The secret(ory) life of megakaryocytes

• Applicant: Dr. Isabelle Becker
• Subject Area: Cell Biology
• Term: from 2022 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 497998118

Megakaryocytes (MKs), the platelet precursors, reside in the bone marrow and develop from hematopoietic stem cells in a complex differentiation process involving DNA replication without cytokinesis (endomitosis) as well as cytoplasmic maturation. Although their main function appears to be the production of platelets, MKs, that only account for 0.05 - 0.1% of all bone marrow cells, are increasingly thought to markedly contribute to the maintenance of marrow homeostasis through the release of cytokines and growth factors. Previous studies already identified MK-derived platelet factor 4 and transforming growth factor β1 (TGFβ1) to be essential for the maintenance of stem cell quiescence, however, whether their release is an active process and which underlying mechanisms induce cytokine secretion still remains a matter of debate. Myelofibrosis, a consequence of some myeloproliferative disorders, is caused by clonal expansion of hematopoietic progenitors and results in impaired blood cell generation, however, treatment options for patients suffering from symptomatic and high-risk myelofibrosis are still limited. Myelofibrosis clusters are characterized by an accumulation of immature MKs, which exhibit an increased release of profibrotic cytokines, such as TGFβ1, which has not only been shown to inhibit matrix metalloproteinase activity, but to also increase collagen and proteoglycan synthesis in MK-adjacent cells. Studies on fibroblasts in the bone marrow, suggested that TGFβ1 secretion is linked to the activity of the Rho GTPase RhoA and dependent on the release of autophagosomes, which were shown to contain TGFβ1. This is in line with other purely cytosolic proteins lacking an N-terminal endoplasmic reticulum signal peptide, which were nonetheless secreted from cells, among them macrophage-derived interleukin 1β, thus suggesting unconventional secretion mechanisms to account for their release.In the proposed project, I aim to identify how autophagy-mediated secretion is regulated in vitro-matured and native MKs and whether it can be modified in order to alter cytokine secretion in vitro. By taking advantage of a variety of autophagy and cytoskeletal activators/inhibitors in combination with coculture assays, I intend to uncover how MKs affect bone marrow homeostasis and whether unconventional secretion from MKs can be targeted therapeutically. Previous data suggests autophagy-dependent secretion to depend on the cytoskeletal protein RhoA as well as the small GTPase Arf6, an endosomal regulator. I will therefore take advantage of a mouse models conditionally lacking Arf6 that I previously identified to exhibit myelofibrosis and analyze disease progression and signaling pathway alterations in MKs. Ultimately, the aim of the proposed project is to 1) identify the signaling mechanisms underlying unconventional secretion of cytokines from MKs and 2) determine how cytokine secretion can be targeted therapeutically to inhibit myelofibrosis progression.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Dr. Joseph E. Italiano