Populism: Utility of its Approaches and the Prospects of the Phenomenon's Resurgence in America

Populism in the twenty-first century became prominent in scholarly circles following Brexit and the 2016 electoral victory of Donald Trump. As democracy and globalization enthusiasts least anticipated these two monumental events in modern history, much emphasis came to be placed on the nature of populism and what conditions led to its manifestation in contemporary times. Working within this background, this study aims to review the ideational, political strategy and discursive approaches to the populism phenomenon and unpack the relative utility of each approach. I offer a thoughtful perspective that while the ideational approach’s “thin-centered” strand has the tendency to blur the boundaries of populism and lead scholars to accept “anything” as populism; it nevertheless enables us to comprehensively capture populism usage in multiple contexts. Further, I advance the notion that the 2020 electoral defeat of Donald Trump should not be misconstrued as the end or weakening of right-wing populism; the present state of American politics makes it ripe for populism resurgence

A flow-cytometry-based pipeline for the rapid quantification of C2C12 cell differentiation.

peer reviewedThe C2C12 cell line represents a simple in vitro model for cell differentiation. Here, we present a flow-cytometry-based pipeline to quantitate C2C12 cell differentiation based on myosin heavy-chain marker expression. We describe steps for cell seeding, transfection, drug treatment, differentiation, and labeling. We then detail procedures for flow cytometry acquisition and introduce the R script FlowFate for automated analysis, including the study of dose-dependent effects of GFP-tagged genes on differentiation. For complete details on the use and execution of this protocol, please refer to Chippalkatti et al. (2023).1

Eliminating oncogenic RAS: back to the future at the drawing board.

RAS drug development has made enormous strides in the past ten years, with the first direct KRAS inhibitor being approved in 2021. However, despite the clinical success of covalent KRAS-G12C inhibitors, we are immediately confronted with resistances as commonly found with targeted drugs. Previously believed to be undruggable due to its lack of obvious druggable pockets, a couple of new approaches to hit this much feared oncogene have now been carved out. We here concisely review these approaches to directly target four druggable sites of RAS from various angles. Our analysis focuses on the lessons learnt during the development of allele-specific covalent and non-covalent RAS inhibitors, the potential of macromolecular binders to facilitate the discovery and validation of targetable sites on RAS and finally an outlook on a future that may engage more small molecule binders to become drugs. We foresee that the latter could happen mainly in two ways: First, non-covalent small molecule inhibitors may be derived from the development of covalent binders. Second, reversible small molecule binders could be utilized for novel targeting modalities, such as degraders of RAS. Provided that degraders eliminate RAS by recruiting differentially expressed E3-ligases, this approach could enable unprecedented tissue- or developmental stage-specific destruction of RAS with potential advantages for on-target toxicity. We conclude that novel creative ideas continue to be important to exterminate RAS in cancer and other RAS pathway-driven diseases, such as RASopathies

Medium-Throughput Detection of Hsp90/Cdc37 Protein-Protein Interaction Inhibitors Using a Split Renilla Luciferase-Based Assay

The protein-folding chaperone Hsp90 enables the maturation and stability of various oncogenic signaling proteins and is thus pursued as a cancer drug target. Folding in particular of protein kinases is assisted by the co-chaperone Cdc37. Several inhibitors against the Hsp90 ATP-binding site have been developed. However, they displayed significant toxicity in clinical trials. By contrast, the natural product conglobatin A has an exceptionally low toxicity in mice. It targets the protein-protein interface (PPI) of Hsp90 and Cdc37, suggesting that interface inhibitors have an interesting drug development potential. In order to identify inhibitors of the Hsp90/Cdc37 PPI, we have established a mammalian cell lysate-based, medium-throughput amenable split Renilla luciferase assay. This assay employs N-terminal and C-terminal fragments of Renilla luciferase fused to full-length human Hsp90 and Cdc37, respectively. We expect that our assay will allow for the identification of novel Hsp90/Cdc37 interaction inhibitors. Such tool compounds will help to evaluate whether the toxicity profile of Hsp90/Cdc37 PPI inhibitors is in general more favorable than that of ATP-competitive Hsp90 inhibitors. Further development of such tool compounds may lead to new classes of Hsp90 inhibitors with applications in cancer and other diseases

Elaiophylin Is a Potent Hsp90/Cdc37 Protein Interface Inhibitor with K-Ras Nanocluster Selectivity

The natural product elaiophylin is a macrodiolide with a broad range of biological activities. However, no direct target of elaiophylin in eukaryotes has been described so far, which hinders a systematic explanation of its astonishing activity range. We recently showed that the related conglobatin A, a protein-protein interface inhibitor of the interaction between the N-terminus of Hsp90 and its cochaperone Cdc37, blocks cancer stem cell properties by selectively inhibiting K-Ras4B but not H-Ras. Here, we elaborated that elaiophylin likewise disrupts the Hsp90/ Cdc37 interaction, without affecting the ATP-pocket of Hsp90. Similarly to conglobatin A, elaiophylin decreased expression levels of the Hsp90 client HIF1 alpha, a transcription factor with various downstream targets, including galectin-3. Galectin-3 is a nanocluster scaffold of K-Ras, which explains the K-Ras selectivity of Hsp90 inhibitors. In agreement with this K-Ras targeting and the potent effect on other Hsp90 clients, we observed with elaiophylin treatment a submicromolar IC50 for MDA-MB-231 and MIA-PaCa-2 3D spheroid formation. Finally, a strong inhibition of MDA-MB-231 cells grown in the chorioallantoic membrane (CAM) microtumor model was determined. These results suggest that several other macrodiolides may have the Hsp90/ Cdc37 interface as a target site

PDE6D Dependent Trafficking of K-Ras to Stemness Promoting Centriolar Organelles

The cancer driving properties of KRAS, NRAS and HRAS appear to correlate with their ability to drive stemness in normal and cancer cells. However, exactly how RAS proteins promote stemness is not understood. The trafficking chaperone PDE6D transports prenylated proteins to the primary cilium (PC), which is found on stem and epithelial cells, and harbours several stemness signalling pathways. Upon re-entry of the cell cycle, the PC is internalized and its basal body becomes the mother centrosome. Asymmetric inheritance of the centrosomes is typically associated with asymmetric divisions, where one cell retains stemness. Given that PDE6D plays a critical role in regulating the sub-cellular distribution of KRAS, we hypothesized that it also facilitates localization of KRAS to the PC, which would enable asymmetric apportioning of KRAS via the mother centrosome. We found that KRAS accumulates more at the PC than NRAS and HRAS, and in a PDE6D-dependent manner in muscle C2C12 cells. Modulation of KRAS on the phosphorylation-site Ser181 either pharmacologically or by introducing mutations decreased its interaction with PDE6D as measured by BRET in HEK cells and reduced KRAS accumulation at the PC in C2C12 cells. Ciliated, stem-like C2C12 myoblasts differentiate into non-ciliated myocytes/ -tubes within a few days upon switching to low serum, which transiently downregulates MAPK-signalling. In line with this, we find that overexpression of dominant-negative KRAS-S17N increases differentiation even under high serum conditions. Intriguingly, the opposite is true for oncogenic KRAS-G12V, which increases the stem-like fraction even under low serum conditions. Additional data suggest that reducing the accumulation of active KRAS on centriolar organelles drives differentiation. Our data indicate that the distribution of KRAS during the cell-cycle to centriolar organelles, such as the PC and centrosomes, may be critical for its normal and aberrant cancer-associated activities.R-AGR-3754 - C19/BM/13673303/PolaRAS2 - part UL (01/09/2020 - 31/08/2023) - ABANKWA Danie

Rapalogs can promote cancer cell stemness in vitro in a Galectin-1 and H-ras-dependent manner

Currently several combination treatments of mTor- and Ras-pathway inhibitors are being tested in cancer therapy. While multiple feedback loops render these central signaling pathways robust, they complicate drug targeting. Here, we describe a novel H-ras specific feedback, which leads to an inadvertent rapalog induced activation of tumorigenicity in Ras transformed cells. We find that rapalogs specifically increase nanoscale clustering (nanoclustering) of oncogenic H-ras but not K-ras on the plasma membrane. This increases H-ras signaling output, promotes mammosphere numbers in a H-ras-dependent manner and tumor growth in ovo. Surprisingly, also other FKBP12 binders, but not mTor- inhibitors, robustly decrease FKBP12 levels after prolonged (> 2 days) exposure. This leads to an upregulation of the nanocluster scaffold galectin-1 (Gal-1), which is responsible for the rapamycin-induced increase in H-ras nanoclustering and signaling output. We provide evidence that Gal-1 promotes stemness features in tumorigenic cells. Therefore, it may be necessary to block inadvertent induction of stemness traits in H-ras transformed cells by specific Gal-1 inhibitors that abrogate its effect on H-ras nanocluster. On a more general level, our findings may add an important mechanistic explanation to the pleiotropic physiological effects that are observed with rapalogs.Peer reviewe

Detection of Ras nanoclustering-dependent homo-FRET using fluorescence anisotropy measurements

The small GTPase Ras is frequently mutated in cancer and a driver of tumorigenesis. The recent years have shown great progress in drug-targeting Ras and understanding how it operates on the plasma membrane. We now know that Ras is non-randomly organized into proteo-lipid complexes on the membrane, called nanoclusters. Nanoclusters contain only a few Ras proteins and are necessary for the recruitment of downstream effectors, such as Raf. If tagged with fluorescent proteins, the dense packing of Ras in nanoclusters can be analyzed by Förster/ fluorescence resonance energy transfer (FRET). Loss of FRET can therefore report on decreased nanoclustering and any process upstream of it, such as Ras lipid modifications and correct trafficking. Thus, cellular FRET screens employing Ras-derived fluorescence biosensors are potentially powerful tools to discover chemical or genetic modulators of functional Ras membrane organization. Here we implement fluorescence anisotropy-based homo-FRET measurements of Ras-derived constructs labelled with only one fluorescent protein on a confocal microscope and a fluorescence plate reader. We show that homo-FRET of both H-Ras- and K-Ras-derived constructs can sensitively report on Ras-lipidation and -trafficking inhibitors, as well as on genetic perturbations of proteins regulating membrane anchorage. By exploiting the switch I/II-binding Ras-dimerizing compound BI-2852, this assay is also suitable to report on the engagement of the K-Ras switch II pocket by small molecules such as AMG 510. Given that homo-FRET only requires one fluorescent protein tagged Ras construct, this approach has significant advantages to create Ras-nanoclustering FRET-biosensor reporter cell lines, as compared to the more common hetero-FRET approaches

A covalent calmodulin inhibitor as a tool to study cellular mechanisms of K-Ras-driven stemness

This article is part of the Research Topic Ras and other GTPases in Cancer: From Basic to Applied Research https://doi.org/10.3389/978-2-88974-081-9Recently, the highly mutated oncoprotein K-Ras4B (hereafter K-Ras) was shown to drive cancer cell stemness in conjunction with calmodulin (CaM). We previously showed that the covalent CaM inhibitor ophiobolin A (OphA) can potently inhibit K-Ras stemness activity. However, OphA, a fungus-derived natural product, exhibits an unspecific, broad toxicity across all phyla. Here we identified a less toxic, functional analog of OphA that can efficiently inactivate CaM by covalent inhibition. We analyzed a small series of benzazulenones, which bear some structural similarity to OphA and can be synthesized in only six steps. We identified the formyl aminobenzazulenone 1, here named Calmirasone1, as a novel and potent covalent CaM inhibitor. Calmirasone1 has a 4-fold increased affinity for CaM as compared to OphA and was active against K-Ras in cells within minutes, as compared to hours required by OphA. Calmirasone1 displayed a 2.5-4.5-fold higher selectivity for KRAS over BRAF mutant 3D spheroid growth than OphA, suggesting improved relative on-target activity. Importantly, Calmirasone1 has a 40-260-fold lower unspecific toxic effect on HRAS mutant cells, while it reaches almost 50% of the activity of novel K-RasG12C specific inhibitors in 3D spheroid assays. Our results suggest that Calmirasone1 can serve as a new tool compound to further investigate the cancer cell biology of the K-Ras and CaM associated stemness activities.Peer reviewe

NRAS is unique among RAS proteins in requiring ICMT for trafficking to the plasma membrane

Isoprenylcysteine carboxyl methyltransferase (ICMT) is the third of three enzymes that sequentially modify the C-terminus of CaaX proteins, including RAS. Although all four RAS proteins are substrates for ICMT, each traffics to membranes differently by virtue of their hypervariable regions that are differentially palmitoylated. We found that among RAS proteins, NRAS was unique in requiring ICMT for delivery to the PM, a consequence of having only a single palmitoylation site as its secondary affinity module. Although not absolutely required for palmitoylation, acylation was diminished in the absence of ICMT. Photoactivation and FRAP of GFP-NRAS revealed increase flux at the Golgi, independent of palmitoylation, in the absence of ICMT. Association of NRAS with the prenyl-protein chaperone PDE6δ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6δ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane.</p