P53-mediated DNA Damage Response Research Articles

399-P: Dapagliflozin Is Protective in a Murine Model of Balkan Nephropathy

Episodes of acute kidney injury (AKI) increase the risk of chronic kidney disease (CKD). Proximal tubular uptake of aristolochic acid (AA) induces aristolactam (AL)-DNA adducts, which induce a p53-mediated DNA damage response and acute tubular injury. Recurrent exposure to AA causes kidney function loss and fibrosis in humans (Balkan nephropathy), and is a model of AKI to CKD transition. SGLT2 inhibitors protect against CKD progression regardless of the presence of diabetes, however, the underlying mechanism is still unclear. Here we elucidated the effects of SGLT2 inhibition on AA-induced kidney injury. 15-week-old C57BL/6J female and male mice were administered vehicle or AA every 3 days for 3 weeks at a dose of 10 and 3 mg/kg i.p., respectively. Application of dapagliflozin (dapa, 0.01 g/kg diet) or vehicle was initiated 7 days prior to AA injections. Urine/plasma were collected after completion of AA injections and 3 weeks later together with kidneys (n=7-11/group). AA-induced increases in plasma creatinine and urinary albumin excretion were robustly attenuated by dapa independent of sex. These effects of dapa were associated with lower urinary levels of kidney-injury molecule 1 (KIM1, normalized to creatinine) after injection and lower kidney mRNA expression of p53 and KIM1 at harvest. Renal up-regulation of pro-inflammatory and pro-fibrotic genes as well as collagen staining by AA were likewise significantly attenuated by dapa. Dapa caused a minor numerical reduction in kidney AL-DNA adduct formation when assessed one day after a single AA injection (by ~10%; P=0.084). In conclusion, dapa attenuated the AA-induced p53-mediated DNA damage response and subsequent nephropathy. SGLT2 inhibitors may protect the kidneys by improving the tubular response to nephrotoxins. Disclosure Y.Oe: None. Y.Kim: None. V.S.Sidorenko: None. S.Kanoo: None. N.Lopez: None. H.A.Goodluck: None. V.Vallon: Research Support; NIH - National Institutes of Health, Gilead Sciences, Inc., Merck Sharp & Dohme Corp., Novo Nordisk, Maze Therapeutics, Boehringer-Ingelheim, Speaker's Bureau; AstraZeneca. Funding National Institutes of Health (R01DK112042, R01DK132690, R01AG061296, P30DK079337)

Smoking Suppresses the Therapeutic Potential of Adipose Stem Cells in Crohn’s Disease Patients through Epigenetic Changes

Patients with Crohn's disease (CD) who smoke are known to have a worse prognosis than never-smokers and a higher risk for post-surgical recurrence, whereas patients who quit smoking after surgery have significantly lower post-operative recurrence. The hypothesis was that smoking induces epigenetic changes that impair the capacity of adipose stem cells (ASCs) to suppress the immune system. It was also questioned whether this impairment remains in ex-smokers with CD. ASCs were isolated from non-smokers, smokers and ex-smokers with CD and their interactions with immune cells were studied. The ASCs from both smokers and ex-smokers promoted macrophage polarization to an M1 pro-inflammatory phenotype, were not able to inhibit T- and B-cell proliferation in vitro and enhanced the gene and protein expression of inflammatory markers including interleukin-1b. Genome-wide epigenetic analysis using two different bioinformatic approaches revealed significant changes in the methylation patterns of genes that are critical for wound healing, immune and metabolic response and p53-mediated DNA damage response in ASCs from smokers and ex-smokers with CD. In conclusion, cigarette smoking induces a pro-inflammatory epigenetic signature in ASCs that likely compromises their therapeutic potential.

Granulocyte Colony-Stimulating Factor Impairs Engraftment of CRISPR-Cas9 Gene Edited Human Hematopoietic Stem Cells By Exacerbating p53 Mediated DNA Damage Response

Granulocyte Colony-Stimulating Factor Impairs Engraftment of CRISPR-Cas9 Gene Edited Human Hematopoietic Stem Cells By Exacerbating p53 Mediated DNA Damage Response

Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells

SummaryLong-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

A developmentally programmed splicing failure contributes to DNA damage response attenuation during mammalian zygotic genome activation.

Transition from maternal to embryonic transcriptional control is crucial for embryogenesis. However, alternative splicing regulation during this process remains understudied. Using transcriptomic data from human, mouse, and cow preimplantation development, we show that the stage of zygotic genome activation (ZGA) exhibits the highest levels of exon skipping diversity reported for any cell or tissue type. Much of this exon skipping is temporary, leads to disruptive noncanonical isoforms, and occurs in genes enriched for DNA damage response in the three species. Two core spliceosomal components, Snrpb and Snrpd2, regulate these patterns. These genes have low maternal expression at ZGA and increase sharply thereafter. Microinjection of Snrpb/d2 messenger RNA into mouse zygotes reduces the levels of exon skipping at ZGA and leads to increased p53-mediated DNA damage response. We propose that mammalian embryos undergo an evolutionarily conserved, developmentally programmed splicing failure at ZGA that contributes to the attenuation of cellular responses to DNA damage.

Impaired p53-Mediated DNA Damage Response Contributes to Microcephaly in Nijmegen Breakage Syndrome Patient-Derived Cerebral Organoids.

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive genetic disorder caused by mutations within nibrin (NBN), a DNA damage repair protein. Hallmarks of NBS include chromosomal instability and clinical manifestations such as growth retardation, immunodeficiency, and progressive microcephaly. We employed induced pluripotent stem cell-derived cerebral organoids from two NBS patients to study the etiology of microcephaly. We show that NBS organoids carrying the homozygous 657del5 NBN mutation are significantly smaller with disrupted cyto-architecture. The organoids exhibit premature differentiation, and Neuronatin (NNAT) over-expression. Furthermore, pathways related to DNA damage response and cell cycle are differentially regulated compared to controls. After exposure to bleomycin, NBS organoids undergo delayed p53-mediated DNA damage response and aberrant trans-synaptic signaling, which ultimately leads to neuronal apoptosis. Our data provide insights into how mutations within NBN alters neurogenesis in NBS patients, thus providing a proof of concept that cerebral organoids are a valuable tool for studying DNA damage-related disorders.

Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking

Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700–2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.

MAGE-a Inhibits Apoptosis in Multiple Myeloma through Regulation of Bim and PUMA

MAGE-a Inhibits Apoptosis in Multiple Myeloma through Regulation of Bim and PUMA

Functional Implication of p53-Mediated DNA Damage Response on the B-Cell Lymphoma Microenvironment Interaction in Chemoimmunotherapy

Resistance against chemotherapy remains a challenge in the current treatment of B cell malignancies. In previous work we could show that macrophages are an essential element in the synergistic response to the administration of the DNA damaging agent Cyclophosphamide and monoclonal antibodies. The combination treatment strongly increases tumor clearance overall survival in the treatment refractory humanized BCL2-MYC-dependent double hit lymphoma hMB mouse model.Here we observed that the alkylating agent Cyclophosphamide is inducing the secretion of distinct cytokines like VEGF-A, CCL4, IL8, IL10 and TNFα by the leukemic cells. This in turn leads to a repolarization of tumor associated macrophages from a suppressive to an activated phenotypic state, which is designated as acute secretory activating phenotype (ASAP). We analyzed the functional implications of the DNA damage response pathway for the generation of the ASAP and synergy in chemoimmunotherapy. When disrupting DNA damage response pathway by knock-down of key elements like ATM, DNA-PK or p53 in the leukemic cells we could prevent the formation of the stimulatory ASAP effect on macrophage phagocytic capacity. Particularly p53 status seems to possess a central regulating role on macrophage mediated malignant cell depletion. TP53 activation via Nutlin-3A treatment of lymphoma cell enhances ADCP in in p53 wildtype cells, while not displaying enhancement in p53-deficient lymphoma cells. Utilizing the hMB model for modeling in vivo treatment of lymphoma bearing mice we could demonstrate diminished ASAP and ADCP for p53-deficient lymphoma treated with cyclophosphamide in vivo . Using primary human CLL patient cells comparing both wildtype and p53-deficient status, the p53-deficient CLL cells failed to induce the stimulatory, cytokine-mediated effect on macrophage phagocytosis in response to combination treatment as seen with the p53 proficient CLL cells. This observation is underlined by in vivo modeling of CLL therapy by treating Eµ-TCL1/p53wt/wt as well as Eµ-TCL1p53-/- mice. Here, low-dose cyclophosphamide treated Eµ-TCL1p53-/- mice failed to induce an antibody mediated stimulatory effect on macrophage phagocytosis capacity as seen with Eµ-TCL1/p53wt/wt mice.In this work, we are able to recapitulate the effect of ASAP mediated increase of macrophage mediated antibody dependent cellular phagocytosis in the Eµ-Tcl1 mouse model and therefore demonstrate that the ASAP is not only restricted to double hit lymphoma, but also shows functional relevance in low-grade lymphoma such as CLL. Moreover, we indicate that p53 functional status influences the generation of the ASAP in independent models in vitro and in vivo as in primary CLL patient cells. Mediated by this process p53 regulates the interraction with tumor asociated macrophages and the tumor microenvironment.Finally our ongoing research offers possibility to reveal and tailor new treatment approaches for chemorefractory patients. DisclosuresHallek:AbbVie: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding.

Bortezomib synergizes with nocodazole in p53 mediated DNA damage response signalling in A549 lung cancer cells

Bortezomib synergizes with nocodazole in p53 mediated DNA damage response signalling in A549 lung cancer cells

RNA oxidation in chromatin modification and DNA-damage response following exposure to formaldehyde

Formaldehyde is an environmental and occupational chemical carcinogen implicated in the damage of proteins and nucleic acids. However, whether formaldehyde provokes modifications of RNAs such as 8-oxo-7,8-dihydroguanine (8-oxoG) and the role that these modifications play on conferring long-term adverse health effects remains unexplored. Here, we profile 8-oxoG modifications using RNA-immunoprecipitation and RNA sequencing (8-oxoG RIP-seq) to identify 343 RNA transcripts heavily enriched in oxidations in human bronchial epithelial BEAS-2B cell cultures exposed to 1 ppm formaldehyde for 2 h. RNA oxidation altered expression of many transcripts involved in chromatin modification and p53-mediated DNA-damage responses, two pathways that play key roles in sustaining genome integrity and typically deregulated in tumorigenesis. Given that these observations were identified in normal cells exhibiting minimal cell stress and death phenotypes (for example, lack of nuclear shrinkage, F-actin alterations or increased LDH activity); we hypothesize that oxidative modification of specific RNA transcripts following formaldehyde exposure denotes an early process occurring in carcinogenesis analogous to the oxidative events surfacing at early stages of neurodegenerative diseases. As such, we provide initial investigations of RNA oxidation as a potentially novel mechanism underlying formaldehyde-induced tumorigenesis.

CEP131 Abrogates CHK1 Inhibitor-Induced Replication Defects and Is Associated with Unfavorable Outcome in Neuroblastoma.

Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. Recently, we reported that CHK1i, PF-477736, induced a p53-mediated DNA damage response. As a result, the cancer cells were able to repair DNA damage and became less sensitive to CHK1i. In this study, we discovered that PF-477736 increased expression of MDM2 oncogene along with CHK1i-induced replication defects in neuroblastoma NB-39-nu cells. A mass spectrometry analysis of protein binding to MDM2 in the presence of CHK1i identified the centrosome-associated family protein 131 (CEP131), which was correlated with unfavorable prognosis of neuroblastoma patients. We revealed that MDM2 was associated with CEP131 protein degradation, whereas overexpression of CEP131 accelerated neuroblastoma cell growth and exhibited resistance to CHK1i-induced replication defects. Thus, these findings may provide a future therapeutic strategy against centrosome-associated oncogenes involving CEP131 as a target in neuroblastoma.

Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance.

CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens using TP53 wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type and TP53 knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells.

Identification of functional regulatory elements in the human genome using pooled CRISPR screens

BackgroundGenome-scale pooled CRISPR screens are powerful tools for identifying genetic dependencies across varied cellular processes. The vast majority of CRISPR screens reported to date have focused exclusively on the perturbation of protein-coding gene function. However, protein-coding genes comprise < 2% of the sequence space in the human genome leaving a substantial portion of the genome uninterrogated. Noncoding regions of the genome harbor important regulatory elements (e.g. promoters, enhancers, silencers) that influence cellular processes but high-throughput methods for evaluating their essentiality have yet to be established.ResultsHere, we describe a CRISPR-based screening approach that facilitates the functional profiling of thousands of noncoding regulatory elements in parallel. We selected the tumor suppressor p53 as a model system and designed a pooled CRISPR library targeting thousands of p53 binding sites throughout the genome. Following transduction into dCas9-KRAB-expressing cells we identified several regulatory elements that influence cell proliferation. Moreover, we uncovered multiple elements that are required for the p53-mediated DNA damage response. Surprisingly, many of these elements are located deep within intergenic regions of the genome that have no prior functional annotations.ConclusionsThis work diversifies the applications for pooled CRISPR screens and provides a framework for future functional studies focused on noncoding regulatory elements.

P53 dynamics in single cells are temperature-sensitive

Cells need to preserve genome integrity despite varying cellular and physical states. p53, the guardian of the genome, plays a crucial role in the cellular response to DNA damage by triggering cell cycle arrest, apoptosis or senescence. Mutations in p53 or alterations in its regulatory network are major driving forces in tumorigenesis. As multiple studies indicate beneficial effects for hyperthermic treatments during radiation- or chemotherapy of human cancers, we aimed to understand how p53 dynamics after genotoxic stress are modulated by changes in temperature across a physiological relevant range. To this end, we employed a combination of time-resolved live-cell microscopy and computational analysis techniques to characterise the p53 response in thousands of individual cells. Our results demonstrate that p53 dynamics upon ionizing radiation are temperature dependent. In the range of 33 °C to 39 °C, pulsatile p53 dynamics are modulated in their frequency. Above 40 °C, which corresponds to mild hyperthermia in a clinical setting, we observed a reversible phase transition towards sustained hyperaccumulation of p53 disrupting its canonical response to DNA double strand breaks. Moreover, we provide evidence that mild hyperthermia alone is sufficient to induce a p53 response in the absence of genotoxic stress. These insights highlight how the p53-mediated DNA damage response is affected by alterations in the physical state of a cell and how this can be exploited by appropriate timing of combination therapies to increase the efficiency of cancer treatments.

I-Silence, Please! An Alternative for Gene Disruption via Adenine Base Editors.

i-Silence, Please! An Alternative for Gene Disruption via Adenine Base Editors.

P53-Mediated Tumor Suppression: DNA-Damage Response and Alternative Mechanisms

The tumor suppressor p53 regulates different cellular pathways involved in cell survival, DNA repair, apoptosis, and senescence. However, according to an increasing number of studies, the p53-mediated canonical DNA damage response is dispensable for tumor suppression. p53 is involved in mechanisms regulating many other cellular processes, including metabolism, autophagy, and cell migration and invasion, and these pathways might crucially contribute to its tumor suppressor function. In this review we summarize the canonical and non-canonical functions of p53 in an attempt to provide an overview of the potentially crucial aspects related to its tumor suppressor activity.

Somatic Niche Cells Regulate the CEP-1/p53-Mediated DNA Damage Response in Primordial Germ Cells

Genome integrity in primordial germ cells (PGCs) is a prerequisite for fertility and species maintenance. In C.elegans, PGCs require global-genome nucleotide excision repair (GG-NER) to remove UV-induced DNA lesions. Failure to remove the lesions leads to the activation of the C.elegans p53, CEP-1, resulting in mitotic arrest of the PGCs. We show that the eIF4E2 translation initiation factor IFE-4 in somatic gonad precursor (SGP) niche cells regulates the CEP-1/p53-mediated DNA damage response (DDR) in PGCs. We determine that the IFE-4 translation target EGL-15/FGFR regulates the non-cell-autonomous DDR that is mediated via FGF-like signaling. Using hair follicle stem cells as a paradigm, we demonstrate that the eIF4E2-mediated niche cell regulation of the p53 response in stem cells is highly conserved in mammals. We thus reveal that the somatic niche regulates the CEP-1/p53-mediated DNA damage checkpoint in PGCs. Our data suggest that the somatic niche impacts the stability of heritable genomes.

Explosively Puffed Ginseng Ameliorates Ionizing Radiation-Induced Injury of Colon by Decreasing Oxidative Stress-Related Apoptotic Cell Execution in Mice.

Although radiation therapy (RT) is a feasible treatment approach for early colorectal cancer, RT is considerably toxic to normal tissues due to the increased reactive oxygen species production, which can induce tissue damage. Ginseng, a natural antioxidant agent, exhibits the protective effects against ionizing radiation (IR)-induced damage in in vitro and in vivo models. The explosive puffing of ginseng has been investigated as a process to improve the efficacy of ginseng due to the resulting physicochemical changes in its functional components. In this study, we provided the evidence for promotion in the beneficial role of puffed ginseng extract (PGE) and associated mechanisms of action, in comparison with white ginseng extract (WGE), against IR-induced colorectal injury, using in vivo study on a mouse model. To study the role of PGE in preventing IR-induced damage, we examined colorectal injury and apoptotic changes in mice exposed to 137Cs at 8 Gy. High-performance liquid chromatography analysis showed that PGE had an increased total ginsenoside concentration with new generation of Rg3, Rg5, and Rk1, compared with the concentrations in WGE. Administering PGE, but not WGE, significantly ameliorated IR-induced colorectal cell death through negative regulation of apoptotic signaling pathways. These antiapoptotic effects of PGE were linked to the capacity to suppress the p53-mediated DNA damage response and NF-κB-mediated apoptotic signaling. Moreover, IR-induced oxidative stress in the colorectal epithelium was markedly reduced by PGE administration. Collectively, this study establishes a mechanism of action by which PGE counteracts IR-induced colorectal injury as a novel radioprotective agent.

PCNA-Mediated Degradation of p21 Coordinates the DNA Damage Response and Cell Cycle Regulation in Individual Cells

To enable reliable cell fate decisions, mammalian cells need to adjust their responses to dynamically changing internal states by rewiring the corresponding signaling networks. Here, we combine time-lapse microscopy of endogenous fluorescent reporters with computational analysis to understand at the single-cell level how the p53-mediated DNA damage response is adjusted during cell cycle progression. Shape-based clustering revealed that the dynamics of the CDK inhibitor p21 diverges from the dynamics of its transcription factor p53 during S phase. Using mathematical modeling, we predict and experimentally validate that S phase-specific degradation of p21 by PCNA-CRL4cdt2 is sufficient to explain these heterogeneous responses. This highlights how signaling pathways and cell regulatory networks intertwine to adjust the cellular response to the individual needs of a given cell.