bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4513: Predicting response of non-small cell lung cancer to the telomerase inhibitor imetelstat
Authors: Frink Robin E. , Girard Luc , Wright Woodring E. , Shay Jerry W. , Minna John D.
Published: June 2012

Telomerase is active in more than 80% of non-small cell lung cancers (NSCLC) but is not active in most somatic cells, making it an attractive target for cancer therapy. Furthermore, telomerase is required for cancer stem cell maintenance so targeting telomerase could affect this chemo-resistant subpopulation. Imetelstat, a 13-mer N3’ P5′-thio-phosphoramidate, inhibits telomerase by binding to the RNA template component leading to shortened telomeres and associated cell death. Because imetelstat is currently in clinical trials for NSCLC, it is important to determine the spectrum of activity of imetelstat in a variety of NSCLCs. To accomplish this, we examined the in vitro effect of short-term imetelstat treatment in both the colony formation assay and a 5 day MTS assay using a panel of NSCLC cell lines. For the colony formation assay, cells were plated at clonal density, treated with 3 μM imetelstat 24 hours later, and allowed to grow into colonies for 2-4 weeks. The end point was determined by untreated control cells in colony formation and was dependent on inherent population doubling time. We found an array of responses ranging up to 96% inhibition of colony formation. The panel can be divided into 3 groups: responders (>70% inhibition), intermediate (20%-70% inhibition), and non-responders (<20% inhibition). These groups may be used to develop a biomarker signature predictive of which patients will be most sensitive to imetelstat. With the 5 day MTS assay, cells were treated with imetelstat concentrations ranging from 2.6 nM to 42.5 μM and allowed to incubate for 5 days. Cell viability was determined by a colorimetric readout using MTS. Because of expected delayed response time due to the mechanism of action of imetelstat, we were surprised to find a cell line sensitive to 1 μM imetelstat in the short-term assay. Only 50% of the cells remained after 5 days of treatment. Although this cell line is not sensitive to the imetelstat mismatch control, further studies are needed to determine if this effect is telomere specific. Our studies indicate a wide range of response of NSCLC to imetelstat emphasizing the importance of predicting which patients are most likely to respond to imetelstat 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 4513. doi:10.1158/1538-7445.AM2011-4513
Abstract 4514: NMR study of XR5944 interaction with diverse estrogen response elements (EREs)
Authors: Mathad Raveendra I. , Zhang Zhenjiang , Shu Feng-jue , Chen Xi , Carver Megan , Kallen Caleb , Sidell Neil , Yang Danzhou
Published: June 2012

ER has emerged as one of the most effective targets for breast cancer therapy since the discovery of the estrogen receptor (ER) as the modulator of estrogen action. However, the current ER-targeting therapies have a main limitation related to resistance. Our previous studies have shown that XR5944, a DNA bis-intercalator is capable of inhibiting the ER activity by its ability to specifically block the binding of ER to its consensus EREs. An DNA intercalator that targets ERE may be a useful therapeutic agent that can overcome the resistance to existing endocrine therapies. To understand the mechanism of the inhibition of ER-ERE interactions by XR5944, we have tested the specificity of XR5944 binding to consensus EREs (AGGTCAnnnTGACCT) with different tri-nucleotide spacer sequences as well as natural EREs using 1H NMR titration studies. Our results suggested that the ERE spacers play a significant role in determining the binding characteristics of the complex. XR5944 binds the ERE sequences in a slow-exchange binding mode on the NMR time-scale. The binding stoichiometry of XR5944 with ERE sequences was shown to be 2:1. Of those tested, the CGG/GCC and AGG/CCT spacers showed the best binding, while the CGT/ACG and TTT/AAA spacers showed the worst binding with XR5944. Results of luciferase reporter assays in MCF-7 cells indicated that XR5944 was significantly more potent in inhibiting the activity of reporters containing ERE-CGG than those containing ERE-TTT, consistent with our NMR results. Natural ERE sequences, including those located in the promoter of ER target genes TFF1, GREB1, Cathepsin D, Lactoferrin, and TGF-α, also appear to bind to XR5944 with different specificities as evident by differences in the 1H NMR spectra of their drug-DNA complexes. Thus, the affinity and specificity of the XR5944-ERE complex is influenced both by the nucleotide sequence of the half-sties and the tri-nucleotide spacer. Of the natural EREs tested, XR5944 appears to bind with the highest specificity to the ERE sequence of TFF1; hence we carried out further NMR studies of the binding of XR5944 to the TFF1 ERE sequence. Using site-specific labeling at each base, we were able to assign both the imino and H8 protons of the free TFF1 DNA and its drug complex. These assignments provide important information on the XR5944 binding sites with TFF1 ERE.

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 4514. doi:10.1158/1538-7445.AM2011-4514
Abstract 4515: A novel obatoclax derivative is a promising agent to overcome bortezomib's resistance in acute leukemia cells
Authors: Liu Chun-Yu , Shiau Chung-Wai , Kuo Hsin-Yu , Cheng Ann-Lii , Tzeng Cheng-Hwai , Chen Kuen-Feng
Published: June 2012

Background: Our previous work showed that bortezomib, a reverse-inhibitor of the 26S proteasome, exerts differential cytotoxic effects to acute leukemia cells through down-regulation of phosphor-Akt (AACR 2010 abstract 1575). Interestingly, some acute leukemia cells (MOLT-3 and K562) are resistant to bortezomib. One of the resistant mechanisms of bortezomib involves the abnormality in intrinsic apoptotic pathway members, the bcl-2 family. Of particular, it has been shown that bortezomib induces upregulation of Mcl-1 and that Mcl-1 accumulation may delay bortezomib-induced apoptosis. Obatoclax, a small molecule inhibitor of pro-survival BCL-2 members, including MCL-1, synergizes with bortezomib in mantle cell lymphoma (Blood 2007) and may overcome bortezomib resistance. Here, we designed a novel Mcl-1 inhibitor deriving from obatoclax, SC-2001, to overcome the resistance. Methods: MOLT-3 and K562 cell lines were used for in vitro studies. Apoptosis was examined by both flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western Blot. Gene silencing was done by small interference RNA (siRNA). Results: MOLT-3 and K562 were resistant to bortezomib up to 100nM. Upregulation of Mcl-1 expression after bortezomib treatment were evident in these cells. Interestingly, the addition of either obatoclax or SC-2001, restored the sensitivity of K562 and MOLT-3 cells to bortezomib. SC-2001, comparing to obatoclax, exerted better efficacy in sensitivity restoration of bortezomib in association with Mcl-1 downregulation in MOLT-3 cells. Moreover, down-regulation of Mcl-1 by siRNA overcame the apoptotic resistance to bortezomib in K562 cells. These data indicate that novel Mcl-1 inhibitors could restore bortezomib sensitivity in resistant acute leukemia cells. Conclusions: a novel Mcl-1 inhibitor SC-2001, is a promising agent to overcome bortezomib's resistance in acute leukemia, further elucidating the drug mechanism regulating Mcl-1 inhibition is needed. Supported by VGHTPE 99DHA0100332 and 100DHA0100591

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 4515. doi:10.1158/1538-7445.AM2011-4515
Abstract 4524: ON015640, a novel anti-cancer agent with tubulin depolymerizing activity
Authors: Hoffman Benjamin S. , Cosenza Stephen C. , Palella Venkat , Reddy Ramana , Reddy EP
Published: June 2012

Oncology drug discovery is constantly evolving by integrating new basic findings with established developmental practice. Two drug discovery platforms currently exist. One is an extension of basic research, rational drug design. Its alternative, one based on cytotoxicity, which qualifies leads by their ability to selectively kill cancer cells. Using this cytotoxicity based approach we recently discovered a novel synthetic small molecule we have called ON015640. This compound is highly effective at killing 15 different cancer cell lines with IC50 ranges from 20-90 nanomolar. The predominant phenotype associated with this compound is that of cellular rounding and detachment from substratum, a similar phenomenon to that observed during treatment of cultured cells with mitotic inhibitors like Taxol and Nocodazole. Using FACS, molecular mitotic indexing, and fluorescent microscopy we have subsequently demonstrated that ON015640 induces mitotic arrest and aberrant spindle formation. This mitotic arrest and spindle disruption can be attributed to this molecule's ability to depolymerize tubulin both in vitro and in vivo in a similar manner to Nocodazole. At effective concentrations this compound commences a program of mitotic catastrophe subsequently initiating effector caspases 3/7 and cleavage of Poly ADP Ribose Polymerase, primary steps in the activation of the intrinsic apoptotic pathway. Unlike classical tubulin depolymerizing agents, ON015640 is not a substrate for the ATP binding cassette family of efflux pumps and is capable of killing Multi Drug Resistant cell lines. This characteristic and the compound's nanomolar activity make it an attractive candidate for further development and characterization. To date, the exact site where ON015640 binds to tubulin remains unknown. Additionally, the pharmacokinetics and efficacy of this molecule have yet to be fully characterized. Thus, current and future efforts will be focused on establishing the dynamics by which ON015640 interacts with tubulin, while also developing this molecule into a clinically relevant drug.

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 4524. doi:10.1158/1538-7445.AM2011-4524
Abstract 4539: Androgen receptor N-term death domains induce apoptosis through a bax-mediated mitochondrial pathway
Authors: Haferkamp Bonnie , Lin Yuting , Godfrey Brad , Xiang Jialing
Published: June 2012

While the DNA- and ligand-binding domains of the androgen receptor (AR) have been extensively studied to elucidate AR transcriptional mechanisms in androgen-independent prostate cancer, the role of the N-terminal is poorly understood. Previously, we showed that AR is required for stress-induced cell death in androgen-independent prostate cancer cells, and that cell death does not require AR transcriptional activity. Here we show that N-term AR is sufficient to induced cell death in androgen-independent prostate cancer PC3 cells without death stimulus. The minimal death domain is located in the first 85 amino acids of AR, rich in polyQ. The pro-death activities of full length AR and its N-term truncated proteins act through the Bax-mitochondrial apoptotic pathway. Electronic microscope imaging revealed mitochondrial swelling in PC3 cells transfected with death-inducing AR truncated constructs, but not with constructs that failed to induce cell death. Cell death induced by AR N-term truncated constructs can be effectively blocked by caspase 3, but not caspase 1 inhibitor. Finally, transfection of AR death-active N-term truncations caused significant cell death in Bax wild-type but not Bax-/- MEFs. Taken together, these results indicate that pro-death AR N-term fragments generated by stress-induced proteolysis cause cell death through a Bax-dependent mechanism involving the mitochondrial apoptosis pathway in androgen-independent prostate cancer cells.

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 4539. doi:10.1158/1538-7445.AM2011-4539
Abstract 4542: Dose-dependent effects of progestins on progesterone receptor activity and target gene expression: less is more
Authors: Wade Hilary , McDonnell Donald , Brown Myles
Published: June 2012

While the molecular mechanisms by which the progesterone receptor (PR) regulates breast tumor growth and metastasis have not been fully elucidated, recent studies highlight the fact that PR has a complex role in breast cancer progression that may be dependent on ligand dose. For example, data from the Women's Health Initiative trial suggest that low-dose progestins used in combination with estrogen are associated with an increased risk of invasive breast cancer compared to estrogen alone. On the other hand, high-dose progestin therapy has been used as an effective front-line and second-line treatment for advanced, metastatic breast cancer. To investigate this paradox, we undertook studies to identify aspects of PR biology that respond differently to treatment with low or high doses of progestins. Interestingly, we found that treatment of T47D breast cancer cells with low-dose progestins generates a significantly more robust induction of a subset of PR target genes, including the cell cycle regulators cyclin D1 and E2F1, than treatment with high-dose progestins. Furthermore, we show that varying doses of progestins have differential effects on the receptor itself, including the subcellular localization, phosphorylation and subsequent turnover of PR. At the level of transcriptional regulation, our ChIP analyses have led us to hypothesize that the strength of PR activation of target gene expression may hinge upon the ratio of phospho-PR versus unmodified PR that is recruited to enhancer elements. This dose-dependent effect may result in the formation of different PR transcriptional complexes and explain the diverse downstream biologies that are regulated by varying levels of progestins throughout the female reproductive cycle and in diseases such as breast cancer. Given the wide range of medical therapies that utilize progestins, our findings may have important clinical implications.

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 4542. doi:10.1158/1538-7445.AM2011-4542
Abstract 455: Adipocyte sTNFR2 secretion and macrophage migration in the breast tumors
Authors: Rego Stephen , Swamydas Muthulekha , Ricci Krista , Dréau Didier
Published: June 2012

The breast tumor microenvironment greatly influences tumor progression and modulates the migratory abilities of both cancer cells and macrophages, especially M2 macrophages, at the tumor site. The pleiotropic inflammatory cytokine TNFα through interactions with TNFR promotes macrophage migration and tumor cell apoptosis. The shedding of TNFR increases the concentration of sTNFR within the tumor microenvironment. We determined whether sTNFR modulates the trafficking of specific macrophage subtypes in the tumor microenvironment. Concentrations of TNFα, sTNFR1 and sTNFR2 secreted by 4T1 and NMuMG mammary epithelial cells, stroma mesenchymal stem cells, and adipocyte-derived cells were measured by cytokine arrays and ELISA. The migration of murine monocytes cultured in media promoting preferential expression of M0, M1, or M2 phenotypes toward tumor or stroma cell conditioned media (CM) was determined by transwell assays. The effects of conditioned media obtained following treatment with the sheddase inhibitor TAPI-0 on cell migration were also assessed. The concentrations of sTNFR2 secreted were drastically higher in the CMs from 4T1 cells compared to NMuMG cells (p<0.05). In addition, adipocytes secreted markedly more sTNF2 compared to D1 mesenchymal stem cells (p<0.05). sTNFR2 concentrations were significantly decreased in the presence of a sheddase inhibitor (p<0.05). The transwell migration of M2 phenotype macrophages toward 4T1-CM was significantly greater than the migration of M0 or M1 phenotype macrophages (p<0.05). Furthermore, only the migration of the M2 macrophage subtype following TAPI-0 treatment toward 4T1 CM was markedly higher (p<0.05). These results strongly suggest a role for sTNFR secreted by tumor cells and adipocytes in the infiltration of M2 phenotype macrophages within the tumor microenvironment. Whether selective prevention of sTNFR shedding may have therapeutic benefit for breast cancer patients remains to be determined.

This work was supported by grants from the Department of Defense Era of Hope program (BC044778) and the National Science Foundation EFRI program (CBE0736007).

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 455. doi:10.1158/1538-7445.AM2011-455
Abstract 4554: Regulation of intracrine androgen synthesis by NF-kappaB2/p52
Authors: Nadiminty Nagalakshmi , Tummala Ramakumar , Chun Jae Yeon , Evans Christopher P. , Gao Allen C.
Published: June 2012

Introduction: Benign prostatic hyperplasia and the initial stages of prostate cancer (CaP) exhibit androgen dependence, but androgen ablation results only in temporary regression of CaP, with progression to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) signaling remains active in CRPC due to aberrant activation of the AR. Synthesis of intracrine androgens has emerged as one of the mechanisms by which AR is activated in CRPC after androgen ablation. Even though levels of androgen biosynthetic enzymes have been shown to be elevated in CRPC, their regulation is not completely understood. In this study, we examined the role of NF-kappaB2/p52 in intracrine androgen synthesis and castration-resistant progression of CaP.

Methods: Expression levels of androgen biosynthetic enzymes were measured in CaP cells with or without expression of NF-kappaB2/p52 using real-time RT-PCR and western blotting. Regulation by NF-kappaB2/p52 was examined using luciferase reporter assays and plasmids containing regulatory elements of androgen biosynthetic enzymes. Intracrine levels of androgens were measured using EIA in tumors obtained from castrated mice.

Results: Expression levels of androgen biosynthetic enzymes including AKR1C3, CYP17A1, HSD3B2, and SRD5A1 were found to be elevated in CaP cells expressing NF-kappaB2/p52. Luciferase assays showed that NF-kappaB2/p52 regulates their expression directly by binding to their promoters and inducing transcription. The levels of total testosterone in CaP cells expressing NF-kappaB2/p52 were approximately 3-fold higher than control cells as measured using EIA. Intraprostatic androgen levels were found to be at 1002 ± 232 pg/g tissue, compared to 377.8 ± 105 pg/g tissue in control tumors obtained by orthotopic implantation of CaP cells expressing NF-kappaB2/p52. These data suggest that CaP cells synthesize detectable levels of testosterone in the absence of exogenous steroid precursors and overexpression of NF-kappaB2/p52 can increase this process, possibly by enhancing the expression of genes encoding steroidogenic enzymes.

Conclusions: Intraprostatic androgen synthesis in recurrent prostate tumors contributes significantly to resistance to androgen ablation and development of CRPC. NF-kappaB2/p52 regulates the expression levels of steroidogenic enzymes and thereby enhances synthesis of intracrine androgens and aberrant activation of the AR. Coupled with our previous studies, these data suggest that antagonizing NF-kappaB2/p52 signaling may prove beneficial in CRPC 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 4554. doi:10.1158/1538-7445.AM2011-4554
Abstract 456: 25-Hydroxyvitamin D3 inhibits clonogenic growth and regulates expression of multiple cytokines/chemokines in co-culture of colon cancer and macrophage-like THP-1 cells
Authors: Peng Xinjian , Alimirah Fatouma , Roy Sarbani , Yuan Liang , Murillo Genoveva , Mehta Rajeshwari R. , Mehta Rajendra G.
Published: June 2012

Colon cancer cells interact with immune cells in vivo, creating a microenvironment which affects both tumor growth and treatment. Macrophages are often the most abundant immune cells in the tumor microenvironment and are involved in inflammation. Since inflammation is tightly associated with colon carcinogenesis, it is important to understand how vitamin D affects the interaction between colon epithelial cells and macrophages. In this study, we used a co-culture system to examine the role of 25-hydroxyvitamin D3 (25(OH)D3) on the clonogenic growth of colon cancer cells and on the profiling of cytokines/chemokines. 25(OH)D3 at 250 nM had minor effect on clonogenic growth in CaCo-2 cells, co-culture of macrophage-like THP-1 cells with CaCo-2 cells significantly stimulated the clonogenic growth of CaCo-2 cells in the absence of 25(OH)D3. However, in the presence of 25(OH)D3, THP-1 cells failed to stimulate the the clonogenic growth of CaCo-2 cells. Cytokine antibody array demonstrated that several cytokines were differentially expressed in the medium after co-culture of the two cell lines for 2 days in the absence of 25(OH)D3. These include increased expression of GROα, MCP-1 and decreased expression of TNFα, Angiogenin; however, in the presence of 25(OH)D3, MCP-2 and MDC were significantly induced and TNFα level was restored. Proliferation assay showed that MCP-2 and MDC had minor or no effect on cell proliferation in CaCo-2 cells, suggesting that these chemokines mainly target macrophages. 25(OH)D3 treatment of THP-1 cells for 24h also induced CYP24, an immediate vitamin D target gene and enhanced MDC and MCP-2 expression as evaluated by qRT-PCR. These results demonstrate that 25(OH)D3 at 250 nM might selectively target macrophages in a co-culture system with colon cells and block the interaction between colon cancer cells and macrophages, which may result in prevention of inflammation. [Supported by NCI R01 CA121157].

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 456. doi:10.1158/1538-7445.AM2011-456
Abstract 4563: Antibody targeting of anaplastic lymphoma kinase induces cytotoxicity of human neuroblastoma
Authors: Carpenter Erica L. , Haglund Elizabeth A. , Chow Adrian K. , Wood Andrew C. , Belcastro Lili T. , Christensen James G. , Vigny Marc , Maris John M. , Lemmon Mark A. , Mosse Yael P.
Published: June 2012

The purpose of this study was to provide the first pre-clinical evidence for antibody targeting of anaplastic lymphoma kinase (ALK) in human neuroblastoma. ALK is a receptor tyrosine kinase expressed by the majority of neuroblastoma tumors. Germline mutations of ALK, shown to account for most cases of familial neuroblastoma, as well as somatically acquired ALK aberrations, induce increased auto-phosphorylation and constitutive ALK activation and increased downstream signaling. Antibody targeting of receptor tyrosine kinases, as monotherapy or together with small-molecule inhibitors, is highly effective in other cancers such as breast and lung cancer. While antibody-mediated immunotherapy targeting of disialoganglioside GD2 has recently been reported to significantly increase 2-year survival in high-risk neuroblastoma patients, over half the patients receiving this therapy ultimately succumb to their disease and therapeutic alternatives are urgently needed to improve survival rates of this devastating pediatric cancer. Therefore, we hypothesized that antibody targeting of ALK in neuroblastoma was a therapeutically appropriate strategy. To first confirm the potential of anti-ALK antibody-mediated immunotherapy, we used in vitro assays to demonstrate enhanced immune-cell induced cytotoxicity of antibody-treated human neuroblastoma-derived cell lines. We next showed that in vitro antibody treatment of neuroblastoma cell lines expressing activated ALK led to growth inhibition and cell death. These effects were enhanced by treatment with PF-02341066, an orally available small-molecule inhibitor of the ALK tyrosine kinase. To identify the mechanism behind this enhanced combined effect, we used flow cytometry to show that PF-02341066 sensitizes cells to antibody treatment by inducing accumulation of cell-surface ALK, thus increasing the accessibility of antigen for antibody binding. Finally, to further predict in vivo cytotoxic mechanisms of dual ALK targeting, we used flow cytometry to demonstrate enhanced apoptosis and proliferation inhibition resulting from combined antibody and inhibitor treatment as compared to either drug alone. Taken together, these findings provide strong evidence for the therapeutic relevance of antibody targeting of ALK in neuroblastoma and argue for the rapid development and further testing of a clinical grade anti-ALK antibody.

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 4563. doi:10.1158/1538-7445.AM2011-4563
Abstract 4567: Polyubiquitin K63-related CD137 signalsomes in T cells stimulated with agonist anti-CD137 monoclonal antibodies
Authors: Forero Ivan Martinez , Azpilicueta Arantza , Villan Estanislao Nistal , Palazon Asis , Rouzaut Ana , Aragon Tomas , Dubrot Juan , Murillo Oihana , Hervás-Stubbs Sandra , Melero Ignacio
Published: June 2012

Agonist anti-CD137 monoclonal antibodies are employed to enhance CD8-mediated antitumor immunity both in mouse models and in clinical trials. A panel of agonist anti-human CD137 mAb was prepared recognizing three different epitopes on the CD137 surface glycoprotein. All mAb costimulated T cell activation irrespectively of interference or not with natural ligand-binding. Interestingly, CD137 perturbation with agonist mAb results in internalization towards a vesicular endosomal compartment derived from the plasma membrane. Such internalization was observed on CD137 transfectants in 293 cellsas well as on primary activated T cells from humans and mice. In vivo treatment with anti-CD137 mAb of immunodeficient mice harboring activated human or mouse T cells results in internalization of CD137 to this vesicular compartment. CD137 is known to associate with TRAF2 at the plasma membrane. TRAF-2 is an adaptor protein endowed with E3 ubiquitin ligase activity that synthesizes k63-polyubiquitin chains. K63 polyubiquitin, as opposed to K48 polyubiquitin, constitute a docking site for downtream signaling proteins that lead to NF-kB and AP-1 activation. CD137 vesicles are decorated with k63-polyubiquitin and CD137 stimulation leads to the formation of K63-polyub containing complexes. Therefore CD137 stimulation under therapy like conditions with mAbs leads to the assembly of an intracellular signaling compartment that we term as CD137 -signalsomes

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 4567. doi:10.1158/1538-7445.AM2011-4567
Abstract 4568: Anti-tumor effect of an anti-human müllerian inhibiting substance type II receptor antibody in mouse models for ovarian cancer: towards a new targeted therapy
Authors: Kersual Nathalie A. , Garambois Veronique , Salhi Imed , Chardès Thierry , Colombo Pierre Emmanuel , Bascoul-Mollevi Caroline , Cambon-Roques Sylvie , Bibeau Frédéric , Pouget Jean Pierre , Behrens Christian , Gaucher Christine , Urbain Rémy , Prost Jean François , Pèlegrin André , Navarro-Teulon Isabelle
Published: June 2012

Often diagnosed at an advanced stage, ovarian cancer is responsible for the highest mortality rate among patients with gynecologic malignancies. Due to the limitations of the current therapeutic approaches, there is a strong need for novel, more efficient, therapies, among which targeted immunotherapy. The monoclonal antibody 12G4 specifically recognizes the human Müllerian Inhibiting Substance type II Receptor (MISRII). Expressed at high levels on most human Granulosa Cell Tumors (GCT) as well as on a majority of human epithelial ovarian cancer (EOC) cells, MISRII represents a good target for antibody-based tumor targeting.

Characterization of murine antibody 12G4 has been previously reported, in particular epitope mapping and in vitro assays showing that 12G4 binding does not affect MIS interaction with MISRII. In the present study we further assess 12G4 activity in vitro and in vivo. 12G4 was shown to induce 37% and 30% apoptosis in vitro (FACS, DNA fragmentation) on the two MISRII expressing cell lines cov434-MISRII and OVCAR3, respectively. Internalization of the antibody-MISRII complex was also observed in vitro on cov434-MISRII. Using 12G4 antibody we confirmed by immunohistochemistry the expression of the MISRII target in all GTC samples and all but one EOC tissue sections (13/14). Furthermore, we have developed several immunodeficient Nude mouse models of ovarian tumor xenografts expressing human MISRII. In these models the efficacy of 12G4 could be clearly demonstrated with a significant effect on tumor growth.

According to our data, MISRII, expressed on a majority of EOCs appears as a good target for ovarian cancer immunotherapy. Furthermore, based on the good efficacy observed in vivo for 12G4, the development of a humanized anti-MISRII antibody, derived from 12G4, has been initiated and represents a new promising targeted therapeutic approach.

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 4568. doi:10.1158/1538-7445.AM2011-4568
Abstract 4569: Cell death-inducing ICAM-1 antibody has broad and potent anti-myeloma activity in vivo
Authors: Veitonmaki Niina , Hansson Markus , Li Zhan-chun , Ljungars Anne , Danielsson Lena , Bjorn Frendeus
Published: June 2012

We previously developed combined target and drug discovery methodology enabling isolation of tumor cell death-inducing antibodies from the human antibody library n-CoDeR®. Among several antibodies targeting different tumor cell associated receptors with documented cell death-inducing properties, antibodies specific for intercellular adhesion molecule-1 (ICAM-1) – a receptor not previously associated with tumor cell death- were obtained.

We here characterize the ICAM-1 specific antibody BI-505 with respect to in vivo anti-tumor activity in different well-established tumor disease models, and explore mechanisms underlying its anti-tumor activity. We demonstrate in different tumor xenograft models that BI-505 is equally or more efficacious in preventing CD20 positive B cell tumor growth and in pro-longing tumor-bearing mouse survival compared to CD20 mAb Rituximab in vivo. Moreover, BI-505 showed significant anti-tumor activity against a panel of CD20 negative myeloma tumors in vivo, and BI-505 conferred enhanced mouse survival compared to the proteasome inhibitor bortesomib – approved for treatment of relapsed refractory multiple myeloma – in an experimental model of advanced multiple myeloma. Preliminary data indicate that the BI-505 epitope is highly expressed on myeloma cells in patients with multiple myeloma.

The differential in vitro and in vivo anti-tumor activities of BI-505 Fc-variants correlated with binding to recombinant human FcγRIIIa and mouse FcγRIV receptors, respectively. These differences could not be explained by altered antigen-binding properties as all Fc-variants retained near identical EC50 values for binding to recombinant or cell surface expressed ICAM-1 protein. These observations are consistent with cell death and FcγR-dependent anti-tumor immunity being important mechanisms underlying BI-505 therapeutic activity.

Collectively, our results demonstrate proof-of-principle for our human antibody based combined target and drug discovery methodology, and provide a rationale for further pre-clinical and clinical evaluation of BI-505 in the treatment of MM. An open multicenter phase I dose-escalation clinical trial with BI-505 in relapsed/refractory MM has recently started in the United States and Sweden.

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 4569. doi:10.1158/1538-7445.AM2011-4569
Abstract 4579: CSPG4 monoclonal antibodies inhibit the growth of human malignant mesothelioma
Authors: Zeyana Rivera , Ferrone Soldano , Wang Xinhui , Jube Sandro , Yang Haining , Gaudino Giovanni , Carbone Michele
Published: June 2012

Malignant Mesothelioma (MM) is a very aggressive cancer, resistant to current chemotherapy, with a median survival of 1 year from diagnosis. The membrane bound Chondroitin Sulphate Proteoglycan 4 (CSPG4) is involved in tumor onset and progression, as well as in angiogenesis. Because of high expression on the surface of cancer cells, CSPG4 represents an attractive target for immunotherapy. CSPG4 was detected in 24 out of 40 MM biopsies, with minimal expression on surrounding normal cells. CSPG4 expression in MM cells was induced upon engagement on extracellular matrix components (ECM). Cell attachment and survival was greater to fibronectin than other ECM. Therefore, we aimed at using CSPG4 monoclonal antibodies (mAb) to impair MM growth. CSPG4-specific TP41.2 mAb inhibited MM cell attachment to ECM, the resulting intracellular signaling and cell survival, with a two-fold increase in apoptosis, compared to isotype IgG control treated cells. Moreover, interfering with the function of CSPG4 by TP41.2 significantly reduced MM cell motility, invasiveness and wound healing. CSPG4 is involved also in the maintenance of the transformed phenotype, as assessed by anchorage-independent growth assay. A significant reduction in soft-agar colony size and numbers was observed when MM cells were treated with TP41.2, compared to control cells. Preliminary studies of in vivo tumorigenesis, performed on SCID mice xenografts, reveal that TP41.2 mAb is able to either prevent MM tumor establishment or reduce tumor mass volume. Our results strongly suggest that CSPG4 mAb-based immunotherapy may represent a novel valid approach for the treatment of MM.

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 4579. doi:10.1158/1538-7445.AM2011-4579
Abstract 4580: A novel role for Junctional Adhesion Molecule-A (JAM-A) in tumor proliferation: modulation by a humanized anti-JAM-A monoclonal antibody
Authors: Goetsch Liliane , Haeuw Jean-François , Broussas Matthieu , Wurch Thierry , Beau-Larvor Charlotte , Robert Alain , Bailly Christian , Corvaia Nathalie
Published: June 2012

Biotherapeutic drugs in oncology are generating a great enthusiasm, and tremendous amount of effort is made to identify new targets for the development of new therapeutics. Functional approaches using tumor cells as immunogens have been performed in order to select on the basis of their function, molecules that, targeted by monoclonal antibodies (Mabs), would induce inhibition of cell proliferation both in vitro and in vivo. Using this type of approaches coupled to a target identification plateform, we had identified an antibody, named 6F4 that was inhibiting in vitro and in vivo tumor growth. A proteomic analysis using 6F4-tagged beads coupled to a MS analysis indicated that 6F4 Mab specifically recognized the human junctional adhesion molecule A (JAM-A). These results indicate that the 6F4 anti-JAM-A Mab is able to induce tumor growth inhibition.

Mouse 6F4 Mab was humanized by conventional CDR-grafting methods. Immunogenetic analysis was performed based on IMGT databases and bioinformatic tools. The human frameworks (FRs) used as the acceptors for CDR grafting were chosen using the “best-fit” selection procedure. Engineered variants were designed and genes were chemically synthetized, produced as human IgG4/kappa antibodies and evaluated by ELISA and competition with biotinylated mouse 6F4 Mab. One humanized antibody (IgG4 isotype) was selected, it efficiently and potently bound to JAM-A ECD and inhibited binding of mouse 6F4. Its germinality index is > 95 % (CDRs excluded). Humanized 6F4 was further tested in vitro and in vivo models and showed ability to induce tumor regression in animal models in particular in A431 and DU145 tumor models. These results confirm that JAM-A is a potential novel target in Oncology and demonstrate that a functional approach coupled to proteomic analysis can be successfull to identify new antibody target molecules that lead to promising new antibody-based therapies against 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 4580. doi:10.1158/1538-7445.AM2011-4580
Abstract 4582: Characterization of a new antibody that reacts with a tumor-specific variant of CEACAM-5 and CEACAM-6
Authors: Du Xiulian , Kantor Judith , Luka Janos , Stafford Lewis J. , Bristol Andrew
Published: June 2012

Introduction: h16C3 is a humanized monoclonal antibody being developed as a novel biological treatment for several types of solid tumor. By mass spectroscopy identification and target validation research, we determined that h16C3 appears to recognize a variant form of CEACAM-5 and CEACAM-6, molecules with a well-known association to cancer. Here we show the pre-clinical activities of this new mAb which shows great potential as a therapeutic agent to treat solid tumors.

Methods: The 16C3 antibody was selected from hundreds of hybridoma clones generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. In vitro assays and in vivo studies were performed to characterize the potential of h16C3 as a therapeutic modality for solid tumors that express the variant CEACAM antigens.

Results: Here we show that unlike most modalities targeting the CEACAM molecules, h16C3 reacts with variant CEACAM-5/6 expressed specifically by human tumor tissues as follows: 98% of human colorectal tumor tissues, 80% of pancreatic tumor tissues, 76% of lung tumor tissues, 43% of uterus tumor tissues, and 29% of breast tumor tissues stained positively with h16C3. Importantly, h16C3 did not cross-react significantly with normal human tissues, except for occasional, weak binding to certain GI tract tissues, which may indicate a pre-malignant state. We showed further that the h16C3 antibody exhibits cell-specific binding and ADCC activity against human colorectal and pancreatic tumor cells, but not against antigen-negative control tumor cell lines. In vivo, the anti-tumor efficacy of h16C3 was tested using pre-established subcutaneous human tumor xenograft models. The data showed significant anti-tumor action compared to control treated mice, including some complete tumor regressions. The bio-distribution of radiolabeled h16C3 in mice with pre-established human tumors exhibited specific accumulation of h16C3 at the tumor site, with little or no binding or accumulation in several major organ systems.

Conclusions: The pre-clinical studies of h16C3 to date indicate that it has significant, specific anti-tumor potency, and suggest that it may have clinical activity in cancer patients whose tumor expresses the variant CEACAM-5/6 epitope. The therapeutic humanized 16C3 antibody is being developed for testing in a Phase I clinical trial.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4582. doi:10.1158/1538-7445.AM2011-4582
Abstract 4583: A blocking antibody to HB-EGF inhibits the growth of tumor xenograft
Authors: Kimura Naoki , Taniguchi Kenji , Hirata Yuichi
Published: June 2012

Heparin-binding epidermal growth factor (HB-EGF), a member of the EGF family of growth factors, is widely expressed in many human cancers compared with normal tissues. HB-EGF is initially produced as a membrane-anchored form (pro-HB-EGF) and later processed to a soluble form (s-HB-EGF), which exert its biological activity through activation of EGFR and EGFR4. A key role of HB-EGF in the acquisition of malignant phenotypes has been well documented, such as tumorigenicity, invasion, metastasis, angiogenesis, and resistance to chemotherapy. In this study, we demonstrate the potential of an anti-HB-EGF mAb for cancer therapy by generating a potent blocking monoclonal antibody, HC-15. The HC-15 mAb recognized both forms of HB-EGF, pro- and s-HB-EGF. HC-15 efficiently blocked the binding of HB-EGF to EGFR-Fc in the ELISA. Furthermore, HC-15 substantially inhibited the HB-EGF-dependent proliferation of the EGFR expressing Ba/F3 transfectant lines. To investigate the potential of HC-15 for cancer therapy, the in vivo antitumor activity of HC-15 was tested. Significant growth inhibition by treatment of HC-15 (10mg/kg once a week) was observed in established human tumor models, such as ovarian cancer (MCAS), pancreatic cancer (BXPC-3), and breast cancer (MDA-MB-231) in athymic nude mice. Thus, we conclude that the anti-HB-EGF mAb HC-15 has strong anti-tumor activity in various models, supporting the inhibition of HB-EGF functions by mAb is useful in cancer treatment.

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 4583. doi:10.1158/1538-7445.AM2011-4583
Abstract 4593: Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in athymic nude mice
Authors: Budhraja Amit , Gao Ning , Zhang Zhou , Son Young-Ok , Ding Songze , Hitron Andrew , Gupta Pooja , Shi Xianglin
Published: June 2012

Purpose of the study: To investigate the functional role of Akt and JNK signaling cascades in apigenin-induced apoptosis in human leukemia cells (in vitro) and anti-leukemic activity of apigenin in U937 xenografts (in vivo).

Experimental Design: Leukemia cells were treated with apigenin in dose and time dependent manners, after which apoptosis, caspases activation, Akt and JNK signaling pathways were evaluated by pharmacologic and genetic approaches. Parallel studies were also conducted in Jurkat, HL60 and normal peripheral blood mononuclear cells. In vivo anti-leukemic activity mediated by apigenin was evaluated using U937 xenograft mouse model.

Results: Exposure of apigenin caused pronounced increase in apoptosis in leukemia cells but not in normal peripheral blood mononuclear cells. Apigenin-induced apoptosis by inactivation of Akt with concomitant activation of JNK, cytochrome c release from mitochondria and caspases activation in dose and time dependent manners. Enforced activation of Akt by a constitutively active myristolated Akt prevented apigenin-induced JNK and caspases activation and apoptosis. Conversely, PI3K (phosphatidyl 3-kinase) inhibitor LY294002 and a dominant negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. In addition, Akt kinase activity was diminished after exposure to apigenin in leukemia cells. Interruption of JNK pathway by pharmacologic (SP600125) and genetic (JNK1 siRNA) approaches showed marked reduction in apigenin-induced caspases activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin via intraperitoneal route resulted in attenuation of tumor growth in U937 xenografts accompanied by apoptosis.

Conclusions: Collectively, these findings suggested a hierarchical model of apigenin-induced apoptosis in human leukemia cells in which apigenin-induced Akt inactivation represents a primary event resulting in JNK activation and culminating in caspases activation and apoptosis. In addition, attenuation of tumor growth in U937 xenografts by apigenin raise the possibility that apigenin may have clinical implications for therapeutic intervention against leukemia.

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 4593. doi:10.1158/1538-7445.AM2011-4593