Département de biologie moléculaire
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Research Projects

Oncogene-induced DNA replication stress

The long-term goal of our research is to obtain a better understanding of cancer, such that effective, non-toxic therapies can be developed. Towards this goal, we aim to identify differences between normal cells and cancer cells. Our laboratory, in collaboration with Vassilis Gorgoulis (University of Athens Medical School) and, independently, the laboratory of Jiri Bartek (Danish Cancer Society) reported that human cancers are characterized by the presence of DNA replication stress (Figure 1). Importantly, normal cells, even from highly replicating tissues, do not have DNA replication stress.

DNA replication stress leads to damaged replication forks and formation of DNA double-strand breaks. Thus, replication stress can explain the high degree of genomic instability that is present in human cancers, as well as why the p53 gene is the most frequently mutated gene in cancer. DNA damage activates p53, which then induces cell death or senescence. Thus, in the face of DNA replication stress, cancer cells can proliferate rapidly only after the p53 gene has been mutated. Our studies have further identified activated oncogenes as the cause of DNA replication stress in human cancers. Interestingly, it appears that most oncogenes induce DNA replication stress.

Figure 1. Model for oncogene-induced DNA replication stress in human cancers selecting for p53 mutations and driving genomic instability.
Figure 1. Model for oncogene-induced DNA replication stress in human cancers selecting for p53 mutations and driving genomic instability.

Mechanism by which activated oncogenes induce DNA replication stress

We recently proposed a mechanism to explain how activated oncogenes induce DNA replication stress (Figure 2 and Movie 1). We found that both normal and cancer cells establish a large number of DNA replication origins, from which DNA replication may initiate. In normal cells, the origins that are located in genes are inactivated by the transcription machinery during the G1 phase of the cell cycle. The inactivation is very efficient, because the G1 phase lasts for ten hours or more. As a result, when cells enter S phase, DNA replication begins only from origins that are located between genes. However, in cancer cells, the G1 phase is shortened and there is not enough time for transcription to inactivate the replication origins within large genes. Initiation of replication from these origins, leads to collisions between the replication and transcription machinery and DNA damage.

The shortening of the length of the G1 phase of the cell cycle in cancer cells is due to the activation of oncogenes, which drive entry of the cancer cells into the cell cycle. Thus, the presence of DNA replication stress in human cancers can be viewed as a necessary side-effect of oncogene activation.

Figure 2. Mechanism to explain how oncogenes induce DNA replication stress.
Figure 2. Mechanism to explain how oncogenes induce DNA replication stress.
Shortening of the length of the G1 phase of the cell cycle by activated oncogenes leads to firing of DNA replication within genes, collisions between the replication and transcription machineries, DNA breaks and genomic instability.
Movie 1. Mechanism to explain how oncogenes induce DNA replication stress.
The DNA corresponding to a gene is shown in red; the DNA replication machinery is in green; and the transcription machinery in yellow.

Oncogene-induced DNA replication stress as a therapeutic target

Since the presence of DNA replication stress distinguishes cancer cells from normal cells, it could serve as a target for cancer-specific therapies. Targeting DNA replication stress has the advantage that cancer cells cannot develop resistance by switching their growth dependency from one activated oncogene to another, since most oncogenes induce replication stress.

We envision two broad approaches that could help the development of novel therapies. First, we could exploit our understanding of how oncogenes induce DNA replication stress to enhance the level of replication stress present in cancer cells. Second, since DNA replication stress leads to damaged replication forks, we could inhibit the relevant DNA repair pathways. We have identified break-induced replication (BIR) as a major pathway for repair of collapsed forks in cancer cells. BIR relies on homologous recombination to restart DNA replication from collapsed forks. BIR-initiated forks retain the D-loop formed during homologous recombination and replicate DNA in a conservative manner (Figure 3). It is very likely that the differences between BIR-initiated and origin-initiated forks could be exploited for the development of cancer therapies. We have already identified genes that function in BIR, for example, RAD52, POLD3 and POLD4, and shown that suppressing these genes inhibits proliferation specifically of cancer cells.

Figure 3. Comparison of BIR-initiated and origin-initiated forks.
Figure 3. Comparison of BIR-initiated and origin-initiated forks.
BIR-initiated forks retain the D-loop formed during strand invasion and replicate DNA in a conservative manner.

Methodology

Research in the laboratory relies heavily on high throughput sequencing approaches to study DNA replication (Figure 4). We also employ siRNA screens to identify genes that function in the response to DNA replication stress and use both cell lines and organoids as model systems.

Figure 4. DNA replication origin firing in U2OS cells, as determined by the EdUseq method.
Figure 4. DNA replication origin firing in U2OS cells, as determined by the EdUseq method.
The top bar indicates replication timing domains (early S, blue; mid S, green; late S, yellow). The lower bar indicates the genes (in color, based on the direction of transcription: green, forward; red, reverse) and intergenic regions (in gray).

Development of SARS-CoV-2 Inhibitors

The COVID-19 pandemic has already had significant impact on the health, life-style and economy of every society worldwide. One can argue that scientists competent to contribute to our understanding of this disease have a moral obligation to do so. Our expertise is best geared towards development of therapeutics. We have therefore initiated efforts to identify chemical compounds that inhibit replication of SARS-CoV-2, the agent responsible for COVID-19.

Mpro (3CLpro) is the main protease of SARS-CoV-2. Mpro cleaves the viral precursor proteins PP1a and PP1ab into functional viral proteins. The inhibition of Mpro would therefore hinder multiple processes essential for viral replication, as it would prevent the production of the mature forms of most viral proteins [1].

Several groups have determined the three-dimensional structure of SARS-CoV-2 Mpro in its apo form or bound to various inhibitors [2-8]. These structures have revealed that the N-terminus of Mpro contains two domains (domain I, residues 10-99; and domain II, residues 100-184) that adopt a chymotrypsin-like fold. The active site maps at the cleft formed between these domains and contains a Cys145-His41 catalytic dyad. A total of five protein segments comprising residues 25-27 and 44-50 from domain I and residues 140-143, 165-168 and 188-190 from domain II form the walls of the active site (Figure 5A).

Comparison of eight Mpro structures determined by different groups has revealed significant flexibility in the conformation of the Mpro active site. Specifically, binding of inhibitors leads to changes in the position of the loop containing Gln189 and of the short alpha-helix containing Ser46, resulting in widening of the active site (Figure 5B).

We intend to pursue various conformers of SARS-CoV-2 Mpro for very high content in silico screening (Fig. 1C-D). Compounds identified by this screening approach will be synthesized and examined for their ability to inhibit Mpro in vitro and viral replication in cells. Collaborators with expertise in drug screening, medicinal chemistry and virology will be assisting us in these efforts.

Figure 5.
Figure 1.
Figure 5. Conformation of the active site of SARS-CoV-2 Mpro and models for in silico screening. A. Secondary structure elements that form of the active site of Mpro. The catalytic dyad Cys145 (C145)-His41 (H41) at the base of the active site is surrounded by secondary structure elements that are contributed by domains I and II of the protease. The side chains of selected residues are shown: Thr25 (T25), Ser46 (S46), Asn142 (N142), Pro168 (P168) and Gln189 (Q189). The structure of Mpro bound to the inhibitor N3 is shown (pdb: 6lu7).
B. Overlay of Mpro active site structures showing that binding of inhibitors induces conformational changes in two secondary structure elements that form the walls of the active site: the loop containing Gln189 and the short alpha-helix containing Ser46 (see panel A). The main chain is colored gray for three structures of free Mpro (pdb codes: 6y2e, 6yb7, 6m2q), purple for the structure of Mpro bound to the inhibitor N3 (pdb: 6lu7), blue for the structure of Mpro bound to the inhibitor 11b (pdb: 6m0k) and green for three other structures of Mpro bound to inhibitors (pdb codes: 6m2n, 6y2g, 7buy). C. Van der Waals surface of the active site of Mpro bound to the inhibitor N3 (pdb: 6lu7), after removing all water and ligand molecules. The side chains of Met49 (M49), Ser46 (S46) and Cys145 (C145) are colored.
D. Van der Waals surface of the active site of Mpro bound to the inhibitor 11b (pdb: 6m0k), after removing all water and ligand molecules (left) and of the same structure after selecting different rotamers for the side chains of Met49, Ser46 and Cys145 (right).
The images in panels A-D show Mpro from the same orientation and were generated using Pymol.
References

1. Wioletta Rut, Katarzyna Groborz, Linlin Zhang, Xinyuanyuan Sun, Mikolaj Zmudzinski, Rolf Hilgenfeld, Marcin Drag. Substrate specificity profiling of SARS-CoV-2 Mpro protease provides basis for anti-COVID-19 drug design. BioRxiv, March 2020. doi 10.1101/2020.03.07.981928

2. Linlin Zhang, Daizong Lin, Xinyuanyuan Sun, Ute Curth, Christian Drosten, Lucie Sauerhering, Stephan Becker, Katharina Rox, and Rolf Hilgenfeld. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors. Science, page eabb3405, March 2020. doi: 10.1126/science.abb3405

3. Zhenming Jin, Xiaoyu Du, Yechun Xu, Yongqiang Deng, Meiqin Liu, Yao Zhao, Bing Zhang, Xiaofeng Li, Leike Zhang, Chao Peng, Yinkai Duan, Jing Yu, Lin Wang, Kailin Yang, Fengjiang Liu, Rendi Jiang, Xinglou Yang, Tian You, Xiaoce Liu, Xiuna Yang, Fang Bai, Hong Liu, Xiang Liu, Luke W. Guddat, Wenqing Xu, Gengfu Xiao, Chengfeng Qin, Zhengli Shi, Hualiang Jiang, Zihe Rao, Haitao Yang. Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature, Apr 2020. doi: 10.1038/s41586-020-2223-y

4. Wenhao Dai, Bing Zhang, Haixia Su, Jian Li, Yao Zhao, Xiong Xie, Zhenming Jin, Fengjiang Liu, Chunpu Li, You Li, Fang Bai, Haofeng Wang, Xi Cheng, Xiaobo Cen, Shulei Hu, Xiuna Yang, Jiang Wang, Xiang Liu, Gengfu Xiao, Hualiang Jiang, Zihe Rao, Lei-Ke Zhang, Yechun Xu, Haitao Yang, Hong Liu. Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease. Science, page eabb4489, Apr 2020. doi: 10.1126/science.abb4489

5. Zhenming Jin, Yao Zhao, Yuan Sun, Bing Zhang, Haofeng Wang, Yan Wu, Yan Zhu, Chen Zhu, Tianyu Hu, Xiaoyu Du, Yinkai Duan, Jing Yu, Xiaobao Yang, Xiuna Yang, Kailin Yang, Xiang Liu, Luke W. Guddat, Gengfu Xiao, Leike Zhang, Haitao Yang & Zihe Rao. Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug carmofur. Nat Struct Mol Biol, May 2020. doi: 10.1038/s41594-020-0440-6

6. Su, H.X., Yao, S., Zhao, W.F., Li, M.J., Zhang, L.K., Ye, Y., Jiang, H.L., Xu, Y.C. Identification of a novel inhibitor of SARS-CoV-2 3CLpro. doi: 10.2210/pdb6M2N/pdb

7. Owen, C.D., Lukacik, P., Strain-Damerell, C.M., Douangamath, A., Powell, A.J., Fearon, D., Brandao-Neto, J., Crawshaw, A.D., Aragao, D., Williams, M., Flaig, R., Hall, D., McAauley, K., Stuart, D.I., von Delft, F., Walsh, M.A. COVID-19 main protease with unliganded active site. doi: 10.2210/pdb6YB7/pdb

8. Su, H.X., Yao, S., Zhao, W.F., Li, M.J., Zhang, L.K., Ye, Y., Jiang, H.L., Xu, Y.C. Identification of a novel inhibitor of SARS-CoV-2 3CLpro. doi: 10.2210/pdb6M2Q/pdb