Telomeric Replication Stress Promotes ALT-Associated Hallmarks in ATRX- and p53-Deficient Cells

Background: Most human tumors maintain telomeres through telomerase activation; however, a subset relies on the telomerase-independent mechanism known as Alternative Lengthening of Telomeres (ALT). ALT-positive tumors are frequently characterized by inactivating alterations in ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-linked) and p53 and by distinct molecular and cellular features. Although chronic telomeric replication stress has been proposed as a trigger for ALT, several aspects of this process remain poorly understood. Recent evidence suggests that ATRX depletion promotes the persistence of DNA secondary structures, including G-quadruplexes (G4), which can impair replication fork progression, leading to fork stalling and DNA damage. Methods: To investigate whether ATRX and p53 deficiency sensitize telomerase-positive cells to develop ALT-associated molecular features in response to telomeric replication stress, we analyzed telomeric DNA damage, telomere sister chromatid exchange (T-SCE), Telomeric Repeat-containing RNA (TERRA) expression, and C-circle formation in HCT116 cells proficient or deficient for ATRX and p53. ATRX was depleted using both transient (siRNA-mediated knockdown) and stable (shRNA-expressing clones) approaches, allowing us to distinguish the early cellular response from the features arising upon persistent ATRX loss. To further assess the contribution of telomeric replication stress, cells were treated with the G4-stabilizing ligand RHPS4. Results: Our data show that G4 stabilization, in the context of ATRX and/or p53 deficiency, promotes the appearance of multiple ALT-associated hallmarks, including telomeric DNA damage, increased T-SCE, and elevated TERRA expression. Moreover, the combined loss of ATRX and p53 permits cell-cycle progression despite the presence of RHPS4-induced telomeric replication stress and telomeric DNA damage. Conclusions: These findings clarify how ATRX and p53 loss, even in telomerase-positive cells, are permissive conditions for the emergence of ALT-associated hallmarks under telomeric replication stress and highlight the G2/M checkpoint as a critical barrier limiting the propagation of cells displaying these features.