bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 634: Combinatorial targeting in melanoma cell lines with defined and distinct mechanisms of acquired resistance to V600EB-RAF inhibition
Authors: Shi Hubing , Kong Xiangju , Ribas Antoni , Lo Roger S.
Published: June 2012

V600EB-RAF mutation is found in 50-60% of melanomas, and its in-human “druggability” has recently been demonstrated using the novel agent PLX4032, which can achieve an unprecedented ∼80% on-target, anti-melanoma response rate. Acquired resistance to PLX4032, however, has been observed in the majority of patients. We have shown previously that melanoma tumors in patients and cell lines in vitro acquire resistance to PLX4032 not by developing secondary mutations in V600EB-RAF but instead by upregulating PDGFRβ (showing MEK inhibitor insensitivity) or N-RAS (showing MEK inhibitor sensitivity). Here we used distinct subsets of melanoma cell lines (resistance derived in vitro) and short-term cultures (resistance derived in vivo) with hitherto characterized mechanisms of acquired resistance to PLX4032 to define the optimal combination of molecular target inhibition necessary to achieve efficient growth inhibition and cytotoxicity. We focused on a set of molecular targets downstream of overexpressed PDGFRβ or mutant N-RAS in each subset and used specific small molecule inhibitors, singly or in combination, to correlate growth inhibitory responses with p-ERK, p-AKT, p-p70S6K levels and apoptotic responses. We found that single inhibitor treatment led to functionally significant compensatory activation or rebound activities, consistent with dynamic network connectivity at key nodes of signaling. In PDGFRβ-upregulated, PLX4032-resistant melanoma cell lines, combined B-RAF, PI3K and TORC1/2 inhibition led to the strongest synergistic growth inhibitory and cytotoxic responses. In N-RAS-upregulated, PLX4032-resistant cell lines, either MEK or dual TORC1/2 inhibition sufficed to yield significant growth inhibition, and combined MEK and TORC1/2 inhibition did not synergize in growth inhibitory or cytotoxic responses. Together, these observations could serve to rationally guide animal model studies and urgently needed clinical testing in pre-identified subsets of patients relapsing on V600EB-RAF inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 634. doi:10.1158/1538-7445.AM2011-634
Abstract 641: Effects of an mTOR inhibitor on immune subpopulations
Authors: Seliger Barbara , Giersberg Corrina , Leich Franziska
Published: June 2012

Immune suppression is frequently detected in renal cell carcinoma (RCC) and might contribute to tumor progression. Recently, receptor tyrosine kinase inhibitors have been shown to revert the frequency of the myeloid-derived suppressor cell (MDSC)-mediated immune suppression, the development of regulatory T cells (Treg), increase the number of NK cells and prevent tumor-specific T cell anergy. The mTOR inhibitors exhibit a unique target profile resulting in both anti-tumor and anti-angiogenic activity. It has been shown that everolimus can induce apoptosis in various cancers of distinct origin. However, the role of everolimus on immune cell subpopulations have not yet been studied in detail. Therefore peripheral blood mononuclear cells as well as human CD3+ T cells obtained from healthy blood donors were either left untreated or stimulated with antibodies or PHA and subjected to multicolour flow cytometry using a large set of antibodies.

We here show that everolimus exerts different effects on peripheral blood mononuclear cells regarding the composition of immune cell subpopulations, their proliferation, activation, cytokine production and degranulation. Upon everolimus treatment the number of stimulated CD3+, CD4+ and CD8+ and CD4+/FoxP3* T cells decreased, whereas the number of NK cells, CD4+/CD8+ CD45RO T cells increased. Furthermore, the T cell proliferation was reduced upon mTOR inhibitor treatment. These data suggest that everolimus modulates the composition of immune cells, their activation and growth properties, which is important for the design of combinatorial therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 641. doi:10.1158/1538-7445.AM2011-641
Abstract 644: Anti-tumor activities of antibodies targeting the RON receptor and a biomarker of response
Authors: Whalen Kerry , Gifford James , Boudrow Andrea , Meetze Kristan , Winston William , Liu Qing , Lerner Lorena , Feng Bin , Jiang Jinwei , Cook Lynette , DiPrima Michael , Chen Ting , Weiler Solly , Gyuris Jeno , Han May
Published: June 2012

RON is a receptor tyrosine kinase of the MET family. Stimulation by its ligand, Macrophage Stimulating Protein (MSP), activates a signaling cascade leading to cell growth, migration, invasion and resistance to apoptosis. In animal models, RON overexpression in breast and lung results in tumor growth and metastasis. RON receptor activation in animal models also play a role in tumor-host interactions such as osteolytic bone destruction and tumor associated macrophage infiltration. RON overexpression has been demonstrated in several solid tumors including pancreatic, breast, ovarian and colon. RON overexpression is correlated with disease progression and shorter survival in ovarian and colon cancer. Several isoforms of RON have been reported, including a potentially oncogenic form, RON Δ160 in CRC. Given the strong evidence for the involvement of RON in numerous aspects of tumor biology, investigating an anti-RON antibody as cancer therapy is warranted. We have identified and characterized a panel of antagonistic murine anti-human RON antibodies. Humanization of two antibodies resulted in Superhumanized™ anti-RON antibodies that are capable of inhibiting MSP dependent RON downstream signaling, cell migration and invasion in vitro. The anti-RON antibodies have subnanomolar binding affinity to wildtype RON and RON Δ160 receptors. The lead antibody is capable of internalizing and degrading the receptor in vitro and in vivo. The antibodies are capable of inhibiting growth of engineered murine models that are driven by wildtype or RON Δ160 receptor, as well as traditional human cancer xenografts.

Given the complex role of RON in tumor biology, identification of response biomarkers is crucial for identifying the patient populations most likely to benefit from treatment. A multi-gene biomarker potentially predictive of tumor response to RON antibody, the RON pathway index, was tested and validated using a panel of human cancer cell line xenografts. Current results demonstrate a statistically significant correlation between the degree of tumor growth inhibition by anti-RON antibody treatment and RON pathway index value. Thus, we have identified a biomarker of tumor response to anti-RON antibody that can potentially help us identify tumor types or tumor subtypes of interest in the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 644. doi:10.1158/1538-7445.AM2011-644
Abstract 66: Epigenetic inactivation of the tumor suppressor BIN1 drives proliferation of SNF5-deficient tumors
Authors: McKenna Elizabeth S. , Tamayo Pablo , Cho Yoon-Jae , Mora-Blanco E. Lorena , Sansam Courtney G. , Tillman Erik J. , Koellhoffer Edward C. , Pomeroy Scott L. , Roberts Charles W. M.
Published: June 2012

SNF5 (SMARCB1/INI1/BAF47), a core subunit of SWI/SNF chromatin remodeling complexes, is a potent tumor suppressor that is specifically inactivated in a variety of aggressive pediatric cancers. We have recently demonstrated that SNF5 acts as a tumor suppressor not genetically via maintenance of genome integrity, but epigenetically via transcriptional regulation. However, few of the target genes essential for oncogenesis following SNF5 loss have been identified. Here, we demonstrate that aberrant epigenetic silencing of the tumor suppressor gene BIN1 is a conserved event in SNF5-deficient cancers and contributes to tumorigenesis. Comparative genomewide expression analysis reveals that BIN1 expression is strongly downregulated in SNF5-deficient malignant rhabdoid tumors (MRT) relative to other human tumors, and we also observe decreased Bin1 expression in murine Snf5-deficient lymphomas. The underlying mechanism is epigenetic silencing as reintroduction of SNF5 into deficient tumor cell lines results in increased SWI/SNF recruitment to the BIN1 promoter and increased BIN1 expression. Furthermore, expression of BIN1 specifically compromises the proliferation of SNF5-deficient MRT cell lines. BIN1 encodes a family of alternatively spliced adaptor proteins that have been implicated in cellular differentiation, tumor suppression, and immune escape of transformed cells, but its regulation has not been well characterized. Identification of BIN1 as a key SNF5 target reveals a novel mechanism by which epigenetically based changes can drive cancer formation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 66. doi:10.1158/1538-7445.AM2011-66
Abstract 664: Polymerization of human βIII-tubulin is distinct from βI-tubulin in a cell-free system
Authors: Yang Chia-Ping H. , Horwitz Susan B.
Published: June 2012

Tubulins are a family of proteins consisting of 7 α- and 6 β-tubulin isotypes. Many studies have demonstrated that tumors overexpressing βIII-tubulin have a poor response to chemotherapy. Suppression of βIII-tubulin sensitizes non-small cell lung cancer (NSCLC) cells to microtubule-interacting agents, such as Taxol. βIII-tubulin has also been shown to mediate the incidence and progression of lung cancer. Tubulin isotype content of βI-, βIII-, βIV- and βV-tubulin in the human lung carcinoma cell line A549 is 50%, 8%, 36.5%, and 5.5%, respectively. In our study, the ability of six microtubule stabilizing agents, Taxol, epothilone B (EpoB), discodermolide (Disco), Ixabepilone, laulimalide and peloruside A, to polymerize tubulin in a 100,000 × g supernatant prepared from A549 cells was examined. The level of different tubulin isotypes in the drug-polymerized tubulin was determined by Western blot analysis with tubulin isotype specific antibodies. In the absence of drug, the level of polymerized βI-, βIII-, βIV- and βV-tubulin was 55%, 9%, 25%, and 48%, respectively. Ten µM Taxol caused a smalll increase (<15%) in βI- and βV- tubulin polymerization, but it induced a 367% and 228% increase in βIII- and βIV-tubulin polymerization. Among the six drugs tested, Disco and EpoB exhibited the greatest ability to polymerize all isotypes. The differential βI- and βIII-tubulin polymerization effects also were observed when intact A549 cells were lysed in the presence of drugs, followed by centrifugation. In the absence of drug, the level of polymerized βI- and βIII-tubulin was 9% and 0.2%, respectively. In the presence of 10 µM Taxol, the level of polymerized βI- and βIII-tubulin was increased to 28% and 7% which corresponded to a 3- and 35-fold increase in βI- and βIII-tubulin polymerization, respectively. However, when purified bovine brain tubulin in the absence of all endogenous proteins was used, there was no differential effect of drugs on the polymerization of βI+II- and βIII-tubulin. The βI-, βII-, βIII-, and βIV-tubulin content in bovine brain is 3%, 58%, 25% and 14%, respectively. Disco and EpoB also have the strongest effects on the polymerization of all bovine brain tubulin isotypes. Our results indicate that polymerization of βIII-tubulin is distinct from that of βI-tubulin in a cell-free system and that endogenous cellular components may influence the polymerization of tubulin isotypes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 664. doi:10.1158/1538-7445.AM2011-664
Abstract 666: Synthesis and structure-activity relationships of compounds that induce methuosis, a novel type of non-apoptotic cell death
Authors: Robinson Michael W. , Young Ashley M. , Overmeyer Jean H. , Erhardt Paul W. , Maltese William A.
Published: June 2012

Methuosis is a caspase-independent form of necrotic cell death in which massive accumulation of vacuoles derived from macropinosomes ultimately causes cells to detach from the substratum and rupture. This form of death was initially described in glioblastoma cells overexpressing activated GTPases, such as Ras or Rac1, but methuosis-like forms of death have since been reported in other types of cells exposed to different stimuli. We have been interested in developing small molecules that can induce methuosis because of the possibility that stimulating this alternative death pathway may represent a useful therapeutic strategy for cancers that are refractory to apoptosis. We now describe the synthesis and biological testing of a class of small molecules that can effectively induce methuosis in glioblastoma cells at low micromolar concentrations. Details of the design and synthesis of a directed library of these novel compounds will be presented, including complete chemical structures and definition of structure-activity relationships. Each compound was evaluated for its ability to induce cytoplasmic vacuolization and reduce cell viability in U251 glioblastoma cells, using phase contrast microscopy, MTT and colony-forming assays as readouts. The results highlight a lead compound with an EC50 of approximately 1-3 micromolar, based on results from both MTT and colony-forming assays. Although the specific molecular target of these compounds is currently unknown, preliminary evidence indicates that they work at the level of endosome trafficking steps mediated by the Rab5 and Rab7 GTPases. The status of work toward target identification will be discussed. The potential significance of these studies is underscored by our recent observation that this class of compounds can induce methuosis in glioblastoma cells resistant to the front-line chemotherapeutic drug, temozolomide. This raises the possibility that the current lead compound may serve as a prototype for development of agents that could be used to trigger death by methuosis in drug-resistant cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2011-666
Abstract 674: Novel combinatorial quinacrine cancer therapies for advanced colon cancer
Authors: Gallant Jean-Nicolas , Wang Wenge , Dolloff Nathan G. , Allen Joshua E. , El-Deiry Wafik S.
Published: June 2012

BACKGROUND: Although treatments have improved patient prognosis in early stages of colorectal cancer, new effective drugs with improved safety profiles are needed for improving the treatment of patients with advanced colon cancer. Quinacrine, a small molecule anti-malarial agent that has activity in giardiasis, lupus, prion disease, and used as a means of non-surgical sterilization, has shown cytotoxic activity across a broad range of cancers. The well characterized safety profile of quinacrine and its anticancer activity hold promise in novel combinatorial cancer therapies.

OBJECTIVES: To evaluate the potential of adding quinacrine to anticancer chemotherapeutics and targeted agents as a potential novel combinatorial therapy for advanced colon cancer and to detail the mechanism of action of quinacrine when combined with cancer chemotherapy and targeted agents.

METHODS: In vitro dose-response curves and isobolograms were constructed for single agent and dual agent therapies using quinacrine, 5-fluorouracil, gemcitabine, and sorafenib using the following colon cancer cell lines: HT-29, HT-29-luc, HCT-15, HCT-116, HCT-116 p53−/−, HCT-116 bax−/−, RKO, DLD-1, SW-480, and SW-620. Western blot analysis and flow cytometry were used to investigate potential mechanisms of action and to examine cell death. In vivo experiments were carried out to investigate toxicity and efficacy of combinatorial quinacrine chemotherapy in an athymic nu/nu mouse tumor xenograft model with human HT-29-luc cells.

RESULTS: Single-agent quinacrine demonstrates potent anticancer activity with an average IC50 of 5.79 ± 0.83 μM across all ten colon cancer cell lines screened. In combination, quinacrine enhanced the anticancer activity of 5-fluorouracil and gemcitabine while significantly synergizing with sorafenib. Western blot analysis revealed that quinacrine's anticancer activity partially arises from its ability to stabilize p53, elevate death receptor levels, and lower anti-apoptotic protein levels. Quinacrine, when combined with sorafenib, dramatically lowers levels of the anti-apoptotic protein MCL-1. In vivo studies have shown that quinacrine monotherapy at a daily dose of 150 mg/kg significantly lowers the tumor load of nu/nu mice.

CONCLUSIONS: Quinacrine is cytotoxic to a broad range of colon cancer cells lines and enhances the anticancer activity of chemotherapies such as 5-fluorouracil, gemcitabine, and sorafenib. The administration of quinacrine in combination with chemotherapy agents and targeted agents in patients with advanced colon cancer should be further explored in the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 674. doi:10.1158/1538-7445.AM2011-674
Abstract 68: Increased H3K27 trimethylation is associated with decreased expression of miRNAs and bivalently modified genes in rhabdoid tumors
Authors: Gadd Samantha L. , Perlman Elizabeth J.
Published: June 2012

Background: Rhabdoid tumors (RT) are aggressive tumors of infancy for which no effective treatment is currently available. These tumors are characterized by genetic loss of the SMARCB1 (SNF5, INI-1) component of the SWI/SNF chromatin remodeling complex. We previously reported an overall pattern of repression of genes associated with bivalent histone modification in embryonic stem cells within RT. We hypothesize that loss of SMARCB1 in RT results in an overall failure to release the repressive (H3K27) histone methylation in genes responsible for early differentiation. To test this hypothesis, we performed chromatin immunoprecipitation (ChIP) using antibodies specific for permissive (H3K4) and H3K27 trimethylation, comparing an RT cell line (G-410) with a human embryonic kidney cell line (HEK293). In addition, we report the global miRNA analysis of RT using the Illumina 96-sample Universal Matrix Array.

Results: Those genes previously shown by others to be direct targets of SMARCB1 (PTN, CDK2NA, ATP1B1, HESR1, and FZD7) and genes previously shown to be down-regulated in RT (CDH2, SPON1, LEF1, SOX11, CDKN1B, SERPINE2, SPOCK1, DOCK4) all showed a significantly higher ratio of H3K27 methylation to H3K4 methylation (1.5-fold to 130-fold) in G410 cells compared with HEK293. In contrast, genes shown to have increased expression in RT (TFRC and DDX21) showed increased H3K4 methylation. Global miRNA analysis demonstrated striking downregulation of 156/158 (p<0.05) miRNAs compared with other pediatric renal tumors. ChIP analysis for H3K4 and H3K27 associated with miRNAs mir127, mir134, mir519e, mir99b, and mir152 likewise showed increased association with H3K27.

Summary: Gene and miRNA expression strongly correlate with histone methylation patterns in the RT cell line. This suggests that loss of SMARCB1 in RTs results in striking repression of gene and miRNA expression which may be due to aberrant repressive (H3K27) histone methylation. The use of histone methylation inhibitors may therefore merit investigation for this pediatric tumor for which there is no effective therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 68. doi:10.1158/1538-7445.AM2011-68
Abstract 682: Design and discovery of novel small molecule modulators of reactive oxygen species-mediated cell signaling
Authors: Pathania Divya , Sechi Mario , Palomba Michele , Cosseddu Alessia , Sanna Vanna , Berrettini Francesco , Sias Angela , Taheri Laleh , Neamati Nouri
Published: June 2012

Oxidative stress is an important hallmark of cancer. Elevated levels of intrinsic reactive oxygen species (ROS) make tumor cells more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm the adaptive antioxidant capacity of cancer cells and trigger ROS-mediated cell death. Increasing ROS production or decreasing ROS scavengers shows potential for selectively targeting tumor cells that are under persistent oxidative stress. Recently, we have discovered novel class of quinazolinediones that exert their anticancer effects by modulating ROS-mediated cell signaling.The sub-micromolar anti-proliferative properties of these compounds were demonstrated by MTT and colony formation assays in a panel of cancer cell lines of different origins. Treatment with these agents induced cell cycle arrest, and increased superoxide production in cancer cells. Furthermore, proteomics analysis of 600 target antibody array revealed that these molecules significantly affected several cell signaling pathways implicated in carcinogenesis, cancer progression, redox signaling, and cell death.In conclusion, quinazolinediones are promising lead compounds that selectively enhance ROS production and induce ROS-mediated cell death in cancer cells, and warrant further preclinical studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 682. doi:10.1158/1538-7445.AM2011-682
Abstract 685: PTX-008 antiproliferative effects in cancer cells are mediated though galectin-1 inhibition and MAPK-dependent G2/M cell cycle arrest
Authors: Astorgues-Xerri Lucile , Bonezzi Katiuscia , Serova Maria , Bieche Ivan , Riveiro Maria E. , Cvitkovic Esteban , Noel Kay , Giavazzi Raffaella , Taraboletti Giulia , Raymond Eric , Faivre Sandrine
Published: June 2012

Background. Galectin-1 is a multifunctional protein involved in various aspects of tumorigenesis and has been described as a promising cancer target. PTX-008, a small molecule interacting with galectin-1, is currently investigated as an anticancer agent in various preclinical models. The aim of the present study was to study molecular mechanism associated with the cellular effects of PTX-008 in cancer cells.

Materials and Methods. Antiproliferative effects were evaluated in a panel of 20 human cancer cell lines by MTT assay. Effects of PTX-008 on cell cycle and protein expression were assessed by flow cytometry and western blot analysis, respectively. mRNA expression was evaluated with qRT-PCR.

Results. PTX-008 induces potent antiproliferative effects in our panel of cell lines at concentrations ranging 1-27µM. Interestingly, exposure to PTX-008 for >48 hours inhibits galectin-1 protein level. Furthermore, we have found a clear correlation between low expression of mRNA and proteins levels of galectin-1 and PTX-008 sensitivity in a panel of 20 cancer cell lines (R2=0.59, p<0.05). However, inhibition of galectin-1 by shRNA in SQ20B cells led to no significantly changed PTX-008 antiproliferative activity suggesting that PTX-008 may not exclusively act thought galectin-1 protein down expression. Previous studies showed that galectin-1 might interact with H-RAS and K-RAS, regulating the magnitude of MAPK signal output. In RAS/RAF wild-type SQ20B cells, exposure to 3µM PTX-008 for >5 hours inhibits pERK1/2, pAKT, and pS6, suggesting the important role of MAPK and AKT/mTOR in downstream signalling of galectin-1 in our model. Cell cycle analysis of SQ20B treated with PTX-008 showed a dose-dependent accumulation in G2/M phase without apoptosis induction. Consistent with the inhibition in G2 to mitosis transition, PTX008-induced cell cycle changes were associated with a decrease of Wee1 and an increase of Cdc2 phosphorylation but no inactivation of cdc25C. Inhibition of MAPK with UO126 abolished PTX-008 inhibition of cdc2 reinforcing the important role of MAPK-inhibition in cellular effects of PTX-008.

Conclusion. Our findings show that PTX-008 inhibits cancer cells proliferation by interacting with galectin-1, blocking G2/M transition, and inhibiting MAPK signalling pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 685. doi:10.1158/1538-7445.AM2011-685
Abstract 694: Cyclosporin-modulated intensified dosage chemotherapy well-tolerated for saving eyes with intraocular retinoblastoma
Authors: Chan Helen S.L , Héon Elise , Dimaras Helen , Gallie Brenda L.
Published: June 2012

PURPOSE: Cyclosporin inhibits the multidrug resistance P-glycoprotein. Since 2000, we tested increased carboplatin and increased etoposide dosages in a cyclosporin-modulated carboplatin-etoposide-vincristine protocol, followed by focal laser/cryotherapy consolidation, to avoid elective radiation of newly diagnosed retinoblastoma (RB) patients, since radiation increases the secondary cancer risk in patients with heritable RB. We report the results for International Classification Groups A, B, C and D eyes.

METHOD: Since Neupogen support became available, we increased the mainly myelotoxic carboplatin to 28 mg/kg (from 18.7 mg/kg in a previous trial) and etoposide to 12 mg/kg (from 7.7 mg/kg in a previous trial), but kept the mainly neurotoxic vincristine unchanged at 0.05 mg/kg, modulated by the same cyclosporin schedule (33 mg/kg over 3 hours on each of the 2 days per cycle). We treated 35 eyes in 23 patients: 2 A eyes, 8 B eyes, 8 C eyes and 17 D eyes. Radiation or enucleation for recurrence was considered failure.

RESULTS: We report the long-term results at median followup of 8.1 years (range 1.7-10.5 years). Eye event-free rates were 100% for both Groups A and B eyes, and 50% for Group C (4/8) and 47% for Group D (8/17) eyes. Four C eyes failed and all had enucleation only. Nine D eyes failed and 6 were enucleated and 3 radiated. No child lost both eyes. Toxicity rates were acceptable with 15.7% fever-and-neutropenia, 0.7% bacterial sepsis, 9.7% blood transfusion and 27.6% platelet transfusion, and no long-term renotoxicity or ototoxicity.

CONCLUSION: This protocol was well well-tolerated, with no child losing both eyes, and most avoiding radiation. The same protocol is being tested in a multicenter trial.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 694. doi:10.1158/1538-7445.AM2011-694
Abstract 701: A mechanistic study of mdr1a gene regulation in vivo using bioimaging
Authors: Gu Long , Lin Min , Higgins Kathleen , Forman Barry , Kane Susan E.
Published: June 2012

The human P-glycoprotein (Pgp) encoded by the MDR1 gene is as a transmembrane drug transporter that mediates the efflux of drugs from cells. It confers multidrug resistance in cancer cells that overexpress MDR1. In addition, basal expression of MDR1 in organs such as liver, kidney, and colon affects the pharmacokinetics of drug uptake and excretion for agents that are substrates for Pgp transport. Nuclear receptors such as SXR and CAR have been suggested as possible master regulators of xenobiotic- and drug-inducible expression of MDR1 and of other genes involved in drug metabolism in drug processing organs. However, the mechanism of MDR1 regulation in tumors or in normal, non-malignant tissue, particularly in response to xenobiotics, has yet to be demonstrated in vivo. To facilitate the mechanistic study of MDR1 gene regulation in vivo, we previously reported a mouse model that allows non-invasive bioimaging of the mdr1 gene expression in vivo and in real time by inserting a firefly luciferase (fLUC) gene into the murine mdr1a genetic locus by homologous recombination.1 To determine the role of PXR, the murine homologue of SXR, in drug-mediated induction of mdr1a, we crossed mdr1a.fLUC mice with PXR knockout mice. Pregnenolone-16α-carbonitrile (PCN), a known SXR substrate, induced intestinal mdr1a.fLUC expression in the PXR wild type background but not the PXR knockout background. Notably, mdr1a.fLUC was re-inducible over three rounds of drug dosing, which could be measured longitudinally in our real-time assessment of gene expression. TCPOBOP, a known CAR ligand, induced mdr1a.fLUC in both the wild type and knockout background, consistent with the idea that mdr1a is also regulated by the CAR nuclear receptor. The chemotherapeutic agent docetaxel induced mdr1a.fLUC in the PXR wild type background but induction was dramatically reduced in the PXR KO background; paclitaxel gave only low-level induction in both genetic backgrounds. This result is consistent with the reported ligand specificity of mouse PXR. Future studies in a CAR-null background will determine if the observed effect of paclitaxel on mdr1a expression is mediated through CAR. These studies demonstrate the utility of our murine imaging model for the in vivo study of mdr1 gene regulation in real time. Studies with additional chemotherapeutic drugs and genetic backgrounds will establish the variety of agents capable of inducing mdr1a at the transcriptional level and the trans-acting factors that are involved in such induction.

1Gu et al. Proc Natl Acad Sci 106:5394-5399 (2009)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 701. doi:10.1158/1538-7445.AM2011-701
Abstract 704: Upregulation of the human equilibrative nucleoside transporter 1 (hENT1) expression by indole-3-carbinol (I3C) in pancreatic cancer cell lines
Authors: Wang Honggang , Word Beverly , Lyn-Cook Berverly
Published: June 2012

Pancreatic adenocarcinoma is the fourth most common cause of death in cancer in the Western world. In the past years, gemcitabine (GEM) has become the standard chemotherapeutic agent for patients with pancreatic cancer. However, a substantial number of patients fail to respond to therapy due to inherent or developed GEM chemoresistance. GEM is transported into cells mainly by the human equilibrative nucleoside transporter 1 (hENT1). Overexpression of hENT1 mRNA and protein in pancreatic carcinoma is known to correlate with better outcomes. In recent years, indole-3-carbinol (I3C), a dietary compound in cruciferous vegetables, has been shown to alter cancer cell survival by inducing apoptosis in vivo. I3C has also been shown to reverse multiple drug resistance by inhibiting efflux transporter, P-glycoprotein (P-gp) expression. In this study, the potential of I3C to modulate hENT1 expression and activity was evaluated in a panel of human pancreatic cancer cell lines, AsPC-1, BxPC-3, MiaPaca2, PANC-1 and PL-45. Our data showed that I3C at 50 and 100 uM significantly increased hENT1 mRNA and protein expression in BxPC-3 cell, MiaPaca2 cells and PANC-1 cells compared with the vehicle controls. In contrast, no such effect was observed in AsPC-1 and PL-45 cells with same treatment. The potential synergistic cytotoxic effect of I3C and GEM was also examined in all five pancreatic cancer cell lines. Pancreatic cancer cells were incubated with or without I3C at 50 uM for 24 h and then treated with GEM at different concentrations with or without I3C at 50 uM and a specific hENT1 inhibitor, NBMPR, at 50nM for 48 hours. MTS assay was used for cell viability measurement. Our data showed that I3C decreased IC50 by 6.8, 8.1 and 8.4 folds in PANC-1, MiaPaca2 and BxPC-3 cells respectively, and only 2.7 and 2.0 folds in AsPC-1 and PL-45 cells respectively, compared with GEM alone treated cells. Investigation also found that NBMPR, a specific inhibitor of hENT1, only abrogated a part of the augmented effect of I3C in response to GEM in all five pancreatic cell lines.

The present study provides evidence that increasing hENT1 expression is a novel mechanism of the antitumor effect of I3C. The combination of GEM and I3C may be an effective strategy to overcome the GEM chemoresistance in pancreatic cancer patients. This affirms our hypothesis that I3C could be an adjuvant therapy improving the response to GEM treatment in patients with pancreatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 704. doi:10.1158/1538-7445.AM2011-704
Abstract 708: Integrated analysis of drug resistance networks in breast cancer
Authors: Komurov Kakajan , Tseng Jen-Te , Muller Melissa , Ram Prahlad T.
Published: June 2012

Targeted inhibition of HER2 tyrosine kinase receptor presents a promising strategy in the treatment of HER2-positive breast cancer patients. However, despite favorable response during the acute phase, majority of patients develop resistance to anti-HER2 drugs during the chronic phase of treatment. Here, we developed a cell culture model of chronic lapatinib (a dual inhibitor of EGFR and ERBB2 receptor tyrosine kinases) treatment by continuous passaging of HER2-overexpressing SKBR3 cells in increasing doses of lapatinib over a year. The resultant cell line, SKBR3-R, is almost 100-fold more resistant to lapatinib-induced growth inhibition when compared to the parental line. We have verified that this difference in lapatinib sensitivity is not due to altered expression or signaling at the level of HER2 or HER3 receptors in SKBR3-R cells. By integrating genome-wide gene expression profiling with our novel network-based genomic data analysis approach, we show that 1) cell toxicity mediated by acute lapatinib treatment in HER2-positive cells is associated with nutrient starvation and energy stress, and 2) chronic lapatinib treatment leads to the activation of compensatory cellular networks involved in the response to nutrient starvation, which confers resistance to lapatinib-induced cell death. Accordingly, while imposing a glucose starvation response phenotype on SKBR3 cells protects from lapatinib-mediated cell death, pharmacological inhibition of the nutrient starvation response in SKBR3-R cells reverses lapatinib resistance. In order to see if these networks play a role in breast cancer pathogenicity within clinical settings, we carried out a detailed network-based correlations of tumor gene expression profiles of breast cancer patients with their clinical data. Importantly, we found that the metabolic networks associated with lapatinib resistance are also associated with poor outcome within clinical settings. This study implicates nutrient deprivation response as a major factor in the acquired resistance of breast cancer cells to lapatinib, and suggests novel targets for combinatorial therapy of HER2-positive breast cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 708. doi:10.1158/1538-7445.AM2011-708
Abstract 717: JAK2/STAT3 induces erlotinib-resistance in lung cancer cells harboring EGFR-activating mutations
Authors: Harada Daijiro , Harada Daijiro , Takigawa Nagio , Ohashi Kadoaki , Ichihara Eiki , Kubo Toshio , Takeda Hiromasa , Kashihara Hiromi , Hotta Katsuyuki , Tanimoto Mitsune , Kiura Katsuyuki
Published: June 2012

Epidermal growth factor receptor (EGFR) inhibitors, including the small molecule tyrosine kinase inhibitors such as gefitinib and erlotinib, have demonstrated promising effects in non-small cell lung cancer harboring EGFR-activating mutation. But even in the patients with an initial dramatic response, acquired resistance to the EGFR tyrosine kinase inhibitor develops almost without exception after varying periods of time, usually 6-12 months. Secondary mutations such as T790M mutation in EGFR and/or amplification of the gene encoding the MET receptor tyrosine kinase account for some of the resistance. However, tumors from approximately 40% of patients with acquired resistance do not have the known mechanisms.

We generated an erlotinib-resistant cell line (PC-9/ER3) from PC-9 that harbored the delE746-A750 mutation, by exposing the cell line to erlotinib for 12 weeks. The cytotoxicity of the drugs for the cells was evaluated using MTT assay. The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) for 96 h exposure was used to evaluate the cytotoxicity. Protein expression was determined by Western blotting.

The PC-9/ER3 cells were 250-fold more resistant to erlotinib than the parental cells, and were stable for 20 weeks or longer without erlotinib exposure. The resistant cells harbored neither T790M mutation nor MET gene amplification. Erlotinib suppressed pEGFR and pMAPK, however, increased pSTAT3 in both PC-9 and PC-9/ER3 cells. No differences were observed in the IC50 values of JSI-124, a JAK2 inhibitor that suppresses STAT3 phosphorylation, between the parental and resistant cells. The PC-9/ER3 cells restored sensitivity to erlotinib when combined with JSI-124 (0.05 µM), even though the IC50 values of erlotinib combined with JSI-124 in the PC-9 cells did not change. Additionally, pSTAT3 increased by erlotinib was suppressed by JSI-124 both in the PC-9 and PC-9/ER3 cells. pAKT expressions treated by erlotinib with or without JSI-124 seemed to be equivalent in the PC-9 cells; however, the combination of erlotinib with JSI-124 more suppressed pAKT compared to erlotinib alone in the PC-9/ER3 cells.

Our results would suggest that the activation of JAK2/STAT3 signaling pathway followed by continuous erlotinib exposure accounts partly for the acquired resistance, and that it might be overcome by the combined treatment with JAK2 inhibitor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 717. doi:10.1158/1538-7445.AM2011-717
Abstract 72: Gene-specific H3K4 trimethylation changes in healthy human subjects exposed to carcinogenic metal components of air particles
Authors: Cantone Laura , Kluz Thomas , Tarantini Letizia , Bonzini Matteo , Marinelli Barbara , Bertazzi Pier Alberto , Baccarelli Andrea , Costa Max
Published: June 2012

Ambient and occupational exposure to air particulate matter (PM) has been consistently associated with increased risk of lung cancer. Epidemiological studies strongly indicate that the metal components of PM may be responsible for PM carcinogenicity, but the underlining mechanisms are poorly understood.

H3K4 trimethylation (H3K4me3) is known to be modified by exposure to carcinogenic metals in-vitro and correlates with transcriptional activation when found in gene promoter regions.

In a pilot study on workers of a steel-plant production, we investigated whether PM metal components, such as nickel and arsenic, had an effect on H3K4me3 in the promoter regions of target genes, including tumor suppressors and genes whose expression is increased following metal exposures, such as NIMA-related kinase 6 (NEK6) genes and carbonic anhydrase IX (CA9).

We obtained blood samples on the first (sample 1) and last (sample 2) day of a working-week from 10 subjects. We used gene-specific analysis by chromatin immunoprecipitation (ChIP)-real-time PCR in blood leukocytes to investigate whether H3K4 trimethylation changes, at different levels of exposure to airborne metals in PM, can influence transcription of CA9 and NEK6 genes.

We found that both for CA9 and NEK6, there was a difference between sample 1 and sample 2 in all subjects, with a higher level in sample 2; furthermore, in sample 2, we found a positive trend for H3K4me3 in CA9 and NEK6 genes with increasing levels of exposure to metals in PM compared to one unexposed control. The observed tendency is consistent with the fact that carcinogenic metals alter patterns of gene expression leading to activation of genes that may be relevant to the carcinogenic process.

These findings indicate that different levels of exposure to PM metal components can induce changes in H3K4 trimethylation that up-regulates transcription of CA9 and NEK6 genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 72. doi:10.1158/1538-7445.AM2011-72
Abstract 73: PELP1 is a novel reader of chromatin modifications: Implications in cancer progression
Authors: Vadlamudi Ratna K. , Mann Monica , Cortez Valerie A.
Published: June 2012

Cancer is caused by abnormalities of the genome and epigenome including the frequently occurring epigenetic changes of histone modifications. Specifically, estrogen-induced breast carcinogenesis is characterized by alterations in histone modifications. Reader proteins that recognize these modifications facilitate modulation of genes and their resulting biological actions. In addition to regulation by several coregulators, estrogen receptor alpha (ERα) transcriptional activation or repression depends on the modulation of histone methylation at target promoters. The ERα coregulator PELP1 plays an important role in ERα signaling, is a proto-oncogene with aberrant expression in hormone-related cancers and a prognostic indicator of decreased survival in breast cancer patients. The molecular mechanisms by which PEPL1 promotes oncogenesis remain unknown; however, previous studies from our lab have shown that PELP1 interacts with histones and is a novel reader of chromatin modifications. To explore the PELP1 epigenetic interactome, we used a histone peptide array containing 384 unique combinations of histone modifications including methylation, phosphorylation, acetylation and citrullination. Our results show that PELP1 uniquely recognizes histones modified by arginine dimethylation, arginine citrullination and lysine dimethylation. Phosphorylation of residues adjacent to a methyl modification affects the ability of PELP1 to recognize histone methylation. Using various deletions of PELP1 peptides, we have found that PELP1 acts as a module for recognition of a specific histone modification through the carboxyl-terminal glutamic acid rich region. Reporter gene assays showed that PELP1 functionally interacts with arginine methyltransferases including CARM1 and PRMT6, both shown to be dysregulated in human cancers, and synergistically enhances ERα-transactivation. Chromatin immunoprecipitation assays revealed that PELP1 has the potential to alter histone H3 arginine methylation status at ERα target gene promoters. The critical role of PELP1 status in modulating arginine methylation status was also observed through in vivo studies which show PELP1 knockdown results in decreased tumorigenesis and arginine dimethylation. Our findings suggest that PELP1 is a reader of histone methyl modifications and deregulation may have implications on tumor proliferation via epigenetic alterations at ERα target promoters. Targeting these epigenetic alterations through inhibition of PELP1 and the arginine methyltransferases could be a promising cancer therapeutic. This study was funded by NIH grant CA095681.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 73. doi:10.1158/1538-7445.AM2011-73
Abstract 730: Identifying the mechanisms of action and resistance to the novel multi-kinase inhibitor ENMD-2076 in leukemia
Authors: Failes Timothy , Mitic Gorjana , Kavallaris Maria
Published: June 2012

Background: Molecularly targeted agents such as kinase inhibitors have offered great promise in treatment regimens for cancer. However, the rapid development of resistance to kinase inhibitors mediated through adaptations such as point mutations in the target kinase domain has emerged as a major clinical problem. Accordingly, multi-kinase inhibitors are a promising class of chemotherapeutic agents that can target multiple cytotoxic pathways and potentially be less susceptible to the onset of resistance. ENMD-2076 is a multi-kinase inhibitor that targets Aurora A and multiple receptor tyrosine kinases involved in angiogenesis. ENMD-2076 is showing much promise in Phase I/II clinical trials, yet the specific pathways of sensitivity and resistance are still unclear.

Methods: Acute T-cell leukemia CCRF-CEM cells were selected for resistance in 6 µM of ENMD-2076. The resultant resistant cells were designated CEM/AKA6. The level of resistance to the selecting agent was characterized using drug treated cell viability assays. Screening for mutations in Aurora A was conducted using full-length gene sequencing. Real time PCR was used for gene and western blotting for protein expression studies. Phosphoprotein analysis was conducted using Full Moon Biosystems Inc, antibody arrays.

Results: CEM/AKA6 cells were 10-fold resistant to ENMD-2076. No significant changes in the gene and protein expression of Aurora A were found compared to the parental cells. Moreover, full-length sequencing of the Aurora A or Aurora B gene failed to detect mutations. CEM/AKA6 cells were as equally sensitive to a specific Aurora A inhibitor, MLN8237, as the parental cells, further suggesting that resistance to ENMD-2076 is not mediated through an Aurora A dependent pathway. ENMD-2076 is known to target the anti-apoptotic PI3K/Akt signaling pathway and CCRF-CEM cells have a deletion in PTEN that leads to constitutive activation of this pathway. Analysis of the Akt pathway using phospho-specific antibody arrays showed that phosphorylation of PDK1, upstream from Akt, was enhanced in CEM/AKA6 cells compared to parental cells, whilst phosphorylation of proteins downstream from Akt such as p53 and c-Jun was decreased. Validation, localization and functional characterization of these protein changes are planned.

Conclusions: The PI3K/Akt signaling pathway is altered in leukemia cells resistant to the multikinase inhibitor ENMD-2076. Understanding the interplay of this specific pathway in mediating resistance and/or sensitivity to ENMD-2076 will shed light on the mechanism of this promising agent and may enable optimization of its clinical use.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 730. doi:10.1158/1538-7445.AM2011-730