ACKR3 in Health and Disease: From Fundamental Insights To Drug Discovery

Introduced in Chapter 1, Cxcl12 and Cxcr4 play crucial roles during development, adult life and pathophysiology, in particular in hematopoiesis, inflammatory disease and cancer progression. Hematopoiesis relies on a complex interplay between hematopoietic cells and supporting stromal cells within specialized bone marrow (BM) niches. Cxcl12 and Cxcr4 have been extensively investigated in this contexts, whereas the function of Ackr3, a second receptor for Cxcl12, remains partly understood. In chapter 2, we provide a comprehensive overview of Ackr3 expression in mammalian tissue and explore potential functions in the immune system, focusing on the myeloid lineage. Ackr3 has been reported to be expressed in bone tissue and mesenchymal cells, therefore we aimed to further investigate the role of Ackr3 in hematopoiesis, from early development to adult life. In chapter 3, we further investigate Ackr3 expression in BM during development and adult life. Our findings show that Ackr3 is highly expressed in the B cell lineage as well as in multipotent bone mesenchymal stromal cells (MSCs) within the metaphysis and epiphysis. Its expression in the bone stroma is associated with mesenchymal multipotency and is downregulated during osteogenic differentiation. To continue, in chapter 4, we established a robust and universal flow cytometry-based functional assay studying the scavenging capabilities of Ackr3 by measuring the Cxcl12 uptake in several cell systems. This assay has been applied to study the potential of small molecules to inhibit the Cxcl12 scavenging activity of Ackr3. We further applied the assay to primary murine bone-derived cells and provided evidence that these cells are capable of scavenging Cxcl12 through Ackr3, confirming Ackr3 is functionally expressed in these cells. Nanobodies are powerful tools in research and medicine with many beneficial and easily modified properties. Due to their high specificity, nanobodies can distinguish between human and mouse receptors. To study targeting CXCR4 and ACKR3 in cancer, in which both receptors are reported to be expressed in the primary tumor as well as in the tumor microenvironment, therapeutic agents are required that bind and modulate the murine protein. Therefore in chapter 5, we developed and characterized nanobodies targeting murine Cxcr4 and Ackr3, optimized their format, and characterized them in vitro. Their therapeutic potential was tested in a syngeneic mouse model of colorectal carcinoma, which resulted in reduced tumor growth. The broader implications of the key findings will be discussed in Chapter 6, as well as outstanding questions and directions for future research. Understanding Ackr3 in health and disease is crucial for advancing targeted therapies. This work contributes to understanding Ackr3 in bone development, hematopoiesis and cancer and provides translational tools for drug discovery and future research.