Hardeman/Gunning Group: Cytoskeleton Therapeutics Research Unit (CTRU)

Hardeman & Gunning Group: Cytoskeleton Therapeutics Research Unit (CTRU)

The CTRU studies the building blocks of cell architecture and develops therapeutic strategies based on drug-targeting these building blocks. Our focus is the actin cytoskeleton that is responsible for the internal scaffolds of cells, the generation and reaction to force exerted by the environment and the movement of cells throughout the body. We study the role of cell architecture in conditions as diverse as cell signalling, biophysical properties of cells, cancer and the extracellular environment, immune targeting of foreign material, linkage between cells and generation of platelets from megakaryocytes.

Our research spans from bench to bedside and encompasses the disciplines of cell biology, mechanobiology, biophysics, drug discovery and therapeutics. We are world leaders in the field of the actin cytoskeleton and have defined an underlying principle that governs the composition and diverse functions of actin filaments. Actin filament diversification is largely achieved by the tropomyosin (Tpm) isoforms that form co-polymers with actin and directly regulate the function of individual filaments. Tropomyosins are implicated in a range of human conditions and have significant therapeutic potential.

Currently, our in-house projects are focused on:

Understanding the role of the actin/tropomyosin cytoskeleton in cancer cells and associated stromal cells.
Defining the mechanism of tropomyosin regulation of platelet biogenesis.
Defining the role of tropomyosin in eye lens opacification and fibrosis.
Development of novel therapeutics for the treatment of tropomyosin-based human conditions including cancers, platelet disorders, cataract formation and fibrosis in collaboration with industry partners.

We have generated the most comprehensive set of antibodies to visualise the location of the different tropomyosins in the cytoskeleton which are distributed by Sigma-Merck-Millipore. We have generated tropomyosin isoform-specific transgenic, knock-in, knock-out and tagged (GFP, neon green, APEX) mouse lines.

We use state-of-the-art imaging modalities to visualise the actin/tropomyosin cytoskeleton including confocal, live cell, TIRF, intravital subcellular, single molecule intravital subcellular, HILO, AFM, super resolution STED and PALM, correlative cryo-electron tomography and have developed single molecule intravital subcellular microscopy.

Current projects

Oncology
Cardiovascular Disease
Neuroscience
Immunology
Drug Discovery

Anti-tropomyosin Drugs / Anti-cancer Drugs

A potential target for anti-cancer drugs is the actin cytoskeleton; however, it has been unattainable because the toxicity associated with disabling this target, especially in the heart, has been unacceptable. The actin cytoskeleton is a desirable target because it controls many fundamental processes in the cancer cell such as cell growth, migration and interaction with the surrounding environment. We have identified how drugs that target specific isoforms of the actin-associated protein tropomyosin expressed in cancer cells disrupt cancer cell division and response to chemotherapeutic agents.

Cytoskeleton Combination Cancer Treatment

Cancer cells have an unexpected reliance on the tropomyosin Tpm3.1. Actin/Tpm3.1 filaments are the major actin cytoskeleton structures in all types of cancer cells. We have demonstrated that compounds that target the cancer tropomyosin Tpm3.1 synergise with microtubule-targeting drugs to reduce tumour growth in a broad range of cancers (Mol Cancer Ther, 2017; Mol Cancer Res, 2020; Br J Cancer, 2021). This approach also has the potential to selectively disable specific actin filament populations involved in a range of cellular functions. 

Tumour Cytoarchitecture in Pancreatic Cancer

Pancreatic cancer remains one of the great challenges of oncology. New treatment strategies are urgently required. We used Tissue Micro Arrays (TMAs) to identify candidate therapy targets. In collaboration with the Pancreatic Cancer Group at ��, we have identified multiple tropomyosin targets (Tpms 3.1, 1.6, 1.8) that regulate different properties of pancreatic tumours. We are testing the ability of different anti-tropomyosin drugs to both improve drug access to the tumour and to kill the tumour cells.

Chemoresistance in Ovarian Cancer

Epithelial ovarian cancer (EOC) is the leading cause of death In gynaecological malignancies. In collaboration with researchers at Erasmus Medical Center in The Netherlands, we have shown that the tropomyosin isoforms Tpm1.8/9 are expressed in the ascites of patients, drive invasion by activating EMT and Wnt signalling, and underpin resistance to chemotherapeutic agents. Using small molecule inhibitors developed by us, we showed that inhibiting Tpm1.8/9 resensitises EOC cells to anti-microtubule agents. The development of drugs targeting Tpm1.8 and Tpm1.9 could potentially restore patient response to therapy and enhance the efficacy of existing chemotherapeutic regimens.

Platelets are key players in blood clotting and tropomyosin Tpm4.2 has an important role in the generation of platelets from their precursor cell, the megakaryocyte (J Clin Invest, 2017). A novel mechanism of platelet generation was recently discovered that involves membrane budding from megakaryocytes and together with collaborators we have shown that Tpm4.2 plays a crucial role in this process. This finding has sparked interest in developing drugs that target Tpm4.2 to regulate platelet production and reduce stroke risk in patients with thrombocythaemia, a condition marked by excessive platelets, as well as in patients at risk for secondary stroke. Defining the precise role of Tpm4.2 in the budding process will aid in understanding this fundamental biological process, inform drug development for this target, and reveal other potential therapeutic targets that act in concert with Tpm4.2.

The actin cytoskeleton plays a pivotal role in the formation and function of tunneling nanotubes (TNTs) in the nervous system. These remarkable structures facilitate direct cell-to-cell communication, enabling the exchange of proteins, organelles, and genetic material between neurons and glia. The actin cytoskeleton's dynamic nature drives the extension and retraction of TNTs through actin polymerization and depolymerization, regulated by actin-binding proteins. Additionally, actin-based motor proteins facilitate cargo transport along the length of these intricate intercellular bridges.

Significantly, the actin cytoskeleton's involvement in TNT formation intersects with signaling pathways implicated in neuronal development, plasticity, and pathology. In collaboration, we are exploring the role of tropomyosins in TNT-mediated communication in neurodegenerative disorders and neuronal regeneration. Beyond the nervous system, targeting the actin cytoskeleton could disrupt or modulate TNT-mediated processes implicated in pathogen spread and cancer progression, offering potential therapeutic interventions.

The immune synapse, a critical junction between T cells and antigen-presenting cells, plays a vital role in orchestrating an effective immune response. At the heart of this intricate structure lies the dynamic reorganization of the actin cytoskeleton, and our collaborators have shown that actin filaments containing tropomyosin 3.1/2 (Tpm3.1/2) play a pivotal role.

Tpm3.1/2 isoforms are key regulators of actin filament dynamics, influencing the assembly and disassembly of actin networks within the immune synapse. This precise control over actin filament organization is crucial for the proper segregation of signaling molecules, enabling efficient signal transduction and subsequent T cell activation or suppression. Disruptions in this delicate balance can lead to aberrant immune responses.

Given the pivotal role of Tpm3.1/2 in modulating the immune synapse, our collaborators are actively evaluating drugs targeting these actin-binding proteins as potential regulators of the immune response. In autoimmune conditions, where the immune system mistakenly attacks healthy tissues, modulating Tpm3.1/2 activity could potentially dampen excessive T cell activation and mitigate the autoimmune response, alleviating symptoms and preventing tissue damage. Conversely, enhancing Tpm3.1/2 function may boost immune surveillance and T cell responses against pathogens or cancer cells, augmenting the body's ability to combat infections and malignancies.

The Challenge - Discovery and development of therapeutic leads is generally focused only on known molecular targets. This imposes a narrow, ‘target-biased’ approach exploiting only a small part of the universe of potential therapeutic opportunities. This contributes to severe bottlenecks in drug development, further exacerbated by the very high costs (~$200 million USD) and slow progression (1-3 years) of therapeutic lead discovery (Biomarker Development and Advanced R&D Landscape Overview 2019/Q1). New technology platforms are thus urgently needed to bring better therapies to patients, faster and more cheaply.

The Solution - We have developed CellaSense; an outcome-orientated approach to therapeutic lead discovery using phenotypic analysis to identify novel, mechanistically unbiased, structurally diverse therapeutic leads. CellaSense also allows rapid identification of compounds that ‘pheno-copy’ known treatments, supporting accelerated identification of Bio-betters and Bio-similars.

The mechanistically and structurally agnostic nature of CellaSense expands the exploitable universe of therapeutic molecules to include targets not predictable with current biological knowledge, while also providing diversified starting points (Makush structures) for molecular derivatisation. Leveraging a strategy that progressively transitions from experimental to digital lead discovery, CellaSense constitutes a new paradigm that broadens, accelerates and de-costs therapeutic lead identification.

Team – Dr John Lock, Prof Peter Gunning, Prof Edna Hardeman

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Highlighted publications

Xu T; Verhagen MP; Teeuwssen M; Sun W; Joosten R; Sacchetti A; Ewing-Graham PC; Jansen MPHM; Boere IA; Bryce NS; Zeng J; Treutlein HR; Hook J; Hardeman EC; Gunning PW; Fodde R, 2024, 'Tropomyosin1 isoforms underlie epithelial to mesenchymal plasticity, metastatic dissemination, and resistance to chemotherapy in high-grade serous ovarian cancer', Cell Death and Differentiation, 31, pp. 360 - 377,
Wang D; Wang Y; Di X; Wang F; Wanninayaka A; Carnell M; Hardeman EC; Jin D; Gunning PW, 2023, 'Cortical tension drug screen links mitotic spindle integrity to Rho pathway', Current Biology, 33, pp. 4458 - 4469.e4,
Cagigas ML; Bryce NS; Ariotti N; Brayford S; Gunning PW; Hardeman EC, 2022, 'Correlative cryo-ET identifies actin/tropomyosin filaments that mediate cell–substrate adhesion in cancer cells and mechanosensitivity of cell proliferation', Nature Materials, 21, pp. 120 - 128,
Wang Y; Stear JH; Swain A; Xu X; Bryce NS; Carnell M; Alieva IB; Dugina VB; Cripe TP; Stehn J; Hardeman EC; Gunning PW, 2020, 'Drug targeting the actin cytoskeleton potentiates the cytotoxicity of low dose vincristine by abrogating actin-mediated repair of spindle defects', Molecular Cancer Research, 18, pp. 1074 - 1087,
Bryce NS; Failes TW; Stehn JR; Baker K; Zahler S; Arzhaeva Y; Bischof L; Lyons C; Dedova I; Arndt GM; Gaus K; Goult BT; Hardeman EC; Gunning PW; Lock JG; Arndt G, 2019, 'High-Content Imaging of Unbiased Chemical Perturbations Reveals that the Phenotypic Plasticity of the Actin Cytoskeleton Is Constrained', Cell Systems, 9, pp. 496 - 507.e5,
Pleines I; Woods J; Chappaz S; Kew V; Foad N; Ballester-Beltrán J; Aurbach K; Lincetto C; Lane RM; Schevzov G; Alexander WS; Hilton DJ; Astle WJ; Downes K; Nurden P; Westbury SK; Mumford AD; Obaji SG; Collins PW; BioResource N; Delerue F; Ittner LM; Bryce NS; Holliday M; Lucas CA; Hardeman EC; Ouwehand WH; Gunning PW; Turro E; Tijssen MR; Kile BT, 2017, 'Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia', Journal of Clinical Investigation, 127, pp. 814 - 829,
Gunning PW; O'Neill G; Hardeman EC, 2008, 'Tropomyosin-based regulation of the actin cytoskeleton in time and space', Physiological Reviews, 88, pp. 1 – 35.