bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2579: Bax345/BLyS: A novel, completely human fusion construct targeting malignant B cells and delivering a unique mitochondrial toxin
Authors: Lyu Mi-Ae , Cheung Lawrence H. , Marks John W. , Rosenblum Michael G.
Published: February 2011

B lymphocyte stimulator (BLyS) is crucial for B-cell survival and the biological effects of BLyS are mediated by three cell surface receptors BAFF-R, TACI, and BCMA. Increased expression of BLyS and its receptors has been identified in diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). DLBCL and MCL are an aggressive sub-type of B-cell, non-Hodgkin's lymphoma (NHL). Despite recent advances in the understanding of the molecular and cellular basis for their pathogenesis, these tumors are still associated with poor response to treatment and a fatal outcome. Bcl-2 associated x protein (Bax) is a proapoptotic protein of the Bcl-2 family and is a gateway protein to mitochondria dependent apoptosis. We generated a highly cytotoxic fusion protein designated Bax345/BLyS for receptor-mediated delivery to malignant B cells. The Bax345/BLyS fusion protein has the BH3 and BH1 domains of Bax and contains full-length BLyS. This fusion protein was generated in bacteria and purified by IMAC. The final material was shown to be highly cytotoxic to activated B cell (ABC)-subtype DLBCL (IC50 ∼ 0.1 nM) and mantle cell lymphoma lines (IC50 ∼ 0.1-1 nM). The cytotoxic effects of Bax345/BLyS were inhibited by pretreatment with free BLyS demonstrating that the effects of the fusion construct are mediated through interaction with the BLyS receptors. Treatment of cells with Bax345/BLyS induced down-regulation of Akt, p-Akt, Mcl-1, and survivn and triggered accumulation of cells in the sub G1 phase of the cell cycle. In addition, treatment of cells with Bax345/BLyS induced up-regulation of Bax and apoptosis through caspase-3 activation, PARP cleavage, and DNA fragmentation. Our results suggest that BLyS has the potential to serve as an excellent targeting ligand for the specific delivery of the pro-apoptotic molecule Bax345 to neoplastic B cells expressing the BLyS receptors, and Bax345/BLyS may be an excellent candidate for the treatment of the NHLs which are resistant to conventional chemotherapeutic regimens. This work was supported in part by the Clayton Foundation for Research and by LLS 6234-07 Translational Research Award from the Leukemia and Lymphoma Society.

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 2579.
Abstract 2584: The CEA/CD3-bispecific antibody MEDI-565 (MT111) binds a nonlinear epitope present in the full-length but not a short splice variant of CEA
Authors: Peng Li , Oberst Michael , Huang Jiaqi , Brohawn Philip , Lekstrom Kristen , Amann Maria , Baeuerle Patrick , Jallal Bahija , Trail Pamela , Wu Herren , Yao Yihong , Coats Steven , Dall”Acqua William , Damschroder Melissa , Hammond Scott A.
Published: February 2011

MEDI-565 (MT111) is a novel bispecific single-chain antibody of the BiTE (Bi-specific T-cell engager) class that transiently links carcinoembryonic antigen (CEA) on tumor cells with human CD3 on T cells. Engagement of CD3 and CEA by MEDI-565 induces T cells to kill CEA-positive cancer cells using similar elements and cellular machinery as required for T cells to kill through classic T cell receptor/major histocompatibility complex-peptide signaling. Examination of DNA sequence databases revealed a canonical CEA sequence (full length CEA; NCBI RefSeq NP_004354.2; 702 amino acids) and a previously uncharacterized shorter mRNA splice variant (short CEA; NCBI RefSeq CR749337420 amino acids) that lacks the second and third IgC-like domains and part of the N-terminal IgV-like domain. Expression of full-length CEA mRNA in colorectal, pancreatic, lung, and breast tumors was more widespread than that of short CEA; however, concordant expression of short CEA with the full length version was always observed. Mapping of the MEDI-565 binding region of CEA revealed a nonlinear, conformational epitope in the third immunoglobulin C-like domain. With a combination of deletion and swap variants, site directed mutagenesis, and computational homology modeling, we further identified the critical residues for MEDI-565 binding comprised of Phe292, Thr294, Asn299, Val354, Gly355, Pro356, Glu358, Ile374, and Asn376. CHO cells expressing only the short variant of CEA lacking this epitope failed to bind MEDI-565 and did not undergo T cell-directed lysis in the presence of MEDI-565. These studies suggest that MEDI-565 can discriminate between the full-length and short splice variant of CEA and may broadly target CEA-positive colorectal, pancreatic, lung, and breast tumors without regard for expression of the short splice variant of CEA.

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 2584.
Abstract 2586: Adnectins as a platform for multi-specific targeted biologics: A novel bispecific inhibitor of EGFR and IGF-IR growth factor receptors
Authors: Emanuel Stuart L. , Engle Linda J. , Cao Carolyn , Chao Ginger , Lin Zheng , Zhu Rong-Rong , Yamniuk Aaron P. , Hosbach Jennifer , Brown Jennifer , Fitzpatrick Elizabeth , Gokemeijer Jochem , Morin Paul , Morse Brent , Carvajal Irvith , Fabrizio David , Wright Martin , DasGupta Ruchira , Gosselin Mike , Ryseck Rolf , Doyle Michael L. , Wong Tai W. , Camphausen Ray , Cload Sharon T. , Marsh Nick , Furfine Eric S. , Gottardis Marco M.
Published: February 2011

The epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor-1 (IGFR) are transmembrane receptor tyrosine kinases that mediate proliferative and invasive cell signaling in cancer. Inhibition of either receptor reduces tumor growth in both mouse models and in human clinical studies. Blocking the EGFR pathway can induce compensatory activation of the IGFR pathway to drive tumor growth and IGFR inhibition can result in activation of EGFR signaling in preclinical models. Therefore, blocking both receptors simultaneously may achieve superior efficacy to blocking either pathway alone. We developed individual optimized Adnectins™ specific for blocking either EGFR or IGFR signaling and engineered them into a single protein that linked both Adnectins together to construct a bi-specific Adnectin targeting the EGFR and IGFR (EI-tandem). The bifunctional molecule blocked activation of EGFR and IGFR, inhibited both EGF and IGF-induced down-stream cell signaling (MAPK and AKT pathways) and was antiproliferative in human cancer cell lines. Potency of the EI-tandem was comparable to anti-EGFR and anti-IGFR antibodies. The EI-tandem demonstrated a synergistic inhibition of IGFR phosphorylation and down-stream cell signaling compared to Adnectins specific for only EGFR or IGFR alone. Although Adnectins bound to the EGFR at a site distinct from the clinically approved anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, they still blocked binding of EGF to the EGFR. PEGylated EI-tandem inhibited the growth of human tumor xenografts driven by both EGFR and IGFR signaling, degraded EGFR and IGFR, and reduced phosphorylation of EGFR in tumors. Treatment of mice with EI-tandem caused increases in levels of the circulating ligands TGFα and IGF1 resulting from blockade of their respective receptors and provided convenient soluble biomarkers of target suppression. These results show that a bifunctional Adnectin can confer improved biological activity compared to monospecific biologics in tumors where growth is driven by multiple growth factors.

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 2586.
Abstract 2588: Affitoxin - A novel approach to HER2-targeted therapy
Authors: Zielinski Rafal J. , Lyakhov Ilya , Kuban Monika , Capala Jacek
Published: February 2011

Expression of the human epidermal growth factor receptor 2 (HER2) is amplified in 25 −30% of breast cancers and has been associated with unfavorable prognosis. In spite of the development of new HER2-targeted therapies, such as trastuzumab (Herceptin®; Genentech, Inc., South San Francisco, CA), which has revolutionized the treatment of HER2-overexpressing breast cancers, there is a significant number of patients with HER2-positive tumors who do not respond or acquire resistance to these therapies. An alternative treatment maybe constituted by immunotoxins. Immunotoxins are hybrid proteins composed of a targeting moiety such as an antibody, antibody fragment or ligand directed to an antigen or a receptor on the surface of tumor cells, and a toxic domain derived form plant (ricin) or bacteria (diphtheria toxin or Pseudomonas aeruginosa exotoxin A). The targeting moiety directs the toxin moiety to the tumor cell and, then, the active domain induces apoptosis by inhibition of protein synthesis. The PE38, which is a truncated version of Pseudomonas exotoxin A (PE), is preferred for construction of immunotoxin because its high toxic potential has been well documented and cytotoxic pathways described and understood.

Affitoxins are immunotoxin derivatives containing Affibody molecule as a targeting moiety instead of antibodies or antibodies fragments. Affibody molecules are a new class of relatively small (∼7-kDa) affinity proteins. Structurally, they are based on a 58-amino-acid scaffold, derived from the Z domain of the Staphylococcus aureus protein, and were obtained by combinatorial protein engineering.

Previously we reported construction, overxpression and characterization of Affitoxin targeting tumors with HER2 overexpression. Receptor-dependent binding and tumorocidal activity was confirmed on cell lines varying with receptor number. Both binding and toxicity was reverted when excess of Affibody molecules (acting as a competitor) was applied.

Here we report in vivo characterization of HER2 -Affitoxin. First we show that Affitoxin is well tolerated in athymic mice. Pharmacokinetic analysis indicates relatively short half-life of Affitoxin in the circulation most probably due to rapid renal clearance which seems to be compatible with fast binding of Affitoxin to HER2. Finally we show that HER2-Affitoxin is potent agent that leads to elimination of breast and ovarian xenografts (BT474 and SKOV3 s.c. tumors), while no such an effect was observed for off-target Affitoxin applied in the same concentration. Immunogenic potential of HER2 Affitoxin was also evaluated.

We conclude that our HER2-Affitoxin has potential to become an attractive alternative in treatment of tumors associated with expression of HER2 receptors.

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 2588.
Abstract 2606: Absence of transcriptomic signature of response to chemotherapy in metastatic colorectal carcinoma patients
Authors: Laroche-Clary Audrey , Smith Denis , Laurand-Quancard Armelle , Evrard Serge , Robert Jacques , Morvan Valerie Le
Published: February 2011

Using whole genome tumour gene expression profiling in patients treated for metastatic colorectal cancer, we attempted to define a signature able to discriminate between responders and non-responders to first-line chemotherapy.

A total of 42 patients were prospectively included between 2003 and 2006. They all suffered from histologically proven colorectal adenocarcinoma and were metastatic at the time of diagnosis. All of them gave informed consent to the study. They were treated in first line with 5-fluorouracil (according to the de Gramont regimen in most cases) associated either with irinotecan (FolFiri regimen, 180 mg/m2 every two weeks, 22 pts) or with oxaliplatin (FolFox 4 regimen, 85 mg/m2, 20 pts) according to physician's decision. Response was evaluated using WHO criteria every 2 months after initiation of chemotherapy and patients were assigned to complete response (1 pt), partial response (24 pts), stable disease (13 pts) or progressive disease (4 pts).

From a tumour sample obtained at surgery (18 pts) or by biopsy (24 pts) and kept in RNA later, we extracted and purified total RNA. Gene expression profiling was performed by Cogenics using microarrays from Applied Biosystems (Human Genome Survey Microarray v2.0). We used three different methods for supervised data analysis: analysis of variance, SAM (statistical analysis of microarrays) and Limma (Bioconductor). We compared responders to non-responders in the whole set of patients and in the subsets of patients who received either irinotecan or oxaliplatin. With none of the three methods we were able to obtain a stable signature, i.e. a group of genes consistently associated with response to chemotherapy. On the contrary, we obtained very homogeneous gene expression profiles which were even difficult to clusterise using a non-supervised approach. These negative results show that the determinants of response to chemotherapy should be sought not only in the tumour molecular characteristics, but also among the processes leading to drug availability to the tumour (drug dose, drug disposition, etc.). Especially, the relatively low dose of irinotecan which is of standard use in Europe does not allow to recruit all the responders who could be identified at higher dosage, given the sharp dose-efficacy curve that has been identified long ago for this drug.

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 2606.
Abstract 2615: Amplification and/or high expression of Myc family genes sensitizes tumor cells to aurora kinase inhibitors
Authors: Hook Kenneth E. , Pavlicek Adam , Garza Scott J. , Lira Maruja E. , Ching Keith , Kan Julie , Srinivasa Sreesha P. , Christensen James G.
Published: February 2011

The identification of patient populations predicted to derive clinical benefit from systemic treatment regimens is of critical importance for development of targeted therapies. Collective clinical data available for the Aurora kinase inhibitor class of targeted therapies has indicated that broad single agent clinical activity is not readily apparent. We screened a panel of established lung (mostly non-small cell) and colon cell lines for growth inhibitory sensitivity to PF-03814735, an Aurora family kinase inhibitor, revealing a potential correlation with Myc family amplification or expression. The Aurora kinases A and B have been shown to be functionally linked with Myc family oncoproteins in a number of tumor types. Myc family gene amplification events have been reported in about 30% of small cell lung cancer (SCLC) primary tumors and around 50% of cell lines established from SCLC. We next screened a panel of around 20 SCLC lines for sensitivity to PF-03814735 and its relationship to Myc family gene copy number and gene expression levels. Sensitivity to PF-03814735 in vitro was strongly correlated with amplification events in at least one of the Myc family genes (c-Myc, L-Myc, N-Myc) as well as mRNA expression levels of those genes. Myc family expression demonstrated a significant correlation with gene copy number. Follow up studies to evaluate antitumor efficacy in two SCLC xenograft models, H82 (c-Myc) and H69 (N-Myc), indicated significant tumor growth inhibition by PF-03814735. However, the SCLC models were not appreciably more sensitive to PF-03814735 than several non-Myc amplified tumors studied previously (HCT-116, COLO-205, MDA-MB-231) indicating that further study of optimal dose schedule and/or the molecular basis of sensitivity is warranted.

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 2615.
Abstract 265: EGFR positively regulates GBP1 expression to promote glioma cell invasion
Authors: Li Ming , Furnari Frank , Cavenee Webster
Published: February 2011

Glioma is the most common and deadly human brain tumor. Epidermal growth factor receptor (EGFR) amplification or constitutively active mutation (EGFRVIII) is one of the most frequent genetic lesions in gliomas. To identify target genes regulated by EGFR signaling, we performed expression array analysis and found that human guanylate binding protein-1 (GBP1), a large GTPase that is induced by inflammatory cytokines, is one of the predominant genes induced by EGFR/EGF or EGFR VIII kinase activity. Our results demonstrate that EGF/EGFR signaling could dramatically induce GBP1 expression at both the mRNA and protein level through the p38 MAPK signaling cascade that is distinct from Stat1 signaling used by interferon γ. Moreover, we identified IRF1 and YY1 as downstream transcriptional regulator for GBP1 enhancement by EGFR/p38 signaling. Interestingly, we show that GBP1 is required for EGFR-mediated matrix metallopeptidase-1 (MMP1) expression, and plays a role in glioma cell invasion in vitro. Overexpression of GBP1 enhances glioma cell invasion through MMP1 induction, which was independent of GBP1 GTPase activity. In contrast, siRNA-mediated knockdown of GBP1 inhibited MMP1 expression and in turn blocked cell invasion as well. Consistently, we found that GBP1 is overexpressed and positively correlated with EGFR/EGFRVIII and MMP1 expression in human glioma clinical samples and glioma cell lines, particularly in neural subtypes of GBM as demonstrated by TCGA database analysis. Therefore, Our results suggest that the dysregulation of EGFR-mediated GBP1 expression may contribute to glioma invasion.

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 265.
Abstract 267: The effect of amphiregulin knockdown on the expression of transformed phenotypes in triple-negative breast cancer
Authors: Baillo Andrea J. , Ethier Stephen P.
Published: February 2011

Amphiregulin (AREG) and epidermal growth factor receptor (EGFR) co-expression leads to aggressive growth phenotypes observed in some triple negative breast tumors. In SUM-149 cells, a triple negative breast cancer cell line with an AREG/EGFR autocrine loop, we have shown that these cells are more invasive and motile than MCF10A cells grown with EGF and that altered signaling occurs when AREG is the stimulating ligand. To provide additional insight into the role of AREG in the SUM-149 cells, we have successfully knocked down AREG expression in the SUM-149 cells. Q-RT-PCR analysis showed an approximately 40-fold knock-down of AREG mRNA following stable transduction of the most effective shRNA vector. In these knock-down cells, secreted AREG was undetectable by ELISA, and cellular AREG was undetectable by western blot. Knock-down of AREG in SUM-149 cells resulted in a dramatic decrease in the growth rate of these cells, resulting in a doubling time of about half that of the parental SUM149 cell line. Soft agar assays comparing the anchorage-independent growth potential of SUM-149 cells with AREG knock-down SUM-149 cells demonstrated an approximately 50% decrease in soft agar colony forming ability. To examine the migration and invasion capacity of the AREG knock-down cells relative to parental cells, Matrigel invasion assays were carried out and showed that knocking down AREG resulted in a 40-fold decrease in the cells ability to invade the matrix. Our lab has previously shown that in SUM-149 cells, NF-κB DNA binding activity is initiated as a result of AREG activation of EGFR. NF-κB activity in the AREG knock-down cells was altered suggesting that AREG affects cellular signaling by altering transcription factors. Addressing the continued proliferation of the AREG knock-down cells, we examined the requirement for EGFR activation for growth of these cells. Phosphotyrosine western blot analysis demonstrated detectable EGFR activation in the AREG knock-down cells. Growth assays where these cells were treated with an EGFR tyrosine kinase inhibitor or with a neutralizing EGFR antibody demonstrated the dependency of EGFR for growth of these cells. Q-RT-PCR analysis for expression of all seven EGFR family ligands suggested that epigen or EGF may be factors contributing to the continued growth of these cells, in the absence of AREG. In summary, knock-down of AREG expression in SUM-149 cells has a dramatic affect on the cells ability to proliferate, invade, and form colonies in soft agar, which links this growth factor with the aggressive properties associated with the SUM-149 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 267.
Abstract 2676: Unusual binding of TMP in two different sites in the binary complex with the Q35S/N64F double mutant of human dihydrofolate reductase
Authors: Cody Vivian , Piraino Jennifer , Pace James , Queener Sherry F.
Published: February 2011

In an effort to understand the role of specific active site residues in modulating the binding selectivity of inhibitors against dihydrofolate reductase (DHFR) from different species, a series of single and double mutants of DHFR were made at positions 35 and 64 of human (h) DHFR. To explore sequence differences between human and the pathogenic organisms Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) DHFR, the causative agent of Pneumocystis pneumonia (PcP) in patients with AIDS, kinetic data were measured for trimethoprim, used for treatment of AIDS-related pneumonia. Crystal structure data are reported for the Q35S/N64F double mutant of hDHFR as a binary complex with trimethoprim (TMP). These substitutions make the human enzyme resemble a cross between pjDHFR which has Ser/Ser at these positions and pcDHFR which has Lys/Phe at these sites. Kinetic data show enhanced affinity for the binding of TMP to this double mutant (Ki 604 nM) compared to wild type hDHFR (Ki 1757 nM). The Ki of TMP is 43 nM for pjDHFR and 222 nM for pcDHFR. Structural data for the binary complex of TMP with this double mutant of hDHFR revealed the unexpected binding of two copies of TMP; one bound in the normal binding site while the other occupies a shallow pocket different from that involved in binding the γ-glutamate of folate substrates or inhibitors. The trimethoxyphenyl ring of the second TMP makes a series of hydrophobic contacts with the trimethoxyphenyl ring of the first TMP and with the phenyl ring of residue F31 and the mutated F64 in this structure. Structural data for the binary complex of wild type hDHFR with TMP does not indicate such binding. The conformation of the two TMP in the double mutant complex differ significantly from each other and from that observed for the wild type hDHFR binary complex. Analysis of all DHFR TMP complexes reveals the bridge torsion angles fall into two broad ranges and the two TMP in the mutant hDHFR complex fall into the less populated group. These data suggest that the combination of hydrophobic contacts from the normal F31 in hDFHR and the mutant F64 provide a unique environment to create sufficient van der Waals contact energy to stabilize binding in this unusual site. Supported in part by NIH GM51670 (VC).

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 2676.
Abstract 2678: Molecular signature of drug action for microtubule stabilizing agents
Authors: Khrapunovich-Baine Marina , Menon Vilas , Fiser Andras , Angeletti Ruth Hogue , Horwitz Susan Band , Xiao Hui
Published: February 2011

Microtubule-stabilizing agents (MSAs) comprise a class of drugs that bind to polymerized microtubules (MTs), thereby inhibiting their dynamic properties and leading to arrest in mitosis. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling studies led to the identification of the binding site for Taxol in the β-tubulin subunit of the MT polymer, the effect of the drugs on MT conformation was only recently deciphered in our laboratory using hydrogen deuterium exchange (HDX) coupled to high-pressure liquid chromatography and mass spectrometry (HPLC-MS). We have also utilized this technique to determine the binding mode and conformational effects of discodermolide, another MSA with potential to become a useful anti-tumor drug. We found that in tubulin isolated from chicken erythrocytes, discodermolide bound to an overlapping binding site with Taxol, but had a distinct pose within the pocket, such that it oriented itself away from the M-loop and toward the N-terminal H1-S2 loop of β-tubulin. Evidence of conformational effects on the C-terminal H12 helix in the α-tubulin subunit and of complementary MT stabilization between Taxol and discodermolide was found. These results provide the molecular basis for the ability of the two drugs to modulate interactions with endogenous microtubule associated proteins (MAPs) and for the synergy observed between them in vivo. Preliminary analysis of the data with four other MSAs, epothilone B, ixabepilone, peloruside A, and laulimalide, suggests a very similar mode of MT stabilization by all the drugs in this class, with some drug-specific, and more importantly, binding-mode-specific MT-stabilizing effects. Careful examination and qualitative comparison of the conformational effects of all the aforementioned MSAs is currently in progress, and will allow us to determine a molecular signature for drug action that may correlate with the corresponding therapeutic benefits.

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 2678.
Abstract 2681A: Structure-guided design of potent and selective inhibitors of B-Raf kinase displaying on-mechanism in vivo activity
Authors: Kim Joseph L. , Smith Adrian L. , Carnahan Josette , Beltran Pedro J. , Whittington Douglas A. , Rose Mark J. , DeMorin Frenel , Doherty Elizabeth , Huang Qi , Ncube Mquele , Paras Nick A. , Petkus Jeffrey K. , Tasker Andrew S. , Lee Matthew R. , Babij Carol , Fernando Manory , Hess Kristen , Le Quynh , Epstein Linda F. , Yakowec Peter S.
Published: February 2011

The Ras-Raf-MAPK signaling pathway plays a central role in diverse cellular processes including differentiation, proliferation and survival. This pathway is activated in a large percentage of cancers as a result of mutations in Ras or in B-Raf Ser/Thr kinase. The activating mutation V600E in B-Raf is present in approximately 60% of melanomas and occurs with lower, yet still significant, frequency in human colon and thyroid cancers. Hence, V600EB-Raf has received considerable interest as a small-molecule drug discovery target within the pharmaceutical industry. A high-throughput screen of Amgen's internal kinase preferred library against a recombinant V600EB-Raf kinase domain enzyme identified a class of biarylamide compounds as potent inhibitors of this kinase, and potential starting points for medicinal chemistry efforts. However, these compounds suffered from poor kinase selectivity, particularly against tyrosine kinases from the Src-, VEGFR and PDGFR-families. Crystal structures of representative examples of this scaffold in B-Raf and other tyrosine kinases identified a unique pocket in B-Raf that could be accessed to achieve selective inhibitors of this enzyme. Consequently a novel series of isoquinoline-based compounds was derived which demonstrated potent inhibition against V600EB-Raf enzyme and inhibition of MAPK pathway signaling in cell lines harboring V600EB-Raf. Further optimization within this inhibitor class yielded compounds with favorable in vivo properties as demonstrated by robust inhibition of ERK phosphorylation in V600EB-Raf driven pharmacodynamic models. Crystallographic work supporting the optimization of this series of selective B-Raf inhibitors will be presented.

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 2681A.
Abstract 2698: The parametric response map as an imaging biomarker for identifying progression from pseudoprogression in glioma patients
Authors: Tsien Christina , Galban Craig J. , Chenevert Thomas L. , Johnson Timothy D. , Hamstra Daniel A. , Sundgren Pia C. , Junck Larry , Meyer Charles R. , Rehemtulla Alnawaz , Lawrence Theodore S. , Ross Brian D.
Published: February 2011

We assessed whether a new method of quantifying therapeutic-associated hemodynamic alterations may help to distinguish pseudoprogression from true progression in patients with high grade glioma. Patients from a prospective IRB-approved trial with high grade glioma received concurrent chemoradiation. Relative cerebral blood volume (rCBV) and blood flow (rCBF) maps were acquired prior and at week 3 mid-treatment. Pseudoprogression was defined as imaging changes 1-3 months after chemoradiation that mimic tumor progression but stabilized or improved without change in treatment or for which resection revealed radiation effects only. Clinical and conventional MR parameters, including average percent change of rCBV and CBF, were evaluated as potential predictors of pseudoprogression. Parametric response map (PRM), an innovative, voxel-by-voxel method of image analysis, was also performed. Median radiation dose was 72 Gy (range: 60-81). Of 27 patients, stable disease/partial response was noted in 13 and apparent progression in 14. Adjuvant temozolomide was continued in all patients. Pseudoprogression occurred in 6 patients. Based upon PRM analysis, a significant portion of the tumor volume had reduced rCBV (PRMrCBV) at week 3 in patients with progressive disease compared to those with pseudoprogression (p<0.01). In contrast, change in average percent rCBV or rCBF, MR tumor volume changes, age, extent of resection, and RTOG RPA classification did not distinguish progression from pseudoprogression. PRMrCBV at week 3 during chemoradiation is a potential early imaging biomarker of response that may be helpful in distinguishing pseudoprogression from true progression in glioma patients.

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 2698.
Abstract 2701: Clinical implications of UGT1A1*28 genotype testing in colorectal cancer patients
Authors: Shulman Katerina , Cohen Ilana , Barnett-Griness Ofra , Gruber Stephen B. , Lejbkowicz Flavio , Rennert Gad
Published: February 2011

Purpose: Metastatic colorectal cancer is frequently treated with Irinotecan (CPT-11), a topoisomerase-I inhibitor. Irinotecan toxicity is relatively commonly observed. The gene UGT1A1 encodes an enzyme that catalyzes the glucuronidation of the active irinotecan metabolite SN-38 which is eliminated in the liver by metabolic alteration to an inactive form SN-38G. We retrospectively evaluated the association between UGT1A1 genetic variation, prevalence of severe toxicity and colorectal survival.

Patients and Methods: In the Molecular Epidemiology of Colorectal Cancer (MECC) Study we identified 213 patients with metastatic colorectal cancer treated by irinotecan-based chemotherapy (FOLFIRI, IFL). DNA extracted from blood was used for genotyping either by simple determination of TATA box sequence in patient DNA or by implementation of fragment analysis that detects the difference in the number of nucleotides in the specific region analyzed. The polymorphism UGT1A1*28 is characterized by the presence of an additional TA repeat in the TATA sequence of the UGT1A1 promoter, ((TA)7TAA, instead of (TA)6TAA). Data on side effects and survival were extracted from full follow-up oncology records.

Results: UGT1A1*28 genotypes 6/6, 6/7, 7/7 were detected in 41.8%, 46.5%, and 11.7% of the patients respectively. Observed grade 3-4 toxicity included: diarrhea 22.4%, leucopenia 9.3%, neutropenia 8.9%, neutropenic fever 3.7%, and mucositis 4%. No significant difference was observed in the rate of diarrhea in patients with UGT1A1*28 7/7 genotype (20%) as compared to these with the 6/6 genotype (27.5%). Hematological toxicity was significantly more common in patients with the 7/7 genotype. Neutropenic fever was observed in 24% of the 7/7 genotype but only in 2% of the 6/7 genotype and 0% of the 6/6 genotype. Oncological hospitalization rate was very high among the 7/7 group (45.8%) and significantly higher than among the 6/6 group (14.4%). The Median overall survival of patients with 7/7 was significantly lower (2.10 years) than that of patients with the 6/6 variant (4.13 years, p=0.002).

Conclusion: UGT1A1*28 7/7 genotype is strongly associated with severe toxicity (mainly neutropenic fever) and oncological hospitalizations and with lower survival. These data support the FDA recommendation and product labeling to tailor treatment plans for colorectal cancer patients.

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 2701.
Abstract 2702: Renal cell carcinomas lack arginosuccinate synthetase expression and respond to pegylated arginine deiminase treatment
Authors: He Wei , Chen Jianhe , Zheng Yuhui , Dai Zaoming , He Yan , Wu Bor-Wen , Bomalaski John
Published: February 2011

Biomarkers may be predictive of benefit from therapy. As a result, there is increasing interest in determining new biomarkers and new agents that may target specific pathways that are abnormal in a certain cancer. Normal cells produce arginine internally via the urea cycle. However, certain cancer cells lack one of the urea cycle enzymes, argininosuccinate synthetase (ASS) and become dependent upon an exogenous supply of arginine for growth and survival. For these ASS-deficient cancers, preferential killing of tumor cells induced by arginine deprivation may be effective and safe. ADI-PEG20 is a microbial enzyme, arginine deiminase conjugated with polyethylene gycol. The primary mechanism of action of ADI-PEG20 is arginine deprivation. Thus cancers that are deficient in ASS expression and require arginine could benefit from ADI-PEG20. ADI-PEG20 has been in phase 2 clinical trials for the treatment of hepatocellular carcinoma and metastatic melanoma. Patients whose cancers were deficient in ASS expression have demonstrated medical benefit from ADI-PEG20. Thus at least in these cancers, lack of ASS expression could serve as a biomarker to predict clinical benefit from ADI-PEG20. Other investigators have noted that human renal cell carcinoma (RCC) tissue specimens lack expression of ASS. To confirm these observations and investigate the extent of ASS deficiency in RCC, we analyzed biopsy samples obtained from 31 patients for ASS expression by immunohistochemistry and found all to be ASS negative. We also established three primary RCC cell lines and tested them for response to ADI-PEG20 treatment. Dose dependent growth inhibition was observed after 48 hours for all three cell lines using the MTS proliferation assay. We further tested three ATCC RCC cell lines, 786-O, ACHN and Caki-1 using the MTS assay and observed similar growth inhibition after 48 hours. Cell cycle analysis using the three primary RCC lines revealed that G1 arrest was apparent in samples treated with ADI-PEG20. After 48 hours of ADI-PEG20 treatment, the percentage of cells in G1 increased from 33% to 62%. These results suggest that ADI-PEG20 can potentially be an effective therapeutic agent for the treatment of RCC. They also suggest the potential utility of ASS expression as a biomarker in identifying patients that are most likely to benefit from the ADI-PEG20 treatment.

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 2702.
Abstract 2704: Efficacy and candidate biomarker evaluation for the anti-MN immunoconjugate BAY 79-4620, MN-IC in MN (CAIX) positive preclinical xenograft models
Authors: Schatz Christoph A. , Peña Carol E. , Petrul Heike , Röse Lars , Kopitz Charlotte
Published: February 2011

BAY 79-4620 is a novel immunoconjugate consisting of a fully human monoclonal antibody directed against MN (carbonic anhydrase IX, CAIX) conjugated with the auristatin derivative MMAE currently in Phase I clinical testing. MN is overexpressed in a range of tumor types, such as gastric cancer, non-small cell lung cancer, pancreatic cancer or colorectal cancer. CAIX expression is regulated by HIF-1α and therefore associated with tumor hypoxia.

Here we report on the correlation of tumor CAIX antigen expression levels, as determined by a newly developed CAIX IHC staining protocol, and the efficacy of BAY 79-4620 in six preclinical tumor models. Further, CAIX levels in xenograft tumors were compared to those in clinical samples of human tumors. Efficacy of BAY 79-4620 in tumor models increased with their CAIX expression levels. HeLa-MaTu, HT29 and PC3mm2 models characterized by the highest expression of CAIX showed 56-92 % tumor growth inhibition, when treated with 1 mg/kg BAY 79-4620. Hs746T, the tumor model with the lowest CAIX level, did not show tumor growth inhibition at 1 mg/kg MN-IC, but responded to higher doses. Analysis of CAIX expression by IHC using human tumor samples (gastric, CRC, NSCLC and breast) demonstrated that tumor cell CAIX expression varied both within and between tumor types. IHC scoring revealed that 95% of colon cancer tumors, 55% of NSCLC tumors, and 35% of breast cancer tumors assayed had ≥10% of cells scoring 2+ or 3+ by IHC. In preclinical models analysis of serum CAIX extracellular domain (ECD) by ELISA revealed an initial rise in plasma CAIX ECD as early as 2 days after 30 mg/kg BAY 79-4620 followed by a decrease to below-baseline levels. In two out of three models tested, changes or increased levels of the apoptosis marker CK18 were observed following BAY 79-4620 administration. Changes in serum CAIX ECD levels as well as CK18 will be explored as an early pharmacodynamic (PD) response markers in clinical trials for BAY 79-4620.

In summary, these results demonstrate that targeted delivery of MMAE resulted in higher efficacy of BAY 79-4620 in tumor models with high CAIX expression. Responsive xenograft models were representative of clinical samples with respect to CAIX expression scored by IHC. Quantification of MN (CAIX) by IHC is a candidate stratification biomarker for the immunoconjugate BAY 79-4620 currently in Phase I testing.

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 2704.
Abstract 2705: Quantitative targeted proteomics in pancreatic cancer: Combinations of enzyme and transporter protein expression are novel indicators for gemcitabine sensitivity
Authors: Kawaguchi Kei , Motoi Fuyuhiko , Ohmine Ken , Ohtsuka Hideo , Ohtuki Sumio , Rikiyama Toshiki , Katayose Yu , Egawa Shinichi , Terasaki Tetsuya , Unno Michiaki
Published: February 2011

Background and Aims: Gemcitabine (GEM) is widely used as a first line agent for the treatment of pancreatic cancer. In some patients, it provides a long-term survival, while there are many non-responders. Recent advances in proteomics technology made it possible to quantitate trace proteins such as transporters and metabolic enzymes at high sensitivity enough to reflect the function of proteins in living organisms. In this study, we exhaustively quantitated the protein profiles of GEM related transporters and metabolic enzymes in pancreatic cancer cell lines and resected specimen using LC-MS/MS technique. We created the GEM sensitivity equation based on the profiles and the IC50 values of cell lines. This equation was validated using surgical specimens of pancreatic cancer.

Materials and Methods: We used 6 pancreatic cancer cell lines for profiling. Surgical specimens from 9 patients from March 2004 to January 2006 were classified GEM respond group (RS), GEM non-respond group (NR) and surgery effect group (SE) according to post-operative CA 19-9 value. The expression levels of 90 membrane proteins and 15 enzymes were quantified in 6 pancreatic cancer cell lines and 9 surgical specimens, by means of multiplexed MRM analysis with LC-MS/MS.

Results: Significant differences in the expression levels of dCK and Purine-5-NT were observed between responsive and non-responsive groups of patients, although no correlation of the expression levels of these proteins with IC50 of gemicitabine was found in the cell lines. The expression levels of ten membrane proteins in the cell lines were correlated to IC50 of gemcitabine, but the expression levels of these proteins failed to predict the gemcitabine sensitivity of cancer tissues. Next, expression ratios of multiple proteins (transporters and enzymes) that were present in cancer tissues were calculated for the cell lines. dCK/(MRP1+MRP4), dCK/(MRP1+MRP4+Purine-5NT), dCK/(MRP1+MRP4+CTPS1) and dCK/(MRP1+MRP4+Purine-5NT+CTPS1) showed high correlation with IC50 values. For all four combinations, the protein expression ratio in the RS group was significantly higher than those in NR and SE groups.

Conclusion: Applying these equations, we can predict GEM sensitivity and tailor-made chemotherapy can be implemented.

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 2705.
Abstract 2706: Discovery of predictive biomarkers based on differential chemosensitivity of colorectal cell lines
Authors: Unger Florian T. , Meyer Markus , Harasym Agnieszka , Spangenberg Jörg , Sullivan Kevin , Juhl Hartmut , David Kerstin A.
Published: February 2011

Chemoresistance is the main reason for therapeutic ineffectiveness and recurrence in colorectal cancer. Furthermore, the choice of optimal chemotherapy is complicated by an interpatient variability in drug response caused by the individual geno- and phenotype. To promote a more individualized therapy, predictive biomarkers which indicate individual drug response of patients are urgently needed.

In this study, using a combination of HPLC and mass spectroscopy, we discovered differentially expressed proteins which might be linked to specific phenotypes of intrinsic chemoresistance in colorectal cancer cell lines. Several commercially available cell lines as well as primary mixed cultures and clonal cell lines established from colorectal cancer patients were treated with different drugs, such as 5-Fluorouracil, Oxaliplatin and drug combinations e.g. FOLFOX. Two different assays, the ATPliteTM-(Perkin Elmer) and FMC-assay (according to Larsson et al.) were used in parallel to measure cytotoxic and cytostatic drug effects. Both assays showed time and dose dependent drug responses which differed significantly among the 18 colorectal cell cultures. The analysis of obtained in vitro chemosensitivity data allowed us to cluster sensitive versus resistant cell lines by determining IC50-values.

From all 18 cell cultures, differential protein expression was analyzed using a combination of reversed phase high performance liquid chromatography and matrix assisted laser desorption/ionization mass spectrometry (RP-HPLC MALDI MS) workflow. Up to 3500 m/z ion signals in the molecular mass range between 2500-35000 m/z were detected reflecting a complex pattern of small proteins and peptides. For protein identification, RP-HPLC fractionated samples were digested by trypsin and fragments of interest were subjected to MS/MS analysis in the TOF/TOF mode. The corresponding MS/MS spectra were used to search the NCBInr database using MASCOT software. Based on these data, we hope to gain more understanding of the biological processes of chemoresistance concerning important cellular drug responses (e.g. signal transduction pathways) and thereby obtain insight into new drug targets for future therapeutic intervention.

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 2706.