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Several options exist for the diagnosis and management of suspected common duct stones. We hypothesized that a protocol-directed approach would shorten length of stay in this patient population.Patients from four participating institutions with a peak bilirubin4 mg/dL underwent surgery as the initial procedure, whereas patients with a bilirubin ≥4 mg/dL underwent endoscopy. The primary endpoint was length of stay. Analysis involved chi square and Wilcoxon-Mann-Whitney test with significance at p 0.05.214 patients were managed under the protocol during six-month study period. 111 patients (52%) required endoscopy and surgery. Length of stay and the number of MRCPs performed pre-operatively significantly decreased following protocol implementation (p 0.05)."Surgery first" approach in patients with bilirubin4 ml/dL resulted in low morbidity and mortality, reduced MRCP, and length of stay.

Background We hypothesized that the universal adoption of closed wounds with negative pressure wound therapy (NPWT) in emergency general surgery patients would result in low superficial surgical infection (SSI) rates. Study Design We performed a retrospective observational study using primary wound closure with external NPWT, from May 2017 to May 2018. Patients with active soft tissue infection of the abdominal wall were excluded. Data were analyzed by Fisher's exact tests and Wilcoxon-Mann-Whitney tests, with significance is set at a value of p Results Eighty-five patients (53% female) with a median age of 65 years (range 19 to 98 years) underwent laparotomies. Four patients were excluded for active soft tissue infection. Wounds were classified as dirty (n = 18), contaminated (n = 52), and clean contaminated (n = 11). Median BMI was 27 kg/m2 (interquartile range [IQR] 23.4 to 33.0 kg/m2). Median antibiotic therapy was 4 days (IQR 1 to 7 days). Twenty-six patients had open abdomen management. Patient follow-up was a median of 20 days (range 14 to 120 days). Six patients (7%) developed superficial SSI requiring conversion to open wound management. No patients developed fascial dehiscence. There were no statistically significant associations between SSI and wound class (p = 0.072), antibiotic duration (p = 0.702), open abdomen management, or preoperative risk factors (p Conclusions Primary closure of high risk incisions combined with NPWT is associated with acceptably low SSI rates. Due to the low morbidity and decreased cost associated with this technique, primary closure with NPWT should replace open wound management in the emergency general surgery population.

Falls are the leading cause of trauma-related mortality in geriatric patients. We hypothesized that frailty and anticoagulation status are risk factors for readmission and mortality following falls in patients >80 years. A retrospective review was performed on patients over 80 years old who presented to our level 1 trauma center for a fall and underwent a computed tomography of the head between January 2014 and January 2016. Frailty was assessed via the Rockwood Frailty Score. Clinical outcomes were death, readmission, recurrent falls, and delayed intracranial hemorrhage. Of 803 fall-related encounters, 173 patients over 80 years old were identified for inclusion. The 30-day readmission rate was 17.5% and was associated with an increased 6-month mortality (P = 0.01). One-year and 2-year mortality rates were 28% and 47%, respectively. Frailty was the strongest predictor of 6-month and overall mortality (P

Background Massive transfusion protocols (MTPs) are associated with severe hypocalcemia, contributing to coagulopathy and mortality in severely injured patients. Severity of hypocalcemia following massive transfusion activation and appropriate treatment strategies remain undefined. Study design and methods This was a retrospective study of all MTP activations in adult trauma patients at a Level 1 trauma center between August 2016 and September 2017. Units of blood products transfused, ionized calcium levels, and amount of calcium supplementation administered were recorded. Primary outcomes were ionized calcium levels and the incidence of severe ionized hypocalcemia (iCa ≤1.0 mmol/L) in relation to the volume of blood products transfused. Results Seventy-one patients had an MTP activated during the study period. The median amount of packed red blood cells (PRBCs) transfused was 10 units (range 1-52). A total of 42 (59.1%) patients had periods of severe hypocalcemia. Patients receiving 13 or more units of PRBC had a greater prevalence of hypocalcemia with 83.3% having at least one measured ionized calcium ≤1.0 mmoL/L (p = .001). The number of ionized calcium levels checked and the amount of supplemental calcium given in patients who experienced hypocalcemia varied considerably. Discussion Severe hypocalcemia commonly occurs during MTP activations and correlates with the number of packed red blood cells transfused. Monitoring of ionized calcium and amount of calcium supplementation administered is widely variable. Standardized protocols for recognition and management of severe hypocalcemia during massive transfusions may improve outcomes.

α7-nAChR is a nicotinic acetylcholine receptor [specifically expressed on hepatic stellate cells (HSCs), Kupffer cells, and cholangiocytes] that regulates inflammation and apoptosis in the liver. Thus, targeting α7-nAChR may be therapeutic in biliary diseases. Bile duct ligation (BDL) was performed on wild-type (WT) and α7-nAChR−/− mice. We first evaluated the expression of α7-nAChR by immunohistochemistry (IHC) in liver sections. IHC was also performed to assess intrahepatic bile duct mass (IBDM), and Sirius Red staining was performed to quantify the amount of collagen deposition. Immunofluorescence was performed to assess colocalization of α7-nAChR with bile ducts (costained with CK-19) and HSCs (costained with desmin). The mRNA expression of α7-nAChR, Ki-67/PCNA (proliferation), fibrosis genes (TGF-β1, fibronectin-1, Col1α1, and α-SMA), and inflammatory markers (IL-6, IL-1β, and TNF-α) was measured by real-time PCR. Biliary TGF-β1 and hepatic CD68 (Kupffer cell marker) expression was assessed using IHC. α7-nAChR immunoreactivity was observed in both bile ducts and HSCs and increased following BDL. α7-nAChR−/− BDL mice exhibited decreased (i) bile duct mass, liver fibrosis, and inflammation, and (ii) immunoreactivity of TGF-β1 as well as expression of fibrosis genes compared to WT BDL mice. α7-nAChR activation triggers biliary proliferation and liver fibrosis and may be a therapeutic target in managing extrahepatic biliary obstruction.

The oncologic benefit of a central lymph node dissection (CLND) at the time of modified radical neck dissection (MRND) in patients with papillary thyroid cancer who have previously undergone a total thyroidectomy (TT) has not been studied. Patients with lateral cervical metastases were divided into two treatment groups: the concurrent cohort (TT with CLND and MRND), and the interval cohort (CLND and MRND after prior TT). Primary outcomes were lymph node metastases, skip metastases, level VI cancer recurrence, hypoparathyroidism and recurrent laryngeal nerve injury. Treatment groups consisted of 63 and 16 patients in the concurrent and interval groups, respectively. More central lymph nodes were removed (15.4 ± 8.4 to 10.1 ± 5.2 ( P = 0.02)), but similar level VI lymph node metastasis occurred (92.0–93.8% ( P = 0.99)) in the concurrent group compared with the interval group, respectively. Skip metastases were identified in only 7.6 per cent of patients. The incidence of level VI recurrence and recurrent laryngeal nerve injury was 1.2 per cent. Three patients developed hypoparathyroidism (3.7%). All permanent morbidities occurred in the concurrent group. CLND at the time of MRND for metastatic papillary thyroid cancer frequently identifies level VI metastases and can be done with low operative morbidity by experienced endocrine surgeons, even in patients who have undergone a prior TT.

Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome with neuroendocrine tumorigenesis of the parathyroid glands, pituitary gland, and pancreatic islet cells. TheMEN1gene codes for the canonical tumor suppressor protein, menin. Its protein structure has recently been crystallized, and it has been investigated in a multitude of other tissues. In this review, we summarize recent advancements in understanding the structure of the menin protein and its function as a scaffold protein in histone modification and epigenetic gene regulation. Furthermore, we explore its role in hepatobiliary autoimmune diseases, cancers, and metabolic diseases. In particular, we discuss how menin expression and function are regulated by extracellular signaling factors and nuclear receptor activation in various hepatic cell types. How the many signaling pathways and tissue types affect menin’s diverse functions is not fully understood. We show that small-molecule inhibitors affecting menin function can shed light on menin’s broad role in pathophysiology and elucidate distinct menin-dependent processes. This review reveals menin’s often dichotomous function through analysis of its role in multiple disease processes and could potentially lead to novel small-molecule therapies in the treatment of cholangiocarcinoma or biliary autoimmune diseases.

Cholangiocytes, a small population of cells within the normal liver, have been the focus of a significant amount of research over the past two decades because of their involvement in cholangiopathies such as primary sclerosing cholangitis and primary biliary cholangitis. This article summarizes landmark studies in the field of cholangiocyte physiology and aims to provide an updated review of biliary pathogenesis. The historical approach of rodent extrahepatic bile duct ligation and the relatively recent utilization of transgenic mice have led to significant discoveries in cholangiocyte pathophysiology. Cholangiocyte physiology is a complex system based on heterogeneity within the biliary tree and a number of signaling pathways that serve to regulate bile composition. Studies have expanded the list of neuropeptides, neurotransmitters, and hormones that have been shown to be key regulators of proliferation and biliary damage. The peptide histamine and hormones, such as melatonin and angiotensin, angiotensin, as well as numerous sex hormones, have been implicated in cholangiocyte proliferation during cholestasis. Numerous pathways promote cholangiocyte proliferation during cholestasis, and there is growing evidence to suggest that cholangiocyte proliferation may promote hepatic fibrosis. These pathways may represent significant therapeutic potential for a subset of cholestatic liver diseases that currently lack effective therapies.

Purpose Cholangiocarcinoma (CCA) is a malignancy of the biliary epithelium that is associated with low five-year survival. The apelin receptor (APLNR), which is activated by the apelin peptide, has not been studied in CCA. The purpose of this study is to determine if inhibition of the apelin/APLNR axis can inhibit CCA growth. Methods Immunohistochemistry, rtPCR, immunofluorescence, flow cytometry, and ELISA was used to measure APLNR expression in human CCA cells and tissues. Mz-ChA-1 cells were treated with increasing concentrations of apelin and ML221, an APLNR antagonist. Expression of proliferative and angiogenic genes were measured via rtPCR. In vivo , Mz-ChA-1 cells were injected into the flanks of nu/nu mice, which were treated with ML221 (150 μg/kg) via tail vein injection. Results Expression of the apelin/APLNR axis was increased in CCA. In vitro , CCA proliferation and angiogenesis was inhibited by ML221 treatment. ML221 treatment significantly decreased tumor growth in nu/nu mice. Conclusion The apelin/APLNR axis regulates CCA proliferation and angiogenesis. Inhibition of the apelin/APLNR axis decreases tumor growth in our xenograft model. Targeting APLNR signaling has the potential to serve as a novel, tumor directed therapy for CCA.

Este trabajo analizó la producción del fonema /r/ de los adolescentes anglo-ingleses y punjabí-ingleses bilingües en el oeste de Yorkshire a partir de los datos recogidos en el año 2000. Se encontró una clara diferencia entre los hablantes anglo-ingleses que usan exclusivamente el /r/ británico estándar, [ɹ̠], y los hablantes bilingües punjabí-ingleses que usan el fonema /r/ británico tanto como otras variantes influenciadas por la vibrante retrofleja Punjabi /ɽ/. La influencia de esta última se demuestra en el movimiento formántico y la duración resultantes, así como por las observaciones cualitativas de los espectrogramas de los hablantes. Se predice que la preferencia de los hablantes punjabíes hacia los róticos punjabíes o los róticos británicos depende de si se identifican como "británicos asiáticos" integrados culturalmente o como asiáticos alienados culturalmente. Este estudio también considera la posibilidad de una progresión en la producción del rótico punjabi en los angloparlantes del oeste de Yorkshire en los últimos quince años.

Background This study compares the pathological outcomes and operative morbidity for papillary thyroid cancer (PTC) patients undergoing a primary total thyroidectomy (TT) with central lymph node dissection (CLND), to those undergoing an interval CLND following a previous thyroid operation, or for the unsuspected diagnosis of PTC. Methods Single-institution, retrospective review of PTC patients from 2000 to 2015 was performed. Three treatment groups were identified: primary TT/CLND, interval prophylactic CLND, and interval therapeutic CLND. Primary outcome measures were number of lymph nodes removed, hypoparathyroidism and recurrent laryngeal nerve (RLN) injury. Results Results for 30 prophylactic and 35 therapeutic interval CLND were compared with 218 patients undergoing primary TT/CLND. Interval CLND was associated with similar rates of cervical metastases, complications, and a trend towards decreased lymph node recovery. Conclusion Reoperative CLND for incidental PTC frequently identifies cervical lymph node metastases, potentially reduces recurrence, and can be performed with similar morbidity to a primary lymphadenectomy.

Background Liver transplantation remains the primary treatment for primary sclerosing cholangitis (PSC). Mdr2 −/− mice provide a reliable in vivo model of PSC and develop characteristic biliary inflammation and fibrosis. We tested the hypothesis that the tumor suppressor protein menin is implicated in the progression of liver fibrosis and that menin expression can be regulated in the liver via microRNA-24 (miR-24). Materials and methods Menin expression was measured in human PSC and Mdr2 −/− mice. Twelve-week-old FVB/NJ wild-type (WT) and Mdr2 −/− mice were treated with miR-24 Vivo-Morpholino to knockdown miR-24 expression levels. Liver fibrosis was evaluated by Sirius Red staining and quantitative polymerase chain reaction ( q PCR) for genes associated with liver fibrosis, such as fibronectin 1, collagen type 1 alpha 1, transforming growth factor-β1 (TGF-β1), and α-smooth muscle actin. Studies were also performed in vitro using immortalized murine cholangiocyte lines treated with miR-24 hairpin inhibitor and mimic. Results Menin gene expression was increased in Mdr2 −/− mice and late-stage human PSC samples. Treatment of FVB/NJ WT and Mdr2 −/− mice with miR-24 Vivo-Morpholino increased menin expression, which correlated with increased expression of fibrosis genes. In vitro , inhibition of miR-24 also significantly increased the expression of fibrosis genes. Conclusions Inhibition of miR-24 increases menin and TGF-β1 expression, subsequently increasing hepatic fibrosis in FVB/NJ WT and Mdr2 −/− mice. Modulation of the menin/miR-24 axis may provide novel targeted therapies to slow the progression of hepatic fibrosis into cirrhosis in PSC patients by altering TGF-β1 expression.

Cholangiocytes are the targets of several liver diseases termed cholangiopathies that result in cholestasis and the development of hepatic fibrosis and carcinogenesis. There are numerous studies investigating the role of cholangiocyte death during the progression of cholangiopathies to end-stage liver disease. This chapter reviews the pathophysiology of primary sclerosing cholangitis, primary biliary cholangitis, autosomal dominant polycystic kidney disease, and biliary atresia. We will also discuss current animal models used to study these cholangiopathies and review the mechanisms of cell death in these disease processes, including the roles of innate immune system, apoptosis, senescence, autophagy, and lipoapoptosis. A thorough understanding of cholangiocyte death during cholestatic liver disease is critical to develop targeted therapies against these disease processes.

Laparoscopic cholecystectomy (LC) is routinely performed as an outpatient operation. NSQIP tracks acute or symptomatic congestive heart failure (CHF) within 30 days of the index operation. This study aims to quantify adverse events after LC and determine if patients with CHF may benefit from pre-operative optimization or post-operative admission.This is a retrospective NSQIP database review of all adults undergoing LC between 2008 and 2012. Comorbidities examined were acute or decompensated CHF, along with coronary artery disease, chronic obstructive pulmonary disease, diabetes, dyspnea, obesity, and smoking status. Bivariate and multivariate analyses determined the impact of these conditions on complications.LCs were performed electively in 131,081 patients and emergently in 12,680 patients. Pneumonia, reintubation or death in CHF patients occurred in 9% and 18% of these operations, respectively. The odds ratios, among those with CHF compared to those without, for pulmonary complications was 4.7 (p 0.01, 95%CI: 3.38-6.6) in the elective and 3.7 (p 0.01, 95%CI: 1.89-7.07) in the emergent populations.Patients with acute or decompensated CHF may benefit from pre-operative cardiac optimization and post-operative admission to decrease the risk of pulmonary complications.

Nicotine, the main addictive substance in tobacco, is known to play a role in the development and/or progression of a number of malignant tumors. However, nicotine's involvement in the pathogenesis of cholangiocarcinoma is controversial. Therefore, we studied the effects of nicotine on the growth of cholangiocarcinoma cells in vitro and the progression of cholangiocarcinoma in a mouse xenograft model. The predominant subunit responsible for nicotine-mediated proliferation in normal and cancer cells, the α7 nicotinic acetylcholine receptor (α7-nAChR), was more highly expressed in human cholangiocarcinoma cell lines compared with normal human cholangiocytes. Nicotine also stimulated the proliferation of cholangiocarcinoma cell lines and promoted α7-nAChR–dependent activation of proliferation and phosphorylation of extracellular-regulated kinase in Mz-ChA-1 cells. In addition, nicotine and PNU282987 (α7-nAChR agonist) accelerated the growth of the cholangiocarcinoma tumors in our xenograft mouse model and increased fibrosis, proliferation of the tumor cells, and phosphorylation of extracellular-regulated kinase activation. Finally, α7-nAChR was expressed at significantly higher levels in human cholangiocarcinoma compared with normal human control liver samples. Taken together, results of this study suggest that nicotine acts through α7-nAChR and plays a novel role in the pathogenesis of cholangiocarcinoma. Furthermore, nicotine may act as a mitogen in cholestatic liver disease processes, thereby facilitating malignant transformation.

The diagnosis and treatment of liver disease remain a major health concern worldwide because of the diverse etiologies of this disease. For this reason, new therapeutic targets are greatly needed to halt the progression of this damaging disease. Upon initiation of liver injury by viral infection, autoimmune disease or toxin, and/or hepatitis, chronic disease may develop, which can progress to cirrhosis, hepatocellular carcinoma (HCC), cholangiocarcinoma, liver failure, or death. The Lin28/lethal-7 (let-7) molecular switch has emerged as a central regulator of multiorgan injuries and cancer development. Lin28 is a stem cell marker vital to initiation or maintenance of a stem cell phenotype. Lin28 has not been extensively studied in the liver, despite its ability to induce tissue regeneration via reprogramming of oxidative enzymes in other tissues and its involvement with numerous upstream regulators and downstream targets in liver disease. Theoretically, overexpression of Lin28 in certain forms of liver disease could be a potential treatment that aids in liver regeneration. Alternatively, Lin28 has been implicated numerous times in the progression of diverse cancer types and is associated with increased severity of disease. In this case, Lin28 could be a potential inhibitory target to prevent malignant transformation in the liver. This review seeks to characterize the role of Lin28 in liver disease.

Kupffer cells are the resident macrophages in the liver. They are located in hepatic sinusoid, which allows them to remove foreign materials, pathogens, and apoptotic cells efficiently. Activated Kupffer cells secrete various mediators, including cytokines and chemokines, to initiate immune responses, inflammation, or recruitment of other liver cells. Bile duct ligation (BDL) surgery in rodents is often studied as an animal model of cholestatic liver disease, characterized by obstruction of bile flow. BDL mice show altered functional activities of Kupffer cells compared with sham-operated mice, including elevated cytokine secretion and impaired bacterial clearance. Various mediators produced by other liver cells can regulate Kupffer cell activation, which suggest that Kupffer cells orchestrate with other liver cells to relay inflammatory signals and to maintain liver homeostasis during BDL-induced liver injury. Blocking or depletion of Kupffer cells, an approach for the treatment of liver diseases, has shown controversial implications. Procedures in Kupffer cell research have limitations and may produce various results in Kupffer cell research. It is important, however, to reveal underlying mechanisms of activation and functions of Kupffer cells, followed by hepatic inflammation and fibrosis. This review summarizes present Kupffer cell studies in cholestatic liver injury.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by life-threatening ventricular arrhythmias and fibrofatty replacement of the cardiac tissue. Desmosomes are prominent cell-cell junctions found in a variety of tissues that resist mechanical stress, including the heart, and recruit the intermediate filament cytoskeleton to sites of cell-cell contact. Mutations in several desmosomal components including plakophilin-2 have been identified in ARVC patients; however, the molecular interactions disrupted by plakophilin-2 mutations are currently unknown. To understand the pathological basis of ARVC, the authors analyzed desmosome assembly and stability in epithelial cell lines expressing mutants of plakophilin-2 found in ARVC patients. Mutant plakophilin-2 proteins were unable to disrupt established desmosomes when expressed in an E-cadherin-expressing epithelial cell model; however, they were unable to initiate de novo assembly of desmosomes in an N-cadherin-expressing epithelial cell model. These studies expand our understanding of desmosome assembly and dynamics.

Background Treatment strategies for papillary thyroid cancer remain controversial due to the lack of large, randomized controlled trials. The purpose of this study was to review the benefit of routine bilateral central lymph node dissection (CLND) by analyzing local recurrence and complication rates from a single institution over a 15-year period. Methods A retrospective, institutional review board–approved review of the Baylor Scott & White Tumor Registry was performed on all patients who underwent operation for papillary thyroid cancer between 2000 and 2015. Patients were evaluated by age, sex, tumor size, operation performed, pathologic findings, adjuvant therapy, and date of recurrence. Primary outcomes were cancer recurrence, recurrent laryngeal nerve injury, and hypoparathyroidism. Results Total thyroidectomy with CLND was performed in 266 patients. Metastases to level VI lymph nodes were present in 106/266 (39.8%) patients. Average follow-up after thyroidectomy was 46 months (range 1–125 months). Papillary thyroid cancer recurred in 4 patients after thyroidectomy with CLND for primary tumors with mean size of 1.6 cm (range 1.0–2.0 cm). Two patients with T4 tumors had local recurrence in the paratracheal soft tissues, and 2 patients presented with recurrence in the lateral neck. Temporary nerve injuries occurred in 9/266 (3.4%) and permanent nerve injuries in 1/266 (0.4%) of CLND. Permanent hypoparathyroidism occurred in 4/266 (1.5%) patients. Conclusion Total thyroidectomy with CLND can safely be performed routinely for treatment of papillary thyroid cancer in the hands of experienced endocrine surgeons. Dissection of level VI lymph nodes does not increase the risk of recurrent laryngeal nerve injury when performed routinely. Bilateral CLND with total thyroidectomy for papillary thyroid cancer potentially minimizes recurrence in the level VI compartment.

Purpose: The cardiac glycoside family of steroidal drugs has been shown to possess novel and potent antitumor activities in rodents. Yet most likely due to their cardiotoxic nature, these drugs have failed to show clinical efficacy at doses deemed safe to humans. In order to determine if this drug class could be made safer and more efficacious, we assessed the antitumor activity of a new antibody drug conjugate system in which these steroidal compounds are tethered to antibodies that direct them to protein complexes found on various types of cancer cells. Experimental design: The activity and safety profiles of these novel antibody drug conjugates were examined using multiple cancer cell lines, normal cells, xenograft models in immunodeficient mice and non-human primates. Results: We identified multiple monoclonal antibodies that when conjugated to cardiac glycosides through long stable linkers, could provide cell killing activity independent of effector functions. When tested in vitro on a number of cancer cell lines, these active antibody drug conjugates termed Extracellular Drug Conjugates or EDCs, displayed potent cytotoxic activities with half maximal effective concentration (EC50) in sub-nanomolar ranges via a pathway resembling apoptosis and/or necroptosis. These activities were dependent on the expression of the corresponding antibody, linker length and steroid chemistry. In vivo using various tumor xenograft models, the EDCs were able to partially or completely regress tumor growth when 1 to 10 mgs/kg were administered intravenously. The EDCs were also found to assist various clinically approved therapies regress tumor growth. Finally, because cynomolgus monkeys respond to cardiac glycosides is similar to humans, 5mgs/kg of EDC was slowly infused intravenously into a cynomolgus monkey with no adverse effects observed even though the relative blood concentration of the cardiac glycoside attached reached 1500 nanomolar. Conclusion: These results support efforts to further evaluate these unique targeting antibody drug conjugates for the treatment of human cancers. Citation Format: James R. Prudent, David Marshall, John Murphy, Chad Hall, Scott Harried. Antibody targeted steroids for the treatment of cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A125.

RESULTS: The resection margins were clearly visualized. The average surgical time of group A (19.2 min) was significantly longer than group B (12.3 min) and C (13.1 min). Hemorrhage volume was less than a gauze. The average AST/ALT level 3-day postoperation were 124.2/107.6, 88.6/73.3 and 76.9/64.1 respectively. The rest of the laboratory data were close. Angiogram showed effective occlusion at the target region after the gel administration. Angiogram and necropsy showed no evidence of abnormities 3 months after the operation.

In numerous experimental cancer models, overexpression of eukaryotic translation initiation factor 4E (eIF4E) can drive oncogenic transformation, enable invasiveness and facilitate metastasis. eIF4E binds the 7-methylguanosine cap structure at the 5’ terminus of cellular mRNAs, recruiting these mRNAs into the multi-protein translation complex known as eIF4F. The eIF4F complex is comprised of eIF4E, the eIF4G scaffolding protein and the ATP-dependent RNA helicase, eIF4A. In addition, accessory factors (e.g. the Mnk kinases) can interact with this complex, further refining and regulating the function of the complex and the translation of mRNAs. A wealth of literature has accumulated over the past 20 years demonstrating that enhanced eIF4E function, a common event in many human cancers and experimental cancer models, selectively and disproportionately upregulates the translation of potent growth and survival factors implicated in all aspects of malignancy- dysregulated cellular growth control (c-myc, cyclinD1), enhanced cellular survival (survivin, BCL-2, MCL-1), angiogenesis (VEGF, FGF-2), invasiveness and metastasis (MMP9, osteopontin). The oncogenic function of eIF4E is critically dependent upon the phosphorylation of eIF4E at serine 209- an event exclusively controlled by the Mnk1/2 kinases and dependent upon the binding of both eIF4E and the Mnk kinases to the eIF4G scaffolding protein. We sought to understand better the dynamics of eIF4F complex assembly and eIF4E phosphorylation. Using pharmacologic inhibitors of eIF4F assembly (rapamycin, 4EGI-1, Torin, AZD8055), we now show that eIF4E phosphorylation can be induced- independent of the assembly of the eIF4F complex. This increase in eIF4E phosphorylation was blocked by addition of the Mnk inhibitor cercosporamide, suggesting the event remains Mnk-dependent. Further supporting this notion, eIF4E phosphorylation could not be induced by any treatment in Mnk double knockout mouse embryo fibroblasts (DKO-MEFs). To delineate definitively whether eIF4E could be phosphorylated independent of the eIF4F complex, we transfected glioma cells and H293T cells with eIF4E mutants that cannot bind eIF4G (W73F, G139D and the W73F + G139D double mutants). Phosphorylation of these mutants was evident in all cells tested. In DKO-MEFs, these eIF4E mutants were also phosphorylated when co-transfected with wild-type Mnk1 or Mnk2. Importantly, these eIF4E mutants that cannot bind eIF4G could also be phosphorylated in the DKO-MEFS when co-transfected with Mnk 1 or 2 mutants that also fail to bind eIF4G. These data indicate that the eIF4E interaction with Mnk does not require binding to the eIF4G scaffolding protein and, collectively, suggest a revised model for eIF4E phosphorylation- one dependent upon the Mnk kinases but not necessarily dependent on eIF4F complex assembly. Citation Format: Chad Hall, Chad Dumstorf, Bruce Konicek, Nathaniel Robichaud, Ann McNulty, Steve Parsons, Jerry Pelltier, Nahum Sonenberg, Jeremy R. Graff. eIF4E phosphorylation is Mnk-dependent but does not require assembly of the eIF4F translation initiation complex. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-302. doi:10.1158/1538-7445.AM2014-LB-302

Abstract 1336 Enzastaurin (Enza) is an ATP-competitive inhibitor of PKCβ and related AGC family kinases and blocks signaling through the PI3K/AKT/TOR pathway. Accordingly, in preclinical tumor models, clinically achievable Enza doses suppress phosphorylation of AKT, GSK3β, ribosomal protein S6, mTOR, p70S6 Kinase and 4EBP1. Enza has shown significant activity in Diffuse Large B Cell Lymphoma (DLBCL) patients under clinical study. 4/55 DLBCL patients treated with Enza were progression- free after prolonged, continuous oral enza therapy, with 3/4 confirmed as complete responders (Robertson et al., JCO, 2007). Furthermore, preliminary analyses of intermediate or high-risk DLBCL patients treated with R-CHOP vs. Enza + R-CHOP suggest that Enza improves both progression-free survival and complete response rates (Hainsworth et al., JCO, 29: 2011, suppl; abstract 8016). Enza is currently being investigated in a phase 3 registration trial for DLBCL patients at high risk of relapse following R-CHOP therapy. Gene expression profiling studies have revealed molecular alterations involved in DLBCL. For example, overexpression of PKCβ is particularly evident in recurrent DLBCL and has been associated with reduced patient survival. Similarly, BCL-6, a transcriptional repressor likely involved in the corruption of appropriate B Cell differentiation, is overexpressed in ∼ 70% of DLBCL and the target of translocation in nearly 40% of DLBCL. From these studies, distinct molecular subtypes of DLBCL have emerged. The Activated B Cell (ABC) subtype, which carries the poorest prognosis, is defined by constitutive activation of NFκB whereas the Germinal Center (GC) subtype, which carries a better prognosis, is not. 10% of ABC DLBCLs harbor CARD11 mutations leading to constitutive NFκB activation, while another 20% have mutations in CD79A/B, enabling chronic B Cell receptor signaling. In both ABC and GC subtypes, chronic activation of the B Cell receptor complex signals through PKCβ and the PI3K/AKT pathway. Given its activity in DLBCL patients and its molecular mechanism of action, we chose to investigate the molecular basis for the direct anti-cancer activity of Enza, specifically in DLBCL, and to discern whether sensitivity to Enza is associated with distinct DLBCL subtypes. In multiple DLBCL lines, we show that Enza treatment at clinically achievable concentrations significantly reduces BCL-6 expression. We also show that Enza robustly induces apoptosis in both GC and ABC models, blocking signaling throughout the PI3K/AKT pathway. Moreover, we show in the same DLBCL models that the primary metabolite of enza (LY326020.HCl), which comprises ∼50% of circulating drug levels in patients, more potently inhibits these same signaling pathways and more robustly induces apoptosis. Importantly, we have also identified cell lines that are resistant to both enza and its metabolite. Profiling differences between these resistant and sensitive DLBCL lines, we now show that the pro-apoptotic activity of Enza and LY326020 requires control of the eIF4F translation initiation complex- the complex responsible for enabling translation of critical, malignancy-related mRNAs (e.g. VEGF, BCL-2, c-myc). In insensitive DLBCL cells, 4EBP1 expression levels are low to non-existent, even prior to treatment. In sensitive DLBCL cells, Enza and LY326020 reduce 4EBP1ser65 phosphorylation in a dose and time-dependent manner, increasing 4EBP1: eIF4E binding thereby precluding eIF4E:eIF4G binding and preventing eIF4F complex assembly. Furthermore, selection of DLBCL cells for resistance yields cells with reduced 4EBP1 expression, increased eIF4G expression, or both, highlighting the critical importance of this complex for Enza activity. Collectively, these data show that: 1) Enza and LY326020 have direct anti-cancer activity in DLBCL cells regardless of ABC or GC subtype; 2) Enza and LY326020 directly impact numerous molecular alterations driving DLBCL (BCL-6 expression, PKCβ and PI3K-AKT pathway signaling); 3) the ability to control eIF4F complex assembly is critical for enza and LY326020 –induced apoptosis; and 4) LY326020 is more potent than Enza at blocking signaling and inducing apoptosis. Moreover, these data suggest that baseline expression levels of 4EBP1 and/or levels of phosphorylated eIF4E (an indicator of active eIF4F complex) may be particularly informative in delineating patients most sensitive to treatment. Disclosures: Graff: Eli Lilly and Company: Employment. McNulty:Eli Lilly and Company: Employment. Dumstorf:Eli Lilly and Company: Employment. Konicek:Eli Lilly and Company: Employment. Hall:Eli Lilly and Company: Employment. Parsons:Eli Lilly and Company: Employment.

8082 Background: Enzastaurin (enza) is in ph 3 registration trials for DLBCL patients at high risk of relapse following R-CHOP therapy. In a phase 2 DLBCL study, 4 of 55 treated patients were progression- free after prolonged, continuous oral enza therapy with 3 of these 4 confirmed as complete responders (Robertson et al., JCO, 2007). The molecular mechanism for this differential response is unclear. Methods: In clinical trials, Enza yields 2-4 µM total circulating drug, comprised of ~50% enza, ~50% primary metabolite, LY326020. We therefore evaluated the sensitivity of a DLBCL cell panel representing both Activated B Cell (ABC) and Germinal Center (GC) subtypes to enza and LY326020. Gene expression analyses, western blotting to explore intracellular signaling and mRNA cap analogue co-capture assays were used to identify the critical effectors of drug sensitivity. Results: For the first time, we show the profound biological activity of LY326020, the primary metabolite that accounts for ~ 50% of circulating drug in patients. Like Enza, though more potently, LY326020 inhibits PKC and PI3K-AKT-TOR pathway signaling and robustly induces apoptosis in both ABC and GC DLBCL cells. In both sensitive and resistant cells, enza and LY326020 reduced phosphorylation of numerous proteins in the PI3K-AKT-TOR pathway (e.g. pGSK3βser9) in a dose and time-dependent manner. However, only sensitive DLBCL cells showed reduced 4EBP1ser65 phosphorylation. Accordingly, we show a dose and time-dependent increase in 4EBP1: eIF4E binding. This increase is most pronounced by LY326020. Moreover, cells selected for resistance to enza show reduced 4EBP1 expression and cells lacking 4EBP1 are insensitive to the pro-apoptotic effects of enza and LY326020. Conclusions: These data demonstrate that sensitivity of DLBCL to both enza and LY326020 is critically dependent upon 4EBP1 modulation and subsequent disruption of the eIF4F translation complex. Moreover, these data are the first to show the potent biologic activity of LY326020, the primary metabolite of enza that accounts for ~50% of total circulating drug in patients and in preclinical models.

In a wide variety of malignancies, progressive disease and reduced patient survival have been linked to increased function of eukaryotic translation initiation factor 4E (eIF4E), which selectively enhances the translation of potent growth and survival factors and oncoproteins (e.g. c-myc, VEGF, cyclin D1, Mcl-1, MMP-9, etc.). Recent published reports have shown that the oncogenic activity of eIF4E is critically dependent on the activity of the MNK kinases (Map kinase interacting kinase 1 and 2), which phosphorylate eIF4E at S209 (p4ES209). Increased p4ES209 has been linked to a variety of advanced cancers, notably head and neck and non-small cell lung cancers. Herein, we report that p4ES209 is significantly elevated in human prostate cancer (CaP) tissues (n=133) and associated with reduced patient survival. p4ES209 is elevated In both low grade (Gleason score ≤ 6) and high grade (Gleason score ≥ 7) CaP vs. adjacent normal prostate tissue (BPH) (p < 0.02 and p < 0.0001, respectively). Elevated p4ES209 in invasive CaP is related to significantly reduced patient survival (p = 0.0216) and increased risk of death from CaP (hazard ratio = 2.729). Moreover, p4ES209 is evident in the LNCaP, LNAI, CWR22Rv1 CaP cell lines, with levels being highest in the castrate-resistant cells (LNAI and CWR22Rv1). shRNA-mediated knock-down of MNK 1 and 2 reduced p4ES209 levels and specifically triggered apoptosis. Similarly, treatment of these cell lines with the novel, orally-bioavailable MNK inhibitor, cercosporamide, reduced p4ES209 in a dose-dependent manner and induced apoptosis coincident with reduced expression of key translationally controlled proteins (e.g. Mcl-1). Further, oral administration of this inhibitor can suppress p4ES209 in normal mouse tissues and in xenografted human cancers within 30 minutes, lasting at least 4 hours in tumor tissue. These data demonstrate the utility of cercosporamide in probing MNK function in vitro and in vivo and highlight the potential utility of targeting MNK for prostate cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A237.

Menin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated invitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24–dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression.