Cytokine Secretion from Megakaryocytes Is Governed By Small GTPASES through Autophagy

Megakaryocytes (MKs) are large cells primarily residing in the bone marrow. While it was previously assumed that the sole function of MKs is to produce platelets, recent studies using single cell RNA sequencing have revealed novel subsets of MKs that serve immune functions and maintain stem cell quiescence through the secretion of cytokines such as platelet factor 4 (PF4) and transforming growth factor β1 (TGFβ1). However, while the secretion of these bioactive molecules from platelets requires activation, little is known about what triggers cytokine release from MKs in health and disease. During neoplastic transformation, e.g. upon mutation of the thrombopoietin receptor MPL or its downstream effector kinase JAK2, immature MKs accumulate in the bone marrow, where they release high levels of PF4 and TGFβ1 ultimately causing myelofibrosis and pancytopenia. While current treatment strategies for myeloproliferative neoplasms aim to prevent MK hyperproliferation, an understanding of why cytokine secretion is dysregulated in neoplastic MKs and whether it can be specifically targeted is lacking.

Previous studies have suggested TGFβ1 secretion from fibroblasts occurs via secretory autophagy, the release rather than degradation of autophagosomes, which prompted us to hypothesize that upregulation of autophagy in MKs might promote enhanced TGFβ1 release in myeloproliferative neoplasms. Using an MPL^W515L-driven transplant model of myelofibrosis, we found increased levels of the autophagosome marker light chain (LC) 3B in platelets from diseased mice. In line with this, we identified that TGFβ1 strongly co-localized with the autophagy marker light chain (LC) 3B in native MKs. The GTPase RhoA and its downstream effector Rho kinase 1 (ROCK1) are major regulators of basal autophagy in fibroblasts and upregulated in MKs in myelofibrosis, while loss of the GTPase dynamin 2 from MKs was previously demonstrated to induce bone marrow fibrosis due to increased TGFβ1 release, overall suggesting an important role of small GTPases in regulating cytokine secretion from MKs. To test this hypothesis in vitro, we treated cultured MKs with inhibitors of RhoA or ROCK1, which reduced TGFβ1 secretion and caused an intracellular accumulation of both TGFβ1 as well as the autophagy marker LC3B. In contrast, MKs treated with a small molecule inhibitor of the dynamin 2-associated GTPase Arf6 exhibited reduced intracellular TGFβ1 and LC3B suggesting opposing roles of the GTPases RhoA and Arf6 in governing TGFβ1 secretion.

To investigate this regulation in vivo, we analyzed the bone marrow of mice with a selective deletion of Arf6 in MKs and platelets (Arf6^fl/fl,Pf4Cre). Arf6^fl/fl,Pf4Cre exhibited an expansion of their hematopoietic stem and progenitor cell compartment as well as a 2-fold increase in MK numbers. In line with dynamin 2-deficient mice, which develop progressive bone marrow fibrosis, Arf6^fl/fl,Pf4Cre mice displayed increased levels of TGFβ1 in their bone marrow fluid, which was accompanied by collagen deposition in the bone marrow. Together with the in vitro data, these findings suggest an important role of Arf6 as a negative regulator of autophagy-induced TGFβ1 secretion. To investigate whether inhibition of RhoA/ROCK1-induced secretory autophagy in MKs on the other hand might ameliorate myelofibrosis pathogenesis, we generated mice with a selective deletion of Rhoa in MKs and utilized the MPL^W515L-driven transplant model to induce disease. Recipient mice transplanted with MPL^W515L-transduced RhoA-deficient cells displayed reduced MK clustering and collagen deposition in the bone marrow. Moreover, treatment of MPL^W515L-transplanted mice with the JAK2 inhibitor ruxolitinib in combination with the ROCK inhibitor Y27632 significantly reduced spleen size and collagen deposition beyond ruxolitinib treatment alone, thus attenuating disease hallmarks. Most importantly, only combination treatment with Y27632 significantly reduced TGFβ1 levels in the bone marrow strongly suggesting secretory autophagy as its main secretion pathway.

In summary, our data suggest that TGFβ1 secretion from MKs is regulated by small GTPases through secretory autophagy, a targetable pathway with therapeutic potential for the treatment of myelofibrosis.

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