An Inhibitor of Nonhomologous End-Joining Abrogates Double-Strand Break Repair and Impedes Cancer Progression

Srivastava, Mrinal; Nambiar, Mridula; Sharma, Sheetal; Karki, Subhas S.; Goldsmith, Gunaseelan; Hegde, Mahesh; Kumar, Sujeet; Pandey, Monica; Singh, Ram Kumar; Ray, Pritha; Natarajan, Renuka; Kelkar, Madhura; De, Abhijit; Choudhary, Bibha; Raghavan, Sathees C.

doi:10.1016/j.cell.2012.11.054

Cited by 334 publications

(415 citation statements)

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“…Three repair inhibitors were used: two of them modify NHEJ (NU7026 and SCR7) [39][40][41] and IR-1 disrupts HR (supplemental Materials and methods). 42 To investigate the effect of the combination treatment of melphalan with an inhibitor, we first selected the optimal concentrations of the inhibitors that, when administered alone, showed minimal decrease in cell viability.…”

Section: Resultsmentioning

confidence: 99%

DNA repair of myeloma plasma cells correlates with clinical outcome: the effect of the nonhomologous end-joining inhibitor SCR7

Γκοτζαμανίδου

Terpos

Bamia

et al. 2016

Blood

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Key Points• Responders to melphalan therapy are characterized by slower rates of NER and DSB/R mechanisms and higher apoptotic rates.• The DSB/R inhibitor SCR7 enhances cytotoxicity of melphalan against myeloma plasma cells.DNA repair activity of malignant cells seems to influence therapeutic outcome and patients' survival. Herein, we investigated the mechanistic basis for the link between DNA repair efficiency and response to antimyeloma therapy. Nucleotide excision repair (NER), interstrand cross-links repair (ICL/R), double-strand breaks repair (DSB/R), and chromatin structure were evaluated in multiple myeloma (MM) cell lines (melphalan-sensitive RPMI8226; melphalan-resistant LR5) and bone marrow plasma cells (BMPCs) from MM patients who responded (n 5 17) or did not respond (n 5 9) to subsequent melphalan therapy. The effect of DSB/R inhibition was also evaluated. Responders' BMPCs showed slower rates of NER and DSB/R (P < .0022), similar rates of ICL/R, and more condensed chromatin structure compared with nonresponders. Moreover, apoptosis rates of BMPCs were inversely correlated with individual DNA repair efficiency and were higher in responders' cells compared with those of nonresponders (P 5 .0011). Similarly, RPMI8226 cells showed slower rates of NER and DSB/R, comparable rates of ICL/R, more condensed chromatin structure, and higher sensitivity than LR5 cells. Interestingly, cotreatment of BMPCs or cell lines with DSB/R inhibitors significantly reduced the rates of DSB/R and increased melphalan sensitivity of the cells, with the nonhomologous end-joining inhibitor SCR7 showing the strongest effect. Together, responders' BMPCs are characterized by lower efficiencies of NER and DSB/R mechanisms, resulting in higher accumulation of the extremely cytotoxic ICLs and DSBs lesions, which in turn triggers the induction of the apoptotic pathway. Moreover, the enhancement of melphalan cytotoxicity by DSB/R inhibition offers a promising strategy toward improvement of existing antimyeloma regimens. (Blood. 2016;128(9):1214-1225

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Section: Resultsmentioning

confidence: 99%

DNA repair of myeloma plasma cells correlates with clinical outcome: the effect of the nonhomologous end-joining inhibitor SCR7

Γκοτζαμανίδου

Terpos

Bamia

et al. 2016

Blood

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“…In mice, Maruyama et al [14] have shown that the frequency of HDR is improved by microinjection of a DNA ligase IV inhibitor, SCR7, into mouse zygotes with sgRNA/ Cas9/ssODN [15]. Another way to improve the efficiency is enhancement of the HDR pathway.…”

Section: Hdr-mediated Production Of Nucleotide Substitution In Micementioning

confidence: 99%

Genome editing for the reproduction and remedy of human diseases in mice

Hara

Takada

2017

J Hum Genet

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With the recent progress in genome-editing technologies, such as the CRISPR/Cas9 system, genetically modified animals carrying nucleotide substitutions or large chromosomal rearrangements can be produced rapidly and at low cost. Such genome-editing techniques have been applied in the generation of animal models, especially mice, for reproducing human disease mutations, such as single-nucleotide polymorphisms (SNPs) or large chromosomal rearrangements identified by genome-wide screening analyses. While application methods are under development for various complex mutations involving genome editing for mimicking human disease-causing mutations in mice, functional studies of mouse models carrying replicated human mutations are gradually being published. In this review, we discuss the recent progress in application methods of the CRISPR/Cas9 system, focusing on the production of mouse models of diseases.

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“…2A and detailed in Methods), NHEJ proteins were incubated with linearized plasmid dsDNA, allowing assembly of NHEJ proteins on the DNA, facilitating NHEJ and formation of intermediates in that pathway. This reaction was performed in the absence or presence of the LigIV-specific inhibitor SCR7 (43). A cross-linking reagent [4% (wt/vol) paraformaldehyde (PFA)] was then added to the reactions to preserve structural intermediates.…”

Section: Sr Imagingmentioning

confidence: 99%

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Reid

Keegan

Leo‐Macias

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

157

168

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Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by singlemolecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.genomic integrity | DNA repair | nonhomologous end-joining | super-resolution microscopy | single-molecule FRET C hromosomal double-strand breaks (DSBs), considered the most cytotoxic form of DNA damage, occur as a result of normal cellular processes (1, 2) as well as cancer therapies (3-5). The cellular DNA damage response (DDR) and repair pathways responsible for maintaining genomic integrity are highly regulated and synchronized processes, both temporally and spatially, involving the coordinated recruitment, assembly, and disassembly of numerous macromolecular complexes (6, 7). In mammalian cells, nonhomologous end-joining (NHEJ) is the primary DSB repair pathway; it is active throughout the cell cycle and is crucial for viability. Dysfunctional NHEJ is associated with several clinical conditions, including LIG4 syndrome and severe combined immunodeficiency (1,8). Despite its importance, however, the details of how the NHEJ complex assembles at DSBs, brings together a pair of breaks, and organizes subsequent catalytic repair steps remain unknown.In NHEJ, DSBs are initially recognized by the Ku 70/80 heterodimer (Ku), which encircles dsDNA ends (Ku:DNA) and serves as a molecular scaffold for recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4 like factor), and DNA ligase IV (LigIV) (1,(9)(10)(11)(12)(13)(14). Previous NHEJ models suggested that after binding of Ku to DNA ends, DNA-PKcs binds Ku:DNA to form a DNA-PK holoenzyme and bridges the broken ends (15-18); however, recent experiments indicate that DNAPKcs may have different roles in NHEJ, such as the stabilization of core NHEJ factors, recruitment and retention of accessory factors, involvement in the DDR signaling cascade, and repair of complex and clustered . In addition, recent structural studies have shown that XRCC4 and XLF form filamentous structures in vitro (26-28). Whether such filaments mediate repair in vivo has not yet been determined.Our present understanding of the cellular NHEJ response to DSBs ...

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An Inhibitor of Nonhomologous End-Joining Abrogates Double-Strand Break Repair and Impedes Cancer Progression

Cited by 334 publications

References 56 publications

DNA repair of myeloma plasma cells correlates with clinical outcome: the effect of the nonhomologous end-joining inhibitor SCR7

DNA repair of myeloma plasma cells correlates with clinical outcome: the effect of the nonhomologous end-joining inhibitor SCR7

Genome editing for the reproduction and remedy of human diseases in mice

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

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