CXCL10 potentiates immune checkpoint blockade therapy in homologous recombination-deficient tumors.

Background: Homologous recombination deficiency (HRD) is a common molecular characteristic of genomic instability, and has been proven to be a biomarker for target therapy. However, until now, no research has explored the changes in the transcriptomics landscape of HRD tumors. Methods: The HRD score was established from SNP array data of breast cancer patients from the cancer genome atlas (TCGA) database. The transcriptome data of patients with different HRD scores were analyzed to identify biomarkers associated with HRD. The candidate biomarkers were validated in the gene expression omnibus (GEO) database and immunotherapy cohorts. Results: Based on data from the gene expression profile and clinical characteristics from 1310 breast cancer patients, including TCGA database and GEO database, we found that downstream targets of the cGAS-STING pathway, such as CXCL10, were upregulated in HRD tumors and could be used as a predictor of survival outcome in triple-negative breast cancer (TNBC) patients. Further comprehensive analysis of the tumor immune microenvironment (TIME) revealed that the expression of CXCL10 was positively correlated with neoantigen load and infiltrating immune cells. Finally, in vivo experimental data and clinical trial data confirmed that the expression of CXCL10 could be used as a biomarker for anti-PD-1/PD-L1 therapy. Conclusions: Together, our study not only revealed that CXCL10 is associated with HRD but also introduced a potential new perspective for identifying prognostic biomarkers of immunotherapy.

Congenital Diseases of DNA Replication: Clinical Phenotypes and Molecular Mechanisms.

Deoxyribonucleic acid (DNA) replication can be divided into three major steps: initiation, elongation and termination. Each time a human cell divides, these steps must be reiteratively carried out. Disruption of DNA replication can lead to genomic instability, with the accumulation of point mutations or larger chromosomal anomalies such as rearrangements. While cancer is the most common class of disease associated with genomic instability, several congenital diseases with dysfunctional DNA replication give rise to similar DNA alterations. In this review, we discuss all congenital diseases that arise from pathogenic variants in essential replication genes across the spectrum of aberrant replisome assembly, origin activation and DNA synthesis. For each of these conditions, we describe their clinical phenotypes as well as molecular studies aimed at determining the functional mechanisms of disease, including the assessment of genomic stability. By comparing and contrasting these diseases, we hope to illuminate how the disruption of DNA replication at distinct steps affects human health in a surprisingly cell-type-specific manner.

RNF8 Dysregulation and Down-regulation During HTLV-1 Infection Promote Genomic Instability in Adult T-Cell Leukemia.

The genomic instability associated with adult T cell leukemia/lymphoma (ATL) is causally linked to Tax, the HTLV-1 viral oncoprotein, but the underlying mechanism is not fully understood. We have previously shown that Tax hijacks and aberrantly activates ring finger protein 8 (RNF8) - a lysine 63 (K63)-specific ubiquitin E3 ligase critical for DNA double-strand break (DSB) repair signaling - to assemble K63-linked polyubiquitin chains (K63-pUbs) in the cytosol. Tax and the cytosolic K63-pUbs, in turn, initiate additional recruitment of linear ubiquitin assembly complex (LUBAC) to produce hybrid K63-M1 pUbs, which trigger a kinase cascade that leads to canonical IKK:NF-κB activation. Here we demonstrate that HTLV-1-infected cells are impaired in DNA damage response (DDR). This impairment correlates with the induction of microscopically visible nuclear speckles by Tax known as the Tax-speckle structures (TSS), which act as pseudo DNA damage signaling scaffolds that sequester DDR factors such as BRCA1, DNA-PK, and MDC1. We show that TSS co-localize with Tax, RNF8 and K63-pUbs, and their formation depends on RNF8. Tax mutants defective or attenuated in inducing K63-pUb assembly are deficient or tempered in TSS induction and DDR impairment. Finally, our results indicate that loss of RNF8 expression reduces HTLV-1 viral gene expression and frequently occurs in ATL cells. Thus, during HTLV-1 infection, Tax activates RNF8 to assemble nuclear K63-pUbs that sequester DDR factors in Tax speckles, disrupting DDR signaling and DSB repair. Down-regulation of RNF8 expression is positively selected during infection and progression to disease, and further exacerbates the genomic instability of ATL.

The aberrant expression of rhythm genes affects the genome instability and regulates the cancer immunity in pan-cancer.

Although emerging studies showed that certain rhythm genes regulate cancer progression, the expression and roles of the vast majority of rhythm genes in human cancer are largely unknown, and the hallmarks of cancer regulated by rhythm genes have not been detected. In this study, we detected the expression changes of rhythm genes in pan-cancer and found that almost all rhythm genes mutated in all cancer types, and their expression level was significantly altered partially due to abnormal methylation, and several rhythm genes regulate the expression of other rhythm genes in various cancer types. Furthermore, we revealed that rhythm genes are significantly enriched in genome instability and the expression of certain rhythm genes is correlated with the tumor mutation burden, microsatellite instability, and the expression of DNA damage repair genes in most of the detected cancer types. Moreover, rhythm genes are associated with the infiltration of immune cells and the efficiency of immune blockade therapy. This study provides a comprehensive understanding of the roles of rhythm genes in cancer immunity, which may provide a novel method for the diagnosis and treatment of cancer.

RADIATION-INDUCED BYSTANDER EFFECT - MODELING, MANIFESTATION, MECHANISMS, PERSISTENCE, CANCER RISKS (literature review). / RADIATsIY̆NO-INDUKOVANYY̆ EFEKT SVIDKA ­ MODELIuVANNIa, PROIaVY, MEKhANIZMY ROZVYTKU, PERSYSTENTsIIa, ONKOLOGIChNI RYZYKY (ogliad literatury).

The review summarizes and analyzes the data of world scientific literature and the results of the own research con- cerning one of the main non-targeted effects of ionizing radiation - the radiation induced bystander effect (RIBE) - the ability of irradiated target cells to induce secondary biological changes in non-irradiated receptor cells. The his- tory of studies of this phenomenon is presented - it described under various names since 1905, began to study from the end of the twentieth century when named as RIBE and caused particular interest in the scientific community during recent decades. It is shown that the development of biological science and the improvement of research methods allowed to get new in-depth data on the development of RIBE not only at the level of the whole organism, but even at the genome level. The review highlights the key points of numerous RIBE investigations including mod- eling; methodological approaches to studying; classification; features of interaction between irradiated and intact cells; the role of the immune system, oxidative stress, cytogenetic disorders, changes in gene expression in the mechanism of development of RIBE; rescue effect, abscopal effect, persistence, modification, medical effects. It is emphasized that despite the considerable amount of research concerning the bystander response as the universal phenomenon and RIBE as one of its manifestations, there are still enough «white spots¼ in determining the mech- anisms of the RIBE formation and assessing the possible consequences of its development for human health.

Nbn-Mre11 interaction is required for tumor suppression and genomic integrity.

We derived a mouse model in which a mutant form of Nbn/Nbs1mid8 (hereafter Nbnmid8) exhibits severely impaired binding to the Mre11-Rad50 core of the Mre11 complex. The Nbnmid8 allele was expressed exclusively in hematopoietic lineages (in Nbn-/mid8vav mice). Unlike Nbnflox/floxvav mice with Nbn deficiency in the bone marrow, Nbn-/mid8vav mice were viable. Nbn-/mid8vav mice hematopoiesis was profoundly defective, exhibiting reduced cellularity of thymus and bone marrow, and stage-specific blockage of B cell development. Within 6 mo, Nbn-/mid8 mice developed highly penetrant T cell leukemias. Nbn-/mid8vav leukemias recapitulated mutational features of human T cell acute lymphoblastic leukemia (T-ALL), containing mutations in NOTCH1, TP53, BCL6, BCOR, and IKZF1, suggesting that Nbnmid8 mice may provide a venue to examine the relationship between the Mre11 complex and oncogene activation in the hematopoietic compartment. Genomic analysis of Nbn-/mid8vav malignancies showed focal amplification of 9qA2, causing overexpression of MRE11 and CHK1 We propose that overexpression of MRE11 compensates for the metastable Mre11-Nbnmid8 interaction, and that selective pressure for overexpression reflects the essential role of Nbn in promoting assembly and activity of the Mre11 complex.

The ESC: The Dangerous By-Product of V(D)J Recombination.

V(D)J recombination generates antigen receptor diversity by mixing and matching individual variable (V), diversity (D), and joining (J) gene segments. An obligate by-product of many of these reactions is the excised signal circle (ESC), generated by excision of the DNA from between the gene segments. Initially, the ESC was believed to be inert and formed to protect the genome from reactive broken DNA ends but more recent work suggests that the ESC poses a substantial threat to genome stability. Crucially, the recombinase re-binds to the ESC, which can result in it being re-integrated back into the genome, to cause potentially oncogenic insertion events. In addition, very recently, the ESC/recombinase complex was found to catalyze breaks at recombination signal sequences (RSSs) throughout the genome, via a "cut-and-run" mechanism. Remarkably, the ESC/recombinase complex triggers these breaks at key leukemia driver genes, implying that this reaction could be a significant cause of lymphocyte genome instability. Here, we explore these alternate pathways and discuss their relative dangers to lymphocyte genome stability.

Association of BRCA1- and BRCA2-deficiency with mutation burden, expression of PD-L1/PD-1, immune infiltrates, and T cell-inflamed signature in breast cancer.

Immune checkpoint inhibitors have demonstrated effective anti-tumour response in cancer types with high mutation burden (e.g. melanoma) and in subset of cancers with features of genomic instability (e.g. mismatch-repair deficiency). One possible explanation for this effect is the increased expression of immune checkpoint molecules and pre-existing adaptive immune response in these cancers. Given that BRCA1 and BRCA2 are integral in maintaining genomic integrity, we hypothesise that the inactivation of these genes may give rise to breast cancers with such immunogenic phenotype. Therefore, using two large series of publicly available breast cancer datasets, namely that from The Cancer Genome Atlas and Wellcome Trust Institute, we sought to investigate the association between BRCA1- and BRCA2-deficiency with features of genomic instability, expression of PD-L1 and PD-1, landscape of inferred tumour-infiltrating immune cells, and T-cell inflamed signature in breast cancers. Here, we report that BRCA1 and BRCA2-deficient breast cancers were associated with features of genomic instability including increased mutation burden. Interestingly, BRCA1-, but not BRCA2-, deficient breast cancers were associated with increased expression of PD-L1 and PD-1, higher abundance of tumour-infiltrating immune cells, and enrichment of T cell-inflamed signature. The differences in immunophenotype between BRCA1- and BRCA2-deficient breast cancers can be attributed, in part, to PTEN gene mutation. Therefore, features of genomic instability such as that mediated by BRCA1- and BRCA2- deficiency in breast cancer were necessary, but not always sufficient, for yielding T cell-inflamed tumour microenvironment, and by extension, predicting clinical benefit from immunotherapy.

Apoptosis and genome instability in children with autoimmune diseases.

As apoptosis and genome instability in children with autoimmune diseases (AIDs) are insufficiently investigated, we aimed to analyse them in peripheral blood lymphocytes (PBLs) of children and adolescents with Hashimoto's thyroiditis (HT), Graves' disease (GD) and type 1 diabetes mellitus (T1DM), including possible factors that could affect their occurrence. The study population included 24 patients and 19 healthy controls. Apoptotic cells were detected using an Annexin V-FITC/7-AAD kit. Genome instability was measured as micronuclei (MNs) frequency using the cytokinesis-block MN assay. In addition, comet assay was performed for determination of genome instability as genome damage index (GDI) in new subpopulation of patients with T1DM. The percentage of apoptotic PBLs in patients with AID was significantly lower than in control subjects. There was a positive correlation between thyroid-stimulating homone (TSH) concentration and the proportion of cells in late stage apoptosis in patients with autoimmune thyroid diseases (AITDs). The MN frequency in patients was significantly higher than in controls. Individuals with HT or T1DM had a significantly higher MN frequency than those with GD. Similarly, the value of GDI in patients with T1DM was significantly higher than in controls. The level of apoptosis was positively correlated with MN frequency as well as with GDI in patients with AID. In conclusion, children with AITD (HT and GD) and T1DM have a significantly lower level of apoptosis in PBLs and significantly higher MN frequency as GDI than healthy subjects. Apoptosis and the level of genome instability in these patients with AID are positively correlated.

Immune signatures correlate with L1 retrotransposition in gastrointestinal cancers.

Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons are normally suppressed in somatic tissues mainly due to DNA methylation and antiviral defense. However, the mechanism to suppress L1s may be disrupted in cancers, thus allowing L1s to act as insertional mutagens and cause genomic rearrangement and instability. Whereas the frequency of somatic L1 insertions varies greatly among individual tumors, much remains to be learned about underlying genetic, cellular, or environmental factors. Here, we report multiple correlates of L1 activity in stomach, colorectal, and esophageal tumors through an integrative analysis of cancer whole-genome and matched RNA-sequencing profiles. Clinical indicators of tumor progression, such as tumor grade and patient age, showed positive association. A potential L1 expression suppressor, TP53, was mutated in tumors with frequent L1 insertions. We characterized the effects of somatic L1 insertions on mRNA splicing and expression, and demonstrated an increased risk of gene disruption in retrotransposition-prone cancers. In particular, we found that a cancer-specific L1 insertion in an exon of MOV10, a key L1 suppressor, caused exon skipping and decreased expression of the affected allele due to nonsense-mediated decay in a tumor with a high L1 insertion load. Importantly, tumors with high immune activity, for example, those associated with Epstein-Barr virus infection or microsatellite instability, tended to carry a low number of L1 insertions in genomes with high expression levels of L1 suppressors such as APOBEC3s and SAMHD1 Our results indicate that cancer immunity may contribute to genome stability by suppressing L1 retrotransposition in gastrointestinal cancers.

The Effect of Light Exposure at Night (LAN) on Carcinogenesis via Decreased Nocturnal Melatonin Synthesis.

In mammals, a master clock is located within the suprachiasmatic nucleus (SCN) of the hypothalamus, a region that receives input from the retina that is transmitted by the retinohypothalamic tract. The SCN controls the nocturnal synthesis of melatonin by the pineal gland that can influence the activity of the clock's genes and be involved in the inhibition of cancer development. On the other hand, in the literature, some papers highlight that artificial light exposure at night (LAN)-induced circadian disruptions promote cancer. In the present review, we summarize the potential mechanisms by which LAN-evoked disruption of the nocturnal increase in melatonin synthesis counteracts its preventive action on human cancer development and progression. In detail, we discuss: (i) the Warburg effect related to tumor metabolism modification; (ii) genomic instability associated with L1 activity; and (iii) regulation of immunity, including regulatory T cell (Treg) regulation and activity. A better understanding of these processes could significantly contribute to new treatment and prevention strategies against hormone-related cancer types.

DNA Damage Response Signals Transduce Stress From Rheumatoid Arthritis Risk Factors Into T Cell Dysfunction.

Rheumatoid arthritis (RA) is an autoimmune-mediated disease that is associated with significant cartilage damage and immunosenescence. Despite decades of research, the major signal pathways that initiate RA are still unclear. The DNA damage response (DDR) is a specific and hierarchical network that includes cell cycle checkpoints, DNA repair, and DNA-damage tolerance pathways. Recent studies suggest that this condition is associated with deficits in telomere maintenance and overall genomic instability in the T cells of RA patients. Analysis of the underlying mechanisms has revealed defects in DDR pathways. Particularly, the DNA repair enzyme, ataxia telangiectasia mutated (ATM), is downregulated, which leaves the damaged DNA breaks in RA-associated T cells unrepaired and pushes them to apoptosis, exhausts the T cell pool, and promotes the arthritogenesis effector function of T cells. This review discusses recent advancements and illustrates that risk factors for RA, such as viral infections, environmental events, and genetic risk loci are combat with DDR signals, and the impaired DDR response of RA-associated T cells, in turn, triggers disease-related phenotypes. Therefore, DDR is the dominant signal that converts genetic and environmental stress to RA-related immune dysfunction. Understanding the orchestration of RA pathogenesis by DDR signals would further our current knowledge of RA and provide novel avenues in RA therapy.

cGAS surveillance of micronuclei links genome instability to innate immunity.

DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP-AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.

DNA Damage and Repair Biomarkers of Immunotherapy Response.

DNA-damaging agents are widely used in clinical oncology and exploit deficiencies in tumor DNA repair. Given the expanding role of immune checkpoint blockade as a therapeutic strategy, the interaction of tumor DNA damage with the immune system has recently come into focus, and it is now clear that the tumor DNA repair landscape has an important role in driving response to immune checkpoint blockade. Here, we summarize the mechanisms by which DNA damage and genomic instability have been found to shape the antitumor immune response and describe clinical efforts to use DNA repair biomarkers to guide use of immune-directed therapies.Significance: Only a subset of patients respond to immune checkpoint blockade, and reliable predictive biomarkers of response are needed to guide therapy decisions. DNA repair deficiency is common among tumors, and emerging experimental and clinical evidence suggests that features of genomic instability are associated with response to immune-directed therapies. Cancer Discov; 7(7); 675-93. ©2017 AACR.

IFN-γ is required for cytotoxic T cell-dependent cancer genome immunoediting.

Genetic evolution that occurs during cancer progression enables tumour heterogeneity, thereby fostering tumour adaptation, therapeutic resistance and metastatic potential. Immune responses are known to select (immunoedit) tumour cells displaying immunoevasive properties. Here we address the role of IFN-γ in mediating the immunoediting process. We observe that, in several mouse tumour models such as HA-expressing 4T1 mammary carcinoma cells, OVA-expressing EG7 lymphoma cells and CMS5 MCA-induced fibrosarcoma cells naturally expressing mutated extracellular signal-regulated kinase (ERK) antigen, the action of antigen-specific cytotoxic T cell (CTL) in vivo results in the emergence of resistant cancer cell clones only in the presence of IFN-γ within the tumour microenvironment. Moreover, we show that exposure of tumours to IFN-γ-producing antigen-specific CTLs in vivo results in copy-number alterations (CNAs) associated with DNA damage response and modulation of DNA editing/repair gene expression. These results suggest that enhanced genetic instability might be one of the mechanisms by which CTLs and IFN-γ immunoedits tumours, altering their immune resistance as a result of genetic evolution.

Immune and inflammatory responses to DNA damage in cancer and aging.

Genome instability is a hallmark of both cancer and aging processes. Beyond cell-autonomous responses, it is known that DNA damage also elicits systemic mechanisms aimed at favoring survival and damaged cells clearance. Among these mechanisms, immune activation and NF-κB-mediated inflammation play central roles in organismal control of DNA damage. We focus herein on the different experimental evidences that have allowed gaining mechanistic insight about this relationship. We also describe the functional consequences of defective immune function in cancer development and age-related alterations. Finally, we discuss different intervention strategies based on enhancing immunity or on the modulation of the inflammatory response to improve organism homeostasis in cancer and aging.

Role of Genomic Instability in Immunotherapy with Checkpoint Inhibitors.

AIM: We evaluated whether tumor genome sequencing to detect the number and type of alterations could be used as a valuable biomarker for judging the potential utility of immune checkpoint inhibitors in patients with advanced cancers. MATERIALS AND METHODS: We identified patients with solid tumors who were treated with checkpoint inibitors and had received commercially available next generation sequencing (NGS). Tumors profiled by Caris Life Sciences, Foundation Medicine and Guardant360 between 2013 and 2015. Patients were divided into 5 quintiles based on mutational load (pathogenic mutations plus variants of undetermined significance). RESULTS: Fifty patients with solid tumors on immunotherapy that had NGS reports available were identified. Top quintile patients had more genomic alterations (median=16.5) than the others (median=2) and had more pathogenic mutations in cell-cycle regulatory genes (100% versus 48%). The overall survival (OS) was significantly superior for patients in the top quintile (722 days) versus the others (432 days). We found no significant difference in progression-free survival (PFS) between the two groups. The objective response rate was numerically higher for the top quintile (50%) vs. others (20%). Programmed cell death protein 1 (PD1) and programmed death-ligand 1 (PDL1) status by immunohistochemistry was not associated with outcomes. CONCLUSION: The use of immune checkpoint blockade in tumors with higher mutational load was associated with improved OS. Our results suggest that the evaluation of tumor genomes may be predictive of immunotherapy benefit.

Role of Pro-inflammatory Cytokines in Radiation-Induced Genomic Instability in Human Bronchial Epithelial Cells.

Inflammatory cytokines have been implicated in the regulation of radiation-induced genomic instability in the hematopoietic system and have also been shown to induce chronic DNA damage responses in radiation-induced senescence. We have previously shown that human bronchial epithelial cells (HBEC3-KT) have increased genomic instability and IL-8 production persisting at day 7 after exposure to high-LET (600 MeV/nucleon (56)Fe ions) compared to low-LET (320 keV X rays) radiation. Thus, we investigated whether IL-8 induction is part of a broader pro-inflammatory response produced by the epithelial cells in response to damage, which influences genomic instability measured by increased micronuclei and DNA repair foci frequencies. We found that exposure to radiation induced the release of multiple inflammatory cytokines into the media, including GM-CSF, GROα, IL-1α, IL-8 and the inflammation modulator, IL-1 receptor antagonist (IL-1RA). Our results suggest that this is an IL-1α-driven response, because an identical signature was induced by the addition of recombinant IL-1α to nonirradiated cells and functional interference with recombinant IL-1RA (Anakinra) or anti-IL-1α function-blocking antibody, decreased IL-8 production induced by radiation exposure. However, genomic instability was not influenced by this pathway as addition of recombinant IL-1α to naive or irradiated cells or the presence of IL-1 RA under the same conditions as those that interfered with the function of IL-8, did not affect micronuclei or DNA repair foci frequencies measured at day 7 after exposure. While dose-response studies revealed that genomic instability and IL-8 production are the consequences of targeted effects, experiments employing a co-culture transwell system revealed the propagation of pro-inflammatory responses but not genomic instability from irradiated to nonirradiated cells. Collectively, these results point to a cell-autonomous mechanism sustaining radiation-induced genomic instability in this model system and suggest that while molecules associated with these mechanisms could be markers for persisting damage, they reflect two different outcomes.