First-generation bromodomain extra-terminal protein (BETP) inhibitors (BETi)  that disrupt binding of BETP BRD4 to chromatin transcriptionally attenuate AML-relevant progrowth and prosurvival oncoproteins. BETi treatment induces apoptosis of AML BPCs, reduces in vivo AML burden and induces clinical remissions in a minority of AML patients. Clinical efficacy of more potent BETis, e.g., ABBV-075 , is being evaluated. Venetoclax and A-1210477 bind and inhibit the antiapoptotic activity of BCL2 and MCL1, respectively, lowering the threshold for apoptosis. BETi treatment is shown here to perturb accessible chromatin and activity of enhancers/promoters, attenuating MYC, CDK6, MCL1 and BCL2, while inducing BIM, HEXIM1, CDKN1A expressions and apoptosis of AML cells. Treatment with venetoclax increased MCL1 protein levels, but cotreatment with ABBV-075 reduced MCL1 and Bcl-xL levels. ABBV-075 cotreatment synergistically induced apoptosis with venetoclax or A-1210477 in patient-derived, CD34+ AML cells. Compared to treatment with either agent alone, cotreatment with ABBV-075 and venetoclax was significantly more effective in reducing AML cell-burden and improving survival, without inducing toxicity, in AML-engrafted immune-depleted mice. These findings highlight the basis of superior activity and support interrogation of clinical efficacy and safety of cotreatment with BETi and BCL2 or MCL1 inhibitor in AML. BRD4 promotes pTEFb-mediated phosphorylation of serine 2 in the heptad repeats within the CTD of RNAP2, as well as of the negative transcription elongation factors, NELF and Sept5, which induces promoter-proximal pause release of RNAP2 and RNA transcript elongation6. This has been shown to occur at the enhancers and promoters of oncogenes that promote growth and survival of cancer cells, including acute myeloid leukemia (AML) stem-progenitor cells9. Consistent with this, knockdown of BRD4 by RNAi, or disruption of its binding to acetylated chromatin by BET inhibitors (BETi) leads to lethality in AML blast progenitor cells (BPCs), associated with down regulation of AML-relevant progrowth and prosurvival oncogenes13. BETis, including JQ1 and OTX015, have been documented to reduce AML burden and improve survival of mice engrafted with human AML BPCs13. Whereas treatment with BETi was shown to induce clinical responses in AML, refractoriness to BETi therapy and resistance with disease progression is uniformly observed16. This has prompted the development and testing of more potent and effective BETis, e.g., ABBV-07520. Since BETi treatment attenuated expressions of several BCL2 family of antiapoptotic proteins21, to further lower the threshold for apoptosis and enhance clinical anti-AML efficacy of BETi, a logical approach would be to concomitantly target and inhibit activity of the antiapoptotic proteins.The bromodomain extra-terminal (BET) protein (BETP) BRD4 interacts with transcription factors as well as cofactors, including mediator protein complex, lysine methyltransferase NSD3, arginine demethylase JMJD6, and pTEFb (a heterodimer of CDK9 and cyclin T), to regulate RNA pol II (RNAP2)-mediated transcript elongation23. They bind proapoptotic BCL2 family members BAX and BAK  and BH3 domain-only proapoptotic activator proteins, to inhibit intrinsic mitochondria-induced pathway of apoptosis24. The first, highly selective BCL2 inhibitor venetoclax (ABT-199) binds specifically to BCL2 and displaces BH3 domain-only proteins to trigger BAX/BAK-mediated mitochondria-induced apoptosis of cancer, including AML cells26. Venetoclax treatment alone showed anti-AML in vivo efficacy in the mouse xenograft models27. Although effective in inducing clinical remissions in AML, innate or acquired resistance to venetoclax alone is commonly observed28. The best predictor of sustained response to venetoclax is the lack of readily accessible resistance mechanisms provided by Bcl-xL and MCL128. In venetoclax-resistant cells, increased MCL1 and/or Bcl-xL levels was observed29. Preclinically, dual targeting of BCL2 and MCL1, but not either alone, was also shown to prolong survival of AML or lymphoma bearing mice31. Combining venetoclax with other anti-AML drugs such as cytarabine or DNA hypomethylating agent has yielded higher remission rates33. However, a full assessment of their clinical efficacy has not been conducted. In present studies we determined the effects of the BETi on cis-regulatory DNA elements and on mRNA and protein expressions of AML-relevant oncoproteins, including MYC, BCL2, Bcl-xL, MCL1, and CDK6. We also determined whether the BETi ABBV-075-induced perturbations in protein levels of these oncoproteins are associated with synergistic in vitro activity of ABBV-075 and venetoclax or the MCL1 inhibitor A-1210477. Finally, we also determined whether combination of ABBV-075 and venetoclax exerted superior in vivo efficacy against mouse xenograft models of AML BPCs.BCL2, Bcl-xL, and MCL1 are members of multi-BCL-2 homology (BH) domain (BH1−BH4) containing family of antiapoptotic proteinsIn vivo grade ABBV-075 and ABT-199 for mouse xenograft experiments were kindly provided by AbbVie, Inc. In vitro grade A-1210477 , ABBV-075  and ABT-199  were purchased from Selleck Chemicals  and utilized for in vitro experiments. All compounds were prepared as 10 mM stocks in 100% dimethyl sulfoxide (DMSO) and frozen at −80 °C in 5–10 µL aliquots to allow for single use, thus avoiding multiple freeze-thaw cycles that could result in compound decomposition and loss of activity. Anti-BRD4 (RRID:AB_2620184) antibody was obtained from Bethyl Labs . Anti-c-Myc (RRID: AB_1903938), anti-HEXIM1 (Cat #12604), anti-p21 (RRID: AB_823586), anti-p-Histone H2AX (Ser139) (RRID: AB_2118010), anti-Bcl-xL (RRID: AB_10695729), anti-BAX (RRID:AB_2744530), anti-BAK (RRID:AB_2290287), anti-Cleaved PARP (RRID:AB_331426), anti-BIM (RRID:AB_1030947) and anti-MCL1 (RRID:AB_2281980) antibodies were obtained from Cell Signaling Technologies . Anti-CDK6 (RRID: AB_10610066), anti-Bcl2 (RRID: AB_626733), Alexa488-conjugated anti-BAX(6A7) (RRID:AB_626728) and anti-β-Actin (RRID: AB_626630) antibodies were obtained from Santa Cruz Biotechnologies . Anti-BAK(NT) (RRID:AB_310159) antibody was obtained from Millipore Sigma .Human AML cell lines MOLM13 (RRID:CVCL_2119), SET2 (RRID:CVCL_2187), SKM1 (RRID:CVCL_0098) and OCI-AML5 (RRID:CVCL_1620) were obtained from the DSMZ . MV4-11 cells were obtained from the ATCC . All experiments with cell lines were performed within 6 months after thawing or obtaining from ATCC or DSMZ. The cell lines were also authenticated in the Characterized Cell Line Core Facility at M.D. Anderson Cancer Center, Houston TX. SKM1 and MOLM13 cells were cultured in RPMI media with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin and 1% non-essential amino acids. OCI-AML5 cells were cultured in MEM alpha media with 20% FBS and 1% penicillin/streptomycin and 1% non-essential amino acids. OCI-AML5 cultures were also supplemented with 10 ng/mL of GM-CSF . MV4-11 cells were cultured in ATCC-formulated Iscove's Modified Dulbecco's Medium (IMDM) with 20% FBS 1% penicillin/streptomycin and 1% non-essential amino acids. Luciferase-expressing MOLM13 cells were created by transducing cells with Luc-ZSGreen(GFP). pHIV-Luc-ZsGreen was a gift from Bryan Welm (Addgene plasmid #39196). Logarithmically growing, mycoplasma-negative cells were utilized for all experiments. Following drug treatments, cells were washed free of the drug(s) prior to the performance of the studies described.Patient-derived AML cells samples were obtained with informed consent as part of a clinical protocol approved by the Institutional Review Board of The University of Texas, M.D. Anderson Cancer Center. Normal hematopoietic progenitor cells (HPCs) were obtained from delinked, de-identified cord blood samples. Mononuclear cells were purified by Ficoll Hypaque density centrifugation. Mononuclear cells were washed with complete RPMI media containing 20% FBS. CD34+ AML BPCs and normal HPCs were purified by immunomagnetic beads conjugated with anti-CD34 antibody  prior to utilization in the cell viability assays and immunoblot analyses.MYC, BCL-2, BCL2L1 (Bcl-xL), CDK6, HEXIM1, and CDKN1A (p21) was performed on cDNA using TaqMan probes from Applied Biosystems . Relative mRNA expression was normalized to the expression of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and compared to the untreated cells.Following the designated treatments with ABBV-075, total RNA was isolated from AML cells utilizing a PureLink RNA Mini kit from Ambion, Inc.  and reverse transcribed. Quantitative real-time PCR analysis for the expression of Seventy five micrograms of total cell lysate were used for SDS-PAGE. Western blot analyses were performed on total cell lysates using specific antisera or monoclonal antibodies. Blots were washed with 1× PBST, then incubated in IRDye 680RD goat anti-mouse (RRID:AB_10956588) or IRDye 800CW goat anti-rabbit (RRID:AB_621843) secondary antibodies  for 1 h, washed three times in 1× Phosphate Buffered Saline with Tween®20 (PBST) and scanned with an Odyssey CLX Infrared Imaging System utilizing Image Studio 5.0 Software (RRID:SCR_015795) . The expression levels of β-Actin in the cell lysates were used as the loading control for the western blots. Immunoblot analyses were performed at least twice. Representative immunoblot images were subjected to densitometry analysis.Untreated or drug-treated cells were stained with Annexin V-FITC  and TO-PRO-3 iodide  and the percentages of apoptotic cells were determined by flow cytometry. To analyze in vitro synergism between ABBV-075 and ABT-199 or A-1210477 or synergy between A-1210477 and ABT-199, cells were treated with in vitro grade single agents and combinations for 48 h and the percentages of annexin V-positive, apoptotic cells were determined by flow cytometry. The combination index (CI) for each drug combination was calculated by median dose effect and isobologram analyses  utilizing the commercially available software Compusyn. CI values of less than 1.0 represent a synergistic interaction of the two drugs in the combination. The CI values were input into GraphPad V7.0 to create Box and Whisker plots of the range of the CI values for each cell line and drug combination.(PD)-AML cells were stained with trypan blue . The numbers of nonviable cells were determined by counting the cells that exhibited trypan blue uptake in a hemocytometer, and were reported as a percentage of the untreated control cells. Alternatively, cells were washed with 1× PBS, stained with TO-PRO-3 iodide and analyzed by flow cytometry on an Accuri CFlow6 flow cytometer.Following designated single agent or combination treatments, primary, patient-derived null ; The Jackson Laboratory, Bar Harbor, ME) mice (n = 7) which had received a preconditioning dose of radiation (2.5 Gy) 24 h prior to injection of cells. All the mice were monitored for 4 days and imaged to document engraftment. Mice were randomly assigned to treatment cohorts. Following this, mice were treated daily with vehicle , 50 mg/kg of ABT-199 , 1 mg/kg of ABBV-075  or ABBV-075 + ABT-199 for 2 weeks. Mice were injected with 75 mg/kg of d-Luciferin and imaged once per week by Xenogen camera to monitor disease status and treatment efficacy. Mice that became moribund or experienced hind limb paralysis were euthanized according to the approved IACUC protocol. Investigators were not blinded to the experimental conditions; however, veterinarians and veterinary staff assisting in determining when euthanasia was required were blinded to the experimental conditions of the study. The survival of the mice is represented by a Kaplan-Meier plot. The variance between cohorts was similar. To determine the antileukemia effects of ABBV-075 and/or ABT-199 against human AML patient-derived xenograft (PDX) models, NSG mice (n = 8 per cohort) were injected with patient-derived AML blast cells following a preconditioning dose of radiation. Mice were monitored for engraftment by flow cytometry. Upon engraftment, mice were treated with ABBV-075 and/or ABT-199 for 22 days. At the end of the treatment period, mice were humanely euthanized and the % of engraftment was documented in the mice.All animal studies were performed under a protocol approved by the IACUC at M.D. Anderson Cancer Center, an AAALAC-accredited institution. To determine the in vivo effects of ABT-199 and/or ABBV-075 on leukemia progression and engraftment, 2 million MOLM13/GFP-Luc cells were injected with a 26-gauge needle into the lateral tail vein of 4–6-week-old female NOD-scid IL2Rgammat test. For the in vivo mouse models, a two-tailed t test or a Mantel–Cox Rank sum test was utilized for group comparisons. p values of <0.05 were assigned significance.Significant differences between values obtained in a population of AML cells treated with different experimental conditions were determined using the Student’s cis-regulatory regions of genes, including MYC, Bcl-xL (BCL2L1), MCL1, BIM (BCL2L11), HEXIM1/2, PIM1, BAD, CDKN1A, ITGAM (CD11b), and CEBPα/β, while reducing chromatin accessibility at cis-regulatory regions of other genes, including IL7 receptor (IL7R), MYB, and CDK6  mRNA levels were induced, mRNA levels of MYC, CDK6, BCL2L1 (Bcl-xL), BCL2, and PIM1 were downregulated. We next confirmed by qPCR analysis whether the more potent BETi ABBV-075 induces similar mRNA perturbations in AML cells, including patient-derived (PD) CD34+ AML BPCs. As shown in Fig. MYC, BCL2, Bcl-xL, and CDK6, while inducing HEXIM1 and p21 mRNA levels in MV4-11, OCI-AML5, MOLM13, and PD CD34+ AML BPCs. Genetic alterations detected by NextGen sequencing (NGS) of an AML-associated 28-gene panel, conducted in the AML cell lines, is presented in Fig. p < 0.05). Following ABBV-075 treatment, similar effects on the protein levels were observed in three samples of PD CD34+ AML BPCs, including a decline in the levels of MCL1 , as shown in the Kaplan-Meier plot depicting the survival of the mice  and the ensuing execution phase of apoptosis in AML BPCslls Fig. 29,30. Th-xL Fig. 29–31,42,35. Stability of MCL1 is controlled by ubiquitylation by the E3 ligase MULE (MCL1 ubiquitin ligase E3) and deubiquitinase USP9X53. By displacing BH3 domain-containing MULE from MCL1, A-1210477 is known to stabilize and increase MCL1 levels35. However, as shown here and previously in other reports, treatment with BETi, including ABBV-075, inhibits RNAP2-mediated transcription and levels of MCL1 and Bcl-xL, while simultaneously inducing BIM levels46. Together these perturbations due to superior lethal activity of cotargeting MCL1 and BCL2 is also underscored here by our findings that cotreatment with A-1210477 and venetoclax induces synergistic lethal effects against AML BPCs. Similar to the effects of ABBV-075, previous reports have also documented that cotreatment with CDK9 inhibitor that transcriptionally reduces short-lived MCL1 levels also enhances lethal effects of BCL2 inhibition54.Consistent with previous reports, our findings also show that A-1210477 treatment increased MCL1 levels32, cytarbine and idarubicin30, which induce mitochondrial priming by inducing the expressions of the BH3 domain-only proapoptosis proteins such as BIM, NOXA, and PUMA in a p53-dependent or independent manner56. Combinations of venetoclax with novel anti-AML agents include inhibitors targeting MEK, PI3K/mTOR, NEDD8, HDM2, and IDH1/259. There is evidence to suggest that venetoclax may selectively target AML stem cells that depend on mitochondrial oxidative phosphorylation60. If so, how combining each of these novel agents modulates this important activity of venetoclax would have to be evaluated in future studies.Several recent reports have also presented evidence for superior activity of combining venetoclax with other conventional or novel anti-AML agents, besides the BETi described here, that further reduce the threshold for apoptosis in AML BPCs. These include combinations with standard anti-AML agents, e.g., azacytidineSupplemental Data FiguresSupplemental Figure LegendsExtended and Supplemental Methods


We previously demonstrated that H2O2‐induced endothelial nitric oxide synthase (eNOS) activation and excessive NO production contribute to vascular cell injury and increases in microvessel permeability. However, the mechanisms of excessive NO‐mediated vascular injury and hyperpermeability remain unknown. This study aims to examine the functional role of NO‐derived peroxynitrite (ONOO−) in H2O2‐induced vascular barrier dysfunction by elucidating the interrelationships between H2O2‐induced NO, superoxide, ONOO−, and changes in endothelial [Ca2+]i and microvessel permeability. Experiments were conducted on intact rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca2+]i, NO, and O2− were assessed with fluorescence imaging. Perfusion of vessels with H2O2 (10 µmol/L) induced marked productions of NO and O2−, resulting in extensive protein tyrosine nitration, a biomarker of ONOO−. The formation of ONOO− was abolished by inhibition of NOS with NG‐Methyl‐L‐arginine. Blocking NO production or scavenging ONOO− by uric acid prevented H2O2‐induced increases in endothelial [Ca2+]i and Lp. Additionally, the application of exogenous ONOO− to microvessels induced delayed and progressive increases in endothelial [Ca2+]i and microvessel Lp, a pattern similar to that observed in H2O2‐perfused vessels. Importantly, ONOO− caused further activation of eNOS with amplified NO production. We conclude that the augmentation of NO‐derived ONOO− is essential for H2O2‐induced endothelial Ca2+ overload and progressively increased microvessel permeability, which is achieved by self‐promoted amplifications of NO‐dependent signaling cascades. This novel mechanism provides new insight into the reactive oxygen and/or reactive nitrogen species‐mediated vascular dysfunction in cardiovascular diseases.Elevated H We first measured H2O2‐induced changes in EC [Ca2+]i, NO production, and microvessel Lp. Under the same experimental conditions, H2O2‐induced O2− production was assessed using dihydroethidium. The formation of ONOO− was examined by fluorescent immunostaining of nitrotyrosine, a biomarker of ONOO−, in H2O2‐perfused vessels. The functional relationship between ONOO−, and changes in EC [Ca2+]i and microvessel permeability was further investigated using uric acid, an endogenous ONOO− scavenger. The direct effects of exogenously applied ONOO− on EC [Ca2+]i, NO production, as well as microvessel Lp, were also examined.This study is designed to examine the functional roles of ONOOAll animal experiments were conducted on female Sprague–Dawley rats . All procedures and animal use were approved by the Animal Care and Use Committee at West Virginia University. Rats were anesthetized with pentobarbital sodium (65 mg/kg body wt) administered subcutaneously. A midline surgical incision (1.5–2 cm) was made in the abdominal wall and the mesentery was gently taken out from the abdominal cavity and spread over a glass coverslip attached to an animal tray for studies. The upper surface of the mesentery was continuously superfused with mammalian Ringer's solution at 37°C. Each experiment was performed on one microvessel per animal.2O), controlled by a water manometer, is applied through the micropipette to the vessel lumen, which allows the perfusate to continuously flow through the vessel. For each measurement, the perfused vessel was occluded briefly downstream with a glass rod. The initial water flux/unit area of microvessel wall (Jv/A) was calculated from the velocity of the marker cell after vessel occlusion, the vessel radius, and the distance between the marker cell and the occlusion site. Lp was calculated as the slope of the relationship between Jv/A and the pressure difference across the vessel wall. In each experiment, the baseline Lp and the Lp after the application of testing solutions were measured in the same vessel, and the changes in Lp were expressed as the ratio of Lptest/Lpcontrol. All testing agents were added to the perfusate and delivered into the vessel lumen through the cannulation pipette. To prevent the marker red blood cells from interacting with H2O2 and ONOO− in the perfusate, marker cells were absent during the perfusion period and added back for Lp measurements after designated duration of perfusion.Microvessel permeability was assessed by measuring hydraulic conductivity, Lp, using modified Landis technique, which measures the volume flux of water across the microvessel wall. Details have been described previously , and the emission was separated with a dichroic mirror (DM400) and a wide‐band interference filter . The corresponding FI values  were collected with a 0.25‐sec exposure at each wavelength. Baseline EC [Ca2+]i was measured after fura 2‐AM was removed from the vessel lumen with albumin‐Ringer perfusion for 10 min. Then the same vessel was recannulated with pipette containing test reagents  and the changes in EC [Ca2+]i were measured immediately after the recannulation. At the end of the experiment, the microvessel was superfused with a modified Ringer solution (5 mmol/L of Mn2+ without Ca2+) and perfused with the same solution that contained ionomycin (10 µmol/L) to bleach the Ca2+‐sensitive form of fura 2. The background FI due to unconverted fura 2‐AM and other Ca2+‐insensitive forms of fura 2 were subtracted from FI340 and FI380 values. The ratios of the two FI values were converted to Ca2+ concentrations using an in vitro calibration curve  was used for image acquisition. The excitation wavelength for DAF‐2 DA was selected by an interference filter (480/40 nm), and emission was separated by a dichroic mirror (505 nm) and a band‐pass barrier (535/50 nm). To minimize photo bleaching, a neutral density filter (0.5 N) was positioned in front of the interference filter and the exposure time was minimized to 0.12 sec at 1‐min intervals. All the images were acquired and analyzed using Metafluor software . During the experiment, each vessel was continuously perfused with albumin‐Ringer solution containing DAF‐2 DA (5 µmol/L). NO production was measured after the DAF‐2 loading reached the steady state . The rate of FIDAF change was derived by the first differential conversion of cumulative FIDAF over time. DAF‐2 specificity for NO detection was examined by mixing DAF‐2 (10 µmol/L) with albumin‐Ringer solution, H2O2 , SNP (20 and 200 µmol/L), hypoxanthine/xanthine oxidase , or peroxynitrite  using the same experimental settings as those used for NO measurement on intact vessels. No significant increase in FIDAF‐2 was observed for all the test agents except SNP at 200 µmol/L that caused an accumulative increase in FIDAF‐2, indicating the specificity of DAF‐2 as a NO fluorescent indicator. Peroxynitrite at 500 µmol/L caused a small increase in FIDAF, but that concentration is beyond the range we used in the experiments. The representative measurements with each agent are shown in Fig Levels of endothelial NO were quantified at cellular level in individually perfused microvessels using a fluorescence imaging system and 4,5‐diaminofluorescein diacetate (DAF‐2 DA). The experimental rigs were the same as that used for Ca2O2‐perfused vessels. Images were acquired using Leica objective ×63  with ×1.5 electronic zoom, and the vertical step was 0.3 μm. The mean FI of each stack of ROIs that cover the vessel wall was quantified using Leica confocal software. The averaged FI of the perfused vessels was calculated. The ratio of the FI over control value was used to present the changes in the nitrated tyrosine content. The absolute mean FI in arbitrary unit from each vessel was calculated for DHE images to semi‐quantify the magnitude of superoxide production. Nitrotyrosine immunostaining was conducted in fixed vessels. The mesentery bearing each perfused vessel was fixed with paraformaldehyde, followed by 10‐min permeabilization with 0.1% Triton X‐100 and 1 h blocking with 5% bovine serum before overnight exposure to the mouse Anti‐Nitro tyrosine antibody  at 4°C on an automatic laboratory shaker. The tissue was then washed with BSA‐Ringer solution for three times before incubation with Alexa 488‐conjugated goat anti‐mouse secondary antibody  at room temperature for 3 h. For detection of superoxide, each vessel was loaded with DHE (10 µmol/L) for 30 min followed by perfusion with either BSA‐Ringer alone or containing H2O2 (10 µmol/L) for 30 min. The images were acquired with excitation at 488 nm and emission at 590–700 nm. To determine the source of superoxide, diphenylene iodonium  was applied to the vessel for 20 min after DHE loading, followed by perfusion with H2O2 (10 µmol/L) in the presence of DPI.Leica confocal system (SP2) was used to detect fluorescent immunostaining for nitrotyrosine and superoxide production with dihydroethidium (DHE) in H2, 1.2 MgSO4, 5.5 glucose, 5.0 NaHCO3, 20 N‐2‐hydroxyethylpiperazine‐N’‐2‐ethanesulfonic acid (HEPES), and Na‐HEPES. All perfusates used for control and test perfusion contained BSA (10 mg/ml). Fura‐2 AM was purchased from Invitrogen. H2O2 (30%), DAF‐2, DAF‐2 DA, LaCl3, L‐NMMA, DPI, and DHE were purchased from Sigma. All the fluorescent dyes except for Alexa488‐conjugated secondary antibody (in BSA‐Ringer solution) were prepared in DMSO for stock solution, and at least 1:1000 dilution was made for the final working solutions. ONOO− and degraded ONOO− were purchased from EMD Millipore and were diluted in the BSA‐Ringer solution into final concentrations of 10 or 50 µmol/L. All perfusates containing the test reagents were freshly prepared before each cannulation.Mammalian Ringer solution . Paired t‐test was used for paired data analysis, that is, control and treatment were conducted in the same vessel. Unpaired t‐test with Welch's correction was used for data comparison between two groups, and one‐way or two‐way ANOVA with Tukey post hoc test was applied for multiple group comparison. A probability value of P < 0.05 was considered as statistically significant, and the exact or the range of p values is presented in figure legends.All values are presented as means ± SD. Each “2O2 at pathologically relevant concentrations induced delayed and progressive increases in microvessel Lp, which was initiated by the activation of eNOS and excessive NO production . Since the FIDAF increase in reaction to NO is cumulative , the greater slope of the FIDAF increase indicates a higher NO production rate and the plateau indicates no further increase in NO. When H2O2 (10 µmol/L) was perfused into the microvessel, there was an immediate increase in FIDAF that reached 425 ± 18.1% of the baseline level in 5 min, followed by a lower sustained level. A second increase in FIDAF was observed after 20 min of H2O2 perfusion with 280 ± 41% additional increase in FIDAF, and reached a plateau level at 41 min of H2O2 perfusion with a total of 830 ± 85% increase in FIDAF. In another four DAF‐2 DA‐loaded vessels, perfusion of albumin‐Ringer solution as a control showed approximately 1% increase per minute in FIDAF, representing basal NO production. Preperfusion of vessels with NOS inhibitor, L‐NMMA (2 mmol/L), for 30 min followed by the administration of H2O2 in the presence of L‐NMMA completely abolished H2O2‐induced NO production . Then Lp was measured under the same experimental conditions that NO was measured. The application of L‐NMMA nearly abolished H2O2 (10 µmol/L)‐induced increases in microvessel Lp with decrease in mean Lp from 9.3 ± 1.3 to 1.3 ± 0.16 times of the control after 2‐h perfusion of H2O2 . The mean baseline EC [Ca2+]i was 80 ± 10.5 nmol/L. Perfusion of venules with H2O2 (10 µmol/L) did not cause immediate increase in EC [Ca2+]i. A slow but progressive increase in EC [Ca2+]i occurred after 15 min of H2O2 perfusion and reached a significant increase (207 ± 47.1 nmol/L) from control levels at 36 min of perfusion (p = 0.004), and plateaued at 436 ± 52.7 nmol/L with H2O2 perfusion for ~84 min , prevented H2O2‐induced increase in EC [Ca2+]i. The mean EC [Ca2+]i before and after 2‐hour perfusion of H2O2 in the presence of LaCl3 were 80 ± 10.5 nmol/L and 96 ± 7.2 nmol/L, respectively ‐induced delayed increases in Lp  to be responsible for increased Lp.The effect of H2O2‐induced NO production is immediate and EC [Ca2+]i did not increase until 15 min later, we predict that, if they are related, the delayed increase in EC [Ca2+]i could be the result of the initial large amount of NO production in H2O2‐perfused vessels. To test this relationship, we measured EC [Ca2+]i after blocking NO production using L‐NMMA in four vessels. L‐NMMA (2 mmol/L), without affecting the baseline EC [Ca2+]i (67 ± 7.8 vs. 75 ± 10.8 nmol/L), completely abolished H2O2‐induced increase in EC [Ca2+]i  induced an extensive tyrosine nitration in the cytosol and cell membrane , and the application of L‐NMMA before and during H2O2 perfusion (2 h) reduced the FI to 1.3 ± 0.1 times to that of the control , indicating H2O2‐induced ONOO− formation is NO dependent.To examine whether ONOO−. To examine the role of H2O2‐induced NO in the formation of ONOO−, we examined the intracellular superoxide levels in H2O2‐perfused vessels with DHE staining. As shown in Fig. 2O2 (10 µmol/L), which was abolished by pretreatment of the vessels with an inhibitor of NADPH oxidase, diphenylene iodonium . The mean FIs of DHE were 48 ± 2.5, 141 ± 15.6, and 59 ± 0.5 AU in BSA‐Ringer , H2O2 (n = 5), and H2O2 plus DPI‐perfused vessels (n = 6), respectively .NO reacts quickly with superoxide to form ONOO− accounts for the H2O2‐induced increases in EC [Ca2+]i and microvessel Lp, the effects of H2O2 on EC [Ca2+]i and microvessel Lp were investigated with the application of uric acid, a ONOO− scavenger. Uric acid (50 µmol/L) perfusion for 20 min did not alter the baseline EC [Ca2+]i (69 ± 4.7 vs. 85 ± 4.1 nmol/L) and Lp , but significantly reduced H2O2‐induced increase in EC [Ca2+]i from a mean peak value of 403 ± 43.0 (n = 5) to 139 ± 30.6 nmol/L , and completely prevented the H2O2‐induced increase in Lp (Lptest/ Lpcontrol) from 9.3 ± 1.34 (n = 5) to 1.0 ± 0.08 times the control level . All values are compared after 2‐h perfusion of H2O2 (10 µmol/L). Fig. To examine whether NO‐derived ONOOric acid 0 µmol/L ric acid 0 µmol/L − on microvessel permeability, we perfused ONOO− to microvessels and measured the changes in Lp. The mean baseline Lp was 2.1 ± 0.16 × 10−7 cm s−1 cmH2O−1 (n = 25). No changes in Lp were observed with ONOO− perfusion for 30 min at 10 and 50 µmol/L  and 5.9 ± 0.44 (P = 0.003) times the control at 10 µmol/L, and to 6.0 ± 0.57 and 12.0 ± 0.96 times the control at 50 µmol/L (P < 0.001) after 1 and 2 h of ONOO− perfusion, respectively . We also examined the effect of degraded peroxynitrite on microvessel Lp in two vessels. No changes in Lp were observed after 1‐h perfusion of degraded peroxynitrite (50 µmol/L). Fig To investigate the direct effect of ONOO− and EC [Ca2+]i was further examined in seven vessels loaded with fura‐2. Baseline EC [Ca2+]i was 82 ± 12.0 nmol/L. A significant increase in EC [Ca2+]i occurred at 20 ± 4.0 min of ONOO− perfusion (50 µmol/L), and progressively reached to a sustained level of 375 ± 30.0 nmol/L at 78 ± 2.3 min of ONOO− perfusion . Preperfusion of vessels with LaCl3 (50 µmol/L) for 20 min did not affect baseline EC [Ca2+]i, but prevented ONOO−‐induced increases in EC [Ca2+]i ‐perfused vessesls. The Lptest/Lpcontrol reduced from 6.0 ± 0.57 (n = 6) to 1.1 ± 0.18 times the control ‐perfused vessels (n = 4). Perfusion of vessels with ONOO− induced an immediate increase in NO production with an initial peak rate at 31.9 ± 6.43% min‐1 for 2 min followed by a rate of 6.5 ± 2.30% min‐1 till 10 ± 1.4 min  was 332 ± 25% of baseline control. With continuous ONOO− perfusion, a second peak of NO production occurred at ~18 min with a rate of 16.6 ± 4.50% min‐1 and fell to 1.9 ± 0.10% min−1 at ~ 62 min  only showed basal NO production at a rate of 0.1%/min . As described above , 20 min before and during ONOO− perfusion significantly decreased ONOO−‐induced NO production. FIDAF reduced from 570 ± 141.0% to 154 ± 50.0 % of control,   induce both Ca2+‐independent and ‐dependent eNOS activation and the excessively produced NO triggers caspase activation and the impairment of intracellular Ca2+ homeostasis, leading to vascular cell apoptosis and consequently delayed and progressive increases in microvessel permeability , coordinated with decreased phosphorylation at Thr495  H2O2 induced concomitant NO and superoxide production, the two elements required to form ONOO−, (2) H2O2 induced NO‐dependent extensive protein tyrosine nitration (a commonly recognized biomarker for ONOO−) in the vascular wall, (3) uric acid, an endogenous ONOO− scavenger  exogenously applied ONOO− induced a delayed and progressive increases in EC [Ca2+] and microvessel Lp in a dose‐ and time‐dependent manner, a pattern similar to that observed in H2O2‐perfused vessels.Although NO has been reported to have potential cytotoxic effects, many of them are attributed to its oxidation product, particularly ONOO2O2‐induced superoxide production has been considered as an important feed‐forward mechanism for ROS‐mediated pathogenesis of cardiovascular diseases H oxidase) prevented the H2O2‐induced increases in superoxide production in vascular endothelial cells H oxidase inhibitor, prevented the increases in superoxide production in both endothelium and pericytes in H2O2‐perfused intact vessels  increased O2‐.production, rather than that of NO  were used in order to show some of the responses  in the perfusate and affects the effectiveness of the treatment. That is why we perfuse the vessel without marker cells for a certain time period and only add the marker cells during Lp measurements. This also explains why we can get the responses with pathological range of plasma H2O2 concentrations (10 µmol/L). Based on our current , and NO. Under diseased conditions, measured plasma peroxide levels are close to 10 µmol/L , and ONOO− in solution using the same imaging system when NO was measured in intact microvessels. The results shown in Fig − in the absence of NO within the reagents' concentrations we used in the experiments, although at this point we may not exclude the possibility that the presence of oxidants may facilitate the DAF‐2T formation with increased flux of NO and enhance DAF‐2 FI. Our results show that the use of NOS inhibitor completely blocked the H2O2‐ or ONOO−‐induced increases in DAF‐2 FI indicating a strong association between DAF‐2T and increased NO production. We consider that DAF‐2 is the most reliable specific NO indicator comparing with other up to date methods. Since the H2O2‐ and ONOO−‐induced DAF‐2 FI changes are in large magnitude, some contributions from oxidant effect will not change the nature of our findings. Especially, our previous studies have provided the validation of the NO measurements with DAF‐2 by conducting parallel studies with immunostaining of eNOS activation . The formed ONOO− is able to further activate eNOS and augment NO production, resulting in a self‐promoted augmentation of nitroxidative stress and subsequent vascular cell damage and vascular barrier dysfunction. The signaling cascades and sequential functional changes illustrated in Fig. In conclusion, our study uncovered a novel mechanism of elevated HThe authors declare no competing financial interests.


FLT3-ITD, and absence of mutant NPM1. Moreover, ABCB1 activity was associated with worse overall- and event-free survival. However, ABCB1 activity did not directly lead to ex vivo drug resistance to anthracyclines. We found that ABCB1 was highly correlated with gene expressions of BAALC, CD34, CD200, and CD7, indicating that ABCB1 expression maybe a passenger characteristic of high-risk AML. Furthermore, ABCB1 was inversely correlated to HOX cluster genes and CD33. Thus, low ABCB1 AML patients benefited specifically from anti-CD33 treatment by gemtuzumab ozogamicin in addition to standard chemotherapy. We showed prognostic importance of ABCB1 gene expression, protein expression, and activity. Furthermore, ABCB1 was not directly linked to drug resistance, ABCB1 inhibition did not improve outcome of high ABCB1 AML patients and thus high ABCB1 may represent a passenger characteristic of high-risk AML.ABCB1 is a member of the ATP binding cassette transporter family and high ABCB1 activity is considered as a poor prognostic factor in acute myeloid leukemia (AML) treated with intensive chemotherapy, its direct relation with drug resistance remains unclear. We evaluated ABCB1 activity in relation with clinical parameters and treatment response to standard chemotherapy in 321 patients with de novo AML. We assessed multiple clinical relationships of ABCB1 activity—ex vivo drug resistance, gene expression, and the ABCB1 inhibitor quinine were evaluated. ABCB1 activity was observed in 58% of AML and was linked to low white blood cell count, high expression of CD34, absence of  Acute myeloid leukemia (AML) is a heterogeneous disease and chemotherapy resistance is the major cause of leukemia related deaths . During In this study, we aimed to comprehensively evaluate the clinical relevance of ABCB1 activity in AML; firstly, by determining its association with clinical parameters , gene expression, and ex vivo drug resistance in patients with de novo AML. Furthermore, we examined clinical outcome in high ABCB1 activity AML patients treated with the combination of the ABCB1 inhibitor quinine with standard chemotherapy and we investigated the role of ABCB1 in AML patients treated with GO plus chemotherapy. We used global gene expression to gain new insights into why high ABCB1 maybe associated to worse clinical outcome in AML.A positive ABCB1 phenotype was defined as a V/E ratio  greater than 1.5 within the blast population and this was detected in 58% of AML (94 of 161). A representative flow cytometry analysis of an ABCB1 negative and positive phenotype is shown in p = 0.038; NPM1 or FLT3-ITD, and in AML with both markers. ABCB1 activity is highly variable within AML with adverse and favorable cytogenetics as well as in AML without known prognostic genetics .CR after remission induction chemotherapy was evaluated in 87 patients and was achieved in 86% of patients. Although not significant, lower CR rates (81%) were obtained in patients with positive ABCB1 AML compared to those with negative ABCB1 activity (91%) and ABCB1 activity was higher in non-responders vs. patients in CR  and OS  = 1.79, 95% confidence interval (CI) = 1.06–3.03, In summary, high ABCB1 activity demonstrated an independent negative impact on event-free and overall survival of AML treated with standard chemotherapy.n = 57). The median age was 57 years (range: 37–78). Toxicities related to the ABCB1 inhibitor quinine occurred in the majority of patients and included nausea and vomiting , mild tinnitus (77%), diarrhea , and mucositis . As this was a single arm pilot study, we used a subset of 35 of the 161 AML patients with positive ABCB1 activity, treated with standard chemotherapy matched for cytogenetics and molecular markers as a control cohort for outcome comparison. The median age was 56 years (range: 22–76). In the standard chemotherapy plus quinine pilot cohort 70% achieved CR, compared to 77% in the matched standard chemotherapy only control cohort. Neither CR rate, DFS, EFS, nor OS were significantly different when comparing standard chemotherapy plus quinine vs. standard chemotherapy alone  < 2% and absolute r > 0.3)  . We obseWe found no significant association between ABCB1 activity and ex vivo drug resistance in primary AML blast cells. However, correlative gene expression analysis pointed to new insights as to why ABCB1 may be linked to poor prognosis.ABCB1 expression and 21 had high ABCB1 expression. In patients with lower ABCB1 expression, addition of GO to standard chemotherapy was associated with significantly better DFS (p = 0.049) and EFS (p = 0.011) (p = 0.118) (ABCB1 expression (ABCB1 expression maybe useful to guide selection of GO responsive AML cases.The cell surface protein CD33 is targeted by the anti-CD33 drug conjugate gemtuzumab ozogamicin (GO). Because ABCB1 was negatively correlated to CD33 expression, we examined whether his had any effect on clinical response to GO. Among the 194 patients of the ALFA-0701 trial, 173 patients had low to intermediate = 0.011) A,B. Howe= 0.118) C. Converpression . We showBecause ABCB1 was highly associated with CD34 and CD33 we postulated that ABCB1 may be a bystander effect inherent to a respective leukemia stem cell (LSC) phenotype. For some samples, we performed the CD34 and CD38 staining in combination with ABCB1 activity. We found that if the progenitor population defined as CD34+ and CD38− was ABCB1 positive the total blast population was also ABCB1 positive and vice versa D,E. FurtWe found that ABCB1 expression of the leukemia bulk is related to its hematopoietic progenitor cell ABCB1 expression.Identification of new prognostic markers, remains important and only three molecular markers  are currently used in clinical practice . Here, wwww.cbioportal.org).In our study in adult AML patients treated with standard chemotherapy, the median ABCB1 activity was not significantly higher in AML patients not achieving CR after remission induction therapy. Similarly, disease relapse did not occur more frequently in positive ABCB1 AML. This finding indicates that apart from drug resistance to chemotherapy other co-operative mechanisms likely exist explaining the negative effect of positive ABCB1 activity on long-term treatment outcome of AML. It has been reported that multiple transporters of the ABC family may confer resistance to chemotherapy, among them ABCB1, ABCC1, and ABCG2 have been described most frequently ,4. Of noFLT3-ITD and NPM1 mutations and this was not described in previous studies where mutational status of these genes was available [Yet, ex vivo drug resistance testing of primary AML cells revealed no correlation between high ABCB1 activity and neither daunorubicin nor idarubicin resistance. In addition, the impact of ABCB1 activity and expression was more prominent on later clinical events like OS and EFS and was neither significant for CR nor DFS. In particular, as inherent drug resistance is thought to rather impact the latter two. This would explain why most associations between ABC transporters and clinical outcome are correlative and have not been shown to be causal ,4. Nevervailable . These aSince newer generation and more selective ABCB1 inhibitors have failed to prove clinical relevance, it was suggested that possibly larger spectrum ABC efflux transporters maybe more effective as multiple efflux transporters maybe involved to form the MDR phenotype . To asseABCB1, CD200, and BAALC. Similar to ABCB1, the latter two had also been associated with an adverse prognosis in AML [BAALC was not associated in AML with high ABCB1 activity in another study, probably due to its restriction to CN-AML [CD200 was involved in the development of an impaired immune system in response to AML [We then hypothesized that evaluation of ABCB1 co-expressed genes may uncover potential mechanisms explaining how high ABCB1 leads to treatment failure in AML. Our transcriptomic analysis revealed a positive correlation among s in AML ,23,25,26o CN-AML . More ine to AML . This prCD33 was negatively correlated with ABCB1 expression. CD33 is expressed on myeloid progenitors and is the therapeutic target of the drug GO. Treatment with GO was beneficial in terms of DFS and EFS for patients with lower ABCB1 expression. Our findings are in line with previous studies showing high ABCB1 activity was associated with a poorer response to GO [Our transcriptomic analysis showed that se to GO  and thatse to GO . Notablyse to GO . FurtherPatients were classified according to ELN 2010 by cytogenetics and molecular markers into favorable: t, inv(16), normal karyotype/mutant NPM1/wild-type FLT3, normal karyotype/mutant CEBPA; adverse: inv(3), t, −5, −7, abnormal 17p, complex, MLL rearranged; intermediate—II: t, other non-normal karyotypes; intermediate—I: normal karyotype/mutant NPM1/FLT3-ITD, normal karyotype/wild-type NPM1/FLT3-ITD, other normal karyotypes.ABCB1 activity was measured in a cohort of 161 adult AML patients, diagnosed and treated at the Lille Hospital between 2000 and 2010, their median age was 60.7 years (range: 21–88). Clinical features are summarized in The single arm quinine pilot study included 57 AML patients recruited at the local Lille Hospital between 2000 and 2008 and treated with intensive chemotherapy (cytarabine/idarubicin) plus the ABCB1 inhibitor quinine. The clinical protocol MAquinine was approved by the local hospital review board. Inclusion criteria were high ABCB1 activity, non-favorable disease characteristics, and absence of any signs of heart failure or abnormal kidney or liver function. Median age was 57.0 (range: 37–78). Quinine treatment was administrated as previously described .ABCB1 gene expression and ex vivo drug resistance were performed in 103 adults diagnosed with de novo AML at Lille Hospital between 2010 and 2014. The median age was 55.4 years (range: 20–82). Finally, we included a subset of 194 AML patients treated with or without GO plus chemotherapy from the ALFA 0701 clinical trial for which ABCB1 gene expression data was available [For ABCB1 activity, vailable ,29. MediAll studies were approved by the local ethics committee and informed consent was obtained at diagnosis. A CONSORT diagram outlines the different cohorts of patients and associated datatypes .All patient studies were approved by the local ethics committee and informed consent was obtained at diagnosis.The local ethics committee approved all studies and all patients signed an informed consent at diagnosis .6 cells to 0.2 µg/mL rhodamine 123 for 45 min in the dark at room temperature. The cells were then washed twice with RPMI-FBS at 4 °C. Secondly, evaluation of the rhodamine 123 efflux (E) was performed after a 2-h incubation at 37 °C with and without 10 µg/mL Verapamil . The cells were washed twice with RPMI-FBS at 4 °C and stained with anti-CD45-PC5.5  for 30 min in the dark at room temperature, washed twice, and analyzed by MFC. Blast cells were gated as CD45dim and SSClow. Two ratios of mean fluorescence intensities were calculated: E/A (rhodamine 123 efflux) and V/E . Experiments were performed on a Navios flow cytometer and analyzed with Kaluza software (Beckman-Coulter). The cytometer settings were tested daily for optical alignment, fluidic stability, and optical detector sensitivity using standard fluorospheres .ABCB1 activity was measured by multiparametric flow cytometry (MFC). Blast cells were washed twice at 37 °C with RPMI-1640 supplemented with 10% fetal bovine serum (RPMI-FBS) . Firstly, accumulation (A) of rhodamine-123 , in the cells was determined by exposing 1.5 × 10Five patients were analyzed with an anti-CD34-AA700  and an anti-CD38-PB  simultaneously with the measurement of ABCB1 activity to evaluate the CD34+CD38- population known to be enriched for leukemia stem cells.https://www.ncbi.nlm.nih.gov/geo/.Gene expression analysis was performed on Affymetrix GeneChip™ U133 Plus 2.0 array according to the manufacturer’s directions (Thermo Fisher), using total RNA extracted from cryopreserved AML samples and log2 gene expression values were obtained as described previously . We filt50).The MTT  assay was used to study drug resistance in conditions as previously described . The folThe association of the MDR phenotype with biological factors was assessed with Student t- or Mann–Whitney U (MWU) test and qualitative variables were compared using Chi-square or Fisher exact test. Outcome analysis was performed as previously described . Time waFLT3-ITD and/or NPM1 mutational status. Thus, assessment of the ABCB1 phenotype may help to refine AML treatment as GO may not be effective in AML when ABCB1 expression is high.We showed that ABCB1 activity and expression was linked to worse prognosis and therefore evaluating its activity or expression at diagnosis may still be important. However, ABCB1 did not directly mediate chemotherapy resistance ex vivo and in vivo and therefore we hypothesize that ABCB1 may be a bystander effect. We provide several potential candidate genes associated with high ABCB1 which are directly involved in hematopoiesis or affect immunologic response. Finally, ABCB1 activity had an independent prognostic role and was not solely associated with


Mentoring programmes are commonplace, yet little is known about the circumstances in which they operate. This study aimed to gain insight into the context surrounding youth mentoring programmes by asking programme managers and experts in the United Kingdom about their experiences. Telephone interviews with twenty-three programme managers and five experts were undertaken. Interviews were recorded, transcribed verbatim and analysed iteratively using thematic analysis. Contextual influences at the individual-, interpersonal-, organisational-, community-, policy-, and societal-level were identified to impact on programme's development, delivery and maintenance and were summarised in a model. This study further found that youth mentoring programmes operate within a complex context. This context provides challenges and opportunities that impact on programme's sustainability; resulting in many externally-funded programmes to ‘hang by a thread’. It is important for service providers, commissioners and academics to recognise the complexity surrounding mentoring programmes to ensure that programmes are delivered as intended and evaluated appropriately. •Formal mentoring programmes in the United Kingdom operate within a complex context•Individual-, interpersonal-, organisational-, community-, policy-, and societal-level influences were identified•A conceptual model was established highlighting the range of contextual influences on formal mentoring programmes•The context provides challenges and opportunities that impact on programme's sustainability Previous evaluations have highlighted the potential benefits of such programmes in improving young people's health, social and educational outcome's . HoweverContext can be defined as the “circumstances or events that form the environment within which something exists or takes place” , p. 59. Having a thorough understanding of the context surrounding an intervention is therefore crucial for: (i) understanding the intervention's possible reach, efficacy, adoption, implementation and maintenance , (ii) deUnderstanding the context of public health interventions is important, yet there is an overall lack of knowledge of the contextual factors at play . A CochrIn sum, knowledge about how interventions operate in different contexts and the extent to which contextual factors can impact on the evaluation of a given intervention is incomplete, which might partly be explained by the limited reporting of context required by existing guidelines.Contextual factors in youth mentoring programmes have also been neglected so far in most previous evaluations . This isOnly a handful of studies have specifically focussed on contextual influences within youth mentoring programmes, only one of which was within the evaluation of mentoring programmes . For exaStudies that focussed on the context of mentoring programmes are limited in that they were oftentimes focussed on programmes other than youth mentoring programmes  and did With recent developments and a rise of mentoring in the UK and other developed countries, assessing the generalisability of USA-based studies and programmes is vital. Developments in mentoring in the UK have been widely influenced by the USA as most evidence comes from there . It is nWe undertook interviews with programme managers of youth mentoring programmes and experts in the field of mentoring in the United Kingdom about their experiences of the development, delivery and maintenance of formal mentoring programmes for young people and the context within which formal mentoring programmes operate.22.1Programme managers from purposefully sampled mentoring organisations and selected experts in the field were invited to take part in semi-structured telephone interviews. Eligibility criteria for mentoring organisations were that they had to be involved in the delivery of one or more formal mentoring programmes to young people aged 11–16 years that were currently enrolled in secondary schools in the UK. Mentoring in the research was defined as ‘any programme between an identified young person and other individual(s), aimed at the support of the young person’. Therefore, mentoring organisations that provided any type of mentoring, any type of mentor and any type of aims and objectives were eligible for inclusion. An overview of common terms related to mentoring used in this paper is provided in A thorough search of national mentoring websites, charitable organisations and turst registers were undertaken to identify current organisations and programmes. From a total of 815 organisastions that were identified from the search, 163 met the eligibility criteria for inclusion in the study, namely that they were provided for young people and that that they entailed mentoring programmes rather than other types of intervention programmes such as befriending, coaching or counselling. Maximum variation sampling was used to recruit a purposeful sample of programme managers from within the eligible organisations. Mentoring organisations were purposefully selected by country within the UK and by the type of mentoring provided to ensure that different perspectives and experiences were captured. Participants who took part in the research study were asked whether they knew of other mentoring programmes that met study eligibility criteria. As such, snowball sampling was used in combination with maximum variation sampling to obtain a varied sample of organisations. Programme managers were selected as participants because they were seen to be best placed to talk about the operational aspects of the programme, the programme's historical development as well as challenges, opportunities and concerns experienced at an organisational level. Of the 163 eligible organisations, a total of 29 eligible and varied organisations were contacted in three stages to ensure that different programmes were sampled. For example, the initial 10 interviews did not capture any peer-mentoring programmes or programmes provided by school staff.Experts in the field were identified through the literature and via consultation with other researchers and then also purposefully selected and invited to take part in semi-structured interviews. The aim was to identify experts from both academic and practitioner backgrounds who could share their experiences of having run or worked with a range of different mentoring programmes and who could therefore help gain insight into broader contextual aspects of mentoring programmes.All participants were contacted with an invitation letter or email, containing an information sheet explaining the research study. Invitations were followed up with a further letter, email or telephone call. Following participants' expressions of interest to take part, telephone interviews were arranged. There were no financial incentives to taking part. These interviews were collected as part of a wider study with the aim of developing a typology of mentoring programmes and findings from this have been reported elsewhere .2.2Telephone interviews were carried out by the main author of the study and facilitated using a semi-structured topic guide. The topic guide included open questions about the history, structure, organisation and characteristics of the mentoring programme, perceived challenges and opportunities and about any changes to the organisation or programme. Interviews were audio-recorded using an encrypted audio recorder (Olympus DS-3400). Interviews were conducted concurrent with data analysis in three waves between September 2015 and March 2017. Interviews lasted on average one hour.Interview recordings were transcribed verbatim by the main author of the study and then anonymised. All data were stored on a secure, password protected, electronic database. Hard copy data such as reply slips and consent forms were held in locked filing cabinets at the University of Bristol, United Kingdom.2.3Transcripts were read and re-read to allow familiarisation with the dataset and to begin the analytical process. Whilst doing so, initial thoughts and impressions were noted. Segments of text were summarised in the document margins and any thoughts about the data itself or occurring concepts were noted. Where necessary, certain parts of audio-recordings were listened back to in order to clarify meaning. In line with inductive analysis techniques, when summarising parts of texts, the participants' original wordings were used wherever possible . Data anA selection of transcripts was shared with two other members of the team who were not involved in the data collection. Where there was disagreement, original interview data were scrutinised against the coding and discussed until agreement was reached. Data from all transcripts was labelled or indexed accordingly, by identifying relevant parts of text in transcripts and assigning them to one or more of the categories in the conceptual framework in NVivo10. All researchers agreed on the final emerging themes and the representations of such.2.4Ethics approval for the study was granted by the Faculty of Medicine and Dentistry Research Ethics Committee (FREC) in 2015 (reference number 24341). All participants provided written consent to take part. Experts in this study were asked for their consent to be acknowledged by name as it would have been difficult to preserve their anonymity.33.1Twenty-three of 29 programme managers approached (79% response rate) and all 5 experts in the field of mentoring took part in interviews. Managers worked in organisations such as secondary schools, universities, mentoring or youth service organisations and were involved in running a total of 28 diverse youth mentoring programmes. Programmes were run within schools, communities or online and these were located within one or more of the four nations of the UK, had been in existence from less than two to over 16 years and varied in the number of young people supported each year (from 25 to over 5000 youths per year). The majority of programmes were delivered face-to-face using adult mentors, whereas some programmes were provided online or used peer mentors. Programmes differed in the frequency and length of mentoring sessions, time of delivery and overall programme duration. Mentoring provision by secondary schools or universities were usually internally-funded whereas programmes delivered by mentoring organisations and youth organisations were externally-funded. A typology of programmes has been previously published by authors .Experts taking part in interviews were from both academic and practitioner backgrounds and had worked in the field of mentoring for several years, in all but one case over ten years. Some were involved in delivering mentoring programmes themselves.Participants spoke in detail about how their programmes were developed, delivered, maintained, and sustained and emphasised the range of challenges and opportunities that they perceived. Each of these areas will be discussed in turn. When speaking about their programmes, participants highlighted that these areas are informed by contextual factors and, as such, that wider contextual factors impact on the development, delivery and maintenance of programmes. Quotations from programme managers have the reference M and experts the reference E. 3.2Development of a mentoring programme involved four key aspects: (i) generating the idea for a programme, (ii) gaining support from others, (iii) deciding upon a programme model and (iv) obtaining the necessary funding to run the programme. Managers explained that programmes were established because of the perceived or actual need for certain young people to get extra support and guidance to help them cope with challenges. By providing young people with an older, more experienced role model, through a flexible and individualised ‘amenable’ intervention, participants felt that this need could be met. Experts noted a rise in the overall number of programmes available in the UK in last three decades and observed that mentoring programmes were now more embedded in schools. The support for mentoring programmes in the UK was seen to have arisen out of changing family structures and the decline of other sources of support for young people, such as youth and social work. Similarly, participants noted that there were fewer places for young people to meet. Programmes were also seen to provide an outlet for adults in the community who wanted to help young people who they saw struggling.“I suppose some of it was in settings where the adult mentors were striving for that kind of relationship […] they were trying to sort of be someone who could help a young person.” “Most organisations deliver mentoring programmes to the need that they are looking for.” .Participants pointed out that, compared to statutory interventions, mentoring programmes offered a different, possibly ‘better’, approach to working with young people. This was because mentoring programmes were voluntary and young-person led. An underlying belief in mentoring as a beneficial and valuable intervention was expressed by many managers:“The key reason that we use mentoring is because, I'd say it's a youth work approach, and it's not a statutory based intervention so its voluntary […] that young people engage in the service” (M2).Some managers drew upon the fact that young people desired mentoring themselves. They also highlighted the interest of schools in mentoring, explaining that it helped teachers deal with increasing pressures, that it relieved them from having to deal with difficult students and that programmes could provide individual support for students that would otherwise have been missed by the school.Once the idea for a mentoring programme arose, support and trust both from within and outside the organisation had to be gained. Gaining trust was described as essential to the success of a programme, yet difficult to obtain and potentially time-consuming.“If you haven't got the support of your senior team in the school, it's [mentoring programme] gonna fail.” (M15).Furthermore, organisations had to decide upon a type of mentoring programme which involved acquiring knowledge about the circumstances, available resources and preferences in the given locality. Moreover, a decision had to be made about the type of mentor, setting, management and procedure of referrals, referral criteria, voluntary nature of the programme and targeting criteria. Decisions about the type of programme were made in collaboration with local partners and undertaken in an iterative and collaborative fashion.Finally, managers addressed the need to obtain funding for the programme which was described as a key challenge for many organisations. Interviewees acknowledged that funders had a key influence on the chosen programme model, approach and aim to mentoring, programme size and structures:“Our funders then kind of dictate the area and the types of young people that we work with.” (M6).The support for mentoring by schools was shown in schools' willingness to pay for the mentoring, oftentimes by spending their ‘pupil premium’ funding . Likewise, participants emphasised that businesses and universities had an interest in mentoring programmes to align with their corporate social responsibility and referred to the availability of government funding for mentoring programmes.In sum, opportunities arose because of widespread support for programmes from a range of institutions, willingness of these institutions to fund and invest in programmes and overall support and an underlying belief in mentoring as valuable for young people.3.3Managers highlighted seven challenges and opportunities of programme delivery: (i) setting up the programme, (ii) developing programme infrastructure and policies, (iii) risk management, (iv) working with external partners, (v) working with schools, (vi) making the programme work for a specific locality and (vii) managing the end of the mentoring relationship.Managers primarily talked about the necessity and challenge of recruiting mentees and mentors to take part in the programme. Whilst recruiting peers to act as mentors was not perceived as difficult, recruiting ‘good’ mentors in volunteer-based programmes was a challenge:“The number-one challenge is getting enough good volunteers […]” (M5).Setting up a programme further included consideration of how the programme, people and participants would be managed and monitored. In most cases, organisations appointed programme staff, such as programme managers, to oversee the programme. Developing suitable guidelines and policies was regarded as critical to the delivery, yet time-consuming.One key concern for all organisations was to ensure that risks of the programme to those involved were appropriately managed. This was a particularly pertinent issue as many programmes worked with vulnerable young people and were delivered on a one-to-one basis in sometimes isolated areas.“Putting everything into place for safeguarding young people, and our mentors, that's a massive challenge.” (M10).The initial mentor training typically covered safeguarding and specific instructions for mentors to pass on any concerns or issues to programme staff or teachers. Some managers reflected that this might involve breaking confidentiality, in other words, telling the mentee that something raised confidentially as part of mentoring had to be passed on. This was regarded as a particularly challenging aspect as programmes wanted young people to trust the programme and mentor.“If the [organisation] says you must disclose this kind of stuff where does that leave someone who feels, you know, where the young person has said, I am telling you, because I can't tell anyone else?” (E5).To mitigate risks within school-based programmes, interviewees spoke about having coordinators and other staff nearby to oversee the mentoring, and the ‘private but public’ nature of mentoring within the school context. Despite a few programmes being run with close monitoring structures, most programmes worked on a “managing by exception” rule which stipulated that mentors were expected to inform programme staff only in the case of child protection related issues or concerns:“We have a kind of ‘managing by exception’ rule, so they need to detail if there are any concerns they have got, ok, and […] they will all have a monthly review.” (M3).All programmes except internally-run school-based programmes required partnership working. Partners were essential within the process of making the mentoring programme happen and could involve schools, social workers, health services, police and local authorities. Working in partnership took different formats: from an institution solely being involved as an organisation to refer young people or mentors into the programme to being responsible for the  delivery of the programme.Many managers spoke about their experiences of working with secondary schools. Practical considerations mentioned included lacking space and facilities within schools to undertake mentoring, needing security clearance for mentors to work within schools and having difficulty organising mentoring. Managers acknowledged that schools are pressured environments and that the school's overall focus is on learning, which took priority over mentoring and led to sudden changes or cancellations of mentoring sessions.“We have occasions where we are all set up to go into a school and then at the last minute the school suddenly just says, ‘sorry, we can't do it, we got this happening’.” (M24).Furthermore, one expert noted that some individuals, such as teachers, might not like mentors coming in to deliver mentoring:“And I think often teachers resent mentors coming in from outside because they are able to do things that they feel they can't to.” (E5).Managers revealed that some schools might have different agendas from mentoring organisations that would need to be negotiated. Some schools chose students despite them not meeting the referral criteria:“They [mentees] are definitely not the most dysfunctional, though, saying that, some of them are because the school is so desperate for some support for these children, they put them on the scheme.” (M22).To make the programme work for a given locality, managers emphasised the importance of partnership working and being flexible in the delivery of a programme. This meant possibly considering altering the programme model and structures:“We do a bespoke package for each child […] what we noticed is that not one size fits all.” (M16).Attention was also given to the end of the mentoring relationship, with different practices in different programmes including “phasing out” the mentoring, stopping mentoring altogether or allowing mentor and mentee to continue to meet see .3.4Five components to maintaining a mentoring programme were identified: (i) ensuring continued funding, (ii), managing partnerships, (iii) engaging mentees and mentors, (iv) assessment and evaluation and (v) adapting the programme for long-term sustainability.Differences were observed with internally and externally-funded programmes. In general, these challenges were greater and more central to the experience of externally-funded compared to internally-funded programmes.One key factor emphasised by many participants of externally-funded programmes was the need to continuously receive funding to maintain and sustain their programmes long-term. Participants referred to occasions when funding was changed, cut or withdrawn, which emphasised the insecurity faced with short-term funding.“Insecure funding is, it really does affect everything.” (M10).Funding insecurity led to worries and concerns of those working in mentoring organisations. Whilst the challenge of funding was a recurrent theme, some benefits from funding uncertainty were given: the need for organisations to think critically about their programme and programme theory, to develop structures for evaluation, ultimately leading to more evidence-based practice and expanded knowledge about ‘what works’.Accommodating the needs of external partners was another major factor in enabling successful programme delivery. Managers described actively managing external relationships, emphasised the importance of not overburdening their partners and referred to changes made in the programme to accommodate partners' needs.Moreover, the need to engage mentees and mentors and manage their expectations was emphasised. Whereas peer-mentoring programmes within secondary schools were typically oversubscribed, providers of other programmes using volunteer adults spoke about difficulties in recruiting and retaining enough mentors. Strategies to foster continuation in mentoring and to manage expectations were also highlighted see .To maintain programmes long-term, most participants and all experts acknowledged the importance of assessing and evaluating programme outcomes. Interviewees emphasised that this was particularly of priority for externally-funded programmes.“We know that grant-funders now are more interested in evidence-based approaches so what we had to do was introduce a standardised evaluation framework” (M23).However, in many cases evaluations were not a priority and only undertaken if problems arose. Outcomes and evaluation for programmes were regarded as a particularly pertinent issue in the sector and one that organisations struggled with:“If there is something wrong, then there'd be a bit of research or investigation.” (M10).Participants acknowledged that despite a range of mentoring programmes in existence, the actual evidence on mentoring programmes was weak and potentially not in line with the impact that was expected, making the evaluation of programmes a difficult one.“There is very little research […] about the impact mentoring programmes have. It's terribly popular, everyone loves it, but when they actually try to track hard impact, it's always been a little bit disappointing.” (M5).Participants spoke about what outcomes they measured, how, and that this differed widely between programmes. Outcomes measured ranged from proximal to distal and spanned a range of topics, including programme experiences, educational outcomes, and risk behaviour outcomes. Less interest seemed to be shown in measuring health outcomes. The type of outcomes measured were generally based upon the aim of the programme. Participants drew a distinction between what was referred to as ‘hard’ and ‘soft’ outcomes: hard ones being relatively direct and tangible to assess by looking at educational records, while soft ones referred to those outcomes which were more challenging to assess and often based on self-report such as changes in self-esteem or confidence. Methods for capturing outcomes included qualitative interviews, questionnaires and surveys, monitoring forms, school records or other available data.Generally, outcome evaluation and assessment seemed to be an ‘area in progress’ as many providers described currently looking for ways in which they can best measure programme outcomes. Some participants acknowledged having limited experience in evaluation and were hesitant about what to measure and how.Another part of programme maintenance included the adaptation of the programme and programme structure. All participants mentioned having undertaken programme changes over the years and this was regarded as inevitable to keep the programme relevant, acceptable and valuable for everyone involved.“We like to change that [mentoring programme] every year because we feel the community changes every year” (M16).Participants gave insight into changes that were made in the past to their programme, including changes in referral criteria, referral organisations, setting, programme model, programme structures, size and staffing. The potential dependency of mentee and mentor was voiced as an issue by a handful of participants who explained that, to overcome this, they changed the frequencies at times, made programmes less frequent towards the end, and, in the case of one programme, having mentees swap mentors.Most participants intended to expand their programmes, highlighting the necessity and importance given to ensuring the continued operation and sustainability of programmes.“Over the next kind of two years we are […] looking to roll the programme out to different regions.” (M12).3.5When speaking about the development, delivery and maintenance of programmes, programme managers and experts recognised that formal mentoring programmes operated within a challenging and complex field overall.“The field is a little more complicated than a straight forward thinking, is mentoring good or bad? It's the context in which it is provided in.” (E4).(i)individual-level (i.g. mentee and mentor's own background)(ii)interpersonal-level (iii)community-level (i.e. available resources and places to meet)(iv)organisational–level (i.e. programme structures and regulations)(v)policy-level (vi)societal factors (i.e. norms and perceptions of mentoring).In their description of programme structures, it became apparent that formal mentoring programmes interacted with a range of contextual influences; factors that were external to the programme but that influenced how programmes were developed, delivered and maintained. Contextual factors were found to encompass various levels of influence:Taken together, these factors had significant bearing on how programmes were developed, delivered and maintained and, as such, the programme's overall sustainability. These influences were understood to be related and interlinked.Safeguarding requirements, a policy-level contextual factor, influenced the development of programmes by informing who was working with whom. For example, many programmes typically refrained from matching female mentees with male mentors and safeguarding policies were used to explain the lack of male mentors in many community-based programmes. Safeguarding requirements also influenced the delivery of programmes, for instance, as this was covered in the training with mentors, as this influenced programme infrastructure and monitoring and the setting in which the mentoring programme was provided. Finally, safeguarding policies also played a role with regard to the maintenance of programmes, as programme managers continuously assessed the risks of programmes. An overview of different influences specific to formal mentoring programmes is provided in Due to the challenging context within which programmes were developed, delivered and maintained, participants raised concerns that the challenges experienced could impact on the effectiveness of programmes. More specifically, experts were concerned about the possibility of mentoring programmes to do harm:“They are aiming to tackle long-term issues but […] their funding is precarious, they're embeddedness in, you know, the life of the young people is very uncertain, […] one of the things that we were concerned about was the capacity of mentoring programmes to do more harm than good.” (E5).“I think there is pressure to sell programmes, […] and make matches at all costs. […] if a young person is having difficulty in their life and it's decided that a mentor would be good for this young person, and you form a match and […] then for whatever reason the mentor packs it in […] closing that match can be very difficult. You know? So we need to be aware of the dangers.” (E4).Potential harms alluded to were the fact that taking part in a mentoring programme might impact on other circumstances in the life of the young person, such as undermine their relationship with other adults in their life or their access to other services.“Can mentoring undermine those sort of relationships? What I mean by that is […] by the introduction of a child to a mentor […] are you ruling out the possibility that an aunt or an uncle could provide that support if they were encouraged?” (E4).“And the question that we have is, if that mentor had stepped away, would that [better living situation] have happened sooner? Or equally […] would it not have happened and that person would be in an even greater, more vulnerable situation? So questions without answers.” (M8).In fact, some managers acknowledged that programme models or structures were not followed at times because of contextual constraints.“I mean to a certain extent we try to match them. But in reality, it comes down to timetabling and who is available.” (M7).“We ask the students if they prefer male or female because we want them to feel comfortable, and […] we accommodate that as much as we possibly can” (M22).Experts raised concerns about the fact that organisations often had a vested interest in the delivery of programmes for the organisation's benefit and emphasised the need to look at external agendas.“There might be sanctions if the young person didn't behave in an appropriate way, then the mentoring would be taken away from them. “(E5).One expert, in particular, highlighted contradictions with regard to the field of mentoring. One contradiction was the frequently short duration and short-term funding of programmes compared to the long-term outcomes programmes aimed to achieve. Another contradiction was programmes wishing to help young people but removing access to mentoring if a young person did not attend and by programmes typically recruiting successful mentors to work with what were regarded as ‘unsuccessful’ mentees.Some participants expressed frustrations with some of these contextual factors and external requirements and particularly drew attention to the insecurity of funding available for programmes. Securing the sustainability of a mentoring programmes seemed to be a ‘juggling act’ for many providers; having to cope with pressures and accommodating wishes and interests of partner institutions, having to engage mentees and mentors, managing expectations, and needing to keep partner organisations happy. This required organisations to be flexible in their approach and to be continuously evaluating programmes structures in order to keep the programme relevant, acceptable and valuable for all stakeholders involved.4Formal mentoring programmes for young people in secondary schools in the UK are faced with a range of challenges and contextual influences in their development, delivery and maintenance. Key challenges and opportunities included funding, managing risks and expectations, partnership working, engaging mentees, mentors and stakeholders with the programme and adapting the programme to ensure long-term sustainability. Given the range of challenges experienced, securing the sustainability of a mentoring programme seemed to be a ‘juggling act’ for many mentoring providers. This study identified a range of interactive influences, occurring at the individual, interpersonal-, organisational-, community-, policy and cultural-level, which are summarised in a model. This highlights how mentoring programmes operate within a specific local and national context.Differences were found between internally- and externally-funded programmes. Those running externally-funded programmes experienced the challenges to a higher degree, particularly concerning the funding and consequent need to assess and evaluate their programmes. Many of such programmes found themselves in a position whereby they could be seen to ‘hang by a thread’. This comparison between internally- and externally-funded programmes has not been of focus in the literature and deserves further exploration.The current model of youth mentoring, which is used as a guiding conceptual framework for programmes by Rhodes , acknowlWe reported that there are circumstances in which potential harm can arise in mentoring programmes. Specific areas that were highlighted were the end of the mentoring relationship and mentor characteristics, which are in line with the previous literature . Due to Some contradictions within mentoring were described, highlighting the complex and possibly conflicting needs of programmes and participants. Programme managers spoke less about these potential conflicts and generally spoke in positive terms about their own mentoring programmes. Matching often unrelated and similar mentors and mentees has been previously alluded to in studies of successful mentoring relationships, where, for instance cultural awareness and understanding have been noted as key to establishing a trusting mentoring relationship .Some of the named challenges such as the need to manage expectations have been discussed in the literature or past evaluation studies . Country4.1To our knowledge, this is the first time that programme managers and experts themselves have explicitly given voice to their experiences and the challenges and opportunities faced in the development, delivery and maintenance of mentoring programmes. This work is based on the participants' first-hand accounts of running mentoring programmes and the experiences of experts in the field.The generalisability and representativeness of our findings with regard to countries outside the UK is uncertain. This is particularly the case as our study identified that specific contextual factors impact on the development, delivery and maintenance of programmes. Whereas the research aimed to provide a general overview, since each programme is operating within a specific setting and context, other mentoring organisations might face different or additional challenges not mentioned. Due to the sampling framework for our study, the experiences of smaller programmes or programmes without a web-presence were not included and their experiences might differ. The application of this research to other types of mentoring programmes, such as those working with adults, or primary school children, or in other countries, may be limited for similar reasons. The conceptual model  has not 4.2Mentoring programmes are situated within a specific and highly complex context. This study underlines the importance of taking into account contextual factors within programme descriptions and evaluations and for contextual factors to be captured as part of programme evaluations. For these reasons, this research suggests that mentoring programmes might work in one, but not necessarily in another context, meaning that findings from evaluations in the USA might not be easily transferable to the UK context.Our study carries implications for mentoring researchers and practitioners in the field internationally as well as in the UK, the latter in which a recent rise of youth mentoring programmes has been noted . In ordeService managers need to provide a clear description of the programme logic and work towards evidence-based programme structures and characteristics. Whereas many programmes seemed to follow what is regarded as ‘good practice’, such as allowing the relationship to develop over time  and compAs some managers expressed a lack of knowledge regarding their outcome assessment, this might be one area for collaboration between practitioners and academics. Academics need to acknowledge the complexity surrounding programmes and recognise that programme models and structures may change over time. Service commissioners should closely consider programme factors and contexts and commit secure funding to evidence-based, cost-effective programmes.Future research is needed to explore how contextual factors can impact on programme effectiveness to identify what works, for whom and under which circumstances. Given the lack of evidence of effectiveness of mentoring programmes in the UK, there is a need for a future effectiveness trial. We recommend that this includes the use of a cost-effectiveness and process evaluation to capture the contextual factors within which mentoring is provided. It would further be of interest to explore which programme-level moderators of effectiveness and organisational practices and contexts which lead to more effective programmes.Research is needed to extend the present investigation and examine how the perceived challenges and opportunities relate to others involved in mentoring programmes, such as mentor/mentee, parents/carers or other stakeholders. Future research is also required to test the generalisability of the conceptual model presented both within the UK and internationally and to further extend the model to identify additional contextual factors not captured by it.This study found that mentoring programmes undergo processes of adaptation and changes over the years, thereby changing what is delivered. Future research is required to explore reasons for programme adaptation and the extent to which this is documented and consequent implications for the theoretical stance of programmes and logic models.5This study highlights that the development, delivery and maintenance of formal mentoring programmes to operate within a complex context. This study identified a range of interactive influences, occurring at the individual, interpersonal-, organisational-, community-, policy and cultural-level. Findings from this study will help service providers, commissioners and academics think about the contextual influences on mentoring programmes to ensure that programmes are delivered as intended, sustained long-term and evaluated appropriately.Centre for the Development and Evaluation of Complex Interventions for Public Health Improvement (DECIPHer), a UKCRC Public Health Research Centre of Excellence. Joint funding (MR/KO232331/1) from the British Heart Foundation, Cancer Research UK, Economic and Social Research Council, Medical Research Council, the Welsh Government and the Wellcome Trust, under the auspices of the UK Clinical Research Collaboration, is gratefully acknowledged.The work was undertaken with the support of The Declarations of interest: none.