Erratum: [Corrigendum] Inhibition of human oral squamous cell carcinoma proliferation and migration by prodrug­activating suicide gene therapies.

[This corrects the article DOI: 10.3892/etm.2023.11790.].

Selective C9orf72 G-Quadruplex-Binding Small Molecules Ameliorate Pathological Signatures of ALS/FTD Models.

The G-quadruplex (G4) forming C9orf72 GGGGCC (G4C2) expanded hexanucleotide repeat (EHR) is the predominant genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Developing selective G4-binding ligands is challenging due to the conformational polymorphism and similarity of G4 structures. We identified three first-in-class marine natural products, chrexanthomycin A (cA), chrexanthomycin B (cB), and chrexanthomycin C (cC), with remarkable bioactivities. Thereinto, cA shows the highest permeability and lowest cytotoxicity to live cells. NMR titration experiments and in silico analysis demonstrate that cA, cB, and cC selectively bind to DNA and RNA G4C2 G4s. Notably, cA and cC dramatically reduce G4C2 EHR-caused cell death, diminish G4C2 RNA foci in (G4C2)29-expressing Neuro2a cells, and significantly eliminate ROS in HT22 cells. In (G4C2)29-expressing Drosophila, cA and cC significantly rescue eye degeneration and improve locomotor deficits. Overall, our findings reveal that cA and cC are potential therapeutic agents deserving further clinical study.

Quantitative evaluation and comparison of two prodrug-activating suicide gene therapies on oral squamous cell carcinoma.

Prodrug-activating suicide gene therapy (PA suicide gene therapy for short) for cancer is to introduce cancer cells with suicide genes. The enzyme encoded by suicide gene is not toxic but is able to kill cancer cells by converting a non-toxic prodrug into a toxic compound. This approach is a promising cancer gene therapy that could reduce non-specific toxicity to normal tissue. However, there is no quantitative method to evaluate efficacy of suicide gene therapy in preclinical study. The aim of this study is to develop a new method to quantitatively evaluate and compare prodrug-activating suicide gene therapies. This study was carried out on an oral squamous cell carcinoma (OSCC) cell line CAL-27. Suicide genes were integrated into ROSA26 locus of CAL-27 by CRISPR-Cas9. CAL-27 cell lines stably expressing herpes simplex virus-thymidine kinase (TK) or yeast cytosine deaminase (CD) were used to evaluate and compare PA suicide gene therapies. The efficacies of PA suicide gene therapies were quantitatively evaluated from three aspects: effective prodrug concentration, prodrug treatment time, and bystander effect. This method also could be used for different types of suicide gene therapies and different types of cancer. When the prodrug concentration, treatment time, and rate of suicide gene-positive cells (related to bystander effect) are fixed, anti-cancer effects could be quantitatively measured. This information is important for suicide gene therapy preclinical development.

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence.

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)2 that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)2 associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5'-to-5' arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3'-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.

Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA.

The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

The crystal structure of an antiparallel chair-type G-quadruplex formed by Bromo-substituted human telomeric DNA.

Human telomeric guanine-rich DNA, which could adopt different G-quadruplex structures, plays important roles in protecting the cell from recombination and degradation. Although many of these structures were determined, the chair-type G-quadruplex structure remains elusive. Here, we present a crystal structure of the G-quadruplex composed of the human telomeric sequence d[GGGTTAGG8GTTAGGGTTAGG20G] with two dG to 8Br-dG substitutions at positions 8 and 20 with syn conformation in the K+ solution. It forms a novel three-layer chair-type G-quadruplex with two linking trinucleotide loops. Particularly, T5 and T17 are coplanar with two water molecules stacking on the G-tetrad layer in a sandwich-like mode through a coordinating K+ ion and an A6•A18 base pair. While a twisted Hoogsteen A12•T10 base pair caps on the top of G-tetrad core. The three linking TTA loops are edgewise and each DNA strand has two antiparallel adjacent strands. Our findings contribute to a deeper understanding and highlight the unique roles of loop and water molecule in the folding of the G-quadruplex.