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Introduction: Our current understanding of the mechanism of action (MoA) of many cancer drugs is still incomplete. This hampers efforts in establishing efficient personalised medicine regimens, the optimisation of combination therapies, and the development of next generation drugs. Results: Here, we apply a high-resolution implementation of the proteome-wide cellular thermal shift assay (CETSA) to follow protein interaction changes induced by the antimetabolite 5-fluorouracil (5-FU) and related nucleosides. We focus on the relatively early drug effects, up to 12 hours, when this time frame is less accessible to other omics methods, while critical for defining drug MoAs. We confirm anticipated effects on the known main target, thymidylate synthase (TYMS), and enzymes in pyrimidine metabolism and DNA damage pathways. However, most interaction changes we see are for proteins previously not associated with the MoA of 5-FU, including wide-ranging effects on RNA modification and processing pathways. Attenuated responses of specific proteins in a resistant cell model identify key components of the 5-FU MoA, where intriguingly the abrogation of TYMS inhibition is not required for cell proliferation. Conclusions: When 5-FU is one of the most used and studied cancer drugs, this novel information significantly expands our understanding of the 5-FU MoA and will be important to direct further work towards dissecting its complete MoA. Furthermore, the CETSA responses of several proteins now provide ideal readouts for directly monitoring whether the required 5-FU effects are accomplished and can now be examined as CETSA-based candidate biomarkers in clinical studies. Together, this work also validates an efficient strategy for dissecting the MoA of cancer drugs, which could be broadly applicable to other cancer drugs in clinical use or in development. Citation Format: Smaranda Bacanu, Ying Yu Liang, Anderson D. Ramos, Nayana Prabhu, Pär Nordlund. CETSA interaction proteomics define specific RNA modification pathways as key components of fluorouracil based cancer drug cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1130.

The optimal use of many cancer drugs is hampered by a lack of detailed understanding of their mechanism of action (MoA). Here, we apply a high-resolution implementation of the proteome-wide cellular thermal shift assay (CETSA) to follow protein interaction changes induced by the antimetabolite 5-fluorouracil (5-FU) and related nucleosides. We confirm anticipated effects on the known main target, thymidylate synthase (TYMS), and enzymes in pyrimidine metabolism and DNA damage pathways. However, most interaction changes we see are for proteins previously not associated with the MoA of 5-FU, including wide-ranging effects on RNA-modification and -processing pathways. Attenuated responses of specific proteins in a resistant cell model identify key components of the 5-FU MoA, where intriguingly the abrogation of TYMS inhibition is not required for cell proliferation.

The use of taxanes has for decades been crucial for treatment of several cancers. A major limitation of these therapies is inherent or acquired drug resistance. A key to improved outcome of taxane-based therapies is to develop tools to predict and monitor drug efficacy and resistance in the clinical setting allowing for treatment and dose stratification for individual patients. To assess treatment efficacy up to the level of drug target engagement, we have established several formats of tubulin-specific Cellular Thermal Shift Assays (CETSAs). This technique was evaluated in breast and prostate cancer models and in a cohort of breast cancer patients. Here we show that taxanes induce significant CETSA shifts in cell lines as well as in animal models including patient-derived xenograft (PDX) models. Furthermore, isothermal dose response CETSA measurements allowed for drugs to be rapidly ranked according to their reported potency. Using multidrug resistant cancer cell lines and taxane-resistant PDX models we demonstrate that CETSA can identify taxane resistance up to the level of target engagement. An imaging-based CETSA format was also established, which in principle allows for taxane target engagement to be accessed in specific cell types in complex cell mixtures. Using a highly sensitive implementation of CETSA, we measured target engagement in fine needle aspirates from breast cancer patients, revealing a range of different sensitivities. Together, our data support that CETSA is a robust tool for assessing taxane target engagement in preclinical models and clinical material and therefore should be evaluated as a prognostic tool during taxane-based therapies.

The cellular thermal shift assay (CETSA) is a biophysical technique allowing direct studies of ligand binding to proteins in cells and tissues. The proteome-wide implementation of CETSA with mass spectrometry detection (MS-CETSA) has now been successfully applied to discover targets for orphan clinical drugs and hits from phenotypic screens, to identify off-targets, and to explain poly-pharmacology and drug toxicity. Highly sensitive multidimensional MS-CETSA implementations can now also access binding of physiological ligands to proteins, such as metabolites, nucleic acids, and other proteins. MS-CETSA can thereby provide comprehensive information on modulations of protein interaction states in cellular processes, including downstream effects of drugs and transitions between different physiological cell states. Such horizontal information on ligandmodulation in cells is largely orthogonal to vertical information on the levels of different proteins and therefore opens novel opportunities to understand operational aspects of cellular proteomes.

Fe65 is a brain enriched multi domain adaptor protein involved in diverse cellular functions. One of its binding partners is the amyloid-β (Aβ) precursor protein (APP), which after sequential proteolytic processing by secretases gives rise to the Alzheimer's Aβ peptide. Fe65 binds to the APP intracellular domain (AICD). Several studies have indicated that Fe65 binding promotes the amyloidogenic processing of APP. It has previously been shown that expression of APP increases concomitantly with a shift of its processing to the non-amyloidogenic pathway during neuronal differentiation. In this study we wanted to investigate the effects of neuronal differentiation on Fe65 expression. We observed that differentiation of SH-SY5Y human neuroblastoma cells induced by retinoic acid (RA), the phorbol ester PMA, or the γ-secretase inhibitor DAPT resulted in an electrophoretic mobility shift of Fe65. Similar effects were observed in rat PC6.3 cells treated with nerve growth factor. The electrophoretic mobility shift was shown to be due to phosphorylation. Previous studies have shown that Fe65 phosphorylation can prevent the APP-Fe65 interaction. We propose that phosphorylation is a way to modify the functions of Fe65 and to promote the non-amyloidogenic processing of APP during neuronal differentiation.

Fe65 is an adaptor protein involved in both processing and signaling of the Alzheimer-associated amyloid-β precursor protein, APP. Here, the subcellular localization was further investigated using TAP-tagged Fe65 constructs expressed in human neuroblastoma cells. Our results indicate that PTB2 rather than the WW domain is important for the nuclear localization of Fe65. Electrophoretic mobility shift of Fe65 caused by phosphorylation was not detected in the nuclear fraction, suggesting that phosphorylation could restrict nuclear localization of Fe65. Furthermore, both ADAM10 and γ-secretase inhibitors decreased nuclear Fe65 in a similar way indicating an important role also of α-secretase in regulating nuclear translocation.

A key step of the action of most drugs is their binding (engagement) of the target protein(s). However, limitations in the available methods for directly accessing this critical step have added uncertainties in many stages of drug development. We have developed a generic method for evaluating drug binding to target proteins in cells and tissues (Martinez Molina et al. Science, 341:84). The technique is based on the physical phenomenon of ligand-induced thermal stabilization of target proteins; the method is therefore called the cellular thermal shift assay (CETSA). The technique allows for the first time to directly measure the biophysical interactions between a drug and protein target in non- engineered cells and tissues. We show that using CETSA a range of critical factors for drug action can be addressed at the target engagement level, including drug transport and activation, off-target effects, drug resistance as well as drug distribution in cells, patient and animal tissues. Although CETSA was first developed for evaluating drug binding to target proteins in cells it is also useful for characterizing physiological interactions, including protein-protein, protein-metabolite and protein-nucleic acid interactions (Martinez Molina & Nordlund, Ann Rev Pharm Toxic 2016 56:141). In the proteome-wide CETSA experiment, interactions with more than 7000 proteins can be measured in parallel (Savitski, Science 2014 346;6205). This strategy therefore constitutes a novel mean for discovering key interactions determining the fate of cancer cells during therapy and resistance – interactions that can serve as biomarkers or candidates for novel drug targets in cancer therapy. Citation Format: Ying Yu Liang, Anderson Ramos, Henriette Laursen, Olga Surova, Johan Lengqvist, Sara Lööf, Anette Langebäck, Smaranda Bacanu, Jonas Bergh, Pär Nordlund. Biophysics in the cell—CETSA to study drug binding and cellular processes in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5655.

Selective CDK4/6 inhibition is now part of standard treatment for HR+ breast cancer. There is a pressing need for a practical biomarker that can provide early indication of the biologic activity of these agents and correlate with clinical outcome. Expression of thymidine kinase (TK) occurs in actively proliferating cells, is E2F-dependent and is downregulated after CDK4/6 inhibitor-mediated G1 arrest. Here, we have investigated TK activity (TKA) as a pharmacodynamic marker for CDK4/6 inhibition in both preclinical and clinical contexts and demonstrate the potential of this assay as CDK4/6 inhibitors are further developed. TKA in response to the CDK 4/6 inhibitor palbociclib (palbo) was studied in cell culture, mouse models and samples from clinical studies. TKA was determined by the DiviTum Assay (Biovica, Sweden). In culture, the intracellular TKA and TK release in response to palbo were determined by analysis of cellular extracts and tissue culture media. In vivo, mice bearing human xenografts were treated with vehicle or palbo to determine effects on TKA in serum and in tumor extracts. Finally, serum TKA (s-TKA) was assayed in patient samples procured before and after palbo treatment in a Phase 1 study of palbo and the MEK inhibitor PD0325901 (NCT02022982). Palbo was administered on 3/4 (21 of every 28 days) or 4/4-week (continuous) schedules. In K562 cells, intracellular TKA levels exhibited a clear dose response to palbo. Reductions in TKA were seen at lower drug concentrations than those affecting cell viability. Similar results were observed in MCF-7 xenografts, where lower TKA in serum and tumor was observed after palbo treatment, whereas in E0771 xenografts, no change in TKA was observed. Clinically, serum samples were obtained before and at various time points after palbo exposure in 20 cancer patients. One patient, who discontinued study treatment at the end of cycle 1, demonstrated an increase in s-TKA. In contrast, for the other 19 patients, there was a marked decrease in s-TKA at day 21. Pre-treatment s-TKA values were higher than C1D21 (paired t-test, p Citation Format: Geoffrey Shapiro, Magnus Neumuller, Sara Lööf, Smaranda Bacanu, Takahiro Seki, John Hilton, Khan Do, Nicole Chau, Leena Gandhi, Joseph W. Gibson, Robert Distel, Pawel Niekrasz, Edward M. Suh, Mattias Bergqvist. Thymidine kinase activity as a response marker for CDK 4/6 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2340. doi:10.1158/1538-7445.AM2017-2340