Stabilization of quadruplex DNA perturbs telomere replication leading to the activation of an ATR-dependent ATM signaling pathway

Abstract

Functional telomeres are required to maintain the replicative ability of cancer cells and represent putative targets for G-quadruplex (G4) ligands. Here, we show that the pentacyclic acridinium salt RHPS4, one of the most effective and selective G4 ligands, triggers damages in cells traversing S phase by interfering with telomere replication. Indeed, we found that RHPS4 markedly reduced BrdU incorporation at telomeres and altered the dynamic association of the telomeric proteins TRF1, TRF2 and POT1, leading to chromosome aberrations such as telomere fusions and telomere doublets. Analysis of the molecular damage pathway revealed that RHPS4 induced an ATR-dependent ATM signaling that plays a functional role in the cellular response to RHPS4 treatment. We propose that RHPS4, by stabilizing G4 DNA at telomeres, impairs fork progression and/or telomere processing resulting in telomere dysfunction and activation of a replication stress response pathway. The detailed understanding of the molecular mode of action of this class of compounds makes them attractive tools to understand telomere biology and provides the basis for a rational use of G4 ligands for the therapy of cancer.

PARP1 is activated at telomeres upon G4 stabilization: possible target for telomere-based therapy

“…Thus, telomeric DNA strand breaks may activate and recruit PARP1 to help repair PARP inhibition increases the efficacy of telomere-based therapy E Salvati et al PARP inhibition increases the efficacy of telomere-based therapy E Salvati et al enzymes and/or act as the architecture of the damaged telomeres. The apparent contradiction regarding the inhibition of phosphorylation, activation of ATM by PARP1 inactivation and the concomitant increase of the phosphorylation of the ATM substrate H2AX can be justified by the fact that, as already reported in our previous paper, phosphorylation of H2AX is dependent on the ATR kinase (Salvati et al, 2007;Rizzo et al, 2009). The biomedical relevance of PARP1 activation at telomeres upon RHPS4 treatment has also been highlighted in this paper by showing that the PARP inhibitor GPI 15427 enhanced RHPS4 antitumoral activity both in vitro and in xenograft models of human tumors.…”

“…In agreement with previous studies (Lindahl et al, 1995), the poly-ADP ribosylation level had greatly increased in cells exposed to CPT and ionizing radiation (Figures 1a and b). Interestingly, PARs were also detected in cells treated with the telomere-damaging agent RHPS4 (Figures 1a-c and Supplementary Figure 2) and they are strictly dependent on S-phase (Supplementary Figure 3) as a result of the alteration in telomere replication (Rizzo et al, 2009). More importantly, most of the PAR spots co-localized with the telomeric marker TRF1 in RHPS4 cells (Figure 1d), suggesting that poly-ADP ribosylation is activated as a consequence of telomere damage.…”

“…RHPS4 is one of the most selective G4 ligands with anticancer activity both as a single agent and in combination with CPTs in pre-clinical models of solid tumors (Salvati et al, 2007;Leonetti et al, 2008). This agent, behind its classical telomerase-inhibitory properties, impairs fork progression and/or telomere processing, resulting in telomere dysfunction and activation of an ATR/ATM signaling pathway (Rizzo et al, 2009). Overall, these studies show us how an agent of this novel class may be used clinically in combination with chemotherapeutic agents and warrant further investigations.…”

“…The fact that both RHPS4 and CPT interfere PARP inhibition increases the efficacy of telomere-based therapy E Salvati et al with DNA replication, together with the known PARP activation by stalled replication forks, suggest that PARP1 activation is caused by telomere replication defects (Sugimura et al, 2008;Rizzo et al, 2009).…”

“…RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate) is one of the most effective G4 ligands showing a high selectivity for quadruplex DNA structure (Gavathiotis et al, 2003). We revealed that this agent, by stabilizing G4 DNA at telomeres, impairs fork progression and/or telomere processing, resulting in telomere dysfunction and activation of an ATR-dependent ATM pathway (Rizzo et al, 2009). Interestingly, in view of clinical application, RHPS4 is active in vivo as a single agent with a good toxicological profile and potentiates the antitumoral efficacy of TOPOI inhibitors in preclinical models of solid tumors (Salvati et al, 2007;Leonetti et al, 2008).…”