Component Of DNA Damage Response Research Articles

Mechanistic insights into DNA damage recognition and checkpoint control in plants.

The plant DNA damage response (DDR) pathway safeguards genomic integrity by rapid recognition and repair of DNA lesions that, if unrepaired, may cause genome instability. Most frequently, DNA repair goes hand in hand with a transient cell cycle arrest, which allows cells to repair the DNA lesions before engaging in a mitotic event, but consequently also affects plant growth and yield. Through the identification of DDR proteins and cell cycle regulators that react to DNA double-strand breaks or replication defects, it has become clear that these proteins and regulators form highly interconnected networks. These networks operate at both the transcriptional and post-transcriptional levels and include liquid-liquid phase separation and epigenetic mechanisms. Strikingly, whereas the upstream DDR sensors and signalling components are well conserved across eukaryotes, some of the more downstream effectors are diverged in plants, probably to suit unique lifestyle features. Additionally, DDR components display functional diversity across ancient plant species, dicots and monocots. The observed resistance of DDR mutants towards aluminium toxicity, phosphate limitation and seed ageing indicates that gaining knowledge about the plant DDR may offer solutions to combat the effects of climate change and the associated risk for food security.

IL-22 is secreted by proximal tubule cells and regulates DNA damage response and cell death in acute kidney injury

Acute kidney injury (AKI) affects over 13 million people worldwide annually and is associated with a 4-fold increase in mortality. Our lab and others have shown that DNA damage response (DDR) governs the outcome of AKI in a bimodal manner. Activation of DDR sensor kinases protects against AKI, while hyperactivation of DDR effector proteins, such as p53, induces cell death and worsens AKI. The factors that trigger DDR to switch from pro-repair to pro-cell death remain to be resolved. Here we investigated the role of interleukin 22 (IL-22), an IL-10 family member whose receptor (IL-22RA1) is expressed on proximal tubule cells (PTCs), in DDR activation and AKI. Using cisplatin and aristolochic acid (AA) induced nephropathy as models of DNA damage, we identified PTCs as a novel source of urinary IL-22. Functionally, IL-22 binding IL-22RA1 on PTCs amplified the DDR. Treating primary PTCs with IL-22 alone induced rapid activation of the DDR. The combination of IL-22 and either cisplatin- or AA-induced cell death in primary PTCs, while the same dose of cisplatin or AA alone did not. Global deletion of IL-22 protected against cisplatin- or AA-induced AKI, reduced expression of DDR components, and inhibited PTC cell death. To confirm PTC IL-22 signaling contributed to AKI, we knocked out IL-22RA1 specifically in kidney tubule cells. IL-22RA1ΔTub mice displayed reduced DDR activation, cell death, and kidney injury compared to controls. Thus, targeting IL-22 represents a novel therapeutic approach to prevent the negative consequences of the DDR activation while not interfering with repair of damaged DNA.

Loss of Function of SETD2 Tumor Suppressor in Chronic Myeloid Leukemia (CML) Progenitors Fosters Genomic Instability and Enhances Clonogenic Potential

BACKGROUND AND AIMS - Genomic instability is a hallmark of chronic myeloid leukemia (CML) cells since the chronic phase (CP) of the disease, and results in BCR::ABL1 mutations and/or additional genetic and genomic aberrations that may drive resistance to tyrosine kinase inhibitors (TKIs) and progression to blast crisis (BC). Genomic instability is also a feature of CML stem and progenitor cells and may contribute to their persistence. The SETD2 tumor suppressor codes for a protein that trimethylates histone H3 at lysine 36 (H3K36me3). In solid tumors, SETD2 loss of function has been shown to impair H3K36me3-mediated recruitment of DNA damage response components. We have recently reported that non genomic loss of function of SETD2 is a feature of BC CML and results from premature proteasome-mediated degradation of the SETD2 protein triggered by Aurora kinase A phosphorylation and MDM2 ubiquitination. In the present study, we aimed to assess SETD2/H3K36me3 status in CD34+ progenitors of CP CML patients (pts) and whether SETD2/H3K36me3 deficiency may play a role in genomic instability in CML models. METHODS - Western blotting (WB) was used to assess SETD2 protein expression and H3K36me3 as a surrogate marker of SETD2 function in the CD34+ cell fraction isolated from the bone marrow of 20 newly diagnosed CP CML pts and from a pool of healthy donors (HD). SETD2 forced expression in CD34+ progenitors from newly diagnosed CP CML pts and in the SETD2-deficient KCL22 cell line was performed by nucleofection. SETD2 knock-down in the SETD2-proficient LAMA84 cell line was performed by RNAi. Clonogenic capacity was evaluated by clonogenic assays. Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K36me3 was performed on an Illumina HiSeq2000 with a min 50 million 150-bp single-end reads per replicate. High resolution karyotyping wias perfomed with Cytoscan HD arrays. DNA damage and DNA repair activation were assessed in primary samples and cell lines by WB and immunofluorescence (IF) using antibodies specific for phospho-H2AX, mismatch repair (MMR) and homologous recombination repair (HR) proteins. RESULTS - WB demonstrated a marked down-modulation of SETD2 expression, paralleled by H3K36me3 deficiency, in the CD34+ cells of all newly diagnosed CP CML pts as compared to the total mononuclear fraction and to CD34+ cells from HDs. To investigate whether SETD2 loss affects the activation and proficiency of HR and MMR, we used chronic exposure to UV rays or a single exposure to hydrogen peroxide (1mM for 30 and 60 min). Both induced DNA damage in SETD2 siRNA-depleted LAMA84 cells. Compared to parental cells, cells silenced for SETD2 failed to activate the ATM-dependent repair pathway. Moreover, we found that ATM inactivation prevents H2AX foci formation, associated with a loss of RAD51 and RAD54 (HR) and MSH6 (MMR) repair foci. To confirm the hypothesis that SETD2 is a tumor suppressor implicated in maintaining genomic stability in CML, we transfected SETD2-deficient KCL22 cells with an ectopic SETD2 plasmid. SETD2 forced expression was able to restore DNA damage response, as demonstrated by WB and IF detection of ATM, p95 and H2AX phosphorylation, BRCA1, BRCA2 and CtIP expression and finally RAD51 and RAD54 localization on HR repair foci observed after UV or hydrogen peroxide exposure. High-resolution karyotyping after DNA damage showed increased rate of DNA breakpoints in SETD2-deficient cells, preferentially occuring at loci where H3K36me3 marks were lost as assessed by ChiP-seq. In line with the effects observed in cell line models, forced expression of SETD2 in CD34+ cells from 5 CP CML pts was found to restore proliferation control, since a >50% reduction in clonogenic potential was observed after nucleofection. CONCLUSIONS - Our findings demonstrate that SETD2 is a bona fide tumor suppressor in CML progenitors and establish a functional link between SETD2 loss of function and genomic instability. SETD2 inactivation may thus play a pivotal role in the harmful cascade of events that may foster drug resistance and ultimately lead to disease progression. Since SETD2 loss of function in CML is mediated by post-translation mechanisms, hence is reversible, therapeutic strategies aimed at interfering with these mechanisms may restore proliferation control and interrupt this cascade. Supported by AIRC IG 2019 (23001) and by Italian Ministry of Health, “Bando Ricerca Finalizzata 2016”, project GR-2016-02364880.

Studying the DNA damage response pathway in hematopoietic canine cancer cell lines, a necessary step for finding targets to generate new therapies to treat cancer in dogs.

Dogs present a significant opportunity for studies in comparative oncology. However, the study of cancer biology phenomena in canine cells is currently limited by restricted availability of validated antibody reagents and techniques. Here, we provide an initial characterization of the expression and activity of key components of the DNA Damage Response (DDR) in a panel of hematopoietic canine cancer cell lines, with the use of commercially available antibody reagents. The techniques used for this validation analysis were western blot, qPCR, and DNA combing assay. Substantial variations in both the basal expression (ATR, Claspin, Chk1, and Rad51) and agonist-induced activation (p-Chk1) of DDR components were observed in canine cancer cell lines. The expression was stronger in the CLBL-1 (B-cell lymphoma) and CLB70 (B-cell chronic lymphocytic leukemia) cell lines than in the GL-1 (B-cell leukemia) cell line, but the biological significance of these differences requires further investigation. We also validated methodologies for quantifying DNA replication dynamics in hematopoietic canine cancer cell lines, and found that the GL-1 cell line presented a higher replication fork speed than the CLBL-1 cell line, but that both showed a tendency to replication fork asymmetry. These findings will inform future studies on cancer biology, which will facilitate progress in developing novel anticancer therapies for canine patients. They can also provide new knowledge in human oncology.

MiR-4796 enhances the sensitivity of breast cancer cells to ionising radiation by impairing the DNA repair pathway.

MicroRNAs (miRNAs) are important regulators of DNA damage response (DDR) through post-transcriptional regulation on their target genes, which are implicated in DDR and DNA repair (DR). In this study, we investigated the functional roles and target genes of miR-4796 and miR-1287 in breast cancer cells in response to radiation. The molecular mechanism of miR-4796 in regulating the radiosensitivity of breast cancer cells was also elucidated. Real-time polymerase chain reaction detected miR-4796 and miR-1287 expression; colony formation assay and irradiation therapy tumour xenograft in vivo examined radiosensitising effect; comet assay assessed DNA damage; immunofluorescence imaging determined the formation of γ-H2AX foci; targetscan and RegRNA predicted target mRNAs; luciferase reporter and mutation assays validated target genes; western blotting detected the expression of genes at the protein level; and flow cytometry quantified the activities of nonhomologous end-joining (NHEJ) and homologous recombination (HR). The expressions of miR-4796 and miR-1287 were acutely fluctuated in response to ionising radiation. In the absence of radiation, overexpression of miR-1287 dramatically promoted growth of breast cancer cells in vitro and in vivo, whereas overexpression of miR-4796 did not affect cell growth. When under the treatment with radiation, overexpression of miR-4796 suppressed DR and sensitised cancer cells to radiation both in vitro and in vivo. However, such effect was only observed in cell assays in the overexpressed miR-1287 group, and not confirmed in vivo. We therefore further explored the molecular mechanism of action of miR-4796, and found that miR-4796 targeted multiple components of DDR and DR, including ATM, BRCA1, PARP and RAD51. Moreover, overexpression of miR-4796 inhibited the expression of these DDR components at the protein level. In addition, miR-4796 inhibited HR and NHEJ repair pathways and aggravated radiation-induced DNA damage. The findings here suggest that miR-4796 can enhance radiation-induced cell death by directly targeting multiple DDR components, and repress NHEJ and HR DNA repair pathways. miR-4796 can act as an effective radiation sensitising agent.

Involvement of ATR-CHK1 pathway in fish megalocytivirus infection induced DNA-damage response in vitro

Increasing evidence has demonstrated that megalocytiviruses cause severe disease with high mortality in both freshwater and marine species. However, the molecular events employed in megalocytivirus infection in vitro still remained largely uncertain. Here, using an in vitro infection model of sea perch iridovirus (SPIV), we investigated the transcriptomic profiles of sea perch fry (SPF) cells upon SPIV infection, and elucidated the roles of crucial molecular events during infection using the specific inhibitors. Our data showed that almost all the differentially expressed genes (DEGs) associated with DNA-damage response (DDR), including DNA replication, mismatch repair, homologous recombination, nucleotide excision repair and Fanconi anemia (FA) pathway exhibited the up-regulation expression patterns during SPIV infection. As expected, DNA damage was detected in infected SPF cells, as evidenced by the DNA migration and the increased phosphorylation levels of H2AX along with the infection time. Furthermore, the key components of DDR, ataxia telangiectasia mutated and Rad3 related (ATR) and checkpoint kinase 1 (CHK1) kinases were elucidated to be essential for SPIV replication. Meanwhile, we also found that SPIV infection induced cell cycle arrest at G2/M phase, and 45 genes related to cell cycle pathway were up-regulated during infection. Inhibition of ATR activity induced cell cycle arrest of the infected cells at S phase, perhaps leading to the inhibitory effects on SPIV replication. Together, our data demonstrated for the first time that fish megalocytivirus induced DDR in vitro, and the ATR-CHK1 pathway exerted crucial roles in viral replication via regulating the cell cycle progression. These findings also provided new insights into the understanding of the interactions between fish megalocytivirus and host cells in vitro.

Abstract 1218: Mercury(II)-induced abnormalities in processing of topoisomerase I-mediated DNA damage

Abstract Mercury (Hg) is a persistent environmental pollutant that has long been associated with neurotoxic and other noncancer health effects in exposed populations. Recent large epidemiological studies using different internal indices of Hg exposure have found significantly elevated risks for skin cancers in both men and women. To understand potential carcinogenic mechanisms for Hg(II), we examined its mutagenicity and impact on processing of different types of DNA damage. In agreement with its lack of DNA reactivity, we found that Hg(II) was not mutagenic at the mammalian Hprt locus and did not change DNA incorporation of rNMPs which are the most abundant endogenous nucleotide abnormalities in the genome. We next tested the impact of Hg(II) at nontoxic or only mildly toxic concentrations on sensitivity of cells to several genotoxicants producing specific classes of DNA damage. We found that even nontoxic concentrations of Hg(II) impaired cellular resistance to topoisomerase I-mediated DNA damage in human epidermal keratinocytes. Hg(II) had no significant effect on cytotoxicity of topoisomerase II-mediated DNA lesions and examination of numerous components of DNA damage response activated by topoisomerase II-targeting drugs showed no major differences between control and Hg(II)-exposed cells. In contrast, Hg(II)-treated keratinocytes displayed a distorted DNA damage response to topoisomerase I-produced DNA damage, manifested in a misbalanced activation of ATM and ATR targets. Consistent with their lower survival, Hg(II)-treated cells accumulated higher levels of toxic DNA double-strand breaks (DSBs). Further analyses indicated that Hg(II) caused impartment of DNA repair of replication-associated DSBs. Thus, although Hg(II) is not directly genotoxic, it can increase genomic instability by interfering with accurate processing of endogenous DNA damage produced by topoisomerase I. Citation Format: Giorgiana Madalina Ursu, Anna M. Cyran, Anatoly Zhitkovich. Mercury(II)-induced abnormalities in processing of topoisomerase I-mediated DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1218.

Phosphorylation-dependent regulation of the transcriptional activity of Arabidopsis SOG1.

Eukaryotic organisms rely on the evolutionary conserved DNA damage response (DDR) mechanism to maintain genome integrity. Despite sharing many DDR components, plants lack the key mammalian DDR regulator called tumour suppressor P53. They use the functionally equivalent but otherwise unrelated plant-specific transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) instead. Outside the conserved folded DNA-binding NAC domain, SOG1 is disordered and carries a net negative charge in its C-terminal tail. Using a high-throughput screening method in yeast, we located three putative SOG1 transcription activation domains (TAD). Two out of three are located in a C-terminal region and both comply with the Acidic Exposure Model of TAD function. In addition, those TADs are flanked by phosphorylation sites whose phosphorylation state contribute to the TAD's activity. The TADs biophysical properties are being determined and linked to their putative role as protein-protein interaction interfaces. Special interest goes out to the potential formation of transient structures in the disordered SOG1 C-terminus and its functional implications.

DNAR-05. A ROLE FOR TAZ/YAP IN DNA DAMAGE REPAIR IN GLIOBLASTOMA

Abstract Previous studies have shown that glioma stem-like cells (GSCs) evade conventional cytotoxic therapies and repopulate as recurrent tumors. A fuller understanding of the molecular mechanisms underlying the biology and therapy resistance of GSCs is required. Our group has shown that TAZ, a transcriptional co-factor is highly expressed in about 70% of glioblastomas (GBMs). TAZ and its paralog YAP are oncogenic drivers of brain tumor progression. GSCs overexpressing TAZ undergo a proneural (PN) to mesenchymal (MS) subtype transition, which is accompanied by aggressive phenotypes such as increased grade and treatment resistance in GSC xenografts. Although the oncogenic functions of TAZ/YAP are well established, the exact molecular mechanisms underlying TAZ/YAP mediated cell fate transition and therapy resistance in GBM are not fully understood. Here, we present novel functional interaction between TAZ/YAP and DNA-dependent protein kinase (DNA-PK) in response to exposure to ionizing radiation in GSCs. Mass-spectrometric analyses of TAZ-binding proteins showed that DNA-PK complex composed of the catalytic subunit (DNA-PKcs) and a Ku heterodimer of Ku70 and K80 interacted with TAZ. Immunoprecipitation of DNA-PK showed interaction of both YAP as well. Interaction of YAP/TAZ with DNA-PK complex and TEAD was enhanced by ionizing radiation. DNA-PKcs phosphorylated both YAP and TAZ, and phosphorylation level of these proteins were increased after irradiation, consistent with an increased interaction of these proteins. Intriguingly, siRNA knockdown of YAP and TAZ resulted in inactivation of DNA-PKcs, although protein stability of DNA-PKcs was not affected. Pharmacological inhibition of TAZ/YAPTEAD resulted in increased DNA damage and suppressed cell proliferation, and this effect was synergized by irradiation. We conclude that TAZ and YAP are integral components of DNA damage response and that pharmacological targeting of TAZ/YAP/TEAD pathway can overcome radiation resistance in GSCs.

Targeting DNA damage response components induces enhanced STING-dependent type-I IFN response in ATM deficient cancer cells and drives dendritic cell activation

ABSTRACT The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example, ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However, additional antitumor benefits from targeting the DDR pathways, which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal, inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here, we demonstrate that ATM −/− FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However, STING deficiency in tumor cells does not prevent DC activation, suggesting that transactivation of the STING pathway occurs within DCs. Furthermore, depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells, indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary, in this study, we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation, which is enhanced in ATM-deficient cells.

Quercetin Augments Cisplatin-Induced Apoptosis, DNA Damage Response, and MiR-22 Expression While It Prevents DNA Repair in Osteosarcoma Cells.

Osteosarcoma (OS) patients are commonly treated with chemotherapeutic agents like cisplatin (Cis). Quercetin with fewer side effects can improve the potency of chemotherapy and be used in combinational therapies. Herein, we aimed to evaluate the effects of Cis plus quercetin on DNA damage response (DDR), DNA repair, and apoptosis in Saos-2 cells. The effects of Cis and quercetin single or in combination on Saos-2 cell viability and the cytotoxicity of the drugs were measured by MTT assay. The expression of DDR and repair components including P53, ATM, ATR, RAD51, and H2AX, and also miR-22 were analyzed by real-time PCR. The rate of apoptosis was measured by flow cytometry. Quercetin potentiated the cytotoxic effects of Cis in Saos-2 cells. The IC50 of Cis reduced from 6.12 µM to 4.25 µM. The combination of quercetin and Cis was associated with the up-regulation of miR-22 and DDR components, including P53, ATM, ATR, and H2AX as well as the down-regulation of RAD51. Moreover, this combined regimen significantly induced apoptosis in Saos-2 cells compared to mono drugs. The co-treatment of quercetin and Cis can accelerate DNA damage, DNA damage response, and apoptosis while interfering with the DNA repair process in Saos-2 cells. Moreover, this combination provokes the tumor suppressor miR-22 expression in these cells.

Abstract 151: Mixed-lineage kinase 4 is involved in the activation of the ATM-NEMO signaling axis in response to DNA damage and confers chemoresistance in triple-negative breast cancer

Abstract Resistance to chemotherapy leads to high rates of relapse and poor outcomes in patients with triple-negative breast cancer (TNBC). Therefore, there is a need to identify novel molecular targets that could be exploited to overcome TNBC chemoresistance. Mixed-lineage kinase 4 (MLK4) is a member of MLK family of serine/threonine kinases. Large-scale genomic and transcriptomic data indicated that MLK4 gene (MAP3K21/KIAA1804) is amplified and overexpressed in approximately 50% of triple-negative tumors. We have recently demonstrated that MLK4 promotes the aggressive growth and metastatic potential of TNBC cells. However, the functional role of MLK4 in resistance to chemotherapy has not been investigated so far. In our recent study, we have shown that MLK4 contributes to TNBC chemoresistance by regulating the pro-survival response to DNA-damaging therapies. MLK4 knock-down or inhibition sensitized TNBC cell lines to doxorubicin and etoposide in vitro. Moreover, loss of MLK4 enhanced sensitivity of HCC1806 TNBC cell line towards doxorubicin in a xenograft mouse model. Our data indicated that MLK4 silencing induced persistent DNA damage accumulation and enhanced apoptosis in TNBC cells treated with chemotherapeutics. To further determine cellular mechanisms responsible for the identified MLK4-dependent chemoresistance in TNBC, we performed a quantitative phosphoproteomic analysis of control and MLK4-depleted HCC1806 cells, either untreated or treated with doxorubicin. We found that the doxorubicin-induced phosphorylation of several core DNA damage response components, including ATM, TRIM28 (KAP-1), MDC1 and TP53BP1, was impaired in cells with MLK4 knock-down. These findings were further confirmed using HCC1806 cells with CRISPR/Cas9-mediated deletion of MLK4, which showed markedly reduced phosphorylation of ATM kinase at Ser1981 - a site critical for ATM monomerization and activation. ATM was shown to activate NF-κB transcription in response to DNA damage through the well-established nuclear to cytoplasmic signaling pathway involving NEMO. Interestingly, we observed diminished phosphorylation of NEMO and reduced expression of several NF-кB-associated pro-survival cytokines (IL6, IL8, CXCL1, CXCL6) in MLK4-depleted TNBC cells treated with doxorubicin. These data, along with the additional immunoprecipitation and kinase assay experiments, indicate that MLK4 is involved in the regulation of DNA damage response signaling and ATM-NEMO pathway activation. Moreover, our recent results suggest that MLK4 controls autocrine/paracrine signaling loops in cancer cells and tumor microenvironment (TME) upon treatment with chemotherapy. We currently aim to further determine how MLK4-dependent crosstalk between TNBC cells and TME contributes to chemoresistance and tumor progression. Citation Format: Dawid Mehlich, Michal Lomiak, Aleksandra Sobiborowicz, Alicja Mazan, Dagmara Dymerska, Lukasz M. Szewczyk, Anna Mehlich, Agnieszka Borowiec, Monika K. Prelowska, Dominika Nowis, Anna A. Marusiak. Mixed-lineage kinase 4 is involved in the activation of the ATM-NEMO signaling axis in response to DNA damage and confers chemoresistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 151.

Function and Molecular Mechanism of the DNA Damage Response in Immunity and Cancer Immunotherapy.

The DNA damage response (DDR) is an organized network of multiple interwoven components evolved to repair damaged DNA and maintain genome fidelity. Conceptually the DDR includes damage sensors, transducer kinases, and effectors to maintain genomic stability and accurate transmission of genetic information. We have recently gained a substantially improved molecular and mechanistic understanding of how DDR components are interconnected to inflammatory and immune responses to stress. DDR shapes both innate and adaptive immune pathways: (i) in the context of innate immunity, DDR components mainly enhance cytosolic DNA sensing and its downstream STimulator of INterferon Genes (STING)-dependent signaling; (ii) in the context of adaptive immunity, the DDR is needed for the assembly and diversification of antigen receptor genes that is requisite for T and B lymphocyte development. Imbalances between DNA damage and repair impair tissue homeostasis and lead to replication and transcription stress, mutation accumulation, and even cell death. These impacts from DDR defects can then drive tumorigenesis, secretion of inflammatory cytokines, and aberrant immune responses. Yet, DDR deficiency or inhibition can also directly enhance innate immune responses. Furthermore, DDR defects plus the higher mutation load in tumor cells synergistically produce primarily tumor-specific neoantigens, which are powerfully targeted in cancer immunotherapy by employing immune checkpoint inhibitors to amplify immune responses. Thus, elucidating DDR-immune response interplay may provide critical connections for harnessing immunomodulatory effects plus targeted inhibition to improve efficacy of radiation and chemotherapies, of immune checkpoint blockade, and of combined therapeutic strategies.

Impact of DNA Damage Response-Targeted Therapies on the Immune Response to Tumours.

Simple SummaryTargeting tumour-specific defects in the DNA damage response (DDR) presents an opportunity for new therapeutic approaches to selectively kill cancer cells. Although the therapeutic rationale of DDR-targeted agents initially focused on their actions against tumour cells, these agents might also alter the crosstalk between tumour cells and the immune system. Here, we discuss recent data showing that DDR-targeted agents affect the antitumour immune response both through direct actions on the immune system components and through indirect effects on expression of different molecules and pathways in tumour cells that underpin the tumour cell–immune system.The DNA damage response (DDR) maintains the stability of a genome faced with genotoxic insults (exogenous or endogenous), and aberrations of the DDR are a hallmark of cancer cells. These cancer-specific DDR defects present new therapeutic opportunities, and different compounds that inhibit key components of DDR have been approved for clinical use or are in various stages of clinical trials. Although the therapeutic rationale of these DDR-targeted agents initially focused on their action against tumour cells themselves, these agents might also impact the crosstalk between tumour cells and the immune system, which can facilitate or impede tumour progression. In this review, we summarise recent data on how DDR-targeted agents can affect the interactions between tumour cells and the components of the immune system, both by acting directly on the immune cells themselves and by altering the expression of different molecules and pathways in tumour cells that are critical for their relationship with the immune system. Obtaining an in-depth understanding of the mechanisms behind how DDR-targeted therapies affect the immune system, and their crosstalk with tumour cells, may provide invaluable clues for the rational development of new therapeutic strategies in cancer.

DNA Damage Response Inhibitors as Potentiators of Radiation and Immunogenicity in Merkel Cell Carcinoma

<h3>Purpose/Objective(s)</h3> Both Merkel cell polyomavirus positive (VP) and negative (VN) Merkel cell carcinoma (MCC) are highly immunogenic with the highest response rates to immune checkpoint inhibitors (ICI) among solid tumors. Approximately 50% of patients with advanced VP or VN MCC attain durable disease control with ICI; but, patients with MCC (or other cancers) refractory to ICI have limited options necessitating new immune stimulating therapies. Cell cycle checkpoints and the DNA damage response (DDR) are promising targets, and selective inhibitors of key DDR components, including ATR, ATM & DNA-PK, are in early clinical trials. Deregulation of early cell cycle components (e.g., p53 & Rb inactivation paired with Myc protein upregulation) promotes rapid MCC cell division, suggesting high replication stress and susceptibility to DDR inhibitors (DDRi) or DNA damaging agents. Indeed, MCC is very radiosensitive. Animal studies show DDRi + radiation synergize to induce antitumor immunity. The mechanisms for how DDRi promote antitumor immunity are not well understood; however, they may drive immunogenic tumor cell death. Here, we assess potential synergy of DDRi + radiation to upregulate MCC immune markers. <h3>Materials/Methods</h3> MCC-VP (WaGa & MKL2) and VN (MCC13-HL) MCC cell lines were treated with various doses of single fraction radiation (0, 2, 4, 8, 16, 24 Gray; 160 kV x-rays) +/- inhibitors of ATR, ATM or DNA-PK. Cells were assessed for viability, expression of IFNβ (ELISA), and MHC-I expression (flow cytometry at 3 days post-radiation). <h3>Results</h3> In VN-MCC cells, ATRi and ATMi potentiated low-dose radiation (2-4 Gy) to induce IFNβ release and MHC-I upregulation compared to drug or radiation treatment alone at equivalent doses. DNA-PKi was the most potent radiosensitizer inducing rapid cell death when combined with low-dose radiation (2-4 Gy), but early cell death limited IFNβ production relative to VN-MCC treated with radiation alone. DNA-PKi + low-dose radiation to upregulated MHC-I in VN-MCC. All DDRi tested abrogated peak IFNβ production in VN-MCC at radiation doses ≥8 Gy. The Merkel cell polyomavirus inhibits cGAS/STING mediated IFNβ production, and radiation + DDRi did not induce detectable IFNβ release in two VP cell lines. All MCC cell lines were viable when treated with DDRi alone. <h3>Conclusion</h3> Given inherent immunogenicity, cell cycle checkpoint deficiencies and radiosensitivity, MCC is an ideal tumor to test the potential of DDRi to overcome ICI-resistance. Our preliminary data suggest that ATRi and ATMi may effectively potentiate immunogenic cell death in a VN-MCC cell line when paired with 2-4 Gy radiation, yet abrogate IFNβ production with higher radiation doses <b>in vitro</b> suggesting higher doses coupled with DDRi induce non-immunogenic cell death. DNA-PKi was the most potent radiosensitizer in 3 MCC cell lines by cell viability assays but less effective at inducing IFNβ. <b>In vitro</b> studies are ongoing to support future animal studies as we explore a potential trial for DDRi in ICI-resistant MCC.

Deficient DNA mismatch repair and persistence of SARS-CoV-2 RNA shedding: a case report of hereditary nonpolyposis colorectal cancer with COVID-19 infection

BackgroundSeveral independent risk factors have been reported to influence viral shedding following COVID-19 infection, but the influence of host-related molecular factors has not yet been described. We report a case of a cancer patient with Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) who manifested SARS-CoV-2 PCR (polymerase chain reaction) positivity for at least 54 days after contracting mild COVID-19 illness. We propose that deficient mismatch repair (MMR) may play a role in the prolonged SARS-CoV-2 RNA shedding.Case presentationA patient with Lynch syndrome was under surveillance for metastatic adenocarcinoma after completing palliative chemotherapy in October 2019. Between the period of April 2020 to June 2020, he was admitted multiple times to address several clinical needs mainly related to his underlying malignancy. These included progressive disease observed in the aortocaval lymph nodes leading to recurrent episodes of upper gastrointestinal bleeding, dehydration resulting in acute kidney injury and a short-lived episode of pyrexia. A SARS-CoV-2 PCR of the nasopharyngeal swab (NPS) was positive at his initial admission with mild COVID-19 symptoms. He remained positive on subsequent admissions when tested routinely for SARS-CoV-2 without demonstrating any apparent clinical features of COVID-19 infection.The MMR pathway, a component of DNA damage response (DDR), is impaired in Lynch syndrome due to an inherited genetic mutation. This pathway is also required for viral clearance from the host cells following certain RNA viral infections like influenza virus and other coronaviridae. Here we provide a current understanding of the importance of DDR deficiencies in the clearance of RNA virus and suggest how this may play a similar role in the clearance of COVID-19, as evident in our case that demonstrated persistent positivity.ConclusionThe importance of understanding the scientific basis of extended viral shedding during the COVID-19 pandemic is now centre-stage in the establishment of robust track and trace services to allow the recovery and function of societies and economies. This patient with Lynch syndrome recovered from infection but had prolonged viral positivity, which might merit further investigation to better understand the effect of this condition on infection duration and outcome.

Oxymestane, a cytostatic steroid derivative of exemestane with greater antitumor activity in non-estrogen-dependent cell lines

A new promising steroid derivative of Exemestane (Exe), the drug used for the treatment of estrogen-dependent breast cancer, was synthesized and evaluated against a set of human cancer cell lines. The new compound (Oxymestane-D1, Oxy) was tested comparatively with Exe against colon (C2BBe1, WiDr), liver (HepG2, HuH-7), lung (A549, H1299) and prostate (LNCaP, PC3) human cancer cell lines. Likewise, its effect on human colon normal cells (CCD-841 CoN) and human normal fibroblast cells (HFF-1) was studied. The cytostatic activity of Oxy was also compared with that of the reference cytostatic drugs used in chemotherapy protocols, namely carboplatin, cisplatin, doxorubicin, epirubicin, etoposide, flutamide, 5-fluorouracil, irinotecan, oxaliplatin and sorafenib. In all cell lines tested, Oxy proved to be more powerful cytostatic than Exe. Additionally, the IC50 at 72 h showed a three-fold activity greater than 5-fluorouracil in the WiDr cell line, twice as high as cisplatin for cell line A549 and five times higher than cisplatin for cell line H1299. Also, Oxy surprisingly revealed to induce DNA damage and inhibit the DNA damage response (DDR) proteins ATM, ATR, CHK1 and CHK2. The results obtained allow concluding that Oxy can be a promising anticancer agent to be used in chemotherapy protocols. Furthermore, its ability to inhibit crucial components of DDR can also be useful for the monotherapy or for combination with chemo and/or radiotherapy of cancer.

Very long intergenic non-coding (vlinc) RNAs directly regulate multiple genes in cis and trans

BackgroundThe majority of the human genome is transcribed in the form of long non-coding (lnc) RNAs. While these transcripts have attracted considerable interest, their molecular mechanisms of function and biological significance remain controversial. One of the main reasons behind this lies in the significant challenges posed by lncRNAs requiring the development of novel methods and concepts to unravel their functionality. Existing methods often lack cross-validation and independent confirmation by different methodologies and therefore leave significant ambiguity as to the authenticity of the outcomes. Nonetheless, despite all the caveats, it appears that lncRNAs may function, at least in part, by regulating other genes via chromatin interactions. Therefore, the function of a lncRNA could be inferred from the function of genes it regulates. In this work, we present a genome-wide functional annotation strategy for lncRNAs based on identification of their regulatory networks via the integration of three distinct types of approaches: co-expression analysis, mapping of lncRNA-chromatin interactions, and assaying molecular effects of lncRNA knockdowns obtained using an inducible and highly specific CRISPR/Cas13 system.ResultsWe applied the strategy to annotate 407 very long intergenic non-coding (vlinc) RNAs belonging to a novel widespread subclass of lncRNAs. We show that vlincRNAs indeed appear to regulate multiple genes encoding proteins predominantly involved in RNA- and development-related functions, cell cycle, and cellular adhesion via a mechanism involving proximity between vlincRNAs and their targets in the nucleus. A typical vlincRNAs can be both a positive and negative regulator and regulate multiple genes both in trans and cis. Finally, we show vlincRNAs and their regulatory networks potentially represent novel components of DNA damage response and are functionally important for the ability of cancer cells to survive genotoxic stress.ConclusionsThis study provides strong evidence for the regulatory role of the vlincRNA class of lncRNAs and a potentially important role played by these transcripts in the hidden layer of RNA-based regulation in complex biological systems.

Interfaces between cellular responses to DNA damage and cancer immunotherapy

The DNA damage response (DDR) fulfils essential roles to preserve genome integrity. Targeting the DDR in tumors has had remarkable success over the last decade, exemplified by the licensing of PARP inhibitors for cancer therapy. Recent studies suggest that the application of DDR inhibitors impacts on cellular innate and adaptive immune responses, wherein key DNA repair factors have roles in limiting chronic inflammatory signaling. Antitumor immunity plays an emerging part in cancer therapy, and extensive efforts have led to the development of immune checkpoint inhibitors overcoming immune suppressive signals in tumors. Here, we review the current understanding of the molecular mechanisms underlying DNA damage-triggered immune responses, including cytosolic DNA sensing via the cGAS/STING pathway. We highlight the implications of DDR components for therapeutic outcomes of immune checkpoint inhibitors or their use as biomarkers. Finally, we discuss the rationale for novel combinations of DDR inhibitors with antagonists of immune checkpoints and current hindrances limiting their broader therapeutic applications.

Genome instability and loss of protein homeostasis: converging paths to neurodegeneration?

Genome instability and loss of protein homeostasis are hallmark events of age-related diseases that include neurodegeneration. Several neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis are characterized by protein aggregation, while an impaired DNA damage response (DDR) as in many genetic DNA repair disorders leads to pronounced neuropathological features. It remains unclear to what degree these cellular events interconnect with each other in the development of neurological diseases. This review highlights how the loss of protein homeostasis and genome instability influence one other. We will discuss studies that illustrate this connection. DNA damage contributes to many neurodegenerative diseases, as shown by an increased level of DNA damage in patients, possibly due to the effects of protein aggregates on chromatin, the sequestration of DNA repair proteins and novel putative DNA repair functions. Conversely, genome stability is also important for protein homeostasis. For example, gene copy number variations and the loss of key DDR components can lead to marked proteotoxic stress. An improved understanding of how protein homeostasis and genome stability are mechanistically connected is needed and promises to lead to the development of novel therapeutic interventions.