Expression of CD47 and Calr in Myeloproliferative Neoplasms and Myelodysplastic Syndrome: Potential New Therapeutical Targets

Myelodysplastic neoplasms (MPN) and myelodysplastic syndrome (MDS) are myeloid malignancies characterized by either overproduction of mature blood cells or hyperplastic bone marrow and peripheral cytopenia, but both with a tendency to evolve into acute myeloid leukaemia. 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.

Aims: Investigate the expression and cellular localisation of CALR and 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: Total CALR and CD47 protein expression increased in MPN samples compared with controls (CALR- 7.9 vs 5.1; CD47- 2.7 vs 2.2 fold, respectively). 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%), suggesting an anti-phagocytic effect being induced by cytotoxic drugs in MF and PV cells. In ET cell membranes however, CD47 expression is reduced after cyto-reductive treatment (22% vs 16.6%), suggesting instead a prophagocytic effect. Similarly, 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 - 16.9%, intermediate 2 - 17%, high - 17.9% Vs control - 29.1%).

Summary/Conclusion: 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. This opposes previous studies in solid tumours, which showed significant increases of both CALR and CD47 as disease progresses, suggesting a role for CD47 as a strong antiphagocytic signal responsible for immune survival in MPN and MDS. All MPN patients showed decreased CALR expression during cyto-reduction therapy, but we observed a significant difference in CD47 expression across different MPN subtypes. In fact, treating MF and PV patients with cyto-reductive agents, increases CD47 expression leading to a stronger anti-phagocytic signal, while in ET we observed a reduction, suggesting a more pro-phagocytic reaction to cytotoxic drugs in ET. A larger cohort of patients is required to confirm our findings and allow a further stratification based of type of cyto-toxic drugs. 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.