Biology subjects

MacFarlane, M.

Publications and source records attributed to MacFarlane, M..

8 recordsLinked to original sources

bioRxiv2026

YBX3 overexpression in mesothelioma drives aberrant cell proliferation

Malignant pleural mesothelioma (MpM) is a lethal tumour closely linked to asbestos exposure and is a cancer of unmet clinical need with no known oncogenic drivers. Recent advancements in technologies to identify RNA-binding proteins (RBPs) has uncovered an emerging role for RBP-RNA interactions in cancer progression and we therefore assessed changes in the RBPome of patient-derived MpM cell lines. We identify over 350 RBPs showing altered RNA binding, with functions consistent with key cancer hallmarks, and discovered YBX3 as a potential oncoprotein driving cell proliferation in MpM. Mechanistically we show the impact of YBX3 on cell growth is achieved through its control of the expression of the amino acid transporter SLC7A5/LAT1, with increased amino acid uptake increasing protein synthesis rates. Notably, we show the inhibition of cell growth by YBX3 deletion is recapitulated by the clinically-relevant SLC7A5/LAT1 inhibitor JPH203. Finally, we demonstrate that JPH203 sensitizes MpM cells to radiotherapy, which could provide a promising therapeutic strategy for MpM. TeaserHigher levels of YBX3 expression in mesothelioma increases cell proliferation and protein synthesis rates by upregulation of amino acid uptake.

molecular biology↗

bioRxiv2024

The Discovery of Small Molecule Inhibitors of cFLIP that Sensitise Tumour Cells to TRAIL

The TNF-related apoptosis-inducing ligand (TRAIL) has potential as a therapeutic agent as it has previously been shown to induce apoptosis in triple-negative breast cancer. Recombinant human TRAIL has shown promise in pre-clinical studies of breast cancer. TRAIL exhibits specificity for triple-negative and treatment-resistant disease subsets. However, several studies have demonstrated that patient tumours exhibit resistance to TRAIL and TRAIL-receptor agonists. We have previously demonstrated that suppression of the TRAIL-receptor inhibitor cFLIP can sensitise breast cancer stem cells to apoptosis inducers, but development of pharmacological inhibitors of cFLIP have been impeded by concerns over structural similarities between cFLIP and the pro-apoptotic procaspase-8. We used molecular dynamics to model the interactions between cFLIP, procaspase-8 and the TRAIL-receptor Death Inducing Signalling Complex (TRAIL-DISC), followed by virtual pharmacophore screening and in-cell viability assays to identify a small-molecule (OH14, 3) that selectively inhibited cFLIP binding to the DISC and promoted TRAIL-mediated apoptosis in breast cancer cell lines. When used in combination with TRAIL, OH14 significantly impaired breast cancer cell viability in primary derived and established cell culture. Given the relatively low (micromolar) potency of the initial hit compound inhibitor OH14 (3), limiting its utility as a preclinical development candidate, we carried out structure-activity relationship studies to find a cFLIP inhibitor with more potent cellular activity. Our findings confirm the proof-of-principle that selective pharmacological inhibition of cFLIP can be used to target a vulnerability in breast cancer cells.

cancer biology↗

bioRxiv2023

The antipsychotic medications aripiprazole, brexpiprazole and cariprazine are off-target respiratory chain complex I inhibitors

Antipsychotic drugs are the mainstay of treatment for schizophrenia and provide adjunct therapies for other prevalent psychiatric conditions, including bipolar disorder and major depressive disorder. However, they also induce debilitating extrapyramidal syndromes (EPS), such as Parkinsonism, in a significant minority of patients. The majority of antipsychotic drugs function as dopamine receptor antagonists in the brain while the most recent third-generation, such as aripiprazole, act as partial agonists. Despite showing good clinical efficacy, these newer agents are still associated with EPS in [~]5-15% of patients. However, it is not fully understand how these movement disorders develop. Here, we combine clinically-relevant drug concentrations with mutliscale model systems to show that aripiprazole and its primary active metabolite induce mitochondrial toxicity inducing robust declines in cellular ATP and viability. Aripiprazole, brexpiprazole and cariprazine were shown to directly inhibit respiratory complex I through its ubiquinone-binding channel. Importantly, all three drugs induced mitochondrial toxicity in primary embryonic mouse neurons, with greater bioenergetic inhibition in ventral midbrain neurons than forebrain neurons. Finally, chronic feeding with aripiprazole resulted in structural damage to mitochondria in the brain and thoracic muscle of adult Drosophila melanogaster consistent with locomotor dysfunction. Taken together, we show that antipsychotic drugs acting as partial dopamine receptor agonists exhibit off-target mitochondrial liabilities targeting complex I.

pharmacology and toxicology↗

bioRxiv2023

Metabolic reprogramming provides a novel approach to overcome resistance to BH3-mimetics in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure and highly resistant to chemotherapy, potentially due to upregulated expression of the pro-survival proteins, BCL2/BCL-XL/MCL-1. Using clinically-relevant models of MPM we show that patient-derived primary MPM cell lines and ex-vivo 3D tumour explants are highly resistant to apoptosis induced by the BCL2/BCL-XL inhibitor, ABT-737. Importantly, we discover that 2-deoxyglucose (2DG), a glycolytic inhibitor, can sensitize MPM cells to ABT-737 and show this correlates with loss of the pro-survival protein, MCL-1. siRNA knockdown of MCL-1 (MCL-1 KD) combined with ABT-737 induced BAX/BAK-dependent, but BIM/PUMA-independent apoptosis, mimicking 2DG/ABT-737 treatment. MCL-1 KD/ABT-737 induced mitochondrial cytochrome c release and caspase-independent inhibition of mitochondrial respiration. Moreover, we observed a hitherto unreported caspase-dependent cleavage of glycolytic enzymes and subsequent inhibition of glycolysis. 2DG inhibited ERK/STAT3 activity, decreased MCL-1 mRNA and protein levels, with concurrent activation of AKT, which limited loss of MCL-1 protein. However, co-treatment with a specific AKT inhibitor, AZD5363, and 2DG/ABT-737 potently induced cell death and inhibited clonogenic cell survival, while in MPM 3D tumour explants MCL-1 protein expression decreased significantly following 2DG or 2DG/AZD5363 treatment. Notably, a similar synergy was observed in MPM cell lines and MPM 3D tumour explants using ABT-737 in combination with the recently developed MCL-1 inhibitor, S63845. Importantly, our study provides a mechanistic explanation for the chemoresistance of MPM and highlights how this can be overcome by a combination of metabolic reprogramming and/or simultaneous targeting of MCL-1 and BCL-2/BCL-XL using BH3-mimetics.

cell biology↗

bioRxiv2023

TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

Dysregulated mitochondrial fusion and fission has been implicated in the pathogenesis of numerous diseases. We have identified a novel function of the p53 family protein TAp73 in regulating mitochondrial dynamics. TAp73 regulates the expression of Optic atrophy 1, a protein responsible for controlling mitochondrial fusion, cristae biogenesis and electron transport chain function. Disruption of this axis results in a fragmented mitochondrial network and an impaired capacity for energy production via oxidative phosphorylation. Owing to the role of OPA1 in modulating cytochrome c release, TAp73-/- cells also display an increased sensitivity to apoptotic cell death, e.g., via BH3-mimetics. We also show that the TAp73/OPA1 axis has functional relevance in the upper airway, where TAp73 expression is essential for multiciliated cell differentiation and function. Consistently, ciliated epithelial cells of Trp73-/- (global p73 KO) mice display decreased expression of OPA1 and perturbations of the mitochondrial network, which may drive multiciliated cell loss. In support of this, Trp73 and OPA1 gene expression is decreased in COPD patients, a disease characterised by alterations in mitochondrial dynamics. We therefore highlight a potential mechanism involving the loss of p73 in COPD pathogenesis. This work also adds to the growing body of evidence for growth-promoting roles of TAp73 isoforms.

cell biology↗

bioRxiv2022

Cleavage of cFLIP restrains cell death during viral infection and tissue injury and favors tissue repair

Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. cFLIP is a crucial regulator of cell death and a substrate of Caspase-8. Yet, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we discovered an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to SARS-CoV-induced lethality, impaired skin wound healing and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to TNF-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on Gln(Q)469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress, to ensure the execution of repair programs.

cell biology↗

bioRxiv2021

Characterisation of FADD interactome reveals novel insights into FADD recruitment and signalling at the Death Inducing Signalling Complex (DISC)

Fas-associated death domain protein (FADD) plays a vital role in the extrinsic apoptotic pathway, where it forms an essential component of the death-inducing signaling complex (DISC). However, the precise early molecular events that facilitate recruitment of FADD to the DISC remain poorly defined. Using affinity purification and mass spectrometry we investigated the FADD interactome in untreated cells and following death receptor stimulation to identify novel FADD-interacting proteins. As expected, in death receptor-stimulated samples our analysis identified key components of the DISC such as Caspase-8. In addition, we identified novel binding partners including Transferrin Receptor 1 (TfR1) and Myosin Light Chain Kinase 2 (MYLK2) that are able to modulate FADD recruitment to the DISC and consequently downstream apoptotic signaling. TfR1 is pre-associated with FADD and recruited into the DISC; moreover, our data reveal that TfR1 is also pre-associated with the death receptors, TRAIL-R1 and TRAIL-R2, thereby functioning as a key regulator of DISC formation. In the case of MYLK2, specific binding of FADD to MYLK2 in non-apoptotic cells sequesters FADD from other DISC components ensuring aberrant apoptosis is not initiated. Furthermore, MYLK2 enzymatic activity is required to for it to translocate, in complex with FADD, to sites of DISC-mediated death receptor oligimerization. Taken together, our study highlights the important role that additional novel FADD binding partners play in the regulation of death receptor-mediated apoptotic cell death, in part by modulating FADD recruitment to the DISC.

biochemistry↗

bioRxiv2020

Asbestos accelerates disease onset in a genetic model of Malignant Pleural Mesothelioma

HypothesisAsbestos-driven inflammation contributes to malignant pleural mesothelioma beyond the acquisition of rate-limiting mutations. MethodsGenetically modified conditional allelic mice that were previously shown to develop mesothelioma in the absence of exposure to asbestos were induced with lentiviral vector expressing Cre recombinase with and without intrapleural injection of amosite asbestos and monitored until symptoms required euthanasia. Resulting tumours were examined histologically and by immunohistochemistry for expression of lineage markers and immune cell infiltration. ResultsInjection of asbestos dramatically accelerated disease onset and end-stage tumour burden. Tumours initiated in the presence of asbestos showed increased macrophage infiltration. Single-agent suppression of macrophages in mice with established tumours failed to extend survival. ConclusionAsbestos-driven inflammation contributes to the severity of mesothelioma beyond the acquisition of rate-limiting mutations, however, targeted suppression of macrophages alone in established disease showed no therapeutic benefit.

cancer biology↗