A clinically relevant heterozygous ATR mutation sensitizes colorectal cancer cells to replication stress

Abstract

Colorectal cancer (CRC) ranks third among the most frequent malignancies and represents the second most common cause of cancer-related deaths worldwide. By interfering with the DNA replication process of cancer cells, several chemotherapeutic molecules used in CRC therapy induce replication stress (RS). At the cellular level, this stress is managed by the ATR-CHK1 pathway, which activates the replication checkpoint. In recent years, the therapeutic value of targeting this pathway has been demonstrated. Moreover, MSI + (microsatellite instability) tumors frequently harbor a nonsense, heterozygous mutation in the ATR gene. Using isogenic HCT116 clones, we showed that this mutation of ATR sensitizes the cells to several drugs, including SN-38 (topoisomerase I inhibitor) and VE-822 (ATR inhibitor) and exacerbates their synergistic effects. We showed that this mutation bottlenecks the replication checkpoint leading to extensive DNA damage. The combination of VE-822 and SN-38 induces an exhaustion of RPA and a subsequent replication catastrophe. Surviving cells complete replication and accumulate in G2 in a DNA-PK-dependent manner, protecting them from cell death. Together, our results suggest that RPA and DNA-PK represent promising therapeutic targets to optimize the inhibition of the ATR-CHK1 pathway in oncology. Ultimately, ATR frameshift mutations found in patients may also represent important prognostic factors.

S Phase Duration Is Determined by Local Rate and Global Organization of Replication

“…Similarly, in an osteosarcoma cell line, both FR and IOD increased by approximately 50% as a consequence of knocking down various proteins involved in origin firing, such as Treslin [77]. Inhibiting the checkpoint protein ATR causes a decrease in FR [69] that is thought to be a consequence of the activation of additional origins, further demonstrating the coupling between origin density and FR. The change of IOD as a consequence of modifying the FR can be explained as a passive mechanism-a slowed FR gives more time for dormant origins to be activated [67], while faster forks reduce the likelihood of activation of neighboring origins due to their passive replication by the ongoing fork [65,68,76,78,79].…”

“…Interestingly, the LRR of the cells seems to be robust to changes in either FR or IOD, since FR and IOD are usually coupled and can compensate for each other [65][66][67][68][69]. Decreasing FR, for example by decreasing the nucleotide levels with hydroxyurea (HU) [70][71][72] or by interfering with DNA polymerase activity with aphidicolin [73][74][75], is followed by a decrease in IOD.…”

Identification and Characterisation of Proteins Binding to a G-Quadruplex Origin G-rich Repeated Element in Mammalian Cells

“…Cells were fixed, and nuclei prepared using a pepsin-digestion procedure, as described (Egger et al, 2022). Samples were analysed using a MACSQuant flow cytometer (Miltenyi Biotec) at the Montpellier Ressources Imagerie (MRI) platform.…”

Could Inhibiting the DNA Damage Repair Checkpoint Rescue Immune-Checkpoint-Inhibitor-Resistant Endometrial Cancer?

“…Mutations in the ATR gene are most frequent in endometrial cancer compared with other tumors [125]. ATR heterozygous mutations, which may be associated with tumorigenesis and progression, have been widely demonstrated in MSI tumors [129][130][131]. Fang et al [132] induced genomic instability and chromatin amplification and rearrangement by targeting a single ATR allele in MLH1-deficient HCT116 colon cancer cells and observed the abrogation of CHK1 activation.…”