Periodic Reporting for period 4 - TENSION (Targeting replication stress recovery pathways in oncology)
Periodo di rendicontazione: 2021-02-01 al 2021-07-31
Background:
Cancer is caused by genetic aberrancies. During the development of a tumor, cells progressively accumulate DNA lesions, which is called genomic instability. ‘Replication stress’ is a key cause of genomic instability, and drives tumorigenesis. Detailed mechanisms by which tumor cells adapt to replication stress are largely unknown, precluding the identification and validation of therapeutic targets, and selection of tumors that potentially benefit from targeting replication stress salvation pathways.
Problem:
We do not know how cancer cells can resolve replication stress, and how such mechanisms can be targeted in cancer cells.
Relevance for society:
Replication stress is a cause of genomic instability and drives tumorigenesis. Replication stress and genomic instability are key characteristics in difficult-to-treat cancers, including triple-negative breast cancers and high-grade serous ovarian cancers. However, it remains unclear how these characteristics can be therapeutically targeted. These cancers specifically affect young women, and have profound societal consequences. Identification of novel therapeutics strategies will therefore not only have potential scientific but also large societal impact.
The project will provide a molecular and genetic interaction map of the newly discovered mitotic RSR machinery. The knowledge resulting from experimentally controlled model systems and patient material, will be broadly relevant, particularly for researchers studying DNA metabolism and the biology of cancer. Data and bioinformatics tools have been made publically available. Finally, this project for the first time assessed replication stress in tumors directly, which will be an important step in implementing novel therapeutic targets. The results will therefore be immediately relevant for clinicians and pharmaceutical partners who are developing novel drugs to treat genomically instable cancers.
Objectives:
This project addressed three key objectives, in three integrated work packages:
1. Molecularly define the components and wiring of the mitotic replication stress recovery machinery.
2. Uncover the genetic profiles of cancer subgroups that are sensitive to inactivation of the mitotic RSR machinery.
3. Explore the feasibility of therapeutic inactivation of the replication stress recovery machinery.
Conclusions:
1. We identified a comprehensive proteomic list of the PICH interactome.
2. A chromatin-based proteomics approach revealed extensive re-wiring of the DNA damage response during mitosis.
3. Oncogene expression leads to a targetable dependance on mitotic DNA replication.
4. Inactivation of the C1orf112 is synthetic lethal with loss of the PICH DNA translocase.
5. DNA replication kinetics can be reliably measured in fresh patient material, revealing heterogeneity between breast cancer subtypes.
Cancer is caused by genetic aberrancies. During the development of a tumor, cells progressively accumulate DNA lesions, which is called genomic instability. ‘Replication stress’ is a key cause of genomic instability, and drives tumorigenesis. Detailed mechanisms by which tumor cells adapt to replication stress are largely unknown, precluding the identification and validation of therapeutic targets, and selection of tumors that potentially benefit from targeting replication stress salvation pathways.
Problem:
We do not know how cancer cells can resolve replication stress, and how such mechanisms can be targeted in cancer cells.
Relevance for society:
Replication stress is a cause of genomic instability and drives tumorigenesis. Replication stress and genomic instability are key characteristics in difficult-to-treat cancers, including triple-negative breast cancers and high-grade serous ovarian cancers. However, it remains unclear how these characteristics can be therapeutically targeted. These cancers specifically affect young women, and have profound societal consequences. Identification of novel therapeutics strategies will therefore not only have potential scientific but also large societal impact.
The project will provide a molecular and genetic interaction map of the newly discovered mitotic RSR machinery. The knowledge resulting from experimentally controlled model systems and patient material, will be broadly relevant, particularly for researchers studying DNA metabolism and the biology of cancer. Data and bioinformatics tools have been made publically available. Finally, this project for the first time assessed replication stress in tumors directly, which will be an important step in implementing novel therapeutic targets. The results will therefore be immediately relevant for clinicians and pharmaceutical partners who are developing novel drugs to treat genomically instable cancers.
Objectives:
This project addressed three key objectives, in three integrated work packages:
1. Molecularly define the components and wiring of the mitotic replication stress recovery machinery.
2. Uncover the genetic profiles of cancer subgroups that are sensitive to inactivation of the mitotic RSR machinery.
3. Explore the feasibility of therapeutic inactivation of the replication stress recovery machinery.
Conclusions:
1. We identified a comprehensive proteomic list of the PICH interactome.
2. A chromatin-based proteomics approach revealed extensive re-wiring of the DNA damage response during mitosis.
3. Oncogene expression leads to a targetable dependance on mitotic DNA replication.
4. Inactivation of the C1orf112 is synthetic lethal with loss of the PICH DNA translocase.
5. DNA replication kinetics can be reliably measured in fresh patient material, revealing heterogeneity between breast cancer subtypes.
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
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
- We for the first-time measured replication kinetics in fresh tumor tissues. We anticipate that by the end of the project we will be able to correlate the measurements to genetic features of each individual tumor. We aim to uncover tumor subgroups that could benefit from targeted agents that inactivate replication checkpoint kinases.
- We have uncovered a thus far unknown genetic network, of genes and pathways that -when inactivated – pose a dependence on the mitotic Replication Stress Response. The mitotic Replication Stress Response will likely be a potentially valuable target for anti-cancer strategies in these genetic contexts.
- We have completed an unbiased proteomic analysis that shows how the DNA damage response is re-wired during mitosis.
- We have uncovered a thus far unknown genetic network, of genes and pathways that -when inactivated – pose a dependence on the mitotic Replication Stress Response. The mitotic Replication Stress Response will likely be a potentially valuable target for anti-cancer strategies in these genetic contexts.
- We have completed an unbiased proteomic analysis that shows how the DNA damage response is re-wired during mitosis.