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APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression
Hiroyuki Yamazaki1, Kotaro Shirakawa1, Tadahiko Matsumoto1
1Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Abstract:
Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.
Insights
Researchers developed novel reporter cells to study DNA cytosine deaminase 3B (A3B) in cancer. Conventional therapies increase A3B, while targeted therapies do not, suggesting DNA damage pathways regulate A3B expression.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is implicated in cancer mutagenesis.
- High homology within the APOBEC family complicates specific investigation of A3B in cancer cells.
Purpose of the Study:
- To develop a tool for comprehensive investigation of A3B function in multiple myeloma cells.
- To screen anticancer treatments and understand their impact on A3B expression.
Main Methods:
- CRISPR/Cas9 gene editing was used to create A3B reporter cells with 3xFLAG tag and IRES-EGFP sequences.
- Reporter cell expression was validated using stimuli (PMA) and knockdown (shRNA).
- A panel of anticancer treatments was screened for effects on A3B expression.
Main Results:
- Conventional therapies (antimetabolites, radiation) increased endogenous A3B expression.
- Molecular targeted therapeutics (bortezomib, lenalidomide, elotuzumab) did not increase A3B expression.
- Inhibition of ATM, ATR, and DNA-PK suppressed A3B reporter expression induced by antimetabolites, indicating a role in DNA damage signaling.
Conclusions:
- DNA damage signaling pathways involving ATM, ATR, and DNA-PK regulate A3B expression.
- Novel A3B reporter cells provide a valuable tool for studying A3B biology in cancer.
- Differential effects of conventional versus targeted therapies on A3B highlight distinct mechanisms of action.
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