Using high-throughput screening, Taylor et al. identified actinomycin D (ACTD) to be cytotoxic over a panel of 12 patient-derived glioma stem-like cell (GSC) lines.1,2 Treatment with ACTD significantly reduced tumor growth in 2 recurrent glioblastoma (GBM) patient-derived models and significantly increased survival. ACTD specifically downregulated expression of human stem cell transcription factor SOX2 both in vitro and in vivo. To corroborate these results, we have performed a molecular docking study.
We used the program AutoDock Vina Extended for the docking study.3 AutoDock Vina Extended achieves approximately two orders of magnitude acceleration compared with the molecular docking software AutoDock 4, while also significantly improving the accuracy of the binding mode predictions. Further speed is achieved from parallelism, by using multithreading on multicore machines. AutoDock Vina Extended automatically calculates the grid maps and clusters the results in a way transparent to the user. UCSF Chimera 1.14 was used for molecular visualization.4
The ACTD molecule is from PubChem C62H86N12O16. Solution structure of the HMG (high-mobility group) box DNA-binding domain of human stem cell transcription factor SOX2 was deposited in the RCSB Protein Data Bank (2LE4) 2011-06-16, released: 2011-07-27.
ACTD docked in the region of glutamine 25 in a hydrophilic area of the so-called major wing of the HMG domain. Only one docking position with root mean square deviation (RMSD) of atomic positions = 0 was highly valid, and this position is shown in Fig. 1. The next 6 RMSD docking calculations were >0, indicating that docking was less likely. The binding energy of ACTD to the HMG major wing was −32 kcal/mol and binding constant was 1.63 × 10−18 µmol. Amino acids in the hydrophilic section of the major wing have a large impact on DNA and RNA binding. Several residues in the hydrophobic core of the minor wing have essentially no effect.5
Fig. 1.
Solution structure of the HMG box DNA-binding domain of human stem cell transcription factor SOX2. Actinomycin D (arrow) docks to a hydrophilic (blue) region of the HMG major wing. Hydrophobic regions are red.
The human SOX2 gene is situated on chromosome 3 at position q26.3-27 and encodes for a protein of 317 amino acids. SOX2 is a member of the SOX family of transcription factors, which have been shown to play key roles in many stages of mammalian development.5 This protein family shares a nucleic acid-binding domain, highly conserved among species, HMG box domain, which contains approximately 80 amino acids. HMG binds to RNA and DNA sequences and contains a nuclear localization signal (NLS) and a nuclear export signal (NES). The region of the glutamine 25 residue to which ACTD docked in our study is directly adjacent to these nuclear signals.6 ACTD could inhibit transcription by binding to one of them, thus interfering with its stability. Ideally, further studies should test this phenomenon by site-directed mutagenesis.
Dysregulation of SOX2 expression contributes to cancer development. Amplification of the SOX2 gene locus and elevated SOX2 expression affect cancer progression. SOX2 controls several features of cancer cells such as proliferation, epithelial-to-mesenchymal transition, migration, invasion, metastasis, sphere and colony formation, tumor initiation, cancer stem cell formation as well as resistance to apoptosis and therapy.7
ACTD is used to treat Wilms tumor and rhabdomyosarcomas in children, choriocarcinoma and low-risk gestational trophoblastic neoplasia in women. ACTD for GBM treatment could be a new modality for the large proportion of GBMs that do not respond to temozolomide. Clinical trials are warranted.
Funding
This work was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai. Research reported in this paper was also supported by the Office of Research Infrastructure of the National Institutes of Health under award numbers S10OD018522 and S10OD026880. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflicts of interest statement. The authors declare that they have no competing interests.
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