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Title: Cytokine receptor-like factor 3 (CRLF3) : a novel regulator of platelet biogenesis and potential drug target for thrombocythaemia
Author: Bennett, Cavan
ISNI:       0000 0004 7230 8389
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Thrombocythaemia is defined as a circulating platelet count above 450x10$^9$/L in humans. The major cause of thrombocythaemia is reactive $(secondary)$ thrombocythaemia which occurs secondary to many conditions such as infection, cancer and inflammation. However, acquired clonal mutations in mainly Janus Kinas 2 $(JAK2)$, CALR and MPL cause essential thrombocythaemia $(ET)$. ET is a rare disease that leads to an increased risk of cardiovascular thrombotic events. Current treatment of ET uses combination of low dose aspirin to decrease platelet function and cytoreductive agents to decrease thrombopoiesis. The most commonly used cytoreductive agents are hydroxyurea, anagrelide and interferon-$alpha$ and all have unwanted side effects. Cytokine receptor-like factor 3 $(CRLF3)$ is a 2.4kb gene that is ubiquitously expressed throughout the haematopoietic system. Very little is known about the function of CRLF3, with only one peer reviewed journal article in the literature which shows that CRLF3 may negatively regulate the cell cycle at the G0/G1 phase. However, nothing is known about the role of CRLF3 in platelet biology. Using a Crlf3 knockout mouse $(Crlf3-/-)$ developed by the Wellcome Trust Sanger Institute we show CRLF3’s role in platelet biogenesis and how it could be used as a novel therapeutic target to treat ET. Crlf3-/- mice have an isolated and sustained 25-40$\%$ decrease in platelet count compared to wildtype $(WT)$ controls. Platelet function is unaffected as demonstrated in a range in a range of in vitro assays. The thrombocytopenia is a consequence of abnormalities in hematopoietic cells, as shown by bone marrow transplantations. Megakaryopoiesis is upregulated in Crlf3-/- mice and proplatelet morphology is unaffected, suggesting the thrombocytopenia is due to increased platelet clearance. Indeed, splenectomised Crlf3-/- mice show normalised platelet counts within 7 days, showing rapid splenic removal of platelets is responsible for the thrombocytopenia. Abnormal large platelet structures that resemble proplatelets shafts $(preplatelets)$ are abnormally present in the circulation of elderly Crlf3/- mice. Immunohistochemistry showed increased and aberrant tubulin expression in Crlf3-/- platelets compared to WT controls, especially in the preplatelet forms. Cold induced depolymerisation of microtubules was decreased in Crlf3-/- platelets, suggestive of increased tubulin stability, however, the ratio of detyrosinated to tyrosinated tubulin was not altered. We then crossbred Crlf3-/- mice with JAK2 V617F ET mice, to determine the effect of Crlf3 ablation of thrombocythaemia. Crossbred mice showed restoration of platelet counts to WT values without grossly affecting platelet function or other blood lineages, providing the rational for CRLF3 as a novel therapeutic target for treatment of ET. Finally, we aimed to resolve the crustal structure of CRLF3 and discover its interactome. To this end, we were able to resolve the crystal structure of a C-terminal portion of the full length protein containing the predicted fibronectin type III domain. To shed light on the interactome of CRLF3, endogenous CRLF3 was tagged with a tandem affinity purification $(TAP)$ tag using CRISPR/Cas9 technology in induced pluripotent stem cells $(iPSCs)$. We have been able to produce megakaryocytes from these TAP-tagged iPSCs by forward programming. However, as yet we have not been able to generate enough MKs to have adequate material to perform immunoprecipitation assays. Therefore, the interactome of CRLF3 in MKs remains unknown. In conclusion, we identified a mechanism by which Crlf3 controls platelet biogenesis. Slowed maturation of Crlf3-/- preplatelets in the peripheral circulation potentially due to increased structural stability leads to rapid removal of these immature forms by the spleen and therefore a decrease in platelet count. The isolated effect on platelet numbers and normalisation of platelet count in ET mice deficient of Crlf3 provides the rational for further study on CRLF3 drug targeting as a novel therapeutic strategy for ET.
Supervisor: Ghevaert, Cedric Sponsor: British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: platelets ; preplatelets ; thrombocytopenia ; thrombocythaemia ; essential thrombocythaemia ; megakaryocytes ; proplatelets ; mouse models ; platelet biology