Contributions
Abstract: PB1910
Type: Publication Only
Background
Myelodysplastic syndrome (MDS) and Myeloproliferative neoplasms (MPN) are clonal myeloid disorders with the tendency to progress into acute myeloid leukaemia. Previous studies in solid tumours have shown an increase in expression of both pro-phagocytic calreticulin (CALR) and anti-phagocytic CD47, as they act in response to one another, reflecting a possible apoptosis vs survival mechanism in response to chemotherapy.
Aims
The aim of our study is to assess the changes in CALR and CD47 levels during treatment of MDS and MPN with azacytidine (AZA) or ruxolitinib (RUXO), in a series of model cell systems.
Methods
CALR and CD47 gene and protein expression was measured in MDS cell line models (MOLM-13 and SKM-1), MPN cell line models (HEL-92 and GDM-1) and in an intermediate MDS/MPN cell line (K562) before and after treatment with AZA and RUXO. Drug titrations were completed, resulting in dosing regimens of 0.05µM/ml for both AZA and RUXO, with re-drugging occurring at 24 hours. Cells were then harvested, cDNA was synthesized for use in qPCR and protein levels determined by Western blot analysis.
Results
When treated with AZA, MDS cell models showed a 7-10 fold increase in CALR expression and 4-6 fold increase in CD47 expression. In contrast, the MDS/MPN intermediate cell model (K562) showed a 4.5 fold increase in CALR but only a 0.5 fold increase in CD47 expression. In the MPN model HEL-92, a 9 fold CALR increase and 5 fold CD47 increase was seen, whereas in the other MPN model (GDM-1 cells) expression was more evenly matched between CALR and CD47 (5.3 and 4.8 fold increases, respectively). After treatment with RUXO, MPN models showed a 9.5-16 fold increase in CALR expression and a 6-9 fold increase in CD47, which would be expected as RUXO is used to treat MPN in humans. When the MDS/MPN cell model or pure MDS models were treated with RUXO, the ratio of CALR/CD47 decreased substantially (with CALR expression only increased 2.4-3.7 fold compared to CD47 increasing 4.6-6.9 fold) showing resistance to treatment and a significant anti-phagocytic response. Interestingly one of the MDS cell line models (MOLM-13) showed an unexpectedly good response to RUXO therapy with high CALR/CD47 ratio (8 fold vs. 4.8 fold, respectively).
Conclusion
In line with results in solid tumours, we have shown that treatment for MDS and MPN leads to an up-regulation of CALR and, to a lesser extent, CD47 in cell lines models. The ratio of CALR/CD47 seems to correlate with specific treatment response, significantly increasing when given diseases models are treated with the appropriate drug. We postulate a role of CALR expression in leukaemia cell phagocytosis, with CD47 co-expression in synergy as a protective instinct within the cell to try and prevent apoptosis. Some models showed an excessive rise in CD47 expression and low expression of CALR. This indicates that the CD47 mediated anti-phagocytosis takes control and supresses the CALR expression, leading to cancer cell survival and ineffectiveness of treatment. Those results need to be validated in human samples at different stages of disease to allow a better understanding of treatment response and/or resistance to chemotherapy within these diseases.
Session topic: 9. Myelodysplastic syndromes - Biology
Keyword(s): Myeloproliferative disorder
Abstract: PB1910
Type: Publication Only
Background
Myelodysplastic syndrome (MDS) and Myeloproliferative neoplasms (MPN) are clonal myeloid disorders with the tendency to progress into acute myeloid leukaemia. Previous studies in solid tumours have shown an increase in expression of both pro-phagocytic calreticulin (CALR) and anti-phagocytic CD47, as they act in response to one another, reflecting a possible apoptosis vs survival mechanism in response to chemotherapy.
Aims
The aim of our study is to assess the changes in CALR and CD47 levels during treatment of MDS and MPN with azacytidine (AZA) or ruxolitinib (RUXO), in a series of model cell systems.
Methods
CALR and CD47 gene and protein expression was measured in MDS cell line models (MOLM-13 and SKM-1), MPN cell line models (HEL-92 and GDM-1) and in an intermediate MDS/MPN cell line (K562) before and after treatment with AZA and RUXO. Drug titrations were completed, resulting in dosing regimens of 0.05µM/ml for both AZA and RUXO, with re-drugging occurring at 24 hours. Cells were then harvested, cDNA was synthesized for use in qPCR and protein levels determined by Western blot analysis.
Results
When treated with AZA, MDS cell models showed a 7-10 fold increase in CALR expression and 4-6 fold increase in CD47 expression. In contrast, the MDS/MPN intermediate cell model (K562) showed a 4.5 fold increase in CALR but only a 0.5 fold increase in CD47 expression. In the MPN model HEL-92, a 9 fold CALR increase and 5 fold CD47 increase was seen, whereas in the other MPN model (GDM-1 cells) expression was more evenly matched between CALR and CD47 (5.3 and 4.8 fold increases, respectively). After treatment with RUXO, MPN models showed a 9.5-16 fold increase in CALR expression and a 6-9 fold increase in CD47, which would be expected as RUXO is used to treat MPN in humans. When the MDS/MPN cell model or pure MDS models were treated with RUXO, the ratio of CALR/CD47 decreased substantially (with CALR expression only increased 2.4-3.7 fold compared to CD47 increasing 4.6-6.9 fold) showing resistance to treatment and a significant anti-phagocytic response. Interestingly one of the MDS cell line models (MOLM-13) showed an unexpectedly good response to RUXO therapy with high CALR/CD47 ratio (8 fold vs. 4.8 fold, respectively).
Conclusion
In line with results in solid tumours, we have shown that treatment for MDS and MPN leads to an up-regulation of CALR and, to a lesser extent, CD47 in cell lines models. The ratio of CALR/CD47 seems to correlate with specific treatment response, significantly increasing when given diseases models are treated with the appropriate drug. We postulate a role of CALR expression in leukaemia cell phagocytosis, with CD47 co-expression in synergy as a protective instinct within the cell to try and prevent apoptosis. Some models showed an excessive rise in CD47 expression and low expression of CALR. This indicates that the CD47 mediated anti-phagocytosis takes control and supresses the CALR expression, leading to cancer cell survival and ineffectiveness of treatment. Those results need to be validated in human samples at different stages of disease to allow a better understanding of treatment response and/or resistance to chemotherapy within these diseases.
Session topic: 9. Myelodysplastic syndromes - Biology
Keyword(s): Myeloproliferative disorder