CD47 but Not Calreticulin Is over Expressed in Patients with Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are chronic myeloid cancers characterized by the overproduction of mature blood cells, and the tendency to evolve into acute leukaemia. In solid tumours, calreticulin (CALR) overexpression on the cell surface, produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism. In this study, we investigated the expression of CALR and CD47 in patients with MPN and potential changes induced by treatment.

CALR and CD47 gene and protein expression were measured by Real Time PCR and western blotting, in K562 and in mononuclear cells obtained by FICOLL separation, from peripheral blood of 13 MPN patients [4 Polycythaemia Vera, 8 Essential Thrombocythemia, and 1 myelofibrosis; 7 treated (IFN or Hydroxyurea) and 6 untreated] and compared with 4 healthy controls. Cells were also fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus and protein fractions analysed.

We found a significant increase in CD47 protein expression into the membrane of MPN patients comparing with controls (91.9 % vs 19.7%). Interestingly in treated MPN the CD47 expression increases even further in comparison with untreated (92.6% vs 91.9%). No significant differences were found in total CALR expression comparing with controls (3.23, vs 2.95-fold), however in treated patients we observed a reduction in the expression in the membrane comparing with untreated (26.4 % vs 39.5 %) and an increase into cytoplasm (69.4 % vs 54.6 %). These findings suggest CD47 exposure as a mechanism of defence in MPN cells during treatment.

To better understand the potential effects of therapy on CALR and CD47 expression, we incubated K562 with 0.05μM/ml of Ruxolitinib (RUXO), re-dosed at 24 hours and harvested at 48 hours. RUXO induced CALR internalization, reducing its expression into the membrane (RUXO 8.2% vs Untreated: 13.3%), but increasing its expression in cytosol (RUXO 51% vs Untreated 48.4%) and in the cytoplasm (RUXO 13.5% vs Untreated 2.9%). In contrast, CD47 expression remains relatively unmodified, with only slight increases on the membrane (RUXO 43.1% vs Untreated 40.4%) and in the cytoplasm (RUXO 34.5% vs UT 32.5%).

In this study we demonstrated that CD47 but not CALR, is overexpressed on the membrane of patients with MPN. This opposes previous studies in solid tumours, which show significant increases of both CALR and CD47. This suggests a role for CD47 as a strong anti-phagocytic signal responsible for immune survival in MPN. We have also shown a potential mechanism adopted by MPN cells in response to therapy, with the internalization of CALR and the enhanced membrane expression of CD47 which remains strongly expressed on cell surface. The addition of Anti-CD47 compounds in combination with conventional therapies might represent a future therapeutically strategy to counteract MPN cells immune-escape mechanism.