bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 19: Identification of a novel mode of kinase inhibitor resistance: An F604S exchange in FIP1L1-PDGFRA modulates FIP1L1-PDGFRA protein stability in a SHP-2 and SRC-dependent manner
Authors: Sivahari Gorantla P. , Bubnoff Nikolas Von , Illert Ana Lena , Schneider Nicolas , Peschel Christian , Duyster Justus
Published: February 2011

FIP1L1-PDGFR alpha is a constitutively activated protein kinase which was reported in chronic eosinophilic leukemia (CEL) and in cases of hypereosinophilic syndrome and mastocytosis with eosinophilia. Imatinib is clinically active against FIP1L1-PDGFRA positive disease. However, clinical resistance to imatinib has been observed in FIP1L1-PDGFRA positive leukemia and was shown to occur due to a secondary mutation (T674I) in the PDGFR alpha kinase domain. Using a screening strategy to identify imatinib resistant mutations, we generated numerous imatinib resistant cell clones. Analysis of the PDGFRA kinase domain in these cell clones revealed a broad spectrum of resistance mutations including the clinically reported exchange T674I. Interestingly, one of the abundant mutations was a Phe to Ser exchange at position 604 (F604S), which occurred alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S in contrast to D842H and F604+D842H did not increase the biochemical or cellular IC50 value to imatinib when compared to wild-type (wt). However, F604S and F604S+D842H transformed Ba/F3, NIH3T3 and mouse bone marrow more efficiently compared to wt and D842H, respectively. Immunoprecipitation and immunoblotting indicated increased amounts of FIP1L1-PDGFRA protein in F604S versus wt cells. Pulse chase analysis revealed that FIP1L1-PDGFRA/F604S is strongly stabilised compared to wt. SRC coimmunoprecipitated with FIP1L1-PDGFRA in wt, but not F604S cells. Co-expression of SRC in 293T cells augmented degradation of wt, but not F604S FIP1L1-PDGFRA, indicating that SRC is a negative regulator of FIP1L1-PDGFRA protein stability. Importantly both, the SRC inhibitor PD166326 and SRC siRNA mimicked the F604S phenotype and resulted in stabilization of the wt protein. Importantly, phosphatase inhibitor treatment of FIP1L1-PDGFRA/F604S led to destabilization and SRC recruitment indicating that phosphatases might be responsible for the enhanced stability of FIP1L1-PDGFRA/F604S. In fact, coimmunuprecipitaion experiments identified the phosphatase SHP2 as a specific binding partner of F604S mutated FP. Together, these results suggest that stabilization of FIP1L1-PDGFRA/F604S is due to dephosphorylation by SHP-2 leading to lesser activation of the SRC and Cbl mediated ubiquitination machinery. In summary, imatinib resistance screening identified a novel class of resistance mutations in FIP1L1-PDGFRA, that do not act by impeding drug binding to the target, but increased target stability and abundance by interfering with SRC- mediated degradation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 19.
Abstract 191: Azacitidine induces differentiation of acute myeloid leukemia cell lines along the granulocytic/monocytic lineage
Authors: Nguyen Aaron N. , Luna-Moran Antonio , Richard Normand , Belka Igor , Brady Helen , MacBeth Kyle J.
Published: February 2011

Acute myeloid leukemia (AML) is characterized by blast cells that are unable to mature into functional, terminally-differentiated hematopoietic cells. Inducing leukemic cells to differentiate restores a natural cell death program and inhibits proliferation. Azacitidine (AZA; 5-azacytidine; Vidaza) is a cytidine nucleoside analog used clinically for the treatment of myelodysplastic syndromes (MDS) and AML. AZA therapy was recently shown to significantly increase median overall survival in higher-risk MDS and World Health Organization AML (20-30% bone marrow blasts) patients compared with conventional care regimens, and a phase III clinical trial of AZA in patients with more advanced AML has been activated. We have shown previously that AZA induces dose-dependent cytotoxicity to AML cell lines; however, at sub-micromolar AZA concentrations, complete cell kill is not achieved. To explore an additional anti-leukemic mechanism of AZA in AML, we assessed the effect of AZA on induction of AML cell differentiation in vitro. AML cell lines, encompassing several FAB classifications, were evaluated, using all-trans retinoic acid (ATRA) and 1,25-dihydroxyvitamin D3 (VD3), two potent inducers of AML cell differentiation, as control compounds. Differentiation along the granulocytic/monocytic lineage was assayed by CD11b expression (antigen detection by flow cytometry and mRNA by Luminex) and nitroblue tetrazolium (NBT) reduction. ATRA, VD3, and AZA induced CD11b RNA and protein expression and NBT reduction in HL-60 and AML-193 cell lines. Gene expression profiles (GEPs) in HL-60 and AML-193 cells revealed significant overlap in the genes regulated by AZA-treatment vs. VD3- or ATRA-treatment. GEPs of HL-60 and AML-193 cells treated with AZA strongly correlated with publicly-available gene sets representing differentiated eosinophils, neutrophils, and monocytes, but negatively correlated with those of differentiated erythrocytes and megakaryocytes. Similar studies in primary AML cells are ongoing. Our results demonstrate that AZA can induce cellular differentiation of AML cell lines along the granulocytic/monocytic lineage in vitro, and suggest that cellular differentiation may contribute as one of multiple mechanisms of AZA's anti-leukemic activity in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 191.
Abstract 1911: Influence of gap-junction formation on melanoma specific-CD8 T cells function
Authors: Benlalam Houssem , Carré Thibault , Jalil Abdelali , Avril Marie-Françoise , Kieda Claudine , Mami-Chouaib Fathia , Chouaib Salem
Published: February 2011

Gap-junction channels (GJ) constitute a new potential pathway involved in the regulation of the tumor immunity. We previously showed, in a 3D construct model, that connexin43-based-GJ permitted the transfer of antigenic peptide from melanoma cell line to autologous endothelial cells. This antigenic peptide was cross-presented by endothelial cells leading to their specific recognition and lysis by specific autologous CD8+ CTL. We further investigate the potential role of GJ in the interaction between endothelial and melanoma cells. Using histological sections staining, our results indicate that gap-junction formation can occur in vivo from melanoma patients. Morever, we found that GJ are colocalized in the immunological synapse between CD8+ CTL and melanoma or melanoma-derived endothelial cells. Analysis of intracellular traffic of connexin43-GFP, using confocal live imaging, corroborates this observation since a translocation of intracellular connexin43-GFP to the contact area between CTL and target cells was observed. Our data demonstrate that CTL and melanoma cell lines exchange their intracytoplasmic content through GJ formation during the formation of the immunological synapse. The characterization of the GJ role and the influence of transferred molecules between cells will be discussed. These data open a new perspective on gap-junction importance in the anti-tumor activity of CTLs.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1911.
Abstract 1920: Chemo-resistant regulatory T cells inhibit the augmentation of antitumor immunity during homeostatic T cell proliferation
Authors: Baba Junko , Watanabe Satoshi , Ichikawa Kosuke , Koshio Jun , Miyabayashi Takao , Tanaka Junta , Tanaka Hiroshi , Kagamu Hiroshi , Yoshizawa Hirohisa , Narita Ichiei
Published: February 2011

It has been demonstrated that lymphodepletion by cytotoxic regimens, such as chemotherapy and radiotherapy, augments antitumor immune response. Although the mechanism underlying this stimulation of antitumor immunity remains unclear, possible explanations have been proposed; depletion of suppressor cells, improvement of tumor-antigen presentation, and elimination of lymphocytes competing activation cytokines. We previously reported that CD4+CD25+Foxp3+ regulatory T cells were increasing in sublethally (500 rad) irradiated mice. Those radio-resistant regulatory T cells inhibited the development of antitumor immunity during recovery from lymphopenia. Depletion of regulatory T cells after irradiation enhanced generation of antitumor effector T cells and significantly suppressed tumor progression.

We hypothesized that chemotherapy-resistant, as well as radio-resistant, regulatory T cells inhibited antitumor immune response. In this study, C57/BL6 mice were administered 200 or 400 mg/kg of cyclophosphamide to deplete lymphocytes. On the same day, naïve spleen T cells were transferred intravenously followed by subcutaneous injection of MCA205 tumor cells. We found that the percentage of regulatory T cells increased from 4-5% in normal mice to 16% in lymphopenic mice injected with cyclophosphamide. Transfer of naïve T cells into cyclophosphamide injected lymphopenic mice showed some antitumor effects, and further elimination of regulatory T cells by anti-CD25 antibodies (PC61) following administration of cyclophosphamide significantly inhibited skin-tumor growth. It is well known that transforming growth factor-beta (TGF-beta) is critical differentiation cytokine for regulatory T cells. Thus, we examined the effects of anti-TGF-beta antibodies (1D11) on regulatory T cells during recovery from lymphopenia. Injection of anti-TGF-beta antibodies after lymphodepletion stimulated antitumor immune responses and delayed tumor progression.

The results indicate that regulatory T cells are resistant to cytotoxic regimens. The combination of cytotoxic therapy and inhibition of regulatory T cells efficiently increases antitumor immune response.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1920.
Abstract 1924: The paraproteins of patients with familial MGUS / multiple myeloma (MM) target family-specific antigens: Experience with paratarg-7 and paratarg-8
Authors: Grass Sandra , Preuss Klaus-Dieter , Wikowicz Aleksandra , Thome Stephan , Weisenburger Dennis D. , Witt Vinetta , Lynch Jane , Mahlknecht Ulrich , Pfreundschuh Michael , Lynch Henry
Published: February 2011

Background: Antigenic targets of paraproteins in monoclonal gammopathy of undetermined significance (MGUS) and multiple Myeloma (MM) might play a role in the pathogenesis of these neoplasms by chronic antigenic stimulation. However, very few antigens have been identified, of which most were specific for one individual paraprotein only. In contrast, we recently described paratarg-7 (P-7), a protein of unknown function which is expressed in all human tissues as the target of 15% of IgA and IgG paraproteins in MGUS and MM (Grass et al. Lancet Oncology 2009; 10: 950-956). MGUS/MM patients with P-7 specific paraproteins all carry a hyperphosphorylated variant of P-7 (pP-7), which is inherited in a dominant fashion, explaining pP7-posistive cases of familial MGUS/MM. pP-7 is the first molecularly defined autosomal-dominant risk factor for MGUS/MM and is associated with the highest odds ratio (7.9) of all risk factors for MGUS/MM known to date. Since not all cases of familial MGUS/MM have a P-7 specific paraprotein, we set out to identify the antigenic targets of paraproteins in cases of familial MGUS/MM that are not carriers of pP-7. Methods: The sera of 28 members of a family with familial MGUS/MM (Lynch et al. N Engl J Med 2008;359:152-157) were tested for antibody reactivity against antigens represented in a fetal brain derived protein macroarray using a modified SEREX approach (Preuss et al. International J. Cancer 2009;125, 656-661). Results: Sera from 28 family members were analyzed. The sera of all 6 family members affected by MGUS (n=3) and MM (n=3) reacted with paratarg-8, which is encoded by the ATG13 gene, a member of the “autophagy regulatory complex” family of genes. The titers ranged from 1:107 to 1:108. No antibody reactivity was detected in the sera of non-affected family members nor in the paraproteins from >300 other patients tested. Conclusions: The paraproteins of members affected by familial MGUS/MM are directed against family-specific antigenic targets, suggesting that the genetic background shared by these patients entertains a chronic auto-immune response. Paratarg-7 and paratarg-8 are the first family-specific antigens that have been molecularly defined to date. Whether the antigen/paraprotein interaction is an epiphenomeon or invovled in the pathogenesis of familial cases of MGUS/MM can now be addressed using specific molecular tools.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1924.
Abstract 1925: MHC class I-restricted MART-127-35 specific TCR engineered human CD4+ and CD8+ T cells exhibit polyfunctional properties
Authors: Ray Swagatam , Chhabra Arvind , Chakraborty Nitya G. , Ribas Antoni , Economou James S. , Mukherji Bijay
Published: February 2011

The “polyfunctional” property of antigen specific T cells has turned out to be an important element in successful host immune responses in AIDS and in malignancies. We have shown that human CD4+ CD25- T cells engineered to express MHC Class I-restricted MART-127-35 epitope specific TCR show cytolytic activity, secret Th1 type cytokines. We have now carried out an extended analysis of the functional dimension of MART-127-35 epitope specific TCR engineered CD4+ and CD8+ T cells. Freshly isolated circulating CD4+ and CD8+ T cells, transduced with MART-127-35 epitope specific DMF5 TCR containing retrovirus, were tested for proliferative, cytokine secretory and for cytolytic capacity (polyfunctionality analyzed with intracytoplasmic immunofluorescence staining for IL-2, IFN-γ, TNF-α, MIP-1β and CD107a using the SPICE software). The TCR engineered CD4+ T cells were also tested for their potential for “helping” cytolytic T cells response by CD8+ T cells in in vitro CTL generation cultures.

The TCR-engineered CD4+ as well as CD8+ T cells were found to be polyfunctional. For example, they underwent multiple rounds of division following stimulation by peptide pulsed dendritic cells and by melanoma cells, synthesized multiple cytokines (IFN-γ, TNF-α, IL-2, MIP1ß) and lysed target cells. The TCR engineered CD4+ T cells also amplified the CD8+ CTL burst size in vitro. Multiparametric analyses revealed that a single TCR-engineered CD4+ T cell could perform as many as five different functions. Nearly 100% MART-127-35 specific TCR expressing CD4+ as well as CD8+ T cells could be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR-transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1925.
Abstract 193: Dissecting satellite and imprinting genes methylation status in cloned bovine: Aberrant methylation in imprinting loci
Authors: Shen Chih-Jie , Ling Chiao-Chieh , Shen Perng-Chih , Cheng Winston T. K. , Chen Hsiao-Ling , Chen Chuan-Mu
Published: February 2011

In mammals, genome-wide epigenetic reprogramming systems exist in primordial germ cells and zygote. It plays a crucial role in regulating genome functions at critical stages of embryo development and confers stability of gene expression during mammalian development. Inappropriate reprogramming of donor nuclei in somatic cell resulted in neonatal death and preimplantation defects during animal cloning technique. Specific demethylation events in differentiated tissues could then lead to further changes in gene expression as needed. In this study, six cloned bovines created by ear fibroblast nuclear transfer with short life span and multiple organ defects were used as the experimental materials. We focus on three imprinting genes included two growth factor genes IGF-2, H19 and Xist (X chromosome inactive regulated gene) by combined bisulfite and restriction assay (COBRA). Bisulfite sequencing was also applied to analyze the aberrant CpG sites in different imprinted genes loci in these cloned bovine genomes. Our data show that loss of imprinting (LOI) phenomenon was frequently appeared in IGF-2, Xist and H19 imprinted loci in several tissues of these cloned bovines. Furthermore, we detected the cloned bovine NTG-2 expression of these imprinting genes with quantitative real time PCR (Q-PCR). The data suggested that IGF-2 and H19 are extremely overexpressed in liver, vein, ear, skin and uterus. The IGF-2 mRNA expression in uterus and liver of NTG-2 cow are almost 1,200 and 800 folds compared to WT cow. The H19 expression level in liver of NTG-2 also exhibited a 34,000-fold higher than that of WT cow. According to our research, the extensively overexpression of IGF-2 and H19, probably were the major reason to cause the cloned bovine NTG-2 not only organ defects, but also aberrant embryonic development. In conclusion, the death of clones may be due to aberrant DNA methylation at the loci of imprinting genes and disruption of incompletely reprogrammed after nuclear transfer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 193.
Abstract 1937: Enhanced tumor control with adoptive transfer of TCR engineered T cells expressing Tcl1
Authors: Koya Richard C. , Mok Stephen , Chodon Thinle , Teitell Michael , Ribas Antoni
Published: February 2011

Advanced stage melanoma is associated with notable resistance to radio and chemotherapy, warranting intense research for new treatments. Immunotherapy with adoptive transfer of antigen-specific T cells has shown anti-tumor activity in melanoma, but questions remain regarding the biology of T cell responses, and the necessary enhancement strategies to achieve significant impact in the overall clinical outcome. The14 kDa protein T cell leukemia-1 (TCL1) has shown to augment the growth and survival of mature but not immature T cells through enhancement of signals that operate through the PI3K-Akt and protein kinase C (PKC)-MAPK- ERK pathways. Here we engineered TCL1 transgenic lymphocytes to express a T cell receptor (TCR) specific to ovalbumin antigen to be used in an adoptive transfer model for melanoma tumor control in vivo. A bicistronic MSCV-based retroviral vector was engineered to express the alpha and beta chains of a TCR (OT1) specific for SIINFEKL peptide of ovalbumin (OVA) presented on mouse MHC I (Kb). The transgenes were interspaced with picornavirus-derived highly efficient self-cleaving 2A-like sequences to allow balanced protein expression. Transduction of ex vivo activated primary murine T cells with retrovirus stocks demonstrated correct expression of specific TCR as analyzed by flow-cytometry with anti-Valpha2/Vbeta5.1+5.2 staining and OVA (SIINFEKL) tetramer assays, with transduction efficiency of 64.8-76% at M.O.I.=20. OT1 TCR-transduced murine splenocytes from TCL1 transgenic mice stimulated with ovalbumin-expressing murine EL4 lymphoma or B16 melanoma showed high and specific IFNgamma production (30,000 and 16,000 pg/ml/1e6cells/24h respectively) as assessed by ELISA. In vivo studies were carried out in C57BL6 mice implanted with syngeneic B16/OVA murine melanoma. Intravenously adoptive transferred OT1 TCR-transduced splenocytes from TCL1 transgenic mice lead to higher survival and tumor size control in comparison to OT1 TCR-transduced control syngeneic splenocytes (tumor volume: 221.65mm3 and 1095.48mm3, respectively at day 23 post-infusion, p<0.05). In conclusion, TCL1 expression in mature T cells enhanced the potency of adoptively transferred T cells for treatment of melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1937.
Abstract 1948: Pvt1-encoded microRNA miR-1204 in tumorigenesis of human ovarian and breast cancer
Authors: Huang Jing , Collisson Eric A. , Gibb Bill , Jakkula Lakshmi , Martin Steven , Gray Joe w.
Published: February 2011

Amplification at chromosome 8q24 is one of the most frequent genomic abnormalities in human cancers and is associated with reduced survival duration in ovarian and breast cancers. Recently, microRNAs (miRNAs) at 8q24 have been identified in the PVT1 locus. Herein, we explored the biological functions of one miRNA, miR-1204, residing within the exon1B of PVT1. Mature hsa-mir-1204 is over-expressed in PVT1 amplified ovarian and breast cancer cell lines (HEY, OVCAR8, SUM-159PT and MDA-MB231). In vitro treatment with antagomir against hsa-mir-1204 inhibited cell proliferation and induced apoptosis in the four PVT1 amplified ovarian and breast cancer lines. Conversely, over-expression of miR-1204 promoted cell growth in PVT1 non-amplified ovarian and breast cancer lines (OV90 and MCF10A) and cooperated with c-Myc to potentiate Rat1A cell colony growth in soft agar assays. Since an inverse expression pattern is often present between miRNA and its target genes, we used computational approaches (miRanda, TargetScan, Pita) to identify distinct miR-1204 candidate target genes from two independent expression microarray datasets, one with significantly down-regulated genes by PVT1 copy number gain in 147 breast tumors and the other involving genes significantly repressed by miR-1204 overexpression and derepressed by silencing of miR-1204 in ovarian and breast cancer cells. Thirty putative target genes of miR-1204 were empirically verified by Q-RT-PCR method, and this group of genes was enriched with genes regulating cell death, cell growth and proliferation, cell cycle, cellular assembly and organization. Combined, these data suggest that human miR-1204 contributes to ovarian and breast tumor proliferation and survival and down-regulates distinct targets including multiple tumor suppressor genes and serves as a potential therapeutic target against tumors with 8q24 amplification.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1948.
Abstract 1949: Positive feedback regulation between EWS-FLI1 and miR-145 in Ewing's sarcoma
Authors: Ban Jozef , Mestdagh Pieter , Kauer Max , Jug Gunhild , Speleman Frank , Kovar Heinrich
Published: February 2011

The EWS-FLI1 chimeric protein is a potent tumor specific transcriptional regulator and the molecular cause of Ewing's sarcoma family of tumors (ESFT). We had previously hypothesized that, since EWS-FLI1 is the major driver of aberrant gene expression in ESFT, there should be an inverse correlation between the changes in gene expression observed in ESFT cell lines in response to RNAi-mediated EWS-FLI1 knockdown and the corresponding gene expression in primary tumors relative to the tissue of origin. We found that mesenchymal progenitor cells (MPC) fit this assumption best and established an ESFT specific EWS-FLI1 transcriptional signature on the mRNA level. Following the same approach, we now studied the EWS-FLI1 dependent regulation of microRNAs (miRNAs) in ESFT. Genome-wide miRNA analysis was performed after shRNA-mediated stable (5 cell lines) and inducible (1 cell line) EWS-FLI1 knockdown, as well as in 6 primary ESFT and 6 MPC samples using the stem-loop reverse transcription quantitative PCR platform. Among miRNAs consistently affected by EWS-FLI1 silencing and inversely expressed in primary tumors compared to MPCs was hsa-miR-145 which we found upregulated upon EWS-FLI1 silencing. Since several target sites exist for this miRNA within the FLI1 3′UTR, we asked if hsa-miR-145 is capable of modulating EWS-FLI1 expression in ESFT cells. Upon ectopic expression of pri-miR-145 in wildtype p53 ESFT cells, strong modulation of EWS-FLI1 protein levels, but not RNA levels, was observed. In p53 mutant cell lines this effect was less pronounced consistent with a recently reported role of p53 in miR-145 maturation. Preliminary studies suggest that hsa-miR-145 is indirectly regulated by EWS-FLI1 by a mechanism that may involve the NOTCH signaling pathway. Our results identify a positive feed-back loop between EWS-FLI1 and hsa-miR-145 expression in ESFT.

Supported by the 6th framework program of the European Commission, (STREP “E.E.T. Pipeline“ contract LSHC-CT-2006-037260) and grant P20665-B12 of the Austrian Science Fund FWF.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1949.
Abstract 195: Functional epigenetics identifies a protein phosphatase-1 regulatory subunit gene as a candidate tumor suppressor frequently silenced by promoter CpG methylation in multiple tumors
Authors: Leung Ching Hei , Ng Ka Man , Wong Ada Ho Yan , Ying Jian ming , Srivastava Gopesh , Leung Sing Fai , Chan Anthony Tak Cheung , Tao Qian
Published: February 2011

Protein Phosphatase 1 regulatory subunits (PPP1R) are a family of small molecules which modify the function and protein targeting of protein phosphatase-1 upon their interactions. Deregulated expression of PPP1R members could often be found in cancer cells. For example, ASPP family is well known for its necessity of p53 function while ASPP silencing leads to carcinogenesis or poor prognosis in cancer patients. Here, one of the PPP1R members (TUSC7) was identified which its role in tumor suppression had been investigated

Using semi-quantitative RT-PCR, expression profile of TUSC7 in cancer cell lines was obtained. TUSC7 was frequently silenced in cancer cells. Bisulfite treated tumor DNA was subjected to Methylation-specific PCR (MSP) using primers flanking across the ∼130bp CpG island of the TUSC7 promoter. It was revealed that silencing of TUSC7 was due to promoter hypermethylation in most of the corresponding cancer cell lines which had been further confirmed by bisulfite genomic sequencing (BGS). Treatment with azacytidine and trichostatin restored TUSC7 expression through demethylating the hypermethylated promoter. To quantitatively determine the extensiveness of cancer cell growth inhibition by TUSC7, colony formation assay was used. It was found that ectopic expression of TUSC7 could inhibit the proliferation of tumor cells, Caski (cervical carcinoma), HCT116 (colon carcinoma) and KYSE150 (esophageal squamaus carcinoma cells) by 40-50%. In addition, ectopic expression of TUSC7 increased the amount of phosphorylated GSK-3beta (pSer9-GSK-3beta) as shown in the western blot analysis. pSer-9 GSK-3beta is the inactive form of GSK-3beta. It has been reported that inactive GSK-3beta may affect p53 activities. To summarize, TUSC7 was found to be a tumor suppressor frequently silenced in cancer cells through epigenetic mechanism. TUSC7 may be significant in regulating the activity of GSK-3beta whose signaling is important in carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 195.
Abstract 1956: A novel mouse model of breast cancer metastasis to bone
Authors: Shaw Aubie K. , Johnson Rachelle , Sterling Julie , Mundy Greg , Moses Hal
Published: February 2011

Patients with advanced stage breast cancer develop bone metastasis, which leads to hypercalcemia, fractures, nerve compression, paralysis and pain. Breast cancer bone lesions can be osteolytic or osteoblastic, however osteolytic lesions are predominant. Currently, there is no experimental model available to examine osteotropism or trafficking of breast cancer cells to the bone from the primary tumor. TGFβ signaling has been suggested to be responsible for osteotropism. TGFβ stimulates Gli2 and PTHrP expression, which have been shown to be responsible for osteolysis. We have shown that MCF10A, AT-1 and CA1d human breast epithelial cells are responsive to TGFβ and express very high levels of Gli2 and PTHrP. Orthotopic grafting to the cleared fatpad of SCID mice shows that MCF10A and AT-1 cells do not form tumors, even when grafted with normal fibroblasts. CA1d carcinoma cells that are co-grafted with normal fibroblasts show rapid tumor formation with 100% incidence, whereas CA1d cells grafted alone rarely form tumors in the orthotopic site. The tumors can be localized real-time and non-invasively using optical imaging of GFP. X-ray imaging revealed loss of bone density in sacral/caudal vertebrae and hindlimbs. Ca1d grafts generate undifferentiated primary tumors that metastasize to lung and bone. Future studies will determine if CA1d cells stimulate osteolysis. This model may provide a valuable model to examine osteotropism of breast cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1956.
Abstract 1961: Ikzf1 haploinsufficiency contributes to the pathogenesis of BCR-ABL1 positive acute lymphoblastic leukemia
Authors: Collins-Underwood J Racquel , Miller Christopher B. , Downing James R. , Mullighan Charles G.
Published: February 2011

Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), an expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. We recently identified a near-obligate deletion of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. To test this hypothesis, we have examined the effect of Ikaros haploinsufficiency in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from either Ikzf1 wild type mice or mice heterozygous for an Ikzf1 null allele was transduced with MSCV bicistronic retrovirus expressing GFP and p185 BCR-ABL1 and transplanted into lethally irradiated C57BL/6 recipients. Recipients of MSCV-GFP-p185 BCR-ABL1-transduced Ikzf1+/− marrow developed leukemia with markedly increased penetrance and reduced latency in comparison to recipients of wild-type marrow (46 vs. 122 d, P<0.0001). The resulting leukemias resulted in bone marrow replacement by lymphoblasts, pancytopenia, extramedullary infiltration, and frequent clinical and histologic central nervous system involvement. The leukemias were of pre-B immunophenotype and uniformly transplantable into sublethally irradiated secondary recipients. Importantly, detailed enumeration of lymphoid subsets in Ikzf+/+ and +/− marrow did not demonstrate an expansion of lymphoid progenitors in Ikzf1+/− marrow, suggesting that the observed increased penetrance of B-ALL in Ikzf1+/− animals was not simply due to the expansion of targets of BCR-ABL1 transformation. Thus, Ikaros haploinsufficiency cooperates with BCR-ABL1 to induce an aggressive pre-B leukemia with clinical and pathologic features recapitulating human BCR-ABL1 ALL. These results support genomic analyses of human leukemia suggesting that IKAROS loss is important in the pathogenesis of this disease. Experiments evaluating the interaction of Ikzf1 loss with other targets of genomic alteration in BCR-ABL1 ALL (e.g. CDKN2A/B, PAX5), studies examining the role of Ikzf1 in responsiveness to kinase inhibitors, and genomic profiling studies are ongoing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1961.
Abstract 1973: Targeting activated alleles of EGFR derived from glioma and lung cancer: Correlating kinase active-site occupancy with efficacy
Authors: Nicolaides Theodore P. , Barkovich Krister , Hariono Sujatmi , Garske Adam , Fan Qi Wen , Shokat Kevan , Weiss William A.
Published: February 2011

Introduction: The Epidermal Growth Factor Receptor (EGFR) features prominently in malignant glioma (MG), yet attempts to exploit this target in patients with MG have been somewhat disappointing. Unlike the highly sensitizing kinase site alleles of EGFR derived from patients with non-small cell lung cancer (NSCLCA), mutationally activated alleles of EGFR derived from patients with MG respond poorly to the currently available reversible clinical EGFR inhibitors. We hypothesize that the anti-tumor effects of EGFR inhibitors in MG-associated alleles of EGFR require more efficient blockade to achieve anti-tumor responses, in comparison with NSCLCA-associated alleles of EGFR, and that thisincreased efficiency can be achieved using irreversible inhibitors.

Methods: We generated glioma and fibroblast cell lines that expressed wild-type or tumor-associated alleles of EGFR derived either from MG (EGFRvIII) or NSCLCA (L858R or del 747-753). Using an EGFR affinity probe (an irreversible EGFR inhibitor tethered to a flourophore), we quantified occupancy of the kinase active site in response to erotinib, a clinical reversible inhibitor of EGFR, correlating occupancy with proliferation and apoptosis. Next, we repeated these measurements using the tool compound and irreversible EGFR inhibitor PD-168394.

Results: Using the affinity probe in pulse-chase experiments, we quantified active site occupancy of EGFR int response to increasing doses of inhibitors. In cells expressing MG-derived alleles, irreversible EGFR inhibitors were significantly more potent than reversible agents in abrogating signaling downstream of EGFR and in decreasing viability. Using the affinity probe, we found that irreversible inhibitors showed nearly complete occupancy of the active site, whereas treatment with reversible EGFR inhibitors resulted in only partial occupancy. Cells containing NSCLCA-derived EGFR alleles showed similar signaling and growth responses to both reversible and EGFR inhibitors, despite a large difference in EGFR kinase site occupancy with the two types of inhibitors. Flow cytometry studies correlating kinase site occupancy with downstream signaling in individual cells will also be presented.

Conclusions: Activated alleles of EGFR derived from MG require significantly higher levels of kinase site blockade to achieve an anti-proliferative threshold, as compared with activated alleles derived from NSCLCA. This higher degree of kinase site blockade can be achieved using irreversible inhibitors of EGFR. These studies support the initiation of clinical trials using irreversible inhibitors of EGFR in MG.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1973.
Abstract 1978: The poly-SUMO protein specific E3 ubiquitin ligase RNF4 is induced in multiple myeloma and reduces bortezomib-induced cell killing
Authors: Driscoll James J. , Gootenberg Jonathan , Amin Samir , Pelluru Dheeraj , Avet-loiseau Herve , Minville Stephane , Anderson Kenneth C. , Munshi Nikhil C. , Annunziata Christina M.
Published: February 2011

Multiple Myeloma (MM) is a fatal neoplasm of B-cell origin characterized by the clonal proliferation and accumulation of malignant plasma cells in the bone marrow. While recent advances in mechanistic understanding and treatment modalities have extended median survival to >6 years and 10% of patients survive >10 years, the vast majority of MM remains incurable with conventional, high dose therapy or stem cell transplantation. Bortezomib is a selective, reversible inhibitor of the 26S proteasome that inhibits protein degradation and is now FDA-approved for the treatment of newly diagnosed, relapsed and refractory MM. Though the catabolism of ubiquitinated substrates has been targeted therapeutically with significantly improved prognosis, patient response to bortezomib remains highly variable and cannot be predicted accurately. E3 ligases confer specificity on target selection for Ub+proteasome degradation. We therefore analyzed the expression of individual E3's using a microarray dataset obtained from MM patient tumor samples and found a striking variability in the expression level of individual E3 ligases between normal plasma cells and patients MM cells. RNF4, an E3 specific for poly-sumoylated proteins, was induced in MM patients and correlated with decreased patient response to the proteasome inhibitor bortezomib. Expression profiling of pretreatment tumor samples obtained from MM patients in independent clinical trials were used to generate a signature that correlated expression of SUMO+Ub+Proteasome pathway components with clinical outcome to predict patient response to bortezomib. Experimental validation by overexpression of RNF4-wt rendered myeloma cell lines relatively resistant to bortezomib while RNF4 depletion by shRNA enhanced sensitivity to bortezomib. Transfection of HA-tagged SUMO followed by bortezomib exposure led to the accumulation of HA-SUMO∼conjugates that were immunoreactive with Ub and proteasome components to further link the pathways. In summary, RNF4 an E3 ligase specific for SUMO-conjugates and induced in myeloma, modulates the cellular response to proteasome-based therapy and promotes bortezomib resistance. Our results support regulators of the sumoylation pathway as biomarkers to predict clinical response to bortezomib and provide evidence for targeting SUMO pathway to improve therapeutic outcome in myeloma in general and bortezomib specifically.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1978.
Abstract 1982: Analyzing TCGA data: A novel, systems-oriented method for diagnosing batch effects and adjusting for them
Authors: Zhang Nianxiang , Akbani Rehan , Zhang Li , Broom Bradley , Baggerly Keith A. , Mills Gordon B. , Yung W.K. Alfred , Weinstein John N.
Published: February 2011

The Cancer Genome Atlas (TCGA) project aims at multi-faceted molecular characterization of up to 25 different types of clinical cancers over the next 5 years. Included are profiles of single-nucleotide mutations, indels, loss of heterozygosity, DNA copy number, DNA methylation, mRNA expression, and microRNA expression. For each type of measurement, an absolutely critical initial question is whether the batches of tumors obtained from different sources and/or processed at different times show batch effects that might confound our ability to draw valid conclusions about the biology or about clinical outcomes. As PIs and members of a newly designated TCGA Genome Data Analysis Center B, we have developed and applied a novel, systems-oriented approach to the problem of batch differences. The method is based on a scalar quantity that we call the “correlation of correlations” (CR). CR reflects the difference between batches, not in terms of characteristics of the individual genes but in a more systems-oriented way based on the pattern of relationships among the various genes in the data set. If two batches of samples are identical, CR = 1; if they are randomly related to each other in their properties, CR = 0. To assess the statistical robustness (i.e., p-value) of any apparent difference between two batches, we apply a permutation test, keeping the batch sizes constant. That analysis is complemented by principal component analysis and hierarchical clustering. In addition to diagnosing batch effects, we have tested five different algorithms (empirical Bayes with parametric and non-parametric prior, median polish, batch-wise median polish, ANOVA, and ANOVA with variance shrinkage) to correct in part for any batch effects that do exist. We first applied the new approach to TCGA Affymetrix U133A mRNA expression data on 237 high-grade serous ovarian cancers. One batch (designated # 9) out of 6 stood out as different from the others, particularly from batch #14 (permutation test two-tail p <0.001). Of the 5 adjustment methods, batch-wise median polish produced the greatest uniformity of the final data set and the greatest decrease in hierarchical clustering batch effects (Fisher's exact p <0.0001). Similar analysis of 327 TCGA glioblastoma samples showed batch #4 to be the most statistically significant outlier out of 10 batches, with batch #3 also significantly different. We are using the same methods to assess global similarities and differences among disparate classes of data, for example mRNA and microRNA expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1982.
Abstract 1984: A novel logistic regression model for microRNA microarray normalization
Authors: Wang Bin , Howell Paul , Wang Xiaofeng , Qian Xuemin , Huang Kun , Riker Adam , Ju Jingfang , Xi Yaguang
Published: February 2011

MicroRNA (miRNA) is a set of newly discovered non-coding small RNA molecules. It regulates gene expression at the translational level through binding to the 3′-UTR of the target gene. Its significant effects have contributed to a number of critical biological events including cell proliferation, apoptosis, development as well as tumorigenesis. High throughput genomic discovery platforms (e.g. microarray) have been employed to evaluate the important roles of miRNAs by analyzing their expression profiling. However, because of the small total number of miRNAs and the absence of well known endorse controls, the traditional normalization methods for messenger RNA (mRNA) profiling analysis could not offer a suitable normalization solution for miRNA analysis. The establishment of new adaptive normalization methods has come to the forefront. In this study, Locked Nucleic Acid (LNA) based miRNA array was employed to profile miRNAs using colorectal cancer cell lines HCT116 under different drug treatments (5FU, CPT-11, and oxaliplatin). We randomly selected approximate 10% of total microRNAs from the microarray (37 miRNAs) and determined their expression patterns using quantitive real-time PCR (qRT-PCR), which has become the gold standard for relative gene expression analysis and one of the major validation methods after high throughput discovery. Then, a logistic regression model was built to normalize the miRNA expression data from miRNA array, by utilizing the results from qRT-PCR and some auxiliary variables. Twenty additional miRNAs were selected to validate the predicted results by the novel model. The result shows that the consistency between the qRT-PCR and array results was significantly improved with the new normalization method. Compared to other popularly used normalization methods, the logistic regression model efficiently calibrates the variance across arrays and improves miRNA microarray discovery accuracy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1984.
Abstract 1985: Identification of differentially expressed genes using annealing control primer (ACP)-based GeneFishing PCR technique in serous ovarian carcinoma of Korean women
Authors: Choi Dae Han , Bae Sumi , Kim Jeong Shick , Bae Dong Han , Baek Kwang-Hyun , Ahn Woong Shick
Published: February 2011

Object : Ovarian cancer is the most lethal among gynecologic neoplasmas in women more than 60% of ovarian cancer is serous cystadenocarcinoma. The aim of this study is to discover different genes expression profiles between serous cystadenocarcinoma of ovary in Korean women and normal ovarian tissue.

Methods : Twenty ovarian serous cystadenocarcinoma specimens were obtained from the Department of Obstetrics and Gynecology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea. We used a common reference as a control Stratagene (www.stratagene.com). The profiles of expressed genes were discovered from the ovarian cancer using annealing control primer (ACP)-based GeneFishing polymerase chain reaction (www.seegene.co.kr), screened by a BLAST search, and confirmed by quantitative reverse transcription-PCR (qRT-PCR).

Results : In the study, about 173 differential expressed genes were identified between normal and ovarian serous cystadenocarcinoma samples, 105 of differentially expressed genes were identified more than 2-folds, 68 up-regulated and 37 down-regulated genes were sequenced. Up-regulated genes were DC_UbP, mitochondrial respiratory proteins, secretoglobin, gelsolin, actinin, cox2, sarcoglycan, VTCN1, DYNEIN, S100 A10, IFI27, EIF4A1, IFITM1, B-cell translocation protein 1, PPA1, NDRG1, HLA-DRA, COL11A1, BAZ2B, RAD54L, PRDX1, SMAD5, ELF3, SSBP1, HSBP1, TFG, SP3 etc. and down-regulated genes were MYH11, TIMP1, CYP21A2, Myo15, complement component1(C1S), FOXP1, Ku etc. Among 105 clones selected by ACP-based GeneFishing PCR, 57 genes (46 up-regulated and 11 down-regulated genes) with different expression patterns were determined and all these were confirmed by quantitative real time RT-PCR. Especially qRT-PCR analysis showed that SSBP1, HSBP1, TFG and SP3 genes were up expressed, and C1S gene was down expressed in 100% (20/20) patients with serous ovarian carcinoma.

Conclusion : We identified the five highly expressed genes in serous ovarian adenocarcinoma using ACP-based GeneFishing PCR technique in Korean women. The detail functions of those genes and their products remain to be determined, the genes will provide insights into mechanisms of carcinogenesis or tumor progression in serous ovarian carcinoma in Korean women.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1985.