Therapeutic targeting of PRMT5 and mutant ACVR1 in paediatric glioma
Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal paediatric brainstem tumour with a median overall survival of 9-11 months. The unique genetic, anatomical and developmental identity of the tumour means that there are currently no effective chemotherapies or targeted therapies. This the...
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| Format: | Thesis |
| Language: | English |
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2021
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| _version_ | 1826316154285588480 |
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| author | Brown, EJ |
| author2 | Bullock, A |
| author_facet | Bullock, A Brown, EJ |
| author_sort | Brown, EJ |
| collection | OXFORD |
| description | Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal paediatric brainstem tumour with a median overall survival of 9-11 months. The unique genetic, anatomical and developmental identity of the tumour means that there are currently no effective chemotherapies or targeted therapies. This thesis aimed to identify and characterise novel targets for the treatment of DIPG. Potential therapeutic opportunities arise from the frequent mutations found in histone H3 (H3K27M) and kinase ACVR1 found in DIPG tumours. A screen of epigenetic protein inhibitors revealed that the viability of the patient-derived DIPG cell line HSJD-DIPG-007 was highly sensitive to arginine methyltransferase (PRMT) inhibitors, particularly in ACVR1 mutant lines. The PRMT5 inhibitor LLY-283 was identified as the most promising lead for its potent inhibition of DIPG viability and the recognised role of PRMT5 in the regulation of the cell cycle and stemness. RNA-sequencing and phenotypic analyses revealed that LLY-283 did not induce cell death, but reduced stemness and invasiveness, altered metabolism and restored H3K27me3 levels in DIPG cells. Through collaboration with Ángel Montero Carcaboso, LLY-283 was tested in an orthotopic murine xenograft model of DIPG. LLY-283 reduced infiltration of DIPG cells into the murine forebrain, but did not prolong survival, perhaps reflecting inadequate drug exposure. In parallel, the CRISPR/Cas9 system was used to correct the causative G328V ACVR1 mutation in DIPG cells. The resultant isogenic DIPG cells demonstrated slowed proliferation after correction of ACVR1 mutations, rather than a complete growth arrest as observed with published shRNA mediated ACVR1 knockdown. Surprisingly, the isogenic cell lines also showed comparable sensitivity (GI50) to PRMT5 and ACVR1 inhibition, suggesting other genetic differences contribute to the variation in treatment sensitivity between DIPG cell lines. My findings highlight the value of isogenic DIPG cell lines for interrogating the effects of individual tumorigenic mutations. It remains incredibly challenging to develop new strategies to treat DIPG. This thesis supports the future design of combination treatments for DIPG and provides new experimental tools to aid their development. |
| first_indexed | 2024-03-07T07:10:24Z |
| format | Thesis |
| id | oxford-uuid:2c8ee194-05e1-4f33-be7b-538c8ee07ed4 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:38:44Z |
| publishDate | 2021 |
| record_format | dspace |
| spelling | oxford-uuid:2c8ee194-05e1-4f33-be7b-538c8ee07ed42024-12-07T10:26:10ZTherapeutic targeting of PRMT5 and mutant ACVR1 in paediatric gliomaThesishttp://purl.org/coar/resource_type/c_db06uuid:2c8ee194-05e1-4f33-be7b-538c8ee07ed4Intracranial tumorsIntracranial tumors in childrenBrainCancer in childrenGliomasCentral nervous systemTumors in childrenCancer cellsCancerEnglishHyrax Deposit2021Brown, EJBullock, AFarnie, GDiffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal paediatric brainstem tumour with a median overall survival of 9-11 months. The unique genetic, anatomical and developmental identity of the tumour means that there are currently no effective chemotherapies or targeted therapies. This thesis aimed to identify and characterise novel targets for the treatment of DIPG. Potential therapeutic opportunities arise from the frequent mutations found in histone H3 (H3K27M) and kinase ACVR1 found in DIPG tumours. A screen of epigenetic protein inhibitors revealed that the viability of the patient-derived DIPG cell line HSJD-DIPG-007 was highly sensitive to arginine methyltransferase (PRMT) inhibitors, particularly in ACVR1 mutant lines. The PRMT5 inhibitor LLY-283 was identified as the most promising lead for its potent inhibition of DIPG viability and the recognised role of PRMT5 in the regulation of the cell cycle and stemness. RNA-sequencing and phenotypic analyses revealed that LLY-283 did not induce cell death, but reduced stemness and invasiveness, altered metabolism and restored H3K27me3 levels in DIPG cells. Through collaboration with Ángel Montero Carcaboso, LLY-283 was tested in an orthotopic murine xenograft model of DIPG. LLY-283 reduced infiltration of DIPG cells into the murine forebrain, but did not prolong survival, perhaps reflecting inadequate drug exposure. In parallel, the CRISPR/Cas9 system was used to correct the causative G328V ACVR1 mutation in DIPG cells. The resultant isogenic DIPG cells demonstrated slowed proliferation after correction of ACVR1 mutations, rather than a complete growth arrest as observed with published shRNA mediated ACVR1 knockdown. Surprisingly, the isogenic cell lines also showed comparable sensitivity (GI50) to PRMT5 and ACVR1 inhibition, suggesting other genetic differences contribute to the variation in treatment sensitivity between DIPG cell lines. My findings highlight the value of isogenic DIPG cell lines for interrogating the effects of individual tumorigenic mutations. It remains incredibly challenging to develop new strategies to treat DIPG. This thesis supports the future design of combination treatments for DIPG and provides new experimental tools to aid their development. |
| spellingShingle | Intracranial tumors Intracranial tumors in children Brain Cancer in children Gliomas Central nervous system Tumors in children Cancer cells Cancer Brown, EJ Therapeutic targeting of PRMT5 and mutant ACVR1 in paediatric glioma |
| title | Therapeutic targeting of PRMT5 and mutant ACVR1 in paediatric glioma |
| title_full | Therapeutic targeting of PRMT5 and mutant ACVR1 in paediatric glioma |
| title_fullStr | Therapeutic targeting of PRMT5 and mutant ACVR1 in paediatric glioma |
| title_full_unstemmed | Therapeutic targeting of PRMT5 and mutant ACVR1 in paediatric glioma |
| title_short | Therapeutic targeting of PRMT5 and mutant ACVR1 in paediatric glioma |
| title_sort | therapeutic targeting of prmt5 and mutant acvr1 in paediatric glioma |
| topic | Intracranial tumors Intracranial tumors in children Brain Cancer in children Gliomas Central nervous system Tumors in children Cancer cells Cancer |
| work_keys_str_mv | AT brownej therapeutictargetingofprmt5andmutantacvr1inpaediatricglioma |