Etoposide quinone is a covalent poison of human topoisomerase IIβ

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Etoposide is a widely used chemotherapy drug for various cancers.
• A subset of patients develop acute myeloid leukemias (t-AMLs) after etoposide treatment, often with MLL gene rearrangements.
• Etoposide metabolites, like etoposide quinone, and topoisomerase IIβ are suspected contributors to t-AML development.

Purpose of the Study:

• To investigate the effects of etoposide quinone on human topoisomerase IIβ activity.
• To compare the activity of etoposide quinone against topoisomerase IIβ and topoisomerase IIα.
• To elucidate the mechanism by which etoposide quinone interacts with topoisomerase IIβ.

Main Methods:

• In vitro assays to measure enzyme-mediated DNA cleavage.
• Comparative analysis of etoposide quinone's potency against topoisomerase IIα and IIβ.
• Assessment of etoposide quinone's reaction kinetics and ATP dependence.
• Evaluation of the effect of reducing agents and pre-incubation on enzyme activity.

Main Results:

• Etoposide quinone induced approximately 4 times more DNA cleavage than etoposide.
• Etoposide quinone was about 2 times more potent against topoisomerase IIβ than IIα and reacted faster with the β isoform.
• Etoposide quinone acted as a covalent poison, with activity abolished by reducing agents and capable of inactivating the enzyme prior to DNA addition.
• The metabolite showed a higher ratio of double- to single-stranded DNA breaks and less ATP dependence compared to etoposide.

Conclusions:

• Etoposide quinone is a potent covalent poison of human topoisomerase IIβ.
• The interaction of etoposide quinone with topoisomerase IIβ provides a molecular mechanism for etoposide-related leukemogenesis.
• These findings highlight the critical role of etoposide metabolites in chemotherapy-induced secondary malignancies.