CRISPR-Cas9–based target validation for p53-reactivating model compounds

Wanzel, Michael; Vischedyk, Jonas B.; Gittler, M.; Gremke, Niklas; Seiz, Julia R.; Hefter, Mirjam; Noack, Magdalena; Savai, Rajkumar; Mernberger, Marco; Charles, Joël P.; Schneikert, Jean; Bretz, Anne Catherine; Nist, Andrea; Stiewe, Thorsten

doi:10.1038/nchembio.1965

Cited by 78 publications

(77 citation statements)

References 50 publications

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“…Further analysis showed RITA-dependent activation of p38 and the JNK/SAPK pathway in p53-null cells that resulted in mitochondrial apoptosis [25]. RITA has also been shown to be effective in a multiple myeloma cells independently of p53 [79,80] and in cell types expressing mutant p53 protein [81]. These results are in line with our observations that RITA can induce architectural remodeling independently of p53 status in cancer cells [82].…”

Section: Discussionsupporting

confidence: 86%

“…p53 is highly sensitive to post-translational modifications which can profoundly modulate the specific activities of this gene [83]. It is also known that conformational changes in p53 can affect DNA binding and transactivation activity, which is often seen in tumors expressing mutant forms of p53 [81]. The differential effects of RITA-stabilized p53 and Nutlin-3-stabilized p53 have been demonstrated directly through differences in gene expression profiles between the two, with RITA-bound p53 showing a preference for transcriptional programs leading to apoptosis rather than growth arrest [24,83,84].…”

Section: Discussionmentioning

confidence: 99%

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p53 CRISPR Deletion Affects DNA Structure and Nuclear Architecture

Rangel‐Pozzo

Booth

et al. 2020

JCM

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The TP53 gene is a key tumor suppressor. Although the tumor suppressor p53 was one of the first to be characterized as a transcription factor, with its main function potentiated by its interaction with DNA, there are still many unresolved questions about its mechanism of action. Here, we demonstrate a novel role for p53 in the maintenance of nuclear architecture of cells. Using three-dimensional (3D) imaging and spectral karyotyping, as well as super resolution microscopy of DNA structure, we observe significant differences in 3D telomere signatures, DNA structure and DNA-poor spaces as well gains or losses of chromosomes, between normal and tumor cells with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-deleted or wild-type TP53. Additionally, treatment with Nutlin-3 results in differences in nuclear architecture of telomeres in wild-type but not in p53 knockout MCF-7 (Michigan Cancer Foundation-7) cells. Nutlin-3 binds to the p53-binding pocket of mouse double minute 2 (MDM2) and blocks the p53-MDM2 interaction. Moreover, we demonstrate that another p53 stabilizing small molecule, RITA (reactivation of p53 and induction of tumor cell apoptosis), also induces changes in 3D DNA structure, apparently in a p53 independent manner. These results implicate p53 activity in regulating nuclear organization and, additionally, highlight the divergent effects of the p53 targeting compounds Nutlin-3 and RITA.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

p53 CRISPR Deletion Affects DNA Structure and Nuclear Architecture

Rangel‐Pozzo

Booth

et al. 2020

JCM

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“…Recently, it was reported that DNA damage response also contributes to the cellular response to RITA relative to Nutlin-3a (61). Furthermore, RITA may impair p21 function (47), which could account for the low number of genes downregulated by RITA treatment in our meta-analysis (Supplementary Figure S5A).…”

Section: Resultsmentioning

confidence: 99%

Integration of TP53, DREAM, MMB-FOXM1 and RB-E2F target gene analyses identifies cell cycle gene regulatory networks

et al. 2016

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Cell cycle (CC) and TP53 regulatory networks are frequently deregulated in cancer. While numerous genome-wide studies of TP53 and CC-regulated genes have been performed, significant variation between studies has made it difficult to assess regulation of any given gene of interest. To overcome the limitation of individual studies, we developed a meta-analysis approach to identify high confidence target genes that reflect their frequency of identification in independent datasets. Gene regulatory networks were generated by comparing differential expression of TP53 and CC-regulated genes with chromatin immunoprecipitation studies for TP53, RB1, E2F, DREAM, B-MYB, FOXM1 and MuvB. RNA-seq data from p21-null cells revealed that gene downregulation by TP53 generally requires p21 (CDKN1A). Genes downregulated by TP53 were also identified as CC genes bound by the DREAM complex. The transcription factors RB, E2F1 and E2F7 bind to a subset of DREAM target genes that function in G1/S of the CC while B-MYB, FOXM1 and MuvB control G2/M gene expression. Our approach yields high confidence ranked target gene maps for TP53, DREAM, MMB-FOXM1 and RB-E2F and enables prediction and distinction of CC regulation. A web-based atlas at www.targetgenereg.org enables assessing the regulation of any human gene of interest.

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“…Two p53-MDM2 blockers, RITA (Issaeva et al, 2004) and nutlin (Vassilev et al, 2004), were discovered in 2004. Furthermore, recently Wanzel and coworkers used CRISPR-Cas9 based target validation to reveal the actual mechanisms of two p53-MDM2 blockers (Wanzel et al, 2016). Nutlin was designed to blocks the p53-binding pocket of Mdm2 and RITA was to bind the Mdm2-interacting N terminus of p53, but on the contrary, results showed that the activity of nutlin strictly depends on functional p53 while the sensitivity of RITA correlates with induction of DNA damage but not with p53 (Wanzel et al, 2016).…”

Section: P53mentioning

confidence: 99%

The Drug-Resistance Mechanisms of Five Platinum-Based Antitumor Agents

et al. 2020

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Platinum-based anticancer drugs, including cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin, are heavily applied in chemotherapy regimens. However, the intrinsic or acquired resistance severely limit the clinical application of platinum-based treatment. The underlying mechanisms are incredibly complicated. Multiple transporters participate in the active transport of platinum-based antitumor agents, and the altered expression level, localization, or activity may severely decrease the cellular platinum accumulation. Detoxification components, which are commonly increasing in resistant tumor cells, can efficiently bind to platinum agents and prevent the formation of platinum-DNA adducts, but the adducts production is the determinant step for the cytotoxicity of platinum-based antitumor agents. Even if adequate adducts have formed, tumor cells still manage to survive through increased DNA repair processes or elevated apoptosis threshold. In addition, autophagy has a profound influence on platinum resistance. This review summarizes the critical participators of platinum resistance mechanisms mentioned above and highlights the most potential therapeutic targets or predicted markers. With a deeper understanding of the underlying resistance mechanisms, new solutions would be produced to extend the clinical application of platinum-based antitumor agents largely.

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CRISPR-Cas9–based target validation for p53-reactivating model compounds

Cited by 78 publications

References 50 publications

p53 CRISPR Deletion Affects DNA Structure and Nuclear Architecture

p53 CRISPR Deletion Affects DNA Structure and Nuclear Architecture

Integration of TP53, DREAM, MMB-FOXM1 and RB-E2F target gene analyses identifies cell cycle gene regulatory networks

The Drug-Resistance Mechanisms of Five Platinum-Based Antitumor Agents

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