Academic literature on the topic 'Fingolimod (FTY720)'

FTY720 {fingolimod hydrochloride, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride}, a novel immunosuppressant, was discovered by chemical modification based on the structure activity relationships of ISP-I (myriocin), a metabolite of the fungus Isaria­ sinclairii. This short perspective provides insights to the various strategies available in the literature for the synthesis of FTY720 and its analogues.1 Introduction2 Classification of Immunosuppressive Drugs3 The Rise of FTY7204 Different Synthetic Strategies for FTY7205 Analogues of FTY7206 Binding Studies of FTY7207 Mode of Action8 Conclusion

Abstract FTY720 (Fingolimod; Gilenya®) is an immune-modulatory prodrug which, after intracellular phosphorylation by sphingosine kinase 2 (SphK2) and export, mimics effects of the endogenous lipid mediator sphingosine-1-phosphate. Fingolimod has been introduced to treat relapsing-remitting multiple sclerosis. However, little has been published about the immune cell membrane penetration and subcellular distribution of FTY720 and FTY720-P. Thus, we applied a newly established LC-MS/MS method to analyze the subcellular distribution of FTY720 and FTY720-P in subcellular compartments of spleen cells of wild type, SphK1- and SphK2-deficient mice. These studies demonstrated that, when normalized to the original cell volume and calculated on molar basis, FTY720 and FTY720-P dramatically accumulated several hundredfold within immune cells reaching micromolar concentrations. The amount and distribution of FTY720 was differentially affected by SphK1- and SphK2-deficiency. On the background of recently described relevant intracellular FTY720 effects in the nanomolar range and the prolonged application in multiple sclerosis, this data showing a substantial intracellular accumulation of FTY720, has to be considered for benefit/risk ratio estimates.

FTY720 (fingolimod; Gilenya®), a sphingosine 1-phosphate (S1P) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing–remitting multiple sclerosis. FTY720 is rapidly converted in vivo to the active S-fingolimod-phosphate, which binds to S1P receptors. This action inhibits egress of lymphocytes from the lymph nodes, preventing entry into the blood and thus infiltration into the central nervous system. More recent studies, however, convincingly show that FTY720 crosses the blood–brain barrier, where it is thought to act on S1P receptors on cells within the central nervous system, such as astrocytes, oligodendrocytes or microglia. Here we discuss the evidence showing that FTY720 also plays a role in remyelination and repair within the brain. While the mechanisms of action still require firm elucidation, it is clear that FTY720 could also be reparative, extending its therapeutic potential for multiple sclerosis.

Background: FTY720 (Fingolimod) is a drug having immune-regulatory properties. It is a structurally analogous to Sphingosine-1-phosphate. S1P is a biologically active lipid mediator in various inflammatory pathways. Aim: To investigate the effects of FTY720 on myocardial ischemia reperfusion (IR) injury in cardiac surgery. Study design: Experimental study Place and duration of study: Aga Khan University, Karachi and Khyber Medical University, Peshawar Pakistan from 1st January 2017 to 31st December 2020 with collaboration of University of Verona, Italy. Methodology: Twenty Sprague-Dawley rats were segregated into two groups; treatment and control. In treatment group received FTY720 at 1 mg/kg, intravenously 15 minutes prior to the experiment. Both groups were exposed to myocardial ischemia (30 m) reperfusion (2 h). Blood gas analysis, lymphocyte count, myeloperoxidase assay and TUNEL assay were performed and analyzed to observe the effect of FTY720 on neutrophil infiltration and apoptosis. Results: FTY720 treated group had improvement in blood gas levels in contrast to the control, treatment group also experienced decrease in neutrophil infiltration, lymphocyte count and myeloperoxidase enzyme expression. Conclusions: FTY720 pre-treatment reduce lymphocyte count that leads to reduce the level of apoptosis and salvage the myocardial damage incurred by ischemia reperfusion. Keywords: Fingolimod, Cardiopulmonary bypass, Lymphocyte, Neutrophil, Myeloperoxidase

Abstract FTY720 is the functional analogue of sphingosine and is the first oral treatment for relapsing remitting MS. We investigated the immune regulatory effects of Fingolimod (FTY720) on T cells in Multiple Sclerosis (MS) and controls. FTY720 function by blocking immune cell trafficking and prevents relapses in Multiple Sclerosis. We found that T cells activated in the presence of FTY-720 were less inflammatory with decreased proliferation and cytokines expression. FTY720 treated T cells have decreased expression of granzyme B and induced less neuronal cytotoxicity. We showed, FTY720 treated T cells have unique anti-inflammatory immune gene profile. The anti-inflammatory effect of FTY720 on T cells is dependent upon increased expression of a regulatory transcription factor TCF1 (Transcription factor 1). TCF1 has been previously linked to T- cell differentiation and lineage specification in mice. We show that the TCF1 could regulate human T cell differentiation. Knock down of TCF1 gene expression in human T cells switched anti-inflammatory T cells to pro-inflammatory T cell phenotype. In summary, we showed that FTY720 could regulate human T cells differentiation and its immune regulatory function is dependent upon the expression of regulatory transcription factor TCF1.

Fingolimod (FTY720, Gilenya) was the first US Food and Drug Administration–approved oral therapy for relapsing forms of multiple sclerosis (MS). Research on modified fungal metabolites converged with basic science studies that had identified lysophospholipid (LP) sphingosine 1-phosphate (S1P) receptors, providing mechanistic insights on fingolimod while validating LP receptors as drug targets. Mechanism of action (MOA) studies identified receptor-mediated processes involving the immune system and the central nervous system (CNS). These dual actions represent a more general theme for S1P and likely other LP receptor modulators. Fingolimod's direct CNS activities likely contribute to its efficacy in MS, with particular relevance to treating progressive disease stages and forms that involve neurodegeneration. The evolving understanding of fingolimod's MOA has provided strategies for developing next-generation compounds with superior attributes, suggesting new ways to target S1P as well as other LP receptor modulators for novel therapeutics in the CNS and other organ systems.

Aim. Abnormal scars such as hypertrophic scars (HSs) and keloids are excessively growing scars that exhibit chronic inflammation and capillary vasculogenesis. The lipid mediator sphingosine-1-phosphate (S1P) is important in inflammatory cell recruitment and angiogenesis. Fingolimod (FTY720) is an analog of S1P and thus functionally antagonizes S1P receptors and inhibits the enzyme that produces S1P. We examined the effects of topical FTY720 injections on mechanical force-induced HS progression. Methods. Mechanical force-induced HSs were generated in C57BL6/J mice by suturing a dorsal incision and applying a stretching device on Days 6, 8, 10, and 12. On Days 8, 10, and 12, intracutaneous FTY720 (10 μM) or control vehicle injections were performed. On Day 14, scar tissues and blood were procured and subjected to histology and flow cytometry. Results. Flow cytometry showed that FTY720 decreased the frequencies of macrophages with M2 predominance in the scars but had no effect on total, CD4+, or CD8a+ T cell frequencies. FTY720 also decreased the vascular endothelial cell frequencies in the scar along with the microvessels, as determined by immunohistochemistry. Compared to the vehicles, FTY720 treatment significantly reduced the gross scar area and the cross-sectional scar area on histology. On the other hand, FTY720 tended to reduce white blood cells and significantly reduced the lymphocyte frequencies in the blood. Conclusion. Topical FTY720 induces M2 predominance and impairs angiogenesis. Therefore, its local immunosuppressive mechanisms differ from those of conventional immunosuppressive agents. Topical FTY720 can be a novel therapeutic option for abnormal scars that are difficult to control with corticosteroids. Its lymphocytopenic effects may be limited by careful optimization of the treatment regimen.

Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in models of cardiovascular disease. We previously suggested sphingosine-1-phosphate (S1P) activates volume-sensitive Cl- current (ICl,swell) by ROS-dependent signaling. S1P and its analog, FTY720 (fingolimod), primarily act via G-protein coupled receptors (S1PR; S1PR1-3 in heart), but several intracellular S1P ligands are known. We investigated how these agents regulate ICl,swell. ICl,swell was elicited by bath S1P (500 nM), FTY720 (S1PR1,3 agonist; 10 μM), and SEW2871 (S1PR1 agonist; 10 μM) and was fully inhibited by DCPIB, a specific blocker. These data suggested role of S1PR in activation of ICl,swell. Surprisingly, neither CAY10444 (S1PR3 antagonist; 10 μM) nor VPC23019 (S1PR1,3 antagonist; 13 μM) blocked FTY720-induced ICl,swell. Also, gallein a pan Gbeta-gamma inhibitor, failed to block the S1P-induced current. Moreover, 100 nM FTY720 applied via the pipette evoked a larger, faster activating current than 10 μM bath FTY720. Similarly, 500 nM S1P gave larger, faster activating ICl,swell when added to the pipette than when added in the bath. In contrast to FTY720, bath S1P-induced ICl,swell was blocked by CAY10444, but a 3-fold higher concentration failed to eliminate the response to pipette S1P, and VPC23019 failed to suppress bath and pipette S1P-induced currents. Taken together, inconsistencies in the responses to S1PR agents and the greater sensitivity to pipette than bath S1P and FTY720 support the notion that intracellular ligands rather than sarcolemmal S1PR activated ICl,swell. Next we tested if S1P and FTY720, like osmotic swelling, require both NADPH oxidase and mitochondrial ROS production to evoke ICl,swell. S1P- and FTY720-induced ICl,swell were blocked by rotenone but were insensitive to gp91ds-tat, suggesting only mitochondrial ROS production was needed. One possibility is that S1P and FTY720 elicit ICl,swell by binding to mitochondrial prohibitin-2, an S1P ligand whose knockdown augments mitochondrial ROS productions. These data suggest ICl,swell may be activated by S1P accumulation in ischemia-reperfusion and CHF. Understanding S1P-signaling that elicits ICl,swell may provide insight into electrophysiological mechanisms of cardiac pathology and help identify novel targets for therapy.

Les lymphomes non hodgkiniens (LNH) regroupent un panel hétérogène de pathologies originaires de cellules lymphatiques. Parmi les LNH à cellules B matures, la leucémie lymphoïde chronique (LLC) constitue la forme de leucémie de l’adulte la plus fréquente en Occident. La physiopathologie des LNH à cellules B matures est marquée par l’inhibition des mécanismes de la mort cellulaire, notamment via la surexpression de la protéine MCL-1. Une première partie de ce travail de thèse a été de déterminer quelles pouvaient être les enzymes de déubiquitination (DUBs) impliquées dans la survie des LNH à cellules B matures et la stabilisation de MCL-1. Notre étude a permis d’identifier la DUB USP14, qui est liée au système ubiquitine-protéasome, comme capable de réguler MCL-1 et la survie cellulaire. Nos travaux montrent également pour la première fois que l’activité DUB des cellules, ainsi que l’activité d’USP14, sont directement régulées par la signalisation du BCR via l'activité de la tyrosine kinase SYK. Le FTY720, un analogue de la sphingosine utilisé comme immunosuppresseur dans la sclérose en plaques, a montré un effet cytotoxique dans des hémopathies malignes sans toutefois que son mécanisme d’action soit clairement expliqué. Une deuxième partie de ce travail de thèse a été de caractériser la mort induite par le FTY720. Notre étude montre que la caractérisation de la morphologie cellulaire et des marqueurs induits par la mort due au FTY720 dans les LLC correspond en fait à une nécrose cellulaire programmée indépendante de RIPK1, mais dépendante d'une enzyme régulatrice de la fission mitochondriale, DRP1<br>Non-Hodgkin lymphoma (NHL) include a diverse range of pathologies originate from the lymphatic cells. Among the mature B-cell NHL, chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. The pathophysiology of mature B-cell NHL is marked by the inhibition of cell death mechanisms, particularly through the overexpression of MCL-1 protein. The first part of this thesis was to determine which deubiquitinating enzymes (DUBs) are involved in the survival of mature B-cell NHL and in the stabilization of MCL-1. Our study identified the DUB USP14, which is linked to the ubiquitin-proteasome system, as able to regulate MCL-1 and cell survival. Our work also shows for the first time that the DUB activity of the cells and the activity of USP14 are directly regulated by BCR signaling through the activity of the SYK tyrosine kinase. FTY720, a sphingosine analog used as an immunosuppressive drug in multiple sclerosis, showed a cytotoxic effect in hematological malignancies but its mechanism of action is not well understood. A second part of this thesis was to characterize the death induced by FTY720. Our study shows that the characterization of the cellular morphology and markers induced by death due to FTY720 in the LLC corresponds in fact to a programmed RIPK1-independent necrosis cell death, but dependent on DRP1, a regulatory enzyme of the mitochondrial fission

Les neuropathies à anticorps anti-MAG sont secondaires à une gammapathie monoclonale IgM dirigée contre la MAG des gaines de myéline des nerfs périphériques. Le traitement est celui de l'hémopathie sous‐jacente. Même si les thérapeutiques sont de plus en plus efficaces, les hémopathies restent le plus souvent incurables. Le rituximab est couramment utilisé dans le traitement des neuropathies à anticorps anti‐MAG, mais son efficacité n'a pas pu être clairement démontrée dans deux études contrôlées. Le FTY720 ou fingolimod est un sphingolipide, analogue de la sphingosine, qui inhibe les récepteurs de la sphingosine-1-phosphate (S1P). Il est utilisé comme immunosuppresseur dans la Sclérose en Plaques. Des études ont également rapporté un effet cytotoxique du FTY720 dans des hémopathies sans toutefois clairement expliquer son mécanisme d'action. L'objectif de ce travail est d'élucider les mécanismes moléculaires de l'effet cytotoxique du FTY720 dans un modèle d'hémopathie B, la leucémie lymphoïde chronique (LLC). Des cellules leucémiques primaires de LLC et une lignée cellulaire MEC1 ont été utilisées comme modèle expérimental in vitro. Le FTY720, comme la sphingosine, entraîne une cytotoxicité dose‐dépendante dans la LLC. Cet effet, médié par la forme non phosphorylée de FTY720, est indépendant des récepteurs au S1P. Le FTY720 induit l'expression de marqueurs d'apoptose: exposition de la phosphaJdylsérine, clivage de PARP et de caspase 3. Cependant sa toxicité apparaît indépendante des caspases. La lipidation accrue de LC3 et la formation d'autophagolysosomes indiquent que le FTY720 augmente également le flux autophagique. Cependant, des inhibiteurs de l'autophagie ne permettent pas de bloquer la mort cellulaire induite par le FTY720, suggérant que l'autophagie a ici un rôle protecteur vis à vis de la toxicité du FTY720. Plusieurs éléments permettent de conclure que le FTY720 est responsable d'une nécrose cellulaire : aspect morphologique de nécrose en microscopie électronique, perméabilisation membranaire précoce avec relocalisation cytoplasmique de HMGB1, libération extracellulaire de LDH, perméabilisation de la membrane lysosomale associée à une activation des cathepsines. Au niveau moléculaire, l'action du FTY720 n'est pas bloquée par la nécrostatine 1, indiquant que la nécrose induite par le FTY720 est indépendante de RIPK1 (receptor interacJng protein 1), une kinase clef des voies extrinsèques de nécrose cellulaire programmée. Par contre, nos travaux ont établi l'implication de DRP1 (dynamin related protein), une enzyme régulatrice de la fission mitochondriale, dans le processus de nécrose induite par le FTY720. En plus d'une relocalisation précoce de DRP1 à la mitochondrie accompagnée d'une augmentation de sa phosphorylation sur des sites régulateurs de son activité, nos expériences montrent que la suppression de son expression par interférence à ARN dans les cellules leucémiques réduit fortement la mort cellulaire induite par le FTY720. Le FTY720 est donc responsable dans la LLC d'une nécrose cellulaire programmée dépendante de DRP1. Nos résultats illustrent l'implication des sphingolipides dans la régulation de la survie cellulaire et dans les voies de nécrose programmée. Le FTY720 a un mode d'action original différent de l'apoptose induite par les chimiothérapies classiques. Le FTY720 pourrait donc être une alternative thérapeutique dans les néoplasies B résistantes aux chimiothérapies usuelles et dans certaines manifestations auto‐immunes des hémopathies comme les neuropathies à anticorps anti‐MAG.

Background and purpose: Ischemic Heart Diseases (IHD) are the most common cause of morbidity and mortality. Incidence and prevalence is continuously growing. There is an escalating risk for revascularization or resuscitation in patients with IHD. Recently, it has been reported that sphingosine 1-phosphate receptor agonist plays anti-apoptotic and anti-inflammatory role in ischemia-reperfusion injury. The aim of our study is to investigate the cardioprotective effects of sphingosine 1-phosphate receptor agonist fingolimod (FTY720) in global ischemia-reperfusion injury related to the cardiac arrest, cardiopulmonary bypass, and heart transplantation. Methods: In all the three experimental studies, global ischemia-reperfusion was achieved by cardiac arrest either by Ventricular fibrillation or cardioplegia (CPB HT) in anesthetized (sodium pentobarbital, 60 mg/ml/kg i.v) and ventilated male Sprague-Dawley rats (300 - 350 g). The global ischemic period lasted 10 min in the cardiac arrest and cardioplegic arrest, but in transplantation, ischemia time was 60 min while reperfusion times were maintained for either 60 min or 24 hours. Where applicable, monitoring was done using electrocardiogram and hemodynamic data recorded at baseline, 1 hour and 24 hours of reperfusion. The level of high energy phosphates was measured. Apoptotic, inflammatory and oxidative markers were analyzed. The statistical significance was considered as p ≤ 0.05. Experimental design: In all the above mentioned protocols, 10 animals were used in each of the control and treated groups. The first study examined the cardioprotective potential of fingolimod using the following treatment strategy, treatment administered (post ischemia) immediately at the early phase of reperfusion (fingolimod 1mg/kg i.v) compared to saline controls (0.9% saline). In next two experimental models, treatment administration was done 15 min before ischemic phase (fingolimod 1mg/kg i.v.) compared to controls (0.9% saline), followed by 10 min ischemia and 60 min reperfusion in cardioplegic arrest while 60 min ischemic phase remained from heart explantation to reperfusion, and reperfusion phase lasted for 24 hours following blood and tissue collection Results: Three experimental models demonstrated significant myocardial protection in Fingolimod treated groups as compared to control groups. Hemodynamic assessment showed improved cardiac function at late phase. Reduced frequency of apoptotic cells and inflammatory mediators were found in the treated group. High energy phosphates were preserved in the treated as compared to control groups. Reactive Oxygen Species (ROS) were also attenuated in the fingolimod-treated group. Increased phosphorylation of Akt and Erk1/2 signaling pathways found in fingolimod treated group as compared to control, these are important part of Reperfusion Injury Salvage Kinase (RISK) and Survivor Activating Factor Enhancement(SAFE) pathways. Conclusions: The intravenous administration of fingolimod in global ischemia-reperfusion was cardioprotective. Fingolimod cardioprotection appears to be mediated through preservation of high energy phosphates, reduction in oxidative stress, inhibition of apoptosis and inflammation leading to preserved cardiac functions. In summary, targeting sphingosine 1-phosphate receptors modulation may offer a new potential therapeutic cardioprotective agent to attenuate myocardial damage during global myocardial ischemia and reperfusion.

Research Doctorate - Doctor of Philosophy (PhD)<br>Breast cancer is a leading cause of morbidity and mortality worldwide, and despite advancements made in early diagnosis and new treatments over the past few decades, disease progression and therapy resistance remains an unmet challenge. A better understanding of the mechanisms involved in disease progression and resistance to therapies is essential in order to develop improved treatment strategies. Reversible phosphorylation is controlled by the balanced activities of protein kinases and phosphatases, and is a central regulator of the signal transduction pathways required for cell proliferation, differentiation and survival. As such, dysregulation of this balance can result in loss of cellular differentiation and sustained proliferation and survival – key characteristics of cell transformation. While the role of protein kinases in tumourigenesis has been extensively studied, the role protein phosphatases is less well understood. Protein phosphatase 2A (PP2A), is a family of serine/threonine phosphatases that is inactivated in many cancers, including breast cancer, and as such is considered a tumour suppressor. PP2A controls over 50% of serine/threonine phosphatase activities in cells, and regulates numerous growth and survival signalling pathways including the PI3K-AKT and MAPK pathways. PP2A is a trimeric protein complex consisting of a structural subunit (PP2A-A), a catalytic subunit (PP2Ac) and a regulatory subunit (PP2A-B), of which there are at least 4 families each with multiple isoforms. While the dimeric complex of PP2A-AC can dephosphorylate multiple proteins, it is the PP2A-B regulatory subunit that provides substrate specificity and subcellular localization of PP2A. Recent large scale genomic analyses have identified recurrent loss of heterozygosity (LOH) at the PPP2R2A gene locus, which encodes the PP2A-B55α regulatory subunit. PPP2R2A LOH was most common in estrogen receptor positive (ER⁺) luminal breast tumours, and in particular in the aggressive Luminal B subtype. However, the functional role of PPP2R2A loss in breast cancer is not known. Therefore, the overall goal of this thesis was to characterise the gene and protein expression of PP2A subunits, including PPP2R2A (PP2A-B55α), in human breast tumours, and to identify the specific functional role of reduced PP2A-B55α in luminal breast cancer. The first aim of this thesis was to use in silico analysis of publically available gene expression databases, and immunohistochemistry (IHC) of human breast tumours, to compare the gene and protein expression of PP2A subunits and associating proteins between normal breast tissues and breast tumours, and to examine the association of PP2A subunit expression with breast tumour subtypes and disease outcome. This analysis, presented in chapter 3, revealed that low gene and protein expression of the PP2A-B55α and PP2Ac subunits was associated with high grade tumours, and with Luminal B, Human epidermal growth factor positive (HER2⁺), and triple negative breast (ER⁻, progesterone receptor (PR)⁻, HER2⁻; TNBC) molecular subtypes, which are more aggressive than the Luminal A breast tumour subtype. Thus, low PP2A-B55α and PP2Ac protein expression is associated with aggressive breast tumours, and this is likely regulated at the gene expression level. Low PPP2R2A (PP2A-B55α) was further shown to predict for poor relapse-free and overall survival in breast cancer patients, most notably for patients with luminal (ER⁺) tumours. Reduced gene expression of the PP2A regulators SETBP1 and alpha-4, was also associated with aggressive subtypes and worse outcome, suggesting that they may function as tumour suppressors. In contrast, high expression of a number of PP2A-B56 family subunits, and the PP2A inhibitors SET and CIP2A, were associated with more aggressive breast tumours, and thus may play an oncogenic role and be targets for breast cancer therapy. To determine if the reduced PPP2R2A expression observed in aggressive poor outcome tumours, is functionally important, I next investigated the effects of molecular inhibition of PPP2R2A in the human luminal breast cancer cell lines, ZR751, MCF7 and BT474. The data in chapter 4 shows that short-hairpin RNA (shRNA) mediated inhibition of PPP2R2A, resulting in reduced PP2A-B55α protein expression, increased breast cancer cell proliferation, migration and invasion, and increased tumour growth in an orthotopic xenograft mouse model. This was associated with increased activation of ER and AKT signalling, and evidence of an epithelial-to-mesenchymal transition (EMT) phenotype. These results suggest that functional inactivation of PP2A-B55α complexes is important for breast cancer progression, and supports a tumour suppressive role for PPP2R2A. The introduction of anti-estrogen therapies, such as Tamoxifen, to ER⁺ breast cancer patients has led to remarkable improvements in survival. However, many patients are either intrinsically resistant to therapy, or develop resistance and later relapse with therapy-resistant disease. Given that low PPP2R2A expression was associated with poor outcome in ER⁺ patients, in chapter 5 I explored whether inhibition of PP2A-B55α mediated tamoxifen resistance. Analysis of publically available datasets showed that the loss of PPP2R2A was a strong predictor of earlier relapse and distant metastasis in tamoxifen treated breast cancer patients. Furthermore, molecular knockdown of PP2A-B55α induced resistance to tamoxifen in ER+ breast cancer cells. In contrast, ER⁺ breast cancer cells selected for resistance to tamoxifen, expressed reduced PP2A-B55α compared to parental drug sensitive cells, demonstrating a functional role for PP2A-B55α in ER signalling and therapy resistance. In addition, PP2A-B55α knockdown in the HER2⁺ breast cancer cell line, BT474, induced resistance to the anti-HER2 therapeutics, Trastuzumab and Lapatinib. Therefore, the poor outcome observed in patients with low PPP2R2A expression may be mediated by intrinsic resistance to standard therapies. Importantly however, breast cancer cells with low PP2A-B55α were highly sensitive to pharmacological activators of PP2A. Clonogenic and cytotoxicity assays showed that PP2A-B55α knockdown cells were just as sensitive, and in some cases were more sensitive, than control cells, to sphingolipid PP2A agonists (FTY720 and derivatives) and small molecule activators of PP2A (SMAPs). This suggests that PP2A activities can still be enhanced in tumours with low PPP2R2A, and thus is a potential therapeutic strategy for poor outcome breast cancer patients. In chapter 6 I further showed that treating breast cancer cells with PP2A activating drugs can increase the sensitivity of breast cancer cells to targeted therapies, including tamoxifen, Lapatinib and the CDK4/6 inhibitor, Palbociclib. Importantly, the addition of a PP2A activating drug sensitized tamoxifen-resistant breast cancer cells to tamoxifen, providing a strong rationale to combine PP2A activating drugs with standard therapies for the treatment of therapy sensitive and resistant breast tumours. Finally, given that sphingolipid PP2A activators and SMAPs are distinct classes of drugs with different mechanisms of PP2A activation, I examined the effects of combining these two classes of drugs. Intriguingly, I found that the combination displayed highly synergistic cytotoxicity in breast cancer cells with or without PP2A-B55α knockdown. Collectively, the body of work presented in this thesis enhances our understanding of the function of PP2A-B55α in breast cancer signalling and therapy resistance, and suggests that PP2A-B55α expression may be a useful biomarker for predicting disease outcome in luminal breast cancer. Furthermore, these data support the clinical testing of PP2A activating drugs alone and/or in combination, in relapsed/resistant ER⁺ breast cancer patients, with the ultimate goal of improving the survival of breast cancer patients.