Transcription is one of the key processes through which cells control regulation of gene expression. The aberrant transcriptional programs, resulting from the dysregulation at any of the steps at which transcription is regulated, are key to development and maintenance of cancer cells and consequently cancer cells are often hypersensitive to the targeting of the transcriptional machinery. The earlier therapeutic treatment for various cancers often focused on targeting on DNA replication and repair which was effective in killing cancer cells, but often they had very strong side effects as they also killed normal cells. Cancer cells have their own transcriptional program which sustain their proliferation and this specific transcriptional requirement, termed as transcriptional addiction can be specifically targeted to kill cancer cells.
Transcriptional cyclin-dependent kinases (CDK), which phosphorylate key residues of RNA polymerase II (RNAPII) c-terminal domain (CTD), play a major role in sustaining aberrant transcriptional programs that are key to the development and maintenance of cancer cells. We have demonstrated that pharmacological inhibition of transcriptional cyclin-dependent kinases, CDK12 and CDK13 inhibits the proliferation of many cancer. Furthermore, we have found that inhibition of CDK12/CDK13 leads to a genome-wide abrogation of RNAPII CTD phosphorylation in those cancer causing a near-total shutdown of nascent transcription and unexpectedly, DNA synthesis. We aim to characterize the effect of CDK12/13 inhibition induces transcriptional perturbations on DNA replication. We will implement technologies to monitor origin firing and replication fork movement reported by Halazonetis laboratory in papers, Macheret et al, 2018a,b, PMID: 29466339, 30487655.
Implementation of EdU-seq HU protocol
In order to better understand DNA replication initiation following CDK12/13 inhibition, we will implement EdU-seq-HU to study origin firing genome wide, which is a recently described method for mapping early S-phase replication origins. Cells, synchronized by mitotic shake-off, are released in medium containing 5-ethynyl-2′-deoxyuridine (EdU; to label nascent DNA) and hydroxyurea (HU; to limit fork progression after origin firing). After using click chemistry to tag the EdU label with a biotin conjugate that is cleavable under mild conditions, the nascent DNA is captured on streptavidin beads. One variant of EdUseq-HU allows mapping of DNA replication origins on the genome at a resolution of 10 kb, and a second variant monitors progression of replication forks.
Choice of cellular model
We have found that CDK12/CDK13 inhibitors rapidly abrogate replication in many cancer cells. We will perform these experiments in pancreatic cancer cells, PANC-1 which are very strongly affected by the inhibition of CDK12/CDK13. Additionally, because the original papers describing EdU-seq-HU are done using U2OS cells, therefore we will use U2OS cells to setup and benchmark the technology. We will perform following experiments:
- Optimization and characterization of cell cycle synchronization protocol in PANC-1 cells. We will use nocodazole to synchronize the cells in mitosis and monitor the synchronization using flow cytometry by using the lowest concentration of nocodazole. Following a successful setup, we will optimize the release into EdU and HU-containing media. These will be monitored using flow cytometry (months 1-3).
- We will monitor the effect of CDK12/CDK13 inhibition on DNA replication following release from nocodazole treatment using flow cytometry (months 1-3).
- Using EdU-seq HU, we will identify the changes in replication origins and replication fork movements following CDK12/CDK13 inhibition (months 4-8).
- Validation using qPCR on genomic loci based on published literature (PMID: 29466339, 30487655) will be performed.
- Data analyses and writing the thesis integrating the results obtained in a)-c) (months 6-10)
Implementation
Experiments a)-b) are routine experiments, c) is challenging yet possible to perform during the duration of this project. While setting up the EdU-seq HU, qPCR validations on the selected genomic loci will be performed. d) will start during this project and will continue further.
Milestones
Through the experiments above, we aim to contribute to our understanding of how transcriptional changes may regulate replication.
Current state of development
We have optimized and setup the synchronization protocol for U2OS cells, using which we have investigated the effect of transcriptional perturbations on replication using flow cytometry and have gotten very interesting results. Feel free to come by to discuss the project.
Supervision
We are looking to recruit one or two master students. Two students working together in a team are also welcome. The students will be supervised by Deo Prakash Pandey, project leader at the department of Molecular Microbiology, Oslo University Hospital (OUS), please see https://www.ous-research.no/pandey.
Currently, our group consists of a technician and a postdoctoral fellow and 3-4 talented master students. We have the necessary resources and setup to carry out these experiments. The progress and development are monitored through regular one-to-one, group meetings and there is an opportunity to present the work at the section level.