A PoleP286R mouse model of endometrial cancer recapitulates high mutational burden and immunotherapy response.

Cancer is instigated by mutator phenotypes, including deficient mismatch repair and p53-associated chromosomal instability. More recently, a distinct class of cancers was identified with unusually high mutational loads due to heterozygous amino acid substitutions (most commonly P286R) in the proofreading domain of DNA polymerase ε, the leading strand replicase encoded by POLE. Immunotherapy has revolutionized cancer treatment, but new model systems are needed to recapitulate high mutational burdens characterizing human cancers and permit study of mechanisms underlying clinical responses. Here, we show that activation of a conditional LSL-PoleP286R allele in endometrium is sufficient to elicit in all animals endometrial cancers closely resembling their human counterparts, including very high mutational burden. Diverse investigations uncovered potentially novel aspects of Pole-driven tumorigenesis, including secondary p53 mutations associated with tetraploidy, and cooperation with defective mismatch repair through inactivation of Msh2. Most significantly, there were robust antitumor immune responses with increased T cell infiltrates, accelerated tumor growth following T cell depletion, and unfailing clinical regression following immune checkpoint therapy. This model predicts that human POLE-driven cancers will prove consistently responsive to immune checkpoint blockade. Furthermore, this is a robust and efficient approach to recapitulate in mice the high mutational burdens and immune responses characterizing human cancers.

Chromosomal instability and pro-inflammatory response in aging.

Aging refers to the progressive deterioration of tissue and organ function over time. Increasing evidence points to the accumulation of highly damaged cell cycle-arrested cells with age (cellular senescence) as major reason for the development of certain aging-associated diseases. Recent studies have independently shown that aneuploidy, an abnormal chromosome set, occurs in senescent cells, and that the accumulation of cytoplasmic DNA driven by faulty chromosome segregation during mitosis aids in the establishment of senescence and its associated secretory phenotype known as SASP. Here we review the emerging link between chromosomal instability (CIN) and senescence in the context of aging, with emphasis on the cGAS-STING pathway activation and its role in the development of the SASP. Based on current evidence, we propose that age-associated CIN in mitotically active cells contributes to aging and its associated diseases, and we discuss the inhibition of CIN as a potential strategy to prevent the generation of aneuploid senescent cells and thereby to delay aging.

The immunological consequences of radiation-induced DNA damage.

Historically, our understanding of the cytotoxicity of radiation has centred on tumour cell-autonomous mechanisms of cell death. Here, tumour cell death occurs when a threshold number of radiation-induced non-reparable double-stranded DNA breaks is exceeded. However, in recent years, the importance of immune mechanisms of cell death has been increasingly recognised, as well as the impact of radiotherapy on non-malignant cellular components of the tumour microenvironment. Conserved antiviral pathways that detect foreign nucleic acid in the cytosol and drive downstream interferon (IFN) responses via the cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of IFN genes (cGAS/STING) pathway are key components of the immune response to radiation-induced DNA damage. In preclinical models, acute induction of a type 1 IFN response is important for both direct and abscopal tumour responses to radiation. Inhibitors of the DNA damage response show promise in augmenting this inflammatory IFN response. However, a substantial proportion of tumours show chronic IFN signalling prior to radiotherapy, which paradoxically drives immunosuppression. This chronic IFN signalling leads to treatment resistance, and heterotypic interactions between stromal fibroblasts and tumour cells contribute to an aggressive tumour phenotype. The effect of radiotherapy on myeloid cell populations, particularly tumour-associated macrophages, has an additional impact on the immune tumour microenvironment. It is not yet clear how the above preclinical findings translate into a human context. Human tumours show greater intratumoural genomic heterogeneity and more variable levels of chromosomal instability than experimental murine models. High-quality translational studies of immunological changes occurring during radiotherapy that incorporate intrinsic tumour biology will enable a better understanding of the immunological consequences of radiation-induced DNA damage in patients. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System.

Aneuploidy, a state of karyotype imbalance, is a hallmark of cancer. Changes in chromosome copy number have been proposed to drive disease by modulating the dosage of cancer driver genes and by promoting cancer genome evolution. Given the potential of cells with abnormal karyotypes to become cancerous, do pathways that limit the prevalence of such cells exist? By investigating the immediate consequences of aneuploidy on cell physiology, we identified mechanisms that eliminate aneuploid cells. We find that chromosome mis-segregation leads to further genomic instability that ultimately causes cell-cycle arrest. We further show that cells with complex karyotypes exhibit features of senescence and produce pro-inflammatory signals that promote their clearance by the immune system. We propose that cells with abnormal karyotypes generate a signal for their own elimination that may serve as a means for cancer cell immunosurveillance.

Tumor Cell-Free DNA Copy Number Instability Predicts Therapeutic Response to Immunotherapy.

Purpose: Chromosomal instability is a fundamental property of cancer, which can be quantified by next-generation sequencing (NGS) from plasma/serum-derived cell-free DNA (cfDNA). We hypothesized that cfDNA could be used as a real-time surrogate for imaging analysis of disease status as a function of response to immunotherapy and as a more reliable tool than tumor biomarkers.Experimental Design: Plasma cfDNA sequences from 56 patients with diverse advanced cancers were prospectively collected and analyzed in a single-blind study for copy number variations, expressed as a quantitative chromosomal number instability (CNI) score versus 126 noncancer controls in a training set of 23 and a blinded validation set of 33. Tumor biomarker concentrations and a surrogate marker for T regulatory cells (Tregs) were comparatively analyzed.Results: Elevated CNI scores were observed in 51 of 56 patients prior to therapy. The blinded validation cohort provided an overall prediction accuracy of 83% (25/30) and a positive predictive value of CNI score for progression of 92% (11/12). The combination of CNI score before cycle (Cy) 2 and 3 yielded a correct prediction for progression in all 13 patients. The CNI score also correctly identified cases of pseudo-tumor progression from hyperprogression. Before Cy2 and Cy3, there was no significant correlation for protein tumor markers, total cfDNA, or surrogate Tregs.Conclusions: Chromosomal instability quantification in plasma cfDNA can serve as an early indicator of response to immunotherapy. The method has the potential to reduce health care costs and disease burden for cancer patients following further validation. Clin Cancer Res; 23(17); 5074-81. ©2017 AACR.

A Novel Mutation in a Critical Region for the Methyl Donor Binding in DNMT3B Causes Immunodeficiency, Centromeric Instability, and Facial Anomalies Syndrome (ICF).

PURPOSE: Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is an extremely rare autosomal recessive disease. The immune phenotype is characterized by hypogammaglobulinemia in the presence of B cells. T cell lymphopenia also develops in some patients. We sought to further investigate the immune defect in an ICF patient with a novel missense mutation in DNMT3B and a severe phenotype. METHODS: Patient lymphocytes were examined for subset counts, immunoglobulin levels, T and B cell de novo production (via excision circles) and receptor repertoire diversity. Mutated DNMT3B protein structure was modeled to assess the effect of a mutation located outside of the catalytic region on protein function. RESULTS: A novel homozygous missense mutation, Ala585Thr, was found in DNMT3B. The patient had decreased B cell counts with hypogammaglobulinemia, and normal T cell counts. CD4+ T cells decreased over time, leading to an inversion of the CD4+ to CD8+ ratio. Excision circle copy numbers were normal, signifying normal de novo lymphocyte production, but the ratio between naïve and total B cells was low, indicating decreased in vivo B cell replication. T and B cell receptor repertoires displayed normal diversity. Computerized modeling of the mutated Ala585 residue suggested reduced thermostability, possibly affecting the enzyme kinetics. CONCLUSIONS: Our results highlight the existence of a T cell defect that develops over time in ICF patient, in addition to the known B cell dysfunction. With intravenous immunoglobulin (IVIG) treatment ameliorating the B cell defect, the extent of CD4+ lymphopenia may determine the severity of ICF immunodeficiency.

Chromosomal instability triggers cell death via local signalling through the innate immune receptor Toll.

Chromosomal instability (CIN) is a hallmark of cancer and has been implicated in cancer initiation, progression and the development of resistance to traditional cancer therapy. Here we identify a new property of CIN cells, showing that inducing CIN in proliferating Drosophila larval tissue leads to the activation of innate immune signalling in CIN cells. Manipulation of this immune pathway strongly affects the survival of CIN cells, primarily via JNK, which responds to both Toll and TNFα/Eiger. This pathway also activates Mmp1, which recruits hemocytes to the CIN tissue to provide local amplification of the immune response that is needed for effective elimination of CIN cells.

ATM deficiency promotes development of murine B-cell lymphomas that resemble diffuse large B-cell lymphoma in humans.

The serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M(+) B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell-like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors.

Molecular basis of colorectal cancer: towards an individualized management?

Colorectal cancer (CRC) has become a highly relevant condition nowadays. In this respect, advances in the understanding of its molecular basis are key for an adequate management. From the time when the adenoma-carcinoma sequence was formulated as a carcinogenesis model to this day, when, among other things, three major carcinogenic pathways have been identified, the CRC concept has evolved from that of a single disease to the notion that each CRC is a differentiated condition in itself. The suppressor or chromosome instability pathway, the mutator or microsatellite instability pathway, and the methylator or CpG island methylation pathway allow various phenotypes to be identified within CRC. Similarly, the presence of different changes in certain genes confers several behaviors on CRC from both the prognostic and responsive standpoints to specific therapies. However, this apparent complexity does help develop the clinical management of this disease through the identification of novel, more specific therapy targets, and also markers for various behaviors within the condition, which will most likely lead us to an individualized management for these patients(AU)

ICF syndrome in Saudi Arabia: immunological, cytogenetic and molecular analysis.

BACKGROUND: Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is an extremely rare autosomal recessive disorder. In addition to the juxtacentromeric heterochromatic instability, the disease is characterized by variable reduction in serum immunoglobulin levels which cause most ICF patients to succumb to infectious diseases before adulthood as well as exhibit facial dysmorphism including hypertelorism, epicanthal folds, and low-set ears. SUBJECTS AND METHODS: A case series of five patients with ICF from a major immunodeficiency center in Saudi Arabia were included. Immunological and cytogenetic studies were performed for all the five patients. Molecular data was conducted on three patients. RESULTS: All patients had variable hypogammaglobulinemia and characteristic centromeric instability of chromosomes 1, 16, and sometimes 9. One of the patients had pseudomonas meningitis. Pauciarticular arthritis was noted in one patient, a previously not reported finding in ICF, though it has been reported among patients with humoral immune defect. In addition, we identified a novel homozygous c.2506 G>A (p.V836M) mutation in DNMT3B in one of the three patients tested. CONCLUSIONS: This report describes five patients with ICF Saudi Arabia for the first time. ICF should be suspected in children with facial dysmorphism who present with recurrent infections especially in highly inbred populations.

Pol zeta ablation in B cells impairs the germinal center reaction, class switch recombination, DNA break repair, and genome stability.

Pol zeta is an error-prone DNA polymerase that is critical for embryonic development and maintenance of genome stability. To analyze its suggested role in somatic hypermutation (SHM) and possible contribution to DNA double-strand break (DSB) repair in class switch recombination (CSR), we ablated Rev3, the catalytic subunit of Pol zeta, selectively in mature B cells in vivo. The frequency of somatic mutation was reduced in the mutant cells but the pattern of SHM was unaffected. Rev3-deficient B cells also exhibited pronounced chromosomal instability and impaired proliferation capacity. Although the data thus argue against a direct role of Pol zeta in SHM, Pol zeta deficiency directly interfered with CSR in that activated Rev3-deficient B cells exhibited a reduced efficiency of CSR and an increased frequency of DNA breaks in the immunoglobulin H locus. Based on our results, we suggest a nonredundant role of Pol zeta in DNA DSB repair through nonhomologous end joining.

Microsatellite and chromosomal stable colorectal cancers demonstrate poor immunogenicity and early disease recurrence.

OBJECTIVE: Colorectal cancers may demonstrate chromosomal instability (CSI) or microsatellite instability (MSI-H). A third group of microsatellite and chromosome stable (MACS) colorectal cancer has been described more recently. Patients with MSI-H colorectal cancers demonstrate improved outcome and a pronounced inflammatory infiltrate. Enhanced host immune response and increased immunogenicity might explain these observations. This study aims to further characterize colorectal cancer immunogenicity. METHOD: Microsatellite stability status was determined in resected tumour samples. Microsatellite stable (MSS) tumour samples were stratified by DNA ploidy status, as determined by flow cytometry into aneuploid MSS (CSI) and diploid MSS (MACS) cancers. Lymphocyte proliferation, quantified by bromodeoxyuridine incorporation assays assessed tumour protein immunogenicity and ELISA assays quantified inflammatory cytokine release. Kaplan-Meier survival curves and multivariate analyses were used to determine prognostic value. RESULTS: Patients with MSI-H colorectal cancer had improved outcome but those with MACS cancers undergoing curative surgery had significantly poorer disease-free survival (P = 0.002). The MACS phenotype was an independent predictor of poor outcome (HR = 2.44, 1.33-4.47, P = 0.004). Lymphocyte proliferation assays confirmed enhanced immunogenicity of MSI-H proteins and reduced immunogenicity of MACS proteins (P < 0.0001). In vitro levels of IFN-gamma (P = 0.004) and IL-18 (P < 0.0001) mirrored these differences in lymphocyte activity. CONCLUSIONS: Stratification of colorectal cancer by MSI and ploidy status may have prognostic value in patients undergoing curative surgery. MSI-H cancers display enhanced immunogenic properties but the immune response to MACS cancers appears to be absent and this may contribute to their poor prognosis.

[Transgenerational genomic instability in children of liquidators of the accident at the ChNPP (cytogenetic and immunogenetic characteristics)].

A complex of cytogenetic and of immunogenetic study of the state of the lymphocyte genomes in the liquidators of the ChNPP accident and their unirradiated children has been carried out for the first time. Increased frequencies of the chromosome aberrations, of gene mutations (TCR mutations) and of predictors of apoptosis (cells with CD95+ immunophenotype) have been revealed in both generations. The analysis of correlations between the parameters under study has demonstrated distinctive features characteristic of induction of genomic instability in the organism of unirradiated children as compared to their fathers--liquidators directly exposed to radiation. Individual variability of genome destabilization were observed by all criteria used and manifested themselves in the diverse spectrum of transgenerational mutational effects and in different levels of their expression. The results obtained demonstrate the necessity of integral evaluation of the state of the genome using several genetic criteria to reveal transgenerational genomic instability in children of a special category--the offsprings of irradiated parents.

Defective B-cell-negative selection and terminal differentiation in the ICF syndrome.

Immunodeficiency, centromeric region instability, and facial anomalies (ICF) syndrome is a rare autosomal recessive disease. Mutations in the DNA methyltransferase 3B (DNMT3B) gene are responsible for most ICF cases reported. We investigated the B-cell defects associated with agammaglobulinemia in this syndrome by analyzing primary B cells from 4 ICF patients. ICF peripheral blood (PB) contains only naive B cells; memory and gut plasma cells are absent. Naive ICF B cells bear potentially autoreactive long heavy chain variable regions complementarity determining region 3's (V(H)CDR3's) enriched with positively charged residues, in contrast to normal PB transitional and mature B cells, indicating that negative selection is impaired in patients. Like anergic B cells in transgenic models, newly generated and immature B cells accumulate in PB. Moreover, these cells secrete immunoglobulins and exhibit increased apoptosis following in vitro activation. However, they are able to up-regulate CD86, indicating that mechanisms other than anergy participate in silencing of ICF B cells. One patient without DNMT3B mutations shows differences in immunoglobulin E (IgE) switch induction, suggesting that immunodeficiency could vary with the genetic origin of the syndrome. In this study, we determined that negative selection breakdown and peripheral B-cell maturation blockage contribute to agammaglobulinemia in the ICF syndrome.