Main supervisor: Deo Prakash Pandey, Department of Microbiology, Oslo University Hospital (OUS), Rikshospitalet. d.p.pandey@medisin.uio.no
Internal IBV co-supervisor: Pierre Chymkowitch, University of Oslo.
Introduction/background
Cancer cells frequently depend on aberrant transcriptional programs to sustain their proliferation and survival, a phenomenon referred to as transcriptional addiction. This dependency represents an attractive therapeutic vulnerability, particularly in aggressive cancers such as glioblastoma and pancreatic cancer. Transcriptional cyclin-dependent kinases (tCDKs), including CDK12 and CDK13, regulate RNA polymerase II (RNAPII) activity and productive transcriptional elongation. Our laboratory has demonstrated that pharmacological inhibition of CDK12/CDK13 rapidly suppresses RNAPII phosphorylation and nascent RNA synthesis, leading to impaired DNA replication and proliferation in cancer cells.
To identify genetic determinants of treatment response, we performed genome-wide CRISPR/Cas9 screens with two CDK12/CDK13 inhibitors. The screens identified transcription-associated genes and protein complexes whose loss may either increase sensitivity or confer resistance. This project will validate selected candidates and investigate how they influence transcription and cellular responses to CDK12/CDK13 inhibition. It is a follow-up validation project, not a new genome-wide screening project.
Research aims/questions
The overarching aim is to validate and characterize novel genetic determinants of sensitivity and resistance to CDK12/CDK13 inhibition identified through genome-wide CRISPR screens. The specific objectives are to:
- Validate selected candidate genes whose depletion alters sensitivity to CDK12/CDK13 inhibition.
- Determine how these factors influence RNAPII phosphorylation, nascent transcription and cancer cell proliferation.
- Investigate the molecular mechanisms through which these transcriptional regulators modulate the cellular response to CDK12/CDK13 inhibition.
Methods/approaches
The student will work with established human cell models and initially focus on two or three prioritized candidate genes. The experimental scope will be refined according to initial results and project duration.
- Gene depletion using siRNA, with shRNA or other genetic approaches where appropriate; validation by RT-qPCR, Western blotting and/or immunofluorescence.
- Drug-response and proliferation assays comparing control and depleted cells treated with CDK12/CDK13 inhibitors.
- Analysis of RNAPII CTD Ser2/Ser5 phosphorylation by immunofluorescence and Western blotting; nascent RNA synthesis by 5-ethynyl uridine (EU) incorporation.
- EdU incorporation to examine DNA synthesis where relevant.
- Optional advanced mechanistic work, depending on progress: RNAPII CUT&RUN/CUT&Tag or sequencing-based nascent transcription profiling, supported by other group members.
What will you learn?
The student will gain practical experience in mammalian cell culture, RNA interference, assay optimization, RT-qPCR, immunoblotting, fluorescence microscopy, drug-response experiments and quantitative analysis. The student will learn to interpret CRISPR-screen candidates, plan appropriate controls and biological replicates, critically evaluate experimental evidence, maintain research records, present results and write a scientific thesis. Depending on progress, the student may also gain exposure to functional genomics and sequencing-based approaches.
Timing
The project is designed to follow the normal IBV master's thesis period; the detailed schedule will be agreed with the student and internal supervisor. Laboratory work and access will be scheduled around institutional holidays and supervisor availability.
- Initial phase: literature review, safety training, cell culture and selection of candidate genes.
- Middle phase: optimization and validation of gene depletion, followed by drug-response and transcriptional assays.
- Final phase: mechanistic follow-up where feasible, data analysis, thesis writing and presentation.
Practical arrangements
Research will be conducted primarily in Dr. Pandey's group at Oslo University Hospital, Rikshospitalet. Laboratory training, access to relevant equipment and day-to-day support will be provided within the research group. The student will take part in group meetings, journal clubs and scientific discussions, with regular supervision and coordination with the IBV internal supervisor. Relevant laboratory safety and institutional access requirements must be completed before experimental work.
Relevant recommended courses
Relevant coursework includes molecular biology, cell biology, genetics/genomics, cancer biology and gene regulation, as well as introductory statistics or bioinformatics.
Number of students and project scope
One or two students may work on this project. With one student, the project will focus on a small, prioritized set of genes and a manageable combination of validation and transcriptional assays. With two students, distinct candidate genes or complementary mechanistic questions can be assigned, with shared training but independently defined experimental datasets and thesis objectives. The scope can be adjusted to the student's experience, interests, available time and experimental progress.