bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3446: Inhibition of EZH2 suppresses medulloblastoma cell growth
Authors: Alimova Irina N. , Venkataraman Sujatha , Harris Peter , Vibhakar Rajeev
Published: June 2012

EZH2 (enhancer of zeste homologue2) is a polycomb group protein that mediates repression of gene transcription through its Histone 3 Lysine 27methyltransferase activity. EZH2 is highly expressed in many cancers including metastatic prostate cancer, breast cancer, and glioblastoma multiform. EZH2 expression is associated with tumor cell proliferation, invasive growth and aggressive cancer phenotypes. The fact that EZH2 increases tumor cell proliferation and aggressive subgroups in several cancers may be of practical interest because the polycomb proteins have been suggested as candidates for targeted therapy.

We found EZH2 expression to be increased in medulloblastoma when compared to normal cerebellum. Inhibition of EZH2 by RNAi in medulloblastoma cells resulted in suppression of cell growth. To further examine this phenotype we used a global histone methylation inhibitor, 3-Deazaneplanocin A (DZNep). DZNep preferentially inhibits H3 Lys27me via EZH2.

DZNep showed a dose dependent, anti-proliferative and anti-clonogenic activity in medulloblastoma cells. Synergetic effect was shown in cells transfected with shEZH2 and treated with DZNEP. Furthermore DZNep treatment decreased EZH2 protein levels. We found that DZNep induced differentiation of mouse neural stem cells even when they are grown in proliferation medium. Most critically transformation of these neural stem cells was significantly attenuated by shEZH2 and DZNep. Our results suggest that EZH2 is a target for medulloblastoma treatment and that DZNep is an excellent candidate for further testing as a therapeutic candidate.

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 3446. doi:10.1158/1538-7445.AM2011-3446
Abstract 3480: TNFα accelerates tumor vascular leukocyte formation through the induction of integrin α5 expression and adhesion to fibronectin
Authors: Li Bin , Young Pampee
Published: June 2012

Tumor associated myeloid cells are believed to promote tumor development by stimulating tumor growth, angiogenesis, invasion and metastasis. Tumor associated myeloid cells that co-express endothelial and myeloid markers represent a pro-angiogenic subpopulation known as vascular leukocytes. Recently, we and others have demonstrated that tumor-derived TNFα promotes local tumor growth and vascularity. Our data suggest that tumor growth is in part due to TNFα-mediated increased numbers of tumor-associated vascular leukocytes (ie myeloid-endothelial biphenotypic cells). The work detailed herein explored the mechanism by which TNFα mediates endothelial differentiation of myeloid cells. Our studies showed that fibronectin is a robust facilitator of endothelial differentiation of blood mononuclear cells in vitro. We have found that TNFα treatment of monocytes significantly increased expression of α5β1 integrin, a major fibronectin receptor enriched on endothelial cells, leading to a consequent 4-fold increase in fibronectin adhesion. Furthermore, TNFα treated monocytes upregulated expression of endothelial markers, flk-1(VEGFR2/KDR) and VE-cadherin. Integrin α5 subunit inhibitory antibodies blocked adhesion to fibronectin as well as consequent upregulation of flk-1 and VE-cadherin transcripts, implying a role for outside-in signaling by the α5β1 integrin after binding fibronectin. Finally, treatment of mouse tumors with anti-α5 antibodies reduced accumulation of tumor vascular leukocytes in vivo. Our studies suggest that tumor-cell derived TNFα constitutes a tumor microenvironment signal that promotes differentiation of tumor-associated monocytes towards a proangiogenic/provasculogenic myeloid-endothelial phenotype via upregulation of the fibronectin receptor α5β1.

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 3480. doi:10.1158/1538-7445.AM2011-3480
Abstract 349: Fascin expression in colorectal cancer metastases predicts survival after curative-intent surgery
Authors: Sullivan Catherine , Guo Xiaojie , Wang Zuoheng , Theoharis Constantine , Rimm David L. , Boffa Daniel J.
Published: June 2012

Background: Patients with metastatic colorectal cancer that is limited to a few lesions in the lungs or liver may benefit from the complete surgical removal of all deposits of metastatic cancer (referred to as a curative metastasectomy). Although the outcome after this curative-intent surgery is considerably better than patients with more extensive metastatic cancer, only a third of the surgical patients are actually cured. As a result, there is a great need for a mechanism to predict outcome after surgery for limited metastatic cancer, in order to better match patients with effective therapy.

Fascin is an actin bundling protein linked to epithelial to mesenchymal transformation and metastatic progression. Fascin expression has previously demonstrated prognostic potential in patients with extensive metastatic colorectal cancer. We hypothesized that Fascin expression within surgically removed colorectal cancer metastases would correlate with survival after curative-intent surgery.

Methods: 82 paraffin embedded colorectal metastases collected after curative-intent surgery between 1990 and 2008 were incorporated into tissue microarrays (TMAs) in two fold redundancy. Fascin expression within the metastases was determined by quantitative immunofluorescence using Automated Quantitative Analysis (AQUA), and normalized based on a panel of cell lines (AQUA score range 0-72). The median follow up was 2.8 years and thus far 51 patients have died (62%).

Results: The majority of colorectal cancer metastases had been removed from the liver (70 patients), while 12 represented pulmonary metastases. There were no differences in survival by location of metastases (p = .65). Survival was analyzed based on high (top quartile, n = 21) and low (bottom three quartiles, n = 61) expression of Fascin (AQUA score cut point of 3.4). High expression of Fascin correlated with a worse prognosis, HR 1.97; 95% CI 1.07-3.62 p = .009.

Conclusions: Fascin expression within colorectal cancer metastases may be used to predict outcome after curative-intent surgery. Patients with high Fascin expression may be considered for additional therapy or may be advised against particularly high risk procedures. Further study is warranted to validate the role of Fascin expression analysis in this patient cohort.

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 349. doi:10.1158/1538-7445.AM2011-349
Abstract 3493: Expression of VEGFR and FRGFR on endothelial cells isolated from hepatocellular carcinoma and adjacent liver tissue
Authors: Hui-Chuan Sun , Kong Ling-Qun , Zhu Xiao-Dong , Chai Zong-Tao , Wang Wen-Quan , Wu Wei-Zhong
Published: June 2012

Background Heterogenity in gene expression profiles in tumor endothelial cells (TEC) has been reported reported. Our previous studies showed TEC is also different from endothelial cells isolated from adjacent normal liver tissue (NEC) in term of response to chemotherapeutics and anti-angiogenesis drugs.

Methods TEC and NEC were isolated by CD31 and CD105 antibody conjuaged magnetic beads from tumor and adjacent nontumor liver tissues from 5 patients who were diagnosed as hepatocellular carcinoma with a positive alpha fetoprotein (> 20ng/ml). The purity of ECs was proved by LDL internalization assay, tube formation, immunostaining and RT-PCR to exclude tumor cell contamination. VEGFR1, VEGFR2, FGFR-2, CD31 and CD105 expression on the isolated ECs were assayed by flow cytometry within 3-5 passages. Response of different types of ECs to sorafenib treatment was observed, including cell proliferation, VEGFRs and FGFR expression.

Results The isolated ECs were proved by tumor formation and LDL internalization. More than 97% cells are LDL positive. AFP was not detectable on the isolated ECs in both flow cytometry assay and immunostaining assay. VEGFRs and FGFR expression were different on CD105+TEC and CD31+TEC among different patients, ranging from 18% to 67% for VEGFR1, 1% to 8% for VEGFR2, and 18% to 25% for FGFR-2 on CD105+TEC, and 18% to 90% for VEGFR1, 10% to 40% for VEGFR2, and 18% to 30% for FGFR-2 on CD31+TEC. Expression of VEGFRs and FGFR is higher on CD105+ECs than those on CD31+ECs. While between TEC and NEC, the expression of VEGFRs and FGFR is also heterogeneous. Sorafenib at 5 mM inhibted proliferation of TEC and NEC in a dose-dependent manner, while NEC is more sensitive to sorafenib treatment.

Conclusions VEGFRs and FGFR expression were different among different patients; CD105+EC and CD31+EC are probably two types of ECs with different biological features.

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 3493. doi:10.1158/1538-7445.AM2011-3493
Abstract 3501: Flavopiridol decreases Mcl-1 and XIAP in acute myeloid leukemia cell resulting in augmentation of sorafenib-induced apoptosis
Authors: Xia Lijuan , Gabrilove Janice , Jing Yongkui
Published: June 2012

The PI3K/AKT/mTOR and Raf/MEK/ERK pathways, resulting in increased levels of Mcl-1, XIAP and survivin, have been found to be activated in acute myeloid leukemia cells (AML) due to the mutant receptor tyrosine kinases. FMS-like tyrosine kinase (FLT3) mutations including FLT3-internal tandem duplication (FLT3-ITD) are found in about 30% of cases of acute myeloid leukemia and confer an increased relapse rate and reduced overall survival. Sorafenib, inhibiting Raf-1 of the MAPK pathway resulting in diminished MEK and ERK phosphorylation, is being tested in AML patients with FLT3-ITD and modest responses have been observed: similarly flavopiridol, a CDK and mRNA transcription inhibitor, has been tested in AML patients with some evidence of activity. We hypothesize that sorafenib plus flavopiridol will have enhanced therapeutic effects. The combined apoptotic effects of sorafenib plus flavopiridol were investigated in HL-60, MV4;11 FLT3-ITD+ cells, as well as in primary leukemic cells isolated from AML patients. Sorafenib and flavopiridol indepedently induced apoptosis at 10 μM and 200 nM, respectively, in HL-60 cells which was correlated with the down-regulation of Mcl-1, XIAP and/or survivin. The combination of sorafenib (5 μM) plus flavopiridol (100 nM) produced augmented apoptosis associated with enhanced diminution in levels of Mcl-1. MV4;11 cells were more sensitive than HL-60 cells to sorafenib-induced apoptosis, but not to flavopiridol, with the down-regulation of Mcl-1 and survivin. Addition of flavopiridol to MV4;11 cells enhanced sorafenib-induced apoptosis with augmented down-regulation of XIAP. Primary cells isolated from two AML patients with FLT3-ITD+ cells exhibited augmented apoptosis induction with the combination treatment of sorafenib plus flavopiridol. Our data suggest that the augmented apoptosis induction in AML cells by sorafenib plus flavopiridol is mediated by decreasing levels of complimentary anti-apoptotic proteins. This novel combination therapy is worthy of a clinical trial in FLT3-ITD+ AML patients.

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 3501. doi:10.1158/1538-7445.AM2011-3501
Abstract 3513: MK-2206 combined with ridaforolimus or topoisomerase inhibitors synergistically impede the growth of sarcoma cells
Authors: Cubitt Christopher L. , Menth Jillaina L. , Dawson Jana L. , Reed Damon R. , Sullivan Daniel M.
Published: June 2012

The number of effective drugs available for the treatment of soft tissue and bone sarcomas is limited to a small group of antineoplastic agents. The goal of this study was to determine the effectiveness of MK-2206 (Akt inhibitor) when used in combination with Ridaforolimus (mTor inhibitor), Cisplatin, Topotecan or Etoposide in a panel of ten cell lines. Six different types of sarcomas are represented in the panel, including rhabdo-, osteo-, Ewing, leiomyo-, lipo- and fibrosarcomas.

Cell Titer-Blue cell viability experiments were performed to test dilutions of MK-2206 alone and in combination at various molar ratios over a 72 hour time period. The observed IC50s for MK-2206 or Ridaforolimus alone ranged from 4.2 to 12.9 µM or 23.7 to 62.3 µM, respectively. The observed IC50 range for Etoposide was 0.5 to 7.5 µM. To determine the relative potentiation of drug combinations, Excess over Highest Single Agent (HSA) analysis was performed (Table). The average Excess HSA score for MK-2206 combinations with Ridaforolimus or Etoposide were 0.17 and 0.15 respectively with levels of significance (p value) < 0.032. This was in contrast to MK-2206 combinations containing Cisplatin or Topotecan with average HSA scores of 0.084 and 0.085, respectively, indicating less potentiation.

Using Chou and Talalay combination index (CI) analyses (effective dose levels of 0.7, 0.9 and 0.95), a synergistic to strongly synergistic reduction in cell viability was observed in 5 cell lines for MK-2206 plus Ridaforolimus and MK-2206 plus Etoposide combinations. Ongoing experiments are underway to identify the effects of MK-2206 combinations on apoptosis and the effect of drug sequencing on growth inhibition. The results of this study indicate that a MK-2206 plus Ridaforolimus or a MK-2206 plus Etoposide combination may be an effective treatment regimen for sarcoma patients. This work was funded by the Pediatric Cancer Foundation.

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 3513. doi:10.1158/1538-7445.AM2011-3513
Abstract 3526: Coadministration of nicotinic acid with the Nampt inhibitor MPC-9528 enhances antitumor activity in Naprt deficient cancer cells in culture and in xenografts
Authors: Fleischer Tracey C. , Baichwal Vijay R. , Boniface J Jay , Cimbora Daniel M. , DeMie Lynn , Douce Thomas B. , Gassman Andrew D. , Papac Damon I. , Peterson Ashley , Robinson Rosann , Terry-Lorenzo Ryan T. , Carlson Robert O.
Published: June 2012

Background: The tumoricidal small molecule MPC-9528 is a picomolar inhibitor of nicotinamide phosphoribosyltransferase (Nampt). Nampt catalyzes the first and rate-limiting step in NAD synthesis from nicotinamide. Nicotinic acid phosphoribosyltransferase (Naprt) catalyzes the first and rate-limiting step in an alternate pathway of NAD synthesis from nicotinic acid (NA). Cancer cells are particularly dependent on NAD and many cancer cell lines, but not most normal tissues, are deficient in Naprt activity. Therefore administration of NA could prevent MPC-9528-induced NAD depletion in normal tissues, but not in Naprt-deficient tumors, resulting in greater therapeutic index and efficacy.

Methods: Cell viability was determined based on ATP levels. Naprt protein expresson was quantified by western blot and qRT-PCR. NAD was acid-extracted from cells and quantified by a coupled reaction based on fluorescent resorufin. Xenograft studies were performed in nu/nu mice.

Results: In 44 out of 153 cancer cell lines surveyed, NA did not prevent MPC-9528-induced cell death, which correlated with low to undetectable levels of Naprt. MPC-9528-induced NAD depletion and cell death in HCT116 colon carcinoma cells were prevented by the addition of NA, consistent with high Naprt expression. A single dose of MPC-9528 at the maximum-tolerated dose (MTD) of 75 mg/kg caused tumor regression in HCT116 xenografts and NA coadministration completely blocked this effect. NA also completely blocked mortality in mice induced by 300 mg/kg MPC-9528, consistent with the finding that most mouse tissues have high Naprt expression. In Naprt-deficient MIA PaCa-2 xenografts, NA coadministration allowed tolerance of 200 mg/kg MPC-9528 with a substantially increased anti-tumor response relative to the MTD of 75 mg/kg MPC-9528 alone.

Conclusions: Low Naprt expression correlates with the lack of effect of NA on MPC-9528 tumoricidal activity. Because Naprt deficiency is prevalent in cancer cell lines and in primary tumor specimens, but not in normal tissues, NA coadministration with MPC-9528 should increase the tolerability and efficacy of MPC-9528 in patients with Naprt-deficient tumors. A companion diagnostic designed to measure Naprt expression or activity in tumors could be used to identify tumors that would most likely benefit from such combination 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 3526. doi:10.1158/1538-7445.AM2011-3526
Abstract 3539: Targeting levels and oligomerization of mutant nucleophosmin induces differentiation and loss of survival of human AML cells with mutant NPM1
Authors: Balusu Ramesh , Fiskus Warren , Rao Rekha M. , Venkannagari Sreedhar , Nalabothula Narasimha , Williams Casey , Abhyankar Sunil , McGuirk Joseph , Bhalla Kapil N.
Published: June 2012

NPM1 is an oligomeric, nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids. NPM1 plays multiple roles in cell growth and proliferation by participating in diverse biological processes, including ribosome biogenesis and transport, centrosome duplication, DNA repair, transcriptional regulation and histone chaperoning. NPM1 has an N-terminal conserved, hydrophobic, oligomerization domain (residues, 1-110), which is common to all isoforms of NPM1 and critical for its chaperone activity. Recently, NSC348884 was identified as a small molecule inhibitor that disrupts NPM1 dimer/oligomer formation, inducing apoptosis of cancer cells. NPM1 is mutated in approximately one-third of patients with AML. The mutant NPM1c+ contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal (NES), which causes NPM1c+ to be aberrantly localized in the cytoplasm. Here, we determined the effects of targeting NPM1 in cultured and primary AML cells. Treatment with siRNA to NPM1, which depleted both NPM1c+ and un-mutated NPM1, significantly induced p21, decreased the % of cells in S-phase of the cell cycle, as well as induced differentiation of the AML OCI-AML3 cells that express both NPMc+ and un-mutated NPM1. This was associated with up-regulation of C/EBPα but attenuation of the levels of HOXA9 and MEIS1. Notably, treatment with NPM1 siRNA sensitized OCI-AML3 more than HL-60 cells (expressing only wild-type NPM1) to all-trans retinoic acid (ATRA) and cytarabine. Moreover, inhibition of NPM1 oligomerization by NSC348884-induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+, but not AML or normal CD34+ progenitor cells expressing wild-type NPM1, to ATRA and cytarbine. NSC348884 was also able to sensitize primary AML cells that expressed NPM1c+ as well as FLT3-ITD to ATRA and cytarbine. Addtionally, co-treatment with NSC384884 sensitized these primary AML cells to the lethal effects of the FLT3 kinase inhibitor PKC412 (Novartis Pharma). These results show that attenuating levels or oligomerization of NPM1 selectively induces apoptosis and sensitizes AML cells expressing NPM1c+ alone or with FLT3-ITD to treatment with ATRA, cytarabine and FLT3 kinase 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 3539. doi:10.1158/1538-7445.AM2011-3539
Abstract 3547: In vitro and ex vivo anti-leukemia activity of AR-67, a novel lipophilic camptothecin
Authors: Tsakalozou Eleftheria , Howard Dianna , Leggas Markos
Published: June 2012

BACKGROUND: The overall survival and disease-free rates for Acute Myelogenous Leukemia (AML) patients treated with the gold-standard therapy comprised of Cytarabine (Ara-C) and Daunorubicin (DNR) remain significantly low. Camptothecins are potent anticancer drugs that confer their cytotoxic effect by interfering with Topoisomerase-I (Top1). Semi-synthetic camptothecin analogues have been used in the past to treat AML patients with relatively low response rates. Here, we examined the anti-leukemia activity of AR-67, a novel camptothecin analogue, alone or in combination with standard agents for the treatment of AML.

METHODS: The anti-leukemia activity of AR-67, TPT, Ara-C and DNR was studied in HL60, KG1 and K562 cells and in primary leukemia cell cultures after exposure to increasing drug concentrations for 48 hours. Cell proliferation was determined using a colorimetric assay (MTT) assay and IC50s were obtained using nonlinear-regression analysis. Drug combination treatments were assessed for synergy. The expression of Top1, phosphohistone (γ-H2AX), Bcl-2 and c-PARP were evaluated using biochemical assays.

RESULTS: The potency of AR-67 was in the nM range in the leukemic cell lines. Consistent with the lower proliferation rates of primary cells grown in culture, their IC50 values were variable and in the µM range. IC50s estimated for TPT were higher than the respective ones for AR-67. The IC50 values for Ara-C and DNR were in the μM range for the HL60, K562 and primary cells, and higher than the ones for AR-67. However, the KG-1 cell line appeared to be more sensitive to the AML gold standard therapy, but not as sensitive as to AR-67. AR-67 treatment decreased Top1 expression, increased γ-H2AX expression in a concentration-dependent manner suggesting removal of DNA-Top1 complexes and formation of double strand DNA breaks. Induction of apoptosis was manifested by decreased Bcl-2 and increased c-PARP expression suggesting a caspace-3 mediated effect. AR-67 combination treatments that included DNR and Ara-C showed synergy in HL60 cells. Additionally, pretreatment with Ara-C revealed synergy between AR-67 and DNR in both the KG1 and K562 cells, accompanied by increased γ-H2AX and c-PARP expression.

CONCLUSIONS: Our results indicate AR-67 as a potent anticancer agent that induces DNA damage and apoptosis. Using in vitro leukemia models we have shown that AR-67 is a potentially efficacious agent when combined with standard agents for the treatment of AML and may benefit AML patients resistant to Ara-C.

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 3547. doi:10.1158/1538-7445.AM2011-3547
Abstract 3552: JAK/Stat3 signaling pathway modulates human megakaryocytic differentiation
Authors: Hui-Fen Liao , Peng Hsuan-Yu , Chen Yu-Jen
Published: June 2012

Megakaryocytes (MKs) are differentiated from pluripotent hematopoietic stem cells toward a lineage capable of terminal differentiation to mature platelets. Various hematopoietic growth factors such as thrombopoietin, the ligand of c-mpl-encoded protein, have been identified as important regulators of MK development. Induction of MK differentiation may benefit disorders related to platelet dysfunction or thrombocytopenia. However, both effective inducer and clear mechanism of MK differentiation remain lacking. To clarify the regulatory mechanisms of MK differentiation, distinct inducers staurosporine (STS) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) were used to assess the expression of Janus kinase (JAK) and Signal transducers and activators of transcription 3 (Stat3) signaling molecules in both human chronic myeloid leukemia K562 and erythroleukemia HEL cells. Our results demonstrated that STS and TPA effectively induced cell differentiation toward MK lineage in terms of development of polyploidy population, expression of MK-specific markers CD41 and CD61, and secretion of platelet factor 4. Intriguingly, enhancement in phosphorylation of Stat3 (p-Stat3), nuclear translocation and DNA-binding activity was evident in both STS- and TPA-treated cells. Blockade of Stat3 by Stat3 inhibitor VI and its upstream molecule JAK by JAK inhibitor I obviously declined MK differentiation. Furthermore, knockdown of Stat3 by RNA interference attenuated the expression of p-Stat3 and MK differentiation, indicating a crucial role of Stat3 in MK differentiation. In conclusion, JAK/Stat3 signaling pathway may involve MK differentiation and might be a target for development of therapeutic modulators for platelet disorders.

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 3552. doi:10.1158/1538-7445.AM2011-3552
Abstract 3554: CCT137690, a dual inhibitor of Aurora and FLT3 kinases, sensitizes FLT3-ITD positive acute myeloid leukemia and overcomes resistance to selective FLT3-inhibition
Authors: Moore Andrew S. , Faisal Amir , Bavetsias Vassilios , Sun Chongbo , Atrash Butrus , Valenti Melanie , de Haven Brandon Alexis , Avery Sian , de Castro David Gonzalez , Raynaud Florence I. , Workman Paul , Pearson Andrew DJ , Blagg Julian , Eccles Suzanne A. , Linardopoulos Spiros
Published: June 2012

The Aurora kinases are a family of serine-threonine kinases that play key roles in different stages of mitosis. Over-expression of Aurora kinases has been demonstrated in a range of malignancies including leukemia. Aurora kinase inhibitors are emerging as promising agents in the treatment of acute myeloid leukemia (AML) with a number of compounds currently being assessed in clinical trials (Moore AS et al, Leukemia 2010). CCT137690, an imidazo[4,5-b]pyridine derivative discovered at our Institute, is an orally bioavailable, potent, pan-Aurora and FLT3 inhibitor. CCT137690 showed in vitro and in vivo activity in human colon cancer cell lines and xenograft models (Bavetsias V et al, J Med Chem 2010). Here we report the in vitro and in vivo activity of CCT137690 in FLT3-ITD positive AML and demonstrate that CCT137690 overcomes resistance to the selective FLT3 inhibitor MLN518. In cellular assays using FLT3-ITD positive MOLM-13 cells, CCT137690 inhibits autophosphorylation of Aurora A and Aurora B kinases and phosphorylation of histone H3, a direct target of Aurora B kinase. CCT137690 also inhibits autophosphorylation of FLT3 and its downstream targets phospho-STAT5 and phospho-p44/42 MAPK. Dual inhibition of Aurora and FLT3 kinases in FLT3-ITD positive AML with CCT137690 induces apoptosis and results in a unique cell cycle profile with cells accumulating in G2/M. When given orally to athymic mice, CCT137690 achieved target modulation and potently inhibited the growth of subcutaneous MOLM-13 xenografts, with no obvious toxicity or loss of body weight. Inhibition of MOLM-13 xenograft growth was more pronounced with CCT137690 compared to the selective FLT3 inhibitor MLN518, suggesting that dual inhibition of Aurora and FLT3 kinases may have advantages compared to selective FLT3 inhibition alone. To assess whether dual Aurora-FLT3 inhibition with CCT137690 could overcome FLT3-inhibitor resistance, we successfully cultured MOLM-13 cells in the presence of increasing concentrations of MLN518. Although resistant to MLN518, these cells maintained sensitivity to dual Aurora-FLT3 inhibition with CCT137690. The potent preclinical activity of CCT137690 in FLT3-ITD positive AML models supports the growing body of evidence that dual pan-Aurora and FLT3 kinase inhibitors may be of benefit in the high-risk group of patients with FLT3-ITD positive AML, including those with FLT3-inhibitor resistance.

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 3554. doi:10.1158/1538-7445.AM2011-3554
Abstract 3555: Anti-tumor activity of CAL-101, a potent selective inhibitor of the p110Δ isoform of PI3K, in models of human glioblastoma
Authors: Kashishian Adam , Meadows Sarah , Steiner Bart , Lannutti Brian
Published: June 2012

Phosphatidylinositol 3-kinase (PI3K) activation and Akt phosphorylation play a pivotal role in cell proliferation and survival, transducing signals from several cell surface receptors through PI3K isoforms (designated p110α, β, Δ, and γ). Our previous research has elucidated a significant role of the PI3K p110Δ (PI3KΔ) isoform in hematologic malignancies. Aberrant upregulation of the PI3K/Akt pathway is a frequently observed defect in human malignancies, including glioblastoma, but the role of the PI3KΔ isoform, has not been systematically evaluated. Selective inhibition of PI3KΔ with the orally bioavailable investigational drug, CAL-101, offers the potential for targeting PI3KΔ as a therapeutic approach to glioblastoma while avoiding undesirable side effects associated with pan-inhibitors that broadly block PI3K isoforms in multiple cell types. CAL-101 potently inhibits PI3KΔ (with an EC50 of 65 nM in a whole-blood assay) and shows >200-fold selectivity over other Class I PI3K isoforms and no activity against Class II and III PI3K family members or other PI3K-related proteins, including mTOR and DNA-PK. We examined expression levels of PI3KΔ and constitutive pathway activation in a panel of glioma cell lines (LN18, LN229, SF-539, U87MG, U138MG, and U251). PI3KΔ was consistently expressed and was functionally active, inducing high basal levels of phosphorylated-Akt (pAkt) in all tested cell lines. Treatment with CAL-101 decreased p-Akt levels in all cell lines and also reduced phosphorylation of the downstream pathway target, S6. Furthermore, PI3KΔ inhibition with CAL-101 resulted in an accumulation of cells in G1 and a decrease in the S phase population. Consistent with the effects of CAL-101 on G1 cell-cycle arrest, immunoblotting showed a decrease in cyclin D1 levels. In mice bearing U87MG xenografts, in vivo PI3KΔ inhibition resulted in significant anti-tumor effects, delaying time to tumor progression (tumor volume of >1000 mm3) by 30 days (p<0.001). Taken together, these data suggest that PI3KΔ overexpression and heightened functional activity contribute to glioblastoma proliferation, and that selective targeting of PI3KΔ may offer a novel therapeutic approach for patients with central nervous system tumors.

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 3555. doi:10.1158/1538-7445.AM2011-3555
Abstract 3576: Lapatinib in combination with doxorubicin is highly damaging to cardiac myocytes
Authors: Hasinoff Brian B. , Patel Daywin , Wu Xing
Published: June 2012

Lapatinib (Tykerb) is dual tyrosine kinase inhibitor targeting the HER2 (ERBB2) and EGFR (ERBB1, HER1) pathways. It is used to treat ER+/EGFR+/HER2+ breast cancer patients and patients who have HER2-positive advanced breast cancer that has progressed after previous treatment with other chemotherapeutic agents. Lapatinib, which was initially rated as having no cardiotoxicity, has recently been shown in a clinical trial to produce low levels of cardiotoxicity that is reversible. Using a neonatal rat cardiac myocyte model we have investigated the ability of lapatinib alone and in combination with doxorubicin to damage myocytes. Lapatinib alone at pharmacological concentrations did not significantly cause myocyte damage. However, doxorubicin-induced myocyte damage was greatly increased by the addition of low nanomolar lapatinib concentrations. Previous clinical studies have shown that the combination of trastuzumab (Herceptin) and doxorubicin was highly cardiotoxic. Because trastuzumab also targets HER2 receptors, the lapatinib/doxorubicin combination provides a good model to probe the mechanism of this cardiotoxicity using a rat myocyte model. Lapatinib treatment alone had little or no effect on myocyte mitochondrial membrane potential, on oxidation of intracellular DCF (dichlorofluorescein), on intracellular ATP levels or on caspase 3/7 induction. The doxorubicin cardioprotective agent dexrazoxane was able to greatly reduce doxorubicin-induced myocyte damage. However, dexrazoxane was not able to reduce the increase in myocyte damage produced by adding lapatinib to the doxorubicin treatment. The effect of lapatinib on the change in phosphorylation of ACC (acetyl-coenzyme A carboxylase), which is a measure of a change in cellular energy status, was also determined. Doxorubicin-induced growth inhibitory effects on K562 cells, which contain very low levels of ERBB1/2 protein, were not affected by the addition of lapatinib. This result suggests that inhibition of ERBB1/2 is required for the synergistic toxic effects seen with doxorubicin in myocytes. In conclusion these results suggest that combination therapy employing doxorubicin and lapatinib may produce increased cardiotoxicity. Support: CIHR and a Canada Research Chair in Drug Development to BBH.

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 3576. doi:10.1158/1538-7445.AM2011-3576
Abstract 3610: Potent preclinical anti-tumor activity of MGCD265, an oral Met/VEGFR multitargeted kinase inhibitor in clinical development, in combination with taxanes
Authors: Maroun Christiane R. , Ste-Croix Helene , Beaulieu Normand , Bonfils Claire , Fournel Marielle , Dupont Isabelle , Lemoyne Christian , Wand James , Besterman Jeffrey M.
Published: June 2012

MGCD265, a multitargeted receptor tyrosine kinase inhibitor in clinical development, inhibits the activation of Met, VEGFR1, 2, 3, Tie-2 and Ron. Met is a promising molecular target for the development of cancer therapeutics and is found overexpressed and phosphorylated in a large proportion of NSCLC patients. Several agents targeting Met are currently under clinical investigation in NSCLC including MGCD265, and early clinical results are encouraging. Interestingly, to our knowledge, no Met inhibitor has been explored in NSCLC in combination with a taxane. In support of the clinical development of MGCD265, we have investigated preclinically the anti-tumor activity of MGCD265 in combination with taxanes.

The combination of MGCD265 with docetaxel or paclitaxel resulted in enhanced anti-tumor activities compared to treatment with either agent alone in multiple xenograft models, including human NSCLC, glioblastoma, breast, prostate and gastric carcinomas. Enhanced efficacy occurred in the absence of overt toxicity. Moreover, PK analyses demonstrated lack of drug-drug interactions. Interestingly, in a gastric Met-driven carcinoma model in which MET is amplified (MKN45), treatment with MGCD265 in combination with docetaxel resulted in tumor regression at suboptimal doses of the taxane. Immunohistochemistry analyses in this model showed that this combination altered tumor vascularization as detected by endothelial cells labeling (CD31). While it has been suggested that changes in tumor vascularization may increase tumor exposure to docetaxel, the exposure of either MGCD265 or docetaxel was not altered in tumors treated with the combination. To gain insights into the molecular mechanism through which improved anti-tumor activities were achieved by the combination, we performed gene expression analyses on xenograft tumors. Genes differentially regulated by either agent alone were simultaneously regulated and sometimes enhanced, when the two agents were combined. Confirmations are currently ongoing. Together, these studies provide support for the development of MGCD265 in combination with taxanes for the treatment of 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 3610. doi:10.1158/1538-7445.AM2011-3610
Abstract 3622: An antibody-based multifaceted approach targeting the human transferrin receptor for the treatment of multiple myeloma
Authors: Daniels Tracy R. , Ortiz-Sanchez Elizabeth , Luria-Perez Rosendo , Quintero Rafaela , Helguera Gustavo , Bonavida Benjamin , Martinez-Maza Otoniel , Penichet Manuel L.
Published: June 2012

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, CD71) that demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is due to its ability to decrease TfR1 levels leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fc gamma receptors and the complement component C1q, suggesting that is capable of eliciting Fc-mediated effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). Additionally, in two disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant anti-tumor activity including long-term survival. Interestingly, the parental antibody without avidin (ch128.1) also shows remarkable in vivo anti-cancer activity despite its lack of in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the Long-Term Cell-Initiating Culture (LTC-IC) assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies.

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 3622. doi:10.1158/1538-7445.AM2011-3622
Abstract 3627: Targeted delivery of gelonin to claudin-expressing cancer cells and use of activatable cell penetrating peptides to enhance potency
Authors: Lin Xinjian , Yuan Xiaoqin , Manorek Gerald , Howell Stephen B.
Published: June 2012

Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosome/lysosome compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. Although their functional role in cancer progression remains unclear, the differential expression of these proteins between tumor and normal cells, in addition to their membrane localization, makes them prime candidates for targeted cancer therapy. This study utilized the C-terminal 30 amino acid fragment of C. perfringens enterotoxin (CPE), that binds to claudins 3 and 4, to deliver the toxin gelonin (rGel) and examined a new strategy for enhancing delivery to the cytoplasm. CPE was fused to rGel at its N-terminal end via a flexible G4S linker. This molecule was internalized into vesicles from which location it produced little cytotoxicity. Arginine-rich peptides have been extensively used to enhance cellular uptake of various types of cargo, and there is a reasonable presumption that they can deliver cargo out of subcellular compartments as well. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R9) was introduced between the CPE and the rGel. CPE-R9-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. Given that the R9 moiety was so effective at enhancing uptake, we reasoned that it would likely also be good at releasing rGel from the compartment into which CPE delivered it. However, to make use of the R9 sequence in this manner it is necessary to mask the R9 until after CPE-mediated and claudin-dependent endocytotic uptake. To this end an E9-G4S-R9-rGel fusion protein was produced and tested for cytotoxicity. The addition of the E9 through a G4S linker to R9-rGel increased the IC50 by 5.3-fold from 38 to 202 nM indicating that the inhibitory E9 domain can largely neutralize the R9. Addition of CPE to the E9-G4S-R9-rGel fusion protein further reduced the IC50 to 482 nM consistent with the concept that the CPE directs the CPE-E9-G4S-R9-rGel to intracellular vesicles from which it has difficulty in fully escaping. However, addition of endosomolytic reagent chloroquine at a non-toxic concentration increased the inhibition of growth produced by 31.25 nM CPE-E9-G4S-R9-rGel from 10% to 76%. Thus, while CPE provides targeting to claudin-expressing tumor cells, R9 plus chloroquine provide a mechanism for getting out of intracellular compartment, and E9 offers an approach to masking the non-specific toxicity of R9-rGel.

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 3627. doi:10.1158/1538-7445.AM2011-3627
Abstract 3649: Contribution of fatty acid accumulation to myeloid-derived suppressor cell function in cancer
Authors: Cao Wei , Gabrilovich Dmitry
Published: June 2012

MDSCs are a heterogeneous population of cells that consists of myeloid progenitor cells and immature myeloid cells (IMCs). In healthy individuals, IMCs that are generated in the bone marrow quickly differentiate into mature granulocytes, macrophages or dendritic cells (DCs). By contrast, under pathological conditions, such as cancer, a partial block in the differentiation of IMCs and activation of these cells results in the expansion of the population with potent immune suppressive activity – myeloid-derived suppressor cells. We investigated the mechanism of MDSC effect on immune system and focused our attention on possible role of fatty acids (FA) in this process. Recently, we found FA accumulation in dendritic cells (DCs) from tumor bearing mice or cancer patients. It was caused by increased uptake of extracellular lipids due to up-regulation of scavenger receptor A. FA accumulation in DCs resulted in the dysfunction of DCs. Pharmacological normalization of lipid abundance in DCs with an inhibitor of acetyl-CoA carboxylase restored the functional activity of DCs and substantially enhanced the effects of cancer vaccines. In parallel, we also observed dramatic lipid accumulation in MDSC from tumor bearing mice. Interestingly, MDSCs don't have up-regulation of scavenger receptor A, but instead have increased expression of fatty acid transporter FATP4. Over-expression of FATP4 in MDSCs induced lipid accumulation. This suggested that FATP4 may be involved in the accumulation of lipid in these cells. MDSCs with lipid overload exerted stronger inhibitory effect on immune cells than MDSC with normal lipid level. MDSCs treated with unsaturated FA linoleic acid displayed stronger inhibitory effect than MDSCs treated with saturated FA palmitic acid. Since MDSCs has strong oxidizing environment, the data suggested that oxidized FA may be involved in the inhibitory effect of MDSCs. Elucidation of this mechanism in detail will be helpful for the development of potential therapeutic targets regulating immune function in tumor-bearing hosts.

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 3649. doi:10.1158/1538-7445.AM2011-3649
Abstract 3665: Enzymatic depletion of pericellular HA from tumor cell surface sensitizes Erbitux dependent immune cell-mediated cytotoxicity
Authors: Singha Netai C. , Jiang Ping , Frost Gregory I. , Shepard H Michael
Published: June 2012

Hyaluronan (HA), a ubiquitous high-molar-mass linear glycosaminoglycan (GAG) plays a critical role in cell adhesion, motility, growth and differentiation. HA accumulation is associated with many human cancers. Human tumor cells can form HA rich pericellular matrix which might favor tumor growth by preventing the access of host immune cells to malignant cells. Monoclonal antibody (MAb) based therapeutics are emerging as an important component of therapy for human malignancies. Despite the very high specificity of monoclonal antibodies, resistance to MAb therapy is common. Antibody Dependent Cell-mediated Cytotoxicity (ADCC) is an important effector for the clinical efficacy of MAbs and relies on effective interaction between the Fc domain of antigen bound antibody and FcγRIII positive immune cells. We hypothesize that pericellular HA coating may contribute to resistance of HA-rich malignant cells to MAb mediated therapy by preventing efficient access of effector cells to their tumor targets. Enzymatic depletion of HA with PEGPH20 hyaluronidase could potentially sensitize such tumors to MAb mediated therapy. remove ell surface and ated therapy by preventing tumor cells contact

EGFR positive breast carcinoma cell line MDA-MB-231luc and genetically engineered HAS2 over expressing MDA-MB-231HAS2 were used to investigate the role of HA-rich pericellular matrix in inhibition of ADCC. ADCC assay was performed by incubating Calcein AM labeled monolayer cells with monoclonal antibody (1h at 37°C) followed by co-incubation of recombinant human IL-2 (rhIL-2) activated human peripheral blood mononuclear cells (PBMC, 5h at 37°C, 5% CO2) with or without PEGPH20. Live cells from each treatment were assessed by fluorescence readings at Ex/Em 488nm/537nm (SpectraMax M2e plate reader) at the end of the assay. Percentages of cell killing were calculated against control. The student's t test was used to compare the difference between treatments.

Particle exclusion assay demonstrated that HAS2 over expressing MDA-MB-231HAS2 cells formed a pericellular coat that inhibited access of the rhIL-2 activated PBMC to tumor cells. Incubation with PEGPH20 removed the HA-rich pericellular matrix and promoted contact of PBMC to tumor cells. Incubation of Erbitux with rhIL-2 activated PBMC induced a dose dependent killing of both MDA-MB-231luc and MDA-MB-231HAS2 cells. PEGPH20 increased ADCC by 10% on MDA-MB-231luc (HAlow) and 25% on MDA-MB-231HAS2 (HAhigh) tumor cells. Our results show that the HA-rich pericellular matrix on adherent tumor cells inhibits ADCC and PEGPH20 depletion of this coat sensitizes the tumor cells to ADCC.

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 3665. doi:10.1158/1538-7445.AM2011-3665