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Figure S3 - PDF file 26K, Figure S3. AD-01 is not cytotoxic. No change in surviving fraction following AD-01 treatment in both MDA-231 and MCF-7 cells was observed.

Table S2 - PDF file 86K, A: Clinical information and the average MFE for every solid primary breast tumour received; tumours of similar grade and status were combined and the average MFE calculated. B: Tumour initiation data following implantation of MCF-7 cells and AD-01 or PBS treatment from day 1 of implantation

Supplementary Data Legends - PDF file 63K, Data legends for supplemental figs S1 to S7

Purpose: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action.Experimental Design: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status.Results: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype.Conclusion: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy. Clin Cancer Res; 17(5); 1044–56. ©2011 AACR.

Purpose: FK506-binding protein like (FKBPL) and its peptide derivative, AD-01, have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here, we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC).Experimental Design: Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples, and xenografts. Delays in tumor initiation were evaluated in vivo. The anti–stem cell mechanisms were determined using clonogenic assays, quantitative PCR (qPCR), and immunofluorescence.Results: AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESA+/CD44+/CD24− or aldehyde dehydrogenase (ALDH)+ cell subpopulations in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01–mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4, and Sox2, were also significantly reduced. Furthermore, we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy-induced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signaling.Conclusions: AD-01 has dual antiangiogenic and anti-BCSC activity, which will be advantageous as this agent enters clinical trial. Clin Cancer Res; 19(14); 3881–93. ©2013 AACR.

Supplementary Figures S1-S10; Supplementary Table S1.

Supplementary Figure 3 from FKBPL Regulates Estrogen Receptor Signaling and Determines Response to Endocrine Therapy

Supplementary Figure 2 from FKBPL Regulates Estrogen Receptor Signaling and Determines Response to Endocrine Therapy

Supplementary Figure 1 from FKBPL Regulates Estrogen Receptor Signaling and Determines Response to Endocrine Therapy

The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor α (ERα) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERα phosphorylation on Ser118 in response to 17β-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome. Cancer Res; 70(3); 1090–100

BackgroundConsumption of unhealthy foods may have changed during the COVID-19 pandemic. This study explored how dietary fat intake was impacted in a sample of the UK public who were social distancing during the COVID-19 pandemic.MethodsData were collected from a UK COVID-19 online survey. Fat intake was measured using the Dietary Instrument for Nutrition Education questionnaire. Anxiety and depressive symptoms were assessed using Becks’ Anxiety and Depression Inventories, while the short-form Warwick-Edinburgh Mental Well-being Scale assessed mental well-being. Differences between individuals who increased versus decreased fat intake were explored using chi-square or independent sample t-tests. Association between fat intake and mental health was explored using adjusted linear regression models.ResultsEight hundred and eighty-seven adults were included. Approximately, 34% recorded medium-to-high levels of fat consumption during social distancing. Around 48% reported decreased fat intake during social distancing compared to usual levels, while 41.3% documented increased fat intake. Fat intake was not significantly associated (P > 0.05) with any measures of mental health.ConclusionsA higher proportion of a sample of UK adults social distancing during the COVID-19 pandemic recorded decreased fat intake when compared to levels prior to social distancing. There appeared to be no associations between fat intake and mental health.

Public health restrictions, in response to the COVID-19 pandemic, have had potentially wide-ranging, unintended effects on health-related behaviours such as diet and physical activity and also affected mental health due to reduced social interactions. This study explored how healthrelated behaviours and mental health were impacted in a sample of the UK public during the first set of COVID-19 public health restrictions. Two online surveys were administered in the UK, one within the first three months of the restrictions (Timepoints 1 (T1—involving pre-pandemic recall) and 2/T2) and another ten weeks later (Timepoint 3/T3). Moderate–vigorous physical activity (MVPA), outdoor time, sitting time, screen time and sexual activity were self-reported. Diet was assessed using the Dietary Instrument for Nutrition Education questionnaire. Mental health was measured using the short-form Warwick–Edinburgh Mental Wellbeing Scale and Becks’ Anxiety and Depression Inventories. Differences between timepoints were explored using the Friedman, Wilcoxon signed-rank, McNemar and McNemar–Bowker tests. Two hundred and ninety-six adults (74% under 65 years old; 65% female) provided data across all timepoints. Between T1 and T2, MVPA, time outdoors and sexual activity decreased while sitting, and screen time increased (p < 0.05). Between T2 and T3, saturated fat intake, MVPA, time outdoors, and mental wellbeing increased while sitting, screen time and anxiety symptoms decreased (p < 0.05). This study found that depending on the level of COVID-19 public health restrictions in place, there appeared to be a varying impact on different health-related behaviours and mental health. As countries emerge from restrictions, it is prudent to direct necessary resources to address these important public health issues. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

This chapter is informed by the work of researchers that report research outcomes and case studies regarding the adoption of AT solutions in educational environments. The aim is to provide school staff and other professionals in the field of education an insight of state of art research and related practice innovation including different examples of technologies for the empowerment of learners with disabilities are presented. A detailed description of six case studies is provided by their respective authors, each of whom has highlighted specific aspects of the AT practices and experiences implemented. The common feature of these case studies consists of enabling learners to achieve a major sense of autonomy, independence, belonging to the community as well as to overcome some cultural and social barriers through the use of innovative solutions. In detail, case studies are structured in a way that captures the general description of the setting, the persons involved, the main barriers, needs and opportunities identified. Also, case studies reported in this chapter highlight how technology was made helpful and effective in addressing the needs and opportunities, what were the outcomes of the use of technology, and what conditions have to be met in order to make a similar experience possible and replicable on the base of lessons learned and authors’ recommendations. These experiences come from different sources. Four of the cases consist research studies developed in the framework of the ASSISTID Programme. The first one explores the challenges faced in using virtual reality in special education, the second ASSISTID case report is on adapting the Irish Matching Person with Technology tool through a Universal Design approach, the third one on assistive technology for reading and comprehension for learners with autism and intellectual disability and the fourth one of this programme is on assistive technology for inclusive learning in non classroom settings. In addition, the fifth case report of this chapter concerns the use of a digital solution for helping people with cognitive disabilities to learn new skills and achieve independence, and the last one reports the experience of robot-based activities for creating an inclusive learning scenario developed by the Assistive Technology Center ofAIAS Bologna Onlus.

Highlights • More than one in six UK adults increased their alcohol consumption during lockdown. • Increased alcohol consumption was associated with poor overall mental health. • These findings highlight the importance of planning targeted support., Background The aim was to examine the correlates of increased alcohol consumption during the COVID-19 pandemic-related restrictions that were implemented in a sample of UK adults. Methods This paper presents analyses of data from a cross-sectional study. Adults aged 18 years and over, residing in the UK and self-isolating from others outside their own household were eligible to participate. Participants reported increase or no increase in their level of alcohol consumption from before to during lockdown, as well as symptoms of anxiety, depression and mental wellbeing. Socio-demographic characteristics were compared between adults with and without reported increased alcohol consumption. The associations between reported increased alcohol consumption and mental health outcomes were investigated using logistic and linear regression analyses. Results 691 adults (61.1 % women; 48.8 % aged 35−64 years) were included in the analysis. Of these, 17 % reported increased alcohol consumption after lockdown. A higher proportion of 18−34-year olds reported increased alcohol consumption compared to older groups. The prevalence of poor overall mental health was significantly higher in individuals with increased alcohol consumption (vs. no increase) (45.4 % versus 32.7 %; p-value = 0.01). There was a significant association between increased alcohol consumption and poor overall mental health (OR = 1.64; 95 % CI = 1.01, 2.66), depressive symptoms (unstandardized beta = 2.93; 95 % CI = 0.91, 4.95) and mental wellbeing (unstandardized beta=-1.38; 95 % CI=-2.38, -0.39). Conclusions More than one in six UK adults increased their alcohol consumption during lockdown and a higher proportion of these were younger adults. Increased alcohol consumption was independently associated with poor overall mental health, increased depressive symptoms and lower mental wellbeing. These findings highlight the importance of planning targeted support as we emerge from lockdown and plan for potential second and subsequent waves.

A breakdown in vascular integrity and excessive inflammation are hallmarks of serious pathological conditions including sepsis, acute respiratory distress syndrome (ARDs) and most recently, severe COVID-19. FK506 – binding protein like (FKBPL) is a member of the immunophilin protein superfamily with potent anti-tumor activity through inhibition of angiogenesis and cancer stemness. An FKBPL-based 23mer peptide, ALM201, displayed a good safety and pharmacokinetic profile in a Phase 1a oncology clinical trial and was subsequently designated orphan drug status by the FDA in ovarian cancer. Here we describe a novel role for FKBPL and its peptides in regulating vascular integrity and cytokine production though modulating NF-κB signaling. FKBPL knockdown promoted endothelial cell barrier permeability, which was further exacerbated upon stimulation with lipopolysaccharide (LPS) and accompanied by increased expression of TNF mRNA and phosphorylation of p65(RelA). Whilst treatment with the FKBPL based pre-clinical peptide, AD-01, increased VE-cadherin endothelial tight junctions following LPS stimulation. Bone marrow derived macrophages (BMDM) from FKBPL haploinsufficient mice (Fkbpl+/−) also demonstrated increased phosphorylation of p65(RelA) in response to LPS stimulation compared to wild-type mice. Furthermore, treatment with AD-01 inhibited p65(RelA) phosphorylation following LPS stimulation resulting in reduced NF-κB target gene expression and proinflammatory cytokine production. In an in vivo LPS survival model, Fkbpl+/− mice have reduced survival compared to wild-type mice. Moreover, treatment of wild-type mice with the clinical FKBPL-based peptide, ALM201, following LPS injection resulted in a 100% survival rate in mice at experimental endpoint, as well as an abrogation of production of pro-inflammatory cytokines, TNF and IL-6, in peritoneal lavage washings. Analysis of human genetic biobanks found an association between common genetic variants associated with FKBPL and traits associated with inflammatory disorders such as psoriasis, rheumatoid arthritis and high lymphocyte count. In summary, for the first time, we describe a novel role for FKBPL as a regulator of inflammation and vascular integrity through modulating NF-κB signaling and FKBPL based therapies demonstrate potent anti-inflammatory activity.

Aim The aim of the present study was to investigate the cross-sectional association between physical activity levels with depressive symptoms, anxiety symptoms, and positive mental well-being in a sample of the UK public social distancing owing to COVID-19. Method This paper presents pre-planned interim analyses of data from a cross-sectional epidemiological study. Levels of physical activity during COVID-I9 social distancing were self-reported. Mental health was measured using the Beck Anxiety and Depression Inventory. Mental wellbeing was measured using The Short Warwick-Edinburgh Mental Well-being Scale. Participants also reported on sociodemographic and clinical data. The association between physical activity and mental health was studied using regression models. Results 902 adults were included in this study (63.8% of women and 50.1% of people aged 35–64 years). After adjusting for covariates, there was a negative association between moderate-to-vigorous physical activity per day in hours and poor mental health (OR = 0.88, 95% CI = 0.80–0.97). Similar findings were obtained for moderate-to-severe anxiety symptoms, moderate-to-severe depressive symptoms and poor mental wellbeing. Conclusions In the present sample of UK adults social distancing owing to COVID-19 those who were physically active have better overall mental health. Owing, to the cross-sectional design of the present study the direction of the association cannot be inferred., Highlights • We investigated the association between physical activity and mental health in the UK public during COVID-19 “lockdown”. • In this sample of UK adults those who were physically active had better overall mental health. • Owing, to the cross-sectional design of the present study the direction of the association cannot be inferred.

Background On 23rd March 2020, the UK government released self-isolation/social distancing guidance to reduce the risk of transmission of SARS-CoV-2. The influence such guidance has on sexual activity is not known. Aim To investigate levels and correlates of sexual activity during COVID-19 self-isolation/social distancing in a sample of the UK public. Methods This paper presents preplanned interim analyses of data from a cross-sectional epidemiological study, administered through an online survey. Outcomes Sexual activity was measured using the following question: “On average after self-isolating how many times have you engaged in sexual activity weekly?” Demographic and clinical data were collected, including sex, age, marital status, employment, annual household income, region, current smoking status, current alcohol consumption, number of chronic physical conditions, number of chronic psychiatric conditions, any physical symptom experienced during self-isolation, and number of days of self-isolation/social distancing. The association between several factors (independent variables) and sexual activity (dependent variable) was studied using a multivariable logistic regression model. Results 868 individuals were included in this study. There were 63.1% of women, and 21.8% of adults who were aged between 25 and 34 years. During self-isolation/social distancing, 39.9% of the population reported engaging in sexual activity at least once per week. Variables significantly associated with sexual activity (dependent variable) were being male, a younger age, being married or in a domestic partnership, consuming alcohol, and a higher number of days of self-isolation/social distancing. Clinical Implications In this sample of 868 UK adults self-isolating owing to the COVID-19 pandemic, the prevalence of sexual activity was lower than 40%. Those reporting particularly low levels of sexual activity included females, older adults, those not married, and those who abstain from alcohol consumption. Strength and Limitations This is the first study to investigate sexual activity during the UK COVID-19 self-isolation/social distancing. Participants were asked to self-report their sexual activity potentially introducing self-reporting bias into the findings. Second, analyses were cross-sectional and thus it is not possible to determine trajectories of sexual activity during the current pandemic. Conclusion Interventions to promote health and well-being during the COVID-19 pandemic should consider positive sexual health messages in mitigating the detrimental health consequences in relation to self-isolation/social distancing and should target those with the lowest levels of sexual activity.

ObjectiveTo investigate the levels and correlates of physical activity during COVID-19 social distancing in a sample of the UK public.MethodsThis paper presents analyses of data from a cross-sectional study. Levels of physical activity during COVID-19 social distancing were self-reported. Participants also reported on sociodemographic and clinical data. The association between several factors and physical activity was studied using regression models.ResultsNine hundred and eleven adults were included (64.0% were women and 50.4% of the participants were aged 35–64 years). 75.0% of the participants met the physical activity guidelines during social distancing. Meeting these guidelines during social distancing was significantly associated with sex (reference: male; female: OR=1.60, 95% CI 1.10 to 2.33), age (reference: 18–34 years; ≥65 years: OR=4.11, 95% CI 2.01 to 8.92), annual household income (reference: ConclusionDuring COVID-19, social distancing interventions should focus on increasing physical activity levels among younger adults, men and those with low annual household income. It should be noted in the present sample that women and younger adults are over-represented.

Background ALM201 is a therapeutic peptide derived from FKBPL that has previously undergone preclinical and clinical development for oncology indications and has completed a Phase 1a clinical trial in ovarian cancer patients and other advanced solid tumours. Methods In vitro, cancer stem cell (CSC) assays in a range of HGSOC cell lines and patient samples, and in vivo tumour initiation, growth delay and limiting dilution assays, were utilised. Mechanisms were determined by using immunohistochemistry, ELISA, qRT-PCR, RNAseq and western blotting. Endogenous FKBPL protein levels were evaluated using tissue microarrays (TMA). Results ALM201 reduced CSCs in cell lines and primary samples by inducing differentiation. ALM201 treatment of highly vascularised Kuramochi xenografts resulted in tumour growth delay by disruption of angiogenesis and a ten-fold decrease in the CSC population. In contrast, ALM201 failed to elicit a strong antitumour response in non-vascularised OVCAR3 xenografts, due to high levels of IL-6 and vasculogenic mimicry. High endogenous tumour expression of FKBPL was associated with an increased progression-free interval, supporting the protective role of FKBPL in HGSOC. Conclusion FKBPL-based therapy can (i) dually target angiogenesis and CSCs, (ii) target the CD44/STAT3 pathway in tumours and (iii) is effective in highly vascularised HGSOC tumours with low levels of IL-6.

Background Optimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease. Methods In this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett’s multiple comparison test. Results We demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p

The autism spectrum is a neurodevelopmental disability covering a wide range of abilities and complexities [1]. The individuals experience varying levels of difficulties in communication, social, sensory regulation skills and engage in repetitive behaviours. Autism can also be accompanied by intellectual disability (ID) and or other comorbidities. Individuals on the autism spectrum often encounter difficulties with reading and comprehension and students with a co-occurring intellectual disability (ID) acquire limited reading skills by the end of their school education. The ability to read influences not only the academic success but impacts on social and communication aspects and acquisition of literacy skills can have long-term impacts on individuals' quality of life. Given the wide range of strengths and challenges experienced by individuals with autism and co-occurring ID, it is necessary to develop individualised intervention. Here we discuss a user-centred, serious game based, reading intervention package [2], using touch screen device and the online available Headsprout® reading program [3] and reading assessment tool [4] to suit students with autism and moderate/severe ID.

Individuals with autism and intellectual disability (ID) have complex learning needs and often have difficulty in acquiring reading comprehension skills using conventional teaching tools. Evidence based reading interventions for these learners and the use of assistive technology and application of behaviour analysis to develop user-centered teaching is discussed in this paper.

Games and virtual worlds have many potential benefits for people with intellectual disabilities (ID) and autism spectrum disorder (ASD), in terms of training, education, and rehabilitation. However, because this population presents a wide range of specific needs and abilities, it can be difficult to design games which are engaging and present optimum levels of challenge to players. By including individuals with ID and ASD in the design phase we can help meet their specific needs and preferences by personalizing an intervention through the exploration of experimental techniques, methods and assistive technologies. By embracing the Responsible Research and Innovation approach, we bring science and society closer together to shape the world for future generations. A number of approaches for achieving such inclusion have been described, such as User Sensitive Inclusive Design, Universal Design, and Design for All. Here we discuss three specific examples of the design of games and virtual worlds for people with ID/ASD and illustrate how they attempt to meet their needs. Namely 1) a blended approach of computerised program and applied behaviour analysis for reading skills 2) immersive gameplay for employment and transferable skills training and 3) virtual reality training to enhance communication skills.

FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identified a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-β-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-α (ERα). Furthermore, FKBPL and RBCK1 associate with ERα at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.

FKBPs (FK506-binding proteins) have long been recognized as key regulators of the response to immunosuppressant drugs and as co-chaperones of steroid receptor complexes. More recently, evidence has emerged suggesting that this diverse protein family may also represent cancer biomarkers owing to their roles in cancer progression and response to treatment. FKBPL (FKBP-like) is a novel FKBP with roles in GR (glucocorticoid receptor), AR (androgen receptor) and ER (oestrogen receptor) signalling. FKBPL binds Hsp90 (heat-shock protein 90) and modulates translocation, transcriptional activation and phosphorylation of these steroid receptors. It has been proposed as a novel prognostic and predictive biomarker, where high levels predict for increased recurrence-free survival in breast cancer patients and enhanced sensitivity to endocrine therapy. Since this protein family has roles in a plethora of signalling pathways, its members represent novel prognostic markers and therapeutic targets for cancer diagnosis and treatment.

FKBPL is a secreted protein with well-established anti-angiogenic and anti-cancer stem cell activity. A novel therapeutic peptide, ALM201, derived from this protein has successfully completed a phase I clinical trial and was granted orphan drug status, by the FDA, for the treatment of ovarian cancer. Ovarian cancer is the most lethal gynecological cancer and there are no prognostic biomarkers currently used in the clinic. It is associated with a high incidence of recurrent chemo-resistant disease and this has been attributed to a treatment-resistant subpopulation of CD44+ cancer stem cells (CSCs). Previously, we presented evidence that CD44 is a potential target of ALM201 and here, for the first time, we investigate its ability to target both angiogenesis and CSCs in HGSOC. Tumorsphere and flow cytometry assays demonstrated that ALM201 is effective at reducing CSCs in a range of HGSOC cell lines and primary patient samples in vitro. Clonogenic assays demonstrated that ALM201 is not cytotoxic, but rather mediates ovarian CSC differentiation. In vitro, ALM201 displayed potent anti-CSC activity in the HGSOC cell line, OVCAR3. However, OVCAR3 tumor xenografts are not dependent on angiogenesis for their growth and therefore ALM201 did not elicit a strong response in this model. Furthermore, OVCAR3 xenografts dramatically upregulated the inflammatory cytokines IL-6 and IL-8, which is consistent with the lack of anti-CSC activity of ALM201 in vivo compared to in vitro. In contrast, treatment of highly angiogenic Kuramochi tumor xenografts, another HGSOC cell line, resulted in a statistically significant delay in both tumor growth and tumor initiation. This was associated with disruption of the CD31+ vascular network and a 10 fold decrease in the CSC population. Finally, using HGSOC tissue microarrays we demonstrated that high endogenous expression of FKBPL correlated with increased progression free interval; indicating the potential for FKBPL to be used as a prognostic biomarker in HGSOC. HGSOC cell lines display marked differences in tumor vascularisation in vivo. ALM201, a peptide fragment of FKBPL, has potent anti-CSC and anti-angiogenic activity in an HGSOC xenograft which is dependent on angiogenesis for growth; an important finding for the clinical development of ALM201. Our data also strongly suggest that FKBPL is a potential novel prognostic biomarker in HGSOC, supporting its endogenous anti-tumor activity. Citation Format: Stephanie Annett, Gillian Moore, Amy Short, Neermeen Moustafa, Sudipto Das, Darran O'Connor, Cian McCrudden, Adrien Kissenpfenning, Laura Nelson, Ian Harley, Ken Arthur, Anita Yakkundi, Glen McCluggage, Andrea Marshall, Fiona Furlong, Helen O. McCarthy, Graham Cotton, Timothy Harrison, Lana McClements, Tracy Robson. FKBPL as a novel therapeutic target and prognostic biomarker in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-054.

The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor α (ERα) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERα phosphorylation on Ser118 in response to 17β-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome. Cancer Res; 70(3); 1090–100

Aims: RALA is a novel 30 mer bioinspired amphipathic peptide that is showing promise for gene delivery. Here, we used RALA to deliver the FK506-binding protein like – FKBPL gene (pFKBPL) – a novel member of the immunophilin protein family. FKBPL is a secreted protein, with overexpression shown to inhibit angiogenesis, tumor growth and stemness, through a variety of intra- and extracellular signaling mechanisms. We also elucidated proangiogenic activity and stemness after utilizing RALA to deliver siRNA (siFKBPL). Materials & methods: The RALA/pFKBPL and RALA/siFKBPL nanoparticles were characterized in terms of size, charge, stability and toxicity. Overexpression and knockdown of FKBPL was assessed in vitro and in vivo. Results: RALA delivered both pFKBPL and siFKBPL with less cytotoxicity than commercially available counterparts. In vivo, RALA/pFKBPL delivery retarded tumor growth, and prolonged survival with an associated decrease in angiogenesis, while RALA/siFKBPL had no effect on tumor growth rate or survival, but resulted in an increase in angiogenesis and stemness. Conclusion: RALA is an effective delivery system for both FKBPL DNA and RNAi and highlights an alternative therapeutic approach to harnessing FKBPL's antiangiogenic and antistemness activity.

Peptidyl prolyl isomerases (PPIases) are proteins belonging to the immunophilin family and are characterised by their cis-trans isomerization activity at the X-Pro peptide bond, in addition to their tetratricopeptide repeat (TPR) domain, important for interaction with the molecular chaperone, Hsp90. Due to this unique structure these proteins are able to facilitate protein-protein interactions which can impact significantly on a range of cellular processes such as cell signalling, differentiation, cell cycle progression, metabolic activity and apoptosis. Malfunction and/or dysregulation of most members of this class of proteins promotes cellular damage and tissue/organ failure, predisposing to ageing and age-related diseases. Many individual genes within the PPIase family are associated with several age-related diseases including cardiovascular diseases (CVDs), atherosclerosis, type II diabetes mellitus (T2D), chronic kidney disease (CDK), neurodegeneration, cancer and age-related macular degeneration (AMD), in addition to the ageing process itself. This review will focus on the different roles of PPIases, and their therapeutic/ biomarker potential in these age-related vascular diseases.

FK506-binding protein-like (FKBPL) has established roles as an anti-tumor protein, with a therapeutic peptide based on this protein, ALM201, shortly entering phase I/II clinical trials. Here, we evaluated FKBPL's prognostic ability in primary breast cancer tissue, represented on tissue microarrays (TMA) from 3277 women recruited into five independent retrospective studies, using immunohistochemistry (IHC). In a meta-analysis, FKBPL levels were a significant predictor of BCSS; low FKBPL levels indicated poorer breast cancer specific survival (BCSS) (hazard ratio (HR) = 1.30, 95% confidence interval (CI) 1.14-1.49, p < 0.001). The prognostic impact of FKBPL remained significant after adjusting for other known prognostic factors (HR = 1.25, 95% CI 1.07-1.45, p = 0.004). For the sub-groups of 2365 estrogen receptor (ER) positive patients and 1649 tamoxifen treated patients, FKBPL was significantly associated with BCSS (HR = 1.34, 95% CI 1.13-1.58, p < 0.001, and HR = 1.25, 95% CI 1.04-1.49, p = 0.02, respectively). A univariate analysis revealed that FKBPL was also a significant predictor of relapse free interval (RFI) within the ER positive patient group, but it was only borderline significant within the smaller tamoxifen treated patient group (HR = 1.32 95% CI 1.05-1.65, p = 0.02 and HR = 1.23 95% CI 0.99-1.54, p = 0.06, respectively). The data suggests a role for FKBPL as a prognostic factor for BCSS, with the potential to be routinely evaluated within the clinic.

Supplemental Digital Content is available in the text., Objective— The antitumor effects of FK506-binding protein like (FKBPL) and its extracellular role in angiogenesis are well characterized; however, its role in physiological/developmental angiogenesis and the effect of FKBPL ablation has not been evaluated. This is important as effects of some angiogenic proteins are dosage dependent. Here we evaluate the regulation of FKBPL secretion under angiogenic stimuli, as well as the effect of FKBPL ablation in angiogenesis using mouse and zebrafish models. Approach and Results— FKBPL is secreted maximally by human microvascular endothelial cells and fibroblasts, and this was specifically downregulated by proangiogenic hypoxic signals, but not by the angiogenic cytokines, VEGF or IL8. FKBPL’s critical role in angiogenesis was supported by our inability to generate an Fkbpl knockout mouse, with embryonic lethality occurring before E8.5. However, whilst Fkbpl heterozygotic embryos showed some vasculature irregularities, the mice developed normally. In murine angiogenesis models, including the ex vivo aortic ring assay, in vivo sponge assay, and tumor growth assay, Fkbpl+/− mice exhibited increased sprouting, enhanced vessel recruitment, and faster tumor growth, respectively, supporting the antiangiogenic function of FKBPL. In zebrafish, knockdown of zFkbpl using morpholinos disrupted the vasculature, and the phenotype was rescued with hFKBPL. Interestingly, this vessel disruption was ineffective when zcd44 was knocked-down, supporting the dependency of zFkbpl on zCd44 in zebrafish. Conclusions— FKBPL is an important regulator of angiogenesis, having an essential role in murine and zebrafish blood vessel development. Mouse models of angiogenesis demonstrated a proangiogenic phenotype in Fkbpl heterozygotes.

Immunohistochemical studies on formalin-fixed, paraffin-embedded (FFPE) tissue utilizing polyclonal antibodies form the cornerstone of many reports claiming to demonstrate erythropoietin receptor (EPOR) expression in malignant tissue. Recently, Elliott et al. (Blood 2006;107:1892–1895) reported that the antibodies commonly used to detect EPOR expression also detect non-EPOR proteins, and that their binding to EPOR was severely abrogated by two synthetic peptides based on the sequence of heat shock protein (HSP) 70, HSP70-2, and HSP70-5. We have investigated the specificity of the C20 antibody for detecting EPOR expression in non-small cell lung carcinoma (NSCLC) utilizing tissue microarrays. A total of 34 cases were available for study. Antibody absorbed with peptide resulted in marked suppression of cytoplasmic staining compared with nonabsorbed antibody. Four tumors that initially showed a membranous pattern of staining retained this pattern with absorbed antibody. Positive membranous immunoreactivity was also observed in 6 of 30 tumors that originally showed a predominantly cytoplasmic pattern of staining. Using the C20 antibody for Western blots, we detected three main bands, at 100, 66, and 59 kDa. Preincubation with either peptide caused abolition of the 66-kDa band, which contains non-EPOR sequences including heat shock peptides. These results call into question the significance of previous immunohistochemical studies of EPOR expression in malignancy and emphasize the need for more specific anti-EPOR antibodies to define the true extent of EPOR expression in neoplastic tissue.

Drug metabolizing transgene products, which activate bioreductive cytotoxins, can be used to target treatment-resistant hypoxic tumors. The prodrug AQ4N is bioreduced in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Previously we have shown that intra-tumoral injection of CYP3A4 and CYP2B6 transgenes with AQ4N and radiation inhibits tumor growth. Here we examine the ability of other CYPs, in particular CYP1A1, to metabolize AQ4N, and to enhance radiosensitization. Metabolism of AQ4N was assessed using microsomes prepared from baculovirus-infected cells transfected with various CYP isoforms. AQ4N metabolism was most efficient with CYP1A1 (66.7 nmol/min/pmol) and 2B6 (34.4 nmol/min/pmol). Transient transfection of human CYP1A1+/-CYP reductase (CYPRED) was investigated in hypoxic RIF-1 mouse cells in vitro using the alkaline comet assay. There was a significant increase in DNA damage following transient transfection of CYP1A1 compared to non-transfected cells; inclusion of CYPRED provided no additional effect. In vivo, a single intra-tumoral injection of a CYP1A1 construct in combination with AQ4N (100 mg/kg i.p.) and 20 Gy X-rays caused a 16-day delay in tumor regrowth compared to tumors receiving AQ4N plus radiation and empty vector (P=0.0344). The results show the efficacy of a CYP1A1-mediated GDEPT strategy for bioreduction of AQ4N.

Background AQ4N is metabolised in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Most solid tumours are known to contain regions of hypoxia whereas levels of CYPs have been found to vary considerably. Enhancement of CYP levels may be obtained using gene-directed enzyme prodrug therapy (GDEPT). We have therefore examined the potential of a CYP2B6-mediated GDEPT strategy to enhance the anti-tumour effect of the combination of AQ4N with radiation or cyclophosphamide (CPA). Methods In vitro and in vivo transient transfection of human CYP2B6 +/- CYP reductase (CYPRED) was investigated in RIF-1 mouse tumours. Efficacy in vitro was assessed using the alkaline comet assay (ACA). In vivo, the time to reach 4x the treatment volume (quadrupling time; VQT) was used as the end point. Results When CYP2B6 was transfected into RIF-1 cells and treated with AQ4N under hypoxic conditions there was a significant increase in DNA damage (measured by the ACA) compared with non-transfected cells. In vivo, a single intra-tumoural injection of a CYP2B6 vector construct significantly enhanced tumour growth delay in combination with AQ4N (100 mg/kg) and 10 Gy X-rays. AQ4N (100 mg/kg) and CPA (100 mg/kg) with CYP2B6 and CYPRED also enhanced tumour growth delay; this effect became significant when the schedule was repeated 14 days later (p = 0.0197). Conclusions The results show the efficacy of a CYP2B6-mediated GDEPT strategy for bioreduction of AQ4N; this may offer an additional approach to target radiation- and chemo-resistant hypoxic tumours that should enhance overall tumour control. Copyright (c) 2005 John Wiley & Sons, Ltd.

The bgl operon of Escherichia coli, which encodes the genes necessary for transport and catabolism of beta-glucosides, is silent in wild-type cells and is activated by the transposition of IS elements. The silent form of the operon appears to be the stable state. We isolated Bgl(-) revertants of an activated strain after growth under nonselective conditions to understand whether activation of the cryptic operon by IS elements is reversible. Genetic and molecular analyses revealed that a majority of revertants carry deletions of the bgl structural genes, indicating that an irreversible alteration has occurred in the operon. Implications of these results for the evolution and maintenance of cryptic genes are discussed.

Purpose: FK506-binding protein like (FKBPL) and its peptide derivative, AD-01, have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here, we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC). Experimental Design: Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples, and xenografts. Delays in tumor initiation were evaluated in vivo. The anti–stem cell mechanisms were determined using clonogenic assays, quantitative PCR (qPCR), and immunofluorescence. Results: AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESA+/CD44+/CD24− or aldehyde dehydrogenase (ALDH)+ cell subpopulations in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01–mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4, and Sox2, were also significantly reduced. Furthermore, we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy-induced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signaling. Conclusions: AD-01 has dual antiangiogenic and anti-BCSC activity, which will be advantageous as this agent enters clinical trial. Clin Cancer Res; 19(14); 3881–93. ©2013 AACR.

FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity in vitro and in vivo and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development in vivo suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks. © 2013 Yakkundi et al.

Purpose: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action. Experimental Design: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status. Results: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype. Conclusion: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy. Clin Cancer Res; 17(5); 1044–56. ©2011 AACR.

Approximately 40% of patients with oestrogen receptor (ER)-positive breast cancers do not respond to endocrine therapies; furthermore, most responsive tumours eventually become resistant. We have identified a novel oestrogen-responsive Hsp90 co-chaperone and immunophilin, FKBPL, which affects the stability and signalling of ER with implications for breast cancer growth and sensitivity to endocrine therapies. MCF7 cells stably overexpressing FKBPL demonstrate a slower rate of proliferation and become highly dependent on oestrogen for their growth. This dependence on oestrogen renders these cells dramatically more sensitive to tamoxifen and fulvestrant. FKBPL overexpressing cells also exhibit decreased levels of ER and an oestrogen-responsive gene, cathepsin D, critical for breast cancer growth, survival and invasion. Moreover, knockdown of FKBPL using a targeted siRNA approach dramatically increases both ER and cathepsin D protein levels and cell resistance to tamoxifen. FKBPL has been previously implicated in the stabilisation of the cyclin-dependent kinase inhibitor, p21 [1]. Loss of p21 has been associated with a tamoxifen growth-inducing phenotype and hyperphosphorylation of ER at Ser118, with increased expression of ER-regulated genes. Following FKBPL knockdown, we have observed a fall in p21 levels and subsequent increase in Ser118 phosphorylation following treatment with 17β-estradiol or tamoxifen while FKBPL overexpressing cells exhibit the reverse effects. Our in vitro data support a model in which high levels of FKBPL would stabilise p21, decrease ER phosphorylation and abrogate tamoxifen-induced agonist potency, thereby increasing drug sensitivity, and suggest that FKBPL may have prognostic value that might impact upon tumour proliferative capacity and improve patient outcome. In addition, analysis of two publically available breast cancer microarray patient cohorts demonstrated that high FKBPL expression was correlated with increased overall and distant metastasis-free survival.

The bioreductive drug, AQ4N, is metabolized under hypoxic conditions and has been shown to enhance the antitumor effects of radiation and chemotherapy drugs. We have investigated the role of cytochrome P450 3A4 (CYP3A4) in increasing the metabolism of AQ4N using a gene-directed enzyme prodrug therapy (GDEPT) strategy. RIF-1 murine tumor cells were transfected with a mammalian expression vector containing CYP3A4 cDNA. In vitro AQ4N metabolism, DNA damage, and clonogenic cell kill were assessed following exposure of transfected and parental control cells to AQ4N. The presence of exogenous CYP3A4 increased the metabolism of AQ4N and significantly enhanced the ability of the drug to cause DNA strand breaks and clonogenic cell death. Cotransfection of CYP reductase with CYP3A4 showed a small enhancement of the effect in the DNA damage assay only. A single injection of CYP3A4 into established RIF-1 murine tumors increased the metabolism of AQ4N, and when used in combination with radiation, three of nine tumors were locally controlled for >60 days. This is the first demonstration that CYPs alone can be used in a GDEPT strategy for bioreduction of the cytotoxic prodrug, AQ4N. AQ4N is the only CYP-activated bioreductive agent in clinical trials. Combination with a GDEPT strategy may offer a further opportunity for targeting radiation-resistant and chemo-resistant hypoxic tumor cells.

Hsp90 chaperone complexes are involved in maintaining the stability and signalling of Hsp90 client proteins such as the oestrogen receptor (ER). The ER is the primary mediator of breast cancer proliferation in response to oestrogen. Since increased ER levels and transcriptional activation are associated with over 50% of breast cancers, the ER is an attractive target for cancer treatment strategies. Hsp90 inhibitors such as 17AAG are known to destabilize these complexes by promoting proteasome-mediated degradation of the steroid hormone receptor leading to tumour growth inhibition [1] and sensitization to chemotherapy [2] and radiotherapy [3]. Using protein interaction assays, we have identified FKBPL, a novel gene that codes for an immunophilin-like protein, as an Hsp90 cochaperone associated with the ER and dynein motor protein complex. Overexpression studies have demonstrated that FKBPL modulates ER signalling and affects breast cancer growth and survival. Since most tumours become refractory to current hormonal therapies within a year of starting treatment, FKBPL represents a novel drug target that would enable the disruption of signalling pathways integral in maintaining ER-mediated tumour growth and survival.