A versatile modular vector system for rapid combinatorial mammalian genetics

Albers, Joachim; Danzer, Claudia; Rechsteiner, Markus; Lehmann, Holger; Brandt, Laura P; Hejhal, Tomáš; Catalano, Antonella; Busenhart, Philipp; Gonçalves, Ana Filipa; Brandt, Simone; Bode, P.; Bode-Lesniewska, Beata; Wild, Peter J.; Frew, Ian J.

doi:10.1172/jci79743

Cited by 66 publications

(87 citation statements)

References 60 publications

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“…HA-HIF-1α (28), HRE-Luc (29), ODD-Luc (30), CMV-Luc, and SV40-Luc (31) constructs were purchased from Addgene. The ODD-Luc (P564A) mutant was created using a Quikchange site directed mutagenesis kit (Agilent).…”

Section: Methodsmentioning

confidence: 99%

Hypoxia-Inducible PIM Kinase Expression Promotes Resistance to Antiangiogenic Agents

Casillas

Toth

Sainz

et al. 2018

Clinical Cancer Research

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Purpose: Patients develop resistance to anti-angiogenic drugs, secondary to changes in the tumor microenvironment, including hypoxia. PIM kinases are pro-survival kinases and their expression increases in hypoxia. The goal of this study was to determine whether targeting hypoxia-induced PIM kinase expression is effective in combination with VEGF-targeting agents. The rationale for this therapeutic approach is based on the fact that anti-angiogenic drugs can make tumors hypoxic, and thus more sensitive to PIM inhibitors. Experimental Design: Xenograft and orthotopic models of prostate and colon cancer were used to assess the effect of PIM activation on the efficacy of VEGF-targeting agents. Immunohistochemistry and in vivo imaging were used to analyze angiogenesis, apoptosis, proliferation, and metastasis. Biochemical studies were performed to characterize the novel signaling pathway linking PIM and HIF-1. Results: PIM was upregulated following treatment with anti-VEGF therapies, and PIM1 overexpression reduced the ability of these drugs to disrupt vasculature and block tumor growth. PIM inhibitors reduced HIF-1 activity, opposing the shift to a pro-angiogenic gene signature associated with hypoxia. Combined inhibition of PIM and VEGF produced a synergistic antitumor response characterized by decreased proliferation, reduced tumor vasculature, and decreased metastasis. Conclusions: This study describes PIM kinase expression as a novel mechanism of resistance to anti-angiogenic agents. Our data provide justification for combining PIM and VEGF inhibitors to treat solid tumors. The unique ability of PIM inhibitors to concomitantly target HIF-1 and selectively kill hypoxic tumor cells addresses two major components of tumor progression and therapeutic resistance.

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Section: Methodsmentioning

confidence: 99%

Hypoxia-Inducible PIM Kinase Expression Promotes Resistance to Antiangiogenic Agents

Casillas

Toth

Sainz

et al. 2018

Clinical Cancer Research

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“…Recently, CRISPR-Cas9 has been used to knock out tumor suppressor genes cyclin-dependent kinase inhibitor 2A ( CDKN2A ), P53 , and PTEN in mice to establish three murine sarcoma models [2]. First, scientists developed a multiple lentiviral expression (MuLE) viral system which allows multiple sgRNAs to be expressed together with hCas9 from a single viral construct expression [2].…”

Section: Potential Applications Of Crispr-cas9 In Sarcomamentioning

confidence: 99%

Section: Potential Applications Of Crispr-cas9 In Sarcomamentioning

confidence: 99%

“…First, scientists developed a multiple lentiviral expression (MuLE) viral system which allows multiple sgRNAs to be expressed together with hCas9 from a single viral construct expression [2]. To model human sarcoma, a series of MuLE vectors were designed to systematically investigate the single and combinatorial effects of gain of H-RAS function and loss of CDKN2A , P53 , and PTEN functions [2]. To investigate the single effects of the driven gene, researchers generated tricistronic MuLE vectors expressing sgRNAs targeting exon 7 or exon 8 of the P53 locus or exon 2 of the CDKN2A locus together with expression of hCas9 and puromycin resistance [2].…”

Section: Potential Applications Of Crispr-cas9 In Sarcomamentioning

confidence: 99%

“…To model human sarcoma, a series of MuLE vectors were designed to systematically investigate the single and combinatorial effects of gain of H-RAS function and loss of CDKN2A , P53 , and PTEN functions [2]. To investigate the single effects of the driven gene, researchers generated tricistronic MuLE vectors expressing sgRNAs targeting exon 7 or exon 8 of the P53 locus or exon 2 of the CDKN2A locus together with expression of hCas9 and puromycin resistance [2]. To demonstrate that cooperative genetic tumor modeling can be achieved using CRISPR-Cas9, they generated tetracistronic MuLE vectors designed to express either scrambled sgRNA or sgRNA targeting P53 exon 7 or exon 8, as well as H-RAS G12V , hCas9 , and puromycin resistance[2].…”

Section: Potential Applications Of Crispr-cas9 In Sarcomamentioning

confidence: 99%

See 2 more Smart Citations

Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma

Liu

Shen

et al. 2016

Cancer Letters

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Sarcomas include some of the most aggressive tumors and typically respond poorly to chemotherapy. In recent years, specific gene fusion/mutations and gene over-expression/activation have been shown to drive sarcoma pathogenesis and development. These emerging genomic alterations may provide targets for novel therapeutic strategies and have the potential to transform sarcoma patient care. The RNA-guided nuclease CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein-9 nuclease) is a convenient and versatile platform for site-specific genome editing and epigenome targeted modulation. Given that sarcoma is believed to develop as a result of genetic alterations in mesenchymal progenitor/stem cells, CRISPR-Cas9 genome editing technologies hold extensive application potentials in sarcoma models and therapies. We review the development and mechanisms of the CRISPR-Cas9 system in genome editing and introduce its application in sarcoma research and potential therapy in clinic. Additionally, we propose future directions and discuss the challenges faced with these applications, providing concise and enlightening information for readers interested in this area.

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Tonabersat enhances temozolomide‐mediated cytotoxicity in glioblastoma by disrupting intercellular connectivity through connexin 43 inhibition

Schmidt,

Evert,

Pregler

et al. 2024

Molecular Oncology

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Glioblastoma cells rely on connexin 43 (Cx43)‐based gap junctions (GJs) for intercellular communication, enabling them to integrate into a widely branched malignant network. Although there are promising prospects for new targeted therapies, the lack of clinically feasible GJ inhibitors has impeded their adoption in clinical practice. In the present study, we investigated tonabersat (TO), a blood–brain‐barrier‐penetrating drug with GJ‐inhibitory properties, in regard to its potential to disassemble intercellular connectivity in glioblastoma networks. Fluorescence‐guided measurements of calcein cell‐to‐cell transfer were used to study functional intercellular connectivity. Specific DNA fragmentation rates of propidium iodide‐stained nuclei were measured as a surrogate readout for cell death using flow cytometry. CRISPR/Cas9‐mediated gene editing of Cx43 served as a validation tool of cellular effects related to Cx43 GJ inhibition. 3′ mRNA sequencing was performed for molecular downstream analysis. We found that TO reduced intercellular GJ‐mediated cytosolic traffic and yielded a significant reduction of tumor microtube (TM) length. TO‐mediated inhibition of cellular tumor networks was accompanied by a synergistic effect for temozolomide‐induced cell death. CRISPR/Cas9 Cx43‐knockout revealed similar results, indicating that TO‐mediated inhibitory effects rely on the inhibition of Cx43‐based GJs. Gene set enrichment analyses found that GJ‐mediated synergistic cytotoxic effects were linked to a significant upregulation of cell death signaling pathways. In conclusion, TO disrupts TM‐based network connectivity via GJ inhibition and renders glioblastoma cells more susceptible to cytotoxic therapy. Given its previous use in clinical trials for migraine therapy, TO might harbor the potential of bridging the idea of a GJ‐targeted therapeutic approach from bench to bedside.

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A versatile modular vector system for rapid combinatorial mammalian genetics

Cited by 66 publications

References 60 publications

Hypoxia-Inducible PIM Kinase Expression Promotes Resistance to Antiangiogenic Agents

Hypoxia-Inducible PIM Kinase Expression Promotes Resistance to Antiangiogenic Agents

Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma

Tonabersat enhances temozolomide‐mediated cytotoxicity in glioblastoma by disrupting intercellular connectivity through connexin 43 inhibition

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