53BP1-positive Foci Research Articles

Germline Runx1 Mutations Induce Inflammation in Hematopoietic Stem and Progenitor Cells and Predispose to Hematologic Malignancies

Patients with Familial Platelet disorder (FPD) have a germline RUNX1 mutation and are at high risk to developing hematologic malignancies (HM), primarily myelodysplastic syndrome and acute myeloid leukemia (lifetime risk~40%). To understand how germline RUNX1 mutations predispose to HM in vivo, we developed a Runx1 R188Q/+ mouse strain , mimicking the FPD-associated R201Q missense mutation.Analysis of the bone marrow cells in Runx1 R188Q/+ mice revealed a significant increase in the total number of bone marrow cells. Immunophenotypic analysis using Sca-1 and Cd86 markers revealed a significant increase in Sca-1 expression in hematopoietic stem and multi-potential progenitor cells, indicating a systemic inflammation in the bone marrow. In addition, the frequency of common-myeloid, granulocytic-monocytic and granulocytic progenitor cells were found significantly increased in the Runx1 R188Q/+ bone marrow. Accordingly, their colony-forming unit capacity was increased when compared to wildtype controls (wt/Runx1 R188Q/+ CFU average = 45/85), indicating a myeloid bias.The number and size of platelets were not altered in Runx1 R188Q/+ mice. However, platelet function was significantly reduced. The activation of the Cd41/Cd61 fibrinogen receptor complex in membrane after thrombin treatment was reduced in Runx1 R188Q/+ platelets. Similarly, the translocation of P-selectin by alpha granules and the secretion of serotonin by the dense granules were also reduced.Hematopoietic progenitor cells isolated from Runx1 R188Q/+ mice revealed a significant reduction in DNA-damage repair response in vitro. Quantitative analysis of nuclei with 53bp1-positive foci in response to ionizing radiation showed a marked increase in 53bp1-positive foci in Runx1 R188Q/+ nuclei, suggesting that Runx1 R188Q/+ cells have a defective repair of double strand DNA breaks. Furthermore, expression of DNA-damage repair pathway-associated Pmaip1 (Noxa) was significantly reduced in irradiated Runx1 R188Q/+ hematopoietic progenitor cells.To understand underlying mechanism responsible for the observed myeloid bias in Runx1 R188Q/+ cells, transcription profiling analysis was performed in myeloid progenitors from wildtype and Runx1 R188Q/+ mice, utilizing RNA-sequencing. A total of 39 genes were significantly deregulated (> 1.5 FC; FDR<0.05), including 8 up- and 31 down-regulated genes. The expression of three repressed genes with important function in hematopoietic differentiation and malignancy (Cdh1, Gja1, and Fcer1a) were validated by qRT-PCR.To study the FPD-associated pre-leukemic process in vivo, wildtype and Runx1 R188Q/+ mice were monitored for 20 months. Although Runx1 R188Q/+ mice remained healthy for 18 months, somatic mutations in their leukocytes were evident at 12 months. Targeted sequencing of 578 cancer genes (mIMPACT panel) in leukocyte DNA of two Runx1 R188Q/+ mice identified somatic mutations in Kdm6a, Setd1b, Amer1, and Esco1 (variant allele frequencies between 0.5% and 2.8%). These mutations were confirmed at stable frequency for eight following months. Since loss of the second Runx1 allele is a frequent somatic event in progression to FPD/HM, we evaluated the predisposition to HM in Mx1Cre-Runx1 R188Q/fl mice over time. Unlike Runx1 R188Q/+ mice, Runx1 R188Q/Δ mice succumbed to myeloid leukemia with a median latency of 37.5 weeks and full penetrance. In addition, the expression of oncogenic Nras-G12D, in Runx1 R188Q/Δ mice reduced the median latency to 14.7 weeks.These studies demonstrate that FPD-associated Runx1 germline mutations induce inflammation in hematopoietic stem cells, induce myeloid expansion with defective DNA-damage response and predispose to HM over time. These studies suggest that anti-inflammatory therapies in pre-symptomatic FPD patients may reduce clonal expansion and predisposition to HM. DisclosuresEbert: Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Skyhawk Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Deerfield: Research Funding; GRAIL: Consultancy. Levine: Isoplexis: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Lilly: Honoraria; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Incyte: Consultancy; Astellas: Consultancy; Amgen: Honoraria.

Detection of DNA damage response in nonalcoholic fatty liver disease via p53-binding protein 1 nuclear expression

Nonalcoholic fatty liver disease is a major liver disease that leads to cirrhosis and/or hepatocellular carcinoma in a subset of patients. The mechanism underlying disease progression is largely unknown. p53-binding protein 1 (53BP1) is a DNA damage response protein that rapidly localizes at the site of DNA double-strand breaks. In this study, we investigated nuclear 53BP1-positive foci formation as an indicator of DNA double-strand breaks in human nonalcoholic fatty liver disease liver tissues by immunofluorescence microscopy. A total of 52 liver tissue samples, including 43 nonalcoholic fatty liver disease samples and 9 controls, were studied. Our results show that the number of abnormal 53BP1-positive foci in hepatocytes (defined as three or more discrete nuclear foci and/or large foci greater than 1 μM) was significantly increased in nonalcoholic fatty liver disease patients compared to that in controls, both in nonalcoholic fatty liver (p < 0.01) and nonalcoholic steatohepatitis patients (p < 0.01). The number of large foci was significantly increased in the nonalcoholic steatohepatitis cases compared to that in the nonalcoholic fatty liver cases (p < 0.05) and correlated with increased stage of fibrosis. The number of large-foci-expressing hepatocytes was positively correlated with increased age (p < 0.01) and negatively correlated with serum platelet count (p < 0.05). In addition, we performed an in vitro assay using rat hepatocytes treated with the saturated free fatty acid palmitate. Treatment appeared to augment the number of abnormal foci, indicating an induction of double-strand breaks in the hepatocytes through free fatty acid treatment in a caspase-dependent manner. This study demonstrates that 53BP1-positive nuclear foci formation is associated with disease progression in nonalcoholic fatty liver disease patients. Analysis of 53BP1 expression might be a feasible technique to estimate genomic instability in nonalcoholic fatty liver disease.

Radiation‐induced cataracts

SummaryThe radiosensitive nature of the lens has been increasingly reported, although the exact mechanistic details of the radiation response pathways for cataractogenesis are unclear. Radiation‐induced DNA damage and the subsequent impairment of repair pathways within the lens epithelium are involved. Here, two distinct regions of the murine lens epithelium have been analysed for their differences in double strand break (DSB) repair responses to ionising radiation. The responses of epithelial cells located at the anterior pole (central region) have been compared to those in other locations, including the proliferative compartment, and including the very periphery of the monolayer (peripheral region). Described here are the responses between the two regions, across four strains, over a low dose (0–25 mGy) X‐irradiation range up to 24 hours. Damage visualised through biomarker 53BP1 staining was present across the epithelium, repair kinetics appear non‐uniform. Epithelial cells in the central region have significantly more 53BP1 positive foci. In this study, BALB/c were identified as the most suitable strain for low dose ionising radiation exposure investigation.

Mutations in the TP53 gene affected recruitment of 53BP1 protein to DNA lesions, but level of 53BP1 was stable after γ-irradiation that depleted MDC1 protein in specific TP53 mutants.

53BP1 is a very well-known protein that is recruited to DNA lesions. The focal accumulation of p53 binding protein, 53BP1, is a main feature indicating the repair of spontaneous or irradiation-induced foci (IRIF). Thus, here, we addressed the question of whether mutations in the TP53 gene, which often affect the level of p53 protein, can change the recruitment of 53BP1 to γ- or UVA-irradiated chromatin. In various TP53 mutants, we observed a distinct accumulation of 53BP1 protein to UV-induced DNA lesions: in R273C mutants, 53BP1 appeared transiently at DNA lesions, during 10-30min after irradiation; the mutation R282W was responsible for accumulation of 53BP1 immediately after UVA-damage; and in L194F mutants, the first appearance of 53BP1 protein at the lesions occurred during 60-70min. These results showed that specific mutations in the TP53 gene stand behind not only different levels of p53 protein, but also affect the localized kinetics of 53BP1 protein in UVA-damaged chromatin. However, after γ-irradiation, only G245S mutation in TP53 gene was associated with surprisingly decreased level of 53BP1 protein. In other mutant cell lines, levels of 53BP1 were not affected by γ-rays. To these effects, we conversely found a distinct number of 53BP1-positive irradiation-induced foci in various TP53 mutants. The R280K, G245S, L194F mutations, or TP53 deletion were also characterized by radiation-induced depletion in MDC1 protein. Moreover, in mutant cells, an interaction between MDC1 and 53BP1 proteins was abrogated when compared with wild-type counterpart. Together, the kinetics of 53BP1 accumulation at UV-induced DNA lesions is different in various TP53 mutant cells. After γ-irradiation, despite changes in a number and a volume of 53BP1-positive foci, levels of 53BP1 protein were relatively stable. Here, we showed a link between the status of MDC1 protein and TP53 gene, which specific mutations caused radiation-induced MDC1 down-regulation. This observation is significant, especially with regard to radiotherapy of tumors with abrogated function of TP53 gene.

MMSET/WHSC1 Enhances DNA Damage Repair Leading To An Increase In Resistance To Chemotherapeutic Agents

MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, and is believed to be the driving factor in the pathogenesis of this subtype of MM. Overexpression of MMSET also occurs in solid cancers, including neuroblastoma, colon and prostate. MMSET overexpression in MM and prostate cells leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3). This altered epigenetic landscape is accompanied by changes in proliferation, gene expression, and chromatin accessibility. Prior work linked methylation of histones, including H3K36, to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET might play a role in DNA damage repair and response.To investigate the role of MMSET in DNA damage repair, we transfected U2OS cells with a linearized vector expressing a neomycin-resistant gene. In the presence of G418, only cells that are able to integrate this plasmid through non-homologous end joining (NHEJ) can survive. siRNA knockdown of MMSET led to a decrease in cell survival, suggesting that MMSET is necessary for efficient DNA repair. We also used U2OS cells engineered to express the AsiSI enzyme fused to an estrogen receptor hormone-binding domain. Upon tamoxifen treatment, double strand breaks (DSBs) are induced at multiple AsiSI recognition sites, accompanied by an increase in γH2AX foci. The extent of repair after AsiSI-induced damage was ascertained by the ability of a DNA fragment that spans a specific cut site to be PCR amplified. With MMSET knockdown, there was a >10 fold increase in unrepaired DNA. ChIP analysis showed that with the depletion of MMSET, γH2AX persisted at the cut site. ChIP for specific effectors of DNA damage showed a marked decrease of recruitment of CtIP and RAD51 to the DSB. However, immunoblot analysis showed that CtIP and RAD51 levels were drastically decreased with MMSET depletion, thus explaining the loss of their recruitment to DSBs. In contrast, XRCC4 levels were maintained with MMSET siRNA, but its recruitment to the DSB decreased. CtIP is important for both NHEJ and homologous recombination (HR), RAD51 is critical for HR, and XRCC4 is necessary for NHEJ, suggesting that MMSET is important in multiple pathways of DNA repair.To study the effect of MMSET in MM, we used the t(4;14)+ KMS11 cell line, NTKO, and genetically matched TKO cells in which the overexpressed MMSET allele was knocked out. NTKO cells have elevated levels of DNA damage at baseline, as measured by a comet assay and by the presence of elevated numbers of 53BP1-positive foci. Upon addition of the DNA damaging agent melphalan, NTKO cells showed increased damage as measured by an increase in the tail moment by the comet assay. Paradoxically, upon treatment of these cells with the DNA damaging agents, NTKO cells survived better than TKO cells. NTKO repaired DNA damage at an enhanced rate and continued to proliferate after a significant DNA damage insult, whereas TKO cells accumulated DNA damage and entered cell cycle arrest. We repleted TKO cells with constructs expressing either wild-type MMSET or an HMT-dead (Y1118A) isoform. Upon treatment, cells expressing the wild-type MMSET have showed enhanced DNA repair and continued proliferation after DNA damage, whereas cells expressing the HMT-dead protein repaired DNA damage more slowly and entered cell cycle arrest. The HMT activity of MMSET was critical for the induction of expression of genes required for multiple DNA repair pathways including CHEK2, DDB2, DDIT3, RAD51, and MRE11, again suggesting that MMSET modulates DNA repair by affecting expression of critical components of the repair machinery.The clinical relevance of these finds becomes more apparent in vivo. Luciferase-tagged KMS11 cells harboring doxycycline-inducible MMSET shRNA were injected into nude mice. After one week, mice were treated with doxycycline and injected with melphalan or saline. Knockdown of MMSET or melphalan treatment alone decreased tumor growth but eventually all mice had progressive disease. Only when MMSET was knocked down and chemotherapy given were the mice rendered tumor free. These findings indicate a new mechanism for the ability of MMSET to enhance DNA repair and identify the protein as a potential therapeutic target in MM and other cancers. Disclosures:No relevant conflicts of interest to declare.

Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells

The constant shedding and renewal of epithelial cells maintain the protection of epithelial barriers. Interference with the processes of host cell-cycle regulation and barrier integrity permits the bacterial pathogen Neisseria gonorrhoeae to effectively colonize and invade epithelial cells. Here, we show that a gonococcal infection causes DNA damage in human non-tumor vaginal VK2/E6E7 cells with an increase of 700 DNA strand breaks per cell per hour as detected by an alkaline DNA unwinding assay. Infected cells exhibited elevated levels of DNA double-strand breaks, as indicated by a more than 50% increase in cells expressing DNA damage-response protein 53BP1-positive foci that co-localized with phosphorylated histone H2AX (γH2AX). Furthermore, infected cells abolished their expression of the tumor protein p53 and induced an increase in the expression of cyclin-dependent kinase inhibitors p21 and p27 to 2.6-fold and 4.2-fold of controls, respectively. As shown by live-cell microscopy, flow cytometry assays, and BrdU incorporation assays, gonococcal infection slowed the host cell-cycle progression mainly by impairing progression through the G2 phase. Our findings show new cellular players that are involved in the control of the human cell cycle during gonococcal infection and the potential of bacteria to cause cellular abnormalities.