Pär Nordlund's Group

CETSA

Proteins are targets for the majority of drugs and are involved in most cellular process. However, to study proteins and their interactions in live cells and tissues has been very challenging. Recently our group introduced a new method to meet this challenge: the Cellular Thermal Shift Assay (CETSA) (Science 341(6141):84-87). CETSA is a biophysical assay based on the principle of ligand-induced thermal stabilization of target proteins, meaning that a proteins melting temperature will change upon ligand interaction. Thus, by heating samples (lysate, cells or tissue pieces) to different temperatures, and quantifying proteins in the soluble fraction we can detect altered protein interactions after for example drug treatment. This can either be done for selected proteins of interest by using anti-body based assays or proteome wide by using mass spec. CETSA allows direct monitoring of ligand binding to a specific target (target engagement) in lysate, live cells or even tissue pieces. It can also be used to study downstream effects on protein interaction, providing a novel perspective on protein function in situ.

Using CETSA to study cancer

The current focus in our lab is to explore how CETSA can be used in order to improve cancer treatment. We study key processes in cancer cells as well as the effect of drug treatment on protein interactions. Today, a main drawback of many cytostatics is innate or acquired drug resistance. Thus, we also work on gaining knowledge about the cellular mechanisms during resistance development. These approaches will hopefully lead to the discovery of new biomarkers that in the future can be used to predict treatment outcome and help guiding personalized medicine.

Some of our projects also include studies on tumour associated immune cells and the interaction between the immune system and tumour cells. Novel findings in the immune-oncology field have raised hope that many previously untreatable cancers might be possible to target. For some patient groups such treatment may represent actual cures. However, a large proportion of patients does not respond to treatment or develop resistance. Thus, there is a large interest to gain a better understanding of the intricate interplay between the immune system and the tumour cells. Improved immunotherapies could be a game changer in cancer research and we believe that CETSA has the potential to contribute to this goal.

Taken together, we use CETSA to:

• Provide detailed mechanistic understanding of cellular proteomics in basic cancer processes
• Gain a better understanding of protein interactions in cancer-related immune processes
• Provide in-depth mechanistic understanding of resistance to cytostatics and immunotherapies
• Identify novel candidate drug targets
• Find new protein-based biomarkers that report on therapeutic efficacy and resistance that can help guide personalized therapy

In summary, we use CETSA to study protein interactions in order to acquire data that in has the potential to improve future cancer treatment.

Group members

Pär Nordlund, Professor
Sara Lööf, Project manager 
Anderson Ramos, Research Specialist 
Tamoghna Mandal, Post doc 
Mohd Younis Bhat, Post doc 
Jiawen Lyu, Post doc 
Reto Rufener, Post doc 
Smaranda Bacanu, PhD student
Mindaugas Raitelaitis, PhD student
 

Funding

• Barncancerfonden
• Cancerfonden
• Knut och Alice Wallenbergs stiftelse
• Vetenskapsrådet Medicin och Hälsa

Selected publications

CETSA interaction proteomics define specific RNA-modification pathways as key components of fluorouracil-based cancer drug cytotoxicity.
Liang YY, Bacanu S, Sreekumar L, Ramos AD, Dai L, Michaelis M, Cinatl J, Seki T, Cao Y, Coffill CR, Lane DP, Prabhu N, Nordlund P
Cell Chem Biol 2022 Apr;29(4):572-585.e8

CETSA-based target engagement of taxanes as biomarkers for efficacy and resistance.
Langebäck A, Bacanu S, Laursen H, Mout L, Seki T, Erkens-Schulze S, Ramos AD, Berggren A, Cao Y, Hartman J, van Weerden W, Bergh J, Nordlund P, Lööf S
Sci Rep 2019 12;9(1):19384

Horizontal Cell Biology: Monitoring Global Changes of Protein Interaction States with the Proteome-Wide Cellular Thermal Shift Assay (CETSA).
Dai L, Prabhu N, Yu LY, Bacanu S, Ramos AD, Nordlund P
Annu. Rev. Biochem. 2019 Jun;88():383-408

Modulation of Protein-Interaction States through the Cell Cycle.
Dai L, Zhao T, Bisteau X, Sun W, Prabhu N, Lim Y, et al
Cell 2018 05;173(6):1481-1494.e13

Thermal proximity coaggregation for system-wide profiling of protein complex dynamics in cells.
Tan CSH, Go KD, Bisteau X, Dai L, Yong CH, Prabhu N, et al
Science 2018 03;359(6380):1170-1177

Dual blockade of the lipid kinase PIP4Ks and mitotic pathways leads to cancer-selective lethality.
Kitagawa M, Liao PJ, Lee KH, Wong J, Shang SC, Minami N, et al
Nat Commun 2017 12;8(1):2200

CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil.
Almqvist H, Axelsson H, Jafari R, Dan C, Mateus A, Haraldsson M, et al
Nat Commun 2016 Mar;7():11040

The Cellular Thermal Shift Assay: A Novel Biophysical Assay for In Situ Drug Target Engagement and Mechanistic Biomarker Studies.
Martinez Molina D, Nordlund P
Annu. Rev. Pharmacol. Toxicol. 2016 ;56():141-61

The cellular thermal shift assay for evaluating drug target interactions in cells.
Jafari R, Almqvist H, Axelsson H, Ignatushchenko M, Lundbäck T, Nordlund P, et al
Nat Protoc 2014 Sep;9(9):2100-22

Tracking cancer drugs in living cells by thermal profiling of the proteome.
Savitski MM, Reinhard FB, Franken H, Werner T, Savitski MF, Eberhard D, et al
Science 2014 Oct;346(6205):1255784

Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell.
Asial I, Cheng YX, Engman H, Dollhopf M, Wu B, Nordlund P, et al
Nat Commun 2013 ;4():2901

Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay.
Martinez Molina D, Jafari R, Ignatushchenko M, Seki T, Larsson EA, Dan C, et al
Science 2013 Jul;341(6141):84-7