{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/385567"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/385567","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Mechanisms driving the expansion and progression of splicing factor-mutant clonal haematopoiesis","abstract":"Clonal haematopoiesis (CH) describes the expansion of haematopoietic stem cells (HSCs) and their progeny due to the acquisition of somatic mutations. CH can be detected almost ubiquitously with advancing age and is associated with an increased risk of incident haematological malignancy and several non-haematological conditions. Mutations in splicing factor genes (namely SF3B1, SRSF2, and U2AF1) are frequent drivers of CH, particularly in the elderly, and confer a high risk of progression to haematological malignancy. Despite over a decade of research into CH, we still lack an understanding of the mechanisms through which splicing factor mutations drive CH and how cells harbouring these mutations can be targeted therapeutically to prevent or treat haematological malignancies. Here, we set out to investigate the mechanism(s) underpinning the clonal expansion of splicing factor-mutant cells and to identify vulnerabilities of these cells that could be targeted therapeutically, with a particular focus on SF3B1, the most frequently mutated splicing factor in CH. First, we introduced the CH-associated SF3B1-K700E mutation into haematopoietic cell lines and showed that they recapitulated the characteristic mis-splicing events seen in SF3B1-mutant myelodysplastic syndromes (MDS). We then performed high-throughput drug screening to identify therapeutic vulnerabilities of SF3B1-mutant cells. Alongside this, we developed a pipeline for the collection of bone marrow aspirate samples from individuals with SF3B1-mutant blood disorders, and reprogrammed cells from a subset of those patients to human induced pluripotent stem cells (hIPSCs). Splicing factor mutations lead to the widespread mis-splicing of a large number of transcripts, although the functional consequences of many of these aberrant splicing events remains unclear. To investigate this, we performed parallel RNA-sequencing and proteomics on SF3B1-K700E cells, characterising the relationship between splicing alterations, RNA expression, and protein levels. We found that the majority of splicing alterations in SF3B1-mutant cells have minimal effects on RNA/protein expression and observed no change in the global relationship between these two modalities. We also used this data to identify novel transcripts specific to SF3B1-mutant cells and showed that these can be detected at the protein level. This approach could be used to identify neoantigens specific to SF3B1-mutant cells that could be targeted through immunotherapy. Next, we evaluated the differential expression of proteins in SF3B1-mutant cells. This revealed a striking dysregulation of metabolic proteins which, alongside the previously described mitochondrial defects in SF3B1-mutant cells, demanded further investigation. We identified splicing alterations leading to the downregulation of several key proteins within the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) in SF3B1-mutant cells. This was associated with decreased aerobic metabolism and the accumulation of TCA cycle intermediates such as 2-oxoglutarate and the oncometabolite 2-hydroxyglutarate (2HG), which has previously been linked to leukaemogenesis following acquisition of IDH1/2 mutations. We also observed an enhanced sensitivity to glycolysis inhibition in SF3B1-mutant cells, representing a potential vulnerability of these cells that could be exploited therapeutically. Finally, we investigated the unique association between splicing factor-mutant CH and age. Splicing factor mutations are rarely detected before the 6th decade of life but rise dramatically in prevalence beyond this. However, the mechanism underlying this is unclear. Recent work in our group has indicated that telomere length may play a role, demonstrating that individuals with inherited polymorphisms associated with short telomeres possess an increased risk of developing splicing factor-mutant CH. In this work, we adapted methods to measure telomere length by qPCR and flow cytometry and applied these tools to samples from individuals with splicing factor-mutant blood disorders. This revealed that splicing factor mutations appear to promote telomere maintenance, allowing cells to avoid replicative senescence and undergo clonal expansion in old age. Collectively, the work in this thesis sheds light on the forces acting on HSCs in both normal and malignant haematopoiesis and may reveal new ways to prevent, delay, or treat haematological malignancies harbouring splicing factor mutations.","abstract_html":"Clonal haematopoiesis (CH) describes the expansion of haematopoietic stem cells (HSCs) and their progeny due to the acquisition of somatic mutations. CH can be detected almost ubiquitously with advancing age and is associated with an increased risk of incident haematological malignancy and several non-haematological conditions. Mutations in splicing factor genes (namely SF3B1, SRSF2, and U2AF1) are frequent drivers of CH, particularly in the elderly, and confer a high risk of progression to haematological malignancy. Despite over a decade of research into CH, we still lack an understanding of the mechanisms through which splicing factor mutations drive CH and how cells harbouring these mutations can be targeted therapeutically to prevent or treat haematological malignancies. Here, we set out to investigate the mechanism(s) underpinning the clonal expansion of splicing factor-mutant cells and to identify vulnerabilities of these cells that could be targeted therapeutically, with a particular focus on SF3B1, the most frequently mutated splicing factor in CH. First, we introduced the CH-associated SF3B1-K700E mutation into haematopoietic cell lines and showed that they recapitulated the characteristic mis-splicing events seen in SF3B1-mutant myelodysplastic syndromes (MDS). We then performed high-throughput drug screening to identify therapeutic vulnerabilities of SF3B1-mutant cells. Alongside this, we developed a pipeline for the collection of bone marrow aspirate samples from individuals with SF3B1-mutant blood disorders, and reprogrammed cells from a subset of those patients to human induced pluripotent stem cells (hIPSCs). Splicing factor mutations lead to the widespread mis-splicing of a large number of transcripts, although the functional consequences of many of these aberrant splicing events remains unclear. To investigate this, we performed parallel RNA-sequencing and proteomics on SF3B1-K700E cells, characterising the relationship between splicing alterations, RNA expression, and protein levels. We found that the majority of splicing alterations in SF3B1-mutant cells have minimal effects on RNA/protein expression and observed no change in the global relationship between these two modalities. We also used this data to identify novel transcripts specific to SF3B1-mutant cells and showed that these can be detected at the protein level. This approach could be used to identify neoantigens specific to SF3B1-mutant cells that could be targeted through immunotherapy. Next, we evaluated the differential expression of proteins in SF3B1-mutant cells. This revealed a striking dysregulation of metabolic proteins which, alongside the previously described mitochondrial defects in SF3B1-mutant cells, demanded further investigation. We identified splicing alterations leading to the downregulation of several key proteins within the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) in SF3B1-mutant cells. This was associated with decreased aerobic metabolism and the accumulation of TCA cycle intermediates such as 2-oxoglutarate and the oncometabolite 2-hydroxyglutarate (2HG), which has previously been linked to leukaemogenesis following acquisition of IDH1/2 mutations. We also observed an enhanced sensitivity to glycolysis inhibition in SF3B1-mutant cells, representing a potential vulnerability of these cells that could be exploited therapeutically. Finally, we investigated the unique association between splicing factor-mutant CH and age. Splicing factor mutations are rarely detected before the 6th decade of life but rise dramatically in prevalence beyond this. However, the mechanism underlying this is unclear. Recent work in our group has indicated that telomere length may play a role, demonstrating that individuals with inherited polymorphisms associated with short telomeres possess an increased risk of developing splicing factor-mutant CH. In this work, we adapted methods to measure telomere length by qPCR and flow cytometry and applied these tools to samples from individuals with splicing factor-mutant blood disorders. This revealed that splicing factor mutations appear to promote telomere maintenance, allowing cells to avoid replicative senescence and undergo clonal expansion in old age. Collectively, the work in this thesis sheds light on the forces acting on HSCs in both normal and malignant haematopoiesis and may reveal new ways to prevent, delay, or treat haematological malignancies harbouring splicing factor mutations.","abstract_has_math":false,"creators":["McLoughlin, Matthew"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Vassiliou, George"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-10-19","date_published":"2024-10-19","updated_at":"2026-07-22T22:24:21Z","subjects":["Haematopoietic Stem Cells","Cancer","Clonal Haematopoiesis","SF3B1","Myelodysplastic Syndromes"],"languages":[],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/30b51f9f-e870-42c1-b6fd-5bbeaf6189b1/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.119130","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Vassiliou, George"]},{"key":"dc:creator","label":"Author","values":["McLoughlin, Matthew"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-10-19"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/385567"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Haematopoietic Stem Cells","Cancer","Clonal Haematopoiesis","SF3B1","Myelodysplastic Syndromes"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/30b51f9f-e870-42c1-b6fd-5bbeaf6189b1/download","http://purl.org/NET/rdflicense/allrightsreserved"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.119130"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/6883914b-8bb2-4e45-b5c1-0b0ac35c80b4/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Clonal haematopoiesis (CH) describes the expansion of haematopoietic stem cells (HSCs) and their progeny due to the acquisition of somatic mutations. 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First, we introduced the CH-associated SF3B1-K700E mutation into haematopoietic cell lines and showed that they recapitulated the characteristic mis-splicing events seen in SF3B1-mutant myelodysplastic syndromes (MDS). We then performed high-throughput drug screening to identify therapeutic vulnerabilities of SF3B1-mutant cells. Alongside this, we developed a pipeline for the collection of bone marrow aspirate samples from individuals with SF3B1-mutant blood disorders, and reprogrammed cells from a subset of those patients to human induced pluripotent stem cells (hIPSCs). Splicing factor mutations lead to the widespread mis-splicing of a large number of transcripts, although the functional consequences of many of these aberrant splicing events remains unclear. To investigate this, we performed parallel RNA-sequencing and proteomics on SF3B1-K700E cells, characterising the relationship between splicing alterations, RNA expression, and protein levels. We found that the majority of splicing alterations in SF3B1-mutant cells have minimal effects on RNA/protein expression and observed no change in the global relationship between these two modalities. We also used this data to identify novel transcripts specific to SF3B1-mutant cells and showed that these can be detected at the protein level. This approach could be used to identify neoantigens specific to SF3B1-mutant cells that could be targeted through immunotherapy. Next, we evaluated the differential expression of proteins in SF3B1-mutant cells. This revealed a striking dysregulation of metabolic proteins which, alongside the previously described mitochondrial defects in SF3B1-mutant cells, demanded further investigation. We identified splicing alterations leading to the downregulation of several key proteins within the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) in SF3B1-mutant cells. This was associated with decreased aerobic metabolism and the accumulation of TCA cycle intermediates such as 2-oxoglutarate and the oncometabolite 2-hydroxyglutarate (2HG), which has previously been linked to leukaemogenesis following acquisition of IDH1/2 mutations. We also observed an enhanced sensitivity to glycolysis inhibition in SF3B1-mutant cells, representing a potential vulnerability of these cells that could be exploited therapeutically. Finally, we investigated the unique association between splicing factor-mutant CH and age. Splicing factor mutations are rarely detected before the 6th decade of life but rise dramatically in prevalence beyond this. However, the mechanism underlying this is unclear. Recent work in our group has indicated that telomere length may play a role, demonstrating that individuals with inherited polymorphisms associated with short telomeres possess an increased risk of developing splicing factor-mutant CH. In this work, we adapted methods to measure telomere length by qPCR and flow cytometry and applied these tools to samples from individuals with splicing factor-mutant blood disorders. This revealed that splicing factor mutations appear to promote telomere maintenance, allowing cells to avoid replicative senescence and undergo clonal expansion in old age. Collectively, the work in this thesis sheds light on the forces acting on HSCs in both normal and malignant haematopoiesis and may reveal new ways to prevent, delay, or treat haematological malignancies harbouring splicing factor mutations."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["92c6489b8cf8d54e6ccf4de1e0f4ef4f","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["Mechanisms driving the expansion and progression of splicing factor-mutant clonal haematopoiesis"]}]}],"canonical_facts":{"dc:contributor.advisor":["Vassiliou, George"],"dc:creator":["McLoughlin, Matthew"],"dc:date.issued":["2024-10-19"],"dc:description.abstract":["Clonal haematopoiesis (CH) describes the expansion of haematopoietic stem cells (HSCs) and their progeny due to the acquisition of somatic mutations. 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First, we introduced the CH-associated SF3B1-K700E mutation into haematopoietic cell lines and showed that they recapitulated the characteristic mis-splicing events seen in SF3B1-mutant myelodysplastic syndromes (MDS). We then performed high-throughput drug screening to identify therapeutic vulnerabilities of SF3B1-mutant cells. Alongside this, we developed a pipeline for the collection of bone marrow aspirate samples from individuals with SF3B1-mutant blood disorders, and reprogrammed cells from a subset of those patients to human induced pluripotent stem cells (hIPSCs). Splicing factor mutations lead to the widespread mis-splicing of a large number of transcripts, although the functional consequences of many of these aberrant splicing events remains unclear. To investigate this, we performed parallel RNA-sequencing and proteomics on SF3B1-K700E cells, characterising the relationship between splicing alterations, RNA expression, and protein levels. We found that the majority of splicing alterations in SF3B1-mutant cells have minimal effects on RNA/protein expression and observed no change in the global relationship between these two modalities. We also used this data to identify novel transcripts specific to SF3B1-mutant cells and showed that these can be detected at the protein level. This approach could be used to identify neoantigens specific to SF3B1-mutant cells that could be targeted through immunotherapy. Next, we evaluated the differential expression of proteins in SF3B1-mutant cells. This revealed a striking dysregulation of metabolic proteins which, alongside the previously described mitochondrial defects in SF3B1-mutant cells, demanded further investigation. We identified splicing alterations leading to the downregulation of several key proteins within the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) in SF3B1-mutant cells. This was associated with decreased aerobic metabolism and the accumulation of TCA cycle intermediates such as 2-oxoglutarate and the oncometabolite 2-hydroxyglutarate (2HG), which has previously been linked to leukaemogenesis following acquisition of IDH1/2 mutations. We also observed an enhanced sensitivity to glycolysis inhibition in SF3B1-mutant cells, representing a potential vulnerability of these cells that could be exploited therapeutically. Finally, we investigated the unique association between splicing factor-mutant CH and age. Splicing factor mutations are rarely detected before the 6th decade of life but rise dramatically in prevalence beyond this. However, the mechanism underlying this is unclear. Recent work in our group has indicated that telomere length may play a role, demonstrating that individuals with inherited polymorphisms associated with short telomeres possess an increased risk of developing splicing factor-mutant CH. In this work, we adapted methods to measure telomere length by qPCR and flow cytometry and applied these tools to samples from individuals with splicing factor-mutant blood disorders. This revealed that splicing factor mutations appear to promote telomere maintenance, allowing cells to avoid replicative senescence and undergo clonal expansion in old age. Collectively, the work in this thesis sheds light on the forces acting on HSCs in both normal and malignant haematopoiesis and may reveal new ways to prevent, delay, or treat haematological malignancies harbouring splicing factor mutations."],"dc:format.checksum.md5":["92c6489b8cf8d54e6ccf4de1e0f4ef4f","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.119130"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/6883914b-8bb2-4e45-b5c1-0b0ac35c80b4/download"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/385567"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/30b51f9f-e870-42c1-b6fd-5bbeaf6189b1/download","http://purl.org/NET/rdflicense/allrightsreserved"],"dc:subject":["Haematopoietic Stem Cells","Cancer","Clonal Haematopoiesis","SF3B1","Myelodysplastic Syndromes"],"dc:title":["Mechanisms driving the expansion and progression of splicing factor-mutant clonal haematopoiesis"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:21Z"}