Investigating and targeting metabolic dependencies in myeloid leukaemia

Robert de Beauchamp, Lucie (2020) Investigating and targeting metabolic dependencies in myeloid leukaemia. PhD thesis, University of Glasgow.

Due to Embargo and/or Third Party Copyright restrictions, this thesis is not available in this service.

Abstract

Leukaemias, which include chronic and acute myeloid leukaemia (CML and AML), represent the tenth deadliest type of cancer, claiming over 300,000 lives each year worldwide. CML and AML are clonal haematological malignancies caused by mutations and chromosomal rearrangements. These occur in stem and progenitor haematopoietic cells, giving rise to leukemic stem cells (LSCs). While CML is characterised by the presence of the Philadelphia chromosome and the constitutively active BCR-ABL protein, AML can develop from a multitude of genomic aberrations. CML patients have greatly benefited from the introduction of BCR-ABL-targeting tyrosine kinase inhibitors (TKIs), which have brought the disease’s five-year survival rate above 80 %. TKIs however, are not a cure. Indeed, to avoid relapse most CML patients need to remain under treatment their whole lives, with associated toxicities and the risk of developing treatment resistance. In comparison to CML, the survival rate of AML patients has remained stubbornly low: below 40 % for patients under 60 years old, falling below 15 % for older ones. While a number of targeted therapies have been approved in recent years, the first line of treatment in AML remains aggressive chemotherapy and, for eligible patients, haematopoietic stem cell (HSC) transplantation. In addition, up to half of AML patients going through therapy will experience resistance and/or relapse.

While therapies informed by genomic data have transformed the treatment of some cancers, they have also shown their limits. This is especially apparent in our current inability to drug certain targets, and the frequency of treatment resistance. Research into alternative vulnerabilities of cancer cells is therefore of great interest, including in myeloid leukaemia. Critically, a rewired cellular metabolism is now considered a hallmark of cancer. Early work in oncometabolism suggests that cancer cells are more glycolytic than their non-malignant counterparts, a mechanism known as the Warburg effect. In the past decade however, a more complex picture of cell metabolism in myeloid leukaemia has emerged. Indeed, oxidative phosphorylation (OXPHOS) now appears to be of importance in treatment-resistant cells and CML LSCs, while the metabolic profile of AML LSCs has yet to be fully characterised. Thus, in the present study, we aimed to determine whether the energy metabolism of AML LSCs differ from that of less primitive AML cells, and aimed to identify novel OXPHOS inhibitors for application in myeloid leukaemia.

We therefore provided the first in-depth investigation of the energy metabolism of transcriptionally defined AML LSCs, performed in CD34- NPM1-mutated AML. This required the optimisation of media composition and cytokine supplementation for the metabolomic-based study of patient-derived AML samples. Analysis of RNA sequencing data, combined with stable isotope tracing and live-cell metabolic functional assays, revealed the distinct metabolic profile of patient-derived AML samples carrying a stem-like signature. Specifically, stem-like samples were less metabolically active than their differentiated-like counterparts and, noticeably, less glycolytic, potentially rendering them more sensitive to OXPHOS inhibition.

In parallel, addressing our second aim, we performed a metabolism-specific drug repurposing screen. To identify safe and effective OXPHOS inhibitors, over 1,300 compounds were tested in CML and AML cell lines. Following initial validation of the screen’s top hits in cell lines, calcium channel inhibitor lomerizine, fatty acid amide hydrolase inhibitor PF-3845 and antifungal itraconazole were selected for further investigation. Metabolomic-based experiments in OCI-AML3 cells revealed that all three drugs inhibited OXPHOS, and that PF-3845 and itraconazole did so without significantly disrupting other central carbon metabolism pathways. In addition, all three compounds inhibited mitochondrial respiration in patient-derived AML cells, and presented minimal toxicity towards HSCs. Having shown activity against primitive AML cells in vitro, the efficacy of itraconazole was further tested in two in vivo models. In a patient-derived xenograft experiment, itraconazole in combination with cytarabine decreased the percentage of AML LSCs in the bone marrow (BM) of immunocompromised NRG-3GS mice. In a secondary transplant experiment, this combination treatment increased the survival of engrafted mice, although this did not reach statistical significance. In another xenograft model, itraconazole sensitised THP-1 cells to cytarabine treatment, decreasing their survival in mice BM.

Overall, these results indicate that NMP1-mutated, transcriptionally defined CD34- AML LSCs are characterised by low metabolic activity and low glycolysis. This is of clinical interest, considering LSC signatures are associated with poor prognosis in AML patients. In addition, as HSCs are glycolytic, this could indicate a metabolic therapeutic window. Also translationally relevant, several safe and effective OXPHOS inhibitors were identified in a repurposing screen, highlighting the value of this drug development strategy to target oncometabolism. These compounds were then further validated in patient-derived AML samples. Finally, we showed that antifungal itraconazole, already used in routine infection prophylaxis, represents a clinically relevant candidate to target OXPHOS metabolism in chemo-resistant AML cells.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: leukaemia metabolism OXPHOS stem cells resistance.
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences > Paul O'Gorman Leukemia Research Centre
Supervisor's Name: Helgason, Dr. Vignir and Maddocks, Dr. Oliver
Date of Award: 2020
Embargo Date: 22 October 2023
Depositing User: Ms Lucie Robert de Beauchamp
Unique ID: glathesis:2020-81718
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 26 Oct 2020 08:59
Last Modified: 26 Oct 2020 08:59
URI: http://theses.gla.ac.uk/id/eprint/81718

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