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

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

Glioblastoma extracellular vesicles influence glial cell hyaluronic acid deposition to promote invasiveness

Background. Infiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo- and radio-therapy. A deeper understanding of the mechanisms invoked by GMB to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this. Methods. CRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterised EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived extracellular matrix (ECM) to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice. Results. GBM cells expressing a p53 mutant (p53 273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion of PODXL opposes infiltration of GBM in vivo. Conclusions This work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue. Conclusions. This work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue

Leukaemia exposure alters the transcriptional profile and function of BCR::ABL1 negative macrophages in the bone marrow niche

Macrophages are fundamental cells of the innate immune system that support normal haematopoiesis and play roles in both anti-cancer immunity and tumour progression. Here we use a chimeric mouse model of chronic myeloid leukaemia (CML) and human bone marrow (BM) derived macrophages to study the impact of the dysregulated BM microenvironment on bystander macrophages. Utilising single-cell RNA sequencing (scRNA-seq) of Philadelphia chromosome (Ph) negative macrophages we reveal unique subpopulations of immature macrophages residing in the CML BM microenvironment. CML exposed macrophages separate from their normal counterparts by reduced expression of the surface marker CD36, which significantly reduces clearance of apoptotic cells. We uncover aberrant production of CML-secreted factors, including the immune modulatory protein lactotransferrin (LTF), that suppresses efferocytosis, phagocytosis, and CD36 surface expression in BM macrophages, indicating that the elevated secretion of LTF is, at least partially responsible for the supressed clearance function of Ph- macrophages

Apoptotic stress-induced FGF signalling promotes non-cell autonomous resistance to cell death

Damaged or superfluous cells are typically eliminated by apoptosis. Although apoptosis is a cell-autonomous process, apoptotic cells communicate with their environment in different ways. Here we describe a mechanism whereby cells under apoptotic stress can promote survival of neighbouring cells. We find that upon apoptotic stress, cells release the growth factor FGF2, leading to MEK-ERK-dependent transcriptional upregulation of pro-survival BCL-2 proteins in a non-cell autonomous manner. This transient upregulation of pro-survival BCL-2 proteins protects neighbouring cells from apoptosis. Accordingly, we find in certain cancer types a correlation between FGF-signalling, BCL-2 expression and worse prognosis. In vivo, upregulation of MCL-1 occurs in an FGF-dependent manner during skin repair, which regulates healing dynamics. Importantly, either co-treatment with FGF-receptor inhibitors or removal of apoptotic stress restores apoptotic sensitivity to cytotoxic therapy and delays wound healing. These data reveal a pathway by which cells under apoptotic stress can increase resistance to cell death in surrounding cells. Beyond mediating cytotoxic drug resistance, this process also provides a potential link between tissue damage and repair

Arginine dependency is a therapeutically exploitable vulnerability in chronic myeloid leukaemic stem cells

To fuel accelerated proliferation, leukaemic cells undergo metabolic deregulation, which can result in specific nutrient dependencies. Here, we perform an amino acid drop-out screen and apply pre-clinical models of chronic phase chronic myeloid leukaemia (CML) to identify arginine as a nutrient essential for primary human CML cells. Analysis of the Microarray Innovations in Leukaemia (MILE) dataset uncovers reduced ASS1 levels in CML compared to most other leukaemia types. Stable isotope tracing reveals repressed activity of all urea cycle enzymes in patient-derived CML CD34+ cells, rendering them arginine auxotrophic. Thus, arginine deprivation completely blocks proliferation of CML CD34+ cells and induces significantly higher levels of apoptosis when compared to arginine-deprived cell lines. Similarly, primary CML cells, but not normal CD34+ samples, are particularly sensitive to treatment with the arginine-depleting enzyme, BCT-100, which induces apoptosis and reduces clonogenicity. Moreover, BCT-100 is highly efficacious in a patient-derived xenograft model, causing > 90% reduction in the number of human leukaemic stem cells (LSCs). These findings indicate arginine depletion to be a promising and novel strategy to eradicate therapy resistant LSCs