FR900359, an inhibitor of guanine nucleotide dissociation, effectively blunts signaling of GTPase-deficient Gq : mechanism of action and relevance for treating Gq-driven cancers

Uveal melanoma (UM), the most common cancer of the adult eye, is known to have poor prognosis. Over 50% of patients are likely to develop metastasis. In more than 80% of the cases UM is driven by an activating mutation in the Gq/11 protein. Thus, many attempts have been made to target downstream signaling of this oncogene. Unfortunately, none of these approaches have succeeded so far in clinical trials. Targeting only one or even two downstream effectors in combination, may not be sufficient to blunt the oncogenic dynamics of the Gq/11 protein. Direct inhibition of the oncogene itself was believed to be ineffective, since the mode of action of the only known inhibitors for Gq/11 proteins, FR900359 (FR) and YM-254890 (YM) respectively, was considered unsuitable for targeting the constitutively active oncogenes. FR is a macrocyclic depsipeptide, produced by symbiotic bacteria. Recent studies have shown that by binding to Gα-subunits of Gq/11 proteins FR inhibits the nucleotide exchange that is crucial for their activation. Because FR interferes with the rate-limiting step of Gα activation, it was reasoned that this approach might be unsuitable to target a protein, which is already trapped in the active state. Accidentally, however, Evelyn Gaffal, Thomas Tüting and coworkers found that FR inhibited diverse cancer hall marks in melanoma cells, harboring an activating mutation in the G11 protein. These surprising results prompted us to further investigate the inhibition properties of FR on the oncogenic Gq/11 protein. In the frame of this work, we demonstrate FRs’ ability to suppress mitogenic pro-survival pathways over the canonical phospholipase C (PLC) effector cascade. Taking advantage of genome edited HEK293 cells lacking Gαq and Gα11 proteins, we were furthermore able to show direct interaction of the molecule with its target protein. Results of FRET experiments together with molecular modeling gave first hints regarding the mechanism underlying the inability to target PLC-Gqmut interaction. Whole cell label-free technology unveiled a possible mode of action of FR on the mutated protein. Most importantly, we were able to proof inhibition of the oncogene in a therapeutically relevant system. By using different uveal melanoma cell lines, we demonstrated in vitro and in vivo the inhibition of tumor progression by FR treatment. Further, direct comparison of FR with the other well described Gq-inhibitor YM, revealed the long-term superiority of FR, as we observed different drug vulnerability between the two molecules

Synthesizing Bidirectional Temporal States of Knee Osteoarthritis Radiographs with Cycle-Consistent Generative Adversarial Neural Networks

Knee Osteoarthritis (KOA), a leading cause of disability worldwide, is challenging to detect early due to subtle radiographic indicators. Diverse, extensive datasets are needed but are challenging to compile because of privacy, data collection limitations, and the progressive nature of KOA. However, a model capable of projecting genuine radiographs into different OA stages could augment data pools, enhance algorithm training, and offer pre-emptive prognostic insights. In this study, we trained a CycleGAN model to synthesize past and future stages of KOA on any genuine radiograph. The model was validated using a Convolutional Neural Network that was deceived into misclassifying disease stages in transformed images, demonstrating the CycleGAN's ability to effectively transform disease characteristics forward or backward in time. The model was particularly effective in synthesizing future disease states and showed an exceptional ability to retroactively transition late-stage radiographs to earlier stages by eliminating osteophytes and expanding knee joint space, signature characteristics of None or Doubtful KOA. The model's results signify a promising potential for enhancing diagnostic models, data augmentation, and educational and prognostic usage in healthcare. Nevertheless, further refinement, validation, and a broader evaluation process encompassing both CNN-based assessments and expert medical feedback are emphasized for future research and development.Comment: 29 pages, 10 figure

Adaptive Variance Thresholding: A Novel Approach to Improve Existing Deep Transfer Vision Models and Advance Automatic Knee-Joint Osteoarthritis Classification

Knee-Joint Osteoarthritis (KOA) is a prevalent cause of global disability and is inherently complex to diagnose due to its subtle radiographic markers and individualized progression. One promising classification avenue involves applying deep learning methods; however, these techniques demand extensive, diversified datasets, which pose substantial challenges due to medical data collection restrictions. Existing practices typically resort to smaller datasets and transfer learning. However, this approach often inherits unnecessary pre-learned features that can clutter the classifier's vector space, potentially hampering performance. This study proposes a novel paradigm for improving post-training specialized classifiers by introducing adaptive variance thresholding (AVT) followed by Neural Architecture Search (NAS). This approach led to two key outcomes: an increase in the initial accuracy of the pre-trained KOA models and a 60-fold reduction in the NAS input vector space, thus facilitating faster inference speed and a more efficient hyperparameter search. We also applied this approach to an external model trained for KOA classification. Despite its initial performance, the application of our methodology improved its average accuracy, making it one of the top three KOA classification models.Comment: 26 pages, 5 figure

Improving Performance in Colorectal Cancer Histology Decomposition using Deep and Ensemble Machine Learning

In routine colorectal cancer management, histologic samples stained with hematoxylin and eosin are commonly used. Nonetheless, their potential for defining objective biomarkers for patient stratification and treatment selection is still being explored. The current gold standard relies on expensive and time-consuming genetic tests. However, recent research highlights the potential of convolutional neural networks (CNNs) in facilitating the extraction of clinically relevant biomarkers from these readily available images. These CNN-based biomarkers can predict patient outcomes comparably to golden standards, with the added advantages of speed, automation, and minimal cost. The predictive potential of CNN-based biomarkers fundamentally relies on the ability of convolutional neural networks (CNNs) to classify diverse tissue types from whole slide microscope images accurately. Consequently, enhancing the accuracy of tissue class decomposition is critical to amplifying the prognostic potential of imaging-based biomarkers. This study introduces a hybrid Deep and ensemble machine learning model that surpassed all preceding solutions for this classification task. Our model achieved 96.74% accuracy on the external test set and 99.89% on the internal test set. Recognizing the potential of these models in advancing the task, we have made them publicly available for further research and development.Comment: 28 pages, 9 figure

Targeted inhibition of Gq signaling induces airway relaxation in mouse models of asthma

Obstructive lung diseases are common causes of disability and death worldwide. A hallmark feature is aberrant activation of Gq protein–dependent signaling cascades. Currently, drugs targeting single G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) are used to reduce airway tone. However, therapeutic efficacy is often limited, because various GPCRs contribute to bronchoconstriction, and chronic exposure to receptor-activating medications results in desensitization. We therefore hypothesized that pharmacological Gq inhibition could serve as a central mechanism to achieve efficient therapeutic bronchorelaxation. We found that the compound FR900359 (FR), a membrane-permeable inhibitor of Gq, was effective in silencing Gq signaling in murine and human airway smooth muscle cells. Moreover, FR both prevented bronchoconstrictor responses and triggered sustained airway relaxation in mouse, pig, and human airway tissue ex vivo. Inhalation of FR in healthy wild-type mice resulted in high local concentrations of the compound in the lungs and prevented airway constriction without acute effects on blood pressure and heart rate. FR administration also protected against airway hyperreactivity in murine models of allergen sensitization using ovalbumin and house dust mite as allergens. Our findings establish FR as a selective Gq inhibitor when applied locally to the airways of mice in vivo and suggest that pharmacological blockade of Gq proteins may be a useful therapeutic strategy to achieve bronchorelaxation in asthmatic lung disease

Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid– dependent and –independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other G subunits, we here set out to rationally design G16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gq/G11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold– based inhibitors, provided that these inhibitors mimic the interaction of FR with G proteins harboring engineered FR-binding sites

The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq

Kansainvälisyys akateemisen identiteetin muokkaajana: Kansainvälinen henkilökunta suomalaisella tieteen pelikentällä

Tutkimuksen tavoitteena on selvittää, millainen rooli kansainvälisyydellä on akateemisen identiteetin rakentumisessa suomalaisen yliopiston kontekstissa. Kansainvälisyyttä tarkastellaan suomalaisessa monialayliopistossa työskentelevän kansainvälisen henkilökunnan uran näkökulmasta. Tutkimuksen teoreettinen kehys nojautuu Pierre Bourdieun (1986) kenttäteoriaan ja Glynis Breakwellin (1986) uhattujen identiteettien teoriaan. Tutkimusaineisto koostuu kansainväliselle henkilökunnalle tehdyistä haastatteluista (n = 10). Aineisto analysoitiin yhdistäen aineistolähtöistä ja teoriaohjaavaa sisällönanalyysiä. Tulokset osoittavat, että kansainvälisyys toimii keinona hankkia tiedeyhteisön arvostamia pääoman lajeja – kulttuurista, sosiaalista, taloudellista ja symbolista pääomaa. Kansainvälistymisellä uskotaan olevan yhteys parempaan urakehitykseen, auttaen näin selviytymään ja menestymään tieteen pelikentällä. Kansainvälinen ura näyttäytyy tutkimuksessa ristiriitaisena, myös uhkaa aiheuttavana tekijänä akateemiselle identiteetille. Tutkimuksessa uhkat näyttäytyvät pääomien kääntöpuolena ja siten pääomien keräämisen esteenä. Kansainvälistä uraa suomalaisen yliopiston kontekstissa kuvataan menestyksenä ja menetyksenä, ja identiteetin rakentumiseen sisältyy samanaikaisesti sekä toivo voittamisesta, että pelko häviämisestä

Lack of Gαi2 leads to dilative cardiomyopathy and increased mortality in β1-adrenoceptor overexpressing mice

Aims Inhibitory G (Gi) proteins have been proposed to be cardioprotective. We investigated effects of Gαi2 knockout on cardiac function and survival in a murine heart failure model of cardiac β1-adrenoceptor overexpression. Methods and results β1-transgenic mice lacking Gαi2 (β1-tg/Gαi2 -/-) were compared with wild-type mice and littermates either overexpressing cardiac β1-adrenoceptors (β1-tg) or lacking Gαi2 (Gαi2 -/-). At 300 days, mortality of mice only lacking Gαi2 was already higher compared with wild-type or β1-tg, but similar to β1-tg/Gαi2 -/-, mice. Beyond 300 days, mortality of β1-tg/Gαi2 -/- mice was enhanced compared with all other genotypes (mean survival time: 363 ± 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, β1-tg, and Gαi2 -/- mice, but significant impairment for β1-tg/Gαi2 -/- mice (e.g. ejection fraction 14 ± 2 vs. 40 ± 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 ± 0.06 vs. 0.48 ± 0.02% in wild-type mice), left ventricular size (length 0.82 ± 0.04 vs. 0.66 ± 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. Gαi3 was significantly up-regulated in Gαi2 knockout mice (protein compared with wild type: 340 ± 90% in Gαi2 -/- and 394 ± 80% in β1-tg/Gαi2 -/-, respectively). Conclusions Gαi2 deficiency combined with cardiac β1-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, β1-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gαi2. We propose an enhanced effect of increased β1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or a concentration-dependent mechanism.Fil: Keller, Kirsten. Universitat Zu Köln; AlemaniaFil: Maass, Martina. University Hospital of Cologne; AlemaniaFil: Dizayee, Sara. Universitat Zu Köln; AlemaniaFil: Leiss, Veronika. Eberhard Karls University Hospitals and Clinics; AlemaniaFil: Annala, Suvi. Universitat Zu Köln; AlemaniaFil: Köth, Jessica. Universitat Zu Köln; AlemaniaFil: Seemann, Wiebke K.. Universitat Zu Köln; AlemaniaFil: Müller Ehmsen, Jochen. Asklepios Klinik Altona; AlemaniaFil: Mohr, Klaus. Universitaet Bonn; AlemaniaFil: Nürnberg, Bernd. Eberhard Karls University Hospitals and Clinics; AlemaniaFil: Engelhardt, Stefan. Universitat Technical Zu Munich; AlemaniaFil: Herzig, Stefan. Universitat Zu Köln; AlemaniaFil: Birnbaumer, Lutz. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Matthes, Jan. Universitat Zu Köln; Alemani