Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9

Behrmann, Lena; McComb, Scott; Aguadé-Gorgorió, Júlia; Huang, Yun; Hermann, Mario; Pelczar, Pawel; Aguzzi, Adriano; Bourquin, Jean‐Pierre; Bornhäuser, Beat

doi:10.21769/bioprotoc.2222

Cited by 2 publications

(3 citation statements)

References 26 publications

(4 reference statements)

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“…To dissect the overlapping roles of the downstream effector caspase proteins in executing apoptotic cell death, we generated effector caspase-deficient leukemic cell lines using our multicolor LC approach as we have previously described ( 21 , 25 ). We first transduced the acute lymphoblastic leukemia cell lines NALM6, 658w, and Jurkat cells with LC–enhanced green fluorescent protein targeting caspase-7, LC-mCherry targeting caspase-3, and LC-RFP657 targeting caspase-6 (fig.…”

Section: Resultsmentioning

confidence: 99%

Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7

et al. 2019

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Apoptosis is a complex multi-step process driven by caspase-dependent proteolytic cleavage cascades. Dysregulation of apoptosis promotes tumorigenesis and limits the efficacy of chemotherapy. To assess the complex interactions among caspases during apoptosis, we disrupted caspase-8, -9, -3, -7, or -6 and combinations thereof, using CRISPR-based genome editing in living human leukemia cells. While loss of apical initiator caspase-8 or -9 partially blocked extrinsic or intrinsic apoptosis, respectively, only combined loss of caspase-3 and -7 fully inhibited both apoptotic pathways, with no discernible effect of caspase-6 deficiency alone or in combination. Caspase-3/7 double knockout cells exhibited almost complete inhibition of caspase-8 or -9 activation. Furthermore, deletion of caspase-3 and -7 decreased mitochondrial depolarization and cytochrome c release upon apoptosis activation. Thus, activation of effector caspase-3 or -7 sets off explosive feedback amplification of upstream apoptotic events, which is a key feature of apoptotic signaling essential for efficient apoptotic cell death.

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Section: Resultsmentioning

confidence: 99%

Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7

et al. 2019

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“…Multicolor lentiCRISPR constructs were generated as previously published 3,17 from the lentiCRISPR v1 plasmid 18 (Addgene; cat. no.…”

Section: Generation Of Lenticrispr Constructsmentioning

confidence: 99%

“…Lentivirus was produced, as described previously, 17 by transfecting 293T cells with psPAX2 (Addgene; cat. no.…”

Section: Lentivirus Production and Transductionmentioning

confidence: 99%

TNFR2 is required for RIP1-dependent cell death in human leukemia

et al. 2020

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Despite major advances in the treatment of patients with acute lymphoblastic leukemia in the last decades, refractory and/or relapsed disease remains a clinical challenge, and relapsed leukemia patients have an exceedingly dismal prognosis. Dysregulation of apoptotic cell death pathways is a leading cause of drug resistance; thus, alternative cell death mechanisms, such as necroptosis, represent an appealing target for the treatment of high-risk malignancies. We and other investigators have shown that activation of receptor interacting protein kinase 1 (RIP1)–dependent apoptosis and necroptosis by second mitochondria derived activator of caspase mimetics (SMs) is an attractive antileukemic strategy not currently exploited by standard chemotherapy. However, the underlying molecular mechanisms that determine sensitivity to SMs have remained elusive. We show that tumor necrosis factor receptor 2 (TNFR2) messenger RNA expression correlates with sensitivity to SMs in primary human leukemia. Functional genetic experiments using clustered regularly interspaced short palindromic repeats/Cas9 demonstrate that TNFR2 and TNFR1, but not the ligand TNF-α, are essential for the response to SMs, revealing a ligand-independent interplay between TNFR1 and TNFR2 in the induction of RIP1-dependent cell death. Further potential TNFR ligands, such as lymphotoxins, were not required for SM sensitivity. Instead, TNFR2 promotes the formation of a RIP1/TNFR1-containing death signaling complex that induces RIP1 phosphorylation and RIP1-dependent apoptosis and necroptosis. Our data reveal an alternative paradigm for TNFR2 function in cell death signaling and provide a rationale to develop strategies for the identification of leukemias with vulnerability to RIP1-dependent cell death for tailored therapeutic interventions.

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Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9

Cited by 2 publications

References 26 publications

Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7

Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7

TNFR2 is required for RIP1-dependent cell death in human leukemia

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