Some chemotherapeutics used in colorectal cancer (CRC), such as irinotecan (IRI), exert antitumor effects by disrupting DNA replication and inducing replication stress (RS). However, clinical efficacy is often limited by the emergence of drug‑tolerant persistence, a transient state in which a small fraction of cancer cells survives treatment, contributing to relapse and poor outcomes. Persistence is driven by epigenetic mechanisms, with surviving cells adopting a dormant or slow‑cycling state, fueling minimal residual disease. CRC is characterized by two major genomic instability subtypes, microsatellite instability (MSI) and chromosomal instability (CIN/MSS), whose relevance to drug persistence remains unclear. Here, we aimed to identify RS-inducing limiting CRC cellular persistence across MSI/CIN subtypes.

We used human CRC models, commercial cell lines and patient-derived spheroids and organoids, with defined MSI or MSS/CIN status. Cells were treated with IRI alone or with PARP1 inhibitors to generate drug-tolerant persisters. Gene expression was analyzed by RT-qPCR and RNA-seq. Senescence was assessed by SA-β-gal staining, p16/p21 analysis, and immunofluorescence. Secretory profiles were measured with cytokine arrays, and functional stemness by colony- and sphere-formation assays.

Prolonged IRI-induced RS generated drug-tolerant persisters across all CRC models, characterized by morphological changes, proliferative arrest, and regrowth after drug withdrawal. Screening of RS‑inducing agents identified PARP1 inhibitors (PARPi) as sensitizing all CRC lines to IRI. IRI+talazoparib (TZ) nearly eradicated persisters in MSS/CIN cells via replication-catastrophe and reduced persister formation and recovery in MSI models. Persisters displayed a senescent-like phenotype, including SA-β-gal positivity, p16/p21 induction, reduced proliferation, and a stronger SASP, especially in MSI cells. Senescence emerged early during RS and was enhanced by talazoparib. Senolytic agents reduced persister viability and regrowth, especially after IRI+TZ. Transcriptomic and functional analyses showed no enrichment of CRC stemness markers in RS‑induced persisters, especially in MSI models, whereas EMT and organoid architectural remodeling were increased, and functional stemness was suppressed by IRI+PARPi.

Our data indicate that RS-induced persistence in CRC is driven by adaptive cellular plasticity with senescent-like features and EMT activation rather than CSC expansion. IRI+PARPi reduces persisters, while senolytics further impair survival and regrowth, supporting RS‑based, senescence‑targeted strategies in CRC, particularly MSI tumors.

Funding from: AIRC, IG 2022 ID 27685; CARESS FPRC 5×1000 Grant; European Union-NextGenerationEU-PNRR M6C2, Investimento 2.1 (PNRR-POC-2023-12378288); Startup funds from the IIGM and Compagnia di San Paolo.