DDRE-32. SETD2 HISTONE METHYLTRANSFERASE MUTATION STATUS PREDICTS TREATMENT RESPONSE IN GLIOBLASTOMA: STRATEGIES TO OVERCOME CHEMORESISTANCE

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi81-vi81
Author(s):  
Michael Goldstein ◽  
Nishanth Gabriel ◽  
Matthew Inkman ◽  
Jin Zhang ◽  
Sonika Dahiya
Keyword(s):  
Chemotherapy Resistance ◽  
Dna Lesions ◽  
Chemotherapy Response ◽  
Tumor Resistance ◽  
Dna Double Strand Breaks ◽  
Methyl Transferase ◽  
Strand Breaks ◽  
Msh6 Gene ◽  
Epigenetic Modifiers ◽  
Novel Approach

Abstract A major challenge in GBM treatment is tumor resistance to radiation and chemotherapy. A hallmark of GBM is the frequent mutation of epigenetic modifiers resulting in alteration of epigenetic signaling pathways. However, the effect of epigenetic signaling on chemotherapy response in GBM remains unknown. SETD2 is a histone methyl transferase that facilitates H3K36 tri-methylation. Here, we unveil the role of SETD2 mutations that frequently occur in GBM in tumor resistance to temozolomide chemotherapy. Targeted sequencing of the SETD2 gene in GBM tumor samples revealed that SETD2 mutations are associated with reduced overall survival in patients with methylated MGMT (methyl-guanine methyl transferase) promotor who received temozolomide. Consequently, we demonstrate that loss of SETD2 results in reduced H3K36me3 levels and a profound temozolomide resistance in GBM cells. MGMT-deficient tumors can acquire chemoresistance due to disrupted mismatch repair (MMR), a DNA repair pathway that converts primary temozolomide-induced DNA lesions into toxic DNA double-strand breaks. Strikingly, we found that SETD2 loss abrogates the expression of the MMR factor MSH6 indicating that chemoresistance in SETD2-deficient cells us due to disrupted MMR. Mechanistically, we show that SETD2 regulates MMR by promoting transcription of the MSH6 gene in GBM. Epigenetic modifiers have specific antagonists capable of reversing chromatin alterations induced by these modifiers. This provides a unique opportunity to restore chemotherapy response in SETD2-mutant GBM by targeting the antagonists of SETD2. We demonstrate that combined targeting of H3K36me3-specific histone de-methylases KDM4A and NO66 restores H3K36me3 levels along with MSH6 expression and sensitivity to temozolomide in SETD2-deficient GBM cells. Thus, our findings establish SETD2 mutation as a novel molecular marker predictive of chemotherapy response in GBM and provide a framework for a novel approach to overcome chemotherapy resistance in this malignant brain tumor by targeting an epigenetic pathway.

scholarly journals Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

2021 ◽  
Vol 22 (14) ◽  
pp. 7638
Author(s):  
Yvonne Lorat ◽  
Judith Reindl ◽  
Anna Isermann ◽  
Christian Rübe ◽  
Anna A. Friedl ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dna Damage ◽  
Spatial Clustering ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Stimulated Emission ◽  
Nuclear Chromatin ◽  
Carbon Ions ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

scholarly journals The homologous recombination complex of the Rad51 paralogs Rad55-Rad57 avoids translesion DNA polymerase recruitment and counterbalances mutagenesis induced by UV radiation

2021 ◽  
Author(s):  
Laurent G Maloisel ◽  
Emilie Ma ◽  
Eric Coic
Keyword(s):  
Homologous Recombination ◽  
Uv Radiation ◽  
Dna Lesions ◽  
Template Switching ◽  
Dna Double Strand Breaks ◽  
Dna Damage Tolerance ◽  
Strand Breaks ◽  
Rad51 Paralogs ◽  
Replicative Polymerases ◽  
Stalled Replication Forks

Bypass of DNA lesions that block replicative polymerases during DNA replication relies on several DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway involves specialized DNA polymerases that incorporate nucleotides in front of base lesions. The template switching and the homologous recombination (HR) pathways are mostly error-free because the bypass is performed by using typically the sister chromatid as a template. This is promoted by the Rad51 recombinase that forms nucleoprotein filaments on single-strand DNA (ssDNA). The balance between error-prone and error-free pathways controls the level of mutagenesis. In yeast, the Rad55-Rad57 complex of Rad51 paralogs is required for Rad51 filament formation and stability, notably by counteracting the Srs2 antirecombinase. Several reports showed that Rad55-Rad57 promotes HR at stalled replication forks more than at DNA double-strand breaks (DSB), suggesting that this complex is more efficient at ssDNA gaps and thus, could control the recruitment of TLS polymerases. To address this point, we studied the interplay between Rad55-Rad57 and the TLS polymerases Polζ and Polη following UV radiation. We confirmed that Rad55-Rad57 protects Rad51 filaments from Srs2 dismantling activity but we found that it is also essential for the promotion of UV-induced HR independently of Srs2. In addition, we observed that cell UV sensitivity, but not DSB sensitivity, is synergistically increased when Rad55 and Polζ deletions are combined. Moreover, we found that mutagenesis and HR frequency were increased in rad55∆ mutants and in TLS-deficient cells, respectively. Finally, UV-induced HR was partially restored in Rad55-deficient cells with mutated Polζ or Polη. Overall, our data suggest that the HR and TLS pathways compete for the same ssDNA substrates and that the Rad55-Rad57 complex of Rad51 paralogs prevents the recruitment of TLS polymerases and counterbalances mutagenesis.

scholarly journals Recent advances in the nucleolar responses to DNA double-strand breaks

2020 ◽  
Vol 48 (17) ◽  
pp. 9449-9461
Author(s):  
Lea Milling Korsholm ◽  
Zita Gál ◽  
Blanca Nieto ◽  
Oliver Quevedo ◽  
Stavroula Boukoura ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Ribosomal Dna ◽  
Repetitive Sequences ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

scholarly journals Tetrahymena Meiotic Nuclear Reorganization Is Induced by a Checkpoint Kinase–dependent Response to DNA Damage

2009 ◽  
Vol 20 (9) ◽  
pp. 2428-2437 ◽  
Author(s):  
Josef Loidl ◽  
Kazufumi Mochizuki
Keyword(s):  
Dna Damage ◽  
Kinase Inhibitors ◽  
Signal Transduction Pathway ◽  
Dna Lesions ◽  
Meiotic Pairing ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Nuclear Reorganization ◽  
Bivalent Formation ◽  
Atr Kinase

In the ciliate Tetrahymena, meiotic micronuclei (MICs) undergo extreme elongation, and meiotic pairing and recombination take place within these elongated nuclei (the “crescents”). We have previously shown that elongation does not occur in the absence of Spo11p-induced DNA double-strand breaks (DSBs). Here we show that elongation is restored in spo11Δ mutants by various DNA-damaging agents including ones that may not cause DSBs to a notable extent. MIC elongation following Spo11p-induced DSBs or artificially induced DNA lesions is probably a DNA-damage response mediated by a phosphokinase signal transduction pathway, since it is suppressed by the ATM/ATR kinase inhibitors caffeine and wortmannin and by knocking out Tetrahymena's ATR orthologue. MIC elongation occurs concomitantly with the movement of centromeres away from the telomeric pole of the MIC. This DNA damage–dependent reorganization of the MIC helps to arrange homologous chromosomes alongside each other but is not sufficient for exact pairing. Thus, Spo11p contributes to bivalent formation in two ways: by creating a favorable spatial disposition of homologues and by stabilizing pairing by crossovers. The polarized chromosome orientation inside the crescent resembles the conserved meiotic bouquet, and crescent and bouquet also share the putative function of aiding meiotic pairing. However, they are regulated differently because in Tetrahymena, DSBs are required for entering rather than exiting this stage.

scholarly journals DNA double-strand breaks and ATM activation by transcription-blocking DNA lesions

Cell Cycle ◽  
2010 ◽  
Vol 9 (2) ◽  
pp. 274-278 ◽  
Author(s):  
Olivier Sordet ◽  
Asako J. Nakamura ◽  
Christophe E. Redon ◽  
Yves Pommier
Keyword(s):  
Dna Lesions ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

scholarly journals The role of ubiquitin-dependent segregase p97 (VCP or Cdc48) in chromatin dynamics after DNA double strand breaks

2017 ◽  
Vol 372 (1731) ◽  
pp. 20160282 ◽  
Author(s):  
Ignacio Torrecilla ◽  
Judith Oehler ◽  
Kristijan Ramadan
Keyword(s):  
Dna Repair ◽  
Current Knowledge ◽  
Dna Lesions ◽  
Chromatin Remodelling ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Chromatin Dynamics ◽  
Strand Breaks ◽  
Signalling Molecules

DNA double strand breaks (DSBs) are the most cytotoxic DNA lesions and, if not repaired, lead to chromosomal rearrangement, genomic instability and cell death. Cells have evolved a complex network of DNA repair and signalling molecules which promptly detect and repair DSBs, commonly known as the DNA damage response (DDR). The DDR is orchestrated by various post-translational modifications such as phosphorylation, methylation, ubiquitination or SUMOylation. As DSBs are located in complex chromatin structures, the repair of DSBs is engineered at two levels: (i) at sites of broken DNA and (ii) at chromatin structures that surround DNA lesions. Thus, DNA repair and chromatin remodelling machineries must work together to efficiently repair DSBs. Here, we summarize the current knowledge of the ubiquitin-dependent molecular unfoldase/segregase p97 (VCP in vertebrates and Cdc48 in worms and lower eukaryotes) in DSB repair. We identify p97 as an essential factor that regulates DSB repair. p97-dependent extraction of ubiquitinated substrates mediates spatio-temporal protein turnover at and around the sites of DSBs, thus orchestrating chromatin remodelling and DSB repair. As p97 is a druggable target, p97 inhibition in the context of DDR has great potential for cancer therapy, as shown for other DDR components such as PARP, ATR and CHK1. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

scholarly journals SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Sílvia Carvalho ◽  
Alexandra C Vítor ◽  
Sreerama C Sridhara ◽  
Filipa B Martins ◽  
Ana C Raposo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Strand Break ◽  
Dna Lesions ◽  
Alternative Mechanism ◽  
Dna Double Strand Breaks ◽  
Cell Renal Cell Carcinoma ◽  
Strand Breaks ◽  
Dna Damage Signaling ◽  
Dna Double Strand Break ◽  
Recombination Repair

Histone modifications establish the chromatin states that coordinate the DNA damage response. In this study, we show that SETD2, the enzyme that trimethylates histone H3 lysine 36 (H3K36me3), is required for ATM activation upon DNA double-strand breaks (DSBs). Moreover, we find that SETD2 is necessary for homologous recombination repair of DSBs by promoting the formation of RAD51 presynaptic filaments. In agreement, SETD2-mutant clear cell renal cell carcinoma (ccRCC) cells displayed impaired DNA damage signaling. However, despite the persistence of DNA lesions, SETD2-deficient cells failed to activate p53, a master guardian of the genome rarely mutated in ccRCC and showed decreased cell survival after DNA damage. We propose that this novel SETD2-dependent role provides a chromatin bookmarking instrument that facilitates signaling and repair of DSBs. In ccRCC, loss of SETD2 may afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for additional genetic mutations in TP53.

scholarly journals Mismatch repair systems might facilitate the chromosomal recombination induced by N-nitrosodimethylamine, but not by N-nitrosodiethylamine, in Drosophila

Mutagenesis ◽  
2020 ◽  
Vol 35 (2) ◽  
pp. 197-206
Author(s):  
Tomoe Negishi ◽  
Kenji Yamada ◽  
Keiko Miyamoto ◽  
Emiko Mori ◽  
Kentaro Taira ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Mutation Rate ◽  
Spontaneous Mutation ◽  
Dna Lesions ◽  
Spot Test ◽  
Dna Double Strand Breaks ◽  
Spontaneous Mutation Rate ◽  
Futile Cycle ◽  
Strand Breaks ◽  
Chromosomal Recombination

Abstract Mismatch repair (MMR) systems play important roles in maintaining the high fidelity of genomic DNA. It is well documented that a lack of MMR increases the mutation rate, including base exchanges and small insertion/deletion loops; however, it is unknown whether MMR deficiency affects the frequency of chromosomal recombination in somatic cells. To investigate the effects of MMR on chromosomal recombination, we used the Drosophila wing-spot test, which efficiently detects chromosomal recombination. We prepared MMR (MutS)-deficient flies (spel1(−/−)) using a fly line generated in this study. The spontaneous mutation rate as measured by the wing-spot test was slightly higher in MutS-deficient flies than in wild-type (spel1(+/−)) flies. Previously, we showed that N-nitrosodimethylamine (NDMA)-induced chromosomal recombination more frequently than N-nitrosodiethylamine (NDEA) in Drosophila. When the wing-spot test was performed using MMR-deficient flies, unexpectedly, the rate of NDMA-induced mutation was significantly lower in spel1(−/−) flies than in spel1(+/−) flies. In contrast, the rate of mutation induced by NDEA was higher in spel1(−/−) flies than in spel1(+/−) flies. These results suggest that in Drosophila, the MutS homologue protein recognises methylated DNA lesions more efficiently than ethylated ones, and that MMR might facilitate mutational chromosomal recombination due to DNA double-strand breaks via the futile cycle induced by MutS recognition of methylated lesions.

scholarly journals Poly(ADP-ribosyl)ation mediates early phase histone eviction at DNA lesions

2020 ◽  
Vol 48 (6) ◽  
pp. 3001-3013 ◽  
Author(s):  
Guang Yang ◽  
Yibin Chen ◽  
Jiaxue Wu ◽  
Shih-Hsun Chen ◽  
Xiuhua Liu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Molecular Mechanism ◽  
Parp Inhibitor ◽  
Repair Process ◽  
Dna Lesions ◽  
Histone Chaperones ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Lesion ◽  
Damage Repair

Abstract Nucleosomal histones are barriers to the DNA repair process particularly at DNA double-strand breaks (DSBs). However, the molecular mechanism by which these histone barriers are removed from the sites of DNA damage remains elusive. Here, we have generated a single specific inducible DSB in the cells and systematically examined the histone removal process at the DNA lesion. We found that histone removal occurred immediately following DNA damage and could extend up to a range of few kilobases from the lesion. To examine the molecular mechanism underlying DNA damage-induced histone removal, we screened histone modifications and found that histone ADP-ribosylation was associated with histone removal at DNA lesions. PARP inhibitor treatment suppressed the immediate histone eviction at DNA lesions. Moreover, we examined histone chaperones and found that the FACT complex recognized ADP-ribosylated histones and mediated the removal of histones in response to DNA damage. Taken together, our results reveal a pathway that regulates early histone barrier removal at DNA lesions. It may also explain the mechanism by which PARP inhibitor regulates early DNA damage repair.

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