Below, work performed in the individual work packages is described.
WP1: Molecularly define the components and wiring of the mitotic replication stress recovery machinery.
Using proteomics, we revealed interaction partners of the core mitotic RSR component PICH, in the context of replication stress. Besides established interactors, we identified components of the base-excision repair (BER) pathway other DNA repair proteins (1).
In parallel, we identified proteins that are recruited to chromatin upon DNA damage during mitosis, using Xenopus egg extracts combined with mass spectrometry. Intriguingly, we found a strong enrichment of non-canonical DNA repair factors to damaged DNA during mitosis. Especially, we identified proteins involved in mitotic DNA tethering, DNA methylation, Fanconi anemia, and alternative end-joining. Subsequently, we identified that RAD52 and POLQ have opposite functions in the mitotic processing of incompletely replicated DNA into sister chromatid exchanges (SCEs)(2-3). Also, the tools that were generated in this part of the project were used in other collaborative projects (4).
Output:
1. Dataset: PICH interactome. PRIDE repository. Accession: PXD006414.
2. Heijink et al. BioRxiv. doi.org/10.1101/2021.09.17.460736. Nature Comms, under revision.
3. de Boer et al. Mitotic Chromass: mapping the proteomic response to DNA damage in mitosis. Egmond aan Zee DNA repair meeting, April. 2022.
4. Zwinderman et al. ACS Chem Biol. 2021.
WP2. Genetic profiles of cancer subgroups that are sensitive to inactivation of the mitotic RSR machinery.
To identify the genetic contexts in which cells become dependent on mitotic resolution of replication stress, we developed an mRNA signature of oncogene-induced replication stress (RS)(5). Subsequently, we used a genome-wide loss-of-function screen in PICH-deficient HAP1 cells. Besides genes involved in DNA cohesion, centromere stability and DNA damage repair, we identified C1orf112 to be synthetic lethal with PICH loss (6). In the context of oncogene-induced replication stress, we found that Rad52 is recruited to mitotic chromatin upon Cyclin E1 overexpression. Targeted inactivation of RAD52 during mitosis showed that the mitotic function of RAD52 is vital for suppressing mitotic defects upon Cyclin E1 overexpression (7). In two collaborations, we identified that pediatric neuronal cancer cells with Histone H3.3 mutations (8) or cells with defective transcription-coupled nucleotide excision repair (9) become dependent on mitotic resolution of replication stress.
Output:
5. Guerrero et al. Oncogene 2022.
6. Stok et al.Abcam Recombination meeting, London, 2019. Manuscript in preparation.
7. Kok et al. Egmond Aan Zee DNA repair meeting, April 2022manuscript in preparation.
8. Bočkaj et al. PLoS Genet. 2021
9. Geijer et al. Nature Cell Biology, 2021
WP3. Explore the feasibility of therapeutic inactivation of the replication stress recovery machinery.
We successfully set up a protocol to measure DNA replication in primary breast cancer material (10). We have successfully analyzed 74 patients. Unfortunately, the covid pandemic has blocked inclusion of patients for this study, and significantly delayed this part of the project. We are currently continuing these analyses, and will perform some of the genomic analysis after the completion of the ERC grant. As a separate clinically-relevant consequence of replication stress in cancer cells, we studied inflammatory signaling. We reviewed progress in the field (11-13), and identified how unresolved DNA replication stress leads to mitotic defects and a cGAS-dependent inflammatory response (14).
Output:
10. van den Tempel. 1st Dutch DNA replication meeting. Delft University, 2018.
11. Van Vugt and Parkes. Trends in Cancer. 2022
12. Chen et al. Biophys Acta Rev Cancer. 2022
13. Stok et al. Nucleic Acids Res. 2021
14. Heijink et al. Nature Communications. 2019
