For over seventy years, the chemotherapy drug 6-thioguanine (6-TG) has been a stalwart in the fight against leukemia, a group of blood cancers characterized by the uncontrolled proliferation of white blood cells. While its efficacy in eradicating cancerous cells is well-established, a persistent puzzle has remained: why do some leukemia cells succumb to 6-TG’s assault, while others exhibit remarkable resilience? This long-standing question has now taken a significant turn with the groundbreaking discovery of a protein, NUDT5, and its unexpected, non-enzymatic role in dictating cellular response to this crucial therapeutic agent.
This pivotal research, a collaborative effort between scientists at the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, the University of Oxford, the Weizmann Institute of Science, and the University of Dundee, has illuminated a previously unrecognized layer of molecular complexity underlying drug resistance. The findings, published in a leading scientific journal, challenge conventional understandings of drug action and open new avenues for therapeutic intervention.
The Enigmatic Nature of NUDT5
The journey to uncovering NUDT5’s significance began with a preceding discovery from the Kubicek and Huber laboratories. This earlier work, published in Science in 2025, revealed that NUDT5 possessed a crucial cellular function that transcended its known enzymatic activity. Typically, proteins function as enzymes by catalyzing specific biochemical reactions. However, NUDT5 was found to operate as more than just a catalyst; it also acts as a molecular scaffold, playing a vital role in the intricate organization of cellular metabolism. This dual functionality hinted at a deeper, more nuanced role within the cell than previously appreciated.
The current study sought to investigate whether this non-canonical function of NUDT5 was implicated in the differential response of leukemia cells to 6-TG. The initial hypothesis, as articulated by co-first author Tuan-Anh Nguyen from CeMM, leaned towards NUDT5’s enzymatic activity being the key mediator. "We initially expected that NUDT5 would influence 6-TG through its enzymatic activity," Nguyen stated. "Instead, we found that inhibiting the enzyme had little effect. What mattered was whether the protein itself was present." This observation marked a crucial turning point in the research, shifting the focus from chemical inhibition to the complete removal of the protein.
Targeted Protein Degradation: A New Paradigm in Drug Discovery
To rigorously test the hypothesis that NUDT5’s physical presence, rather than its enzymatic function, dictated cellular response to 6-TG, the researchers employed a cutting-edge strategy known as targeted protein degradation. Unlike traditional drug development, which often aims to block the activity of a target protein, targeted protein degradation seeks to eliminate the protein entirely from the cell. This approach offers a powerful tool to dissect the specific contributions of a protein’s structure and presence versus its catalytic function.
The development of a specialized cell-based platform was instrumental in accelerating the discovery of molecules capable of degrading NUDT5. This platform guided medicinal chemistry efforts, culminating in the creation of "dNUDT5," a highly potent and selective NUDT5 degrader. Anne-Sophie Marques, a first author of the paper and whose work at Oxford was central to these advancements, highlighted the platform’s significance. "We developed a cell-based platform to accelerate the discovery of NUDT5 degraders. This platform helped guide the medicinal chemistry efforts that ultimately produced dNUDT5, our most active degrader," Marques explained.
A dedicated medicinal chemistry program, spearheaded by the Huber laboratory at the University of Oxford, was responsible for synthesizing a diverse library of highly selective NUDT5 degraders. Crucially, alongside these degraders, the team also engineered matched control compounds. These controls were designed to bind to NUDT5, mimicking the initial interaction of a degrader, but crucially, they lacked the ability to trigger the protein’s destruction. This meticulous control allowed researchers to isolate the effect of protein removal from mere binding.
Differentiating Function: Enzyme Inhibition vs. Protein Degradation
The comparative analysis between these novel degraders and conventional NUDT5 inhibitors yielded striking results. When cells were treated with inhibitors designed to block NUDT5’s enzymatic activity, there was no significant alteration in their sensitivity to 6-TG. This outcome reinforced the initial suspicion that the enzyme’s catalytic function was not the primary driver of drug response.
However, when NUDT5 was eliminated from the cells using dNUDT5, a dramatic shift occurred. The cells were significantly protected from the cytotoxic effects of 6-TG, demonstrating a clear increase in drug resistance. These findings were further corroborated by independent genetic experiments, which led to the same definitive conclusion: the physical removal of NUDT5 conferred resistance to 6-TG.
Professor Kilian Huber, from the Centre for Medicines Discovery at the University of Oxford and a co-corresponding author of the study, emphasized the power of this methodology. "Chemical degraders give us a way to separate what a protein does as an enzyme from what it does as a physical presence in the cell," Professor Huber stated. "In this case, that distinction was decisive: removing NUDT5 revealed biology that conventional inhibitors missed." This statement underscores the transformative potential of targeted protein degradation in unraveling complex biological mechanisms that have eluded traditional biochemical approaches.
A Hidden Mechanism Beyond Catalysis
The study’s findings strongly suggest that NUDT5’s influence on thiopurine drug sensitivity operates through a mechanism entirely independent of its catalytic activity. This implies that focusing solely on the biochemical reactions catalyzed by NUDT5 would provide an incomplete picture of its role in cellular responses to drugs like 6-TG.
The experimental results, as they emerged, provided compelling evidence. Ludwig Bauer, another first author of the paper, described the moment of revelation: "As the results came in, it became immediately clear that the dNUDT5 was protecting cells from 6-thioguanine toxicity in a dose-dependent manner. That was an incredibly exciting moment." The dose-dependent nature of this protective effect further solidified the link between NUDT5 presence and drug resistance.
Furthermore, the research unearthed an intriguing interplay between NUDT5 and another protein, NUDT15. NUDT15 is already a well-established factor influencing patient responses to thiopurine drugs, with its deficiency often leading to increased sensitivity to these therapies. The new study revealed that NUDT5 and NUDT15 appear to exert opposing effects on thiopurine response.
Specifically, while the loss of NUDT15 renders cells more susceptible to 6-TG, a reduction in NUDT5 levels, achieved through degradation, confers resistance. This suggests a finely tuned balance, where these two proteins, acting through distinct mechanisms, can push cellular response in opposite directions. This complex regulatory network highlights the intricate molecular dialogue that governs therapeutic outcomes.
Corresponding author Stefan Kubicek, a Principal Investigator at CeMM, summarized the broader implications of this discovery. "Our results show that proteins can have important biological functions that are completely independent of their enzymatic activity," Kubicek explained. "By removing NUDT5 rather than simply inhibiting it, we were able to uncover a hidden layer of biology that helps determine how cells respond to a clinically important drug." This emphasizes the need to move beyond a singular focus on enzymatic function when investigating protein roles in disease and drug response.
Implications for Leukemia Treatment and Beyond
While this research does not immediately translate into a new drug on the market, it provides a critical new understanding of the molecular mechanisms governing the effectiveness of a long-standing leukemia therapy. By demonstrating that NUDT5 influences 6-TG response through a non-catalytic function, the study offers a powerful new lens through which to examine the variability in thiopurine treatment outcomes observed among patients.
The findings also serve as a compelling case study for the utility of targeted protein degradation in biological research. This advanced technique has proven invaluable in revealing biological functions that might otherwise remain obscured when relying solely on conventional enzyme inhibitors. The ability to precisely remove a protein and observe the downstream effects is a powerful method for dissecting complex cellular pathways.
The implications extend beyond leukemia. Many drugs currently in use or under development target specific proteins. The discovery of NUDT5’s non-enzymatic function suggests that other proteins, currently understood primarily through their catalytic roles, might possess similar scaffolding or organizational functions that are crucial for cellular processes and drug responses. This could necessitate a re-evaluation of drug targets and the development of new therapeutic strategies that consider the multifaceted nature of protein function.
The research was generously supported by a consortium of prestigious funding bodies, including the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program, the Austrian Science Fund (FWF), the Vienna Science and Technology Fund (WWTF), the Marie Skłodowska-Curie Actions Postdoctoral Fellowships program, the Innovative Medicines Initiative 2 Joint Undertaking (IMI2 JU), the Wellcome Trust, Merck Sharp & Dohme Corp., and Janssen Pharmaceutica NV. This broad support underscores the international recognition of the significance of this research.
The discovery of NUDT5’s hidden role marks a significant advancement in our understanding of leukemia drug resistance. It not only sheds light on the intricate molecular mechanisms governing 6-TG efficacy but also heralds a new era of molecular investigation, where innovative technologies like targeted protein degradation are crucial for uncovering the full spectrum of protein function and its implications for human health. Future research will likely focus on further elucidating the precise scaffolding role of NUDT5 and exploring whether modulating its function, perhaps in conjunction with NUDT15, could lead to more effective therapeutic strategies for leukemia and potentially other cancers.
