Investigating the roles of FOXO3 and FOXO4 in CLL proliferation and survival

Lees, Jamie (2025) Investigating the roles of FOXO3 and FOXO4 in CLL proliferation and survival. PhD thesis, University of Glasgow.

Abstract

Chronic lymphocytic leukaemia is a B-cell malignancy emanating from the aberrant growth and accumulation of monoclonal lymphocytes whose origins can be traced to naïve or mature B-cell clones. A groundbreaking study revealed a heavy reliance of CLL cells on interactions with accessory cells within a ‘tumour microenvironment’ niche that drive proliferation, survival and drug resistance. As such, a core theme in the development of novel therapies has been perturbing the activity of intrinsic signalling components that are pivotal in driving these events, such as BCR and CD40 signal inhibition with the advent of BTK inhibitors (e.g. ibrutinib). Current small molecule inhibitor therapy has revolutionised CLL treatment. Even still, patient responses and suitability for treatments are highly variable, reinforcing an unmet clinical need for tailored therapeutics to treat CLL on a patient-specific basis.

Malignant cells often exploit proliferative and survival signalling components to aid their rapid, uncontrollable growth. A prime example is the PI3K-AKT-mTOR signalling axis, which is hyperactivated in most cancer contexts. This signalling axis can orchestrate proliferative and pro-survival events via downstream mTORC1/2 and AKT activity. mTOR components can promote cell growth and proliferation via a multitude of events including mTORC1-mediated translation initiation, as well as enhancing AKT activity. AKT itself can regulate the activity of several downstream substrates via phosphorylation, including FOXO transcription factors. Canonically, FOXO transcription factors mediate the expression of tumour-suppressive genes, and are negatively regulated by AKT-mediated phosphorylation and subsequent cytoplasmic sequestration. However, ever-emerging evidence (in B-cell malignancies and in the wider cancer context) suggests a bimodality to FOXO function, where they promote and/or suppress tumour progression in a context-dependent manner. Indeed, FOXO1’s tumour suppressive roles in CLL have been characterised as part of previous investigations within the group. In this work, we aimed to characterise the behaviours of the FOXO3 and FOXO4 isoforms to determine the suitability of targeting FOXO activity as a potential novel therapeutic approach in CLL.

Initially, prominent FOXO3/4 expression was demonstrated in ex vivo patient samples and in MEC1 and HG3 CLL cell lines. Further investigation revealed that, while FOXO3/4 protein and gene expression were negatively regulated by TME-associated signals (BCR ligation or CD40 activation), FOXO3/4 expression persisted in CLL cell nuclear fractions, indicative of a reliance of CLL cells on constitutive FOXO3/4 activity. Furthermore, global transcriptomic analyses of primary patient samples revealed that FOXO4 is heavily regulated by mTORC1/2-mediated signals downstream of CD40 activation, demonstrating a notable association between FOXO4 and mTOR activity in CLL. This was evidenced by tight regulation of FOXO3/4 localisation by BTK and mTOR, as well as the expression of discrete FOXO target genes downstream of CD40 activation. These findings demonstrate the complexity of FOXO biology in proliferating CLL cell populations.

To further characterise FOXO3/4 behaviour in CLL, we conducted shRNA-mediated knockdown of FOXO3 or FOXO4 in CLL cell lines (and primary patient samples: FOXO4). Here, we demonstrated an isoform-specific reliance of CLL cells on FOXO3/4; in both cases, viability and proliferative capacity were adversely affected. Cells lacking FOXO3 exhibited a loss of drug-mediated cell kill, while extensive investigation revealed that FOXO4 depletion sensitised CLL cells to multiple targeted agents including AZD8055, ibrutinib and venetoclax.
Further investigation identified that FOXO4 depletion increased CLL cell susceptibility to DNA damage, coincident with the aberrant expression of GADD45A and BCL2 family members, as well as a dysregulation of mTORC1/2 signalling components; the latter perhaps due to a lack of FOXO4-mediated SESN3 expression.

Together, these findings improve our understanding of the characteristics of FOXO transcription factors in CLL, demonstrating their ability to exhibit discrete behaviours and orchestrate distinct cellular functions. We argue that, while they are inextricably regulated by TME-associated signals and may facilitate tumour-suppressive effects, FOXO transcription factors are also required to promote CLL cell proliferation, survival and drug resistance in a context- and isoform-dependent manner. These data have the capacity to contribute to future pre-clinical investigation as well as the consideration of novel therapeutic strategies in CLL and in the wider context of malignancy.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QH Natural history > QH426 Genetics; Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Michie, Professor Alison
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85228
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Jun 2025 15:17
• Last Modified: 19 Jun 2025 15:20
• Thesis DOI: 10.5525/gla.thesis.85228
• URI: https://theses.gla.ac.uk/id/eprint/85228

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