title: Investigating mTORC2/AKT-mediated regulation of FOXO1: a novel therapeutic strategy for chronic lymphocytic leukaemia?
Creators: name: Moles, Michael William
Publisher: name: University of Glasgow
Year: 2021

description: The pathogenesis of chronic lymphocytic leukaemia (CLL) is inextricably linked to the tumour microenvironment, a ‘sanctuary site’ wherein CLL cells engage B cell receptor (BCR) signalling and form interactions with non-malignant accessory cells. ‘Crosstalk’ within the microenvironment elicits survival and proliferative signals that facilitate therapy resistance and outgrowth of malignant clones. Therefore, eliminating the signals that orchestrate these events is crucial to prevent disease progression. The advent of small molecule inhibitors targeting BCR signalling components have proven clinically effective. However, these treatments are not always available (or indeed suitable) for every patient and drug resistance has been reported. Thus, there is a need to identify novel treatment strategies that have the ability to improve CLL patient outcomes. Several oncogenic pathways emanating from microenvironment communication converge upon the PI3K-AKT-mTOR axis in CLL cells. Surprisingly, little is known about the functional importance of mTOR signalling in CLL pathogenesis. mTOR exists in two protein complexes, mTORC1 and mTORC2, which coordinate growth, survival and proliferation signals downstream of PI3K-AKT signalling. Despite encouraging preclinical data with the mTORC1-selective inhibitor rapamycin, the rapalogue everolimus only had modest anti-tumour activity in a CLL clinical trial. Clinical activity of mTORC1-selective inhibitors is limited due to abrogation of a S6K-mediated negative feedback loop modulating mTORC2 activity, which results in activation of AKT-mediated pro-survival signalling. The development of ‘second generation’ ATP-competitive mTOR inhibitors avoid these issues by inhibiting both mTORC1 and mTORC2. As such, this investigation sought to address whether inhibition of mTORC1/2 with the dual mTOR kinase inhibitor AZD8055 would represent an effective therapeutic approach for CLL. The data presented in this thesis demonstrates that mTOR is an effector of BCR crosslinking in vitro, playing a role in the coordination cellular behaviours emanating from BCR engagement (BCR-PI3K-AKT) in CLL cells. mTORC1 (4E-BP1T37/46 and S6S235/236) and mTORC2 (AKTS473) activities were effectively targeted by the ‘second generation’ mTOR kinase inhibitor AZD8055 (and its clinical analogue AZD2014), which disabled pro-survival feedback loops associated with rapamycin treatment. At the molecular level, AZD8055 inhibited mTOR signalling downstream of F(ab’)2-mediated BCR ligation and stromal cell (NT-L/CD40L) co-cultures, highlighting the ability of this compound to disrupt various microenvironmental stimuli. On a functional level, AZD8055 elicited potent inhibitory effects on CLL growth and proliferation, but only moderately affected cell viability in vitro. For these reasons, AZD8055 anti-tumour activity appeared to be limited as a monotherapy. A synergistic combination of AZD8055 and the BTK inhibitor ibrutinib promoted cell death, augmented cell size contraction and arrested proliferation, indicating that simultaneous inhibition of mTOR kinase and BTK in CLL cells evokes anti-tumour activity via targeted inhibition of multiple oncogenic pathways and at different levels within the same pathway. In search of a mechanism of action, we proposed that the combination treatment conferred a more robust inhibition of AKT kinase activity. Among other methods, AKT promotes cell survival and proliferation via negative regulation of FOXO transcription factors. FOXOs are widely regarded as tumour suppressors, which regulate several cellular behaviours including cell cycle arrest and apoptosis. Our data demonstrated that BCR crosslinking negatively regulated FOXO1 (the most abundant FOXO in CLL cells) by AKT-dependent FOXO1T24 phosphorylation, subsequent nuclear export and reduced DNA-binding activity. Like normal B cells, these data suggested that FOXO1 inactivation was an important consequence of BCR engagement in CLL cells. For this reason, we hypothesised that inhibiting BCR signalling would unleash FOXO1 tumour suppressor activity. We showed that elimination of BCR signal transduction, via AZD8055 or ibrutinib mono- and combination therapy, re-engaged FOXO1 DNA-binding activity by preventing FOXO1 nuclear export, which suggested that FOXO1 was an effector of BCR signalling inhibition that mediated treatment response. Through pharmacological FOXO1 inhibition, we demonstrated that FOXO1 activity contributed to the cytotoxic, cell-contracting and cytostatic effects of the combination treatment, indicating that FOXO1 functions as a tumour suppressor in this context. In conclusion, these studies highlight the potential for AZD8055/2014 as a drug partner for novel combination strategies in the treatment of CLL. Indeed, these data suggest that AZD8055/2014 could be combined with agents targeting proximal BCR signalling components (BTK, PI3K or SYK) or perhaps pro-survival signals (BCL2). Furthermore, reactivation of FOXO1 activity or other inactivated ‘genetically intact’ tumour suppressors (via targeted inhibition of negative regulators) represents an interesting treatment strategy for CLL. Collectively, these data provide valuable information that has the potential to inform subsequent pre-clinical investigations and future CLL clinical trials.