Highlights of the Marie Curie Training Network ONCORNET2.0

Ellen Langemeijer and Martine Smit

The ONCORNET2.0 (Oncogenic GPCR Network of Excellence and Training) MSCA ITN programme (2020–2024) has successfully trained 16 early stage researchers (ESRs) (Figure 1) and delivered important technical and conceptual advances to target oncogenic G protein-coupled receptors (GPCRs) CXCR4 and ACKR3. Despite the impact of the COVID-19 pandemic at the start of this project, the ESRs conducted their PhD research impressively.

CXCR4 and ACKR3 are highly expressed in different types of tumours. Moreover, CXCL12, the only chemokine binding CXCR4, is constitutively expressed at tumour sites. ACKR3 (atypical chemokine receptor 3) also binds CXCL12 and its expression is also enhanced in a variety of tumour cells and on tumour-associated vasculature. ACKR3 is a non-G protein signalling GPCR, classically viewed as a modulator of CXCR4 functions through CXCL12 scavenging. Thus, targeting CXCR4 and/ or ACKR3 function may be a valuable approach for therapeutic intervention in cancer. Within this network, we have developed and characterised various tools and assays to study CXCR4 and ACKR3 and their signalling complexes in new model systems.

Scientific output ONCORNET2.0: new modulators, assays and model systems

Within ONCORNET2.0, we developed and/or characterised modulators with new modes of action (photoswitchable small molecules (Bérenger et al., 2024), inverse agonistic nanobodies (Perez Almeria et al., 2024) and small molecule (Pan et al, 2025), fluorescent small ligands (Dekkers et al., 2023) and multivalent nanobodies (Anbuhl et al., 2024) to assess the role of these two receptors in a (patho) physiological context. Multiplex detection of fluorescent chemokine binding (Adamska et al., 2024) was established, and nanoluciferase-based complementation assays were set up to profile GPCR-GRK interactions (Palmer et al., 2022). Roles of new CXCR4 and ACKR3 interactors Ephrin B1 (Rabbito et al., 2024) and known signalling partners Connexin 43 (Gallego et al., 2023), GRK2 were investigated. We assessed the interactome, post- translational modifications, spatiotemporal compartmentalised signalling and conformational signatures of intracellular proteins in real time with advanced imaging. In addition, cross-talk of CXCR4 with the EGFR (Neves et al., 2022), and ACKR3 with the opioid network (Szpakowska et al., 2023) were assessed.

Different nanobody formats were generated to detect CXCR4 clusters, CXCR4-EGFR heteromers via BRET- based approaches (Comez et al., 2022). Multivalent nanobodies, Fc format, showed enhanced affinity and potency to block CXCR4 function. Combined HDX-MS and MD simulations were used to examine the binding mode and mechanism of action of various small molecules and nanobodies directed at ACKR3 with different efficacy for ß-arrestin recruitment (Otun et al., 2024). GRK and ß-arrestin KO cell lines were used to dissect the contribution of the spatiotemporal signalling of ACKR3. ß-arrestin sensors were used to assess the differential modulation of ACKR3 by ACKR3-directed ligands.

In addition, physiologically relevant cell systems expressing CXCR4/ACKR3, including breast cancer cell lines, 3D epithelial cultures or skin organoids, and HPV-infected primary keratinocytes were characterised or established to study the CXCR4-ACKR3-CXCL12 axis in cancer (Laganà et al., 2024; Cuesta-Margolles, Schlecht-Louf and Bachelerie, 2024). Sophisticated mathematical models were generated based on pharmacokinetic parameters, providing a valuable tool for predicting drug effects and exploring new therapeutic strategies. In addition, unique competencies developed through collaborative networks, like this training network, disclosed the synergies of the four Cs: creativity, critical thinking, cooperation and communication (Saha et al., 2023).

Altogether, this and the previous programme resulted in over 70 joint ONCORNET publications published in Open Access journals (see Dissemination on www.oncornet.eu), and more manuscripts are currently being finalised. The majority of ONCORNET2.0 ESRs have defended their thesis in the meantime or plan to do so in the near future. All ESRs have presented their work during one or more scientific conferences by means of a poster or selected talk. PIs have presented work performed within ONCORNET2.0 during their invited talks. At the end of the programme, the ESRs organised the final ONCORNET2.0 symposium, which was open to the GPCR community in September 2023. The ESRs put together the scientific programme, chaired the sessions and presented their own work. This was a very lively and interactive event that showed the major highlights of ONCORNET2.0.

Training the next generation of multidisciplinary scientists in drug discovery

The ESRs of ONCORNET2.0 received exclusive international, intersectorial interdisciplinary ‘GPCR drug discovery’ training. This included training in a diverse set of scientific and transferable skills (entrepreneurship, academic writing, media training, valorisation) to improve the employability and career prospects of young researchers. The ONCORNET and ONCORNET2.0 ESRs have benefited from: top scientific research performed in renowned GPCR laboratories; dedicated, high-quality courses and workshops in the field of modern drug discovery and development; built a personal international network with GPCR experts throughout Europe; understanding the business and innovation processes in the changing world of pharmaceutical and biotech sciences; exposure to entrepreneurship as a particular skill by doing a simulation course on drug discovery and development; and joining round tables with experts to discuss career paths within industry and academia.

During their training, ESRs took one or more secondments with the participating academic and/or private beneficiaries or participating organisations. ONCORNET2.0 ESRs rated the secondments and transferable skills as most helpful in their career development. Due to their interdisciplinary and international nature and secondments, ESRs have the advantage of building extensive networks during their PhD training. Moreover, this transdisciplinary secondment enabled them to see their project from a new perspective. Such engagements can help students gain broad knowledge, which can be helpful in future roles requiring coordinating and managing a multidisciplinary drug discovery project. Such a holistic view allows researchers to bridge different fields and organisations, a skill deemed to be key to the innovativeness of the industry. Moreover, secondments enabled them to get introduced to working in small (InterAx, QVQ) or larger (Sosei-Heptares) companies and compare doing research in an academic setting.

Alumni network expanded

Former ONCORNET ESRs (2015–2019) joined the ONCORNET alumni network to remain in contact with their former host institutions, expand their network and exchange experiences on career changes. Vladimir Bobkov (arGEN-X– former beneficiary of ONCORNET) gave part of the antibody training in the first workshop. Joyce Heuninck, Vladimir Bobkov, Carmen Gallego and Amos Fumagalli were part of the round tables on ‘Careers in academia and industry’. Two- thirds of the former ESRs were present at the celebration event following our final ONCORNET2.0 meeting in Amsterdam. These activities illustrate that the alumni network is active and sets an example for the new generation of ESRs. More impact of this network, with the new generation of ONCORNET2.0 alumni (2020–2024), in and outside the scientific community, is expected in the coming years.

For this well-trained group of ESRs, we expect, hope for, and foresee careers that will thrive as anticipated. Of the ONCORNET and ONCORNET2.0 alumni, 52% have jobs in pharmaceutical industries (36%) and biotech/SMEs (16%), 45% within academia—several obtained MSCA postdoctoral fellowships, and 3% (one ESR) found a position outside the life science sector (see Figure 2). The ESRs truly enjoyed being part of this training network (see some of their quotes in Box 1). The two cohorts of alumni will ensure that the community of GPCR researchers will further expand in a sustainable way for many years to come.

Extrapolation to other oncogenic GPCRs

Within ONCORNET, we employed the latest multidisciplinary research technologies to understand the role of CXCR4 and ACKR3 in cancer and developed tools targeting these receptors for diagnostic and therapeutic purposes. Gaining refined knowledge of the molecular mechanisms governing the biological functions of these receptors will pave the way for targeted, finely-tuned and innovative therapeutic strategies. Importantly, the approaches we developed can be extrapolated to other oncogenic GPCRs. By joining forces, we also laid the groundwork for another application, the EIC-Pathfinder project UniSens, led by ISAR Bioscience GmbH, with CNRS and VUA as partners. Moreover, we were invited to participate as a consortium during the first virtual GPCR forum (GPCR Forum, 2024), illustrating that we are an important player in the GPCR community.

With the development of new modulators, a better understanding of the signalling networks, and translational models for these two fascinating GPCRs highly expressed in cancer, our studies are not yet completed. We would like to thank the EU for funding ONCORNET2.0 and all consortium partners and ESRs, cohorts 2015–2019 and 2020–2024, for their invaluable collaborative contributions over these years. Our research journey will continue.

Quotes from ONCORNET2.0 alumni

“Being part of ONCORNET2.0 was a great experience with many collaborations, scientific discussions, travels, new connections, and lots of fun!’’
Stephanie Anbuhl, ESR2 at QVQ,
The Netherlands

“ONCORNET 2.0 was an incredible PhD experience, surrounded by a supportive team and filled with inspiring teamwork. It provided fantastic networking and learning opportunities, and I’m grateful to have been a part of it.’’

Noemi Karsai, ESR11,
University of Nottingham

”Networks like ONCORNET2.0 are likely among the greatest strengths of the European scientific community, fostering a spirit of collaboration and generosity that can sometimes be scarce in academia. I was incredibly fortunate to be a part of this consortium which taught me immensely both scientifically and in terms of personal development.’’

Christie Palmer, ESR16, Luxembourg Institute of Health

“ONCORNET2.0 was a unique scientific and collaborative opportunity in state-of- the-art research in the molecular biology of cancer, but also a rich experience on the personal level. It was a great opportunity to collaborate with colleagues and group leaders from different disciplines and backgrounds as we work on a common goal in real-time.”

Noureldine Youssef, ESR8, University of Jena

“ONCORNET2.0 was all about great scientific exchange, networking, learning, advice, support and endless stickers. I don’t think you can offer me a better opportunity to do the PhD. Initiatives like these are what keep the European scientific community together.”

Gabriela Cuesta Margolles, ESR14, INSERM Paris Saclay

“ONCORNET was an amazing life experience. I had the opportunity to do good science, meet a lot of people in the GPCR field, improve my scientific skills and have fun with new friends, all in one.”
Alessandro Rabbito, ESR9, IGF at Montpellier

References

Adamska, J.M., Leftheriotis, S., Bosma, R., Vischer, H.F. and Leurs, R. (2024) ‘Multiplex detection of fluorescent chemokine binding to CXC chemokine receptors by NanoBRET’, International Journal of Molecular Sciences, 25(9), 5018. doi: 10.3390/ijms25095018.

Anbuhl, S.M., Dervillez, X., Neubacher, S., Schriek, A.I., Bobkov, V., de Taeye, S.W., Szpakowska, M., Siderius, M., Grossmann, T.N., Chevigné, A., Smit, M.J. and Heukers, R. (2024) ‘Multivalent CXCR4- targeting nanobody formats differently affect affinity, receptor clustering, and antagonism’, Biochemical Pharmacology, 227, 116457. doi: 10.1016/j.bcp.2024.116457.

Bérenger, S., Adamska, J.M., Deflorian, F., de Graaf, C., Palmer, C.B., Szpakowska, M., Chevigné, A., de Esch, I.J.P., Zarzycka, B., Vischer, H.F., Wijtmans, M. and Leurs, R. (2024) ‘Design, synthesis and pharmacological characterization of the first photoswitchable small-molecule agonist for the atypical chemokine receptor’, bioRxiv Chemistry, Medicine. doi: 10.1101/2024.03.20.585914.

Comez, D., Glenn, J., Anbuhl, S.M., Heukers, R., Smit, M.J., Hill, S.J. and Kilpatrick, L.E. (2022) ‘Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells’, Frontiers in Immunology, 13, 1006718. doi: 10.3389/fimmu.2022.1006718.

Cuesta-Margolles, G., Schlecht-Louf, G. and Bachelerie, F. (2024) ‘ACKR3 in skin homeostasis: an overlooked player in the CXCR4/CXCL12 axis’, Journal of Investigative Dermatology. doi: 10.1016/j.jid.2024.08.022.

Dekkers, S., Comez, D., Karsai, N., Arimont-Segura, M., Canals, M., Caspar, B., de Graaf, C., Kilpatrick, L.E., Leurs, R., Kellam, B., Hill, S.J., Briddon, S.J. and Stocks, M.J. (2023) ‘Small molecule fluorescent ligands for the atypical chemokine receptor 3 (ACKR3)’, ACS Medicinal Chemistry Letters. doi: 10.1021/acsmedchemlett.3c00469.

Gallego, C., Jaracz-Ros, A., Laganà, M., Mercier-Nomé, F., Domenichini, S., Fumagalli, A., Roingeard, P., Herfs, M., Pidoux, G., Bachelerie, F. and Schlecht-Louf, G. (2023) ‘Reprogramming of connexin landscape fosters fast gap junction intercellular communication in human papillomavirus-infected epithelia’, Frontiers in Cellular and Infection Microbiology, 13, 1138232. doi: 10.3389/fcimb.2023.1138232.

GPCR Forum (2024) GPCR Forum Conference 2024. Available at: https://www.gpcrforum.org/events/gpcrforumconference2024

Laganà, M., Margolles, G.C., Jaracz-Ros, A., Mercier-Nomé, F., Roingeard, P., Lambert, P.F., Schlecht- Louf, G. and Bachelerie, F. (2024) ‘Optimized protocol for 3D epithelial cultures supporting human papillomavirus replication’, STAR Protocols, 5(1), p. 102828. doi: 10.1016/j.xpro.2023.102828.

Neves, M., Marolda, V., Mayor, F. Jr and Penela, P. (2022) ‘Crosstalk between CXCR4/ACKR3 and EGFR signaling in breast cancer cells’, International Journal of Molecular Sciences, 23(19), 11887. doi: 10.3390/ijms231911887.

Otun, O., Aljamous, C., Del Nero, E., Arimont-Segura, M., Bosma, R., Zarzycka, B., Girbau, T., Leyrat, C., de Graaf, C., Leurs, R., Durroux, T., Granier, S., Cong, X. and Bechara, C. (2024) ‘Conformational dynamics underlying atypical chemokine receptor 3 activation’, Proceedings of the National Academy of Sciences of the United States of America, 121(30), e2404000121. doi: 10.1073/pnas.2404000121.

Palmer, C.B., D’Uonnolo, G., Luís, R., Meyrath, M., Uchański, T., Chevigné, A. and Szpakowska, M. (2022) ‘Nanoluciferase-based complementation assay for systematic profiling of GPCR-GRK interactions’, Methods in Cell Biology, 169, pp. 309–321. doi: 10.1016/bs.mcb.2022.04.001.

Pan K.S., Wang Z., Pfeil C., Bergkamp N.D., Mobach S., Roth S, Rizk A., Lohse M.J. Annibale P., Siderius M., Zimmermann M. , Smit M.J., Bosma R. (2025) ‘Pharmacological characterisation of a clinical candidate, TG-0054, a small molecule inverse agonist targeting CXCR4’, Molecular Pharmacology, in press.

Perez Almeria, C.V., Otun, O., Schlimgen, R., Lamme, T.D., Crudden, C., Youssef, N., Musli, L., Jenjak, S., Bobkov, V., Drube, J., Hoffmann, C., Volkman, B.F., Granier, S., Bechara, C., Siderius, M., Heukers, R., Schafer, C.T. and Smit, M.J. (2024) ‘Constitutive activity of an atypical chemokine receptor revealed by inverse agonistic nanobodies’, bioRxiv. doi: 10.1101/2024.11.04.621790.

Rabbito, A., Otun, O., Fumagalli, A., Séveno, M., Galant, S., Counson, M., Durroux, T., Bechara, C., Smit, M.J., Granier, S., Szpakowska, M., Chevigné, A., Chaumont-Dubel, S. and Marin, P. (2024) ‘Physical interaction with Ephrin B1 promotes CXCR4 intracellular localization and oncogenic potential’, bioRxiv. doi: 10.1101/2024.07.10.602459.

Saha, S., van der Sijde, P., Langemeijer, E.V., Briddon, S.J., van Muijlwijk-Koezen, J.E. and Smit, M.J. (2023) ‘Unique competencies developed through collaborative networks: synergy of the 4Cs: creativity, critical thinking, cooperation and communication’, The Project Repository Journal, 17, pp. 84–87. doi: 10.54050/ PRJ1720311.

Szpakowska, M., D’Uonnolo, G., Luís, R., Alonso Bartolomé, A., Thelen, M., Legler, D. and Chevigné, A. (2023) ‘New pairings and deorphanization among the atypical chemokine receptor family – physiological and clinical relevance’, Frontiers in Immunology, 14, p. 1133394. doi: 10.3389/fimmu.2023.1133394.

PROJECT SUMMARY

The ONCORNET2.0 (ONCOgenic GPCR Network of Excellence and Training) consortium (2020–2024) aims to consolidate an international training network of early stage researchers (ESRs) focused on drug discovery for oncogenic GPCRs. The project methods span a wide range of techniques and disciplines aimed at furthering our understanding of two receptors heavily involved in oncogenic processes.

PROJECT LEAD

The project coordinator of ONCORNET2.0 is Prof. Martine J. Smit (Professor Target and Systems Biochemistry, VUA). Martine Smit coordinated ONCORNET, has received personal (NWO-Vidi/Vici) and public-private funding. Her expertise focuses on modulating and unravelling the signalling properties of human and viral chemokine receptors. She is supported by Prof. Jacqueline van Muijlwijk (educational research chair and vice dean education, VUA), Dr Ellen Langemeijer (project manager, VUA) and Prof. Steve Hill (UNOTT) as confidential advisor to support and advise ESRs.

CONTACT DETAILS

Ellen Langemeijer, Project Manager

De Boelelaan 1108, 1081 HZ Amsterdam,

Netherlands

Email: e.v.langemeijer@vu.nl
Web: https://oncornet.eu/

FUNDING

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Innovative Training Networks (MSCA-ITN) grant agreement No. 860229.