Investigating and targeting folate metabolism in chronic myeloid leukaemia

Zarou, Martha-Maria (2022) Investigating and targeting folate metabolism in chronic myeloid leukaemia. PhD thesis, University of Glasgow.

Abstract

Chronic myeloid leukaemia (CML) is myeloproliferative disease that arises due to the formation of the fusion oncogene, BCR-ABL, in a haematopoietic stem cell (HSC). BCR-ABL oncogene has a constitutive tyrosine kinase activity and drives myeloid expansion and accumulation of mature blood cells. Due to the introduction of imatinib, a specific tyrosine kinase inhibitor (TKI), treatment of the disease has drastically improved the last 20 years. Nevertheless, imatinib and second/third generation TKIs do not eradicate leukaemic stem cells (LSCs), This population of cells persists and is therapy resistant. Thus, current research is focused on the identification of novel targets to target CML LSCs and ultimately, cure the disease.

Recent work from our lab has demonstrated that CML LSCs have high mitochondrial mass and activity and that they rely on oxidative phosphorylation (OXPHOS) when compared to patient matched progenitor cells or normal HSCs. Folate-mediated one carbon (1C) metabolism plays a crucial role in nucleotide synthesis, energy homeostasis and redox defence. Furthermore, folate metabolism is a metabolic vulnerability for various haematological malignancies. As the role of folate metabolism in CML remains unknown, we aimed to investigate the importance of 1C metabolism in CML as a model of LSC-driven haematological malignancies.

By using publicly available microarray datasets we established that folate metabolism associated genes are upregulated in LSCs compared to normal counterparts. Furthermore, we discovered that expression of those genes does not change in CML stem cell enriched (CD34+) primary cells following a 7-day imatinib treatment. In addition, we revealed that the activity of the pathway is significantly upregulated in CML CD34+ cells when compared to normal CD34+ cells.

We also uncovered that genetic inhibition of the pathway following loss of the mitochondrial serine hydroxymethyl transferase 2 (SHMT2), leads to decreased cell growth and cell cycle arrest of K562 cells. Furthermore, loss of SHMT2 significantly impaired tumour xenograft formation of KCL22 cells.

Metabolically, genetic or pharmacological (SHIN1; SHMT1/2 inhibitor) inhibition of 1C metabolism resulted in decreased de novo purine synthesis and glycolysis. Besides this, inhibition of the folate pathway led to AMPK activation, mTORC1 suppression and autophagy induction. Inhibition of the pathway also altered mitochondrial homeostasis by decreasing mitochondrial reactive oxygen species (ROS), causing hyperpolarisation of the mitochondrial membrane and accumulation of the mitochondrial fission related protein DRP1 and mitophagy receptor NIX. Of note, formate (1C unit donor independent from SHMT1/2 activity) supplementation was sufficient to reverse changes caused by folate metabolism inhibition.

Phenotypically, inhibition of 1C metabolism induced the expression of erythropoiesis markers CD71 and Glycophorin A in K562 cells, which was reversed following formate supplementation. Similar results were obtained in CML CD34+ cells when challenged with erythropoietin (EPO). Furthermore, we uncovered that promotion of maturation of CML cells was AMPK-independent, but autophagy dependent.

Of clinical relevance, pharmacological inhibition of 1C metabolism resulted in impaired cell proliferation and short-term colony formation potential of CML CD34+ cells, with minimum effect on normal counterparts. Lastly, combination treatment of SHIN1 with imatinib significantly increased the sensitivity of primary CML cells to imatinib.

In conclusion, these results describe a novel role of folate metabolism in CML, indicating that the pathway can be a metabolic vulnerability for LSCs and sensitise this population to traditional therapeutic approaches.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Helgason, Dr. Vignir and Vazquez, Dr. Alexei
• Date of Award: 2022
• Depositing User: Theses Team
• Unique ID: glathesis:2022-82783
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 04 Apr 2022 09:30
• Last Modified: 08 Apr 2022 16:38
• Thesis DOI: 10.5525/gla.thesis.82783
• URI: https://theses.gla.ac.uk/id/eprint/82783

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