Exploiting tumour cell dependency on pro-survival BCL2 proteins with BH3 mimetics

The RAS-RAF-MEK1/2-ERK1/2 signalling pathway is one of the most frequently deregulated pathways in human cancer, with mutations in KRAS and BRAF commonly occurring in cancers of the pancreas, skin, and colon, among others. Indeed, BRAF and MEK1/2 inhibitors are approved for the treatment of BRAF-mutant malignant melanoma and are improving patient outcomes. However, the development of acquired resistance to these agents is inevitable, highlighting the need for strategies to enhance their primary efficacy in the clinic. Inhibition of ERK1/2 signalling increases the abundance of pro-apoptotic BH3-only proteins such as BIM and BMF. However, despite this, responses to ERK1/2 pathway inhibitors are typically cytostatic, reflecting the residual activity of pro-survival BCL2 proteins. In this thesis, we determined that melanoma cells had a high MCL1:BCL-XL ratio, and as a result were biased towards MCL1 due to low expression of BCL-XL. Consequently, melanoma cells were uniquely dependent on MCL1 to restrain the pro-apoptotic BH3-only proteins induced by ERK1/2 pathway inhibition. The BH3 mimetic AZD5991, a selective MCL1 inhibitor, combined with ERK1/2 pathway inhibitors to drive striking synergistic apoptosis in vitro and robust tumour regressions in vivo. CRISPR/Cas9 gene editing of BIM and BMF in two melanoma cell lines showed that the cell death was substantially BIM/BMF-dependent, and required BAK and BAX to execute. In contrast to melanoma, colorectal cancer (CRC) cells exhibit increased expression of BCL-XL, with a low MCL1:BCL-XL ratio. The combination of MEK1/2 inhibitors with pan-BH3 mimetics that target BCL2, BCL-w and BCL-XL (such as ABT-263) has previously been explored in CRC cells. However, results in this thesis demonstrated that BCL-XL was the critical pro-survival protein in CRC cells responsible for buffering the pro-apoptotic effects of ERK1/2 pathway inhibition. CRC cell lines were completely refractory to BCL2 inhibition when combined with the MEK1/2 inhibitor selumetinib. MEK1/2 inhibitors primed CRC cells for rapid BAX-dependent apoptosis upon BCL-XL antagonism. BH3 mimetics combined with the ERK1/2 inhibitors SCH772984 or AZ6197 in a model of acquired resistance to BRAF and/or MEK1/2 inhibitors in A375 melanoma cells to drive striking apoptosis in vitro and in vivo. Combined BRAF and MCL1 inhibition delayed the onset of acquired resistance to BRAF inhibitor monotherapy in melanoma cells. In SW620 (CRC) cells, combined MEK1/2 and BCL-XL inhibition caused substantial cell death and did not result in the outgrowth of resistant clones; this combination possibly induced senescence in the residual “persister” cells. Finally, in addition to apoptosis, an alternate temporally-distinct cell death modality occurred in CRC cells treated with combined MEK1/2 and BCL-XL inhibition, and this was amplified when caspases were inhibited. This caspase-independent cell death was subsequently determined to be RIPK1-dependent necroptosis, which was reversed by inhibition of RIPK1 by necrostatin-1. BCL-XL may directly inhibit necroptosis induction at the level of the necrosome through the binding of the effector protein MLKL, which was displaced upon BCL-XL inhibition. BH3 mimetics are effective as single agents for the treatment of haematological malignancies, but their application in solid tumours needs to be refined. The results presented here demonstrate that BH3 mimetics harness the apoptotic potential of ERK1/2 pathway inhibitors to drive striking, synergistic cell death in melanoma and CRC cells. The ratio of pro-survival proteins could represent a potential clinical biomarker, and be informative in the clinic to drive BH3 mimetic combination choices.Cambridge International Trust scholarship AstraZenec

Impact of genotype on EPA and DHA status and responsiveness to increased intakes

At a population level, cardioprotective and cognitive actions of the fish oil (FO) derived long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been extensively demonstrated. In addition to dietary intake, which is limited for many individuals, EPA and DHA status is dependent on the efficiency of their biosynthesis from α-linolenic acid. Gender and common gene variants have been identified as influencing the rate-limiting desaturase and elongase enzymes. Response to a particular intake or status is also highly heterogeneous and likely influenced by genetic variants which impacts on EPA and DHA metabolism and tissue partitioning, transcription factor activity, or physiological end-point regulation. Here available literature relating genotype to tissue LC n-3 PUFA status and response to FO intervention is considered. It is concluded that the available evidence is relatively limited, with much of the variability unexplained, though APOE and FADS genotypes are emerging as being important. Although numerous genotype × LC-n3 PUFA × phenotype associations have been described, few have been confirmed in independent studies. A more comprehensive understanding of the genetic, physiological and behavioural modulators of EPA and DHA status and response to intervention is needed to allow refinement of current dietary LC n-3 PUFA recommendations and stratification of advice to ‘vulnerable’ and responsive subgroups

The role of metabolism (and the microbiome) in defining the clinical efficacy of dietary flavonoids

At a population level there is growing evidence for the beneficial effects of dietary flavonoids on health. However there is extensive heterogeneity in the response to increased intake, which is likely mediated via wide inter-individual variability in flavonoid absorption and metabolism. Flavonoids are extensively metabolized by phase I and II- (which occurs predominantly in the gastrointestinal tract and liver) and colonic microbial- metabolism. A number of factors, including age, gender and genotype may impact on these metabolic processes. In addition food composition and flavonoid source is likely to affect bioavailability and emerging data suggest a critical role for the microbiome. This review will focus on the current knowledge for the main sub-classes of flavonoids, including anthocyanins, flavonols, flavan-3-ols and flavanones, where there is growing evidence from prospective studies for beneficial effects on health. Identifying key factors governing metabolism, and understanding if differential capacity to metabolize these bioactive compounds impacts on health outcomes, will help establish how to optimize intakes of flavonoids for health benefits and in specific subgroups. We identify research areas which need to be addressed in order to further understand important determinants of flavonoid bioavailability and metabolism and to advance the knowledge base required to move towards the development of dietary guidelines / recommendations for flavonoids and flavonoid-rich foods

Meal fatty acids and postprandial vascular reactivity

With increasing recognition of the pivotal role of vascular dysfunction in the progression of atherosclerosis, the vasculature has emerged as an important target for dietary therapies. Recent studies have indicated that chronic fatty acid manipulation alters vascular reactivity, when measured after an overnight fast. However, individuals spend a large proportion of the day in the postprandial (non-fasted) state. Several studies have shown that high fat meals can impair endothelial function within 3-4 h, a time period often associated with peak postprandial lipaemia. Although the impact of meal fatty acids on the magnitude and duration of the postprandial lipaemic response has been extensively studied, very little is known about their impact on vascular reactivity after a meal

Thoughts, Ethics and Actions in EMS photography

“Photography can only represent the present. Once photographed, the subject becomes part of the past.” Berenice Abbott (July 17, 1898 – December 9, 1991). To me photography is about capturing a moment, a single moment in time, a single image. Most of us these days are amateur photographers with our camera phones. A picture tells a thousand words they say. A bride on her wedding day, a child killed by a bombing, a beautiful mountain range, a riot, boats , hurricanes...anything you take a photo of is a moment in time, a split second… then it is history.So what role does photography have in Emergency Medical Services (EMS)? It's about education, history, promoting, documenting, recording. Looking back on old photographs we can see how far we have come in terms of equipment, personnel, and training. Without the photos we would have no reference point. It’s a sobering thought that the photos we take today in good faith may in fact be the warnings of tomorrow. Who doesn’t love to look back at photographs when they first started in EMS? Looking to pick out who is still in the job, who has lost the most hair and maybe who has passed away. Sitting around a table, having a cup of coffee with your colleagues, talking about a call you just did, maybe a bad call, someone breaks the tension; “Time for a photo?” Most will smile and join in, some will refuse - each to their own, but a time will come when you look back on these photos remembering not only the bad call but also remembering who had your back that day.Formal EMS events provide a means to mingle and connect and a chance for a photographer to capture a moment in time, the atmosphere, the faces, the colour, the pomp. But in fact, this is also recording history of the EMS staff at that moment in time.Of course there is a graphic side to EMS photography. Photographers will be held to account to portray individuals and scenes with the utmost respect to the patient and their families.(1) Passers-by can be opportunistic and sometimes thoughtless at crisis scenes.(2) So we ask...is it okay to photograph a person in their last few minutes? Graphic photos taken by EMS personnel can be used as a training tool, a reference point and a visual aid when you get to the emergency department. Like a T- boned car, a bullseye impact in the windscreen… a picture tells a thousand words. But where is the line drawn…or is there a difference?The National Press Photographers Association (NPPA) Code of Ethics Summary guide expresses this nicely as: “Photographic and video images can reveal great truths, expose wrongdoing and neglect, inspire hope and understanding and connect people around the globe through the language of visual understanding. Photographs can also cause great harm if they are callously intrusive or are manipulated”.(3

Apolipoprotein E genotype and hepatitis C, HIV and herpes simplex disease risk: a literature review

Apolipoprotein E is a polymorphic and multifunctional protein with numerous roles in lipoprotein metabolism. The three common isoforms apoE2, apoE3 and apoE4 show isoform-specific functional properties including different susceptibilities to diseases. ApoE4 is an accepted risk factor for Alzheimer's disease and cardiovascular disorders. Recently, associations between apoE4 and infectious diseases have been demonstrated. This review summarises how apoE4 may be involved in the infection incidence and associated pathologies of specific infectious diseases, namely hepatitis C, human immunodeficiency virus disease and herpes simplex

The impact of fatty acid desaturase genotype on fatty acid status and cardiovascular health in adults

The aim of this review was to determine the impact of the fatty acid desaturase (FADS) genotype on plasma and tissue concentrations of the long-chain (LC) n-3 PUFA, including EPA and DHA, which are associated with the risk of several diet-related chronic diseases, including CVD. In addition to dietary intakes, which are low for many individuals, tissue EPA and DHA are also influenced by the rate of bioconversion from α-linolenic acid (αLNA). Δ-5 and Δ-6 desaturase enzymes, encoded for by FADS1 and FADS2 genes, are key desaturation enzymes involved in the bioconversion of essential fatty acids (αLNA and linoleic acid (LA)) to longer chained PUFA. In general, carriers of FADS minor alleles tend to have higher habitual plasma and tissue levels of LA and αLNA, and lower levels of arachidonic acid, EPA and also to a lesser extent DHA. In conclusion, available research findings suggest that FADS minor alleles are also associated with reduced inflammation and CVD risk, and that dietary total fat and fatty acid intake have the potential to modify relationships between FADS gene variants and circulating fatty acid levels. However to date, neither the size-effects of FADS variants on fatty acid status, nor the functional SNP in FADS1 and 2 have been identified. Such information could contribute to the refinement and targeting of EPA and DHA recommendations, whereby additional LC n-3 PUFA intakes could be recommended for those carrying FADS minor alleles

A transgenic Camelina sativa seed oil effectively replaces fish oil as a dietary source of eicosapentaenoic acid in mice

Background: Fish currently supplies only 40% of the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) required to allow all individuals globally to meet the minimum intake recommendation of 500 mg/d. Therefore, alternative sustainable sources are needed. Objective: The main objective was to investigate the ability of genetically engineered Camelina sativa (20% EPA) oil (CO) to enrich tissue EPA and DHA relative to an EPA-rich fish oil (FO) in mammals. Methods: Six-week-old male C57BL/6J mice were fed for 10 wk either a palm oil–containing control (C) diet or diets supplemented with EPA-CO or FO, with the C, low-EPA CO (COL), high-EPA CO (COH), low-EPA FO (FOL), and high-EPA FO (FOH) diets providing 0, 0.4, 3.4, 0.3, and 2.9 g EPA/kg diet, respectively. Liver, muscle, and brain were collected for fatty acid analysis, and blood glucose and serum lipids were quantified. The expression of selected hepatic genes involved in EPA and DHA biosynthesis and in modulating their cellular impact was determined. Results: The oils were well tolerated, with significantly greater weight gain in the COH and FOH groups relative to the C group (P < 0.001). Significantly lower (36–38%) blood glucose concentrations were evident in the FOH and COH mice relative to C mice (P < 0.01). Hepatic EPA concentrations were higher in all EPA groups relative to the C group (P < 0.001), with concentrations of 0.0, 0.4, 2.9, 0.2, and 3.6 g/100 g liver total lipids in the C, COL, COH, FOL, and FOH groups, respectively. Comparable dose-independent enrichments of liver DHA were observed in mice fed CO and FO diets (P < 0.001). Relative to the C group, lower fatty acid desaturase 1 (Fads1) expression (P < 0.005) was observed in the COH and FOH groups. Higher fatty acid desaturase 2 (Fads2), peroxisome proliferator–activated receptor α (Ppara), and peroxisome proliferator–activated receptor γ (Pparg) (P < 0.005) expressions were induced by CO. No impact of treatment on liver X receptor α (Lxra) or sterol regulatory element-binding protein 1c (Srebp1c) was evident. Conclusions: Oil from transgenic Camelina is a bioavailable source of EPA in mice. These data provide support for the future assessment of this oil in a human feeding trial