JAK2V617F Mutated Endothelium Has a Stem Cell Potential in Primary Myelofibrosis: a New Disease Ontogenesis for Chronic Myeloproliferative Disorders

This project stems from our previous original observations that endothelial cells lining the vessel wall of splenic venules and capillaries of PMF patients harbour the JAK2V617F mutation (Piaggio G, Rosti V Blood 2009 and Rosti V Blood 2013) that is found in the hematopoietic cells of patients with myelofibrosis. Subsequently, this finding was further confirmed by other groups (Teofili L Blood 2011 and Farina M Cells 2021), although a definitive agreement on the existence of mutated endothelium in PMF has not been reached and the role of mutated endothelial cells in PMF pathogenesis continues to be questioned.

These observations support the hypothesis that endothelial cells and hematopoietic cells may derive from a common progenitor cell, the hemangioblast, in which the driver mutation(s) arise, thus resulting in mutated endothelial and hematopoietic cells. Whereas in mice evidences that hemangioblast exists have been documented (Fadlullah M Blood 2022), in human a definitive demonstration is still lacking. The demonstration that sorted mature endothelial cells share genetic and functional features with hematopoietic cells and that they retain the capacity to differentiate in culture into hematopoietic progenitor cells will clarify the role and significance of circulating mutated mature endothelial cells in patients with primary myelofibrosis. The significance of the project resides in its novel interpretation of the pathogenesis of PMF and, more in general, of chronic myeloproliferative diseases. The cell of origin of the disease is not anymore identified in a hematopoietic stem/progenitor cell, which acquire a mutation during the adult life and, by its clonal proliferation, results in the development of the disease. In this project the cell of origin of PMF is identified in a cell with hemogenic endothelium potential, that acquires the driver mutation during either fetal or adult life. This cell would give rise to mutated hematopoietic and endothelial cells and, in turn, by its clonal proliferation driven by acquired mutation(s) or an altered microenvironment, to the onset of the disease.

Specific Aim 1 Separation and isolation of mature endothelial cells and hematopoietic progenitor cells from peripheral blood of patients with PMF and normal subjects.

Due to the scant data available in the literature on the frequency of the events that will be investigated in this project, it isnot possible to define a sample size of patients to be included in the study. The Center for the Study of Myelofibrosis at Policlinico San Matteo Foundation assures the accrual of a large number of patients and, in turn, it guarantees that all patients undergoing new diagnostic procedures will be asked to participate to the study. All the procedures will be performed after signing a written informed consent approved by the local IRB of Policlinico San Matteo, Pavia. Peripheral blood samples (20-40 ml) will be obtained from a minimum of 20 patients with a diagnosis of primary myelofibrosis (WHO 2016) and positivity for the JAK2V617F mutation, since this is the most frequently found mutation among the driver mutations in patients. Patients with a previous history of deep vein thrombosis will be included in the study. An equal number of healthy subjects will be used as controls. Hematopoietic progenitor cells (HPCs) and mature endothelial cells will be isolated by sterile cell sorting. Twenty-40 milliliters of PB from patients with PMF or healthy controls will be collected in EDTA tubes; mononuclear cells (MNCs) will be separated by ficoll Hypaque. To allow the sorting of HPCs, half of the MNCs will be stained with the anti-CD45, which will discriminate between the hematopoietic and non hematopoietic cells, and the anti-CD34 monoclonal antibodies. The sorting of circulating mature endothelial cells will be performed by staining the remaining MNCs with endothelial lineage specific markers such as the anti-CD146, also known as the melanoma cell adhesion molecule, and anti-CD105, an endoglin protein expressed by activate endothelial cells, monocytes stromal cells and pre-B cells. The nuclear stain DAPI will be added to all the antibody combination. We expect a purity degree for the sorted cell populations greater than 95%. The sorted HPC and mature circulating endothelial cell populations will be further characterized by molecular studies that will compare by Next Generaation Sequencing (NGS) a panel of myeloidassociated mutations (see below).

Specific Aim 2 Morphological, molecular, and functional characterization of mature circulating endothelial cells in patients with PMF and normal subjects.