Investigation into calcium regulation and mitochondrial metabolism in chronic myeloid leukaemia

Khalaf, Ahmed (2023) Investigation into calcium regulation and mitochondrial metabolism in chronic myeloid leukaemia. PhD thesis, University of Glasgow.

Abstract

Chronic myeloid leukaemia (CML) is a myeloproliferative disease characterised by accumulation of myeloid cells in the bone marrow (BM) and blood circulation. On a molecular level, CML is caused by a chromosomal translocation between chromosome 9 and chromosome 22, resulting in a formation of deformed short chromosome called Philadelphia chromosome. This translocation results in a replacement of a negative regulator domain in Abelson (ABL) tyrosine kinase with breakpoint cluster region (BCR), leading to constitutive ABL kinase protein activity. Since the BCR-ABL kinase inhibitor imatinib was discovered in 2000, the survival rates of CML patients have dramatically improved. Nevertheless, not only mutations within BCR-ABL limit BCR-ABL inhibitor potency but also the fact that CML is a haematopoietic stem cell (HSC) driven disease (BCR-ABL positive HSCs are referred to as leukaemic stem cells; LSCs). LSCs adopt both intrinsic and BM extrinsic factors to resist current therapy and underpin the disease persistence experienced in the clinic.
It has been previously reported that LSCs rely more on mitochondrial OXPHOS for their survival compared to their normal HSCs counterparts. Targeting such metabolic vulnerability sensitised those LSCs to eradication by BCR-ABL inhibitors. Since current OXPHOS inhibitors have drawbacks in the clinic, such as short half-life or residual side effects, screening for novel and safe OXPHOS inhibitors is critical.
To deliver this approach, the Helgason lab conducted a drug repurposing screen to identify novel OXPHOS inhibitors which can be safely used in clinics. The drug screening analysis identified the Ca2+ channel blocker lomerizine as one of the most promising OXPHOS inhibitor. Ca2+ is one of the main ions in BM niche and has been shown to catalyse mitochondrial OXPHOS in HSCs. Our transcriptome analysis of LSCs revealed that Ca2+ channels such as TRPC6 and CACNA1D are upregulated in CD34+CD38- LSCs when compared with their normal counterparts. Also, stem cell enriched CML CD34+ cells have higher possession of ER mass and subsequent mitochondrial Ca2+ level than normal counterparts. Both in vitro and ex vivo studies using CML cell lines and CD34+ patient samples revealed that imatinib therapy does not affect the CACNA1D and TRPC6 mediated Ca2+ influx, which is targeted by lomerizine single treatment or when combined with imatinib. Also, by deleting genes using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9, we confirmed the reliance of CML on TRPC6 and CACNA1D to provide ER and mitochondria with Ca2+ ions as a cofactor for tricyclic acid (TCA) cycle dehydrogenases. We also performed in vitro and ex vivo metabolomics, cell growth, and stem cell functional assays on CML cell lines and stem cell enriched patient samples that supported the impact of lomerizine on CML through inhibition of mitochondrial Ca2+ and impacting metabolism.
Finally, we applied our findings to preclinical models of CML. We confirmed the effective combination between lomerizine and imatinib using well-established CML murine models where combination treatment enhanced survival of mice xenografted with CML KCL22 cells. Also, combination of lomerizine with imatinib significantly decreased level of LSCs in BM of mice transplanted with primary CD34+ CML cells.
Overall, we showed for first time that Ca2+ influx via CACNA1D and TRPC6 is important for OXPHOS in therapy resistant LSCs, rendering them sensitive to the combination of lomerizine, as a Ca2+ channel blocker, and imatinib.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology; R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Helgason, Professor Vignir
• Date of Award: 2023
• Depositing User: Theses Team
• Unique ID: glathesis:2023-83341
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 09 Jan 2023 08:53
• Last Modified: 16 Mar 2023 12:17
• Thesis DOI: 10.5525/gla.thesis.83341
• URI: https://theses.gla.ac.uk/id/eprint/83341