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The antitumor toxin CD437 is a direct inhibitor of DNA polymerase α

Nature Chemical Biology volume 12, pages 511–515 (2016)Cite this article

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Abstract

CD437 is a retinoid-like small molecule that selectively induces apoptosis in cancer cells, but not in normal cells, through an unknown mechanism. We used a forward-genetic strategy to discover mutations in POLA1 that coincide with CD437 resistance (POLA1R). Introduction of one of these mutations into cancer cells by CRISPR-Cas9 genome editing conferred CD437 resistance, demonstrating causality. POLA1 encodes DNA polymerase α, the enzyme responsible for initiating DNA synthesis during the S phase of the cell cycle. CD437 inhibits DNA replication in cells and recombinant POLA1 activity in vitro. Both effects are abrogated by the identified POLA1 mutations, supporting POLA1 as the direct antitumor target of CD437. In addition, we detected an increase in the total fluorescence intensity and anisotropy of CD437 in the presence of increasing concentrations of POLA1 that is consistent with a direct binding interaction. The discovery of POLA1 as the direct anticancer target for CD437 has the potential to catalyze the development of CD437 into an anticancer therapeutic.

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Figure 1: Mutations in POLA1 render resistance to CD437.
Figure 2: POLA1 L764S knock-in using CRISPR/Cas9 technology results in CD437 resistance.
Figure 3: CD437 inhibits DNA replication in vivo and POLA1 activity in vitro.
Figure 4: CD437 binds POLA1 in vitro.

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Acknowledgements

The authors thank J. Ready, S.L. McKnight, and members of the Nijhawan laboratory for helpful comments. We thank the McDermott Sequencing Center at UT Southwestern Medical Center for Illumina sequencing, T. Tahirov for the pFastBac1 POLA1 plasmid, J. McKnight for site-directed mutagenesis of POLA1, and B. Li, H. Yu and L. Beatty for help with Sf9 culture. T.H. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. S.B.P. was supported by a grant to M.K. Rosen from NIGMS (R01-GM56322). J.K. was supported by the Cancer Prevention and Research Institute of Texas (CPRIT) grant RP150596. This research was supported by a Harold C. Simmons Cancer Center Startup Awards, a Disease Oriented Clinical Scholar (DOCS) award, a Damon Runyon Clinical Investigator award (CI-68-13) and a grant from the Welch Foundation (I-1879) to D.N.

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Authors and Affiliations

  1. Department of Biochemistry, UT Southwestern Medical Center, Dallas, Texas, USA

    Ting Han & Deepak Nijhawan

  2. Department of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas, USA

    Maria Goralski, Emanuela Capota & Deepak Nijhawan

  3. Department of Biophysics, UT Southwestern Medical Center, Dallas, Texas, USA

    Shae B Padrick

  4. Department of Clinical Sciences, Quantitative Biomedical Research Center, UT Southwestern Medical Center, Dallas, Texas, USA

    Jiwoong Kim & Yang Xie

  5. Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, Texas, USA

    Jiwoong Kim, Yang Xie & Deepak Nijhawan

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  1. Ting Han
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Contributions

T.H. designed the study, performed most of the experiments, interpreted the results, and wrote the manuscript. M.G. performed the initial screen and contributed to validation studies. E.C. expressed and purified recombinant POLA1 protein. S.B.P. guided the binding assay and analyzed the binding data. J.K. and Y.X. performed all bioinformatics analyses. D.N. designed and supervised the study and wrote the manuscript.

Corresponding author

Correspondence to Deepak Nijhawan.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–7. (PDF 18963 kb)

Supplementary Data Set 1

Mutation results for exome sequencing of HCT-116 clones. A list of all detected non-synonymous, splice site, and insertion/deletion mutations for 19 HCT-116 clones. 6 of these clones were resistant to CD437 (CD437R) and 13 others were sensitive (Control). (XLSX 3448 kb)

Supplementary Data Set 2

Genes with missense mutations in HCT-116 clones. A list of genes for which exome sequencing revealed missense mutations in at least one of the 19 HCT-116 clones. Genes that harbored missense mutations in CD437R clones but not Control clones are highlighted and listed at the top. (XLSX 506 kb)

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Han, T., Goralski, M., Capota, E. et al. The antitumor toxin CD437 is a direct inhibitor of DNA polymerase α. Nat Chem Biol 12, 511–515 (2016). https://doi.org/10.1038/nchembio.2082

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