Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds

“Alkaline Single-Cell Gel Electrophoresis Comet Assay Thecometassaywasperformedaspreviouslydescribed (Singh et al., 1988).”

“Genomic instability is a hallmark of cancer caused by failure of normal DNA replication and/or repair mechanisms (Halazonetis Mo et al., 2008; Negrini et al., 2010).”

“G4 structures have been subsequently detected with several other structure-specific antibodies (Biffi et al., 2013; Henderson et al., 2014; Schaffitzel et al., 2001) and interacting small molecules (Lam et al., 2013; Müller et al., 2010; Rodriguez et al., 2012).”

“RPA accumulation on the chromatin, together with elevated frequency of origin firing and reduced replication rates, represents signatures of replicative stress (Zeman and Cimprich, 2014).”

“Importantly, telomeric G-rich DNA sequences have a high propensity to adopt G4 configurations (Parkinson et al., 2002).”

“A conundrum posed by this quantificationwas that PDS induced approximately fifteen DSBs per metaphase in cells lacking RAD51, yet in silico predictions suggested thatmore than 300,000 genomic sites can adopt G4 configurations (Huppert and Balasubramanian, 2005).”

“…Zimmer, Eliana M.C. Tacconi, Cecilia Folio, ..., Annamaria Biroccio, Julian E. Sale, Madalena Tarsounas Correspondence madalena.tarsounas@oncology.ox.ac.uk In Brief Zimmer et al. discovered that homologous recombination activities of BRCA1 and BRCA2…”

“The ability of G-rich DNA to adopt G4 secondary structures in vitro was reported over 50 years ago (Gellert et al., 1962).”

“HR factors act to stabilize stalled replication forks by preventing their nucleolytic degradation (Hashimoto et al., 2010; Schlacher et al., 2011) to restart arrested forks (Lambert et al., 2010) and to repair double-strand breaks (DSBs) arising from disintegrated forks (Aze et al., 2013).”

“Conversely, G4 configurations can be stabilized by specific ligands that exhibit higher binding specificity for G4s over duplex DNA, with the G4-interacting compound PDS being one example (Chambers et al., 2015).”

“This activity of BRCA1 is antagonized by 53BP1, which protects broken DNA ends and channels their repair into non-homologous end joining (NHEJ) (Bouwman et al., 2010; Bunting et al., 2010).”

“G4 Structure Stabilization Exacerbates the Telomere Fragility in Brca2-Deleted MEFs Telomere fragility indicates a telomere replication defect (Martı́nez et al., 2009; Sfeir et al., 2009), which is thought to stem from the potential of telomere DNA sequences to adopt G4 secondary structures known to obstruct replicati…”

“DNA Fiber Assay DNA fiber assays were performed as described previously (Jackson and Pombo, 1998).”

“In vitro, G-rich ssDNA can adopt secondary structures known as G4s under physiological-like conditions (Lipps and Rhodes, 2009).”

“G4 structures have been subsequently detected with several other structure-specific antibodies (Biffi et al., 2013; Henderson et al., 2014; Schaffitzel et al., 2001) and interacting small molecules (Lam et al., 2013; Müller et al., 2010; Rodriguez et al., 2012).”

“In western blot analyses (Figure 3F), PDS and PhenDC both induced apoptosis specifically in RAD51-deficient cells, detected by cleaved PARP1 and gH2AX expression, a well-established marker for DNA damage that is also induced by apoptosis (Rogakou et al., 2000).”

“Strikingly, olaparib-resistant Brca1-deficient cells lacking REV7 or 53BP1 expression (Brca1 / shREV7; Brca1 / 53BP1-deficient) were hypersensitive to PDS (Figures 7A, 7B, S7A, and S7B).”

“G4 structures have been subsequently detected with several other structure-specific antibodies (Biffi et al., 2013; Henderson et al., 2014; Schaffitzel et al., 2001) and interacting small molecules (Lam et al., 2013; Müller et al., 2010; Rodriguez et al., 2012).”

“HR factors act to stabilize stalled replication forks by preventing their nucleolytic degradation (Hashimoto et al., 2010; Schlacher et al., 2011) to restart arrested forks (Lambert et al., 2010) and to repair double-strand breaks (DSBs) arising from disintegrated forks (Aze et al., 2013).”

“Moreover, ionizing radiation (IR)-induced RAD51 foci assemble in olaparib-resistant Brca1 / , 53BP1deficient cells (albeit not at the same level as in Brca1+/+ cells), but not in olaparib-sensitive Brca1 / tumor-derived cells (Jaspers et al., 2013).”

“Supporting this concept, treatment with G4-stabilizing compounds induces telomere dysfunction (Gomez et al., 2006; Rodriguez et al., 2008; Salvati et al., 2007; Tahara et al., 2006).”

“450 Molecular Cell 61, 449–460, February 4, 2016 a2016 The Author damage at genomic sites with predicted G4-forming potential (Lam et al., 2013; Rodriguez et al., 2012).”

“G4 structures have been subsequently detected with several other structure-specific antibodies (Biffi et al., 2013; Henderson et al., 2014; Schaffitzel et al., 2001) and interacting small molecules (Lam et al., 2013; Müller et al., 2010; Rodriguez et al., 2012).”

“G4 Structure Stabilization Exacerbates the Telomere Fragility in Brca2-Deleted MEFs Telomere fragility indicates a telomere replication defect (Martı́nez et al., 2009; Sfeir et al., 2009), which is thought to stem from the potential of telomere DNA sequences to adopt G4 secondary structures known to obstruct replicati…”

“This suggested a role for HR in fork restart when Pif1 activity is abrogated (Ribeyre et al., 2009).”

“Several lines of evidence support a similar function in vivo for these factors, essential to preserve genome stability during DNA replication (Murat and Balasubramanian, 2014).”

“Plasmid-Based Replication Assay Plasmid-based replication assays were performed as previously described (Sarkies et al., 2010; Szüts et al., 2008) with modifications listed in Supplemental Experimental Procedures.”

“BRCA2 promotes recombination reactions by loading the RAD51 recombinase onto single-stranded DNA (ssDNA) in concert with the family of proteins known as the RAD51 paralogs, of which RAD51C is a member (Suwaki et al., 2011).”

“The same specific elimination by PDSwas observed for BRCA2-deficient V-C8 hamster cells (Kraakman-van der Zwet et al., 2002; Figure S2A) and Brca2 / mouse mammary tumor-derived cells (Figure S2B).”

“Instead, we tested in our cellular models a previously reported G4-stabilizing drug, RHPS4 (Gavathiotis et al., 2003; Gowan et al., 2001; Heald et al., 2002), with well-characterized pharmacological features (Leonetti et al., 2008; Salvati et al., 2007).”

“Chromosome Orientation FISH and IF-FISH For CO-FISH, cells were plated at 50%–60% confluency and treated with 10 mM bromodeoxyuridine (BrdU) for 20 hr. Colcemid (0.2 mg/ml) was added to the cells 4–6 hr before metaphases were processed for CO-FISH as previously described (Bailey et al., 2001).”

“Supporting this concept, treatment with G4-stabilizing compounds induces telomere dysfunction (Gomez et al., 2006; Rodriguez et al., 2008; Salvati et al., 2007; Tahara et al., 2006).”

“Instead, we tested in our cellular models a previously reported G4-stabilizing drug, RHPS4 (Gavathiotis et al., 2003; Gowan et al., 2001; Heald et al., 2002), with well-characterized pharmacological features (Leonetti et al., 2008; Salvati et al., 2007).”

“For IF-FISH, metaphases were spun onto coverslips using a cytospin apparatus (Cytospin 4, Fisher) and subjected to immunofluorescence staining as described (Tarsounas et al., 2004).”

“Supporting this concept, treatment with G4-stabilizing compounds induces telomere dysfunction (Gomez et al., 2006; Rodriguez et al., 2008; Salvati et al., 2007; Tahara et al., 2006).”

“, 2001) and interacting small molecules (Lam et al., 2013; Müller et al., 2010; Rodriguez et al., 2012).”

“Moreover, RAD51 depletion sensitized HEK293T cells to theG4 ligand PhenDC (Figure 3E; Piazza et al., 2010).”

“Instead, we tested in our cellular models a previously reported G4-stabilizing drug, RHPS4 (Gavathiotis et al., 2003; Gowan et al., 2001; Heald et al., 2002), with well-characterized pharmacological features (Leonetti et al., 2008; Salvati et al., 2007).”

“, 2011) to restart arrested forks (Lambert et al., 2010) and to repair double-strand breaks (DSBs) arising from disintegrated forks (Aze et al.”

“The latter process is likely mediated by the shelterin component TRF1, which recruits BLM helicase to telomeres to unwind G4 structures (Zimmermann et al., 2014).”

“HR factors, including BRCA2 and RAD51, are required to facilitate telomere replication and to prevent telomere shortening (Badie et al., 2010).”

“Although G4s are thought to positively regulate key cellular processes, they can also obstruct replication-fork progression, leading to genomic instability (Tarsounas and Tijsterman, 2013).”

“In line with previous publications reporting the antitumor effect of RHPS4 (Leonetti et al., 2008; Salvati et al., 2007), this drug repressed growth of BRCA2-proficient tumors as assessed by tumor weight inhibition (TWI) (22%, Figure 6D).”

“G4-stabilizing compounds are toxic to BRCA1- and BRCA2-deficient cells, highlighting their therapeutic potential in targeting BRCA deficiency.”

“Quantification of fragmented telomeric COFISH signals further demonstrated the bias toward fragility of the G-rich telomeric strand in Cre-treated Brca2F/- and Mo Rad51cF/F MEFs (Figures 1D and 1E) as well as in a Brca2 / mouse mammary tumor-derived cell line (Evers et al., 2010; Figure S1D).”

“To further investigate this concept, we used a plasmid-based replication assay (Szüts et al., 2008) in H1299 cells harboring inducible small hairpin RNA (shRNA) against RAD51C or BRCA2.”

“HR factors act to stabilize stalled replication forks by preventing their nucleolytic degradation (Hashimoto et al., 2010; Schlacher et al., 2011) to restart arrested forks (Lambert et al., 2010) and to repair double-strand breaks (DSBs) arising from disintegrated forks (Aze et al., 2013).”

“BRCA gene abrogation is associatedwith the same responses (Carlos et al., 2013).”

“Preparation of Metaphase Spreads and Telomere FISH Metaphase spread preparation and telomeric FISH were performed as previously described (Badie et al., 2015).”

“HR Is Required for Effective Replication of Genomic Regions with G4-Forming Potential HR factors have previously been implicated in telomere maintenance (Tacconi and Tarsounas, 2015).”

“G4 ligands such as pyridostatin and RHPS4 cause DNA double-strand breaks in cancer cells and activate DNA repair pathways [47,117].”

“Like pyridostatin, CX5461 shows activity against HR-deficient cells and tumors (Xu et al., 2017; Zimmer et al., 2016), suggesting a common mechanism of action.”