Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by the
clonal proliferation of CD34+ progenitor cells. A constitutive activation of the JAK-STAT
pathway, due to an acquired gain of function mutation either in JAK2, MPL or CALR genes,
plays a relevant pathogenetic role. Moreover, a state of chronic inflammation affects the
disease course. Currently, the therapy with the JAK inhibitor Ruxolitinib ameliorates the
symptoms of the disease, with no effects on the pathological cell clone; hematopoietic
progenitor cell (HPC) transplantation is the only curative option. The investigators
previously published that mesenchymal stromal cells (MSCs) from patients with PMF display
functional and genetic abnormalities (low proliferative potential, precocious senescence,
reduced differentiation potential). Regarding Ruxolitinib, direct effects on MSCs have
been described, such as the inhibition of the JAK-STAT signaling, the growth of both
healthy donor (HD)- and PMF-MSCs, and the decrease of fibrosis related gene expression.
Ruxolitinib has also been reported to reduce MSC secretion of inflammatory cytokines
MCP-1 and IL-6, suggesting a role of this drug in modifying the bone marrow (BM)
microenvironment. In PMF, as in other hematological malignancies, MSCs participate in the
communication between microenvironment and CD34+ cells. Recent studies demonstrated in
other pathologies that MSC-derived extracellular vesicles (EVs) and parental MSCs have
similar effects. In fact EVs, containing mRNA, microRNA, lipids, and proteins can be
incorporated into recipient cells and modify their functions. In normal hematopoiesis,
MSC-EV incorporation in CD34+ cells has been shown to induce an activation of the
JAK-STAT pathway through increased levels of phospho-STAT5; in addition, a significant
decrease in apoptosis and a higher colony-forming unit granulocyte/macrophage clonogenic
potential have been reported.
The objectives of this study are:
to isolate MSCs from BM of PMF patients and HDs. MSCs will be incubated with/out
Ruxolitinib. EVs will be obtained following MSC starvation and ultracentrifugations,
and stored at -80°C
to assess EV effects on HD-CD34+ or JAK2V617F+ SET2 cells by evaluation of apoptotic
and activation pathways, ROS production and clonogenic capacity. To this regard,
confirmatory experiments will be performed with JAK2-mutated CD34+ cells from
patients receiving or not Ruxolitinib.
to perform gene expression profile on CD34+ and SET2 cells after incubation with
EVs.
to investigate EV biocargoes both qRT-PCR and proteomics will be performed on
MSC-derived EVs, before and after incubation with Ruxolitinib.
The expected impact will include the identification of modifications in functional
activities of CD34+ or SET2 cells following incubation with PMF-MSC-EVs with respect to
HD-MSC-EVs, reflecting the effect of an altered microenvironment on HPCs. The
investigators also expect the recognition of important targets of severity/progression of
the disease by assessing the gene expression profile in CD34+ or SET2 cells that
incorporate EVs from different sources.
In addition, the identification of the Ruxolitinib effects on PMF BM microenvironment may
be clarified.
This study may allow to act on new targets of disease progression or to support the
future use of HD-EVs as an acellular therapy that favours the survival of healthy CD34+
cells or acts against their clonal counterpart.