Daniel Abankwa

University of Luxembourg · Department of Life Sciences and Medicine

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,...

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...

Bioluminescence resonance energy transfer (BRET) allows to quantitate protein interactions in intact cells. Here we provide a step-by-step protocol for measuring BRET due to transient interactions of oncogenic K-RasG12V in plasma membrane nanoclusters of HEK293-EBNA cells. We describe how to seed, transfect and replate cells, followed by their prep...

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 P...

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...

The Ras-MAPK pathway is aberrantly regulated in cancer and developmental diseases called RASopathies. While the impact of Ras on proliferation is typically assessed on various cancer cell lines, it is poorly established how Ras affects cellular differentiation. Here we implement the C2C12 myoblast cell line to systematically study the effect of Ras...

The Ras-MAPK pathway is critical to regulate cell proliferation and differentiation. Its dysregulation is implicated in the onset and progression of numerous types of cancers. To be active, Ras proteins are membrane anchored and organized into nanoclusters, which realize high-fidelity signal transmission across the plasma membrane. Nanoclusters the...

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 intracellular potency, preventing their clinical development. Here we developed a highly soluble PDE6D inhibitor (PDE6Di)...

Simple Summary Trafficking chaperones facilitate the spatio-temporal distribution pattern of proteins inside cells. In the case of the membrane-anchored protein Ras, trafficking chaperones typically bind to the C-terminal farnesyl-moiety. Thus shielded from the aqueous environment, Ras can diffuse more efficiently through the cytoplasm. The calcium...

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...

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 studie...

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 fe...

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...

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 obvi...

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 o...

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 underst...

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...

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...

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 f...

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 toxicit...

Phenothiazines (PTZ) are well known as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they are used for decades as antipsychotic drugs. In addition, significant anti-cancer properties have been ascribed to them. Several attempts for their repurposing were made, however, their incompletely un...

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 int...

Calmodulin (CaM) is involved in cell cycle regulation and present at higher concentrations in malignant tissues and cancer cells. It was therefore pursued as a cancer drug target in the 1980s. Current data suggest that Ca2+/CaM acts as a trafficking chaperone for K-Ras4B (hereafter K-Ras), the highly mutated Ras isoform and the focus of current Ras...

On the plasma membrane, Ras is organized into laterally segregated proteo-lipid complexes called nanoclusters. The extent of Ras nanoclustering correlates with its signaling output, positioning nanocluster as dynamic signaling gain modulators. Recent evidence suggests that stacked dimers of Ras and Raf are elemental units at least of one type of Ra...

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...

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 f...

The ATP-competitive inhibitors of Hsp90 have been tested predominantly in kinase addicted cancers; however, they have had limited success. A mechanistic connection between Hsp90 and oncogenic K-Ras is not known. Here, we show that K-Ras selectivity is enabled by the loss of the K-Ras membrane nanocluster modulator galectin-3 downstream of the Hsp90...

Cancer stem cells (CSC) may be the most relevant and elusive cancer cell population, as they have the exquisite ability to seed new tumors. It is plausible, that highly mutated cancer genes, such as KRAS, are functionally associated with processes contributing to the emergence of stemness traits. In this review, we will summarize the evidence for a...

The kinetics for the cleavage of the phosphotriester PDE6D inhibitors 1 (Deltaflexin‐2) and 3 (Deltaflexin‐1) and their derivatives 2, 4 and 5 is presented under various conditions in aqueous solutions. The conversion of the phosphotriesters (1–5) into the phosphodiesters 1**–5** was detected to take place as a major degradation process. In the abs...

Ras is the most frequently mutated oncogene and recent drug development efforts have spurred significant new research interest. Here we review progress toward understanding how Ras functions in nanoscale, proteo-lipid signaling complexes on the plasma membrane, called nanoclusters. We discuss how G-domain reorientation is plausibly linked to Ras-na...

Ras is the most frequently mutated oncogene and recent drug development efforts have spurred significant new research interest. Here we will review progress toward understanding how Ras functions in nanoscale, proteo-lipid signaling complexes on the plasma membrane, called nanocluster. We will discuss how G-domain reorientation is plausibly linked...

RAS oncogenes are frequently mutated in human cancers and among the three isoforms (KRAS, HRAS and NRAS), KRAS is the most frequently mutated oncogene. Here we demonstrate that a subset of flavaglines, a class of natural anti-tumour drugs and chemical ligands of prohibitins, inhibit RAS GTP loading and oncogene activation in cells at nanomolar conc...

RAS proteins play a direct causal role in human cancers, with activating mutations in RAS occurring in ∼30% of tumors. Despite comprehensive efforts, efficient targeting of RAS has not yet reached the clinic. Here, we report SHANK3, a synaptic scaffolding protein and a regulator of integrin activity, as a novel tumor suppressor targeting RAS proto-...

The trafficking chaperone PDE6D (also referred to as PDEδ) has been nominated as a surrogate target for K-Ras4B (hereafter K-Ras). Arl2-assisted unloading of K-Ras from PDE6D in the perinuclear area is significant for correct K-Ras localization and therefore activity. However, the unloading mechanism also leads to the undesired ejection of PDE6D in...

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 t...

The KRAS gene is highly mutated in human cancers and the focus of current Ras drug development efforts. Recently the interface between the C-terminus of K-Ras and calmodulin (CaM) was proposed as a target site to block K-Ras driven cancer cell stemness. We therefore aimed at developing a high-throughput amenable screening assay to identify novel Ca...

KRAS is highly mutated in human cancers and the focus of current Ras drug development efforts. Recent work demonstrated that K-ras4B is a much stronger promotor of cancer cell stemness than H-ras, providing an additional rational why to focus on K-ras. We recently demonstrated that ophiobolin A inhibits cancer cell stemness and specifically K-ras m...

T helper (Th)17 cells represent a unique subset of CD4+ T cells and are vital for clearance of extracellular pathogens including bacteria and fungi. However, Th17 cells are also involved in orchestrating autoimmunity. By employing quantitative surface proteomics, we found that the evolutionarily conserved prohibitins (PHB1/2) are highly expressed o...

Introduction PI3K/mTORC1- and Ras/MAPK-signalling pathways are aberrantly regulated in most cancers. Specific resistances to drugs targeting these pathways can emerge during tumour evolution. Preexisting, innate resistance mechanisms, such as stemming from feedback-loops, should ideally be known already during drug-target nomination. However, as th...

Approaches to increase the efficiency in developing drugs and diagnostics tools, including new drug delivery and diagnostic technologies, are needed for improved diagnosis and treatment of major diseases and health problems such as cancer, inflammatory diseases, chronic wounds, and antibiotic resistance. Development within several areas of research...

As a major growth factor transducer, Ras is an upstream activator of mTORC1, which further integrates nutrient and energy inputs. To ensure a contextual coupling of cell division via Ras/MAPK-signalling and growth via mTORC1-signalling, feedback loops from one pathway back to the other are required. Here we describe a novel feedback from mTORC1, wh...

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 i...

The Ras/MAPK signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. The SPRED1 gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear, wh...

Ras-induced senescence mediated through ASPP2 represents a barrier to tumour formation. It is initiated by ASPP2's interaction with Ras at the plasma membrane, which stimulates the Raf/MEK/ERK signaling cascade. Ras to Raf signalling requires Ras to be organized in nanoscale signalling complexes, called nanocluster. We therefore wanted to investiga...

Analysis of protein expression by Western blot. (A) Representative Western blots (n>10) from HEK cells expressing ASPP2- or Gal-1-plasmids (indicated on top). Probing antibodies are shown to the left. (B) Representative anti-ASPP2 Western blot (n>10) from HEK cells transfected with pCMV-Sport6-ASPP2 (lane 2), pcDNA3-ASPP2(1–1128)-V5 (lane 3), pcDNA...

ASPP2 truncation mutants localize similarly to full-length ASPP2. (A) Confocal microscopic images of HEK cells transfected with mGFP-RasG12V (green) isoforms or the full-length and truncated ASPP2 (red). (B-C) Confocal fluorescence microscopy on HEK cells cotransfected with (B) mGFP-K-rasG12V or (C) mGFP-N-rasG12V (green) and full-length or truncat...

ASPP2 truncation mutants block Gal-1 dependent nanoclustering. (A-B) Nanoclustering-FRET analysis in HEK cells coexpressing mGFP- and mCherry-tagged (A) H-rasG12V or (B) K-rasG12V. Cells were analysed after overexpression with the proteins as indicated (mean ± SEM, n = 3). Statistical significance of differences between control and treated samples...

Introduction: The lack of direct Ras inhibitors, has led to the development of inhibitors of downstream components of the Ras pathway albeit with mixed success. Unexpected mechanistic complexity, feedback loops and tumor specific synthetically lethal requirements continue to be major obstacles. In the past we investigated the mechanisms of the Ras...

Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The la...

Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding discovery of compounds with similar anti-CSC activity. Here we show that salinomycin very s...

The growth hormone receptor (GHR) has been the archetype for the class I cytokine receptor family. The original paradigm for how growth hormone (GH) binding to the GHR led to activation of the associated JAK2 was based on biophysical and structural studies over 20 years ago which showed that growth hormone binding to the extracellular domain of the...

Ras isoforms H-, N- and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators...

Fluorescence resonance energy transfer (FRET) is widely used to study conformational changes of macromolecules and protein-protein, protein-nucleic acid, and protein-small molecule interactions. FRET biosensors can serve as valuable secondary assays in drug discovery and for target validation in mammalian cells. Fluorescence lifetime imaging micros...

Thermodynamic and kinetic parameters of H-ras mutant in vitro experiments. Table contains background corrected GEF-dependent Eu3+-GTP association (kon), and dissociation (koff) kinetics of wt H-ras and mutants. The Kon, Koff, and Kd derived from Koff/Kon values are calculated from corrected association and dissociation data as described in ‘Materia...

Distribution of isoform specific coding mutations in the switch III region by cancer tissue type. Table details the frequency of amino acid changes due to coding mutations observed in switch III region of each isoform in different tissue types of cancer. Point mutations leading to different amino acid residue changes at the same coding position hav...

Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ra...

Rab proteins constitute the largest subfamily of Ras-like small GTPases. They are central to vesicular transport and organelle definition in eukaryotic cells. Unlike their Ras counterparts, they are not a hallmark of cancer. However, a number of diseases, including cancer, show a misregulation of Rab protein activity. As for all membrane-anchored s...

GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this s...

The conformational changes required to transmit the GH binding signal from the extracellular domain of the GH receptor to its intracellular domain resulting in activation of JAK2 has been enigmatic. We have recently defined the first complete mechanistic model for JAK2 activation based on an archetypal cytokine receptor, the growth hormone receptor...