PO-288 Replication stress response as a target for eradicating colorectal cancer stem cells

Abstract

IntroductionCancer stem cells (CSCs) are subsets of multipotent SCs responsible for tumour development, propagation and evolution, whose targeting is required for tumour eradication. There is (pre)clinical evidence on a role of CSCs in therapeutic resistance and intra-tumour heterogeneity, which limits the efficacy of antineoplastic regimens. In this context, CSCs reportedly share with embryonic/adult SCs a very robust DNA damage response, which favours the survival and resistance to genotoxins, and can be exploited for therapeutic purposes.Material and methodsWe generated a panel of ~30 CRC patient-derived tumorspheres enriched for CSCs (CRC-SCs) and characterised them at the genetic level. To discover potential monotherapeutic anti-CSC agents, we performed high-throughput screenings on multiple CRC-SCs with a library of clinically-relevant drugs. Flow cytometry, fluorescence microscopy and epistatic analyses were conducted to uncover the mechanism of action of identified compound(s), while genetic, cytogenetic and phosphoproteomic studies were carried out to identify predictive biomarkers. DNA replication stress (RS) levels were evaluated by analysing phosphorylated ATM/RPA foci and by performing COMET and DNA fibre assays, and were modulated by single administration of genome destabilising agents or by prolonged exposure to increased doses of compounds targeting the replication stress response (RSR).Results and discussionsWe demonstrated that the CHK1 inhibitor LY2606368 is a potent anti-CSC agent able to kill more than one third of CRC-SCs, both in vitro and in vivo. Moreover, we provided evidence of high but heterogeneous RS levels in CRC-SCs, showing that, in CRC-SCs, RS is mainly boosted endogenously by p53 deficiency, supernumerary chromosomes and DNA replication abnormalities, which results in high dependency on CHK1-mediated RSR. Accordingly, formerly LY2606368-resistant CRC-SCs were sensitised by boosting DNA replication errors or inducing whole-genome doubling, while formerly LY2606368-sensitive CRC-SCs made resistant by the continuous in vitro or in vivo administration of LY2606368 displayed diminished RS levels due to RSR rewiring, and became independent on CHK1.ConclusionOur results demonstrate that RSR is efficient and rewirable in CSCs thereby constituting a prominent therapeutic target. In particular, we designed dedicated RS-modulating or RSR-targeting strategies for long-term CSC depletion in CRC.