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Functional analyses of ATM, ATR and Fanconi anemia proteins in lung carcinoma : ATM, ATR and FA in lung carcinoma
Jan H Beumer1,2, Katherine Y Fu3, Bean N Anyang4
1Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, USA. beumerjh@upmc.edu.
Background:
ATM and ATR are kinases implicated in a myriad of DNA-damage responses. ATM kinase inhibition radiosensitizes cells and selectively kills cells with Fanconi anemia (FA) gene mutations. ATR kinase inhibition sensitizes cells to agents that induce replication stress and selectively kills cells with ATM and TP53 mutations. ATM mutations and FANCF promoter-methylation are reported in lung carcinomas.
Methods:
We undertook functional analyses of ATM, ATR, Chk1 and FA proteins in lung cancer cell lines. We included Calu6 that is reported to be FANCL-deficient. In addition, the cancer genome atlas (TCGA) database was interrogated for alterations in: 1) ATM, MRE11A, RAD50 and NBN; 2) ATR, ATRIP and TOPBP1; and 3) 15 FA genes.
Results:
No defects in ATM, ATR or Chk1 kinase activation, or FANCD2 monoubiquitination were identified in the lung cancer cell lines examined, including Calu6, and major alterations in these pathways were not identified in the TCGA database. Cell lines were radiosensitized by ATM kinase inhibitor KU60019, but no cell killing by ATM kinase inhibitor alone was observed. While no synergy between gemcitabine or carboplatin and ATR kinase inhibitor ETP-46464 was observed, synergy between gemcitabine and Chk1 kinase inhibitor UCN-01 was observed in 54 T, 201 T and H460, and synergy between carboplatin and Chk1 kinase inhibitor was identified in 201 T and 239 T. No interactions between ATM, ATR and FA activation were observed by either ATM or ATR kinase inhibition in the lung cancer cell lines.
Conclusions:
Analyses of ATM serine 1981 and Chk1 serine 345 phosphorylation, and FANCD2 monoubiquitination revealed that ATM and ATR kinase activation and FA pathway signaling are intact in the lung cancer cell lines examined. As such, these posttranslational modifications may have utility as biomarkers for the integrity of DNA damage signaling pathways in lung cancer. Different sensitization profiles between gemcitabine and carboplatin and ATR kinase inhibitor ETP-46464 and Chk1 kinase inhibitor UCN-01 were observed and this should be considered in the rationale for Phase I clinical trial design with ATR kinase inhibitors.
Insights
ATM and ATR kinases are crucial for DNA damage response. This study found these pathways intact in lung cancer cells, suggesting their posttranslational modifications could serve as biomarkers. Drug combinations require careful consideration for clinical trials.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- ATM and ATR kinases play vital roles in DNA damage response pathways.
- Inhibition of ATM or ATR can selectively target cancer cells with specific mutations.
- ATM mutations and FANCF promoter methylation are implicated in lung cancer development.
Purpose of the Study:
- To functionally analyze ATM, ATR, Chk1, and Fanconi anemia (FA) pathway proteins in lung cancer cell lines.
- To investigate alterations in ATM, ATR, and FA pathway genes within the Cancer Genome Atlas (TCGA) database.
- To evaluate the radiosensitization and selective killing effects of ATM and ATR kinase inhibitors in lung cancer.
Main Methods:
- Functional assays were performed on lung cancer cell lines, including a FANCL-deficient line (Calu6).
- The TCGA database was analyzed for genetic alterations in key DNA damage response genes.
- Cell lines were treated with ATM, ATR, and Chk1 kinase inhibitors, alone and in combination with chemotherapy agents.
Main Results:
- No defects in ATM, ATR, or Chk1 activation, or FANCD2 monoubiquitination were found in the examined lung cancer cell lines or TCGA data.
- ATM kinase inhibition radiosensitized cells, but did not cause cell killing alone.
- Synergistic effects were observed between gemcitabine/carboplatin and a Chk1 inhibitor, but not with an ATR inhibitor.
Conclusions:
- ATM and ATR kinase activation and FA pathway signaling are intact in the studied lung cancer cell lines.
- Posttranslational modifications of ATM, Chk1, and FANCD2 may serve as biomarkers for DNA damage signaling integrity in lung cancer.
- Observed differential drug sensitization profiles necessitate careful consideration in designing Phase I clinical trials involving ATR kinase inhibitors.
