resistant cells compared to wild-type cells. Two DNA damage repair genes were more than two times upregulated (NEIL1 and EME2), and three DNA damage repair genes were more than two times downregulated (PCNA, BRIP1, and BARD1). However, these were not associated with outcome after radiotherapy in the TCGA breast cancer cohort. Genes involved in G1, G1/S, G2, and G2/M phases were lower expressed in tamoxifen-resistant cells compared to wild-type cells. Individual genes that were more than two times upregulated (MAPK13) or downregulated (E2F2, CKS2, GINS2, PCNA, MCM5, and EIF5A2) were not associated with response to radiotherapy in the patient cohort investigated. We assessed the expression of DNA damage repair genes and cell cycle genes in tamoxifen-resistant breast cancer cells. Though several genes in both pathways were differentially expressed, these could not explain the cross-resistance for irradiation in these cells, since no association to response to radiotherapy in the TCGA breast cancer cohort was found.

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DNA Damage-Inducing Anticancer Therapies: From Global to Precision Damage

Cancers ◽

10.3390/cancers12082098 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2098 ◽

Cited By ~ 4

Author(s):

Thom G. A. Reuvers ◽

Roland Kanaar ◽

Julie Nonnekens

Keyword(s):

Dna Damage ◽

Cellular Response ◽

Cellular Proliferation ◽

Dna Damage Repair ◽

Therapy Response ◽

Dna Lesions ◽

Response To Therapy ◽

Novel Therapy ◽

Damage Repair ◽

And Function

DNA damage-inducing therapies are of tremendous value for cancer treatment and function by the direct or indirect formation of DNA lesions and subsequent inhibition of cellular proliferation. Of central importance in the cellular response to therapy-induced DNA damage is the DNA damage response (DDR), a protein network guiding both DNA damage repair and the induction of cancer-eradicating mechanisms such as apoptosis. A detailed understanding of DNA damage induction and the DDR has greatly improved our knowledge of the classical DNA damage-inducing therapies, radiotherapy and cytotoxic chemotherapy, and has paved the way for rational improvement of these treatments. Moreover, compounds targeting specific DDR proteins, selectively impairing DNA damage repair in cancer cells, form a promising novel therapy class that is now entering the clinic. In this review, we give an overview of the current state and ongoing developments, and discuss potential avenues for improvement for DNA damage-inducing therapies, with a central focus on the role of the DDR in therapy response, toxicity and resistance. Furthermore, we describe the relevance of using combination regimens containing DNA damage-inducing therapies and how they can be utilized to potentiate other anticancer strategies such as immunotherapy.

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Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells

Radiation Oncology ◽

10.1186/s13014-017-0941-6 ◽

2017 ◽

Vol 12 (1) ◽

Cited By ~ 31

Author(s):

Moshe Giladi ◽

Mijal Munster ◽

Rosa S. Schneiderman ◽

Tali Voloshin ◽

Yaara Porat ◽

...

Keyword(s):

Dna Damage ◽

Radiation Treatment ◽

Dna Damage Repair ◽

Glioma Cells ◽

Tumor Treating Fields ◽

Damage Repair

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Altered expression of DNA damage repair genes in the brain tissue of mice conceived by in vitro fertilization

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaaa010 ◽

2020 ◽

Vol 26 (3) ◽

pp. 141-153

Author(s):

Minhao Hu ◽

Yiyun Lou ◽

Shuyuan Liu ◽

Yuchan Mao ◽

Fang Le ◽

...

Keyword(s):

Dna Methylation ◽

Dna Damage ◽

Brain Tissue ◽

Dna Damage Repair ◽

Damage Repair ◽

Gene And Protein Expression ◽

The Brain ◽

Repair Genes

Abstract Our previous study revealed a higher incidence of gene dynamic mutation in newborns conceived by IVF, highlighting that IVF may be disruptive to the DNA stability of IVF offspring. However, the underlying mechanisms remain unclear. The DNA damage repair system plays an essential role in gene dynamic mutation and neurodegenerative disease. To evaluate the long-term impact of IVF on DNA damage repair genes, we established an IVF mouse model and analyzed gene and protein expression levels of MSH2, MSH3, MSH6, MLH1, PMS2, OGG1, APEX1, XPA and RPA1 and also the amount of H2AX phosphorylation of serine 139 which is highly suggestive of DNA double-strand break (γH2AX expression level) in the brain tissue of IVF conceived mice and their DNA methylation status using quantitative real-time PCR, western blotting and pyrosequencing. Furthermore, we assessed the capacity of two specific non-physiological factors in IVF procedures during preimplantation development. The results demonstrated that the expression and methylation levels of some DNA damage repair genes in the brain tissue of IVF mice were significantly changed at 3 weeks, 10 weeks and 1.5 years of age, when compared with the in vivo control group. In support of mouse model findings, oxygen concentration of in vitro culture environment was shown to have the capacity to modulate gene expression and DNA methylation levels of some DNA damage repair genes. In summary, our study indicated that IVF could bring about long-term alterations of gene and protein expression and DNA methylation levels of some DNA damage repair genes in the brain tissue and these alterations might be resulted from the different oxygen concentration of culture environment, providing valuable perspectives to improve the safety and efficiency of IVF at early embryonic stage and also throughout different life stages.

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Bortezomib Enhances Melphalan Response by Altering Fanconi Anemia (FA)/BRCA Pathway Expression and Function.

Blood ◽

10.1182/blood.v108.11.840.840 ◽

2006 ◽

Vol 108 (11) ◽

pp. 840-840 ◽

Cited By ~ 1

Author(s):

Danielle N. Yarde ◽

Lori A. Hazlehurst ◽

Vasco A. Oliveira ◽

Qing Chen ◽

William S. Dalton

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Repair ◽

Low Dose ◽

Dna Damage Repair ◽

Myeloma Cell ◽

Myeloma Cells ◽

Multiple Myeloma Cell ◽

Damage Repair ◽

Pre Treatment

Abstract The FA/BRCA pathway is involved in DNA damage repair and its importance in oncogenesis has only recently been implicated. Briefly, 8 FA/BRCA pathway family members facilitate the monoubiquitination of FANCD2. Upon monoubiquitination, FANCD2 translocates to the DNA repair foci where it interacts with other proteins to initiate DNA repair. Previously, we reported that the FA/BRCA pathway is upregulated in multiple myeloma cell lines selected for resistance to melphalan (Chen, et al, Blood 2005). Further, reducing FANCF in the melphalan resistant 8226/LR5 myeloma cell line partially reversed resistance, whereas overexpressing FANCF in the drug sensitive 8226/S myeloma line conferred resistance to melphalan. Others have reported, and we have also verified, that bortezomib enhances melphalan response in myeloma cells; however, the mechanism of enhanced melphalan activity in combination with bortezomib has not been reported. Based on our observation that the FA/BRCA pathway confers melphalan resistance, we hypothesized that bortezomib enhances melphalan response by targeting FA/BRCA DNA damage repair pathway genes. To investigate this hypothesis, we first analyzed FA/BRCA gene expression in 8226/S and 8226/LR5 cells treated with bortezomib, using a customized microfluidic card (to detect BRCA1, BRCA2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, RAD51 and RAD51C) and q-PCR. Interestingly, we found that low dose (5nM) bortezomib decreased many FA/BRCA pathway genes as early as 2 hours, with maximal decreases seen at 24 hours. Specifically, 1.5- to 2.5-fold decreases in FANCA, FANCC, FANCD2, FANCE and RAD51C were seen 24 hours post bortezomib exposure. Moreover, pre-treatment of myeloma cells with low dose bortezomib followed by melphalan treatment revealed a greater than 2-fold reduction in FANCD2 gene expression levels. We also found that melphalan treatment alone enhanced FANCD2 protein expression and activation (monoubiquitination), whereas the combination treatment of bortezomib followed by melphalan decreased activation and overall expression of FANCD2 protein. Taken together, these results suggest that bortezomib enhances melphalan response in myeloma by targeting the FA/BRCA pathway. Further understanding of the role of the FA/BRCA pathway in determining melphalan response may allow for more customized and effective treatment of myeloma.

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