Super-resolution GSDIM microscopy unveils distinct nanoscale characteristics of DNA repair foci under diverse genotoxic stress.

DNA double-strand breaks initiate the DNA damage response (DDR), leading to the accumulation of repair proteins at break sites and the formation of the-so-called foci. Various microscopy methods, such as wide-field, confocal, electron, and super-resolution microscopy, have been used to study these structures. However, the impact of different DNA-damaging agents on their (nano)structure remains unclear. Utilising GSDIM super-resolution microscopy, here we investigated the distribution of fluorescently tagged DDR proteins (53BP1, RNF168, MDC1) and γH2AX in U2OS cells treated with γ-irradiation, etoposide, cisplatin, or hydroxyurea. Our results revealed that both foci structure and their nanoscale ultrastructure, including foci size, nanocluster characteristics, fluorophore density and localisation, can be significantly altered by different inducing agents, even ones with similar mechanisms. Furthermore, distinct behaviours of DDR proteins were observed under the same treatment. These findings have implications for cancer treatment strategies involving these agents and provide insights into the nanoscale organisation of the DDR.

Novel insights into the role of Discoidin domain receptor 2 (DDR2) in cancer progression: a new avenue of therapeutic intervention.

Discoidin domain receptors (DDRs), including DDR1 and DDR2, are a unique class of receptor tyrosine kinases (RTKs) activated by collagens at the cell-matrix boundary interface. The peculiar mode of activation makes DDRs as key cellular sensors of microenvironmental changes, with a critical role in all physiological and pathological processes governed by collagen remodeling. DDRs are widely expressed in fetal and adult tissues, and experimental and clinical evidence has shown that their expression is deregulated in cancer. Strong findings supporting the role of collagens in tumor progression and metastasis have led to renewed interest in DDRs.  However, despite an increasing number of studies, DDR biology remains poorly understood, particularly the less studied DDR2, whose involvement in cancer progression mechanisms is undoubted. Thus, the understanding of a wider range of DDR2 functions and related molecular mechanisms is expected. To date, several lines of evidence support DDR2 as a promising target in cancer therapy. Its involvement in key functions in the tumor microenvironment makes DDR2 inhibition particularly attractive to achieve simultaneous targeting of tumor and stromal cells, and tumor regression, which is beneficial for improving the response to different types of anti-cancer therapies, including chemo- and immunotherapy. This review summarizes current research on DDR2, focusing on its role in cancer progression through its involvement in tumor and stromal cell functions, and discusses findings that support the rationale for future development of direct clinical strategies targeting DDR2.

The DNA Damage Sensor MRE11 Regulates Efficient Replication of the Autonomous Parvovirus Minute Virus of Mice.

Parvoviruses are single-stranded DNA viruses that utilize host proteins to vigorously replicate in the nuclei of host cells, leading to cell cycle arrest. The autonomous parvovirus, minute virus of mice (MVM), forms viral replication centers in the nucleus which are adjacent to cellular DNA damage response (DDR) sites, many of which are fragile genomic regions prone to undergoing DDR during the S phase. Since the cellular DDR machinery has evolved to transcriptionally suppress the host epigenome to maintain genomic fidelity, the successful expression and replication of MVM genomes at these cellular sites suggest that MVM interacts with DDR machinery distinctly. Here, we show that efficient replication of MVM requires binding of the host DNA repair protein MRE11 in a manner that is independent of the MRE11-RAD50-NBS1 (MRN) complex. MRE11 binds to the replicating MVM genome at the P4 promoter, remaining distinct from RAD50 and NBS1, which associate with cellular DNA break sites to generate DDR signals in the host genome. Ectopic expression of wild-type MRE11 in CRISPR knockout cells rescues virus replication, revealing a dependence on MRE11 for efficient MVM replication. Our findings suggest a new model utilized by autonomous parvoviruses to usurp local DDR proteins that are crucial for viral pathogenesis and distinct from those of dependoparvoviruses, like adeno-associated virus (AAV), which require a coinfected helper virus to inactivate the local host DDR. IMPORTANCE The cellular DNA damage response (DDR) machinery protects the host genome from the deleterious consequences of DNA breaks and recognizes invading viral pathogens. DNA viruses that replicate in the nucleus have evolved distinct strategies to evade or usurp these DDR proteins. We have discovered that the autonomous parvovirus, MVM, which is used to target cancer cells as an oncolytic agent, depends on the initial DDR sensor protein MRE11 to express and replicate efficiently in host cells. Our studies reveal that the host DDR interacts with replicating MVM molecules in ways that are distinct from viral genomes being recognized as simple broken DNA molecules. These findings suggest that autonomous parvoviruses have evolved distinct mechanisms to usurp DDR proteins, which can be used to design potent DDR-dependent oncolytic agents.

Prognostication of DNA Damage Response Protein Expression Patterns in Chronic Lymphocytic Leukemia.

Proteomic DNA Damage Repair (DDR) expression patterns in Chronic Lymphocytic Leukemia were characterized by quantifying and clustering 24 total and phosphorylated DDR proteins. Overall, three protein expression patterns (C1-C3) were identified and were associated as an independent predictor of distinct patient overall survival outcomes. Patients within clusters C1 and C2 had poorer survival outcomes and responses to fludarabine, cyclophosphamide, and rituxan chemotherapy compared to patients within cluster C3. However, DDR protein expression patterns were not prognostic in more modern therapies with BCL2 inhibitors or a BTK/PI3K inhibitor. Individually, nine of the DDR proteins were prognostic for predicting overall survival and/or time to first treatment. When looking for other proteins that may be associated with or influenced by DDR expression patterns, our differential expression analysis found that cell cycle and adhesion proteins were lower in clusters compared to normal CD19 controls. In addition, cluster C3 had a lower expression of MAPK proteins compared to the poor prognostic patient clusters thus implying a potential regulatory connection between adhesion, cell cycle, MAPK, and DDR signaling in CLL. Thus, assessing the proteomic expression of DNA damage proteins in CLL provided novel insights for deciphering influences on patient outcomes and expanded our understanding of the potential complexities and effects of DDR cell signaling.

DNA Damage Response-Related Proteins Are Prognostic for Outcome in Both Adult and Pediatric Acute Myelogenous Leukemia Patients: Samples from Adults and from Children Enrolled in a Children's Oncology Group Study.

The survival of malignant leukemic cells is dependent on DNA damage repair (DDR) signaling. Reverse Phase Protein Array (RPPA) data sets were assembled using diagnostic samples from 810 adult and 500 pediatric acute myelogenous leukemia (AML) patients and were probed with 412 and 296 strictly validated antibodies, respectively, including those detecting the expression of proteins directly involved in DDR. Unbiased hierarchical clustering identified strong recurrent DDR protein expression patterns in both adult and pediatric AML. Globally, DDR expression was associated with gene mutational statuses and was prognostic for outcomes including overall survival (OS), relapse rate, and remission duration (RD). In adult patients, seven DDR proteins were individually prognostic for either RD or OS. When DDR proteins were analyzed together with DDR-related proteins operating in diverse cellular signaling pathways, these expanded groupings were also highly prognostic for OS. Analysis of patients treated with either conventional chemotherapy or venetoclax combined with a hypomethylating agent revealed protein clusters that differentially predicted favorable from unfavorable prognoses within each therapy cohort. Collectively, this investigation provides insight into variable DDR pathway activation in AML and may help direct future individualized DDR-targeted therapies in AML patients.

Prognostic and predictive value of tumoral DNA damage repair protein expression in patients with resected pancreatic cancer.

OBJECTIVE: DNA damage repair (DDR) gene mutations gained interest in the treatment of metastatic pancreatic cancer (PC) patients, but their relevance in adjuvant setting is not well characterized. We assessed the prognostic and predictive potential of tumoral expression of DDR proteins along with clinical and tumor characteristics in patients with resected PC. PATIENTS AND METHODS: Patients with PC who underwent pancreatic resection in our institution between 2005 and 2017 were retrospectively retrieved. Tumoral expression of a panel of DDR proteins including BRCA1, BRCA2, ATM, and p53 with immunohistochemistry was evaluated and association with patient and tumor features as well as prognosis was assessed. RESULTS: 130 patients were included in the study. The median age was 61 and 66% were males, 57% had lymph node involvement and 17% had a vascular invasion. 25 patients (19%) had thrombosis at the time of diagnosis. Median overall survival (OS) and disease-free survival (DFS) were 21.6 and 11.8 months, respectively. More advanced disease stage (HR: 3.67 95% CI 1.48-9.12, p = 0.005), presence of thrombosis (HR: 2.01 95% CI 1.04-3.89, p = 0.039), high BRCA1 expression (HR: 2.25, 95% CI 1.13-5.48, p = 0.023) and high post-operative CA 19-9 level (>100 IU/ml) (HR:2.61 95% CI 1.40-4.89, p = 0.003) were associated with shorter DFS. BRCA2, ATM, and p53 expression were not associated with DFS or OS. Adjuvant gemcitabine-cisplatin regimen was not associated with increased DFS or OS in the whole group, neither in low or high expressors of BRCA1, BRCA2, ATM or p53. CONCLUSION: Contrary to BRCA2, ATM, and P53, BRCA1 expression may be beneficial for prognosis in resected pancreatic cancer, while no predictive role was observed in terms of adjuvant platinum efficacy.

Discoidin Domain Receptor-Driven Gene Signatures as Markers of Patient Response to Anti-PD-L1 Immune Checkpoint Therapy.

BACKGROUND: Anti-programmed cell death 1 (anti-PD-1) and PD ligand 1 (PD-L1) immune checkpoint therapies (ICTs) provided durable responses only in a subset of cancer patients. Thus, biomarkers are needed to predict nonresponders and offer them alternative treatments. We recently implicated discoidin domain receptor tyrosine kinase 2 (DDR2) as a contributor to anti-PD-1 resistance in animal models; therefore, we sought to investigate whether this gene family may provide ICT response prediction. METHODS: We assessed mRNA expression of DDR2 and its family member DDR1. Transcriptome analysis of bladder cancer (BCa) models in which DDR1 and 2 were perturbed was used to derive DDR1- and DDR2-driven signature scores. DDR mRNA expression and gene signature scores were evaluated using BCa-The Cancer Genome Atlas (n = 259) and IMvigor210 (n = 298) datasets, and their relationship to BCa subtypes, pathway enrichment, and immune deconvolution analyses was performed. The potential of DDR-driven signatures to predict ICT response was evaluated and independently validated through a statistical framework in bladder and lung cancer cohorts. All statistical tests were 2-sided. RESULTS: DDR1 and DDR2 showed mutually exclusive gene expression patterns in human tumors. DDR2high BCa exhibited activation of immune pathways and a high immune score, indicative of a T-cell-inflamed phenotype, whereas DDR1high BCa exhibited a non-T-cell-inflamed phenotype. In IMvigor210 cohort, tumors with high DDR1 (hazard ratio [HR] = 1.53, 95% confidence interval [CI] = 1.16 to 2.06; P = .003) or DDR2 (HR = 1.42, 95% CI = 1.01 to 1.92; P = .04) scores had poor overall survival. Of note, DDR2high tumors from IMvigor210 and CheckMate 275 (n = 73) cohorts exhibited poorer overall survival (HR = 1.56, 95% CI = 1.20 to 2.06; P < .001) and progression-free survival (HR = 1.77 95%, CI = 1.05 to 3.00; P = .047), respectively. This result was validated in independent cancer datasets. CONCLUSIONS: These findings implicate DDR1 and DDR2 driven signature scores in predicting ICT response.

Discoidin domain receptors orchestrate cancer progression: A focus on cancer therapies.

Discoidin domain receptors (DDR), including DDR1 and DDR2, are special types of the transmembrane receptor tyrosine kinase superfamily. DDR are activated by binding to the triple-helical collagen and, in turn, DDR can activate signal transduction pathways that regulate cell-collagen interactions involved in multiple physiological and pathological processes such as cell proliferation, migration, apoptosis, and cytokine secretion. Recently, DDR have been found to contribute to various diseases, including cancer. In addition, aberrant expressions of DDR have been reported in various human cancers, which indicates that DDR1 and DDR2 could be new targets for cancer treatment. Considerable effort has been made to design DDR inhibitors and several molecules have shown therapeutic effects in pre-clinical models. In this article, we review the recent literature on the role of DDR in cancer progression, the development status of DDR inhibitors, and the clinical potential of targeting DDR in cancer therapies.

Clonal hematopoiesis: mechanisms driving dominance of stem cell clones.

The discovery of clonal hematopoiesis (CH) in older individuals has changed the way hematologists and stem cell biologists view aging. Somatic mutations accumulate in stem cells over time. While most mutations have no impact, some result in subtle functional differences that ultimately manifest in distinct stem cell behaviors. With a large pool of stem cells and many decades to compete, some of these differences confer advantages under specific contexts. Approximately 20 genes are recurrently found as mutated in CH, indicating they confer some advantage. The impact of these mutations has begun to be analyzed at a molecular level by modeling in cell lines and in mice. Mutations in epigenetic regulators such as DNMT3A and TET2 confer an advantage by enhancing self-renewal of stem and progenitor cells and inhibiting their differentiation. Mutations in other genes involved in the DNA damage response may simply enhance cell survival. Here, we review proposed mechanisms that lead to CH, specifically in the context of stem cell biology, based on our current understanding of the function of some of the CH-associated genes.

Predictive biomarkers for drug response in bladder cancer.

Bladder cancer is a heterogeneous disease. Interpatient heterogeneity in response to a drug limits treatment options and impairs improvement of patient survival. For example, approximately half of patients do not respond to cisplatin-based combination chemotherapy, although it is the standard of care for muscle-invasive and metastatic bladder cancer. The development of robust predictive biomarkers is expected to improve outcomes by enabling clinicians to use chemotherapy only in the patients who will benefit from it. Recent advances in the molecular characterization of bladder cancer showed that the basal subtype of bladder cancer and tumors with inactivating mutations in DNA damage repair genes were associated with greater benefit from cisplatin-based chemotherapy. The present review summarizes current efforts to develop predictive biomarkers for drug response in bladder cancer, focusing on those that predict the response to cisplatin-based chemotherapy for advanced bladder cancer. We also review the current situation with regard to the identification of predictive biomarkers for response to intravesical therapy, immune checkpoint inhibitors and molecularly-targeted drugs. We also discuss the future applications of new technologies, including liquid biopsies and patient-derived organoids that will also serve as resources for the identification of biomarkers in bladder cancer.

Multikinase Abl/DDR/Src Inhibition Produces Optimal Effects for Tyrosine Kinase Inhibition in Neurodegeneration.

BACKGROUND AND OBJECTIVES: Inhibition of Abelson (Abl) tyrosine kinase as a therapeutic target has been gaining attention in neurodegeneration. Post-mortem Alzheimer's and Parkinson's disease brains show that the levels of several other tyrosine kinases, including Discoidin Domain Receptors (DDR1/2) are elevated. Knockdown of these tyrosine kinases with shRNA reduces neurotoxic proteins, including alpha-synuclein, beta-amyloid and tau. METHODS: Direct profiling of the pharmacokinetics of multi-kinase inhibitors Nilotinib, Bosutinib, Bafetinib, Radotinib and LCB-03-0110 shows differential levels of brain penetration but the ability of these agents to reduce toxic proteins is independent of brain concentration and selectivity to Abl. RESULTS: Our results indicate that the effective dose of Nilotinib has the lowest plasma:brain ratio (1%) followed by Bosutinib and Radotinib (5%), Bafetinib (12%) and LCB-03-0110 (12%). However, similar doses of multi-kinase Abl/DDR inhibitor Nilotinib, DDR/Src inhibitor LCB-03-0110 and Abl/Src inhibitor Bosutinib were much more effective than the more selective Abl inhibitors Radotinib and Bafetinib. Taken together, these data suggest that a multi-kinase target that includes Abl and other tyrosine kinases (DDRs, and Src) may offer more advantages alleviating neurodegenerative pathologies than the absolute CNS drug concentration and selectivity to Abl. CONCLUSION: DDRs and Src are other potential co-targets with Abl in neurodegeneration.

Initiation of Pulmonary Fibrosis after Silica Inhalation in Rats is linked with Dysfunctional Shelterin Complex and DNA Damage Response.

Occupational exposure to silica has been observed to cause pulmonary fibrosis and lung cancer through complex mechanisms. Telomeres, the nucleoprotein structures with repetitive (TTAGGG) sequences at the end of chromosomes, are a molecular "clock of life", and alterations are associated with chronic disease. The shelterin complex (POT1, TRF1, TRF2, Tin2, Rap1, and POT1 and TPP1) plays an important role in maintaining telomere length and integrity, and any alteration in telomeres may activate DNA damage response (DDR) machinery resulting in telomere attrition. The goal of this study was to assess the effect of silica exposure on the regulation of the shelterin complex in an animal model. Male Fisher 344 rats were exposed by inhalation to Min-U-Sil 5 silica for 3, 6, or 12 wk at a concentration of 15 mg/m3 for 6 hr/d for 5 consecutive d/wk. Expression of shelterin complex genes was assessed in the lungs at 16 hr after the end of each exposure. Also, the relationship between increased DNA damage protein (γH2AX) and expression of silica-induced fibrotic marker, αSMA, was evaluated. Our findings reveal new information about the dysregulation of shelterin complex after silica inhalation in rats, and how this pathway may lead to the initiation of silica-induced pulmonary fibrosis.

The DNA methylome of DDR genes and benefit from RT or TMZ in IDH mutant low-grade glioma treated in EORTC 22033.

The optimal treatment for patients with low-grade glioma (LGG) WHO grade II remains controversial. Overall survival ranges from 2 to over 15 years depending on molecular and clinical factors. Hence, risk-adjusted treatments are required for optimizing outcome and quality of life. We aim at identifying mechanisms and associated molecular markers predictive for benefit from radiotherapy (RT) or temozolomide (TMZ) in LGG patients treated in the randomized phase III trial EORTC 22033. As candidate biomarkers for these genotoxic treatments, we considered the DNA methylome of 410 DNA damage response (DDR) genes. We first identified 62 functionally relevant CpG sites located in the promoters of 24 DDR genes, using the LGG data from The Cancer Genome Atlas. Then we tested their association with outcome [progression-free survival (PFS)] depending on treatment in 120 LGG patients of EORTC 22033, whose tumors were mutant for isocitrate dehydrogenase 1 or 2 (IDHmt), the molecular hallmark of LGG. The results suggested that seven CpGs of four DDR genes may be predictive for longer PFS in one of the treatment arms that comprised MGMT, MLH3, RAD21, and SMC4. Most interestingly, the two CpGs identified for MGMT are the same, previously selected for the MGMT-STP27 score that is used to determine the methylation status of the MGMT gene. This score was higher in the LGG with 1p/19q codeletion, in this and other independent LGG datasets. It was predictive for PFS in the TMZ, but not in the RT arm of EORTC 22033. The results support the hypothesis that a high score predicts benefit from TMZ treatment for patients with IDHmt LGG, regardless of the 1p/19q status. This MGMT methylation score may identify patients who benefit from first-line treatment with TMZ, to defer RT for long-term preservation of cognitive function and quality of life.

Discoidin domain receptor inhibition reduces neuropathology and attenuates inflammation in neurodegeneration models.

The role of cell surface tyrosine kinase collagen-activated receptors known as discoidin domain receptors (DDRs) is unknown in neurodegenerative diseases. We detect up-regulation in DDRs level in post-mortem Alzheimer and Parkinson brains. Lentiviral shRNA knockdown of DDR1 and DDR2 reduces the levels of α-synuclein, tau, and ß-amyloid and prevents cell loss in vivo and in vitro. DDR1 and DDR2 knockdown alters brain immunity and significantly reduces the level of triggering receptor expressed on myeloid cells (TREM)-2 and microglia. These studies suggest that DDR1 and DDR2 inhibition is a potential target to clear neurotoxic proteins and reduce inflammation in neurodegeneration.

Consequences of irradiation on adult spermatogenesis: Between infertility and hereditary risk.

DNA damage response in adult spermatogenic cells should limit the propagation of mutations to the offspring, without being detrimental to fertility. In differentiating spermatogenic cells, the genomic instability is limited in time, whereas in spermatogonial stem cells it can be maintained all along life. Spermatogonial stem cells are long-lived cells that support normal germ cell differentiation and must be preserved throughout life. However after irradiation spermatogenesis recovery can be impaired as a consequence of the radiation-induced decline in spermatogonial stem cell. In this review, we summarize the differential sensitivities to DNA damage of spermatogenic cell populations, and the DNA repair mechanisms activated in these cells that paradoxically might favour the maintenance of cells with impaired genomic integrity. We describe how the testis tissue collapses in response to irradiation and we discuss the molecular pathways involved in the control of DNA damage response and homeostasis in spermatogonial stem cells.