Elucidating the molecular mechanisms of CALR and CD47 in myelodysplastic syndromes, myeloproliferative neoplasms

Boasman, Kristian (2021) Elucidating the molecular mechanisms of CALR and CD47 in myelodysplastic syndromes, myeloproliferative neoplasms. PhD thesis, University of Lincoln.

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Elucidating the molecular mechanisms of CALR and CD47 in myelodysplastic syndromes, myeloproliferative neoplasms
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Abstract

Myelodysplastic syndromes (MDS) and Myeloproliferative neoplasms (MPN) are myeloid malignancies arising from clonal disorders of haematopoietic stem cells, with the tendency to progress into acute myeloid leukaemia (AML). Whilst diagnostic criteria have been established, treatment options remain relatively limited promoting the search for markers which could be used to help predicting disease progression or be targeted therapeutically to provide new or enhanced treatment options. 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. The main aim of this work is to investigate the expression and cellular localisation of CALR and CD47 in MDS and MPN, determine any changes in CALR /CD47 as disease progresses and establish how cellular location of CALR and CD47 alters in response to treatment in MDS and MPN. Initial investigation of cell line models of MDS and MPN indicate that when treated with disease specific cytotoxic drugs, total expression of CALR and CD47 increased. However, CALR was shown to internalise away from the membrane upon treatment whereas CD47 was externalised, suggesting a different mode of action for CALR and CD47 in MDS/MPN compared to solid tumours. Further work investigating whether these changes in CALR and CD47 expression in patient samples found that CALR and CD47 baseline protein expression was increased in both MDS and MPN patients compared with controls. CD47 showed higher overall protein expression on MDS and MPN cell membranes when compared with CALR. As MDS progressed, CD47 expression is seen to increase with the severity of the disease as classified by IPSS subgroups. Interestingly, when investigating MPN subtypes, significant increases in CD47 cell membrane expression, in particular, after treatment was only seen in the MF and PV subtypes, suggesting an anti-phagocytic effect induced by cytotoxic drugs. In the ET subtype, CD47 expression is reduced after cytoreductive treatment, suggesting
3 instead a prophagocytic effect. To better understand the relationship between CALR and CD47 and support any future work looking to use these in a more therapeutic setting, knockdown experiments were undertaken. Knockdown experiments revealed a possible link between CALR and CD47. In fact, it was observed that a mutual reduction in either CALR or CD47 when one or the other is repressed, suggesting the influence on each other pathways. Reduction in CD47 expression resulted in higher rate of cell death when cell were treated with cytotoxic drugs, however due to the concomitant reduction of CALR the overall therapeutical effect is reduced. Leaving both CALR and CD47 intact on the myeloid cancer cells but interfering with the CD47/SIRPα interaction instead, could actively signal cell death messages in these myeloid neoplasms via monoclonal antibodies, and let the pro-phagocytic message conducted by CALR to prevail. The overexpression of CD47 in MDS, PV and MF could represent the novel target to block via monoclonal anti-CD47 antibody in monotherapy or in combination to enhance treatment efficacy in myeloid cancers.

Keywords:CALR, CD47, Myelodysplastic syndromes, myeloproliferative neoplasms
Subjects:C Biological Sciences > C110 Applied Biology
Divisions:College of Science > School of Life Sciences
ID Code:49499
Deposited On:23 May 2022 10:15

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