Modulation of non-canonical CXCR4 and ACKR3 functions

The large family of G protein-coupled receptor (GPCR) family transduce extracellular signals via diverse signaling pathways to control physiological functions. They are implicated, amongst others, in cardiovascular and neurodegenerative diseases and cancer. GPCRs represent the most targeted family of membrane receptors, being targeted by around 34% of the FDA-approved drugs. GPCRs interact with different proteins leading to different intracellular signaling and trafficking. Within the GPCR family, chemokine receptors such as CXCR4 and ACKR3 play important roles in immune surveillance, inflammation, and regulation of cell migration. CXCR4 is widely expressed across tissues and is essential for embryonic development but also highly expressed in various types of cancer. ACKR3 is notable for its role in cancer progression, influencing cell proliferation, migration, and drug resistance. In cancer, CXCR4 and ACKR3, and their shared ligand CXCL12, play critical roles in tumor growth, invasion, and metastasis, making them potential targets for therapeutic intervention in oncology and inflammatory diseases. Research has increasingly focused on GPCRs in oncology, particularly CXCR4 and ACKR3, due to their overexpression in multiple cancers. This thesis explores the non-canonical functional consequences of these receptors in a cancer setting using distinct CXCR4 and ACKR3-directed nanobodies. CXCR4-targeting nanobodies have been used to modulate CXCR4 oligomerization and consequently study the functional consequences of these oligomers. We could disrupt CXCR4 oligomers, monomerizing CXCR4, or further induce CXCR4 oligomers. These tools aided us to validate that CXCR4 oligomers activate the oncogenic JAK2/STAT3 signaling pathway. Different approaches have been taken to study ACKR3. First, different phosphorylation mutants in ACKR3's C-terminal tail were generated to understand the importance of phosphorylation in β-arrestin recruitment and trafficking. We showed that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced β-arrestin recruitment, ACKR3 internalization and trafficking. Second, ACKR3-targeting nanobodies with different modes of action were employed to explore the basal activity of ACKR3. Structural analysis of nanobody-bound ACKR3 is carried out to assess their effects at a molecular level. Lastly, we initiated studies using CRISPR-Cas9 tools in a cancer cell line and Turbo-ID as proximity labelling enzyme, to gain deeper insight into its function in a cancer setting under basal conditions. Here, putative interacting partners of ACKR3 are identified which may play a role in basal activity of ACKR3 in cancer cells. The findings underscore the significance of GPCRs, particularly CXCR4 and ACKR3, in cancer biology, emphasizing their potential as targets for novel therapies and providing insights into their molecular mechanisms in cancer cells.