Abstract LB088: MS-CETSA deep functional proteomics uncovers a new induced DNA-repair programs leading to resistance to DNA-damaging drugs

Abstract Mechanisms for resistance to cytotoxic cancer drugs are dependent on dynamic changes in the biochemistry of cellular pathways, information which is hard to obtain at the systems level. We are using a deep functional proteomics implementation of CETSA (Cellular Thermal Shift Assay) revealing a range of induced biochemical responses to DNA-damaging drugs in resistant and sensitive cell lines. 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 induced by cancer drugs (Liang 2021, Ramos 2024), although this application of CETSA remains relatively unexplored. Using proteome-wide CETSA we have studied the time dependent response to more than 10 DNA damaging drugs some in both resistant and sensitive cells. Responses often reflect some known pathway modulations but also novel components of drug mechanism of action and resistance. As an example, gemcitabine rapidly induced binding to ribonucleotide reductase and CETSA shifts reflecting DNA damage responses. However, after 3-8 hours the responses diverge dramatically where sensitive cells show induction of characteristic CETSA signals for early apoptosis (Ramos, 2024), while resistant cells reveal biochemical modulations reflecting transition through a distinct DNA-damage signaling state, including opening of cell cycle checkpoints and induction of translesion DNA synthesis (TLS) programs, allowing bypass of damaged DNA-adducts. The data also reveal the induction of a new program, labeled the Auxiliary DNA Damage Repair (ADDR) protein ensemble likely supporting DNA replication at damaged sites. We show that the ADDR response can be attenuated in resistant cells by an ATR inhibitor re-establishing gemcitabine sensitivity and demonstrate ATR as a key signaling node of this response. The ADDR response is also seen for several other DNA-damaging drugs (eg cisplatin and cladribine). Prominent biochemical responses are typically seen in several other cellular pathways, where ribosomal biogenesis stands out as frequently affected process for a number of these drugs (Liang, 2021). Together our data illustrate that CETSA can yield highly valuable information on modulation of downstream-of-target pathway biochemistry in cancer drug action and resistance. Proteins discovered in some specific pathway also constitute new candidate drug targets for attenuate resistance responses, as well as potential clinical biomarkers. - Martinez Molina et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science, 2013 341(6141):84-7. - Dai et al. Modulation of Protein-Interaction States through the Cell Cycle. Cell, 2018 173(6):1481-1494. - Liang et al. CETSA interaction proteomics define specific RNA modification pathways as key components of fluorouracil-based cancer drug toxicity. Cell Chemical Biology, 2021, S2451-9456(21)00306-8. - Ramos et al. 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 43(10):114784. Citation Format: Mindaugas Raitelaitis, Marc-Antoine Gerault, Smaranda Bacanu, Ying Yu Liang, Khalidah Khalid, Hai Van Le, Hui Min Vivian Teo, Jia Hui Jane Lee, Jiawen Lyu, Allison Chan, Anand Jeyasekharan, Sara Lööf, Wai Leong Tam, Nayana Prabhu, Pär Nordlund. MS-CETSA deep functional proteomics uncovers a new induced DNA-repair programs leading to resistance to DNA-damaging drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB088.