bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract IA9: The molecular pathology of adenocarcinoma versus histopathological classification
Authors: Brambilla Elisabeth
Published: March 2013

Recent therapeutic advances have led to a revolution in the lung cancer field in discovering therapeutically tractable oncogene dependency with major implications for patient evaluation and approach to diagnosis. A recently published IASLC/ATS/ERS classification of lung adenocarcinoma addresses these issues fully based on the molecular features of histological subtypes of adenocarcinoma (1). As deduced from their molecular signatures, pathological patterns are the most achieved translation of the full genomic space (expression profiles, gene copy number alterations, and likely epigenetics space …). Therefore the functional clusters of genes defining a phenotypic population dictates the survival and contains targets of therapies in adenocarcinoma supporting the contention that each of the pathological and molecular features contains this information (2, 3). The new classification of adenocarcinoma has a high prognostic interest validated on further independent series of the literature (4). Molecular pathology recapitulates both differentiation markers and biomarkers of therapeutic sensitivity.

A stepwise accumulation of genetic and molecular abnormalities is sustaining the morphological transition between preinvasive and invasive and metastatic process in adenocarcinoma.

Now that lung cancer therapies personalized for individual patients is based on the histologic type of lung cancer and molecular status, the pathologist role using the knowledge on molecular pathology is to allow the histomolecular classification on small biopsies and cytology, the only specimens available in advanced treatable cases of adenocarcinoma (5).

A few differentiation markers (TTF-1, P63, P40, mucines) met the need for subclassification of cases with adenocarcinoma components (adenocarcinoma per se, large cell carcinoma, sarcomatoid carcinoma and NSCLC/NOS) driving their molecular analysis (6).

TTF-1 positive solid and cribriform tumor with signet ring are strongly associated with susceptibility for EML4-ALK fusion for ALK positivity candidate for crizotinib therapy, whereas Ras mucinous adenocarcinoma (previously mucinous BAC) are essentially TTF-1 negative, amphiregulin positive with IGFR activated pathway which are strongly resistant to gefitinib alone but are restored to sensitivity by IGFR inhibitors (7). Articulation between molecular pathology and histopathological features will in the future be the first organizers of therapeutical decisions (7).

Conclusion: As the molecular changes directly influence the therapy today and even more in the future, we think this should be reflected in the taxonomy as well as in the term given in the WHO classification. Histopathological classification would tend to predict that the molecular characteristics of the tumors together with their immunohistochemical and morphological appearance will dictate therapy in the future. Gefitinib is less effective in non adenocarcinoma NSCLC harboring EGFR mutations (8).
Abstract PR1: Integrating expression data improves mutational significance analysis of lung squamous carcinoma
Authors: Hernandez Bryan , Hammerman Peter , Imielinski Marcin , Lawrence Michael , Stojanov Petar , Getz Gad , Meyerson Matthew
Published: March 2013

In contrast to lung adenocarcinoma in which a driving and potentially targetable genetic aberration can be identified in excess of 50% of tumors, only 10% can be found in squamous cell lung cancer, the second most common type of lung cancer, none of which are druggable by an approved targeted therapy. Our limited knowledge regarding targetable genetic alterations in squamous cell lung cancer is the result of a lack of detailed genomic study of this disease and the lack of experimental validation of identified putative therapeutic targets. We have analyzed exome sequencing data to identify novel driving mutations in 184 lung squamous cell carcinomas using previously successful methods (Integrated genomic analyses of ovarian carcinoma, Nature 2011), but these analyses are confounded by an unusually high mutation rate, 8-9/Mb on average, some reaching as many as 80/Mb. Many of the most significantly mutated genes in the analysis are unlikely related to the pathogenesis of the disease based on a lack of expression in lung and known role in cancer, suggesting that genes which are not expressed (or minimally expressed) escape negative selection and result in a higher mutation density as compared with expressed genes.

Here, we present a method integrating expression and mutation data in order to more accurately determine the significance of a mutated gene based on the expression subclass in which it falls and the frequency at which it's mutated relative to our expectation; unexpressed genes are likely much more tolerant of mutations and should therefore have a higher expected background mutation rate. Analyses of this kind have removed many of the genes putatively irrelevant in this cancer type and have identified many biologically plausible novel genes that to date are unlinked with lung squamous cell carcinoma. We believe this method refines an already powerful analysis for identifying significantly mutated genes in cancer with a particularly high potential for separating signal and noise in the most highly mutated cancer types.

This abstract is also presented as Poster A17.
Abstract A1: Functional significance of miR-10b in tamoxifen resistance of breast cancer cells
Authors: Ahmad Aamir , Reddy Kaladhar B. , Sarkar Fazlul H.
Published: March 2013

Tamoxifen is a conventional treatment option for breast cancer patients diagnosed with estrogen receptor (ER)-positive tumors. However, a significant number of these patients eventually develop resistance to tamoxifen during therapy, and thus the management of such tamoxifen-resistant patients remains a clinical challenge. In an effort to identify novel targets for the treatment of tamoxifen-resistant tumors, we observed increased expression of microRNA 10b (miR-10b) in ER-positive MCF-7 cells-derived tamoxifen-resistant cells (MCF7TR). The miR-10b has been implicated in invasion and metastasis but its role, if any, in drug resistance of breast cancer cells has never been investigated. To ascertain the mechanistic role of miR-10b in tamoxifen resistance, we downregulated its levels in MCF7TR cells and in addition we also overexpressed miR-10b by transfecting MCF-7 cells with pre-miR-10b. We found that the downregulation of miR-10b in MCF7TR cells led to increased sensitivity to tamoxifen whereas overexpression of miR-10b in MCF-7 cells led to increased resistance to tamoxifen. During our mechanistic investigation, we found that increased expression of miR-10b downregulated the expression of HDAC4 in MCF7TR cells. Inactivation of HDAC4 using HDAC4-specific siRNA transfection in MCF-7 cells led to the acquisition of tamoxifen resistance. Moreover, further reduction of HDAC4 in MCF7TR cells by HDAC4-specific siRNA transfection resulted in further enhancement of tamoxifen resistance. In summary, we have identified miR-10b-HDAC4 nexus as one of the molecular mechanism of tamoxifen resistance, and thus targeting this nexus could become a novel targeted therapeutic approach for the treatment of tamoxifen-resistant breast cancer.
Abstract A20: DARPP-32-mediated EGFR protein stability leads to gefitinib resistance in gastric cancer
Authors: Zhu Shoumin , Belkhiri Abbes , El-Rifai Wael
Published: March 2013

Background: Gastric cancer is an aggressive tumor that is often resistant to chemotherapeutics. The 17q amplicon genes, including dopamine and cAMP-regulated phosphoprotein MW 32 kDa (DARPP-32), are overexpressed in two-thirds of gastric adenocarcinomas and may induce chemotherapy resistance via abrogation of apoptosis. EGFR plays an important role in tumor growth and activation of the PI3K pathway. Gefitinib (Iressa) is a specific and effective EGFR inhibitor that has shown antitumor activity in clinical trials against several gastrointestinal cancers, including gastric cancers. However, molecular mechanisms that mediate resistance to gefitinib remain poorly understood.

Methods and Results: The expression of DARPP-32 in the multistep carcinogenesis cascade was examined using IHC analysis on 533 samples. The composite expression score, calculated from immunostaining patterns, progressively increased significantly from normal or gastritis to metaplasia, dysplasia, and adenocarcinoma (p<0.001). Using clonogenic survival assays, and ATP-GLO cell viability assays, we found that overexpression of DARPP-32 leads to a 3-fold increase in gefitinib resistance in MKN-28 cells (25 μM gefitinib, treated overnight, P<0.01). We also found that DARPP-32 expressing MKN-28 cells had activated the PI3K-AKT pathway as compared to control cells. We next examined the binding of DARPP-32 to EGFR and ERBB3. The three-way co-immunoprecipitation experiments demonstrated the existence of DARPP-32, EGFR, and ERBB3 in the same protein complex. The enhanced EGFR-ERBB3 heterodimerization promotes phosphorylation of ERBB3, then activated the PI3K-AKT pathway, as indicated by p-p85 (Tyr458) subunit of PI3K and p-AKT (Ser473) levels. Following treatment with gefitinib (25 μM) overnight, MKN-28 cells expressing DARPP-32 displayed stable protein levels of EGFR. In contrast, control cells exhibited a significant reduction in EGFR protein levels, suggesting that DARPP-32 promotes stability and suppresses degradation of EGFR protein. After the gefitinib treatment of DARPP-32-expressing MKN-28 cells, p-ERBB3 and activated PI3K-AKT pathway were maintained at higher levels than in control cells. The knockdown of endogenous DARPP-32 by lentiviral DARPP-32 shRNA in SNU-16 cell lines reversed these signaling effects and increased sensitivity to gefitinib (p<0.01).

Conclusion: Our results suggest that DARPP-32 overexpression may participate in the transition to intestinal metaplasia and progression to neoplasia. The in vitro studies indicate that DARPP-32 plays a role in increasing the stability of EGFR protein by delaying gefitinib-induced EGFR degradation; maintaining EGFR-ERBB3 heterodimerization promotes phosphorylation of ERBB3, which regulates the PI3K/Akt pathway. DARPP-32 may have potential as a predictive biomarker in gastric cancer prognosis and clinical response to treatment.
Abstract A21: Dual-kinase inhibition of EGFR/ERBB2 as a therapeutic strategy in prostate cancer
Authors: Jathal Maitreyee K. , Mooso Benjamin A. , D'Abronzo Leandro S. , Ghosh Paramita M.
Published: March 2013

Androgen withdrawal therapy (AWT) remains the cornerstone of advanced prostate cancer (PCa) therapy; however, patients on this therapy eventually relapse, caused by the advent of castration-resistant PCa. This stimulates investigation into additional therapeutic targets that may delay or impede the development of CRPC. The EGFR family of receptor tyrosine kinases is intimately involved in prostate cancer (PCa) development. EGFR/ErbB1 and HER2/ErbB2 have been clinical targets in androgen-dependent PCa but targeting each individually encourages compensatory signaling by a third member, HER3/ErbB3. Activated HER3/ErbB3 contributes to the onset of castrate-resistant PCa (CRPC), presently an incurable condition. We previously showed that dual EGFR/HER2 inhibition sensitizes PCa cells to androgen withdrawal by suppressing ErbB3 (Clin Cancer Res. 2011 Oct 1;17(19):6218–28). Based on these results, we hypothesized that the dual EGFR/HER2 inhibitor lapatinib (‘Tykerb’, Lap) may sensitize PCa cells to AWT. Athymic nu/nu mice were subcutaneously implanted with CWR22 (androgen-dependent) or CWR22Rv1 (CRPC) tumors derived from human prostate tumors. Tumor-bearing mice were either castrated or left intact, and were treated with the FDA-approved, reversible, dual-kinase, small-molecule inhibitor lapatinib or vehicle alone. Castration prevented tumor growth in CWR22 tumor-bearing mice, both those treated with lapatinib or vehicle, but in intact mice, lapatinib actually stimulated tumor growth. In CWR22Rv1 tumor-bearing mice, lapatinib was ineffective, both in intact or castrated animals. Therefore we further investigated the effect of dual EGFR/ErbB2 inhibitors in PCa. Comparison of various cells showed that lapatinib was effective in LNCaP cells and its CRPC derivatives, but was ineffective in other lines, including PC-346C cells which express a wild-type androgen receptor (AR), whereas LNCaP cells and its derivatives express a mutant AR (T877A). In the latter cell line, lapatinib increased cell growth similar to that seen in CWR22 tumors. Hence, we investigated the effect of other dual EGFR/HER2 inhibitors in PCa cells. In contrast to lapatinib, dacomitinib (PF00299804, Pfizer, PF), an irreversible, small-molecule, dual-kinase inhibitor currently in several clinical trials for a variety of tumors, was more effective and completely inhibited cell growth in the majority of cell lines. When combined, PF and Lap decreased tumor cell viability, increased cell cycle arrest, increased apoptosis, and downregulated ErbB3 protein. These preclinical data support the hypothesis that dual-kinase inhibition may be a viable therapeutic strategy. They also indicate that prostate cancer patients must be carefully preselected, based on their tumor type, and offered personalized therapeutic regimens in order to ensure maximum efficacy of dual-kinase inhibitory treatment.
Abstract A33: Modeling clinical resistance to RAF inhibitors in an in vivo melanoma model provides insights into prevention of resistance
Authors: Thakur Meghna Das , Salangsang Fernando , Pryer Nancy , McMahon Martin , Stuart Darrin
Published: March 2013

Small-molecule RAF inhibitors such as vemurafenib have demonstrated proof-of-concept that BRAFV600E is a key driver of proliferation and survival in melanoma, as evidenced by tumor regression and prolonged survival in patients in late stage clinical trials. Unfortunately, in many patients the tumor response can be short lived as resistance to the inhibitor rapidly develops. Understanding the mechanisms that underlie drug resistance will be critical to providing improved treatment options for these patients.

Here we model the emergence of resistance to vemurafenib in an early passage primary human melanoma xenograft model, which recapitulates a more physiologically relevant microenvironment, genetic diversity, and selective pressure compared to in vitro models. The selection of resistance in vivo also provides the opportunity to model drug pharmacokinetics which better replicates the selective pressure observed in patients.

Long-term continuous treatment of the homozygous BRAFV600E primary melanoma xenografts with RAF inhibitors resulted in the appearance of resistant tumors over the course of 4 to 6 weeks. Pharmacodynamic (PD) analysis of individual tumors indicated that the MAPK pathway is suppressed by the compounds in resistant tumors, although the degree and duration of suppression is less than in sensitive tumors. Furthermore, there is heterogeneity in the kinetics of pathway inhibition and recovery between tumors. Biochemical analyses indicate that BRAFV600E protein levels are upregulated across all resistant tumors and in some cases there is DNA amplification of the mutant BRAF. Taken together with the PD data, this suggests there is a great deal of tumor cell heterogeneity, and that tumors are able to rapidly adapt to the selective pressure being applied. Both in vivo drug withdrawal studies as well as in vitro analyses of tumor derived cell lines indicate that the resistant cells have become dependent on the presence of the inhibitor for their survival. Based on these observations it appears that resistant tumor cells are less fit than sensitive tumor cells in the absence of drug and therefore we tested an intermittent dosing regimen. Intermittent dosing (4 weeks on / 2 weeks off) of RAF inhibitors prevented the evolution of resistance for the duration of the study (4 months). These data could have significant implications in optimizing dose schedules to prevent the emergence of clinical resistance.
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

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

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

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

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

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

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

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

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

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

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.
Abstract B26: Drug-Diagnostic co-development to target FLT3-ITD mutations in chemo-resistant acute myeloid leukemia cells
Authors: Ko Ching-Huai , Ting-Shou Chen , Mei-Wei Lin , Chin-Pen Lai , Wen-Hsun Kuo , Chrong-Shiong Hwang , Ling-Mei Wang , SC Tsai Jane
Published: March 2013

FMS-like tyrosine kinase-3 (FLT3) overexpression is the common phenomenon in acute myeloid leukemia (AML). There are two major types of FLT3 mutations: internal tandem duplication (ITD) and the activation-loop point mutation at 835th amino residue. Although these FLT3/ITD and the 835th point mutation only account for only 20–25% and 5–10% in AML patients, they have worse prognosis and are often resistant to conventional chemotherapy. Several drugs including CEP-701, MLN-518, and PKC-412 have been developed to target FLT3 tyrosine kinase activity in AML and have been evaluated in clinical trials and showed different efficacy towards these mutations. Therefore, identification of these mutations at the time of diagnosis during drug development, in the case of our drug candidate, BELK01260, will provide a better prognostic prediction, might guide the treatment selection and follow-up strategies. In the study, we developed a sensitive molecular assay with our drug candidate well-suited for this therapeutic need to detect FLT3 mutations and proposed to be used for future clinical application. 100 AML samples with at least 20 samples with known FLT3 mutations are collected and evaluated. The standardize procedure has met the requirement of clinical pathology laboratory with reasonable cost, and both the sensitivity and specificity of this technology achieve 99.9% compared with commonly used methods.
Abstract B28: Downregulation of vimentin confers resistance to the microtubule stabilizing agents peloruside A and laulimalide in 1A9 human ovarian carcinoma cells
Authors: Kanakkanthara Arun , Rawson Pisana , Northcote Peter T. , Miller John H.
Published: March 2013

Microtubule-targeting agents are an important class of drugs for the treatment of cancer. Although the development of drug resistance in cancer cells often limits the clinical efficacy of these agents, the mechanisms of resistance are not fully understood. Peloruside A (PLA) and laulimalide (LAU) are two marine sponge-derived stabilizing agents of microtubules. PLA and LAU represent the only known class of non-taxane site binding microtubule stabilizing agents and have a number of clinically useful potential advantages over paclitaxel, a widely used tubulin-targeting anticancer drug, including reduced susceptibility to the multidrug resistance P-glycoprotein efflux pump, good efficacy against taxane-resistant cells with mutations in the taxoid site, and more effective antitumor effects in non-small cell lung tumor xenografts. To understand the mechanisms of resistance to PLA and LAU, we used two drug-resistant sublines of the 1A9 human ovarian carcinoma cell line, R1 and L4. R1 cells are 6-fold resistant to PLA but remain fully sensitive to LAU; whereas, the L4 cells are 39-fold resistant to both PLA and LAU. We previously showed that both resistant cell lines exhibit different single point-mutations in their βI-tubulin gene. The L4 cells also have a high abundance of βII- and βIII-tubulin isotypes, which play a partial role in the resistance phenotype. To determine other protein changes that might contribute to the resistant phenotype of the R1 and L4 cells, we examined the proteome of the resistant cells using two-dimensional differential in-gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The proteomic analysis identified several proteins that were significantly altered relative to the parental 1A9 cells, including a decreased expression of vimentin in L4 cells. Using Western blotting and immunocytochemistry, the decrease in vimentin abundance seen in 2D-DIGE was confirmed. Vimentin levels were unchanged in the PLA-resistant R1 cells as well as two paclitaxel/epothilone-resistant sublines of 1A9 cells. Transfection with a full-length human vimentin cDNA partially restored sensitivity of L4 cells to PLA and LAU by 23% and 17% respectively. In vimentin-expressing parental 1A9 cells, tubulin-targeting and DNA-damaging agents caused degradation of vimentin and generation of vimentin degradation products. In conclusion, this study is the first to demonstrate a novel function for vimentin as a cellular determinant of the response to microtubule stabilizing agents, and that this effect is likely to be associated with the mode of cell killing by these agents. Since many of the proteins identified in this study have not previously been shown to play a role in drug resistance, our results also introduce potential new mechanisms by which cancer cells may become resistant to antimicrotubule agents.
Abstract B29: TGF-β2 induction in head and neck squamous cell carcinoma cell lines upon treatment with tyrosine kinase inhibitors as a mechanism of acquired resistance
Authors: Kleczko Emily K. , Heasley Lydia , Heasley Lynn E.
Published: March 2013

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide, with 600,000 new diagnoses each year. Due to the fact that over 90% of HNSCC tumors overexpress the epidermal growth factor receptor (EGFR), molecularly targeted therapy against EGFR was approved to treat HNSCC. However, the 5-year survival rate for HNSCC patients, largely unchanged over the past several decades, remains at a dismal 40–50%, and the use of cetixumab, an EGFR-specific monoclonal antibody, has only had a marginal effect on overall survival. Likewise, the use of tyrosine kinase inhibitors (TKIs) that target EGFR has not resulted in an increased survival among HNSCC patients. The modest effect of these drugs on overall survival in HNSCC patients may be a result of tumors becoming resistant to treatment in responding patients. Acquired drug resistance has become a significant limitation to the efficacy of targeted therapeutics. Previously, our lab has identified fibroblast growth factor 2 (FGF2) and its receptor (FGFR) as being involved in an autocrine loop in gefitinib-insensitive cell lines, indicating that the FGFR pathway plays an important role in HNSCC as well. However, targeting FGFR with TKIs will almost undoubtedly lead to drug resistance in these FGFR-dependent cell lines, just as it does when targeting EGFR. To identify pathways important in acquired resistance to TKIs, our lab performed an Affymetrix GeneChip screen in which the cell lines UMSCC25, 584-A2, and Ca9-22 were treated for 4 days with 0.3μM AZ8010, a TKI selective for FGFRs, and/or 0.1μM gefitinib, an EGFR-selective TKI. TGF-β2 was consistently upregulated after treatment with TKIs that inhibited the dominant growth pathway in the HNSCC cell lines. Both ELISA and qRT-PCR confirmed this induction of TGF-β2 in these three cell lines. Treatment with a TGF-β2 neutralizing antibody and with small-molecule TGF-β receptor inhibitors provided additive reduction of growth in response to TKIs. Additionally, silencing TGF-β2 with shRNA in the cell line UMSCC25 leads to decreased clonogenic growth in combination with TKIs compared to a non-silencing control (NSC). Moreover, we found that an induction of NF-kB activity occurs in response to treatment with TKIs, and this induction is mitigated by the addition of a TGF-β2 neutralizing antibody. Furthermore, the cell lines UMSCC25 and UMSCC22A were chronically adapted to gefitinib over the course of several months, and we have observed a stable, prolonged increase in TGF-β2 mRNA expression in the resistant lines compared to the DMSO control lines. Taken together, this data suggests that TGF-β2 induction may provide a novel mechanism of acquired resistance to TKIs, and possibly contributes to oncogene switching programs in HNSCC.