An investigation into the role of autophagy in mediating leukaemic cell location in the bone marrow niche

Zerbst, Désirée (2024) An investigation into the role of autophagy in mediating leukaemic cell location in the bone marrow niche. PhD thesis, University of Glasgow.

Abstract

Leukaemic stem cell (LSC) persistence is the prevailing issue in curing chronic myeloid leukaemia (CML) with the current gold-standard treatment — tyrosine kinase inhibitors (TKIs). To fully comprehend how LSCs evade targeted treatment, it is essential to study LSCs in their natural environment: the bone marrow (BM) niche. In the last few decades, advances have been made to better understand how the niche influences the maintenance and regulation of healthy haematopoietic stem cells (HSCs), as well as the role of the niche in malignancies. LSCs have been shown to be metabolically adapted to survive within the BM niche and outcompete healthy haematopoiesis.

Previous research in CML aimed to unravel the role of autophagy in LSC survival. This revealed that autophagy inhibition induced LSCs differentiation and sensitization to TKI treatment. As autophagy is influenced by the environment, such as hypoxia, nutrient availability, and inflammation, our study aimed to investigate the complex interplay of CML cells within the BM niche, with a specific focus on the role of autophagy. Moreover, we aimed to unravel the role of mitophagy, the selective degradation of mitochondria, in TKI resistance in vitro.

Through a minimally invasive surgery, high-resolution intravital microscopy (IVM) can be utilised to image cells within the BM in the mouse calvarium. We aimed to assess changes in the niche with leukaemia development and dynamics upon treatment response by developing xenograft and genetically engineered mouse models (GEMM) suitable for confocal IVM.

To model CML, we utilised the SCL-tTA/BCR∷ABL1 mouse model — an inducible mouse model resembling human CML-like disease development upon BCR∷ABL1 expression. To study autophagy in primitive leukaemic cells, we used a fluorescent BCR∷ABL1 mouse expressing the autophagy marker GFP-LC3. While we could observe autophagic flux in vitro, we faced challenges in detecting the GFP signal within the BM niche. Various strategies, including injectable fluorescent antibodies and ex vivo dyes for long-term tracking, were explored to overcome these challenges. Lastly, we generated a fluorescent version of the SCL-tTA/BCR∷ABL1 model by crossing it with the mTmG mouse, which expresses membrane-targeted tdTomato (tdTom). Encouragingly, transplantation of BCR∷ABL1tdTom+ haematopoietic cells allowed us to visualise leukaemic cells within the BM niche in WT recipient mice, providing a solid foundation for future studies.

The investigation extends to xenograft mouse models, aiming to understand the in vivo localisation and interactions of human leukaemic cells in the BM microenvironment. We observed highly variable BM engraftment of different cell lines, particularly concerning localisation within the calvarial BM compared to localisation within the long bones. This posed challenges for attempts to unravel the role of autophagy in BM engraftment and in vivo localisation. Furthermore, we noted different migration patterns of transplanted cells, with extramedullary tumour formation dependent on the mouse’s sex occurring in female mice but not males.

Finally, we investigated the role of mitophagy in response to TKI treatment. High-resolution confocal live-cell microscopy was used with KCL22 and K562 cell lines expressing the reporter gene mCherry-GFP-Fis1, referred to as MitoQC. This fluorescent tandem-dye allows distinction of healthy mitochondria and those undergoing mitophagy due to the pH-sensitivity of GFP. We observed an increase in mitophagy upon TKI treatment, mediated by the autophagy machinery, which, to our knowledge, has not been demonstrated previously. To validate our results, we inhibited autophagy by blocking ULK1 activity and by using ATG7 knockout (KO) cells. We explored different canonical mitophagy pathways, focusing on Nix and BNIP3, key proteins in hypoxia-mediated mitophagy. Our results provide initial insights that Nix and, in a broader sense, BNIP3 may be involved in this process, although further investigation is required to unravel their specific roles in TKI-induced mitophagy.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH301 Biology; 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
• Funder's Name: Medical Research Scotland (MEDRESSC), LifeArc
• Supervisor's Name: Helgason, Professor Vignir and Carlin, Dr. Leo
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84311
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2024 13:07
• Last Modified: 10 May 2024 13:07
• Thesis DOI: 10.5525/gla.thesis.84311
• URI: https://theses.gla.ac.uk/id/eprint/84311