Abstract 5763: How cancer drugs work, and fail: proteome-wide cellular thermal shift assay (CETSA) to define cellular mechanism of action and resistance of clinical cancer drugs

Abstract Resistance to cytotoxic cancer drugs is considered a key cause of cancer deaths worldwide. Although extensive understanding of cancer drug mechanisms of action (MoA) and resistance has been derived through focused research, our understanding of these process still remains fragmented. The Cellular Thermal Shift Assay (CETSA) was originally developed in our lab for accessing drug target engagement in intact cells (Martinez Molina, 2013), and is now widely used for this application in academia and industry. However, CETSA can also detect modulations of cellular biochemistry (Dai, 2018), e.g. effects on downstream-of-target pathways, although this application of CETSA remains relatively unexplored. Using a comprehensive and highly sensitive mass-spectrometry implementation of CETSA, we have studied the cellular biochemistry induced by more than 50 clinical cancer drugs. By applying a systematic approach, including time dependent responses to dissect sequential pathway activation, we can confirm components of previously known MoAs for these drugs but also provide comprehensive new insights into their MoA (Ramos 2024). Moreover, parallel studies in sensitive and resistant cancer cell lines provide differential pathway activation in sensitive and resistant cells (Liang, 2021), giving new insights into how cancer cells acquire drug resistance at the biochemical level. Together our data illustrate that CETSA can yield valuable information on modulation of downstream-of-target biochemistry during cancer drug action, information that is not easily accessed with other omics methods. Proteins discovered in some specific pathways also constitute new candidate drug targets for attenuate resistance responses, as well as potential clinical biomarkers. References:- Martinez Molina D, Jafari R, Ignatushchenko M, Seki T, Larsson EA, Dan C, Sreekumar L, Cao Y and Nordlund Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science, 2013 341:84.- Dai L, Zhao T, Bisteau X, Sun W, Prabhu N, Lim YT, Sobota RM, Kaldis P, Nordlund P. Modulation of Protein-Interaction States through the Cell Cycle. Cell, 2018 173(6):1481-1494. - Liang YY, Bacanu S, Sreekumar L, Ramos AR, Dai L, Michaelis M, Cinatl J, Seki T, Cao Y, Coffill CR, Lane DP, Prabhu N, Nordlund P, CETSA interaction proteomics define specific RNA modification pathways as key components of fluorouracil-based cancer drug toxicity. Cell Chemical Biology, 2021, S2451-9456, 00306.-Ramos AD, Liang YY, Surova O, Bacanu S, Gerault M, Mandal T, Ceder S, Langebäck A, Österroos A, Ward GA, Bergh J, Wiman KG, Lehmann S, Prabhu N, Lööf S, Nordlund P. Proteome-wide CETSA reveals diverse apoptosis-inducing mechanisms converging on an initial apoptosis effector stage focused at the peripheral region of the nucleus. Cell Reports, 2024 Oct 22;43:114784. Citation Format: Par Nordlund, Sara Lööf, Anderson Ramos, smaranda Bacanu, Marc Antoine Gerault, Ying Yu Liang, Wai Leong TAM, Nayana Prabhu. How cancer drugs work, and fail: proteome-wide cellular thermal shift assay (CETSA) to define cellular mechanism of action and resistance of clinical cancer drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5763.