Mikael Altun

Precise and flexible control of gene expression is essential for dissecting gene function in complex biological systems. Although recent developments in genetic engineering and CRISPR/Cas9 technology have expanded tools for gene activation, suppression, and editing, their application in physiologically relevant models remains challenging, time-consuming, and expensive. Here, we present a modular, doxycycline-inducible vector system that integrates gene overexpression, shRNA-mediated knockdown, and CRISPR/Cas9-mediated regulation within a single, lentivirus-compatible system. The modular design allows rapid exchange of selection markers, epitope tags, and reporters via Gateway cloning, providing broad adaptability across experimental settings. In addition to standard fluorescent and luminescent reporters, the system includes advanced sensors, such as Fucci cell cycle reporters, to enable monitoring of cellular processes. By combining fluorescence barcoding with combinatorial genetic perturbations, the platform supports multiplexed analysis of gene function and genetic interactions through phenotypic characterization by fluorescence imaging or flow cytometry. We demonstrate its utility in vivo with breast cancer intraductal xenografts, which suggest that ER+ breast cancer cells (MCF7) rely on androgen, estrogen, and progesterone receptors for in vivo growth. This versatile gene perturbation system provides tight temporal control, streamlined implementation, and high-content phenotyping capacity facilitating efficient in vitro and in vivo studies while reducing the use of animals in in vivo validation experiments. It thus expands the experimental repertoire for dynamic, multigene interrogation in complex systems.

Precise and flexible control of gene expression is essential for dissecting gene function in complex biological systems. Although recent developments in genetic engineering and CRISPR/Cas9 technology have expanded tools for gene activation, suppression and editing, their application in physiologically relevant models remains challenging, time consuming, and expensive. Here, we present a modular, doxycycline-inducible vector system that integrates gene overexpression, shRNA-mediated knockdown, and CRISPR/Cas9-mediated regulation within a single, lentivirus-compatible system. The modular design allows rapid exchange of selection markers, epitope tags and reporters via Gateway cloning, providing broad adaptability across experimental settings. In addition to standard fluorescent and luminescent reporters, the system includes advanced sensors, such as FUCCI cell cycle reporters, to enable monitoring of cellular processes. By combining fluorescence barcoding with combinatorial genetic perturbations, the platform supports multiplexed analysis of gene function and genetic interactions through phenotypic characterization by multiplex fluorescence imaging or flow cytometry. We demonstrate its utility in vivo with breast cancer intraductal xenografts to reveal that ER+ breast cancer cells (MCF7) rely on androgen (AR), estrogen (ER) and progesterone receptors (PR) for in vivo growth. This versatile gene perturbation system provides tight temporal control, streamlined implementation, and high-content phenotyping capacity facilitating efficient in vitro and in vivo studies while reducing the use of animals in in vivo validation experiments. It thus expands the experimental repertoire for dynamic, multigene interrogation in complex systems.

Cancer cells require nucleotides to meet proliferation demands, which has been effectively exploited by nucleoside analog (NA) drugs. Despite their success, we still do not completely understand factors dictating their anticancer effect. Here, we report that a NUDT5 scaffolding function indirectly regulates the global efficacy of anti-cancer NA drugs. Mechanistically, PROTAC- or RNAi-mediated NUDT5 depletion, not inhibition, desensitizes cells to 6-thioguanine (6TG) and other NA drugs proportionally with NUDT5 abundance. NUDT5-depleted cells appear locked into de novo purine biosynthesis (DNPB), thus impairing salvage of nucleotide precursors and NA drug activation. Specifically, NUDT5 interacts with phosphoribosyl amidotransferase (PPAT), the rate-limiting DNPB enzyme, to putatively regulate its activity, DNPB flux, and resultant phosphoribosyl pyrophosphate (PRPP) allocation. Collectively, these results suggest that NUDT5 controls NA drug efficacy by coordinating nucleotide synthesis and PRPP utilization, making it a potential biomarker of clinical efficacy and target for finetuning antimetabolite therapies. Statement of significance: NUDT5 non-enzymatically regulates the global efficacy of anti-cancer nucleoside analog drugs by interacting with PPAT to control DNPB and allocation of PRPP, thereby establishing it as a biomarker of clinical significance.

Targeted protein degradation (TPD) via PROTACs and molecular glues holds significant therapeutic promise but demands detailed mechanistic evaluation in live cells to fully understand compound behavior and optimize efficacy. Here, we present an integrated, cell-first platform that combines a modular degradation assay with E3 ligase target engagement readouts for comprehensive assessment of TPD molecules in cells and use it to evaluate PROTACs towards NUDT5. To mimic endogenous degradation conditions and TPD amenability, we established a fusion protein expression system consisting of a lysine-free FKBP12 F36V PROTAC handle (FKBPV _K0 ) and used a HiBiT/akaLuc dual luciferase reporter to accurately measure degradation dynamics. This set-up identified a VHL-dependent NUDT5 PROTAC, DDD2, that induced robust NUDT5 degradation, despite impaired NUDT5 binding in vitro and in cellulo , but no CRBN-dependent degraders. NUDT5 lysine availability mapping with DDD2 and FKBP12 F36V-directed PROTACs suggested that the CRL4 ^CRBN complex is more sensitive to target lysine accessibility than CRL2 ^VHL , which may have implications for E3 ligase choice and therapeutic resistance. CeTEAM drug biosensors were also established towards CRBN and VHL to quantitatively monitor degrader engagement in living cells and confirmed that the tested CRBN-directed NUDT5 PROTACs poorly engaged the E3. All together, this platform provides a versatile and scalable framework for TPD molecule discovery in a cellular context.

Cellular target engagement technologies enable quantification of intracellular drug binding; however, simultaneous assessment of drug-associated phenotypes has proven challenging. Here, we present cellular target engagement by accumulation of mutant as a platform that can concomitantly evaluate drug-target interactions and phenotypic responses using conditionally stabilized drug biosensors. We observe that drug-responsive proteotypes are prevalent among reported mutants of known drug targets. Compatible mutants appear to follow structural and biophysical logic that permits intra-protein and paralogous expansion of the biosensor pool. We then apply our method to uncouple target engagement from divergent cellular activities of MutT homolog 1 (MTH1) inhibitors, dissect Nudix hydrolase 15 (NUDT15)-associated thiopurine metabolism with the R139C pharmacogenetic variant, and profile the dynamics of poly(ADP-ribose) polymerase 1/2 (PARP1/2) binding and DNA trapping by PARP inhibitors (PARPi). Further, PARP1-derived biosensors facilitated high-throughput screening for PARP1 binders, as well as multimodal ex vivo analysis and non-invasive tracking of PARPi binding in live animals. This approach can facilitate holistic assessment of drug-target engagement by bridging drug binding events and their biological consequences.

PARP inhibitors (PARPi) predominantly targeting PARP1 and PARP2 have revolutionized cancer therapy by selectively killing cancer cells with defective DNA repair. However, achieving PARP1 or PARP2-selective inhibitors is difficult due to their close structural homology. Selectivity profiling is typically done with purified proteins, but these lack the complexity of intracellular environments and could therefore be inaccurate. Here, we duplex PARP1 L713F-GFP and PARP2 L269A-mCherry CeTEAM drug biosensors to systematically characterize binding and cell cycle alterations of 27 PARPi at the single cell level. Our results reveal that most PARPi are generally equipotent for both PARPs, including the next-generation drug, senaparib. However, benzimidazole carboxamide (niraparib) derivatives demonstrated PARP1-selective tendencies, while pthalazinones (olaparib) favored PARP2. AZD5305, a reported PARP1-selective inhibitor with characteristics of both series, was the exception and appears ∼1600-fold more potent towards PARP1. In agreement with current understanding, we see that PARP trapping phenotypes positively correlate with PARP1/2 binding potency, while some potent binders, such as veliparib, did not – likely reflecting their allosteric influence on DNA retention. We also assessed the effect of the PARP1/2 active site component, HPF1, on intracellular PARPi binding and see that HPF1 depletion elicits slight deviations in apparent binding potency, while contributing additively to PARP-DNA trapping phenotypes. The PARP1/2 CeTEAM platform thus provides a structural roadmap for the development of selective PARPi and should facilitate the discovery of better targeted therapies. Furthermore, our results highlight that multiplexing CeTEAM biosensors and layered genetic perturbations can systematically profile determinants of intracellular drug selectivity.

The facultative loss of muscle mass and function during aging (sarcopenia) poses a serious threat to our independence and health. When activities of daily living are impaired (clinical phase), it appears that the processes leading to sarcopenia have been ongoing in humans for decades (preclinical phase). Here, we examined the natural history of sarcopenia in male outbred rats to compare the occurrence of motor behavioral deficits with the degree of muscle wasting and to explore the muscle-associated processes of the preclinical and clinical phases, respectively. Selected metrics were validated in female rats. We used the soleus muscle because of its long duty cycles and its importance in postural control. Results show that gait and coordination remain intact through middle age (40–60% of median lifespan) when muscle mass is largely preserved relative to body weight. However, the muscle shows numerous signs of remodeling with a shift in myofiber-type composition toward type I. As fiber-type prevalence shifted, fiber-type clustering also increased. The number of hybrid fibers, myofibers with central nuclei, and fibers expressing embryonic myosin increased from being barely detectable to a significant number (5–10%) at late middle age. In parallel, TGFβ1, Smad3, FBXO32, and MuRF1 mRNAs increased. In early (25-month-old) and advanced (30-month-old) aging, gait and coordination deteriorate with the progressive loss of muscle mass. In late middle age and early aging due to type II atrophy (>50%) followed by type I atrophy (>50%), the number of myofibers did not correlate with this process. In advanced age, atrophy is accompanied by a decrease in SCs and βCatenin mRNA, whereas several previously upregulated transcripts were downregulated. The re-expression of embryonic myosin in myofibers and the upregulation of mRNAs encoding the γ-subunit of the nicotinic acetylcholine receptor, the neuronal cell adhesion molecule, and myogenin that begins in late middle age suggest that one mechanism driving sarcopenia is the disruption of neuromuscular connectivity. We conclude that sarcopenia in rats, as in humans, has a long preclinical phase in which muscle undergoes extensive remodeling to maintain muscle mass and function. At later time points, these adaptive mechanisms fail, and sarcopenia becomes clinically manifest.

The facultative loss of muscle mass and function during aging (sarcopenia) poses a serious threat to our independence and health. Much of what we know about sarcopenia derive from studies on small rodents which serve as models for the human condition. Here we used outbred male rats to study the natural history of sarcopenia with the aim to compare behavioral motor deficits with degree of muscle wasting and to find processes underlying the preclinical phase of sarcopenia. Selected metrics were validated in female rats. We used the soleus muscle because of its long duty cycles in everyday life and significance for postural control. The results show that gait and coordination remain intact through middle age (12-18 months; 40%-60% of median lifespan) when muscle mass relative to body weight is largely maintained. The muscle, however, show multiple signs of remodeling with a shift in myofiber-type composition towards type I, increased number of hybrid fibers and fibers expressing embryonic myosin. In parallel with the shift in fiber type prevalence, fiber type clustering increased. The latter combined with a transcriptional upregulation of nicotinic acetylcholine receptor subunit  (CHRN ), NCAM and Myogenin (MYOG) suggests a remodeling driven by myofiber denervation. Additional stigmata were increased number of central nuclei, transcriptional upregulation of Smad3, FBXO32, and MuRF1mRNAs; unaltered density of satellite cells (SC) and Catenin mRNA. In early (25-month-old) and advanced (30-month-old) aging, gait and coordination deteriorate as loss of muscle mass progresses. In late middle age and early aging because of a type II atrophy (>50%), followed by type I atrophy (>50%). Number of myofiber did not correlate with this process. At advance age, the atrophy is accompanied by a decrease in SC and Catenin mRNA while several transcripts upregulated earlier-on were downregulated. In contrast, glial-derived nerve growth factor (GDNF) mRNA abundance peaked at endpoint. We conclude that sarcopenia in the rat as in humans, has a long preclinical phase where the muscle undergoes extensive remodeling to preserve muscle mass and function. The data supports the notion that myofiber denervation is the instigating mechanism, initially affecting large type II motor units (MUs) and at more advanced stage also the smaller type I MUs.