Epcr expression and Epcr/Par1 signaling in Mpl-deficient and normal hematopoiesis during transplantation

• Applicant: Professorin Ute Modlich, Ph.D.
• Subject Area: Hematology, Oncology
• Term: from 2012 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 221103421

Hematopoietic stem cells (HSC) can produce all blood cells throughout our lifetime. In hematological disorders with impaired blood cell production, HSC can be transplanted and replacehematopoiesis in the new host. This makes HSC interesting Targets for cell and gene therapy. HSC maintenance and differentiation are controlled by many factors, including cytokines. Among them Thrombopoietin (THPO) controls megakaryopoiesis but also HSC self-renewal and quiescence. In case the THPO signaling pathway is defective, patients present with aplastic anemia. In our previous work we developed a gene therapy approach for the treatment of THPO receptor (myeloproliferative leukemia virus oncogene, MPL) deficiency using the Mpl-/- mouse model. We demonstrated the regeneration of HSC after lentiviral Mpl re-expression in Mpl-/- hematopoiesis. In gene expression studies differentially regulated genes in regenerated HSC were identified. Of those we selected potential Thpo targets and lentivirally expressed the genes in the Mpl-/- transplantation model. Among the tested genes we identified Epcr to support HSC engraftment, to improve HSC numbers and to maintain HSC long term in vivo in Mpl-/- mice. Epcr cell Surface expression on wildtype murine and human HSC with long termrepopulation potential has also been demonstrated. Stimulation of HSPC in vitro with Thpo better maintained Epcr expression. Epcr Acts as co-receptor by facilitating the activation and presenting activated protein C (APC) to the proteinase activated receptor 1 (Par1). Cleavage of Par1 by APC takes place at a non-canonical site inducing cytoprotective effects. We now aim to investigate how Epcr on HSC mediates its effects, what signal transduction is transmitted byEpcr/Par1 in HSC and how this pathway may be utilized to Support engraftment of BM cell after in vitro modification. In detail, we wish to determine whether HSC proliferation in vivo after transplantation is increased or survival improved. Using tetracycline-inducible vectors, we will induce Epcr expression also later after transplantation or switch off early to determine the time window where Epcr mediates ist effect. Using confocal microcopy we wish to determine changes in stem cell polarity possibly influenced by small GTPase activity downstream of Epcr/Par1 signaling. Signaling responses will be further elucidated in vitro in suitable cell line models and Primary HSC. In the final work package we want to analyze whether activationof Epcr/Par1 signaling in vitro before the transplantation or in vivo shortly thereafter can improve engraftment of HSPC that underwent in vitro transduction using barcoded vectors. Barcodes will be PCR amplified from genomic DNA and quantified by sequencing allowing the estimation of clonal distribution and clone sizes in hematopoiesis. In our project, we expect to unravel the mechanisms of Epcr/Par1 supportive effects on HSC transplantation that may be used therapeutically in the future.

• DFG Programme: Research Grants
• International Connection: Switzerland