Expression of CD47 and CALR in myeloproliferative neoplasms and myelodysplastic syndrome: Potential new therapeutical targets.

Abstract

7557 Background: Myelodysplastic neoplasms (MPN) and myelodysplastic syndrome (MDS) are myeloid malignancies tendency to evolve into acute myeloid leukaemia. We investigate the expression and cellular localisation of pro-phagocytic CALR and anti-phagocytic CD47 in untreated and treated patients with essential thrombocythemia (ET), polycythemia vera (PV) myelofibrosis (MF), and in MDS patients in comparison with healthy controls. Methods: Mononuclear cells were collected by Ficoll separation, from peripheral blood of 27 MPN (8 PV, 16 ET, 3 MF); 14 MPN patients received cyto-reductive therapies (Hydroxyurea, Anagrelide or Ruxolitinib); 10 MDS patients and 4 controls. Cells were fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus. Proteins were extracted using TRIzol, with CALR and CD47 protein expression analysed by western blotting. Results: CD47 showed higher expression of its overall protein on MPN cell membranes when compared with CALR (22% vs 13.9%). We observed a significant reduction of CALR expression in all MPN subtypes when patients were treated with cyto-reductive agents (ET- untreated 43.3% vs treated 2%, PV- 3.6% vs 2.2%, ET- 21% vs 11%). Interestingly we have observed a significant increase in CD47 cell membrane expression after treatment in MF and PV (CD47 in MF- untreated 11.8% vs treated 34.3%, PV-11.4% vs 35.9%). In MDS cells CD47 is overexpressed compared with controls (CD47- 11.31 vs 2.2 fold, respectively) and it mainly located to the membrane. Interestingly the degree of CD47 expression correlated to patients IPSS-R, increasing from low risk to high risk (low – 15.7%, intermediate 1 – 41.3%, intermediate 2 – 53.9% and high – 67.6%). CALR expression is also reduced in MDS cells comparing with controls when split by IPSS-R risk score (low – 9%, intermediate 1 – 11.9%, intermediate 2 – 17%, high – 17.9% Vs control - 29.1%). Conclusions: CD47, but not CALR, is overexpressed on the membrane of patients with MPN and MDS. In MDS, we observed a progressive increase in CD47 expression as the MDS evolve in accordance to the IPSS-R risk score. In MPN patients we observed a significant difference in CD47 expression across different MPN subtypes. The use of anti-CD47 antibodies could represent a new strategy to enhance the pro-phagocytic signal via increasing the CALR expression, and in combination with standard cyto-reduction therapy, might represent a new therapeutical strategy in both MPN and MDS.