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WNT-mediated signal relay in stem cells and oncogenesis - from basic biology to applications

Final Report Summary - WNTSAPP (WNT-mediated signal relay in stem cells and oncogenesis - from basic biology to applications)

General summary of project objectives:
Secreted WNT glycoproteins control embryonic development in all multicellular organisms. In adults, these proteins also sustain the critical supply of stem cells that replenish various body tissues. The aim of WntsApp is to address key unresolved questions on how WNT signals are relayed from the membrane to the cytosol in stem cells and cancer. The underlying molecular mechanisms provide attractive drug targets, particularly in regenerative medicine and cancer treatment. By fostering a symbiosis between high-tech GPCR pharmacology, advanced in vivo imaging and peptide chemistry on the one hand and efficient transfer of know-how between academic and private sector partners on the other, WntsApp offered a unique training platform with a strong potential for innovation.
WntsApp is organized by ten international partners from the academic and the private sector to i) Provide a committed training programme for young researchers to bridge the gap between basic scientific knowledge and drug development, ii) Centring research and stimulate synergies on a key cancer-signalling pathway, Wnt signalling, iii) Strengthening the link between international partners and private enterprises to stimulate innovation and facilitate exploitation of results. WntsApp is organised in 4 work packages (WP). The highlights per work package are listed below:

WP1. Basic mechanisms of receptor-mediated signal relay:
In WP1 we aim to deliver mechanistic insight on how Wnt proteins initiate specific signalling cascades. Multiple tools were built to study Wnt receptor structure and function, both in vitro and in vivo. We were able to isolate the Wnt receptor Frizzled (FZD) and visualise it with electron microscopy. We showed that FZD can signal via a less characterized signalling pathway in vitro, and confirmed the usage of this pathway in cells. Moreover, we identified novel regulators of non-canonical Wnt signalling and assigned a role for these regulators in the generation of chronic lymphocytic leukemia.

WP2. Multiprotein complex assembly:
In WP2 we disentangle the molecular basis of the assembly and disassembly of multiprotein complexes in the WNT cascade, investigate the role of chaperones and the impact of cancer mutations on complex formation and activity. A bioinformatics tool was created for predicting chaperone binding partners, which can help us address the role of the chaperones in regulating complex assembly and countering protein aggregation. In addition, techniques were developed to characterize protein aggregates in vitro and to separate different aggregation intermediates and their interactomes. Peptides were identified that can inhibit inappropriate aggregational interactions induced by cancer mutations and restore tumour suppressor activity of a WNT pathway regulator. These peptides will be tested in vivo for their therapeutic potential.

WP3. Modulating receptor signalling in cancer:
In WP3 we set out to address the mechanisms that control FZD endocytosis and activity at the cell surface, assess their role in tumorigenesis and dissect the role of non-canonical Wnt signalling in cancer. A tool was generated to isolate and analyse the endogenous Wnt receptor complex by mass spectrometry. One of the hits was identified as a positive regulator of Wnt signalling that is overexpressed in colorectal cancer cell lines. Moreover, we aimed to gain understanding of the mode of action of the Wnt receptor regulator RNF43 via systematic deletion of its binding partners in intestinal organoids using a novel strategy. One of the interaction partners was identified as a new player in the membrane-proximal events leading to RNF43-mediated Wnt receptor downregulation. To assess the tumour suppressor role of RNF43 and its homolog ZNRF3 in liver, a liver-specific double knock-out was generated. In parallel, genome engineering was used to introduce mutations in multiple genes to more closely mimic the mutational spectra of human cancers and use this system to model cancer ex-vivo. In addition, the presence and potential importance of several proteins similar to RNF43 was analyzed in chronic lymphocytic leukemia. The expression of two of them correlated with shorter overall survival of patients.

WP4. Therapeutic interventions:
In WP4 we applied innovative high diversity screens and advanced peptide micro arrays to generate and optimize lead compounds that modulate WNT pathway activity by interfering with protein interactions at the cell membrane. A number of cyclic and linear peptides as well as camelid VHHs were identified as high affinity binders to a number of WNT receptors, and were demonstrated to block Wnt-dependent growth of intestinal tumour organoids. These findings open new avenues for targeting the Wnt receptor complex in cancer.

The WntsApp network had a dedicated training mission to increase key complementary skills to maximally develop capabilities and career prospects for ESRs/ERs inside and outside academia. An extensive training program was organised including workshops and advanced courses in addition to their local PhD programs. Moverover, the program helped the fellows built an international network and have contacts in industry. The training was organized in a separate work package and covered: (i) specialist training in an international, interdisciplinary and intersectorial research project, (ii) advanced technology training courses, (iii) professional training in transferable skills.
A priority of the network was to publish in high impact journals. During the four-year WntsApp period notable research results were produced, leading to 13 peer-reviewed publications. In addition, all fellows presented their data in both local seminars and during international symposia.
WntsApp established several channels to communicate with the general public on different levels. The WntsApp webpage, http://WntsApp.eu was the main channel to inform about WntsApp events, publications and WntsApp news. Most fellows were also actively involved in teaching or organizing workshops for high school students to improve the link between universities and schools, but also to interest children early on for the fascination of today’s research in life sciences.

Socio-economic impact:
The research focus of the WntsApp network is on the Wnt signalling pathway that mediates critical cell fate decisions and is strongly linked to cancer. The underlying mechanisms provide attractive drug targets, particularly in regenerative medicine and cancer treatment. During the project, notable research results were produced, delivering significant new insights. A highlight is the identification, generation and validation of a set of Wnt signalling inhibitors. These newly developed reagents hold significant therapeutic potential for cancer treatment and offer possibilities for commercialisation and exploitation. WntsApp expertise and technologies will allow preclinical investigation of the use of these compounds in the treatment of chronic lymphocytic leukemia and intestinal and liver cancer. The network ensures knowledge transfer from this project to related research programs as well as to the general public via press releases, social media and lectures at high schools. Moreover, WntsApp will have a lasting impact on European research and entrepreneurship even after the funding period. The close collaborations, exchange of know-how and sharing of intellectual input between partners over the last 48 months has expanded existing and fostered new long-term collaborations. SMEs that contributed to the network also greatly profited from technologies, expertise and intellectual input from academic partners, strengthening collaborations and steering future exploitations of the results.