bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract A38: Resistance to Afatinib and Cetuximab Combination Therapy in EGFR-mutant Lung Adenocarcinomas
Authors: Pirazzoli Valentina , Takezawa Ken , de Stanchina Elisa , Pao William , Politi Katerina 
Published: March 2013

Abstract
The epidermal growth factor receptor (EGFR) T790M mutation confers acquired resistance to tyrosine kinase inhibitors (TKIs) in approximately 50% of drug-resistant EGFR mutant lung adenocarcinomas. Experiments using genetically engineered mouse models of EGFR mutant lung cancer have revealed that T790M-mediated resistance can be overcome by a second-generation TKI, afatinib, in combination with the anti-EGFR antibody, cetuximab. This drug combination is currently in clinical trials in patients with TKI-resistant tumors and is showing a promising ∼40% response rate. Nevertheless, cases of afatinib+cetuximab resistance are beginning to emerge.
To identify the molecular mechanisms that play a role in afatinib+cetuximab resistance and test new therapies to overcome resistance to these drugs, we have developed mouse models of afatinib+cetuximab resistance in mice with inducible expression of the EGFRL858R+T790M mutant in type II pneumocytes. Mice harboring lung tumors were treated five days a week with afatinib and twice a week with cetuximab for four weeks. After the first round of treatment, drug-treatments were interrupted for one month. This on/off drug treatment was repeated until lung tumors no longer responded to treatment, as evidenced by imaging as well as clinical symptoms. Resistance to afatinib+cetuximab treatment was also studied in xenografts. Eight-week-old nu/nu athymic nude mice were injected subcutaneously with human lung adenocarcinoma cells, expressing the erlotinib-resistant EGFRDel19+T790M mutant. Notably, both transgenic and xenografts models harboring EGFRT790M-induced tumors developed resistance to EGFR-dual targeting and preliminary molecular studies on the resistant tumors show the absence of additional mutations in EGFR and in the ERBB2 tyrosine kinase domain in resistant tumors. These findings suggest that an alternative pathway or mechanism is involved in mediating resistance to the afatinib and cetuximab combination. We are further exploring mechanisms of resistance by performing genome-wide and signaling pathway analyses of drug-resistant tumors from these models. Findings from mouse models will be validated in repeat biopsy specimens from patients who have developed resistance to the drug combination. An improved understanding of the molecular mechanisms responsible for acquired resistance to EGFR inhibition will provide new insight into the biology of this subset of lung cancers with immediate therapeutic implications for patients.
Abstract A43: The Pi3K inhibitor GSK2126458 is highly toxic in breast ductal carcinoma
Authors: Albawardi Alia , Almarzooqi Saeeda , Al Bashir Mohamed , Souid Abdul-Kader 
Published: March 2013

Abstract
Introduction: Breast carcinoma has been shown to harbor mutations that up-regulate phosphatidylinositol 3 kinase (PI3K) signaling pathway. Thus, targeting this pathway is expected to impair tumor survival. The aim of this work was to investigate the cytotoxicity of GSK2126458 (a highly potent and selective inhibitor of PI3K) in breast ductal carcinoma from a patient.
Methods: A breast ductal carcinoma was collected immediately following resection. Tumor sections (0.5×0.5×0.1 cm) were immersed in ice-cold RPMI medium gassed with 95% O2:5% CO2. The samples were incubated at 37oC in RPMI (continuously gassed as above) with and without 50 nM GSK2126458 (Selleck Chemicals, cat. #S2658) for 90 min (final volume = 2.0 mL). At the end of the incubation period, 37 μM Ac-DEVD-AMC (a caspase-3 substrate) was added with and without the pan-caspase inhibitor zVAD-fmk (32.1 μM, added 10 min before the substrate). The reactions were allowed to continue at 37oC for 20 min before tissue processing for histology, immunohistochemistry for cleaved caspase -3 (Cell Signaling Technology, kit #8120), cellular respiration and caspase activation. Mitochondrial O2 consumption was determined as function of time from the phosphorescence decay rate (1/T) of Pd(II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. For caspase activity, the Ac-DEVD-AMC cleavage reaction was quenched with tissue disruption and passages through a 27-G needle. The supernatants were collected by centrifugation (∼16,300g for 90 min) through Microcentrifuge Filter (nominal molecular weight limit = 10,000 Dalton, Sigma©), separated on HPLC, and analyzed for the free fluorogenic AMC moiety.
Results: This 73-year-old female had invasive ductal carcinoma (grade 3), measuring 4.5 cm. The tumor expressed estrogen and Her2-neu receptors. Ki-67 score was 70%. Compared to untreated tumor, the treated tumor revealed cellular disintegration with loss of cohesion, cytoplasmic vacuolization and numerous apoptotic bodies. Positive cleaved caspase-3 staining was noted in 10% of untreated tumor and 20% in treated tumor. The initial rate of mitochondrial O2 consumption (kc, in μM O2 min−1 mg−1) was 0.17. At 2 h, the value of kc for untreated tumor was 0.019 and for treated tumor 0.033. The AMC peak area (arbitrary units, reflecting intracellular caspase activation) in untreated tumor was 460,886, in tumor treated with GSK2126458 was 7,234,911 (15.7 fold increment), and in tumor treated with GSK2126458 plus zVAD-fmk was 1,523,682 (79% inhibition), confirming the cleavage of Ac-DEVD-AMC was mainly mediated by caspases.
Conclusions: GSK2126458 induced remarkable necrosis in invasive ductal carcinoma. This preliminarily observation supports the role of PI3K activation in breast carcinogenesis and possible therapeutic utility of PI3K inhibitors in the treatment of invasive ductal carcinoma. The described in vitro methodology provides a rapid assessment of the cytotoxicity of PI3K/PTEN/Akt/mTOR inhibitors.
Abstract A44: The Pi3K inhibitor GSK2126458 is highly toxic in ovarian dysgerminoma
Authors: Almarzooqi Saeeda , Albawardi Alia , Souid Abdul-Kader 
Published: March 2013

Abstract
Introduction: Mutations in PI3K/PTEN/Akt/mTOR have been implicated in the pathogenesis of numerous human carcinomas. Moreover, inhibitors of the phosphatidylinositol 3-kinase (PI3K) pathway are shown to be potent in several tumors, including ovarian adenocarcinomas. The effect of this class of agents on ovarian germ cell tumors, however, has not been previously studied. The aim of this work was to investigate cytotoxicity of the PI3K inhibitor GSK2126458 (a highly potent and selective compound) in ovarian dysgerminoma from a patient.
Methods: The studied ovarian dysgerminoma was collected immediately following resection. Tumor sections (0.5×0.5×0.1 cm) were immersed in ice-cold RPMI medium gassed with 95% O2:5% CO2. The samples were then incubated at 37°C in RPMI (continuously gassed as above) with and without 50 nM GSK2126458 (Selleck Chemicals, cat. #S2658) for 150 min. At the end of the incubation period, specimens were processed for hematoxylin and eosin histologic stain.
Results: Sections from untreated tissue revealed variable-sized tumor nests separated by fibrous stromal bands. Neoplastic cells were characterized by uniformly round central nuclei, clear-to-eosinophilic cytoplasm and prominent single nucleoli. Mitotic figures were abundant. Sections from treated tissue revealed a decrease in cellular density. Neoplastic cells demonstrated cytoplasmic disintegration, vacuolar degeneration and nuclear basophilia. Apoptotic bodies were numerous.
Conclusions: GSK2126458 induced remarkable necrosis in the ovarian dysgerminoma. This preliminarily observation suggests sensitivity of this tumor to PI3K inhibition. The described in vitro methodology provides rapid assessment of the cytotoxicity of PI3K/PTEN/Akt/mTOR inhibitors.
Abstract A49: Combination of bendamustine and the HDAC inhibitor entinostat enhances apoptosis and DNA damage response in multiple myeloma cells
Authors: Cai Bo , Huang Jingcao , Lee Choon-Kee , Liu Bolin 
Published: March 2013

Abstract
Bendamustine (Cephalon, Inc., West Chester, PA), a hybrid molecule of purine analog and alkylator, induces cell death by activation of DNA damage response and apoptosis, inhibition of mitotic checkpoints, and induction of mitotic catastrophe. Entinostat (also known as MS-275 or SNDX-275, Syndax Pharmaceuticals, Inc., Waltham, MA), a selective class I inhibitor of histone deacetylase (HDAC), induces apoptosis in multiple myeloma (MM) cells. We sought to determine whether the combination of bendamustine with entinostat enhances cell death of MM cells, and the potential mechanisms. Cell growth assay showed that bendamustine inhibited proliferation of MM cells in a dose-dependent manner. The IC50 of bendamustine for RPMI8226, U266 and MM1.S cells was approximately 662.6, 295.5, and 159.6 umol/L. Apoptotic-ELISA indicated that either bendamustine or entinostat was able to induce apoptosis in all three myeloma cells, and their combinations exhibited a much more potent activity to promote the MM cells undergoing apoptosis. Further studies with western blot analyses revealed that bendamustine in combination with entinostat dramatically induced PARP cleavage and activation of caspase-3 in all three MM cell lines, and also reduced the expression levels of cyclin D1, phospho-Stat3 (P-Stat3) and Stat3 in U266 cells. Moreover, studies on DNA damage response showed that phosphohistone H2A.X, a hall marker of DNA double strand break, was significantly induced by the combinations of bendamustine and entinostat as compared to either agent alone in all three MM cell lines. Our data suggest that bendamustine and its combination with entinostat possess anti-MM activity in vitro. Regimens consisting of bendamustine and/or entinostat may represent novel therapeutic strategies against MM.
Abstract A65: Combinational siRNA silencing of MCL-1 and P-gp enhances the apoptotic response in Human Breast Cancer Cells
Authors: Aliabadi Hamidreza Montazeri , Uludag Hasan 
Published: March 2013

Abstract
Multidrug resistance (MDR) remains is a major hurdle to overcome in cancer therapy. Enhanced cell proliferation and survival of cancerous cells in solid tumor environment along with survival of stem cells to repopulate the tumor and metastasis complicate the matter even more. Therefore, retardation of cell growth and/or induction of apoptosis by posttranscriptional silencing based on short interfering RNA (siRNA) is promising, but remains challenging due to inability of anionic siRNA to cross cell membrane. We previously reported the safety and efficiency of lipid substitution on small molecular weight, non-toxic polyethylenimine (PEI) as promising approach for siRNA delivery. In this study, we report silencing of myeloid cell leukemia 1 (MCL-1) both as an individual target to induce apoptosis and incorporated in multiple silencing in wild type (WT) MDA-MB-435 human breast cancer cells, as well as cells rendered resistant as a result of exposure to an anticancer agent and MDR version of the same cells. Down-regulation of P-glycoprotein (P-gp), survivin, and MCL-1 with respective siRNAs were studied individually and in combinations. While silencing MCL-1 caused almost 90% cell death in the WT cells, the effect was less significant in the MDR cells. Silencing both MCL-1 and P-gp in the MDR cells showed a synergistic effect. Resistance induction, as confirmed by both IC50 experiments and doxorubicin (DOX) uptake, caused an up-regulation in the expression of not only P-gp, but also BCRP, survivin, and MCL-1. Our double silencing experiments in resistance-induced cells showed a similar pattern to MDR cells; however, in the later stages of resistance induction, prolonged exposure to higher DOX concentrations caused a cell dependency on MCL-1 and a cross-link between MCL-1 and P-gp. MCL-1 silencing was proven to be more efficient in causing cell death in resistance-induced cells compared to MDR cells. A re-sensitization to cytotoxic effect of DOX was observed in resistance-induced cells after silencing of MCL-1. In addition to confirming MCL-1 as a viable target for siRNA silencing, this study indicates that anti-apoptotic proteins such as survivin and MCL-1 may have a role in resistance that could be closely related to efflux proteins.
Abstract B12: PTEN modulates sensitivity to a novel ERK inhibitor in BRAFV600E-mutant melanomas
Authors: Krepler Clemens , Herlyn Meenhard , Villanueva Jessie , Samatar Ahmed , Chen Yuhao , Halloran Molly , Samanta Minu , He Xu , Vultur Adina , Wubbenhorst Bradley , Nathanson Katherine 
Published: March 2013

Abstract
The presence of activating BRAF mutations in approximately 50% of melanomas, the majority of which are BRAFV600E, has prompted the development of selective inhibitors of the BRAF/MAPK/ERK pathway for targeted therapy. RAF inhibitors, including vemurafenib and GSK2118436, have shown striking results in clinical trials, highlighting this pathway as a suitable target for melanoma therapy. A number of other pharmacological agents targeting this pathway are currently in pre-clinical or clinical development. However, therapy with MAPK pathway inhibitors results in a wide range of response patterns in patients with BRAF-mutant tumors. Furthermore, even in patients with measurable responses, tumors eventually develop resistance and patients relapse. It has been previously reported that activation of the PI3K pathway may play a role modulating the response to RAF and MEK inhibitors. Here we report that melanoma cells which harbor the BRAFV600E mutation and express PTEN have low levels of phospho-AKT and are sensitive to a novel ATP-competitive ERK inhibitor. In contrast, BRAFV600E mutant melanoma cell lines that do not express PTEN have higher levels of phospho-AKT and are less sensitive to ERK inhibition. Importantly, we have confirmed these findings in an in vivo xenotransplantation model of melanoma. Additionally, we found that siRNA-mediated PTEN silencing in a PTEN wild-type cell line modestly decreases sensitivity to ERK inhibition. Conversely, PTEN overexpression in a PTEN-null cell line partially sensitizes cells to the ERK small molecule inhibitor. Consistent with these findings, targeting PI3K in melanoma cells lacking functional PTEN sensitizes them to ERK inhibition. Our studies suggest that the presence or absence of functional PTEN can modulate response to ERK inhibitors and warrant further evaluation of combination strategies to treat melanomas refractory to inhibitors of the MAPK pathway.
Abstract B17: Mechanisms of resistance of BRAF fusions to first generation selective BRAF inhibitors
Authors: Sievert Angela Jae , Lang Shih-Shan , Storm Phillip B. , Resnick Adam C. 
Published: March 2013

Abstract
Astrocytomas are the most common type of central nervous system tumor in children. We and others have previously reported that activated BRAF mutations, via the KIAA1549-BRAF fusion gene, are a key feature of low-grade astrocytomas. We generated several different cell lines stably expressing various altered BRAF and BRAF-fusion constructs and evaluated first and second-generation BRAF inhibitors including PLX4720 and PLX-PB compounds. In vitro kinase assays demonstrated the KIAA1549-BRAF fusion kinase, like V600E BRAF, undergoes inhibition in the presence of both first and second-generation selective BRAF inhibitors. However, KIAA1549-BRAF fusion expressing cell lines were resistant to PLX4720 and displayed paradoxal activation to first-generation BRAF inhibitor suggesting distinct and cell-context dependent mechanisms for MAPK activation. In contrast, KIAA1549-BRAF-fusion-mediated MAPK activation was abrogated by PB-3, a second-generation, “paradox-breaking,” selective BRAF inhibitor. This suggests that pediatric low-grade astrocytomas harboring the KIAA1549-BRAF fusion may be responsive to second-generation BRAF inhibitors. Our current studies are focused on understanding how KIAA1549-BRAF fusion signaling is mechanistically different than canonical BRAF V600E mutant and how this impacts the differential responses to selective BRAF inhibition.
Abstract B20: A genome-scale RNA interference screen for resistance mechanisms to BRAF inhibition in melanoma
Authors: Whittaker Steven , Hodis Eran , Wagle Nikhil , Hsiao Jessica , Cowley Glenn , Root David , Garraway Levi 
Published: March 2013

Abstract
Mutation of the serine/threonine protein kinase BRAF is observed in approximately 50% of melanomas. This leads to enhanced activation of the kinase, stimulation of the MAPK pathway and aberrant cell proliferation and tumorigenesis. Small molecule inhibitors such as vemurafenib (PLX4032) block BRAF-mediated cell proliferation and recent clinical trials show an overall response rate of about 50%. Evidently, some patients do not respond to the drug or go on to develop resistance while on treatment. Therefore, we designed an RNA interference screen to identify loss of function events that could drive resistance to growth-inhibitory concentrations of PLX4720. We used a pooled lentiviral shRNA library consisting of approximately 90,000 hairpins targeting over 16,500 genes. shRNA-infected cells were split into two groups, one treated with DMSO, the other treated with PLX4720. Following a period in culture, the abundance of each hairpin was assessed by PCR amplification of barcoded hairpin DNA followed by massively parallel paired-end sequencing. RIGER was used to rank the individual hairpins and identify candidate genes that were required for survival of A375 cells in the presence of DMSO, compared to an early time point control. Strikingly, BRAF emerged as the most depleted and therefore most essential gene, validating our screening approach. We then identified those hairpins that were enriched in the presence of PLX4720 compared to DMSO. A small number of genes were identified as candidate ‘resistance suppressors’, which when knocked down, conferred a survival advantage to BRAF inhibitor-treated cells. Validation of these genes in secondary assays and characterization of the resistance mechanism(s) will be presented. By integrating these findings with other genomic and functional studies we aim to identify clinically relevant genetic events that cause RAF-inhibitor resistance in melanoma.
Abstract B23: Reprogramming of the cancer kinome in response to targeted kinase inhibition
Authors: Duncan James S. , Whittle Marty W. , Graves Lee M. , Johnson Gary L. 
Published: March 2013

Abstract
Kinases are members of a large dynamic and cooperative signaling network, which senses inhibition of key nodal kinases and induces compensatory responses that offset pharmacological intervention. Combination therapies that target multiple growth- and survival promoting kinases are proving to be a better strategy for successful cancer therapy. What is lacking is the ability to measure whole kinome activity and to assess kinome adaptation and resistance to targeted therapies. We have developed a chemical proteomics approach that couples kinase affinity capture with quantitative mass spectrometry, providing a systems biology platform to profile global kinome activity in cancer cells, GEMM tumors and patient biopsies. Our chemical proteomic approach captures the majority of the expressed kinome estimated by RNA-seq and detects altered kinome activity profiles in response to stimulus or kinase inhibitors. Kinases from all major kinome subfamilies are captured with a large percentage representing the understudied kinome. For example, we discovered previously undefined activation of tyrosine and serine/threonine kinases in breast cancer cell lines in response to targeted kinase inhibitors that are currently in clinical trials. Combined kinome activity assessment using chemical proteomics and RNAi synthetic lethal screens predicted a specific kinase inhibitor combination therapy. The combination therapy gave apoptosis and tumor regression in a breast cancer GEMM, where single agents were largely ineffective. Thus we predicted an effective combination therapy based on assessment of kinome response to treatment of cells with drug.
Abstract B25: The Effects of PI3K/Akt/mTOR Pathway Inhibitors on a Panel of Non Small Cell Lung Cancer (NSCLC) Cell Lines
Authors: Heavey Susan , Barr Martin , Davies Anthony , O'Byrne Ken , Gately Kathy 
Published: March 2013

Abstract
Introduction: The purpose of this study is to investigate the effects of two PI3K pathway inhibitors on a panel of NSCLC cell lines, with a view to developing resistant cell line models, through exposure to the inhibitors over an extended period of time.
The PI3K/Akt/ mTOR pathway regulates cell growth and proliferation and is often dysregulated in cancer due to mutation, amplification, deletion, methylation and posttranslational modifications. Multiple novel agents, targeting different regulators within the pathway are currently under development. GDC-0941 is a PI3K targeted inhibitor which is currently in phase Ib trials for combination treatment with bevacizumab, trastuzumab and erlotinib in metastatic NSCLC. GDC-0980 is a selective dual inhibitor of PI3K and mTOR, which is currently in phase I trials for combination treatment with bevacizumab, trastuzumab, and capecitabine in solid tumors.
Methods: The effects of GDC-0941 and GDC-0980 were assessed in a panel of lung cancer cells, including normal human bronchial epithelial cells (HBECs), H460 cells (PI3KCA & KRAS mutant), A549 cells (wild-type PI3KCA & KRAS mutated), cisplatin resistant H460 and A549 cells and H1975 cells (PI3KCA mutant /T790M mutant EGFR TKI resistant). The effect of each inhibitor on pAkt levels was examined by Western blot. Effects of the inhibitors on apoptosis and proliferation were analyzed by high content analysis and BrdU assays respectively. IC25 values obtained from these experiments were then used to begin treating cells over an extended period of time, in order to develop a cell line model of resistance.
Results: Treatment of the panel of cell lines with either GDC-0941 or GDC-0980 resulted in reduced pAkt levels as observed by Western blot, as well as dose-dependent increased apoptosis and decreased proliferation of cell lines. IC25 values are currently being used to treat cell lines over an extended period of time in order to develop models of resistance to the two inhibitors.
Discussion: Both GDC-0941 and GDC-0980 were shown to inhibit the PI3K pathway, as observed by the reduced levels of pAkt present after treatment with the inhibitors. Both inhibitors increase apoptosis and decrease proliferation in a dose-dependent manner, with GDC-0980 (dual PI3K/mTOR inhibitor) showing more significantly increased apoptosis and decreased proliferation than GDC-0941 (PI3K inhibitor). Resistant cell line models are currently being developed as a result of this research which will allow biomarkers of response/resistance to the drugs to be identified in the future.