Contributions
Abstract: PB2261
Type: Publication Only
Background
Myelodysplastic syndromes (MDS) and Myeloproliferative neoplasms (MPN) are a group of bone marrow neoplasms arising from clonal disorders of haematopoietic stem cells, with the tendency to progress into acute 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 in response to chemotherapy. To investigate the role of CALR and CD47 expression during treatment for MDS and MPN we used the intermediate MDS/MPN cell line K562.
Aims
To investigate CALR and CD47 protein expression and location after treatment. Protein expression was analysed within an intermediate MDS/MPN cell line model (K562)
Methods
CALR and CD47 gene expression was measured by Real Time PCR and protein expression by western blotting, before and after treatment with azacytidine (AZA) and ruxolitinib (RUXO). Cells were incubated with 0.05µM/ml drug, re-dosed at 24 hours and harvested at 48 hours. Cell were also fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus and CALR and CD47 expression measured in those compartments before and after treatment.
Results
We demonstrated upregulation of both genes, CALR and CD47 and the concomitant overexpression of both CALR and CD47 protein, after incubation with AZA or RUXO. When we analysed the cell fractions, we observed in untreated cells, the highest CALR protein expression is in the cytosol and nucleus (48.4% and 25.4%, respectively) comparing with the membrane and cytoplasm (13.3% and 12.9% respectively). CD47 expression in untreated cells is higher in the membrane and cytoplasm (43.1% and 33.4% respectively) and lower in the nucleus and cytosol (21.3% and 2.2% respectively). Possibly during fractionation CD47 protein residing on the nuclear envelope have been included not representing a common CD47 location. After incubation, cell survival rates were 81.6% in AZA and 89% in RUXO treated cells respectively, comparing with untreated cells, suggesting a slight more cytotoxic effect of AZA. Both drugs reduced CALR expression in the membrane (AZA 8.1%; RUXO 8.2% vs untreated 13.3%), but increased its expression in cytosol (AZA 54%; RUXO 51% vs untreated 48.4%) and in the cytoplasm (AZA 15.3%; RUXO 13.5% vs untreated 12.9%), indicating that pro-phagocytic CALR moves away from the membrane back into the cell. In contrast, CD47 expression significantly increased on the membrane (64.4% vs 43.1%) and decrease in the cytoplasm (0.9% vs 33.4%) when treated with AZA, while the expression remains virtually unmodified on the membrane (40.4% vs 43.1%) and in the cytoplasm (34.5% vs 32.5%) after treatment with RUXO.
Conclusion
In this study, we observed changes in CALR and CD47 localization after exposure to drugs. We identified a very similar pattern of CALR localization which internalised away from the membrane. This opposes previous studies in solid tumours, which show an increase of both CALR and CD47 on the cell membrane in response to chemotherapy. Interestingly the CD47 expression on the cell surface seems to follow a more drug specific trait, increasing significantly when exposed to AZA but decreasing when exposed to RUXO suggesting different ways of priming the immune-response. Further work is now required in MDS and MPN patient’s cells to confirm our in vitro findings and to evaluate whether the different patterns of CALR and CD47 expression result in different treatment outcomes.
Session topic: 15. Myeloproliferative neoplasms – Biology & Translational Research
Keyword(s): Myeloproliferative disorder
Abstract: PB2261
Type: Publication Only
Background
Myelodysplastic syndromes (MDS) and Myeloproliferative neoplasms (MPN) are a group of bone marrow neoplasms arising from clonal disorders of haematopoietic stem cells, with the tendency to progress into acute 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 in response to chemotherapy. To investigate the role of CALR and CD47 expression during treatment for MDS and MPN we used the intermediate MDS/MPN cell line K562.
Aims
To investigate CALR and CD47 protein expression and location after treatment. Protein expression was analysed within an intermediate MDS/MPN cell line model (K562)
Methods
CALR and CD47 gene expression was measured by Real Time PCR and protein expression by western blotting, before and after treatment with azacytidine (AZA) and ruxolitinib (RUXO). Cells were incubated with 0.05µM/ml drug, re-dosed at 24 hours and harvested at 48 hours. Cell were also fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus and CALR and CD47 expression measured in those compartments before and after treatment.
Results
We demonstrated upregulation of both genes, CALR and CD47 and the concomitant overexpression of both CALR and CD47 protein, after incubation with AZA or RUXO. When we analysed the cell fractions, we observed in untreated cells, the highest CALR protein expression is in the cytosol and nucleus (48.4% and 25.4%, respectively) comparing with the membrane and cytoplasm (13.3% and 12.9% respectively). CD47 expression in untreated cells is higher in the membrane and cytoplasm (43.1% and 33.4% respectively) and lower in the nucleus and cytosol (21.3% and 2.2% respectively). Possibly during fractionation CD47 protein residing on the nuclear envelope have been included not representing a common CD47 location. After incubation, cell survival rates were 81.6% in AZA and 89% in RUXO treated cells respectively, comparing with untreated cells, suggesting a slight more cytotoxic effect of AZA. Both drugs reduced CALR expression in the membrane (AZA 8.1%; RUXO 8.2% vs untreated 13.3%), but increased its expression in cytosol (AZA 54%; RUXO 51% vs untreated 48.4%) and in the cytoplasm (AZA 15.3%; RUXO 13.5% vs untreated 12.9%), indicating that pro-phagocytic CALR moves away from the membrane back into the cell. In contrast, CD47 expression significantly increased on the membrane (64.4% vs 43.1%) and decrease in the cytoplasm (0.9% vs 33.4%) when treated with AZA, while the expression remains virtually unmodified on the membrane (40.4% vs 43.1%) and in the cytoplasm (34.5% vs 32.5%) after treatment with RUXO.
Conclusion
In this study, we observed changes in CALR and CD47 localization after exposure to drugs. We identified a very similar pattern of CALR localization which internalised away from the membrane. This opposes previous studies in solid tumours, which show an increase of both CALR and CD47 on the cell membrane in response to chemotherapy. Interestingly the CD47 expression on the cell surface seems to follow a more drug specific trait, increasing significantly when exposed to AZA but decreasing when exposed to RUXO suggesting different ways of priming the immune-response. Further work is now required in MDS and MPN patient’s cells to confirm our in vitro findings and to evaluate whether the different patterns of CALR and CD47 expression result in different treatment outcomes.
Session topic: 15. Myeloproliferative neoplasms – Biology & Translational Research
Keyword(s): Myeloproliferative disorder