Direct Targeting of MYCN Gene Amplification by Alkylating Pyrrole-Imidazole Polyamide and CRISPR/Cas9 in Neuroblastoma Cells (CHP-134)
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE121530 | GEO | 2018/12/25
REPOSITORIES: GEO
Similar Datasets
Project description:Amplification of MYCN plays a pivotal role in multiple types of tumors and correlates with poor prognosis in high-risk neuroblastoma. Despite recent advances in the treatment of neuroblastoma, no approaches directly target the master oncogene MYCN. Difficulties in targeting the MYCN protein have inspired us to develop a new gene level inhibitory strategy using a sequence-specific gene regulator. Here we generated a MYCN-targeting pyrrole-imidazole (PI) polyamide, MYCN-A3, which directly binds to and alkylates DNA at homing motifs within MYCN transcript. Pharmacological suppression of MYCN inhibited the proliferation of cancer cells harboring MYCN amplification compared with MYCN non-amplified cancer cells. In neuroblastoma xenograft mouse models, MYCN-A3 specifically downregulated MYCN expression and suppressed tumor progression with no detectable adverse effects and resulted in prolonged overall survival. Moreover, we observed the copy number reduction of MYCN in neuroblastoma cells with MYCN amplification upon treatment with MYCN-A3 but not MYCN non-targeting PI polyamide. Expression microarray experiments were also performed to compare the genome-wide effect of MYCN-A3 with CRISPR/Cas9 silencing of MYCN (MYCNcr-a).
2018-12-25 | GSE121528 | GEO
Project description:Direct Targeting of MYCN Gene Amplification by Alkylating Pyrrole-Imidazole Polyamide in Neuroblastoma Cells (CHP-134)
| PRJNA497578 | ENA
Project description:Direct Targeting of MYCN Gene Amplification by Alkylating Pyrrole-Imidazole Polyamide and CRISPR/Cas9 in Neuroblastoma Cells (CHP-134)
| PRJNA497575 | ENA
Project description:Amplification of MYCN plays a pivotal role in multiple types of tumors and correlates with poor prognosis in high-risk neuroblastoma. Despite recent advances in the treatment of neuroblastoma, no approaches directly target the master oncogene MYCN. Difficulties in targeting the MYCN protein have inspired us to develop a new gene level inhibitory strategy using a sequence-specific gene regulator. Here we generated a MYCN-targeting pyrrole-imidazole (PI) polyamide, MYCN-A3, which directly binds to and alkylates DNA at homing motifs within MYCN transcript. Pharmacological suppression of MYCN inhibited the proliferation of cancer cells harboring MYCN amplification compared with MYCN non-amplified cancer cells. In neuroblastoma xenograft mouse models, MYCN-A3 specifically downregulated MYCN expression and suppressed tumor progression with no detectable adverse effects and resulted in prolonged overall survival. Moreover, we observed the copy number reduction of MYCN in neuroblastoma cells with MYCN amplification upon treatment with MYCN-A3 but not MYCN non-targeting PI polyamide. Expression microarray experiments were also performed to compare the genome-wide effect of MYCN-A3 with CRISPR/Cas9 silencing of MYCN (MYCNcr-a).
2018-12-25 | GSE121529 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2016-11-01 | GSE86599 | GEO
Project description:Pyrrole-imidazole polyamides are versatile DNA minor groove binders and attractive therapeutic options against oncological targets, especially upon functionalization with an alkylating agent such as endo-seco-CBI. These molecules also provide an alternative for oncogenes deemed “undruggable” at the protein level, where the absence of solvent-accessible pockets or structural crevices prevent the formation of protein-inhibitor ligands; nevertheless, the genome-wide effect of pyrrole-imidazole polyamide binding remain largely unclear to-date. Here we propose a next-generation sequencing-based workflow combined with whole genome expression arrays to address such issue using a candidate anti-cancer alkylating agent, KR12, against codon 12 mutant KRAS. Biotinylating KR12 enables the means to identify its genome-wide effects in living cells and possible biological implications via a coupled workflow of enrichment-based sequencing and expression microarrays. The subsequent computational pathway and expression analyses allow the identification of its genomic binding sites, as well as a route to explore a polyamide’s possible genome-wide effects. Among the 3,343 KR12 binding sites identified in the human LS180 colorectal cancer genome, the reduction of KR12-bound gene expressions was also observed. Additionally, the coupled microarray-sequencing analysis also revealed some insights about the effect of local chromatin structure on pyrrole-imidazole polyamide, which had not been fully understood to-date. A comparative analysis with KR12 in a different human colorectal cancer genome SW480 also showed agreeable agreements of KR12 binding affecting gene expressions. Combination of these analyses thus suggested the possibility of applying this approach to other pyrrole-imidazole polyamides to reveal further biological details about the effect of polyamide binding in a genome.
2016-11-01 | GSE86598 | GEO
Project description:Pyrrole-imidazole polyamides are versatile DNA minor groove binders and attractive therapeutic options against oncological targets, especially upon functionalization with an alkylating agent such as endo-seco-CBI. These molecules also provide an alternative for oncogenes deemed “undruggable” at the protein level, where the absence of solvent-accessible pockets or structural crevices prevent the formation of protein-inhibitor ligands; nevertheless, the genome-wide effect of pyrrole-imidazole polyamide binding remain largely unclear to-date. Here we propose a next-generation sequencing-based workflow combined with whole genome expression arrays to address such issue using a candidate anti-cancer alkylating agent, KR12, against codon 12 mutant KRAS. Biotinylating KR12 enables the means to identify its genome-wide effects in living cells and possible biological implications via a coupled workflow of enrichment-based sequencing and expression microarrays. The subsequent computational pathway and expression analyses allow the identification of its genomic binding sites, as well as a route to explore a polyamide’s possible genome-wide effects. Among the 3,343 KR12 binding sites identified in the human LS180 colorectal cancer genome, the reduction of KR12-bound gene expressions was also observed. Additionally, the coupled microarray-sequencing analysis also revealed some insights about the effect of local chromatin structure on pyrrole-imidazole polyamide, which had not been fully understood to-date. A comparative analysis with KR12 in a different human colorectal cancer genome SW480 also showed agreeable agreements of KR12 binding affecting gene expressions. Combination of these analyses thus suggested the possibility of applying this approach to other pyrrole-imidazole polyamides to reveal further biological details about the effect of polyamide binding in a genome.
2016-11-01 | GSE86597 | GEO
Project description:Auger electrons can cause nanoscale physiochemical damage to specific DNA sites that play a key role in cancer cell survival. Radio-Pt is a promising Auger-electron source for damaging DNA efficiently because of its ability to bind to DNA. Considering that the cancer genome is maintained under abnormal gene amplification and expression, here, we developed a novel 191Pt-labeled agent based on pyrrole-imidazole polyamide (PIP), targeting the oncogene MYCN amplified in human neuroblastoma, and investigated its targeting ability and damaging effects. A conjugate of MYCN-targeting PIP and Cys-(Arg)3-coumarin was labeled with 191Pt via Cys (191Pt-MYCN-PIP) with a radiochemical purity of >99%. The binding potential of 191Pt-MYCN-PIP was evaluated via the gel electrophoretic mobility shift assay, suggesting that the radioagent bound to the DNA including the target sequence of the MYCN gene. In vitro assays using human neuroblastoma cells showed that 191Pt-MYCN-PIP bound to DNA efficiently and caused DNA damage, decreasing MYCN gene expression and MYCN signals in in situ hybridization analysis, as well as cell viability, especially in MYCN-amplified Kelly cells. 191Pt-MYCN-PIP also induced a substantial increase in cytosolic dsDNA granules and generated proinflammatory cytokines, IFN-α/β, in Kelly cells. Tumor uptake of intravenously injected 191Pt-MYCN-PIP was low and its delivery to tumors should be improved for therapeutic application. The present results provided a potential strategy, targeting the key oncogenes for cancer survival for Auger electron therapy.
| S-EPMC10675227 | biostudies-literature
Project description:PIK3CA is the most frequently mutated oncogene in cervical cancer, and somatic mutations in the PIK3CA gene result in increased activity of PI3K. In cervical cancer, the E545K mutation in PIK3CA leads to elevated cell proliferation and reduced apoptosis. In the present study, we designed and synthesized a novel pyrrole-imidazole polyamide-seco-CBI conjugate, P3AE5K, to target the PIK3CA gene bearing the E545K mutation, rendered possible by nuclear access and the unique sequence specificity of pyrrole-imidazole polyamides. P3AE5K interacted with double-stranded DNA of the coding region containing the E545K mutation. When compared with conventional PI3K inhibitors, P3AE5K demonstrated strong cytotoxicity in E545K-positive cervical cancer cells at lower concentrations. PIK3CA mutant cells exposed to P3AE5K exhibited reduced expression levels of PIK3CA mRNA and protein, and subsequent apoptotic cell death. Moreover, P3AE5K significantly decreased the tumor growth in mouse xenograft models derived from PIK3CA mutant cells. Overall, the present data strongly suggest that the alkylating pyrrole-imidazole polyamide P3AE5K should be a promising new drug candidate targeting a constitutively activating mutation of PIK3CA in cervical cancer.
| S-EPMC7935806 | biostudies-literature
Project description:Side and off-target effects remain a difficult issue in the search of pharmaceutical leads, especially with DNA-binding molecules or genome editing methods where the issue becomes particularly thorny. A particular case is the investigations into the off-target effects of N-methylpyrrole-N-methylimidazole polyamides, a naturally inspired class of DNA binders that discriminately access the minor groove with strong affinity and sequence specificity but the relatively short motif of < 20 bases often imply possible non-unique genomic binding. Multiple binding sites may translate to binding at non-intended loci, potentially leading to off-target effects, issues that very few approaches are able to address to-date. We here report an analytical method of inferring off-target binding from expression profiling based on the relative impact to various biochemical pathways, as well as an accompanying side effect prediction engine to allow candidate polyamides to be systematically screened. This method marks the first attempt in PI polyamide research to identify elements in various biochemical pathways sensitive to the treatment of a candidate polyamide to infer possible off-target effects. Expression changes were then considered for their possible effect on outward phenotypic changes, manifested as side effects, should the same PI polyamide candidate be administered clinically. We also performed a series of animal experiments to validate some of these effects, and found some corroboration in certain side effect manifestations, such as changes in the level of aspartate transaminase in ICR and nude mice after injection of some of the candidate polyamides in this study.
2019-04-09 | GSE124456 | GEO