The role of Alternative Lengthening of Telomeres in human cancer

Activation of a telomere maintenance mechanism is a vital step in the development of most cancers and provides a target for the selective killing of cancer cells. Cancers can use either telomerase or Alternative Lengthening of Telomeres (ALT) to maintain their telomeres and inhibition of either telomere maintenance mechanism can cause cancer cells to undergo senescence or apoptosis. Although telomerase inhibitors are undergoing clinical trials, on commencing this study very little was known about the role of ALT in cancer, what proteins were involved in its mechanism and regulation and how it could be targeted clinically. The primary aim of this thesis was to develop an assay for ALT suitable for examining archived tumour specimens and to begin using it to examine the prevalence and clinical significance of ALT in cancer. This assay and gene expression analysis was also used to identify genes that are involved in or associated with the activation of the ALT mechanism, to contribute towards the overall goal of an ALT cancer therapy. The ALT mechanism involves recombination mediated replication and ALT cells have a marked increase in a range of recombinational events specifically at their telomeres. Presumably, as a consequence of this the telomere lengths of ALT cells are very heterogeneous and on average long. This can be detected by terminal restriction fragment (TRF) Southern analysis, which has been used previously as the definitive test for ALT activity. However, TRF analysis requires intact genomic DNA and is unsuitable for tumour specimens which are commonly archived by paraffin embedding. Another hallmark of ALT is ALT-associated PML bodies (APBs) which are the subset of PML bodies that contain telomeric DNA. Work done in this study to consolidate APBs as a hallmark of ALT, combined with published data, showed 29/31 ALT[+], 3/31 telomerase[+] and 0/10 mortal cell lines/strains are APB[+]. The three APB[+]/telomerase[+] cell lines identified here had an order of magnitude lower frequency of APB[+] nuclei than the ALT[+] cell lines. APBs may be functionally linked to the ALT mechanism and contain the recombination proteins that are thought to be involved in the ALT mechanism. This study, in collaboration with Dr W-Q Jiang, strengthened this functional link by demonstrating that loss of ALT activity (as determined by TRF analysis) coincided with the disruption of APBs. The detection of APBs was developed into a robust assay for ALT in archived tumour specimens using a technique of combined immunofluorescence and telomere fluorescence in situ hybridisation. It was demonstrated that the APB assay concurred exactly with the standard assay for ALT (TRF analysis) in 60 tumours for which TRF analysis gave unequivocal results. The APB assay may be a more appropriate technique in the case of tumour specimen heterogeneity, which may explain why the APB assay was able to give definitive results when TRF analysis was equivocal. We demonstrated that intratumoral heterogeneity for ALT does exist and this could explain why about 3% of tumours in this study were APB[+] but with more than a ten-fold reduction in the frequency of APB[+] nuclei. This study also made the novel discovery of single stranded C-rich telomeric DNA inside APBs which potentially could be used to make the APB assay more suitable for routine pathology laboratory use. The APB assay was used to show that ALT is a significant concern for oncology. ALT was utilised in approximately one quarter of glioblastoma multiforme (GBM), one third of soft tissue sarcomas (STS) including three quarters of malignant fibrous histiocytomas (MFH), half of osteosarcomas and one tenth of non-small cell lung carcinomas (NSCLC). Furthermore, the patients with these ALT[+] tumours had poor survival; median survivals were 2 years for ALT[+] GBM, 4 years for ALT[+] STS including 3.5 years for ALT[+] MFH and 5 years for ALT[+] osteosarcoma. ALT[+] STS and osteosarcomas were also just as aggressive as their ALT[-] counterparts in terms of grade and patient outcome. ALT status was not found to be associated with response to chemotherapy in osteosarcomas or survival in STS. ALT was however, less prevalent in metastatic STS. The APB assay was a prognostic indicator for GBM and was correlated with three fold increased median survival in GBM (although this survival was still poor). ALT was more common in lower grade astrocytomas (88% ALT[+]) than GBM (24% ALT[+]) and ALT[+] GBM had an identical median age at diagnosis to that reported for secondary GBM. It is discussed that these data indicate that ALT was indirectly associated with secondary GBM and is possibly an early event in its progression from lower grade astrocytoma. This is relevant because secondary GBM have distinct genetic alterations that may facilitate activation of the ALT mechanism. Putative repressors of ALT could explain why this study found that ALT varied among the different STS subtypes. ALT was common in MFH (77%), leiomyosarcoma (62%) and liposarcoma (33%) but rare in rhabdomyosarcoma (6%) and synovial sarcoma (9%). ALT was not found in colorectal carcinoma (0/31) or thyroid papillary carcinoma (0/17) which have a high prevalence of telomerase activity and a reduced need for a telomere maintenance mechanism (low cell turnover), respectively. A yeast model of ALT predicts that one of the five human RecQ helicases may be required for ALT. Using the APB assay to test for the presence of ALT in tumours from patients with known mutations in either WRN or RECQL4 it was demonstrated that neither of these RecQ helicases is essential for ALT. Although p53 and mismatch repair (MMR) proteins have been suggested to be possible repressors of ALT, there was no apparent increase in the frequency of ALT in tumours from patients with a germline mutation in p53 codon 273 or in colorectal carcinomas that had microsatellite instability and thus MMR deficiency. Also contrary to being a repressor of ALT but consistent with its ability to interact with a protein involved in the ALT mechanism, the MMR protein MLH1, was demonstrated to be present in the APBs of an ALT[+] cell line. To further test for genes that may be involved in the ALT mechanism or associated with its activation, RNA microarray was used to compare the gene expression of 12 ALT[+] with 12 matched telomerase[+] cell lines; 240 genes were identified that were significantly differentially expressed (p<0.005) between the ALT[+] and telomerase[+] cell lines. Only DRG2 and SFNX4 were significantly differentially expressed after adjusting for the estimated false positive rate. Overall, DRG2, MGMT and SATB1 were identified as most likely to be relevant to the ALT[+] tumours and Western analysis indicated that DRG2 and MGMT levels were down-regulated after activation of ALT and up-regulated after activation of telomerase, whereas SATB1 protein levels appeared to be up-regulated after immortalisation but to a higher degree with activation of ALT compared to telomerase. Since lack of MGMT is known to be a determinant of temozolomide sensitivity in GBM, the possibility that ALT and the APB assay could be used to predict temozolomide sensitivity is discussed. The microarray data was consistent with MGMT expression being suppressed by EGF (p < 0.05), indicating that caution may be needed with combining EGFR inhibitors with temozolomide in ALT cancers. One ALT[+] cell line which did not express MGMT had TTAA sequence in its telomeres. This could possibly have resulted from mutations due to lack of MGMT expression and a possible role for MGMT in the ALT mechanism is discussed. Further analysis of the microarray data identified two groups of co-regulated genes (p < 5x10-5): CEBPA, TACC2, SFXN4, HNRPK and MGMT, and SIGIRR, LEF1, NSBP1 and SATB1. Two thirds of differentially expressed genes were down-regulated in ALT. Chromosomes 10 and 15 had a bias towards genes with lower expression in ALT while chromosomes 1, 4, 14 and X had a bias towards genes with higher expression levels in ALT. This work has developed a robust assay for ALT in tumour specimens which was then used to show the significance of ALT in sarcomas, astrocytomas and NSCLC. It has also identified genes that could possibly be molecular targets for the treatment of ALT[+] cancers

Intestinal Dysbiosis, the Tryptophan Pathway and Nonalcoholic Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) progresses from simple steatosis to steatohepatitis (NASH), which may then progress to the development of cirrhosis and hepatocarcinoma. NASH is characterized by both steatosis and inflammation. Control of inflammation in NASH is a key step for the prevention of disease progression to severe sequalae. Intestinal dysbiosis has been recognized to be an important causal factor in the pathogenesis of NASH, involving both the accumulation of lipids and aggravation of inflammation. The effects of gut dysbiosis are mediated by adverse shifts of various intestinal commensal bacterial genera and their associated metabolites such as butyrate, tryptophan, and bile acids. In this review, we focus on the roles of tryptophan and its metabolites in NASH in association with intestinal dysbiosis and discuss possible therapeutic implications

Tetrahydrocannabinol and cannabidiol medicines for chronic pain and mental health conditions

Combination tetrahydrocannabinol (THC)/cannabidiol (CBD) medicines or CBD-only medicines are prospective treatments for chronic pain, stress, anxiety, depression, and insomnia. THC and CBD increase signaling from cannabinoid receptors, which reduces synaptic transmission in parts of the central and peripheral nervous systems and reduces the secretion of inflammatory factors from immune and glial cells. The overall effect of adding CBD to THC medicines is to enhance the analgesic effect but counteract some of the adverse effects. There is substantial evidence for the effectiveness of THC/CBD combination medicines for chronic pain, especially neuropathic and nociplastic pain or pain with an inflammatory component. For CBD-only medication, there is substantial evidence for stress, moderate evidence for anxiety and insomnia, and minimal evidence for depression and pain. THC/CBD combination medicines have a good tolerability and safety profile relative to opioid analgesics and have negligible dependence and abuse potential; however, should be avoided in patients predisposed to depression, psychosis and suicide as these conditions appear to be exacerbated. Non-serious adverse events are usually dose-proportional, subject to tachyphylaxis and are rarely dose limiting when patients are commenced on a low dose with gradual up-titration. THC and CBD inhibit several Phase I and II metabolism enzymes, which increases the exposure to a wide range of drugs and appropriate care needs to be taken. Low-dose CBD that appears effective for chronic pain and mental health has good tolerability and safety, with few adverse effects and is appropriate as an initial treatment

Enhancing Endocannabinoid Control of Stress with Cannabidiol.

The stress response is a well-defined physiological function activated frequently by life events. However, sometimes the stress response can be inappropriate, excessive, or prolonged; in which case, it can hinder rather than help in coping with the stressor, impair normal functioning, and increase the risk of somatic and mental health disorders. There is a need for a more effective and safe pharmacological treatment that can dampen maladaptive stress responses. The endocannabinoid system is one of the main regulators of the stress response. A basal endocannabinoid tone inhibits the stress response, modulation of this tone permits/curtails an active stress response, and chronic deficiency in the endocannabinoid tone is associated with the pathological complications of chronic stress. Cannabidiol is a safe exogenous cannabinoid enhancer of the endocannabinoid system that could be a useful treatment for stress. There have been seven double-blind placebo controlled clinical trials of CBD for stress on a combined total of 232 participants and one partially controlled study on 120 participants. All showed that CBD was effective in significantly reducing the stress response and was non-inferior to pharmaceutical comparators, when included. The clinical trial results are supported by the established mechanisms of action of CBD (including increased N-arachidonylethanolamine levels) and extensive real-world and preclinical evidence of the effectiveness of CBD for treating stress

MEG-BIDS, the brain imaging data structure extended to magnetoencephalography.

We present a significant extension of the Brain Imaging Data Structure (BIDS) to support the specific aspects of magnetoencephalography (MEG) data. MEG measures brain activity with millisecond temporal resolution and unique source imaging capabilities. So far, BIDS was a solution to organise magnetic resonance imaging (MRI) data. The nature and acquisition parameters of MRI and MEG data are strongly dissimilar. Although there is no standard data format for MEG, we propose MEG-BIDS as a principled solution to store, organise, process and share the multidimensional data volumes produced by the modality. The standard also includes well-defined metadata, to facilitate future data harmonisation and sharing efforts. This responds to unmet needs from the multimodal neuroimaging community and paves the way to further integration of other techniques in electrophysiology. MEG-BIDS builds on MRI-BIDS, extending BIDS to a multimodal data structure. We feature several data-analytics software that have adopted MEG-BIDS, and a diverse sample of open MEG-BIDS data resources available to everyone

The real-world problem of care coordination: a longitudinal qualitative study with patients living with advanced progressive illness and their unpaid caregivers.

OBJECTIVES: To develop a model of care coordination for patients living with advanced progressive illness and their unpaid caregivers, and to understand their perspective regarding care coordination. DESIGN: A prospective longitudinal, multi-perspective qualitative study involving a case-study approach. METHODS: Serial in-depth interviews were conducted, transcribed verbatim and then analyzed through open and axial coding in order to construct categories for three cases (sites). This was followed by continued thematic analysis to identify underlying conceptual coherence across all cases in order to produce one coherent care coordination model. PARTICIPANTS: Fifty-six purposively sampled patients and 27 case-linked unpaid caregivers. SETTINGS: Three cases from contrasting primary, secondary and tertiary settings within Britain. RESULTS: Coordination is a deliberate cross-cutting action that involves high-quality, caring and well-informed staff, patients and unpaid caregivers who must work in partnership together across health and social care settings. For coordination to occur, it must be adequately resourced with efficient systems and services that communicate. Patients and unpaid caregivers contribute substantially to the coordination of their care, which is sometimes volunteered at a personal cost to them. Coordination is facilitated through flexible and patient-centered care, characterized by accurate and timely information communicated in a way that considers patients' and caregivers' needs, preferences, circumstances and abilities. CONCLUSIONS: Within the midst of advanced progressive illness, coordination is a shared and complex intervention involving relational, structural and information components. Our study is one of the first to extensively examine patients' and caregivers' views about coordination, thus aiding conceptual fidelity. These findings can be used to help avoid oversimplifying a real-world problem, such as care coordination. Avoiding oversimplification can help with the development, evaluation and implementation of real-world coordination interventions for patients and their unpaid caregivers in the future

Respiratory mortality of childhood, adolescent, and young adult cancer survivors

BackgroundExposure to radiation and/or chemotherapy during cancer treatment can compromise respiratory function. We investigated the risk of long-term respiratory mortality among 5-year cancer survivors diagnosed before age 40 years using the British Childhood Cancer Survivor Study (BCCSS) and Teenage and Young Adult Cancer Survivor Study (TYACSS).MethodsThe BCCSS comprises 34 489 cancer survivors diagnosed before 15 years from 1940 to 2006 in Great Britain. The TYACSS includes 200 945 cancer survivors diagnosed between 15 years and 39 years from 1971 to 2006 in England and Wales. Standardised mortality ratios and absolute excess risks were used.FindingsOverall, 164 and 1079 respiratory deaths were observed in the BCCSS and TYACSS cohorts respectively, which was 6.8 (95% CI 5.8 to 7.9) and 1.7 (95% CI 1.6 to 1.8) times that expected, but the risks varied substantially by type of respiratory death. Greatest excess numbers of deaths were experienced after central nervous system (CNS) tumours in the BCCSS and after lung cancer, leukaemia, head and neck cancer and CNS tumours in the TYACSS. The excess number of respiratory deaths increased with increasing attained age, with seven (95% CI 2.4 to 11.3) excess deaths observed among those aged 50+ years in the BCCSS and three (95% CI 1.4 to 4.2) excess deaths observed among those aged 60+ years in the TYACSS. It was reassuring to see a decline in the excess number of respiratory deaths among those diagnosed more recently in both cohorts.ConclusionsPrior to this study, there was almost nothing known about the risks of respiratory death after cancer diagnosed in young adulthood, and this study addresses this gap. These new findings will be useful for both survivors and those involved in their clinical management and follow-up.</jats:sec

Synergistic tumor suppression by combined inhibition of telomerase and CDKN1A

Tumor suppressor p53 plays an important role in mediating growth inhibition upon telomere dysfunction. Here, we show that loss of the p53 target gene cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21WAF1/CIP1) increases apoptosis induction following telomerase inhibition in a variety of cancer cell lines and mouse xenografts. This effect is highly specific to p21, as loss of other checkpoint proteins and CDK inhibitors did not affect apoptosis. In telomerase, inhibited cell loss of p21 leads to E2F1- and p53-mediated transcriptional activation of p53-upregulated modulator of apoptosis, resulting in increased apoptosis. Combined genetic or pharmacological inhibition of telomerase and p21 synergistically suppresses tumor growth. Furthermore, we demonstrate that simultaneous inhibition of telomerase and p21 also suppresses growth of tumors containing mutant p53 following pharmacological restoration of p53 activity. Collectively, our results establish that inactivation of p21 leads to increased apoptosis upon telomerase inhibition and thus identify a genetic vulnerability that can be exploited to treat many human cancers containing either wild-type or mutant p53

Pelargonidin-3-O-glucoside and its metabolites have modest anti-inflammatory effects in human whole blood cultures

This study hypothesized that the predominant strawberry anthocyanin, pelargonidin-3-O-glucoside (Pg-3-glc), and three of its plasma metabolites (4-hydroxybenzoic acid, protocatechuic acid and phloroglucinaldehyde [PGA]) would affect phagocytosis, oxidative burst and the production of selected pro- and anti-inflammatory cytokines in a whole blood culture model. For the assessment of phagocytosis and oxidative burst activity of monocytes and neutrophils, whole blood was pre-incubated in the presence or absence of the test compounds at concentrations up to 5 μM, followed by analysis of phagocytic and oxidative burst activity using commercially available test kits. For the cytokine analysis, diluted whole blood was stimulated with lipopolysaccharide in the presence or absence of the test compounds at concentrations up to 5 μM. Concentrations of selected cytokines (tumor necrosis factor-α [TNF-α], interleukin [IL]-1β, IL-6, IL-8 and IL-10) were determined using a cytometric bead array kit. There were no effects of any of the test compounds on phagocytosis of opsonized or non-opsonized E. coli or on oxidative burst activity. Pg-3-glc and PGA at 0.08 μM increased the concentration of IL-10 (P<0.01 and P<0.001, respectively), but there was no effect on TNF-α, IL-1β, IL-6 and IL-8 and there were no effects of the other compounds. In conclusion, this study demonstrated a lack of effect of these compounds on the opsonization, engulfment and subsequent destruction of bacteria. Pg-3-glc and PGA, at physiologically relevant concentrations, had anti-inflammatory properties; however, effects were modest, only observed at the lowest dose tested and limited to IL-10

Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers

ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.This work was supported by the Norwegian Cancer Society and the Research Council of Norway (to P.C.); an Australia Research Council Future Fellowship award (to L.H.W.); National Health and Medical Research Council Program Grant 1053792 (to R.B.P. and R.D.H.), senior research fellowships (to R.B.P. and R.D.H.), and a project grant (to L.H.W.); and a Cure Brain Cancer Foundation Australia project grant (to L.H.W. and H.P.J.V.)