Cell Signaling Technology Research Articles

Outcomes of anti-PD-1 therapy in mesothelioma and correlation with PD-L1 expression.

8514 Background: Early phase trials of anti-programmed death 1 (PD-1) antibodies have demonstrated important responses in malignant mesothelioma (MM). Expression of the ligand, PD-L1, is a potential biomarker for PD-1 directed therapy use and is expressed in a significant proportion of MM. We present results for a cohort treated with PD-1 inhibitory antibodies and assessed for PD-L1 expression. Methods: Patients (pts) with unresectable pleural or peritoneal mm treated with anti-PD-1 antibodies were included. Data was collected retrospectively. Radiological response was assessed using RECIST 1.1. Overall survival (OS) and progression-free survival (PFS) were evaluated. PD-L1 expression was assessed with IHC clone E1L3N (Cell Signaling Technology). PD-L1 positivity was defined as membranous expression on tumour cells: > 5% for PD-L1+ and > 50% for PD-L1hi. Results: Forty-six pts were treated between July 2015 and January 2017. Median age was 66.5 years, ECOG PS was 0/1 in 3/46 (7%) and 35/46 (76%) respectively. Most were male (83%), and 43/46 (93%) had ≥1 prior therapy, with a median of 2 (range 0 - 5). The predominant histology was epithelioid (n = 32/46; 70%). Pembrolizumab was used in 45/46 and BGB-A317 (a PD-L1 antibody) in 1 pt. Of the 46 pts, the overall response rate (ORR) was 15% (7 PR) with 15/46 (33%) achieving stable disease, giving a DCR of 44%. Progression was seen in 24/46 (52%). Median OS for the entire cohort was 8.0 months (95% CI: 2.3 – 11.9). Median duration of response was not yet reached (range 1.5 -19.8). PD-L1 testing was performed in 14 samples, with PD-L1+ in 5 (36%) and PD-L1hi in 4 (29%). The ORR was 40% (2 PR, 3 SD) with PD-L1+, 50% (2 PR, 2 SD) with PD-L1hi and 22% (2/9 patients) with negative expression. PFS and OS were greater with both PD-L1+ (PFS HR: 0.26, 95% CI 0.05 – 1.32, p = 0.10) and PD-L1hi(PFS HR: 0.17, 95% CI 0.02 – 1.47, p = 0.11). PD-L1+ positivity remained a borderline predictor of improved survival on multivariate analysis (p = 0.06). Complete PD-L1 analysis will be presented at the meeting. Conclusions: PD-1 targeted therapy demonstrated a clinically significant response rate in this cohort of mm patients. Initial analysis suggests PD-L1 expression is correlated with improved response and survival.

Method development for detection of PD-L1 in circulating tumor cells in small-cell lung cancer patients.

e20012 Background: Previous studies showed that circulating tumor cells (CTCs), as liquid biopsy, are the alternative for tumor tissue in small-cell lung cancer treatment and study. Programmed death- ligand 1(PD-L1) is the most commonly used biomarker in immune-targeted therapy. The aim of this study is to develop a method to detect PD-L1 in CTCs in small-cell lung cancer patients. Methods: The anti-PD-L1 antibodies were purchased from both BD (clone MOPC-21) and Cell Signaling (clone E1L3N) Technology. Control cell lines were selected by single clone culturing technique and confirmed by immunecytochemistry and flow cytometry. CellSearch system was used to select and optimize antibodies, and to test CTCs in small-cell lung cancer patients. Results: After single clone culturing selection, the purity of PD-L1 positive H446 cells (positive control) was increased to 95% from 70%, and the purity of PD-L1 negative PC3 cells (negative control) increased from 90% to 95%. The dilution of 1:200 is the optimal antibody titer for CellSearch System with 98% positive rate and 3% negative rate. In all patients (n = 4) with CTCs presenting (n = 2), PD-L1was detected and the ratios of PD-L1 positive cells to CTC counting were 1:1 and 2:5, respectively. Conclusions: We developed the method for PD-L1 detection in CTCs and validated it in small-cell lung cancer patients, which paved the way for automatic detection of PD-L1 in liquid biopsy in the future.

Development of an assay for cellular efflux of pharmaceutically active agents and its relevance to understanding drug interactions.

Drug interactions may dictate the failure or success of a treatment. Patients undergoing hematopoietic stem cell transplantation (HSCT) are exposed to various types of drugs, and understanding how these drugs interact is of the utmost importance. The pharmacokinetics of busulfan, melphalan, and cyclophosphamide, drugs commonly used for HSCT, are known to be affected by a variety of other drugs with differing molecular structures. We hypothesized that these structurally unrelated drugs affect the transport of DNA-alkylating agents. To test this hypothesis, we developed a flow cytometry assay that used 5-carboxyfluorescein diacetate acetoxymethyl ester, which is cleaved by nonspecific intracellular esterases to 5-carboxyfluorescein (5-CF), a fluorescent ligand for the drug transporter MRP1. A decreased 5-CF efflux in the presence of a test compound suggests competitive inhibition. We demonstrated that chlorambucil, 4-hydroperoxycyclophosphamide, ketoconazole, ethacrynic acid, everolimus, and sirolimus strongly inhibited 5-CF efflux in lymphoma and leukemia cell lines. The efflux of these drugs partially depends on the glutathione (GSH) level, and their cytotoxicity is synergistic with inhibited GSH synthesis. This is consistent with the hypothesis that their GSH-conjugated products are ligands of a common cellular drug transporter. Our results may explain clinical observations on the effects of various drugs on the pharmacokinetics and pharmacodynamics of alkylating agents, and the assay may be used to deduce interaction mechanisms of drugs transported by a common system.

The Immune Microenvironment, Genome-wide Copy Number Aberrations, and Survival in Mesothelioma

IntroductionResults of recent clinical studies of immune checkpoint inhibitors in malignant pleural mesothelioma (MPM) have dampened initial enthusiasm. However, the immune environment and targets of these treatments such as programmed cell death protein 1 and its ligand programmed death ligand 1 (PD-L1) have not been well characterized in MPM. Using a large cohort of patients, we investigated PD-L1 expression, immune infiltrates, and genome-wide copy number status and correlated them to clinicopathological features. MethodsTissue microarrays were constructed and stained with PD-L1(clone E1L3N [Cell Signaling Technology, Danvers, MA]), cluster of differentiation 4, cluster of differentiation 8, and forkhead box P3 antibodies. PD-L1 positivity was defined as at least 5% membranous staining regardless of intensity, and high PD-L1 positivity was defined as at least 50%. Genomic DNA from a representative subset of 113 patients was used for genome-wide copy number analysis. The percent genome alteration was computed as a proxy for genomic instability, and statistical analyses were used to relate copy number aberrations to other variables. ResultsAmong 329 patients evaluated, PD-L1 positivity was detected in 130 of 311 (41.7%), but high PD-L1 positivity was seen in only 30 of 311 (9.6%). PD-L1 positivity correlated with nonepithelioid histological subtype and increased infiltration with cluster of differentiation 4–positive, cluster of differentiation 8–positive, and forkhead box P3–positive lymphocytes. High PD-L1–positive expression correlated with worse prognosis (hazard ratio = 2.37, 95% confidence interval: 1.57–3.56, p < 0.001) in univariate analysis but not in multivariate analysis. Higher percent genome alteration was associated with epithelioid histological subtype and poorer survival (hazard ratio = 1.59, 95% confidence interval: 1.01–2.5, p = 0.04) but not PD-L1 expression. ConclusionsPD-L1 expression was associated with nonepithelioid MPM, poor clinical outcome, and increased immunological infiltrates. Increased genomic instability did not correlate with PD-L1 expression but was associated with poorer survival.

P1.02-003 ROS1 (D4D6) is Reliable for Immunohisochemistry Detecting of ROS1 Fusion Lung Adenocarcinoma in Malignant Pleural Effusion

ROS1 fusion is of a low frequency genetic alteration in non-small cell lung cancer (NSCLC). The clinical trial showed that NSCLC patients harboring ROS1 fusion could benefit from crizotinib treatment. Several studies reported that immunohistochemistry (IHC) could be employed as a screening method for detecting ROS1 fusion in tumor tissue using Anti-ROS1(D4D6) antibody. Malignant pleural effusion (MPE) is the common sample type in advanced NSCLC, the reliability of ROS1(D4D6) for detecting ROS1 fusion in MPE cell blocks (CBs) should be explored. Anti-ROS1(D4D6) monoclonal antibody (Cell Signaling Technology, Danvers, USA) IHC testing was performed on 227 formalin fixed paraffin embedded (FFPE) MPE CBs from lung adenocarcinoma patients. RT-PCR using ROS1 fusion gene detection kit (AmoyDx) was performed to detected ROS1 gene fusion as a comfirming test. Some other lung cancer driver genes were also detected in both IHC/RT-PCR ROS1 positive samples, among which EGFR, KRAS, BRAF, PIK3CA and HER2 20 exon mutation was tested by amplification refractory mutation system kits(AmoyDx), ALK and RET fusion was tested by RT-PCR kits(AmoyDx). 4 of 227 MPE samples(1.76%) of lung adenocarcinoma were interpreted as ROS1 fusion-positive cases by IHC staining, and the cytoplasmic and membranous granular positive signals were displayed. Comparison with RT-PCR testing results, 3 of 4 IHC positive cases were verified by RT-PCR, the sensitivity was 100% and specificity of was 99.6%. The clinicopathologic features and other genes status of 3 both IHC/RT-PCR positive patients were showed Table 1. One ROS1 IHC/RT-PCR positive lung adenocarcinoma patient received crizotinib therapy and obtained partial response. ROS1 (D4D6) would be a reliable antibody for screening ROS1 fusion-positive lung adenocarcinoma on FFPE MPE CBs by IHC assay and shows high specificity in the FFPE MPE CBs samples.Table 1Clinicopathologic features and genes status of ROS1 IHC/RT-PCR positive patientsGenderAgeSmokingTTF1/ P63E/k/B/ P/HA/RTherapyEfficacyPFS/Follow-upp1Male55Smoker+/-WTWTcrizotinibPR10m+/alivep2Female60Never+/-WTWTNo1m/DeadP3Male62Never+/-WTWTPEM+CISPR9m+/aliveE/K/B/P/H: EGFR/KRAS/BRAF/PIK3CA/HER-2 20 exon mutation, A/R: ALK/RET fusion, WT: wild type, PEM+CIS: Pemetrexed plus Cisplatin, PR: partial response, m: months Open table in a new tab

Programmed cell death ligand 1 protein levels predicted survival of non-small cell lung cancer.

Objective: To investigate the relationship between programmed death ligand 1 (PD-L1) expression using 5%, 25%, 50% cutoffs in tumor cells (TC) and postsurgical survival in non-small-cell lung cancer (NSCLC) patients. For samples with tumor infiltrating lymphocytes (TIL), correlation between PD-L1 expression in TIL using 1% cutoff and postsurgical survival was also evaluated.Methods: Primary NSCLC tumor surgical samples staging I to IIIA of 126 patients who underwent surgical procedures from September 2009 to August 2012 in Shanghai Chest Hospital, Shanghai Jiao Tong University were retrospectively included. PD-L1 protein expression was detected by immunohistochemistry (IHC) assays. A rabbit anti-human PD-L1 (E1L3N) monoclonal antibody (1:300, CST#13684, Cell Signaling Technology) was used for PD-L1 IHC staining. PD-L1 expression was evaluated both on TC and TIL. Univariate and multivariate analyses for postsurgical survival were done using Kaplan-Meier and Cox regression model, respectively.Results: The median postsurgical survival for all patients was 44.1 months [95% confidence interval (CI): 33.9-70.0 months). The median postsurgical survival for PD-L1 expression percentage 0, 1-50% and ≥50% were 51.9 months (95%CI: 33.9-70.0 months), 33.2 months (95%CI: 20.8-45.6 months) and 14.7 months (95%CI: 1.9-27.6 months), respectively (P = 0.002). Clinical stage and PD-L1 expression in TC (25% cutoff or 50% cutoff values) were found to be independent predictors for longer postsurgical survival in all cohort. Ninety (71.4%) of the 126 samples were identified to concurrent TIL. The median postsurgical survival time was 39.6 months (95% CI: 31.8-47.4 months) in patients with TIL. PD-L1 expression in TC (25% cutoff or 50% cutoff values) was found to be the independent predictor for longer postsurgical survival time in patients with TIL.Conclusion: PD-L1 negative expression in TC at 25% or 50% cutoff values was the independent predictor for longer postsurgical survival time in both NSCLC samples and NSCLC samples with TIL. For patients with PD-L1 high expression at 25% or 50% cutoff values, PD-L1 blocking may be considered.

ETS1 Phosphorylation at Threonine-38 Is a Marker of B Cell Receptor Activation, Associating with Cell of Origin and Outcome in Diffuse Large B Cell Lymphoma

Background.Diffuse large B cell lymphoma (DLBCL) represents the most common lymphoma subtype (30%-40% lymphomas). Gene expression profiling (GEP) identifies two main subtypes of DLBCL defined by their postulated cell of origin (COO). Activated B cell-like DLBCL (ABC DLBCL) is characterized by activation of B-cell receptor signaling and the nuclear factor kB pathway: these patients respond worse to standard R-CHOP but better to novel agents (ibrutinib or lenalidomide) than patients with DLBCL of the germinal-center-type (GCB DLBCL). We have previously reported an upregulation of the transcriptional factor ETS1 in up to 25% of DLBCL (Bonetti, Testoni et al. Blood, 2013). ETS1 is involved in many biologic processes, including B cell differentiation. ETS1 undergoes a series of post-translational modifications, and, in particular, ETS1 phosphorylation at Threonine 38 (p-ETS1) is a marker for ETS1 activation. Here, we present the impact of p-ETS1 in DLBCL cellular models and in a large series of clinical specimens.Patients and Methods. Levels of p-ETS1 (ab59179, Abcam) and, as controls, of ETS1 (C-20, Santa Cruz Biotechnology), ERK (93, Santa Cruz Biotechnology), p-ERK-Tyr204 (p-ERK; 7383, Santa Cruz Biotechnology) and IRF4 (4964, Cell Signaling Technology) were analyzed in cell lines derived from ABC (n.=7), GCB (n.=8) and type 3 (n.=4) DLBCL. p-ETS1 was examined by immunohistochemistry on clinical specimens: cases were defined as positive if > 10% of the neoplastic cells nuclei were p-ETS1 positive.Results.p-ETS1 was detected in ABC, not in GCB DLBCL cell lines (100% vs 0%, P<0.05), but also in 3/4 Type 3 (75%). All the cell lines expressed ETS1 and ERK. The ABC marker IRF4 was expressed in all ABC (100%), 1/4 type 3 (25%) and 0/8 GCB cell lines (0%). p-ERK was expressed in 6/7 ABC (86%), 2/4 Type 3 (50%) and 0/8 GCB (0%). Since ETS1 is a transcription factor and p-ETS1 is marker for its activation, we looked at the pattern of expression between nucleus and cytoplasm in five ABC DLBCL cell lines. p-ETS1 was present predominantly in the nucleus of all cell lines, while total ETS1 was in both cytoplasm and nucleus in 4/5, and only in the nucleus in 1/5 (TMD8). To evaluate the mechanisms sustaining p-ETS1, two ABC DLBCL cell lines (U2932, TMD8) were exposed to the PI3K-delta inhibitor idelalisib (1 μM), the BTK inhibitor ibrutinib (0.5 μM) and the MEK inhibitor pimasertib (0.5 μM) after stimulation or no stimulation with anti-IgM. In both cell lines, inhibition of BTK or MEK decreased the baseline levels of p-ETS1, while only inhibition of MEK was able to inhibit the increase in p-ETS1 induced by IgM stimulation. PI3K-delta inhibition only lead to a minimal reduction of the baseline p-ETS1 levels. Similar changes were seen for p-ERK. To understand the clinical significance of our findings, we assessed p-ETS1 expression in DLBCL biopsies. First, on a small series of 14 DLBCL cases classified for their COO using the Hans algorithm, p-ETS1 was mostly detected at nuclear level. The percentage of p-ETS1 positive cells was higher in ABC (no.= 7) than in non-ABC (no.= 7) (P 0.023), in agreement with the preclinical data. We then extended the analysis to a cohort of GEP-classified 315 de novo DLBCL cases treated with R-CHOP regimen collected as part of The International DLBCL Rituximab-CHOP Consortium Program Study (Xu-Monette et al, Blood 2013). The presence of p-ETS1 was further confirmed to be more frequent in ABC than in GCB cases: 79% (123/155) vs 57% (91/160) (P < 0.001). The p-ETS1 positive GCB DLBCL presented an inferior progression free survival (PFS) than p-ETS1 negative cases (P 0.034), while no association with outcome was detected in ABC DLBCL.Conclusions. Our data identify ETS1 phosphorylation at threonine 38 as i) associated with the ABC DLBCL phenotype; ii) associated with poor PFS in GCB DLBCL; iii) downstream to BCR signaling and amenable to pharmacological interventions with BTK and MEK inhibitors. DisclosuresRossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria.

Screening for ROS1 gene rearrangements in non-small-cell lung cancers using immunohistochemistry with FISH confirmation is an effective method to identify this rare target.

To assess the prevalence of ROS1 rearrangements in a retrospective and prospective diagnostic Australian cohort and evaluate the effectiveness of immunohistochemical screening. A retrospective cohort of 278 early stage lung adenocarcinomas and an additional 104 prospective non-small-cell lung cancer (NSCLC) cases referred for routine molecular testing were evaluated. ROS1 immunohistochemistry (IHC) was performed (D4D6 clone, Cell Signaling Technology) on all cases as well as fluorescence in-situ hybridization (FISH) using the ZytoVision and Abbott Molecular ROS1 FISH probes, with ≥15% of cells with split signals considered positive for rearrangement. Eighty-eight cases (32%) from the retrospective cohort showed staining by ROS1 IHC, and one case (0.4%) showed ROS1 rearrangement by FISH. Nineteen of the prospective diagnostic cases showed ROS1 IHC staining, 12 (12%) cases of which were confirmed as ROS1 rearranged by FISH. There were no ROS1 rearranged cases that showed no expression of ROS1 with IHC. The ROS1 rearranged cases in the prospective cohort were all EGFR wild-type and anaplastic lymphoma kinase (ALK) rearrangement-negative. The sensitivity of ROS1 IHC in the retrospective cohort was 100% and specificity was 76%. ROS1 rearrangements are rare events in lung adenocarcinomas. Selection of cases for ROS1 FISH testing, by excluding EGFR/ALK-positive cases and use of IHC to screen for potentially positive cases, can be used to enrich for the likelihood of identifying a ROS1 rearranged lung cancer and prevent the need to undertake expensive and time-consuming FISH testing in all cases.

4E-BP1 antibody | knockout validation | Cell Signaling Technology 9644

4E-BP1 antibody | knockout validation | Cell Signaling Technology 9644

Cell Signaling Technology rabbit monoclonal FoxO1 antibody 2880 was validated in knockout mice

Cell Signaling Technology rabbit monoclonal FoxO1 antibody 2880 was validated in knockout mice

Cell Signaling Technology polyclonal anti integrin b3 antibody was validated in knockout mice

Cell Signaling Technology polyclonal anti integrin b3 antibody was validated in knockout mice

Cell Signaling Technology rabbit monoclonal antibody 8540 was validated in knockout cell line

Cell Signaling Technology rabbit monoclonal antibody 8540 was validated in knockout cell line

Toluene downregulates filaggrin expression via the extracellular signal-regulated kinase and signal transducer and activator of transcription–dependent pathways

Toluene downregulates filaggrin expression via the extracellular signal-regulated kinase and signal transducer and activator of transcription–dependent pathways

ALK Immunohistochemistry in NSCLC: Discordant Staining Can Impact Patient Treatment Regimen

IntroductionDiagnostic immunohistochemistry (IHC) is increasingly accepted as a screening method for anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangements in NSCLC. We have sought to establish an ongoing robust external quality assessment process to gauge quality of anaplastic lymphoma kinase (ALK) IHC, which can have an impact on interpretation of patient samples. MethodsUnstained tissue and cell line samples were distributed on a quarterly basis to participating laboratories from 30 countries. Participants stained the slide using their routine diagnostic ALK IHC method and returned the slide along with their in-house control and methodology details. Slides were assessed by a team of pathologists and scientists. ResultsOverall, there was a mean pass rate of 83% (range 71%–98%), with 38 variations in staining protocol. Methods included the following: the Roche D5F3 assay (65% of users, pass rate 93%); Novocastra 5A4 (15% of users, pass rate 65%); Cell Signaling Technology D5F3 (7% of users, pass rate 91%), and Dako ALK1 (5% of users, pass rate 50%). Choice of methodology directly affected final interpretation of distributed ALK-positive and ALK-negative NSCLC cases, which were correctly identified by 89% and 88% of participants, respectively. Antibody detection method was a contributing factor in false-negative staining results. The choice of laboratory controls was found to be unsuitable, and as such, in-house control recommendations are also provided. ConclusionsALK IHC is a robust screening technique, but there is concern that some diagnostic laboratories are using inadequate staining methods, which has a direct impact on final interpretation. External assessment helps provide laboratories with continued confidence in their ALK IHC testing.

378 - The role of High-Mobility Group Box 1 protein in mesenchymal–epithelial signalling in the tumour microenvironment

Introduction Glucose depravation, hypoxia, and acidosis are characteristic features of the central core most solid tumours. Myofibroblasts are stromal cells present in many such solid tumours including those of the colon, and they are known to be involved in all stages of tumour progression. HMGB1 is a nuclear protein that plays an important role in nucleosome stabilisation and gene transcription. HMGB1 is also released from immune cells and is involved in the inflammatory process. This work reports a novel finding that the microenvironmental condition of glucose depravation is responsible for the active release of HMGB1 from different types of cancer cell lines (HT-29, MCF-7 and A549) under normoxic conditions. Materials and method Human colonic myofibroblasts cells CCD18COCo, and cancer cells, colon HT-29, lung A549 and breast MCF7 were obtained from the ATCC and bladder cancer cells EJ138 from the ECACC. The human recombinant HMGB1, anti-HMGB1 antibody, anti-RAGE primary antibody and anti-TLR4 antibody were purchased from R&D systems. MEK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) were purchased from Cell Signalling Technology (USA). Medium was collected from HT-29, MCF-7, EJ138 and A549 cells after 48h treatment with various conditioned or fresh medium and analysed by western blotting for the presence of HMGB1. The MTT assay was used measure proliferation of myofibroblasts after treatment with conditioned medium with or without an MEK1/2 inhibitor or a PI3K inhibitor. Migration of CCD18Co cells in response to medium previously conditioned on HT-29 cells for 20h was studied using 8µm pore Boyden chamber inserts in 24-well plate system (BD Bioscience, California, USA). In addition, invasion assays of myofibroblast cells were performed using 8µm pore matrigel matrix Biocoat® inserts according to the manufacturer’s instruction (Becton Dickinson). Results and discussion Recombinant HMGB1 (10ng/ml) was shown to trigger proliferation in myofibroblasts cells via activation of PI3K and MEK1/2. Conditioned medium collected from glucose deprived HT-29 colon cancer cells was shown to stimulate migration and invasion of colonic myofibroblasts, and these processes were significantly inhibited by immunoneutralising antibodies to HMGB1, RAGE and TLR4, along with specific inhibitors of PI3K and MEK1/2. Conclusion Together these data suggests that HMGB1 released from the cancer cells under glucose depravation is involved in stimulating colonic myofibroblast migration and invasion and that this was through activation of RAGE and TLR4, resulting in activation of the MAPK and PI3K signalling pathways. Thus, it is proposed that HMGB1 may be released by cancer cells in areas of low glucose in solid tumours with the resulting activation of myofibroblasts. Therefore, HMGB1 may be considered as potential therapeutic target to inhibit solid tumour growth.

BRAIN INSULIN RESISTANCE AND ALZHEIMER'S DISEASE NEUROPATHOLOGY AMONG OLDER AUTOPSIED PERSONS WITH AND WITHOUT DIABETES

Diabetes mellitus increases risk of dementia, yet little is known about the role of insulin resistance in the aging brain and its relation to the most common cause of dementia. We examined the relation of brain phosphorylated insulin receptor substrate 1 (IRS-1) and related markers of the leptin pathway, to Alzheimer’s disease (AD) pathology in community-dwelling elderly individuals with and without diabetes. We used data from 124 deceased and autopsied participants of the Rush Religious Orders Study (mean age-at-death =87 years; 52% female), 62 with and 62 without diabetes. Neuropathologic data (mean post-mortem interval=6.6 hours) included a continuous, overall AD pathology measure based on counts of neuritic plaques, diffuse plaques, and neurofibrillary tangles (NFT) on silver-stained sections, as previously published. We used ELISAs to assess insulin and leptin signaling measures (Cell Signaling Technology, Danvers MA) in frozen midfrontal gyrus cortical specimens. Linear and ordinal logistic regressions were used to examine the relation of brain measures and AD pathology. For analyses, the overall AD outcome measure was square-root transformed and other AD measures were categorized into quartiles. In the total group, IRS-1ps307 and IRS-1 were weakly correlated (rs=0.22; p=0.01), and STAT3pY705 and STAT3 were moderately correlated (rs=0.66; p<0.0001). Adjusting for age, sex, and education, a higher IRS-1ps307/IRS-1 ratio was associated with higher overall AD pathology (estimate=0.52, SE=0.24; p=0.03), and with the specific measure of neuritic plaques (OR=2.40, logOR SE=1.18; p=0.04) but not with diffuse plaques or NFT (both p>0.11). The STAT3pY705/STAT3 ratio, a marker of leptin sensitivity, was not associated with measures of overall AD, or neuritic or diffuse plaques or NFT (all p>0.08). Additional analyses examining for effect modification by diabetes, did not show any interaction of diabetes with either ratio (all p>0.09). This study of individuals with and without diabetes suggests that brain insulin resistance, as indicated by phosphorylated IRS-1, is associated with increased AD pathology, and neuritic plaques in particular. Furthermore, diabetes does not appear to modify observed associations of insulin resistance with AD. Funding: The National Institutes of Health grants P30AG010161, R01AG015819, R01NS084965.

Cardiomyocyte-Specific Human Bcl2-Associated Anthanogene 3 P209L Expression Induces Mitochondrial Fragmentation, Bcl2-Associated Anthanogene 3 Haploinsufficiency, and Activates p38 Signaling

The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which supports multiple critical cellular processes, including critical structural roles supporting desmin and interactions with heat shock proteins and ubiquitin ligases intimately involved in protein quality control. The missense mutation P209L in exon 3 results in a primarily cardiac phenotype leading to skeletal muscle and cardiac complications. At least 10 other Bag3 mutations have been reported, nine resulting in a dilated cardiomyopathy for which no specific therapy is available. We generated αMHC-human Bag3 P209L transgenic mice and characterized the progressive cardiac phenotype invivo to investigate its utility in modeling human disease, understand the underlying molecular mechanisms, and identify potential therapeutic targets. We identified a progressive heart failure by echocardiography and Doppler analysis and the presence of pre-amyloid oligomers at 1 year. Paralleling the pathogenesis of neurodegenerative diseases (eg, Parkinson disease), pre-amyloid oligomers-associated alterations in cardiac mitochondrial dynamics, haploinsufficiency of wild-type BAG3, and activation of p38 signaling were identified. Unexpectedly, increased numbers of activated cardiac fibroblasts were identified in Bag3 P209L Tg+ hearts without increased fibrosis. Together, these findings point to a previously undescribed therapeutic target that may have application to mutation-induced myofibrillar myopathies as well as other common causes of heart failure that commonly harbor misfolded proteins.

Biological implications of somatic DDX41 p.R525H mutation in acute myeloid leukemia.

The DDX41 gene, encoding a DEAD-box type ATP-dependent RNA helicase, is rarely but reproducibly mutated in myeloid diseases. The acquired mutation in DDX41 is highly concentrated at c.G1574A (p.R525H) in the conserved motif VI located at the C-terminus of the helicase core domain where ATP interacts and is hydrolyzed. Therefore, it is likely that the p.R525H mutation perturbs ATPase activity in a dominant-negative manner. In this study, we screened for the DDX41 mutation of CD34-positive tumor cells based on mRNA sequencing and identified the p.R525H mutation in three cases among 23 patients. Intriguingly, these patients commonly exhibited acute myeloid leukemia (AML) with peripheral blood cytopenias and low blast counts, suggesting that the mutation inhibits the growth and differentiation of hematopoietic cells. Data from cord blood cells and leukemia cell lines suggest a role for DDX41 in preribosomal RNA processing, in which the expression of the p.R525H mutant causes a certain ribosomopathy phenotype in hematopoietic cells by suppressing MDM2-mediated RB degradation, thus triggering the inhibition of E2F activity. This study uncovered a pathogenic role of p.R525H DDX41 in the slow growth rate of tumor cells. Age-dependent epigenetic alterations or other somatic changes might collaborate with the mutation to causeAML.

Cholesterol-mediated Degradation of 7-Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis

Cholesterol is detrimental to human health in excess but is also essential for normal embryogenesis. Hence, enzymes involved in its synthesis possess many layers of regulation to achieve balanced cholesterol levels. 7-Dehydrocholesterol reductase (DHCR7) is the terminal enzyme of cholesterol synthesis in the Kandutsch-Russell pathway, converting 7-dehydrocholesterol (7DHC) to cholesterol. In the absence of functional DHCR7, accumulation of 7DHC and a lack of cholesterol production leads to the devastating developmental disorder, Smith-Lemli-Opitz syndrome. This study identifies that statin treatment can ameliorate the low DHCR7 expression seen with common Smith-Lemli-Opitz syndrome mutations. Furthermore, we show that wild-type DHCR7 is also relatively labile. In an example of end-product inhibition, cholesterol accelerates the proteasomal degradation of DHCR7, resulting in decreased protein levels and activity. The loss of enzymatic activity results in the accumulation of the substrate 7DHC, which leads to an increased production of vitamin D. Thus, these findings highlight DHCR7 as an important regulatory switch between cholesterol and vitamin D synthesis.

Sprouty2 regulates proliferation and survival of multiple myeloma byinhibiting activation of the ERK1/2 pathway invitro and invivo.

Multiple myeloma (MM) is an incurable disease, and its pathogenesis remains unclear. MicroRNA (miR)-21 was detected at a high level in MM and plays a key role in the pathogenesis of MM. However, Sprouty2 (spry2), a downstream target of miR-21, has low expression, and its mechanism in MM is unknown. We investigated whether spry2 could exert an antimyeloma effect and further studied the potential pathogenesis and progression of MM. To address the functional consequences of spry2, we assessed the expression levels of spry2 in several myeloma cell lines and detected low expression levels in MMcells. Overexpression of spry2 suppressed growth and colony formation ability and decreased the phosphorylation of extracellular signal-regulated kinases 1 and 2. Spry2 also decreased secretion of vascular endothelial growth factor and partially enhanced the sensitivity of MMcells to an inhibitor of mitogen-activated protein kinases 1 and 2. Additionally, spry2 inhibited the tumorigenesis and angiogenesis of MMcells invivo. In summary, we report for the first time that spry2 can inhibit MMcell growth and survival with a concomitant reduction in phosphorylation of extracellular signal-regulated kinases 1 and 2 invitro and invivo.