Role of Myeloid-derived Suppressor Cells In Primary Myelofibrosis

Primary myelofibrosis (PMF) is a Philadelphia-negative chronic myeloproliferative disorder characterized by: clonal expansion of a malignant hematopoietic stem cell CD34+, bone marrow (BM) fibrosis, splenomegaly, extra-medullary hematopoiesis and an extensive neoangiogenesis in BM and spleen. Ninety-five % of patients have an acquired mutation of JAK2, CALR or MPL genes determining an activation of the JAK-STAT pathway in the myeloid lineage. Inflammation is currently thought to play a relevant role in PMF pathogenesis, as proven by high levels of inflammatory cytokines with prognostic significance and by a state of chronic oxidative stress with elevated reactive oxygen species (ROS).

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that accumulate in patients with malignancies, sepsis or chronic inflammation. In steady state, MDSCs migrate from the BM to different peripheral organs, where they differentiate into macrophages, dendritic cells or granulocytes. Cytokines and chemokines that are produced in tumor microenvironment recruit immature myeloid cells in peripheral blood (PB), spleen, liver and lymphoid organs, prevent their differentiation (through the activation of STAT3 signalling pathway) and induce their activation into MDSCs. This cell population is conventionally divided in two subsets: polymorphonuclear (PMN)- and monocytic (M)-MDSCs. Besides, MDSCs have the strong ability to reduce cytotoxic functions of T/NK cells and the potential to differentiate into endothelial cells and incorporate in tumor endothelium, favoring tumor neoangiogenesis. Finally, Wang et al. found high levels of circulating MDSCs in patients with myeloproliferative neoplasms that were able to inhibit T cell proliferation.

These premises suggest that MDSCs could have a role in the pathogenesis of PMF by inhibiting some functions of immune cells, nurturing chronic inflammation that characterizes the disease and/or boosting the angiogenesis process.

A condition of chronic inflammation is commonly associated with an increase of MDSCs (number and activity) that contributes in maintaining and fueling inflammation through induction of oxidative stress by means of ROS production. In PMF patients, inflammation is thought to play a relevant role in the pathogenesis of the disease, as proven by high levels of inflammatory cytokines with prognostic significance and by a state of chronic oxidative stress. Few years ago, Wang et al. reported in a very limited number of patients with PMF an increase of the frequency of circulating MDSCs; however, they were not able, likely due to the small size of samples, to find any correlation between the number of MDSCs and both the genetic background and the disease phenotype.

The rationale of this project stems from the observations that in PMF all the premises that favor a role for MDSCs in the pathogenesis of the disease exist: a) a status of chronic inflammation, b) a variable degree of dysfunction of the immune system (that, together with activation of the JAK/STAT pathway due to driver mutations, could be responsible of disease progression), and c) extensive neoangiogenic processes.

Based on these considerations, the investigators hypothesize that: 1) an increase in number and/or function of MDSCs is involved in the immunological dysfunction that has been described in PMF patients, 2) MDSCs, thank to their ability to acquire an endothelial phenotype and function, contribute to splenic and marrow neoangiogenesis, 3) MDSCs contribute to the maintenance of inflammation through their capacity to produce ROS and, in turn, oxidative stress at cellular level, 4) JAK-inhibitory therapy (currently the most effective symptomatic drug for reducing splenomegaly and abolish systemic symptoms in PMF) exerts its beneficial effect not only by down-regulating the JAK/STAT pathway in hematopoietic cells of patients, but also by inhibiting ROS production in MDSCs.

The significance of the project resides:

1. in the recognition of a new pathogenetic mechanism that, together and in addition to DNA mutation, contribute to the explanation of phenotypic diversity observed during the clinical course of PMF; ii) in the identification of a new target for personalized treatment of PMF.

The primary objective of this project is to investigate the role of MDSCs in fueling chronic inflammation (through the analysis of cytokines/chemokines together with their receptors), to evaluate their percentage in correlation with clinical/biological parameters, to study modifications after treatment with JAK-inhibitors (JAK-i), to test their participation in the neoangiogenic process.

Primary endpoints:

• to characterize, phenotypically and functionally, MDSC subsets (polymorphonuclear (PMN)- and monocytic (M)-MDSCs) in the PB and BM of PMF patients and healthy subjects. In case of splenectomy, we will evaluate the presence of PMN-MDSCs and M-MDSCs in spleen-derived mononuclear cells freshly obtained from patients (undergoing the procedure for therapeutic reasons) and controls (following splenectomy for traumatic lesions).
• to correlate the data with clinical and biological parameters as well as with the genotype of driver mutations of JAK2, MPL, and CALR genes in PMF patients.
• to assess, in plasma samples from PMF and controls, cytokines involved in the inflammatory process, in the immunosuppressive function, in the angiogenic activity and in the increased production of MDSCs.

Secondary endpoints are:

1. investigate if patient-derived MDSCs, cultured in proangiogenic conditions, are able to differentiate in endothelial cells angiogenic culture conditions (flow cytometry) and/or to modify their gene expression profile (by RealTime -quantitative PCR (RT-qPCR) before-, after- incubation);
2. test ex vivo the hypothesis that MDSCs are involved in the neoangiogenic processes that characterizes PMF, evaluating the presence of endothelial-like MDSCs in PMF tissues, performing immunofluorescence staining in spleen and BM sections and analyzing them by confocal microscopy;
3. verify whether MDSCs are effective targets of JAK-inhibitor drugs, testing in vitro the effects of JAK1/JAK2 inhibitors on MDSC activity and function by flow cytometry, RT-qPCR, ELISA tests.