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1
Jiang, Yuning. "Contribution of Microhomology to Genome Instability: Connection between DNA Repair and Replication Stress". International Journal of Molecular Sciences 23, n. 21 (26 ottobre 2022): 12937. http://dx.doi.org/10.3390/ijms232112937.
Testo completoAbstract (sommario):
Microhomology-mediated end joining (MMEJ) is a highly mutagenic pathway to repair double-strand breaks (DSBs). MMEJ was thought to be a backup pathway of homologous recombination (HR) and canonical nonhomologous end joining (C-NHEJ). However, it attracts more attention in cancer research due to its special function of microhomology in many different aspects of cancer. In particular, it is initiated with DNA end resection and upregulated in homologous recombination-deficient cancers. In this review, I summarize the following: (1) the recent findings and contributions of MMEJ to genome instability, including phenotypes relevant to MMEJ; (2) the interaction between MMEJ and other DNA repair pathways; (3) the proposed mechanistic model of MMEJ in DNA DSB repair and a new connection with microhomology-mediated break-induced replication (MMBIR); and (4) the potential clinical application by targeting MMEJ based on synthetic lethality for cancer therapy.
2
Xu, Yijiang, Hang Zhou, Ginell Post, Hong Zan e Paolo Casali. "Rad52 mediates class-switch DNA recombination to IgD". Journal of Immunology 208, n. 1_Supplement (1 maggio 2022): 112.17. http://dx.doi.org/10.4049/jimmunol.208.supp.112.17.
Testo completoAbstract (sommario):
Abstract While the biology of IgD begins to be better understood, the mechanism of expression of this phylogenetically old and highly conserved Ig remains unknown. In B cells, IgD is mainly expressed together with IgM through alternative splicing of primary VHDJH-Cμ-s-m-Cδ-s-m RNAs, but also independently through IgD class switch DNA recombination (CSR) via double-strand DNA breaks (DSBs) and synapse of Sμ with σδ. How such DSBs, however, are resolved is still unknown. Our previous demonstration of a novel role of Rad52 in a Ku70/Ku86-independent “short-range” microhomology-mediated synapsis of intra-Sμ region DSBs led us to hypothesize that this homologous recombination DNA annealing factor is also involved in short-range microhomology-mediated alternative endjoining (A-EJ) recombination of Sμ with σδ. We found that induction of IgD CSR downregulates Zfp318 and promotes Rad52 phosphorylation and recruitment to Sμ and σδ, leading to alternative end-joining (A-EJ)-mediated Sμ-σδ recombination of resected DSB ends with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52−/− B cells aborted IgD CSR in vitro and in vivo and dampened the specific IgD antibody response to OVA. Rad52 knockdown in human B cells also abrogated IgD CSR. Finally, Rad52 phosphorylation was associated with high levels of IgD CSR and anti-nuclear IgD autoantibodies in lupus-prone mice and patients with lupus. Our findings thus show that Rad52 mediates IgD CSR through microhomology-mediated A-EJ in concert with Zfp318 downregulation. This is a previously unrecognized and critical role of Rad52 in mammalian DNA repair that provides a mechanistic underpinning to CSR A-EJ. Supported by NIH grants AI 079705, AI 105813, AAI 167416 and LRA grant 641363 to PC.
3
Lee-Theilen, Mieun, Allysia J. Matthews, Dierdre Kelly, Simin Zheng e Jayanta Chaudhuri. "CtIP promotes microhomology-mediated alternative end joining during class-switch recombination". Nature Structural & Molecular Biology 18, n. 1 (5 dicembre 2010): 75–79. http://dx.doi.org/10.1038/nsmb.1942.
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4
Francis, Nigel J., Bairbre McNicholas, Atif Awan, Mary Waldron, Donal Reddan, Denise Sadlier, David Kavanagh et al. "A novel hybrid CFH/CFHR3 gene generated by a microhomology-mediated deletion in familial atypical hemolytic uremic syndrome". Blood 119, n. 2 (12 gennaio 2012): 591–601. http://dx.doi.org/10.1182/blood-2011-03-339903.
Testo completoAbstract (sommario):
Abstract Genomic disorders affecting the genes encoding factor H (fH) and the 5 factor H related proteins have been described in association with atypical hemolytic uremic syndrome. These include deletions of CFHR3, CFHR1, and CFHR4 in association with fH autoantibodies and the formation of a hybrid CFH/CFHR1 gene. These occur through nonallelic homologous recombination secondary to the presence of large segmental duplications (macrohomology) in this region. Using multiplex ligation-dependent probe amplification to screen for such genomic disorders, we have identified a large atypical hemolytic uremic syndrome family where a deletion has occurred through microhomology-mediated end joining rather than nonallelic homologous recombination. In the 3 affected persons of this family, we have shown that the deletion results in formation of a CFH/CFHR3 gene. We have shown that the protein product of this is a 24 SCR protein that is secreted with normal fluid-phase activity but marked loss of complement regulation at cell surfaces despite increased heparin binding. In this study, we have therefore shown that microhomology in this area of chromosome 1 predisposes to disease associated genomic disorders and that the complement regulatory function of fH at the cell surface is critically dependent on the structural integrity of the whole molecule.
5
Ahrabi, Sara, Sovan Sarkar, Sophia X. Pfister, Giacomo Pirovano, Geoff S. Higgins, Andrew C. G. Porter e Timothy C. Humphrey. "A role for human homologous recombination factors in suppressing microhomology-mediated end joining". Nucleic Acids Research 44, n. 12 (29 aprile 2016): 5743–57. http://dx.doi.org/10.1093/nar/gkw326.
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Chan, C. Y., M. Kiechle, P. Manivasakam e R. H. Schiestl. "Ionizing radiation and restriction enzymes induce microhomology-mediated illegitimate recombination in Saccharomyces cerevisiae". Nucleic Acids Research 35, n. 15 (11 luglio 2007): 5051–59. http://dx.doi.org/10.1093/nar/gkm442.
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7
Ling, Alexanda K., Clare C. So, Michael X. Le, Audrey Y. Chen, Lisa Hung e Alberto Martin. "Double-stranded DNA break polarity skews repair pathway choice during intrachromosomal and interchromosomal recombination". Proceedings of the National Academy of Sciences 115, n. 11 (22 febbraio 2018): 2800–2805. http://dx.doi.org/10.1073/pnas.1720962115.
Testo completoAbstract (sommario):
Activation-induced cytidine deaminase (AID) inflicts DNA damage at Ig genes to initiate class switch recombination (CSR) and chromosomal translocations. However, the DNA lesions formed during these processes retain an element of randomness, and thus knowledge of the relationship between specific DNA lesions and AID-mediated processes remains incomplete. To identify necessary and sufficient DNA lesions in CSR, the Cas9 endonuclease and nickase variants were used to program DNA lesions at a greater degree of predictability than is achievable with conventional induction of CSR. Here we show that Cas9-mediated nicks separated by up to 250 nucleotides on opposite strands can mediate CSR. Staggered double-stranded breaks (DSBs) result in more end resection and junctional microhomology than blunt DSBs. Moreover, Myc-Igh chromosomal translocations, which are carried out primarily by alternative end joining (A-EJ), were preferentially induced by 5′ DSBs. These data indicate that DSBs with 5′ overhangs skew intrachromosomal and interchromosomal end-joining toward A-EJ. In addition to lending potential insight to AID-mediated phenomena, this work has broader carryover implications in DNA repair and lymphomagenesis.
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Chan, Cecilia Y., e Robert H. Schiestl. "Rad1, rad10 and rad52 Mutations Reduce the Increase of Microhomology Length during Radiation-Induced Microhomology-Mediated Illegitimate Recombination in Saccharomyces cerevisiae". Radiation Research 172, n. 2 (1 agosto 2009): 141. http://dx.doi.org/10.1667/rr1675.1.
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Nagai, Koki, Hirohito Shima, Miki Kamimura, Junko Kanno, Erina Suzuki, Akira Ishiguro, Satoshi Narumi, Shigeo Kure, Ikuma Fujiwara e Maki Fukami. "Xp22.31 Microdeletion due to Microhomology-Mediated Break-Induced Replication in a Boy with Contiguous Gene Deletion Syndrome". Cytogenetic and Genome Research 151, n. 1 (2017): 1–4. http://dx.doi.org/10.1159/000458469.
Testo completoAbstract (sommario):
The Xp22.31 region is characterized by a low frequency of interspersed repeats and a low GC content. Submicroscopic deletions at Xp22.31 involving STS and ANOS1 (alias KAL1) underlie X-linked ichthyosis and Kallmann syndrome, respectively. Of the known microdeletions at Xp22.31, a common approximately 1.5-Mb deletion encompassing STS was ascribed to nonallelic homologous recombination, while 2 ANOS1-containing deletions were attributed to nonhomologous end-joining. However, the genomic bases of other microdeletions within the Xp22.31 region remain to be elucidated. Here, we identified a 2,735,696-bp deletion encompassing STS and ANOS1 in a boy with X-linked ichthyosis and Kallmann syndrome. The breakpoints of the deletion were located within Alu repeats and shared 2-bp microhomology. The fusion junction was not associated with nucleotide stretches, and the breakpoint-flanking regions harbored no palindromes or noncanonical DNA motifs. These results indicate that microhomology-mediated break-induced replication (MMBIR) can cause deletions at Xp22.31, resulting in contiguous gene deletion syndrome. It appears that interspersed repeats without other known rearrangement-inducing DNA features or high GC contents are sufficient to stimulate MMBIR at Xp22.31.
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Meyer, Damon, Becky Xu Hua Fu e Wolf-Dietrich Heyer. "DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae". Proceedings of the National Academy of Sciences 112, n. 50 (25 novembre 2015): E6907—E6916. http://dx.doi.org/10.1073/pnas.1507833112.
Testo completoAbstract (sommario):
Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.
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Xu, Ran, Ziyi Pan e Takuro Nakagawa. "Gross Chromosomal Rearrangement at Centromeres". Biomolecules 14, n. 1 (24 dicembre 2023): 28. http://dx.doi.org/10.3390/biom14010028.
Testo completoAbstract (sommario):
Centromeres play essential roles in the faithful segregation of chromosomes. CENP-A, the centromere-specific histone H3 variant, and heterochromatin characterized by di- or tri-methylation of histone H3 9th lysine (H3K9) are the hallmarks of centromere chromatin. Contrary to the epigenetic marks, DNA sequences underlying the centromere region of chromosomes are not well conserved through evolution. However, centromeres consist of repetitive sequences in many eukaryotes, including animals, plants, and a subset of fungi, including fission yeast. Advances in long-read sequencing techniques have uncovered the complete sequence of human centromeres containing more than thousands of alpha satellite repeats and other types of repetitive sequences. Not only tandem but also inverted repeats are present at a centromere. DNA recombination between centromere repeats can result in gross chromosomal rearrangement (GCR), such as translocation and isochromosome formation. CENP-A chromatin and heterochromatin suppress the centromeric GCR. The key player of homologous recombination, Rad51, safeguards centromere integrity through conservative noncrossover recombination between centromere repeats. In contrast to Rad51-dependent recombination, Rad52-mediated single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) lead to centromeric GCR. This review summarizes recent findings on the role of centromere and recombination proteins in maintaining centromere integrity and discusses how GCR occurs at centromeres.
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Robert, Isabelle, Françoise Dantzer e Bernardo Reina-San-Martin. "Parp1 facilitates alternative NHEJ, whereas Parp2 suppresses IgH/c-myc translocations during immunoglobulin class switch recombination". Journal of Experimental Medicine 206, n. 5 (13 aprile 2009): 1047–56. http://dx.doi.org/10.1084/jem.20082468.
Testo completoAbstract (sommario):
Immunoglobulin class switch recombination (CSR) is initiated by DNA breaks triggered by activation-induced cytidine deaminase (AID). These breaks activate DNA damage response proteins to promote appropriate repair and long-range recombination. Aberrant processing of these breaks, however, results in decreased CSR and/or increased frequency of illegitimate recombination between the immunoglobulin heavy chain locus and oncogenes like c-myc. Here, we have examined the contribution of the DNA damage sensors Parp1 and Parp2 in the resolution of AID-induced DNA breaks during CSR. We find that although Parp enzymatic activity is induced in an AID-dependent manner during CSR, neither Parp1 nor Parp2 are required for CSR. We find however, that Parp1 favors repair of switch regions through a microhomology-mediated pathway and that Parp2 actively suppresses IgH/c-myc translocations. Thus, we define Parp1 as facilitating alternative end-joining and Parp2 as a novel translocation suppressor during CSR.
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Wang, Xiaobin S., Junfei Zhao, Foon Wu-Baer, Zhengping Shao, Brian J. Lee, Olivia M. Cupo, Raul Rabadan, Jean Gautier, Richard Baer e Shan Zha. "CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining". Proceedings of the National Academy of Sciences 117, n. 41 (28 settembre 2020): 25700–25711. http://dx.doi.org/10.1073/pnas.2010972117.
Testo completoAbstract (sommario):
To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.
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Chen, Changming, Xiaoling Xie, Xi Wu, Yeling Lu, Xuefeng Wang, Wenman Wu, Yiqun Hu e Qiulan Ding. "Complex recombination with deletion in the F8 and duplication in the TMLHE mediated by int22h copies during early embryogenesis". Thrombosis and Haemostasis 117, n. 08 (2017): 1478–85. http://dx.doi.org/10.1160/th17-01-0046.
Testo completoAbstract (sommario):
SummaryHaemophilia A (HA) is a common X-linked recessive bleeding disorder and almost one half of patients with severe HA are caused by intron 22 inversion (Inv22) in the F8. Inv22 is considered to be almost exclusively of meiotic origin in germ cells during spermatogenesis and only one mosaic Inv22 female carrier with the mutation possibly occurring during mitosis of the embryo has been reported so far. Previously we have identified a novel complex recombination mediated by int22h copies in a sporadic severe HA pedigree and herein we have localised the sequences flanking the breakpoint region using genome walking technique, AccuCopy technique, gene chip and real-time PCR. The disease causing genetic variant registered an 18.1 kb deletion including part of int22h-1 through the intron 23 of F8 and a 113.3 kb duplication of part of int22h-2 through the intron 1 of TMLHE inserted in the religated region of the F8. Two intrinsically linked mechanisms of recombination-dependent DNA replication: microhomology-mediated break-induced replication (MMBIR) followed by break-induced replication (BIR) might be responsible for the incident of the complex recombination during early embryogenesis of the proband’s mother.Supplementary Material to this article is available online at www.thrombosis-online.com.
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Saribasak, Huseyin, Robert W. Maul, Zheng Cao, Rhonda L. McClure, William Yang, Daniel R. McNeill, David M. Wilson e Patricia J. Gearhart. "XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes". Journal of Experimental Medicine 208, n. 11 (3 ottobre 2011): 2209–16. http://dx.doi.org/10.1084/jem.20111135.
Testo completoAbstract (sommario):
Activation-induced deaminase (AID) deaminates cytosine to uracil in immunoglobulin genes. Uracils in DNA can be recognized by uracil DNA glycosylase and abasic endonuclease to produce single-strand breaks. The breaks are repaired either faithfully by DNA base excision repair (BER) or mutagenically to produce somatic hypermutation (SHM) and class switch recombination (CSR). To unravel the interplay between repair and mutagenesis, we decreased the level of x-ray cross-complementing 1 (XRCC1), a scaffold protein involved in BER. Mice heterozygous for XRCC1 showed a significant increase in the frequencies of SHM in Igh variable regions in Peyer’s patch cells, and of double-strand breaks in the switch regions during CSR. Although the frequency of CSR was normal in Xrcc1+/− splenic B cells, the length of microhomology at the switch junctions decreased, suggesting that XRCC1 also participates in alternative nonhomologous end joining. Furthermore, Xrcc1+/− B cells had reduced Igh/c-myc translocations during CSR, supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID-induced single-strand breaks in Igh variable and switch regions become substrates simultaneously for BER and mutagenesis pathways.
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Ehrenstein, M. R., C. Rada, A. M. Jones, C. Milstein e M. S. Neuberger. "Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination". Proceedings of the National Academy of Sciences 98, n. 25 (20 novembre 2001): 14553–58. http://dx.doi.org/10.1073/pnas.241525998.
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Chen, Xiaojiang S., e Richard T. Pomerantz. "DNA Polymerase θ: A Cancer Drug Target with Reverse Transcriptase Activity". Genes 12, n. 8 (27 luglio 2021): 1146. http://dx.doi.org/10.3390/genes12081146.
Testo completoAbstract (sommario):
The emergence of precision medicine from the development of Poly (ADP-ribose) polymerase (PARP) inhibitors that preferentially kill cells defective in homologous recombination has sparked wide interest in identifying and characterizing additional DNA repair enzymes that are synthetic lethal with HR factors. DNA polymerase theta (Polθ) is a validated anti-cancer drug target that is synthetic lethal with HR factors and other DNA repair proteins and confers cellular resistance to various genotoxic cancer therapies. Since its initial characterization as a helicase-polymerase fusion protein in 2003, many exciting and unexpected activities of Polθ in microhomology-mediated end-joining (MMEJ) and translesion synthesis (TLS) have been discovered. Here, we provide a short review of Polθ‘s DNA repair activities and its potential as a drug target and highlight a recent report that reveals Polθ as a naturally occurring reverse transcriptase (RT) in mammalian cells.
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Bothmer, Anne, Davide F. Robbiani, Niklas Feldhahn, Anna Gazumyan, Andre Nussenzweig e Michel C. Nussenzweig. "53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination". Journal of Experimental Medicine 207, n. 4 (5 aprile 2010): 855–65. http://dx.doi.org/10.1084/jem.20100244.
Testo completoAbstract (sommario):
Class switch recombination (CSR) diversifies antibodies by joining highly repetitive DNA elements, which are separated by 60–200 kbp. CSR is initiated by activation-induced cytidine deaminase, an enzyme that produces multiple DNA double-strand breaks (DSBs) in switch regions. Switch regions are joined by a mechanism that requires an intact DNA damage response and classical or alternative nonhomologous end joining (A-NHEJ). Among the DNA damage response factors, 53BP1 has the most profound effect on CSR. We explore the role of 53BP1 in intrachromosomal DNA repair using I-SceI to introduce paired DSBs in the IgH locus. We find that the absence of 53BP1 results in an ataxia telangiectasia mutated–dependent increase in DNA end resection and that resected DNA is preferentially repaired by microhomology-mediated A-NHEJ. We propose that 53BP1 favors long-range CSR in part by protecting DNA ends against resection, which prevents A-NHEJ–dependent short-range rejoining of intra–switch region DSBs.
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Decottignies, Anabelle. "Microhomology-Mediated End Joining in Fission Yeast Is Repressed by Pku70 and Relies on Genes Involved in Homologous Recombination". Genetics 176, n. 3 (4 maggio 2007): 1403–15. http://dx.doi.org/10.1534/genetics.107.071621.
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Ding, Qiulan, Guoling You, Jing Dai, Xiaodong Xi, Hongli Wang, Xi Wu, Yeling Lu e Xuefeng Wang. "Characterisation of large F9 deletions in seven unrelated patients with severe haemophilia B". Thrombosis and Haemostasis 112, n. 09 (2014): 459–65. http://dx.doi.org/10.1160/th13-12-1060.
Testo completoAbstract (sommario):
SummaryLarge deletions in the F9 gene are detected in approximately 5% of patients with severe haemophilia B, but only a few deletion breakpoints have been characterised precisely until now. In this study we identified a total of seven large F9 deletions in the index patients and nine female carriers by the AccuCopy technique. We also successfully characterised the exact breakpoints for each large deletion including four deletions encompassing the entire F9 gene by the genome walking method combined with primer walking strategy. The extents of deletion regions ranged from 11.1 to 884 kb. Microhomologies ranged from 2 to 6 bp were identified in the breakpoint junctions of six deletions. The other deletion occurred between two highly homologous sequences of the same long interspersed nuclear element 1 (LINE/L1). Non-homologous end joining (NHEJ) and microhomology-mediated break-induced replication (MMBIR) may be the main causative mechanisms for the six large deletions with microhomologies. Non-allelic homologous recombination (NAHR) may mediate the deletion occurred between the two tandem LINEs in the other large deletion. Repetitive elements and non-B DNA forming motifs identified in the junction regions may contribute to DNA breakage leading to large deletions.
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You, Guoling, Kun Chi, Yeling Lu, Qiulan Ding, Jing Dai, Xiaodong Xi, Hongli Wang e Xuefeng Wang. "Identification and characterisation of a novel aberrant pattern of intron 1 inversion with concomitant large insertion and deletion within the F8 gene". Thrombosis and Haemostasis 112, n. 08 (2014): 264–70. http://dx.doi.org/10.1160/th13-10-0892.
Testo completoAbstract (sommario):
SummaryIntron 1 inversion (Inv1) is a recurrent causative mutation of haemophilia A (HA) and is responsible for 1–5% of severe HA. Inv1 occurs as a result of intra-chromosomal homologous recombination between int1h-1 within intron 1 and int1h-2 located in approximately 125 kb telomeric to the F8 gene. In this report, we presented a previously undescribed aberrant type of Inv1 with complex genomic rearrangement in a pedigree with severe HA. The breakpoints of the rearrangement were identified by the genome walking technique; copy number variations (CNVs) of the F8 gene and X chromosome were detected by AccuCopy technique, Affymetrix CytoScan HD CNV assay and quantitative PCR (qPCR); the F8 transcripts related to the aberrant Inv1 were analysed by reverse transcription PCR (RT-PCR). We have characterised the exact breakpoints of the complex rearrangement, and determined the location and size of the insertion and deletion. The rearrangements can be summarised as an aberrant pattern of Inv1 with a deletion of 2.56 kb and a duplication of 227.3 kb inserted in the rejoining junction within the F8 gene. Our results suggested that this complex genomic rearrangement was generated by two distinct repair mechanisms of fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR) and nonallelic homologous recombination (NAHR).
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Kohli, Ajay, Simon Griffiths, Natalia Palacios, Richard M Twyman, Philippe Vain, David A. Laurie e Paul Christou. "Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination". Plant Journal 17, n. 6 (marzo 1999): 591–601. http://dx.doi.org/10.1046/j.1365-313x.1999.00399.x.
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Fiori, Mariangela Stefania, Luca Ferretti, Antonello Di Nardo, Lele Zhao, Susanna Zinellu, Pier Paolo Angioi, Matteo Floris et al. "A Naturally Occurring Microhomology-Mediated Deletion of Three Genes in African Swine Fever Virus Isolated from Two Sardinian Wild Boars". Viruses 14, n. 11 (14 novembre 2022): 2524. http://dx.doi.org/10.3390/v14112524.
Testo completoAbstract (sommario):
African swine fever virus (ASFV) is the etiological agent of a lethal disease of domestic pigs and wild boars. ASF threatens the pig industry worldwide due to the lack of a licensed vaccine or treatment. The disease has been endemic for more than 40 years in Sardinia (Italy), but an intense campaign pushed it close to eradication; virus circulation was last detected in wild boars in 2019. In this study, we present a genomic analysis of two ASFV strains isolated in Sardinia from two wild boars during the 2019 hunting season. Both isolates presented a deletion of 4342 base pairs near the 5′ end of the genome, encompassing the genes MGF 360-6L, X69R, and MGF 300-1L. The phylogenetic evidence suggests that the deletion recently originated within the Sardinia ecosystem and that it is most likely the result of a non-allelic homologous recombination driven by a microhomology present in most Sardinian ASFV genomes. These results represent a striking example of a genomic feature promoting the rapid evolution of structural variations and plasticity in the ASFV genome. They also raise interesting questions about the functions of the deleted genes and the potential link between the evolutionary timing of the deletion appearance and the eradication campaign.
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Feng, Wanjuan, Dennis A. Simpson, Jang-Eun Cho, Juan Carvajal-Garcia, Chelsea M. Smith, Kathryn M. Headley, Nate Hathaway, Dale A. Ramsden e Gaorav P. Gupta. "Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks". Nucleic Acids Research 49, n. 9 (8 maggio 2021): 5095–105. http://dx.doi.org/10.1093/nar/gkab299.
Testo completoAbstract (sommario):
Abstract Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays (‘PathSig-dPCR’) for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.
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Stachler, Aris-Edda, Julia Wörtz, Omer S. Alkhnbashi, Israela Turgeman-Grott, Rachel Smith, Thorsten Allers, Rolf Backofen, Uri Gophna e Anita Marchfelder. "Adaptation induced by self-targeting in a type I-B CRISPR-Cas system". Journal of Biological Chemistry 295, n. 39 (28 luglio 2020): 13502–15. http://dx.doi.org/10.1074/jbc.ra120.014030.
Testo completoAbstract (sommario):
Haloferax volcanii is, to our knowledge, the only prokaryote known to tolerate CRISPR-Cas–mediated damage to its genome in the WT background; the resulting cleavage of the genome is repaired by homologous recombination restoring the WT version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalyzed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centered on the targeting site. Furthermore, we observed naïve spacer acquisition at a low level in WT Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2, and Cas4. Taken together, these findings indicate that naïve adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.
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Dahal, Sumedha, e Sathees C. Raghavan. "Mitochondrial genome stability in human: understanding the role of DNA repair pathways". Biochemical Journal 478, n. 6 (19 marzo 2021): 1179–97. http://dx.doi.org/10.1042/bcj20200920.
Testo completoAbstract (sommario):
Mitochondria are semiautonomous organelles in eukaryotic cells and possess their own genome that replicates independently. Mitochondria play a major role in oxidative phosphorylation due to which its genome is frequently exposed to oxidative stress. Factors including ionizing radiation, radiomimetic drugs and replication fork stalling can also result in different types of mutations in mitochondrial DNA (mtDNA) leading to genome fragility. Mitochondria from myopathies, dystonia, cancer patient samples show frequent mtDNA mutations such as point mutations, insertions and large-scale deletions that could account for mitochondria-associated disease pathogenesis. The mechanism by which such mutations arise following exposure to various DNA-damaging agents is not well understood. One of the well-studied repair pathways in mitochondria is base excision repair. Other repair pathways such as mismatch repair, homologous recombination and microhomology-mediated end joining have also been reported. Interestingly, nucleotide excision repair and classical nonhomologous DNA end joining are not detected in mitochondria. In this review, we summarize the potential causes of mitochondrial genome fragility, their implications as well as various DNA repair pathways that operate in mitochondria.
27
Wang, Wenjie, Kuan Li, Zhuo Yang, Quancan Hou, Wei W. Zhao e Qianwen Sun. "RNase H1C collaborates with ssDNA binding proteins WHY1/3 and recombinase RecA1 to fulfill the DNA damage repair in Arabidopsis chloroplasts". Nucleic Acids Research 49, n. 12 (16 giugno 2021): 6771–87. http://dx.doi.org/10.1093/nar/gkab479.
Testo completoAbstract (sommario):
Abstract Proper repair of damaged DNA is crucial for genetic integrity and organismal survival. As semi-autonomous organelles, plastids have their own genomes whose integrity must be preserved. Several factors have been shown to participate in plastid DNA damage repair; however, the underlying mechanism remains unclear. Here, we elucidate a mechanism of homologous recombination (HR) repair in chloroplasts that involves R-loops. We find that the recombinase RecA1 forms filaments in chloroplasts during HR repair, but aggregates as puncta when RNA:DNA hybrids accumulate. ssDNA-binding proteins WHY1/3 and chloroplast RNase H1 AtRNH1C are recruited to the same genomic sites to promote HR repair. Depletion of AtRNH1C or WHY1/3 significantly suppresses the binding of RNA polymerase to the damaged DNA, thus reducing HR repair and modulating microhomology-mediated double-strand break repair. Furthermore, we show that DNA polymerase IB works with AtRNH1C genetically to complete the DNA damage repair process. This study reveals the positive role of R-loops in facilitating the activities of WHY1/3 and RecA1, which in turn secures HR repair and organellar development.
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Öz, Robin, Sean M. Howard, Rajhans Sharma, Hanna Törnkvist, Ilaria Ceppi, Sriram KK, Erik Kristiansson, Petr Cejka e Fredrik Westerlund. "Phosphorylated CtIP bridges DNA to promote annealing of broken ends". Proceedings of the National Academy of Sciences 117, n. 35 (19 agosto 2020): 21403–12. http://dx.doi.org/10.1073/pnas.2008645117.
Testo completoAbstract (sommario):
The early steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The roles of these proteins in nucleolytic DSB resection are well characterized, but their role in bridging the DNA ends for efficient and correct repair is much less explored. Here we study the binding of phosphorylated CtIP, which promotes the endonuclease activity of MRN, to single long (∼50 kb) DNA molecules using nanofluidic channels and compare it to the yeast homolog Sae2. CtIP bridges DNA in a manner that depends on the oligomeric state of the protein, and truncated mutants demonstrate that the bridging depends on CtIP regions distinct from those that stimulate the nuclease activity of MRN. Sae2 is a much smaller protein than CtIP, and its bridging is significantly less efficient. Our results demonstrate that the nuclease cofactor and structural functions of CtIP may depend on the same protein population, which may be crucial for CtIP functions in both homologous recombination and microhomology-mediated end-joining.
29
Zhao, Yuqin, Kaiping Hou, Yu Liu, Yinan Na, Chao Li, Haoyuan Luo e Hailong Wang. "Helicase HELQ: Molecular Characters Fit for DSB Repair Function". International Journal of Molecular Sciences 25, n. 16 (8 agosto 2024): 8634. http://dx.doi.org/10.3390/ijms25168634.
Testo completoAbstract (sommario):
The protein sequence and spatial structure of DNA helicase HELQ are highly conserved, spanning from archaea to humans. Aside from its helicase activity, which is based on DNA binding and translocation, it has also been recently reconfirmed that human HELQ possesses DNA–strand–annealing activity, similar to that of the archaeal HELQ homolog StoHjm. These biochemical functions play an important role in regulating various double–strand break (DSB) repair pathways, as well as multiple steps in different DSB repair processes. HELQ primarily facilitates repair in end–resection–dependent DSB repair pathways, such as homologous recombination (HR), single–strand annealing (SSA), microhomology–mediated end joining (MMEJ), as well as the sub-pathways’ synthesis–dependent strand annealing (SDSA) and break–induced replication (BIR) within HR. The biochemical functions of HELQ are significant in end resection and its downstream pathways, such as strand invasion, DNA synthesis, and gene conversion. Different biochemical activities are required to support DSB repair at various stages. This review focuses on the functional studies of the biochemical roles of HELQ during different stages of diverse DSB repair pathways.
30
Dohrn, Lisa, Daniela Salles, Simone Y. Siehler, Julia Kaufmann e Lisa Wiesmüller. "BRCA1-mediated repression of mutagenic end-joining of DNA double-strand breaks requires complex formation with BACH1". Biochemical Journal 441, n. 3 (16 gennaio 2012): 919–28. http://dx.doi.org/10.1042/bj20110314.
Testo completoAbstract (sommario):
BACH1 (BRCA1-associated C-terminal helicase 1), the product of the BRIP1 {BRCA1 [breast cancer 1, early onset]-interacting protein C-terminal helicase 1; also known as FANCJ [FA-J (Fanconi anaemia group J) protein]} gene mutated in Fanconi anaemia patients from complementation group J, has been implicated in DNA repair and damage signalling. BACH1 exerts DNA helicase activities and physically interacts with BRCA1 and MLH1 (mutL homologue 1), which differentially control DNA DSB (double-strand break) repair processes. The present study shows that BACH1 plays a role in both HR (homologous recombination) and MMEJ (microhomology-mediated non-homologous end-joining) and reveals discrete mechanisms underlying modulation of these pathways. Our results indicate that BACH1 stimulates HR, which depends on the integrity of the helicase domain. Disruption of the BRCA1–BACH1 complex through mutation of BACH1 compromised errorfree NHEJ (non-homologous end-joining) and accelerated error-prone MMEJ. Conversely, molecular changes in BACH1 abrogating MLH1 binding interfered neither with HR nor with MMEJ. Importantly, MMEJ is a mutagenic DSB repair pathway, which is derepressed in hereditary breast and ovarian carcinomas. Since BRCA1 and BACH1 mutations targeting the BRCA1–BACH1 interaction have been associated with breast cancer susceptibility, the results of the present study thus provide evidence for a novel role of BACH1 in tumour suppression.
31
Truong, L. N., Y. Li, L. Z. Shi, P. Y. H. Hwang, J. He, H. Wang, N. Razavian, M. W. Berns e X. Wu. "Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells". Proceedings of the National Academy of Sciences 110, n. 19 (22 aprile 2013): 7720–25. http://dx.doi.org/10.1073/pnas.1213431110.
Testo completo
32
Elert-Dobkowska, Ewelina, Iwona Stepniak, Wiktoria Radziwonik-Fraczyk, Amir Jahic, Christian Beetz e Anna Sulek. "SPAST Intragenic CNVs Lead to Hereditary Spastic Paraplegia via a Haploinsufficiency Mechanism". International Journal of Molecular Sciences 25, n. 9 (3 maggio 2024): 5008. http://dx.doi.org/10.3390/ijms25095008.
Testo completoAbstract (sommario):
The most common form of hereditary spastic paraplegia (HSP), SPG4 is caused by single nucleotide variants and microrearrangements in the SPAST gene. The high percentage of multi-exonic deletions or duplications observed in SPG4 patients is predisposed by the presence of a high frequency of Alu sequences in the gene sequence. In the present study, we analyzed DNA and RNA samples collected from patients with different microrearrangements in SPAST to map gene breakpoints and evaluate the mutation mechanism. The study group consisted of 69 individuals, including 50 SPG4 patients and 19 healthy relatives from 18 families. Affected family members from 17 families carried varying ranges of microrearrangements in the SPAST gene, while one individual had a single nucleotide variant in the 5′UTR of SPAST. To detect the breakpoints of the SPAST gene, long-range PCR followed by sequencing was performed. The breakpoint sequence was detected for five different intragenic SPAST deletions and one duplication, revealing Alu-mediated microhomology at breakpoint junctions resulting from non-allelic homologous recombination in these patients. Furthermore, SPAST gene expression analysis was performed using patient RNA samples extracted from whole blood. Quantitative real-time PCR tests performed in 14 patients suggest no expression of transcripts with microrearrangements in 5 of them. The obtained data indicate that nonsense-mediated decay degradation is not the only mechanism of hereditary spastic paraplegia in patients with SPAST microrearrangements.
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Tomasini, Paula Pellenz, Temenouga Nikolova Guecheva, Natalia Motta Leguisamo, Sarah Péricart, Anne-Cécile Brunac, Jean Sébastien Hoffmann e Jenifer Saffi. "Analyzing the Opportunities to Target DNA Double-Strand Breaks Repair and Replicative Stress Responses to Improve Therapeutic Index of Colorectal Cancer". Cancers 13, n. 13 (23 giugno 2021): 3130. http://dx.doi.org/10.3390/cancers13133130.
Testo completoAbstract (sommario):
Despite the ample improvements of CRC molecular landscape, the therapeutic options still rely on conventional chemotherapy-based regimens for early disease, and few targeted agents are recommended for clinical use in the metastatic setting. Moreover, the impact of cytotoxic, targeted agents, and immunotherapy combinations in the metastatic scenario is not fully satisfactory, especially the outcomes for patients who develop resistance to these treatments need to be improved. Here, we examine the opportunity to consider therapeutic agents targeting DNA repair and DNA replication stress response as strategies to exploit genetic or functional defects in the DNA damage response (DDR) pathways through synthetic lethal mechanisms, still not explored in CRC. These include the multiple actors involved in the repair of DNA double-strand breaks (DSBs) through homologous recombination (HR), classical non-homologous end joining (NHEJ), and microhomology-mediated end-joining (MMEJ), inhibitors of the base excision repair (BER) protein poly (ADP-ribose) polymerase (PARP), as well as inhibitors of the DNA damage kinases ataxia-telangiectasia and Rad3 related (ATR), CHK1, WEE1, and ataxia-telangiectasia mutated (ATM). We also review the biomarkers that guide the use of these agents, and current clinical trials with targeted DDR therapies.
34
Li, Zhichao, e Ralph Bock. "Rapid functional activation of a horizontally transferred eukaryotic gene in a bacterial genome in the absence of selection". Nucleic Acids Research 47, n. 12 (20 maggio 2019): 6351–59. http://dx.doi.org/10.1093/nar/gkz370.
Testo completoAbstract (sommario):
Abstract Horizontal gene transfer has occurred between organisms of all domains of life and contributed substantially to genome evolution in both prokaryotes and eukaryotes. Phylogenetic evidence suggests that eukaryotic genes horizontally transferred to bacteria provided useful new gene functions that improved metabolic plasticity and facilitated adaptation to new environments. How these eukaryotic genes evolved into functional bacterial genes is not known. Here, we have conducted a genetic screen to identify the mechanisms involved in functional activation of a eukaryotic gene after its transfer into a bacterial genome. We integrated a eukaryotic selectable marker gene cassette driven by expression elements from the red alga Porphyridium purpureum into the genome of Escherichia coli. Following growth under non-selective conditions, gene activation events were indentified by antibiotic selection. We show that gene activation in the bacterial recipient occurs at high frequency and involves two major types of spontaneous mutations: deletion and gene amplification. We further show that both mechanisms result in promoter capture and are frequently triggered by microhomology-mediated recombination. Our data suggest that horizontally transferred genes have a high probability of acquiring functionality, resulting in their maintenance if they confer a selective advantage.
35
Lukashchuk, Natalia, Joshua Armenia, Luis Tobalina, Thomas Hedley Carr, Tsveta Milenkova, Ying L. Liu, Richard T. Penson, Mark E. Robson e Elizabeth Harrington. "BRCA reversion mutations mediated by microhomology-mediated end joining (MMEJ) as a mechanism of resistance to PARP inhibitors in ovarian and breast cancer." Journal of Clinical Oncology 40, n. 16_suppl (1 giugno 2022): 5559. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.5559.
Testo completoAbstract (sommario):
5559 Background: PARP inhibitors exploit synthetic lethality in tumor cells with deficiency in homologous recombination repair (HRR). In line with this, most reported mechanisms of PARP inhibitor resistance restore HRR. Of multiple resistance mechanisms reported preclinically, reversion mutations in BRCA genes are the only confirmed mechanism of resistance to both platinum and PARP inhibitors in patients (pts) to date (Lin K et al. Cancer Discov 2019), with most studies focusing on ovarian cancer. MMEJ is an alternative DNA damage repair pathway, in which DNA polymerase θ (POLθ) has a key role; MMEJ has been suggested to play a role in BRCA reversion mutations (Tobalina L et al. Ann Oncol 2021). Methods: Targeted circulating tumor DNA (ctDNA) sequencing analyzed over 500 plasma samples collected at baseline and at progression to therapy in pts with ovarian or breast cancer and a mutation in BRCA1 and/or BRCA2 (BRCAm) who were treated with olaparib or chemotherapy in one of three Phase II/III clinical studies (LIGHT NCT02983799, SOLO3 NCT02282020, OlympiAD NCT02000622). Only pts with an original pathogenic BRCAm detected in ctDNA were evaluable. BRCA reversion mutations were identified using internal computational framework; DNA sequences surrounding BRCA reversion sites were analyzed for MMEJ signatures. Results: At baseline, in pooled data across treatment arms and across all available samples, BRCA reversion mutations were detected in 4/114 (3.5%) and 6/133 (4.5%) of breast and ovarian cancer pts, respectively, which may have developed on prior platinum therapy. At progression, BRCA reversion mutations were detected in 34/79 (43%) breast cancer pts and in 26/101 (26%) ovarian cancer pts who received olaparib, with at least 2/79 and 4/101 reversions already present at baseline, respectively. At progression, in the chemotherapy arm, BRCA reversion mutations were detected in 3/34 (9%) breast cancer pts and 1/29 (3%) ovarian cancer pts, with 2/34 and 0/29 reversions present at baseline, respectively. Reversion mutations varied in allelic frequency and were either present as single or multiple reversions, suggesting multiple events within the tumor were driving resistance. The location and type of reversion mutations reflected the functional importance of BRCA protein domains. A large proportion of BRCA reversion mutations (47/69 [68%] that were evaluable) were mediated by the MMEJ pathway based on the presence of MMEJ signatures around BRCA reversion sites. Conclusions: We detected BRCA reversion mutations in at least ̃40% of breast and ̃20% of ovarian cancer pts following treatment with olaparib. A large proportion of these reversion mutations are likely to have been mediated by MMEJ repair, suggesting that POLθ inhibitors in combination with platinum or PARP inhibitors might prevent or delay emergence of PARP inhibitor resistance. Clinical trial information: NCT02983799, NCT02282020, NCT02000622.
36
Lukashchuk, Natalia, Joshua Armenia, Luis Tobalina, Thomas Hedley Carr, Tsveta Milenkova, Ying L. Liu, Richard T. Penson, Mark E. Robson e Elizabeth Harrington. "BRCA reversion mutations mediated by microhomology-mediated end joining (MMEJ) as a mechanism of resistance to PARP inhibitors in ovarian and breast cancer." Journal of Clinical Oncology 40, n. 16_suppl (1 giugno 2022): 5559. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.5559.
Testo completoAbstract (sommario):
5559 Background: PARP inhibitors exploit synthetic lethality in tumor cells with deficiency in homologous recombination repair (HRR). In line with this, most reported mechanisms of PARP inhibitor resistance restore HRR. Of multiple resistance mechanisms reported preclinically, reversion mutations in BRCA genes are the only confirmed mechanism of resistance to both platinum and PARP inhibitors in patients (pts) to date (Lin K et al. Cancer Discov 2019), with most studies focusing on ovarian cancer. MMEJ is an alternative DNA damage repair pathway, in which DNA polymerase θ (POLθ) has a key role; MMEJ has been suggested to play a role in BRCA reversion mutations (Tobalina L et al. Ann Oncol 2021). Methods: Targeted circulating tumor DNA (ctDNA) sequencing analyzed over 500 plasma samples collected at baseline and at progression to therapy in pts with ovarian or breast cancer and a mutation in BRCA1 and/or BRCA2 (BRCAm) who were treated with olaparib or chemotherapy in one of three Phase II/III clinical studies (LIGHT NCT02983799, SOLO3 NCT02282020, OlympiAD NCT02000622). Only pts with an original pathogenic BRCAm detected in ctDNA were evaluable. BRCA reversion mutations were identified using internal computational framework; DNA sequences surrounding BRCA reversion sites were analyzed for MMEJ signatures. Results: At baseline, in pooled data across treatment arms and across all available samples, BRCA reversion mutations were detected in 4/114 (3.5%) and 6/133 (4.5%) of breast and ovarian cancer pts, respectively, which may have developed on prior platinum therapy. At progression, BRCA reversion mutations were detected in 34/79 (43%) breast cancer pts and in 26/101 (26%) ovarian cancer pts who received olaparib, with at least 2/79 and 4/101 reversions already present at baseline, respectively. At progression, in the chemotherapy arm, BRCA reversion mutations were detected in 3/34 (9%) breast cancer pts and 1/29 (3%) ovarian cancer pts, with 2/34 and 0/29 reversions present at baseline, respectively. Reversion mutations varied in allelic frequency and were either present as single or multiple reversions, suggesting multiple events within the tumor were driving resistance. The location and type of reversion mutations reflected the functional importance of BRCA protein domains. A large proportion of BRCA reversion mutations (47/69 [68%] that were evaluable) were mediated by the MMEJ pathway based on the presence of MMEJ signatures around BRCA reversion sites. Conclusions: We detected BRCA reversion mutations in at least ̃40% of breast and ̃20% of ovarian cancer pts following treatment with olaparib. A large proportion of these reversion mutations are likely to have been mediated by MMEJ repair, suggesting that POLθ inhibitors in combination with platinum or PARP inhibitors might prevent or delay emergence of PARP inhibitor resistance. Clinical trial information: NCT02983799, NCT02282020, NCT02000622.
37
Hays, Michelle, Katja Schwartz, Danica T. Schmidtke, Dimitra Aggeli e Gavin Sherlock. "Paths to adaptation under fluctuating nitrogen starvation: The spectrum of adaptive mutations in Saccharomyces cerevisiae is shaped by retrotransposons and microhomology-mediated recombination". PLOS Genetics 19, n. 5 (16 maggio 2023): e1010747. http://dx.doi.org/10.1371/journal.pgen.1010747.
Testo completoAbstract (sommario):
There are many mechanisms that give rise to genomic change: while point mutations are often emphasized in genomic analyses, evolution acts upon many other types of genetic changes that can result in less subtle perturbations. Changes in chromosome structure, DNA copy number, and novel transposon insertions all create large genomic changes, which can have correspondingly large impacts on phenotypes and fitness. In this study we investigate the spectrum of adaptive mutations that arise in a population under consistently fluctuating nitrogen conditions. We specifically contrast these adaptive alleles and the mutational mechanisms that create them, with mechanisms of adaptation under batch glucose limitation and constant selection in low, non-fluctuating nitrogen conditions to address if and how selection dynamics influence the molecular mechanisms of evolutionary adaptation. We observe that retrotransposon activity accounts for a substantial number of adaptive events, along with microhomology-mediated mechanisms of insertion, deletion, and gene conversion. In addition to loss of function alleles, which are often exploited in genetic screens, we identify putative gain of function alleles and alleles acting through as-of-yet unclear mechanisms. Taken together, our findings emphasize that how selection (fluctuating vs. non-fluctuating) is applied also shapes adaptation, just as the selective pressure (nitrogen vs. glucose) does itself. Fluctuating environments can activate different mutational mechanisms, shaping adaptive events accordingly. Experimental evolution, which allows a wider array of adaptive events to be assessed, is thus a complementary approach to both classical genetic screens and natural variation studies to characterize the genotype-to-phenotype-to-fitness map.
38
Provasek, Vincent E., Joy Mitra, Vikas H. Malojirao e Muralidhar L. Hegde. "DNA Double-Strand Breaks as Pathogenic Lesions in Neurological Disorders". International Journal of Molecular Sciences 23, n. 9 (22 aprile 2022): 4653. http://dx.doi.org/10.3390/ijms23094653.
Testo completoAbstract (sommario):
The damage and repair of DNA is a continuous process required to maintain genomic integrity. DNA double-strand breaks (DSBs) are the most lethal type of DNA damage and require timely repair by dedicated machinery. DSB repair is uniquely important to nondividing, post-mitotic cells of the central nervous system (CNS). These long-lived cells must rely on the intact genome for a lifetime while maintaining high metabolic activity. When these mechanisms fail, the loss of certain neuronal populations upset delicate neural networks required for higher cognition and disrupt vital motor functions. Mammalian cells engage with several different strategies to recognize and repair chromosomal DSBs based on the cellular context and cell cycle phase, including homologous recombination (HR)/homology-directed repair (HDR), microhomology-mediated end-joining (MMEJ), and the classic non-homologous end-joining (NHEJ). In addition to these repair pathways, a growing body of evidence has emphasized the importance of DNA damage response (DDR) signaling, and the involvement of heterogeneous nuclear ribonucleoprotein (hnRNP) family proteins in the repair of neuronal DSBs, many of which are linked to age-associated neurological disorders. In this review, we describe contemporary research characterizing the mechanistic roles of these non-canonical proteins in neuronal DSB repair, as well as their contributions to the etiopathogenesis of selected common neurological diseases.
39
Zhao, Zhihua, Hanshuo Zhang, Tuanlin Xiong, Junyi Wang, Di Yang, Dan Zhu, Juan Li et al. "Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair". International Journal of Molecular Sciences 21, n. 16 (13 agosto 2020): 5821. http://dx.doi.org/10.3390/ijms21165821.
Testo completoAbstract (sommario):
Homologous recombination (HR) is often used to achieve targeted gene integration because of its higher precision and operability compared with microhomology-mediated end-joining (MMEJ) or non-homologous end-joining (NHEJ). It appears to be inefficient for gene integration in animal cells and embryos due to occurring only during cell division. Here we developed genome-wide high-throughput screening and a subsequently paired crRNA library screening to search for genes suppressing homology-directed repair (HDR). We found that, in the reporter system, HDR cells with knockdown of SHROOM1 were enriched as much as 4.7-fold than those with control. Down regulating SHROOM1 significantly promoted gene integration in human and mouse cells after cleavage by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9), regardless of the donor types. The knock-in efficiency of mouse embryos could also be doubled by the application of SHROOM1 siRNA during micro-injection. The increased HDR efficiency of SHROOM1 deletion in HEK293T cells could be counteracted by YU238259, an HDR inhibitor, but not by an NHEJ inhibitor. These results indicated that SHROOM1 was an HDR-suppressed gene and that the SHROOM1 knockdown strategy may be useful for a variety of applications, including gene editing to generate cell lines and animal models for studying gene function and human diseases.
40
Liu, Yu-Chang, Chih-Hao Huang e Ching-Chun Chang. "A Transcriptomic Analysis of Tobacco Leaf with the Functional Loss of the Plastid rpoB Operon Caused by TALEN-Mediated Double-Strand Breakage". Plants 11, n. 21 (26 ottobre 2022): 2860. http://dx.doi.org/10.3390/plants11212860.
Testo completoAbstract (sommario):
At least two sets of RNA polymerase (RNAP), nucleus (NEP)- and plastid (PEP)-encoded polymerases, recognizing distinct promoters exist in the plastids of land plants. Most plastid genes are regulated by multiple promoters with different strengths in their response to developmental stages and environmental cues. Recently, we applied chloroplast-targeted transcription activator-like effector nuclease (cpTALEN) technology to site-specifically cause double-strand DNA breaks in the rpoB gene of tobacco, which encodes the β-subunit of PEP. The repair of damaged chloroplast DNA (cpDNA) through microhomology-mediated recombination caused the functional loss of the rpoB operon and resulted in the heterotrophic growth of an albino plant. We conducted a genome-wide analysis of the steady state of gene expression in the leaf tissue of PEP-deficient tobacco by RNA-Seq and compared it with that of wild-type plants. The expression of NEP genes was up-regulated in PEP-deficient tobacco; in particular, the level of RpoT3 transcripts encoding the specifically plastid-targeted NEP was significantly increased. Alongside most housekeeping genes, NEP also plays an important role in the regulation of gene expression involved in photosynthesis. In contrast, alongside the photosynthesis-related genes, PEP also plays an important role in the regulation of gene expression involved in some housekeeping functions. Furthermore, the mitochondrial DNA copy number and the level of most mitochondrial protein-coding transcripts were slightly increased in PEP-deficient tobacco. The disruption of PEP function not only affected plastid gene expression, but also nuclear and mitochondrial gene expression. This study demonstrated the intercompartmental retrograde signaling in the regulation of gene expression.
41
Anand, Roopesh, Erika Buechelmaier, Ondrej Belan, Matthew Newton, Aleksandra Vancevska, Artur Kaczmarczyk, Tohru Takaki, David S. Rueda, Simon N. Powell e Simon J. Boulton. "HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51". Nature 601, n. 7892 (22 dicembre 2021): 268–73. http://dx.doi.org/10.1038/s41586-021-04261-0.
Testo completoAbstract (sommario):
AbstractDNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2–4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.
42
Alexander, Jessica L., Kelly Beagan, Terry L. Orr-Weaver e Mitch McVey. "Multiple mechanisms contribute to double-strand break repair at rereplication forks inDrosophilafollicle cells". Proceedings of the National Academy of Sciences 113, n. 48 (14 novembre 2016): 13809–14. http://dx.doi.org/10.1073/pnas.1617110113.
Testo completoAbstract (sommario):
Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. However, the primary mechanism used to repair rereplication DSBs varies across different experimental systems. InDrosophilafollicle cells, developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. We have exploited this system to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. Here we show that a null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase θ/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants,mus308displays normal origin firing but reduced fork progression at two regions of rereplication. We also find that MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, we show that fork progression is enhanced in the absence of bothDrosophilaRad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, our findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context.
43
Tsuji, Hideo, Hiroko Ishii-Ohba, Takanori Katsube, Hideki Ukai, Shiro Aizawa, Masahiro Doi, Kyoji Hioki e Toshiaki Ogiu. "Involvement of Illegitimate V(D)J Recombination or Microhomology-Mediated Nonhomologous End-Joining in the Formation of Intragenic Deletions of the Notch1 Gene in Mouse Thymic Lymphomas". Cancer Research 64, n. 24 (15 dicembre 2004): 8882–90. http://dx.doi.org/10.1158/0008-5472.can-03-1163.
Testo completo
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Haberland, Vivien M. M., Simon Magin, George Iliakis e Andrea Hartwig. "Impact of Manganese and Chromate on Specific DNA Double-Strand Break Repair Pathways". International Journal of Molecular Sciences 24, n. 12 (20 giugno 2023): 10392. http://dx.doi.org/10.3390/ijms241210392.
Testo completoAbstract (sommario):
Manganese is an essential trace element; nevertheless, on conditions of overload, it becomes toxic, with neurotoxicity being the main concern. Chromate is a well-known human carcinogen. The underlying mechanisms seem to be oxidative stress as well as direct DNA damage in the case of chromate, but also interactions with DNA repair systems in both cases. However, the impact of manganese and chromate on DNA double-strand break (DSB) repair pathways is largely unknown. In the present study, we examined the induction of DSB as well as the effect on specific DNA DSB repair mechanisms, namely homologous recombination (HR), non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ). We applied DSB repair pathway-specific reporter cell lines, pulsed field gel electrophoresis as well as gene expression analysis, and investigated the binding of specific DNA repair proteins via immunoflourescence. While manganese did not seem to induce DNA DSB and had no impact on NHEJ and MMEJ, HR and SSA were inhibited. In the case of chromate, the induction of DSB was further supported. Regarding DSB repair, no inhibition was seen in the case of NHEJ and SSA, but HR was diminished and MMEJ was activated in a pronounced manner. The results indicate a specific inhibition of error-free HR by manganese and chromate, with a shift towards error-prone DSB repair mechanisms in both cases. These observations suggest the induction of genomic instability and may explain the microsatellite instability involved in chromate-induced carcinogenicity.
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Jiang, Yuning, e Tarek Abbas. "Abstract 6107: Novel roles for AMBRA1 in regulating DNA double-strand breaks". Cancer Research 83, n. 7_Supplement (4 aprile 2023): 6107. http://dx.doi.org/10.1158/1538-7445.am2023-6107.
Testo completoAbstract (sommario):
Abstract AMBRA1 (Activating Molecule in Beclin-1-Regulated Autophagy) is a tumor suppressor protein whose expression is downregulated in several human malignancies. AMBRA1 was recently found to function as a substrate receptor for the cullin-4-based ubiquitin ligase (CRL4AMBRA1) that promotes the ubiquitin-dependent proteolysis of D-type cyclins (cyclin D1, D2 and D3); this serves to guard against premature S-phase entry and replication stress, providing a potential mechanism for its tumor suppressive activity. Cyclin D1 protein, which is stabilized, along with cyclin D2 and D3, in AMBRA1-deficient cells, has been shown to promote homologous recombination (HR) repair of DNA double-strand breaks (DSBs), suggesting that AMBRA1 may be additionally involved in the repair of DSBs. Using our recently published CRISPR-Cas9-based dual fluorescent reporter assay system, we show that while the loss of AMBRA1 increased D-type cyclins and Rb hyperphosphorylation as expected, it did, surprisingly, inhibit HR-mediated repair and stimulated error-free non-homologous end-joining (NHEJ), and that both of these new activities are independent of cyclin D1 or RB1. High-throughput sequencing of DSB repair junctions confirm these results and shows significant depletion of indels with reduction in microhomology-mediated end-joining in AMBRA1-deficient cells, suggesting that AMBRA1 plays a role in promoting resection at DSBs. In support of this hypothesis, we found that the loss of AMBRA1 reduces RAD51 recruitment to DSBs in cells exposed to ionizing radiation. Consistent with its role in promoting HR, we found that AMBRA1-deficient cells are significantly more sensitive to PARP inhibitors compared to AMBRA1-proficient cells. In summary, we show that AMBRA1 is a novel regulator of DSBs, and that its downregulation in cancer cells and tumors render cancer cells susceptible to inhibition by PARP inhibitors. Citation Format: Yuning Jiang, Tarek Abbas. Novel roles for AMBRA1 in regulating DNA double-strand breaks. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6107.
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Yan, Dan, Yao Zhang, Yan Zhang, Jingxi Zhang, Bin Wang, Huixian Chen, Jingxue Shi, Xiaoling Lin, Jincong Zhuo e Kevin Zhou. "Abstract 4532: Discovery of DAT-1000A, a potent Polθ inhibitor that significantly enhances anti-tumor efficacy in combination with PARP inhibitor in homologous-recombination-deficient tumors". Cancer Research 84, n. 6_Supplement (22 marzo 2024): 4532. http://dx.doi.org/10.1158/1538-7445.am2024-4532.
Testo completoAbstract (sommario):
Abstract Cancers exhibiting homologous recombination (HR) repair deficiencies due to mutations in genes like BRCA1 or BRCA2 rely on alternative DNA damage response (DDR) pathways to uphold genomic integrity. This vulnerability allows for the targeted disruption of these pathways through synthetic lethality approaches. DNA polymerase theta (Polθ, encoded by POLQ) assumes a crucial role in mending DNA double-strand breaks (DSB) via microhomology-mediated end-joining (MMEJ), one of the principal pathways for repairing DNA DSB. Notably, Polθ expression is minimal in normal tissues but becomes upregulated in HR-deficient cancer cells, positioning Polθ as a prime target for synthetic lethality in HRD cancers. Reversion mutations in HR-related genes frequently employ micro-homology deletion mechanisms. Consequently, the inhibition of Polθ holds the potential to thwart the emergence of PARPi resistance, primarily driven by BRCA reversion mutations. In this context, we introduce DAT-1000A, a novel small-molecule Polθ inhibitor exhibiting potent activity with a single-digital nanomolar IC50 value. Its efficacy in MMEJ functional assays validates its on-target cellular activity. Assessments of cell viability in HR-deficient BRCA2-KO DLD-1 cells, compared to their parental counterparts, reveal a striking synthetic lethality window exceeding 500-fold. Additionally, DAT-1000A displays synergistic anti-proliferative effects when combined with PARP inhibitors in BRCA1/2-mutated cells. In BRCA1/2-mutated xenograft models, DAT-1000A demonstrates robust and sustained tumor regression, as evidenced by the level of γH2AX, a common marker for double-strand breaks, which closely correlates with anti-tumor efficacy. Importantly, high-dose DAT-1000A treatment in mice elicits no significant abnormalities, underscoring its excellent tolerability. Collectively, DAT-1000A emerges as a novel Polθ inhibitor, offering both potent activity and a favorable safety profile, and holds promise for advancing therapeutic options in the realm of HRD cancer treatment. Citation Format: Dan Yan, Yao Zhang, Yan Zhang, Jingxi Zhang, Bin Wang, Huixian Chen, Jingxue Shi, Xiaoling Lin, Jincong Zhuo, Kevin Zhou. Discovery of DAT-1000A, a potent Polθ inhibitor that significantly enhances anti-tumor efficacy in combination with PARP inhibitor in homologous-recombination-deficient tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4532.
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Patterson-Fortin, Jeffrey, Heta Jadhav, Constantia Pantelidou, Tin Phan, Carter Grochala, Anita K. Mehta, Jennifer L. Guerriero et al. "Abstract 6190: Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers". Cancer Research 83, n. 7_Supplement (4 aprile 2023): 6190. http://dx.doi.org/10.1158/1538-7445.am2023-6190.
Testo completoAbstract (sommario):
Abstract Cancers deficient in homologous recombination (HR) repair secondary to mutations in genes such as BRCA1 or BRCA2, are dependent on alternative DNA damage response (DDR) pathways to maintain genomic integrity, rendering them susceptible to synthetic lethal targeting of these pathways. Recently, inhibitors of polymerase theta (POLθ, encoded by POLQ), the critical enzyme in microhomology-mediated end-joining (MMEJ), have been shown to be synthetic lethal with HR repair deficiency (Zhou et al. Nature Cancer 2021). Both HR and MMEJ require nucleolytic DNA end-resection to allow for DSB repair, and we have previously shown that MMEJ acts as a barrier to DNA end-resection at DSBs (Patterson-Fortin et al. Cancer Research 2022). Given the synthetic lethality between HR and MMEJ leads to unrestrained DNA end-resection generating chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, we hypothesized that POLθ inhibition in HR-deficient cancers would activate the cGAS/STING innate immune response pathway and facilitate immunotherapy. To investigate the interactions of POLθ inhibition with the immune microenvironment in HR-deficient cancers, we used human cell lines and genetically modified mouse models representative of BRCA1-deficient triple-negative breast cancer (TNBC) and BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). Genetic or pharmacological inhibition of POLθ using novobiocin, a first-in-class inhibitor of the POLθ ATPase domain, induced significantly increased cytosolic dsDNA contained in micronuclei. This free DNA was sensed by the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS), increasing synthesis of cGAMP, in HR-deficient tumor cells but not in HR-proficient tumor cells. Increased cGAMP bound to and activated stimulator of interferon genes (STING), triggering phosphorylation of TBK1 and ultimately of IRF3. Activation of the cGAS/STING pathway by POLθ inhibition drove the expression of type I interferon response elements, including PD-L1. Depletion of STING by siRNA or by CRISPR abrogated this pro-inflammatory signaling and abolished the anti-tumor efficacy of novobiocin-mediated POLθ inhibition. Pharmacologic inhibition of POLθ enhanced Granzyme B+ CD8+ T-cell tumor infiltration. Importantly, antibody-mediated depletion of CD8+ T-cell severely compromised the anti-tumor efficacy of novobiocin-mediated POLθ inhibition, whereas anti-tumor activity of POLθ inhibition was augmented with the addition of either anti-PD-1 or anti-CTLA-4 antibodies. These results demonstrate that POLθ inhibition in HR-deficient cancers mediates a pro-inflammatory response in HR-deficient TNBC or PDAC tumor microenvironments, and that immune checkpoint blockade inhibition enhances the therapeutic efficacy of POLθ inhibition. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Constantia Pantelidou, Tin Phan, Carter Grochala, Anita K. Mehta, Jennifer L. Guerriero, Gerburg M. Wulf, Brian M. Wolpin, Ben Z. Stanger, Andrew J. Aguirre, James M. Cleary, Alan D. D'Andrea, Geoffrey I. Shapiro. Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6190.
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Le, Bac Viet, Umeshkumar Vekariya, Monika Toma, Margaret Nieborowska-Skorska, Marie-Christine Caron, George Vassiliou, Malgorzata Gozdecka et al. "Inactivation of DNA Polymerase Theta (PolΘ) Is Synthetic Lethal in DNMT3A Mutated Myeloid Malignancies - Potential Clinical Applications". Blood 142, Supplement 1 (28 novembre 2023): 580. http://dx.doi.org/10.1182/blood-2023-174333.
Testo completoAbstract (sommario):
Somatic mutations in DNMT3A are associated with unfavorable outcome in patients with AML, MPN and CML. DNMT3A mutations promote resistance to anthracyclines (including daunorubicin, the component of standard “7+3” induction therapy), interferon alpha, and ABL1 kinase inhibitor imatinib. Thus, malignant clones carrying DNMT3A mutations may be difficult to eliminate using standard treatments. AML, MPN and CML cells harbor oncogenic tyrosine kinase (OTK) such as FLT3(ITD), JAK2(V617F) and BCR-ABL1, respectively. We reported before that elevated levels of formaldehyde generated by altered serine/one-carbon cycle metabolism contributed to accumulation of highly lethal DNA double-strand breaks (DSBs) in OTK-positive cells. To protect malignant cells from DSB-induced apoptosis, OTKs regulate DNA damage response (DDR) mechanisms involving DSBs sensing (ATM and ATR kinases) and repairing (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, Polθ-dependent microhomology-mediated end-joining = TMEJ) as well as these activating cell cycle checkpoints (CHK1 and CHK2 kinases). Unfortunately, DNMT3A mutations caused resistance of OTK-positive cells to numerous DDR inhibitors (DDRis). DDRis sensitivity screen and synthetic lethal CRISPR/Cas9 screen revealed that OTK-positive cells with DNMT3A mutations are uniquely sensitive to the inhibition of DNA polymerase theta (Polθ encoded by POLQ gene). This effect was dependent on generation of formaldehyde by serine/one-carbon cycle metabolism. Abrogation of DNMT3A function by CRISPR/Cas9 targeting, Cre-loxP gene deletion, and heterozygous R882H mutation resulted in hypersensitivity of OTK-positive murine AML-like cells and human primary AML cells to Polθ inhibitors (Polθis) due to accumulation of toxic DSBs and activation of cGAS/STING pro-apoptotic pathway. Moreover, simultaneous loss of functional DNMT3A combined with inactivation of Polθ (by CRISPR/Cas9 targeting, insertion of neo-resistance gene, and D2230A+Y2231A polymerase inactive mutant) caused accumulation of DSBs, and reduced OTK-driven clonogenic potential and leukemogenic activity in mice. Polθ is abundantly overexpressed in OTK-positive DNMT3A-deficient cells due to enhanced POLQ mRNA stability and elevated translation of Polθ protein but not due to altered POLQ methylation and Polθ protein stability. Polθ is a key element not only in TMEJ of DSBs with limited end-resection, but also in replication fork restart and in single-strand DNA (ssDNA) gap filling. OTK-positive DNMT3A-deficient cells displayed hyperactivity of Polθ-mediated TMEJ and replication fork restart, but not ssDNA gap filling. These effects were accompanied by increased loading of Polθ on DNA damage detected by Polθ foci formation and chromatin extraction. Moreover, DNMT3A deficiency modulates chromatin architecture at DSBs to limit DNA end-resection thus favoring TMEJ over HR. Furthermore, we tested the effectiveness of Polθis combined with FDA approved drugs (quizartinib, etoposide, cytarabine, azacytidine) against FLT3(ITD)-positive DNMT3A-deficient cells (primary patient cells and cell lines) in vitro and in vivo. The combination of Polθis + quizartinib and Polθis + etoposide completely eradicated clonogenic activity of these cells while Polθis + cytarabine and Polθis + azacytidine exerted modest and weak effects, respectively, when compared to individual compound treatments. These drug combinations were only modestly toxic to normal bone marrow cells. Treatment with Polθi or etoposide reduced the percentage of GFP+ FLT3(ITD)-positive DNMT3A-deficient leukemia cells in peripheral blood of the mice by ~2-fold and prolonged survival time by ~1.5-fold. Remarkably, the combination of Polθi and etoposide eradicated leukemia cells below detectable levels in 6/12 mice with no visible toxicity. Median survival time of the mice will be recorded. Altogether, we discovered that Polθ protects OTK-positive DNMT3A-deficient myeloid malignant cells from the toxic effects of DSBs and identified Polθ as a novel therapeutic target.
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Samnotra, Vivek, Veronica Moroz, Luda Shtessel, Mita Kuchimanchi, Patrick Hanafin, Malar Pannirselvam, Aishwarya Bhaskar et al. "First-in-human, phase 1/2 study of GSK4524101, an oral DNApolymerase theta inhibitor (POLQi), alone or combined with the poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) niraparib in adults with solid tumors." Journal of Clinical Oncology 42, n. 16_suppl (1 giugno 2024): TPS3174. http://dx.doi.org/10.1200/jco.2024.42.16_suppl.tps3174.
Testo completoAbstract (sommario):
TPS3174 Background: Double-stranded DNA breaks (DSBs) in human cells are typically repaired via nonhomologous end joining (NHEJ) or homologous recombination (HR). Deficiency of HR-mediated DNA repair plays a role in the initiation and progression of many tumor types. In tumors with HR deficiency, PARP inhibition leads to generation of DSBs that cannot be effectively repaired because of the HR defect, resulting in synthetic lethality. This finding has led to the clinical development and approval of several PARPi for the treatment of various tumors including certain ovarian, breast, prostate, and pancreatic cancers that are prone to display HR deficiency (HRd). DNA polymerase theta (encoded by POLQ) mediates an alternative DNA repair mechanism, microhomology-mediated end joining (MMEJ). DNA polymerase theta is generally not detectable in normal tissues but is upregulated in many tumor types. In preclinical studies, POLQi plus PARPi treatment demonstrated superior efficacy to PARPi alone in preventing the growth of HRd tumors. To evaluate the clinical potential of combining POLQi and PARPi, this first-in-human study investigates treatment with GSK4524101, an investigational POLQi, with or without the PARPi niraparib, in patients with solid tumors. Methods: This open-label, multicenter, phase 1/2 study (NCT06077877) opened in October 2023 and comprises dose-finding (part 1, including a food-effect cohort) and dose-expansion (part 2) parts. This trial aims to assess the maximum-tolerated dose, pharmacokinetics, safety, and preliminary antitumor activity of oral GSK4524101 with or without niraparib. The primary endpoints are safety (part 1) and confirmed objective response rate (part 2). Secondary endpoints include pharmacokinetics, safety, progression-free survival (part 2), and response duration (part 2). Up to 135 patients may be enrolled. To be eligible, patients must be aged ≥18 years; have an advanced or metastatic solid tumor, an Eastern Cooperative Oncology Group performance status score of 0–2, and a life expectancy of ≥3 months; and have exhausted all standard treatment options. Individuals are ineligible if they have not recovered from chemotherapy-associated adverse events or have symptomatic uncontrolled brain or leptomeningeal metastases, a history of myelodysplastic syndrome or acute myeloid leukemia, uncontrolled hypertension, or a second malignancy that has progressed or required active treatment in the previous 2 years. The study is actively recruiting in the US and Canada; as of January 1, 2024, 1 patient has been dosed. Patients enrolled in part 1 will receive GSK4524101 alone or GSK4524101 plus niraparib; patients enrolled in part 2 will be randomized to either GSK4524101 plus niraparib or niraparib alone. Part 1 of this study is expected to complete in 2025. Data presented on behalf of the original authors with their permission. Presented at the American Association for Cancer Research (AACR) Annual Meeting 2024; April 5-10, 2024; San Diego, CA. Final publication number: 9686. Reused with permission. Clinical trial information: NCT06077877 .
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Toma, Monika, Margaret Nieborowska-Skorska, Adam Karami, Monika Pepek, Tomasz Stoklosa e Tomasz Skorski. "Clonal Targeting of DNA Damage Response Pathways Eradicates Myeloproliferative Neoplasms". Blood 142, Supplement 1 (28 novembre 2023): 120. http://dx.doi.org/10.1182/blood-2023-174280.
Testo completoAbstract (sommario):
Clonal diversity of myeloproliferative neoplasms (MPN) plays a key role in poor therapeutic outcomes. MPN cells usually accumulate spontaneous DNA damage including highly toxic DNA double-strand breaks (DSBs) induced by metabolic products and replication stress. To repair numerous DSBs and survive, MPN cells activate the DNA damage response (DDR) involving the pathways that sense (ATM and ATR kinases) and repair (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, PARP1/Polq-dependent microhomology-mediated end-joining = MMEJ) DSBs. Thus, DDR is a legitimate therapeutic target. Numerous MPN-driving mutations [ JAK2(V617F), TETmut, DNMT3Amut, IDH1mut] regulate DDR and affect the sensitivity of leukemia cells to DDR inhibitors (DDRi). The genetic landscape of malignant clones in a patient may be complicated since individual clones can carry multiple mutations. Thus, individual MPN clones may respond differently to DDRi depending on their mutational profile. In our experimental protocol MPN cells from individual patients were treated with various DDRi followed by single-cell targeted DNA sequencing (sctDNA-seq) to integrate patient's leukemia clonal composition with response to the inhibitors. We developed a sctDNA-seq myeloid platform interrogating up to 1394 genetic variants of 54 known leukemia driver genes, which unraveled the clonal landscape of MPN at a single-cell resolution before and after the treatment. Based on these results we designed a “clonal attack”, a patient-tailored combination of DDRi targeting all MPN clones in a patient sample. Here we show that DDRi simultaneously attacking different clones caused MPN clonal attrition in vitro. For example, phylogenetic tree analysis of MPN patient P349 sample detected linear multi-clonal architecture with 3 Lin-CD34+ clones carrying specific sets of mutations. sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. The clone carrying KMT2A(L2373H) + SETBP1(H1100R) was more sensitive to ATRi than RAD52i, conversely clones with KMT2A(L2373H) alone and KMT2A(L2373H) + SETBP1(H1100R) + FLT3(R834L) were more sensitive to RAD52i than ATRi. Based on this observation, we hypothesized that simultaneous treatment with ATRi+RAD52i should result in elimination of all 3 MPN P349 clones. Remarkably, the combination of RAD52i + ATRi was >100x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells (all clonogenic cells were eradicated). On the other hand, combination of RAD52i + ATMi, the two DDR inhibitors displaying similar pattern of clonal targeting was only 2x better than individual inhibitors. Phylogenetic tree analysis of Lin-CD34+ cells from another MPN patient P350 sample showed linear multi-clonal architecture with 4 clones. Again, sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. All 4 clones displayed similar sensitivity to RAD52i and ATMi, but they responded differently to PARPi and ATRi. Clones carrying TET2(P363L) + NRAS(G12D) were more sensitive to ATRi than PARPi, whereas clones with TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) and TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) + IDH2(R132C) responded better to PARPi than ATRi. Importantly, the combination of PARPi + ATRi was >9x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells, whereas RAD52i + ATMi was only 2x better than individual inhibitors. In conclusion, we postulate that sctDNA-seq combined with in vitro DDRi sensitivity testing (sctDNA-seq/DDRi) is a powerful tool to interrogate clonal sensitivity of MPN to these agents. This clonal medicine approach may become a novel therapeutic regimen to overcome clonal complexity of MPN in a cohort of patients. The “clonal attack” by DDR inhibitors shifts the paradigm of genotoxic therapies from those using non-discriminative cytotoxic drugs to those selectively attacking DDR vulnerabilities in MPN clones with minimal harm to normal cells. Since clonal heterogeneity and DNA damage are hallmarks of cancer, the “clonal attack” may be broadly applicable to the quest for cancer cure.