DNA glycosylases as modulators of chemotherapeutic response

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Median survival is less than two years due to several factors, including challenges in surgical removal and chemotherapy resistance, underlining the need for more effective therapeutic options. To identify genes that contribute to chemotherapy resistance, we conducted a synthetic lethal screen in a chemotherapy-resistant GBM derived cell line (T98G) with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored towards “druggable” targets. This screen for TMZ-sensitizing genes indicated that a subset of genes that were over-expressed in GBM cells increased the cell’s sensitivity to TMZ when knocked down. An ubiquitin ligase, UBE3B, and a DNA glycosylase, UNG, were among the TMZ-sensitizing genes identified using the siRNA library. We demonstrate that UBE3B and UNG are sensitizing genes in the screen validation studies using unique siRNA and shRNA sequences. Although UNG is one of four human DNA glycosylases that remove uracil lesions, UNG was the only uracil removing glycosylase to sensitize GBM cells in the validation studies. Notably, analysis of archived transcription datasets revealed that over-expression of UNG was correlated with poor outcomes in glioma patients. In order to uncover functional groupings of TMZ-sensitizing proteins, we conducted in situ pathway analysis of gene candidates for synthetic lethal functions from our screen. This analysis discovered statistically significant enrichment of ontogeny clusters related to base excision repair (BER), response to DNA damage, cellular proliferation and protein modification. Interestingly, this pathway topography overlapped with TMZ-sensitizing genes identified from similar experiments in yeast and bacteria. In order to facilitate rapid in vitro identification of lesion-specific repair activity in cancer cells, we developed a novel fluorescent assay that extends the state of the art. The molecular beacon assay measures real-time DNA repair rates of specific DNA lesions by defined DNA repair proteins. These studies reveal that GBM up-regulates several TMZ-sensitizing genes that correlate with poor patient survival and inhibiting these genes may increase TMZ cytotoxicity in a tumor specific manner. These TMZ-sensitizing genes are not only potential targets for adjuvant therapy, but also represent potential biomarkers to predict TMZ response

XRCC1 and base excision repair balance in response to nitric oxide

Inflammation associated reactive oxygen and nitrogen species (RONs), including peroxynitrite (ONOO−) and nitric oxide (NOradical dot), create base lesions that potentially play a role in the toxicity and large genomic rearrangements associated with many malignancies. Little is known about the role of base excision repair (BER) in removing these endogenous DNA lesions. Here, we explore the role of X-ray repair cross-complementing group 1 (XRCC1) in attenuating RONs-induced genotoxicity. XRCC1 is a scaffold protein critical for BER for which polymorphisms modulate the risk of cancer. We exploited CHO and human glioblastoma cell lines engineered to express varied levels of BER proteins to study XRCC1. Cytotoxicity and the levels of DNA repair intermediates (single-strand breaks; SSB) were evaluated following exposure of the cells to the ONOO− donor, SIN-1, and to gaseous NOradical dot. XRCC1 null cells were slightly more sensitive to SIN-1 than wild-type cells. We used small-scale bioreactors to expose cells to NOradical dot and found that XRCC1-deficient CHO cells were not sensitive. However, using a molecular beacon assay to test lesion removal in vitro, we found that XRCC1 facilitates AAG-initiated excision of two key NOradical dot-induced DNA lesions: 1,N[superscript 6]-ethenoadenine and hypoxanthine. Furthermore, overexpression of AAG rendered XRCC1-deficient cells sensitive to NOradical dot-induced DNA damage. These results show that AAG is a key glycosylase for BER of NOradical dot-induced DNA damage and that XRCC1's role in modulating sensitivity to RONs is dependent upon the cellular level of AAG. This demonstrates the importance of considering the expression of other components of the BER pathway when evaluating the impact of XRCC1 polymorphisms on cancer risk.Massachusetts Institute of Technology. Center for Environmental Health Sciences (NIEHS P30-ES002109)National Institutes of Health (U.S.) (NIH grant P01-CA026731)National Institutes of Health (U.S.) (NIH grant 2-R01-CA079827-05A1)National Institutes of Health (U.S.) (NIH Grant U01-ES016045)National Institutes of Health (U.S.) (NIH Grant GM087798)National Institutes of Health (U.S.) (NIH Grant CA148629)National Institutes of Health (U.S.) (NIH Grant ES019498)National Institutes of Health (U.S.) (Cancer Center Support Grant P30 CA047904

Molecular analysis of vascular gene expression

Abstract A State of the Art lecture entitled “Molecular Analysis of Vascular Gene Expression” was presented at the ISTH Congress in 2021. Endothelial cells (ECs) form a critical interface between the blood and underlying tissue environment, serving as a reactive barrier to maintain tissue homeostasis. ECs play an important role in not only coagulation, but also in the response to inflammation by connecting these two processes in the host defense against pathogens. Furthermore, ECs tailor their behavior to the needs of the microenvironment in which they reside, resulting in a broad display of EC phenotypes. While this heterogeneity has been acknowledged for decades, the contributing molecular mechanisms have only recently started to emerge due to technological advances. These include high‐throughput sequencing combined with methods to isolate ECs directly from their native tissue environment, as well as sequencing samples at a high cellular resolution. In addition, the newest technologies simultaneously quantitate and visualize a multitude of RNA transcripts directly in tissue sections, thus providing spatial information. Understanding how ECs function in (patho)physiological conditions is crucial to develop new therapeutics as many diseases can directly affect the endothelium. Of particular relevance for thrombotic disorders, EC dysfunction can lead to a procoagulant, proinflammatory phenotype with increased vascular permeability that can result in coagulopathy and tissue damage, as seen in a number of infectious diseases, including sepsis and coronavirus disease 2019. In light of the current pandemic, we will summarize relevant new data on the latter topic presented during the 2021 ISTH Congress

Base Excision Repair and Lesion-Dependent Subpathways for Repair of Oxidative DNA Damage

Nuclear and mitochondrial genomes are under continuous assault by a combination of environmentally and endogenously derived reactive oxygen species, inducing the formation and accumulation of mutagenic, toxic, and/or genome-destabilizing DNA lesions. Failure to resolve these lesions through one or more DNA-repair processes is associated with genome instability, mitochondrial dysfunction, neurodegeneration, inflammation, aging, and cancer, emphasizing the importance of characterizing the pathways and proteins involved in the repair of oxidative DNA damage. This review focuses on the repair of oxidative damage–induced lesions in nuclear and mitochondrial DNA mediated by the base excision repair (BER) pathway in mammalian cells. We discuss the multiple BER subpathways that are initiated by one of 11 different DNA glycosylases of three subtypes: (a) bifunctional with an associated β-lyase activity; (b) monofunctional; and (c) bifunctional with an associated β,δ-lyase activity. These three subtypes of DNA glycosylases all initiate BER but yield different chemical intermediates and hence different BER complexes to complete repair. Additionally, we briefly summarize alternate repair events mediated by BER proteins and the role of BER in the repair of mitochondrial DNA damage induced by ROS. Finally, we discuss the relation of BER and oxidative DNA damage in the onset of human disease. Antioxid. Redox Signal. 14, 2491–2507

Deep models of integrated multiscale molecular decipher the response of vascular endothelial cells to ionizing radiation

International audienceThe vascular endothelium is a hot spot in the response to radiation therapy for both tumors and normal tissues. To improve patient outcomes, interpretable systemic hypotheses are needed to help radiobiologists and radiation oncologists propose endothelial targets that could protect normal tissues from the adverse effects of radiation therapy and/or enhance its antitumor potential. To this end, we captured the kinetics of multi-omics layers – i.e. miRNome, targeted transcriptome, proteome and metabolome – in irradiated primary human endothelial cells cultured in vitro. We then designed a strategy of deep learning as in convolutional graph networks that facilitates unsupervised high-level feature extraction of important omics data to learn how ionizing radiation-induced endothelial dysfunction may evolve over time. Last, we present experimental data showing that some of the features identified using our approach are involved in the alteration of angiogenesis by ionizing radiation