Hyperactive Ras signalling is found in most cancers. Ras proteins are only active in membrane nanoclusters, which are therefore potential drug targets. We previously showed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering via direct interaction with the Ras binding domain (RBD) of Raf. Here, we establish that the B-Raf preference of Gal1 emerges from the divergence of the Raf RBDs at their proposed Gal1-binding interface. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B- and C-Raf-RBDs. Its 23-mer core fragment is sufficient to interfere with H-Ras nanoclustering, modulate Ras-signalling and moderately reduce cell viability. These latter two phenotypic effects may also emerge from the ability of L5UR to broadly engage with several RBD- and RA-domain containing Ras interactors. The L5UR-peptide core fragment is a starting point for the development of more specific reagents against Ras-nanoclustering and -interactors. The 23mer L5URcore peptide is identified, which binds with low micromolar affinity to Ras-binding domains, notably of Raf proteins, thus disrupting their galectin1-interaction, H-Ras-nanoclustering, -signalling and more broadly cell proliferation.

The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where various activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds active KRAS, including mutant forms, competes with RAF and limits oncogenic KRAS downstream signalling, maintaining mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity at an optimal level. SHANK3 depletion breaches this threshold, triggering MAPK/ERK signalling hyperactivation and MAPK/ERK-dependent cell death in KRAS-mutant cancers. Targeting this vulnerability through RNA interference or nanobody-mediated disruption of the SHANK3-KRAS interaction constrains tumour growth in vivo in female mice. Thus, inhibition of SHANK3-KRAS interaction represents an alternative strategy for selective killing of KRAS-mutant cancer cells through excessive signalling.

The RAS-MAPK-pathway is aberrantly regulated in cancer and developmental diseases called RASopathies. While typically the impact of Ras on the proliferation of various cancer cell lines is assessed, it is poorly established how Ras affects cellular differentiation.Here we implement the C2C12 myoblast cell line to systematically study the effect of Ras mutants and Ras-pathway drugs on differentiation. We first provide evidence that a minor pool of Pax7+ progenitors replenishes a major pool of transit amplifying cells that are ready to differentiate. Our data indicate that Ras isoforms have distinct roles in the differentiating culture, where K-Ras depletion increases and H-Ras depletion decreases terminal differentiation. This assay could therefore provide significant new insights into Ras biology and Ras-driven diseases. In line with this, we found that all oncogenic Ras mutants block terminal differentiation of transit amplifying cells. By contrast, RASopathy associated K-Ras variants were less able to block differentiation. Profiling of eight targeted Ras-pathway drugs on seven oncogenic Ras mutants revealed their allele-specific activities and distinct abilities to restore normal differentiation as compared to triggering cell death. In particular, the MEK-inhibitor trametinib could broadly restore differentiation, while the mTOR-inhibitor rapamycin broadly suppressed differentiation. We expect that this quantitative assessment of the impact of Ras-pathway mutants and drugs on cellular differentiation has great potential to complement cancer cell proliferation data.

Bioluminescence resonance energy transfer (BRET) allows to quantitate protein interactions in intact cells. Here, we present a protocol for measuring BRET due to transient interactions of oncogenic K-RasG12V in plasma membrane nanoclusters of HEK293-EBNA cells. We describe steps for seeding, transfecting, and replating cells. We then detail procedures for their preparation for BRET measurements on a CLARIOstar microplate reader and detailed data analysis.For complete details on the use and execution of this protocol, please refer to Steffen et al.1

The trafficking chaperone PDE6D (or PDEδ) was proposed as a surrogate target for K-Ras, leading to the development of a series of inhibitors that block its prenyl binding pocket. These inhibitors suffered from low solubility and suspected off-target effects, preventing their clinical development. Here, we developed a highly soluble, low nanomolar PDE6D inhibitor (PDE6Di), Deltaflexin3, which has the lowest off-target activity as compared to three prominent reference compounds. Deltaflexin3 reduces Ras signaling and selectively decreases the growth of KRAS mutant and PDE6D-dependent cancer cells. We further show that PKG2-mediated phosphorylation of Ser181 lowers K-Ras binding to PDE6D. Thus, Deltaflexin3 combines with the approved PKG2 activator Sildenafil to more potently inhibit PDE6D/K-Ras binding, cancer cell proliferation, and microtumor growth. As observed previously, inhibition of Ras trafficking, signaling, and cancer cell proliferation remained overall modest. Our results suggest reevaluating PDE6D as a K-Ras surrogate target in cancer.

The 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

Most RAS drug targeting strategies focus on KRAS, due to the high KRAS mutation frequency in cancer and its high expression level. Moreover, KRAS4B (hereafter KRAS) but not HRAS is a potent driver of stemness traits in cancer cells. Exactly how KRAS promotes stemness and how this activity can be targeted with drugs is not understood. Here we studied the subcellular localization of KRAS mutants to stemness mediating centriolar organelles and the effect of these mutants on the differentiation of mouse muscle C2C12 cells. This cell line contains a pool of ciliated stem/progenitor cells, which differentiate under low serum conditions, concomitant with a drop in RAS-MAPK activity. Intriguingly, expression of oncogenic KRAS retains the stem-like state, even under differentiating conditions. We hypothesized that KRAS localization at the primary cilium of C2C12 progenitor cells is propagated via the mother centriole to the mother centrosome, which is known to asymmetrically partition into the stemness retaining cell. Using confocal microscopy, we show that the trafficking chaperone PDE6D (or PDEdelta) facilitates trafficking of KRAS not only to the plasma membrane, but also to the primary cilium. Cellular BRET-interaction experiments in HEK cells demonstrate that decreasing the affinity between KRAS and PDE6D by the phosphomimetic S181D-mutation in KRAS, correlates with a reduced KRAS localization in the cilium of C2C12 cells. Likewise, treatment with Sildenafil, which increases the phosphorylation of Ser181 of KRAS, disrupts its binding to PDE6D. We then established a flow-cytometry based C2C12 cell differentiation assay to quantify accurately that mutations and pharmacological manipulations, which decrease ciliary localization of KRAS, indeed promote differentiation. These data lend a new rationale to PDE6D inhibition. By combining computational modelling and in vitro testing, we therefore developed a new, low nanomolar inhibitor, Deltaflexin-3 (structure will be disclosed), which targets the hydrophobic pocket of PDE6D. This compound is highly soluble and displays improved on-target activity as compared to previous PDE6D inhibitors, which is demonstrated by a panel of cellular BRET- and proliferation-assay data. Our results suggest that blocking of the ciliary localization of KRAS by synergistically combining PDE6D inhibitors and Sildenafil could specifically inhibit its stemness driving activity. Citation Format: Pelin Kaya, Rohan Chippalkatti, Elisabeth Schaffner-Reckinger, Bianca Parisi, Ganesh B. Manoharan, Yashar Rouzbahani, Christian Eggeling, Daniel Abankwa. Drug targeting of KRAS accumulation on the cilium inhibits its stemness driving activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3493.

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.

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.

Recent data suggest that K-Ras4B (hereafter K-Ras) can drive cancer cell stemness via calmodulin (CaM)-dependent, non-canonical Wnt-signalling. Here we examined whether another Ca2+-binding protein, the CaM-related centrin1, binds to K-Ras and could mediate some K-Ras functions that were previously ascribed to CaM. While CaM and centrin1 appear to distinguish between peptides that were derived from their classical targets, they both bind to K-Ras in cells. Cellular BRET- and immunoprecipitation data suggest that CaM engages more with K-Ras than centrin1 and that the interaction with the C-terminal membrane anchor of K-Ras is sufficient for this. Surprisingly, binding of neither K-Ras nor its membrane anchor alone to CaM or centrin1 is sensitive to inhibition of prenylation. In support of an involvement of the G-domain of K-Ras in cellular complexes with these Ca2+-binding proteins, we find that oncogenic K-RasG12V displays increased engagement with both CaM and centrin1. This is abrogated by addition of the D38A effector-site mutation, suggesting that K-RasG12V is held together with CaM or centrin1 in complexes with effectors. When treated with CaM inhibitors, the BRET-interaction of K-RasG12V with centrin1 was also disrupted in the low micromolar range, comparable to that with CaM. While CaM predominates in regulating functional membrane anchorage of K-Ras, it has a very similar co-distribution with centrin1 on mitotic organelles. Given these results, a significant overlap of the CaM- and centrin1-dependent functions of K-Ras is suggested.

The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56α is a human tumour suppressor. However, the molecular mechanisms inhibiting PP2A-B56α in cancer are poorly understood. Here, we report molecular level details and structural mechanisms of PP2A-B56α inhibition by an oncoprotein CIP2A. Upon direct binding to PP2A-B56α trimer, CIP2A displaces the PP2A-A subunit and thereby hijacks both the B56α, and the catalytic PP2Ac subunit to form a CIP2A-B56α-PP2Ac pseudotrimer. Further, CIP2A competes with B56α substrate binding by blocking the LxxIxE-motif substrate binding pocket on B56α. Relevant to oncogenic activity of CIP2A across human cancers, the N-terminal head domain-mediated interaction with B56α stabilizes CIP2A protein. Functionally, CRISPR/Cas9-mediated single amino acid mutagenesis of the head domain blunted MYC expression and MEK phosphorylation, and abrogated triple-negative breast cancer in vivo tumour growth. Collectively, we discover a unique multi-step hijack and mute protein complex regulation mechanism resulting in tumour suppressor PP2A-B56α inhibition. Further, the results unfold a structural determinant for the oncogenic activity of CIP2A, potentially facilitating therapeutic modulation of CIP2A in cancer and other diseases.

Disruption of the native membrane organization of Ras by the farnesyltransferase inhibitor tipifarnib in the late 1990s constituted the first indirect approach to drug target Ras. Since then, our understanding of how dynamically Ras shuttles between subcellular locations has changed significantly. Ras proteins have to arrive at the plasma membrane for efficient MAPK-signal propagation. On the plasma membrane Ras proteins are organized into isoform specific proteo-lipid assemblies called nanocluster. Recent evidence suggests that Ras nanocluster have a specific lipid composition, which supports the recruitment of effectors such as Raf. Conversely, effectors possess lipid-recognition motifs, which appear to serve as co-incidence detectors for the lipid domain of a given Ras isoform. Evidence suggests that dimeric Raf proteins then co-assemble dimeric Ras in an immobile complex, thus forming the minimal unit of an active nanocluster. Here we review established and novel trafficking chaperones and trafficking factors of Ras, along with the set of lipid and protein modulators of Ras nanoclustering. We highlight drug targeting approaches and opportunities against these determinants of functional Ras membrane organization. Finally, we reflect on implications for Ras signaling in polarized cells, such as epithelia, which are a common origin of tumorigenesis.

The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where numerous different activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds overactive mutant forms to limit oncogenic KRAS signalling and maintain RAS- activity at an optimal level. Depletion of SHANK3 results in hyperactivation of KRAS/mitogen-activated protein kinase (MAPK) signalling, which in turn selectively induces MAPK/ERK-dependent cell death in KRAS -mutant cancers. Furthermore, targeting of this therapeutic vulnerability through nanobody- or RNA interference- mediated disruption of the SHANK3-KRAS interaction reduces tumour growth in vivo. Thus, inhibition of the SHANK3-KRAS interaction represents a new pan-KRAS-mutant compatible strategy for selective killing of KRAS - mutant cancer cells through excessive signalling. Graphical abstract Schematic model of SHANK3-controlled cell fate in KRAS -mutant cancers. SHANK3 directly interacts with KRAS and competes with RAF for KRAS binding to sustain oncogenic RAS-MAPK/ERK signalling at an optimal level (i.e. below toxic oncogenic signalling) in KRAS -mutant cancers. SHANK3 silencing (1) and inhibition of SHANK3-KRAS interaction (2) drive KRAS -mutant cells into cell death.

The multi-target effects of natural products allow us to fight complex diseases like cancer on multiple fronts. Unlike docking techniques, network-based approaches such as genome-scale metabolic modelling can capture multi-target effects. However, the incompleteness of natural product target information reduces the prediction accuracy of in silico gene knockout strategies. Here, we present a drug selection workflow based on context-specific genome-scale metabolic models, built from the expression data of cancer cells treated with natural products, to predict cell viability. The workflow comprises four steps: first, in silico single-drug and drug combination predictions; second, the assessment of the effects of natural products on cancer metabolism via the computation of a dissimilarity score between the treated and control models; third, the identification of natural products with similar effects to the approved drugs; and fourth, the identification of drugs with the predicted effects in pathways of interest, such as the androgen and estrogen pathway. Out of the initial 101 natural products, nine candidates were tested in a 2D cell viability assay. Bruceine D, emodin, and scutellarein showed a dose-dependent inhibition of MCF-7 and Hs 578T cell proliferation with IC50 values between 0.7 to 65 μM, depending on the drug and cell line. Bruceine D, extracted from Brucea javanica seeds, showed the highest potency.

Ras gene mutations are detected in around 30% of human cancers. These mutations have been linked with polarity disruption in epithelial cells, a hallmark for cancer. Still, the cell depolarization mechanism remains unknown. Here, we investigate polarized cells by means of advanced fluorescence microscopy and spectroscopy approaches, such as superresolution STED microscopy, fluorescence correlation spectroscopy, and spectral imaging, to explore the differences between apical and basal membranes regarding lipid organization and Ras-lipid interactions.

Phenothiazines (PTZ) were developed as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they have been used for decades as antipsychotic drugs. In addition, they possess significant anti-cancer properties and several attempts for their repurposing were made. However, their incompletely understood polypharmacology is challenging. Here we examined the potential of the PTZ fluphenazine (Flu) and its mustard derivative (Flu-M) to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras- and MAPK-pathway, cell viability and features of cancer cell stemness. Consistently, we show that the combination of a CaM inhibitor and the PP2A activator DT-061 synergistically inhibited the 3D-spheroid formation of MDA-MB-231 (K-Ras-G13D), NCI-H358 (K-Ras-G12C) and A375 (B-raf-V600E) cancer cells, and increased apoptosis in MDA-MB-231. We reasoned that these activities remain combined in PTZ, which were the starting point for PP2A activator development, while several PTZ are known CaM inhibitors. We show that both Flu and Flu-M retained CaM inhibitory activity in vitro and in cells, with a higher potency of the mustard derivative in cells. In line with the CaM dependence of Ras plasma membrane organization, the mustard derivative potently reduced the functional membrane organization of oncogenic Ras, while DT-061 had a negligible effect. Like DT-061, both PTZ potently decreased c-MYC levels, a hallmark of PP2A activation. Benchmarking against the KRAS-G12C specific inhibitor AMG-510 in MIA PaCa-2 cells revealed a higher potency of Flu-M than combinations of DT-061 and a CaM inhibitor on MAPK-output and a strong effect on cell proliferation. While our study is limited, our results suggest that improved PTZ derivatives that retain both, their CaM inhibitory and PP2A activating properties, but have lost their neurological side-effects, may be interesting to pursue further as anti-cancer agents.

Among the biomedical efforts in response to the current coronavirus (COVID-19) pandemic, pharmacological strategies to reduce viral load in patients with severe forms of the disease are being studied intensively. One of the main drug target proteins proposed so far is the SARS-CoV-2 viral protease 3CLpro (also called Mpro), an essential component for viral replication. Ongoing ligand- and receptor-based computational screening efforts would be facilitated by an improved understanding of the electrostatic, hydrophobic, and steric features that characterize small-molecule inhibitors binding stably to 3CLpro and by an extended collection of known binders. Here, we present combined virtual screening, molecular dynamics (MD) simulation, machine learning, and in vitro experimental validation analyses, which have led to the identification of small-molecule inhibitors of 3CLpro with micromolar activity and to a pharmacophore model that describes functional chemical groups associated with the molecular recognition of ligands by the 3CLpro binding pocket. Experimentally validated inhibitors using a ligand activity assay include natural compounds with the available prior knowledge on safety and bioavailability properties, such as the natural compound rottlerin (IC50 = 37 μM) and synthetic compounds previously not characterized (e.g., compound CID 46897844, IC50 = 31 μM). In combination with the developed pharmacophore model, these and other confirmed 3CLpro inhibitors may provide a basis for further similarity-based screening in independent compound databases and structural design optimization efforts to identify 3CLpro ligands with improved potency and selectivity. Overall, this study suggests that the integration of virtual screening, MD simulations, and machine learning can facilitate 3CLpro-targeted small-molecule screening investigations. Different receptor-, ligand-, and machine learning-based screening strategies provided complementary information, helping to increase the number and diversity of the identified active compounds. Finally, the resulting pharmacophore model and experimentally validated small-molecule inhibitors for 3CLpro provide resources to support follow-up computational screening efforts for this drug target.

Recently, 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.