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Stalling the Replication Fork via the Impedimental Stabilization of Higher-order DNAs

Periodic Reporting for period 1 - STARFISH DNA (Stalling the Replication Fork via the Impedimental Stabilization of Higher-order DNAs)

Reporting period: 2017-10-01 to 2019-09-30

Cancer continues to claim the lives of millions of patients every year (9.6 million deaths in 2018) despite the recent advances in cancer therapeutics. For some cancers, the 5-year survival rates are over 90% for patients (prostate, skin cancers) while others have far lower survival rates (20% for pancreatic, liver or lung cancers). This highlights the need for new therapeutic strategies, either deviating from classical anticancer drug regiments or exploiting them in an alternative manner.

The panoply of treatments provided to physicians and patients includes radiotherapy, immunotherapy and chemotherapy. The latter includes anticancer drugs the bulk of which targets DNA. This approach aims at creating DNA damage to cancer cells, the cancer-versus-healthy cells specificity relying on the poorest ability of cancer cells to manage DNA damage properly as compared to healthy cells. An alternative to irreversibly damage DNA is to stabilize unusual DNA structures that encompass all DNA structures that deviate from the canonical DNA double helix described by Watson & Crick. Their formation is promoted by DNA transactions (transcription, replication) due to local strand separation and DNA supercoiling related to the DNA/RNA polymerase motion along the genomic duplex-DNA. These unusual DNA structures form topological hindrances to replication and transcription machineries that equally threatens genetic integrity.

The reliability of this approach has already been demonstrated by the wealth of data collected over the past 2 decades with the four-stranded DNA structure named G-quadruplex-DNA. In the STARFISH DNA project (for Stalling the Replication Fork via the Impedimental Stabilization of Higher-order DNAs), we focus our attention on another unusual DNA structure, the three-way DNA junction (TWJ). Our aim was to demonstrate that the non-covalent stabilization of TWJ by specific small molecules (TWJ-ligands) does create roadblocks to polymerase processivity, thereby triggering DNA damage that eventually lead to cancer cell death. We selected TWJ as genetic targets since the ligand binding site is more structurally defined within a TWJ than in a G-quadruplex, making their targeting possibly more specific.

This 2-year multidisciplinary scientific program, performed in collaboration with Dr. Anton Granzhan (Institut Curie, Orsay FR) and Dr. Sébastien Britton (Institute of Pharmacology and Structural Biology, Toulouse, FR) has been undoubtedly successful given that we have validated all critical steps towards the development of a new class of anticancer agents. We have indeed screened chemical libraries to identify TWJ ligands, then further characterized the capability of the most promising candidates to interact with TWJ in vitro, prior to assess their antiproliferative activities in breast cancer cells, as standalone agents or included in drug combinations. Collectively, this program has led to the identification of new chemical weapons to fight against cancers that act according to an innovative strategy.
"STARFISH DNA started with the implementation of a high-throughput screen (HTS) assay to identify promising TWJ-ligands on highly stringent criteria in terms of efficiency and selectivity. This mix-and-measure assay, named TWJ-Screen, was created and implemented to assess the TWJ-interacting properties of more than 1200 compounds. This screen provided a series of 20 promising candidates, whose TWJ-interacting properties were further characterized by an in vitro workflow comprising 4 additional and complementary assays. This workflow allowed for refining the pool of candidates to 5 fully validated TWJ ligands referred to as compounds #466, #471, #586, #589 and #1843 (undisclosed for confidentiality issues) belonging to a series of azacryptands synthesized in the laboratory of Dr. Anton Granzhan, Institut Curie, Orsay, France.

These compounds were then investigated for their toxicity on two breast cancer cell lines, in which they were found highly active. We then established that this cellular toxicity originated from their capacity to damage the DNA of treated cells via a series of immunodetection studies. This approach, which allowed for both visualizing and quantifying DNA damage in treated cells, demonstrated that 3 ligands triggered genotoxic DNA damage (#466, #586 and #589), which mainly occurred at the G1/early-S phases of the cell cycle, indicating that TWJ-ligands induce both transcription- and replication- associated DNA damage. We then investigated whether the action of these ligands could be potentiated by inhibitors of DNA repair, according to an approach known as chemically induced synthetic lethality. This strategy aims at using TWJ-ligands to create DNA damage on one hand and to prevent DNA repair using ad hoc inhibitors on the other hand. We selected inhibitors of DNA repair via the homologous recombination (HR) and the non-homologous end-joining (NHEJ) mechanisms, the two main DNA damage repair pathways. These studies performed in collaboration with Dr. Sébastien Britton (IPBS Toulouse, France) highlighted the excellent performances of the azacryptand #466, which was found to synergistically interact efficiently with DNA repair inhibitors. These results allowed for confirming that TWJ ligands trigger both transcription- and replication-associated DNA damage."
STARFISH DNA is a resolutely fundamental research-oriented program, aiming at increasing the knowledge of a community of scientists searching for ever more efficient anticancer strategies. This project is thus expected to impact oncology science via the validation of novel molecular targets and the identification of novel therapeutic molecules and/or drug cocktails to fight against cancers. Our aim is to generate an impetus for higher-order DNA structures to be considered as new players in the arena of therapeutically valuable targets. This approach has already been demonstrated for another unusual DNA structure, the quadruplex-DNA, making our results invaluable in that they provide the long-awaited demonstration that other unusual DNA structures (the DNA junctions) are strategically relevant as well, thus expanding the repertoire of genomic targets with therapeutic outcomes.

More importantly, the complete chain of validation developed in the framework of STARFISH DNA, from the screening of chemical libraries to synthetic lethality strategies, represents turn-key solutions for scientists eager to enter this area of research. To make this toolkit available to all, the technical details have been thoroughly described in publications either in Gold Open Access (OA) journals (e.g. Nucleic Acids Res.) or in Green OA journals (e.g. J. Med. Chem.) along with freely accessible post-prints deposited in OA archives.

We are now impatiently waiting for new results coming from other labs worldwide, which will contribute to confirm the interest of TWJ as anticancer targets and of TWJ ligands as antiproliferative agents. These advances will lend further credence to this strategy and make this area of research ever more thrilling and promising.
Targeting three-way DNA junction with small molecules to trigger DNA damage