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Updated: Dec 11, 2025
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
In vitro analysis reveals necroptotic signaling does not provoke DNA damage or HPRT mutations
Mark A Miles1, Christine J Hawkins2
1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Victoria, Australia. m.miles@latrobe.edu.au.
Abstract:
Most anticancer drugs provoke apoptotic signaling by damaging DNA or other means. Genotoxic therapies may enhance a patient's risk of developing "therapy-related cancers" due to the accumulation of oncogenic mutations that may occur in noncancerous cells. Mutations can also form upon apoptotic signaling due to sublethal caspase activity, implying that apoptosis activating drugs may also be oncogenic. Necroptosis is a different way of killing cancer cells: this version of caspase-independent cell death is characterized by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase-like domain protein (MLKL) activation, leading to cell membrane rupture and controlled cell lysis. The mutagenic potential of sublethal necroptotic signaling has not yet been directly investigated. Smac mimetics drugs, which activate apoptotic or necroptotic cell death, do not induce mutations but the mechanistic basis for this lack of mutagenic activity has not been determined. In this study, we compared the mutagenic potential of these two cell death pathways by engineering cells to activate either apoptotic or necroptotic signaling by exposing them to Smac mimetics with or without TNFα, and/or enforcing or preventing expression of apoptotic or necroptotic regulators. We discovered that sublethal concentrations of Smac mimetics in contexts that activated apoptotic signaling provoked DNA damage and mutations in surviving cells. Mutagenesis was dependent on executioner caspase activation of the nuclease CAD. In contrast, RIPK3- and MLKL-dependent necroptotic signaling following Smac mimetic treatment was not mutagenic. Likewise, DNA damage was not provoked in cells expressing a lethal constitutively active MLKL mutant. These data reveal that cells surviving sublethal necroptotic signaling do not sustain genomic damage and provide hope for a reduced risk of therapy-related malignancies in patients treated with necroptosis-inducing drugs.
Insights
Anticancer drugs can cause mutations via apoptosis. However, necroptosis, a different cell death pathway, does not induce mutations, suggesting safer cancer therapies with reduced risks of therapy-related cancers.
Area of Science:
- Cellular Biology
- Molecular Oncology
- Cancer Therapeutics
Background:
- Most anticancer drugs induce apoptosis, a programmed cell death pathway that can cause DNA damage and mutations.
- This DNA damage raises concerns about therapy-related cancers and oncogenic mutations in surviving cells.
- Necroptosis is an alternative, caspase-independent cell death pathway involving RIPK3 and MLKL, with its mutagenic potential uninvestigated.
Purpose of the Study:
- To compare the mutagenic potential of apoptotic versus necroptotic cell death pathways.
- To investigate the mechanistic basis for the lack of mutagenicity observed with Smac mimetics.
- To determine if sublethal necroptotic signaling causes genomic damage.
Main Methods:
- Engineered cells to activate either apoptosis or necroptosis using Smac mimetics, TNFα, and regulators of cell death.
- Assessed DNA damage and mutations in surviving cells after sublethal signaling.
- Investigated the role of executioner caspases, RIPK3, MLKL, and CAD in mutagenesis.
Main Results:
- Sublethal Smac mimetic treatment activating apoptosis induced DNA damage and mutations in surviving cells, dependent on caspase-activated DNase (CAD).
- In contrast, RIPK3- and MLKL-dependent necroptotic signaling was not mutagenic.
- Cells expressing a lethal constitutively active MLKL mutant also did not sustain DNA damage.
Conclusions:
- Surviving cells from sublethal necroptotic signaling do not accumulate genomic damage.
- Necroptosis-inducing drugs may offer a reduced risk of therapy-related malignancies compared to apoptosis-inducing agents.
- This study provides a mechanistic understanding of why necroptosis is non-mutagenic, supporting its therapeutic potential.
