Expression of CD47 and CALR in myeloproliferative neoplasms: Potential new therapeutic targets.

Abstract

48 Background: In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism. Increases of both CALR and CD47 on the cell membrane have been observed in response to chemotherapy, however their role in myeloid malignancies is poorly understood. We investigated the expression of CALR and CD47 in patients with essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF). Methods: Mononuclear cells were collected from peripheral blood of 27 MPN patients (8 PV, 16 ET, 3 MF), and from 4 controls. In total 14 patients received cyto-reductive therapies (Hydroxyurea, Anagrelide and Ruxolitinib). 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: Total CALR and CD47 protein expression increased in MPN samples comparing with controls (CALR- 7.9 vs 5.1; CD47- 2.7 vs 2.2 fold). CD47 showed higher expression on MPN cells membranes when compared with CALR (22% vs 13.9%). We observed a significant reduction of CALR when patients are 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 after treatment in MF and PV (CD47 in MF- untreated 11.8% vs treated 34.3%, PV-11.4% vs 35.9%), suggesting an anti-phagocytic effect induced by cytotoxic drugs. In ET however, CD47 expression is reduced after treatment (22% vs 16.6%), suggesting instead a pro-phagocytic effect. Conclusions: CD47, is overexpressed on the membrane of patients with MPN suggesting a role for CD47 as a strong anti-phagocytic signal responsible for immune survival in MPN. MPN patients decreased CALR expression during therapy, but we observed a significant difference in CD47 expression in different MPN types. 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 MPN.