bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C191: The effect of the cyclin-dependent kinase inhibitor PD 0332991 on glioblastoma cells and relationship with p15INK4B/p16INK4A expression
Authors: Cen Ling , Ostrem Jamie L. , Schroeder Mark A. , Mladek Ann C. , Fink Stephanie R. , Jenkins Robert B. , Sarkaria Jann N.
Published: June 2012

Glioblastoma multiforme (GBM) is the most common and highly malignant primary brain tumor in adults with a median survival just over 1 year, and novel therapies are urgently needed. Deregulation of the p16 (INK4A)-CDK4/6-pRb pathway are commonly found in human GBM patients and selective inhibitors of CDK4/6 may be specifically useful to suppress proliferation associated with these alterations. PD0332991 is an orally active small molecule that specifically inhibits CDK4 and CDK6. Therefore, the efficacy of PD0332991 was evaluated in the Mayo panel of primary GBM xenograft lines using short-term explant cultures. We hypothesized that expression of p15 and/or p16, endogenous inhibitors of CDK4/6 activity, would suppress the response of GBM tumors to PD0332991 treatment. To test this hypothesis, 2 GBM xenograft cell lines with wild-type p16/p15 levels (GBM5 and 28) and 2 lines without detectable p16/p15 levels (GBM22 and 43) were selected for further analysis. Tumor cells were treated with graded concentrations (10 nanoM to1 microM) of PD0332991 for 5 days and subjected to CyQUANT cell proliferation assays. Consistent with our hypothesis, treatment with 1 microM PD0332991 effectively inhibited cell proliferation to 56.6±3.5% (P<0.001) and 44.3±3.4% (P<0.001) of control in GBM22 and GBM43 cells respectively, but did not affect cell proliferation in GBM5 and GBM28 cells (98.7±15.6% and 101.8±12.0% of control, respectively). In addition, 1 microM PD0332991 induced G1-phase cell cycle arrest in GBM22 (treated 79.7±0.8% vs. control 63.4±0.5% of cells in G1 phase, P<0.001) and GBM43 (treated 70.6±1.1% vs. control 48.9±4.6% of cells in G1 phase, P<0.001) but not in GBM5 and GBM28 (treated 52.3±2.7% vs. control 54.0±5.1%, treated 52.4±4.7% vs. control 54.6±2.6%, respectively). The status of p15 and p16 was confirmed by RT-PCR, which demonstrated readily detectable expression of p15 and p16 in GBM5 and GBM28, but not in GBM22 and GBM43 cells. Additional comparative genomic hybridization (CGH) data showed that CDKN2A and 2B (genes encode for p16 and p15 respectively) are homozygously deleted in GBM22 and 43, hemizygously deleted in GBM5 and normal in GBM28. We also examined the status of CDK4, CDK6, pRb and other molecules in the p16-CDK4/6-pRb pathway by Western, RT-PCR and CGH in these GBM lines and the data will be presented. We are now testing whether shRNA knockdown and/or re-expression of p15/p16 in the relevant cell lines alters their responsiveness to PD0332991. In conclusion, CDK4/6 inhibitor PD0332991 inhibits cell proliferation and induces G1 cell cycle arrest in p15/p16- null GBM tumors, but is less effective in p15/p16 expressing tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C191.
Abstract C198: CX-4945, a novel small molecule inhibitor of CK2 protein kinase, reduces hyperactivated Akt signaling and synergizes with Akt inhibitors in breast cancer cells
Authors: Drygin Denis , Bliesath Josh , Ho Caroline , Siddiqui-Jain Adam , O'Brien Sean , Omori Mayuko , Huser Nanni , Proffitt Chris , Streiner Nicole , Rice William G. , Anderes Kenna
Published: June 2012

Akt, a critical protein kinase in the PI3K signaling pathway regulates multiple biological processes that are important in tumorigenesis. This “Master Regulator Kinase” is often hyperactivated in cancer through various mechanisms, including mutations or deletions in Akt, PI3K or PTEN tumor suppressor. The last decade witnessed the emergence of another “Master Regulator Kinase” - CK2. Like Akt, CK2 controls the growth, proliferation and survival of cancer cells. Ironically, CK2 regulates Akt through phosphorylation and down regulation of PTEN and via direct and specific phosphorylation of Akt at Ser129. This phosphorylation event by CK2 further stimulates the activity of Akt, thereby enhancing the “driver effect” of Akt in promoting oncogenic signaling. Unlike Akt, CK2 does not require phosphorylation for activation but rather its activity appears to be regulated through expression levels. Due to the unique shape of the ATP-binding site and an incomplete understanding of the regulation of CK2 expression, pharmacological targeting of CK2 has proven to be very challenging. To date only one small molecule inhibitor of CK2, CX-4945, has advanced into clinical development. Herein, we describe the pharmacological characterization of CX-4945, its impact on Akt signaling and implications for combination therapies.

The high incidence of abnormalities found in the PI3K pathway in breast cancers and the distinct roles that CK2 and Akt play in this disease have made it an attractive tumor type to study the effects of CX-4945. A cell viability screen of 16 diverse but genetically well-characterized breast cancer lines revealed that cells carrying mutations causing Akt-activation were significantly more sensitive to CX-4945 than those that did not. Western blot analyses of these cell lines demonstrated good correlation between the phosphorylation of Akt at Ser129 and expression of catalytic subunits of CK2. Treatment with CX-4945 resulted in dramatic reductions of phosphorylation of Akt at Ser129 and reductions in phosphorylation of the downstream targets of Akt, e.g. p21. Upon combination of CX-4945 with inhibitors that targeted the PI3K/Akt pathway, we observed synergistic antiproliferative activity in breast cancer cells. Thus, evaluation of the effects of CX-4945 on the Akt pathway in breast cancer cell lines may allow for the identification of patient populations more sensitive to CX-4945 and guide the selection of more effective combination therapies for cancer patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C198.
Abstract C199: Development of potent, small-molecule inhibitors of ETK
Authors: Vollmer David , Bearss Jared , Jones Carissa , McCarthy Virgil , Shi Chongtie , Tang Chunlin , Gourley Eric , Liu Xiao-Hui , Hewitt William , Vankayalapati Hariprasad , Warner Steven , Olsen Colin , Bearss David
Published: June 2012

Epithelial and endothelial tyrosine kinase (Etk) is a nonreceptor tyrosine kinase that plays a central role in the proliferation, differentiation, apoptosis, and tumorigenicity of epithelial cells. Inhibition of Etk signaling can result in impaired cellular transformation, down-regulation of angiogenesis, and increased apoptosis. Employing our proprietary CLIMB™ technology, a computationally driven drug discovery process, we designed and synthesized approximately 35 small molecules for ETK-inhibition testing in biochemical and cellular assays. Most of these compounds exhibited low nanomolar activity and selectivity across a wide panel of kinases. Five compounds were subsequently chosen for further evaluation in in vivo studies. As predicted from CLIMB™, all of the compounds showed sufficient tolerability and pharmacokinetics in mice to advance into tumor efficacy studies. Endometrial and hepatocellular cancers were selected for these studies based on previous in vitro results indicating high ETK expression and potent compound activity. All five compounds demonstrated marked activity in these models; in one cell line, two of the compounds inhibited tumor growth by more than 50% after less than two weeks of dosing. Using these same tumor lines in pharmacodynamic studies, the compounds also showed significant modulation of cellular transformation and anti-apoptotic markers consistent with ETK inhibition. Moreover, quantitative analysis of microvessel density, a key indicator of angiogenesis, demonstrated clear inhibition of blood vessel formation from tumors excised after treatment with the five compounds. Utilizing our CLIMB™ technology, we have rapidly developed a new class of potent inhibitors that consistently demonstrate in vivo activity against ETK-relevant tumor cell lines.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C199.
Abstract C201: A novel potent and selective inhibitor of PI3K/mTOR, GDC-0980, currently in phase I clinical trials
Authors: Friedman Lori S. , Belvin Marcia , Berry Leanne , Berry Megan , Castanedo Georgette , Edgar Kyle , Folkes Adrian , Guan Jane , Heffron Tim , Lesnick John , Lewis Cris , Nonomiya Jim , Olivero Alan , Patel Sonal , Pang Jodie , Prior Wei Wei , Salphati Laurent , Sampath Deepak , Sutherlin Dan , Wallin Jeff , Zhu Bingyan
Published: June 2012

Introduction: The phosphoinositide-3 kinase (PI3K)/Akt signaling pathway is deregulated in a wide variety of cancers. Somatic activating mutations and amplifications in PI3K are common in multiple cancers including breast, colon, and lung cancer. PTEN, a phosphatase that converts PIP3 to PIP2 and thus has an opposing function to PI3K, is a commonly mutated tumor suppressor in multiple cancers including prostate and glioblastoma. Alterations of this pathway have been implicated in tumor initiation, progression, survival, angiogenesis, and metastasis. PI3K/Akt pathway activation has also been implicated in therapeutic resistance. Thus PI3K is considered to be a promising therapeutic target for cancer.

Summary of Results: Medicinal chemistry efforts resulted in the discovery of GDC-0980, a selective, orally bioavailable inhibitor of PI3 Kinase and mTOR kinase with promising pharmacokinetic and pharmaceutical properties. Here we report the first pre-clinical profile of GDC-0980. This compound is a potent ATP-competitive Class I PI3 kinase inhibitor with an IC50 of 4.8 nM against the PI3K p110apha subunit, 26.8 nM p110beta, 6.7 nM p110delta, 13.8 nM p110gamma, and an mTOR Kiapp of 17.3 nM. GDC-0980 demonstrates selectivity against a large panel of protein kinases as well as selectivity over PIK family kinases including Class II, Class III, and DNA-PK. GDC-0980 inhibits the PI3K signaling pathway in vitro causing a reversible G1 cell cycle arrest, and apoptosis induction in a subset of tumor cell lines. Levels of signaling pathway markers such as phosphorylated AKT (pAKT), PRAS40 (pPRAS40), and S6 (pS6) are rapidly and dramatically reduced following exposure of cells to GDC-0980. Oral dosing of GDC-0980 potently inhibits tumor growth in xenograft models including those mutated in PI3K and PTEN, and elicits an exposure-related concomitant decrease in PD biomarkers.

Conclusion: These preclinical data provide compelling evidence in support of GDC-0980 as a clinical candidate, and Phase I studies are ongoing.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C201.
Abstract C203: Inhibition of TTK activates the p53 pathway
Authors: Williams Brandi L. , Rogers Aaron , Cassiano Jared , Tardif Keith D. , Roth Bruce L. , Saunders Michael , Hoarau Christophe , Kumar D. Vijay
Published: June 2012

Background: TTK, also known as Mps1, is a dual-specificity kinase that is essential for the proper amphitelic attachment of chromosomes to the mitotic spindle during mitosis and for preventing anaphase progression until the chromosomes are properly attached. Given the role of TTK in genome integrity, we evaluated the effect of MPI-0479605, a potent and selective inhibitor of TTK, on the p53 pathway.

Materials and Methods: For image analysis, cells were fixed and stained with Hoechst dye and anti-β-tubulin and anti-pericentrin antibodies. Images were taken on a BD Pathway high content imaging system. For cell cycle analysis, cells were fixed, stained with propidium iodide and analyzed by FACS. For Western blot analysis, cell lysates were run on SDS-polyacrylamide gels and proteins were transferred to PVDF membrane. Membranes were probed with the indicated antibodies.

Results: Inhibition of TTK induced severe defects in chromosome segregation, as shown by the presence of lagging chromosomes and/or anaphase bridges during mitosis and the formation of micronuclei at interphase. Anaphase bridges may result in DNA double-strand breaks. Cell cycle analysis showed that a subpopulation of treated cells exhibited a tetraploid phenotype. Because either DNA damage or tetraploidy may induce the p53 pathway, we examined whether p53 was induced by treatment with MPI-0479605. Expression of p53 was induced in a dose- and time-dependent manner in response to MPI-0479605, with maximal levels occurring between 24 and 48 hours of treatment. Induction of p53 correlated with an increase in p21 expression and a decrease in survivin protein levels, indicating that p53 is transcriptionally active. Using phosphospecific antibodies, we determined that p53 is phosphorylated on Ser15 in response to MPI-0479605 treatment but not Thr18, Ser20, Ser37 or Ser46. Phosphorylation of γH2AX at Ser139, which is a marker for the presence of double-strand DNA breaks, was induced by treatment with MPI-0479605 in a time-dependent manner similar to p53 induction. Phosphorylation of p53 on Ser15 and H2AX on Ser139 can be mediated by the DNA damage checkpoint protein ATM. ATM also phosphorylates Chk2 on Thr68 and induces its activity, but, treatment with MPI-0479605 did not induce an increase in Chk2 Thr68 phosphorylation.

Conclusions: Treatment of cells with the small molecule TTK inhibitor, MPI-0479605, activates p53. This may be due to chromosome segregation defects, which cause subsequent DNA double strand breaks and/or tetraploidy. In response to TTK inhibition, we observe an increase in H2AX phosphorylation, followed by the phosphorylation (Ser15) and activation of p53. Given that inhibition of TTK induces an apoptotic cell death, activation of p53 may be one mechanism by which the apoptotic pathway is induced in response to MPI-0479605.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C203.
Abstract C206: In vivo activity of SGI-1252, a potent, small-molecule dual inhibitor of JAK2 and ALK2
Authors: David Vollmer , Bearss Jared , Jones Carissa , McCarthy Virgil , Gourley Eric , Liu Xiao-Hui , Vankayalapati Hariprasad , Warner Steven , Olsen Colin , Bearss David
Published: June 2012

Activation of JAK2, an intracellular protein tyrosine kinase, is known to induce cell proliferation and apoptosis and it has been shown to be mediated by pro-inflammatory cytokines. JAK2 dysregulation is implicated in myeloproliferative disorders, solid tumor malignancies, and autoimmune diseases. Utilizing our proprietary CLIMB™ technology, a computationally driven drug discovery process, we have rapidly designed and developed, SGI-1252, a potent oral inhibitor which exhibits low nanomolar in vitro activity against all of the JAK kinase family, except JAK3. Additionally, it has low nanomolar activity against ALK2, a high-affinity binding receptor for bone morphogenic protein. As predicted from computational models, SGI-1252 is well tolerated across several rodent species and demonstrates a wide safety margin in both short- and long-term toxicity studies. Acute maximum tolerated doses of greater than 900 mg/kg are observed in mice and rats dosed orally with SGI-1252. Over a 5-week period, daily oral dosing of up to 200 mg/kg SGI-1252 shows no hematological toxicity and no body weight loss in mice. Pharmacokinetic studies of SGI-1252 in rodents also demonstrate excellent systemic exposure, with oral bioavailabilities of greater than 50% in mice and rats. Because inflammatory cytokines are known to play a role in the pathogenesis of pancreatic and lung cancers, tumor xenograft models of those cell lines were evaluated. In BxPC3 and A549 xenografts, three weeks of daily oral dosing of 200 mg/kg SGI-1252 demonstrate exceptional activity, with tumor inhibitions of greater than 50% in both models. Pharmacodynamic studies were also performed to further elucidate the effect of SGI-1252 on pro-inflammatory cytokines related to these models. In several acute-phase mouse models, where turpentine-oil injection are utilized for cytokine stimulation, SGI-1252 is observed to down-regulate IL-6 and VEGF expression 16–24 hours after dosing. In summary, SGI-1252 is a potent oral inhibitor that is well tolerated in rodent models, provides excellent PK exposure, and demonstrates tumor growth inhibition as well as modulation of pro-inflammatory cytokines.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C206.
Abstract C234: A novel microtubule inhibitor BPR0L075 overcomes paclitaxel resistance in ovarian cancer cell lines
Authors: Liu Xinli , Wang Xiaolei , Tedjarati Sean , Wang Tian-Li , Hsieh Hsing-Pang
Published: June 2012

Background: The therapy of choice for women with advanced ovarian cancer involves paclitaxel (Taxol®). However, patients develop resistance to paclitaxel during the course of treatment, which is associated with overexpression of P-glycoprotein and alterations involving class III beta-tubulin. BPR0L075 [6-methoxy-3-(3′,4′,5′-trimethoxy-benzoyl)-1H-indole] is a novel synthetic indole compound that inhibits tubulin polymerization through binding to the colchicines-binding site of tubulin (Kuo et al., Cancer Research , 2004). We investigated the activity of BPR0L075 in highly taxol resistant ovarian cancer cell lines.

Methods: human ovarian cancer cell lines OVCAR-3, SKOV-3, and Taxol resistant subclones OVCAR-3/TR(0.5µM Taxol), SKOV-3/TR(0.25µM Taxol) were used for cytotoxicity and apoptosis evaluations.

Results: BPR0L075 displays potent and broad-spectrum cytotoxic activities at low nanomolar concentrations against parental and drug resistant ovarian cancer cell lines investigated, regardless of the expression levels of the multidrug resistance ABC transporters P-gp and BCRP or class III beta-tubulin. Cell cycle analysis revealed the BPR0L075 induced G2/M cell cycle arrest. Furthermore, western blot analysis shows BPR0L075 treatment increases cyclin B1 levels, cleaves poly(ADP-ribose) polymerase, induces BCl-XL phosphorylation. BPR0L075 may induce apoptosis via a caspase-3 independent mechanism in ovarian cancer cell lines.

Conclusion: The present study shows that BPR0L075 is active in drug resistant ovarian cancer cells, suggesting that BPR0L075 is a promising candidate for development as a novel microtubule therapeutic.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C234.
Abstract C243: B7-H4 protein expression in invasive ductal carcinoma and its association with tumor progression
Authors: Goldberg Allison , Burke Stephanie , Shroyer Kenneth
Published: June 2012

High levels of B7-H4 (DD-O110) protein expression have been previously observed in malignant ductal epithelium of the breast but the mechanisms that mediate B7-H4 up-regulation in tumor progression have not yet been determined. An objective of our research has been to explore the differences in the levels of B7-H4 expression that underlie the transition from benign ductal epithelium to ductal carcinoma in situ (DCIS) and to the earliest stages of invasive ductal carcinoma. B7-H4 is a member of the B7 family of proteins which has been found to play a role in antigen-specific immune response regulation, including the regulation of T cell activation. The potential role of B7-H4 in tumor progression, or in shielding breast cancers from immune surveillance mechanisms, remains relatively unexplored. Previous immunohistochemical (IHC) studies have evaluated only a small subset of available B7-H4 antibody epitopes. The aim of the current study is to determine if there is differential expression of specific B7-H4 epitopes that are associated with benign versus proliferative and malignant breast disease. A panel of monoclonal antibodies to defined B7-H4 epitopes (diaDexus, Inc.) was evaluated in normal breast tissues versus invasive ductal carcinoma, using indirect immunoperoxidase methods, in sections of formalin fixed tissues. The patterns of B7-H4 localization of each antibody were compared with the patterns observed with the previously reported A57.1 antibody (Mugler et al., 2007). B7-H4 IHC scores were determined based on the intensity of staining (0 to 3+), the proportion of positive cells, and the cellular pattern of distribution (apical membranous versus circumferential membranous plus cytoplasmic). Preliminary data suggest that certain B7-H4 epitopes appear to have enhanced cancer selectivity, reflected by differential expression between normal ductal cells versus ductal carcinoma cells. Additionally, these studies confirm that there are differences in the subcellular distribution of B7-H4 expression, with low level, apical membranous expression in benign ductal epithelium and high level circumferential membranous expression in most well to moderately-differentiated ductal carcinomas. Furthermore, there appears to be a marked reduction in the levels of B7-H4 expression in some poorly differentiated carcinomas, despite normal levels of expression in adjacent benign ductal epithelial cells. These results suggest that B7-H4 overexpression may play a role in the progression from benign proliferative disease to low grade carcinomas. Further studies are indicated to refine the association of B7-H4 with tumor histologic grade and stage, and to elucidate the potential role of this protein in shielding breast carcinomas from cell-mediated immune surveillance.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C243.
Abstract C245: Protein switches that activate a prodrug in the presence of HIF-1α
Authors: Wright Chapman M. , Eshleman James R. , Ostermeier Marc
Published: June 2012

We have engineered yeast cytosine deaminase (CD) to be activated by the tumor marker, hypoxia-inducible factor 1-alpha (HIF-1α) - turning on its ability to convert 5-fluorcytosine (5FC) into the anticancer drug 5-fluoruracil (5FU). HIF-1α is overexpressed in many types of cancer but is virtually undetectable in normal tissue due to rapid degradation. We sought a “tumor-sensing” protein whose prodrug converting activity is activated in the presence of HIF-1α. Such a protein could be used in a therapeutic treatment similar to gene-directed enzyme prodrug therapy (GDEPT). However, our approach would have an advantage over GDEPT because the “suicide” gene could be delivered using very efficient methods for gene delivery, whether or not they were selective, since the selectivity occurs at the protein level and not the gene delivery level.

To create our desired protein, we constructed a library of fusion proteins between CD and the CH1 domain of p300 - a domain known to interact with HIF-1α. This library was subjected to a two-tiered genetic selection in E. coli designed to identify genes that conferred increased sensitivity to 5FC when HIF-1α was coexpressed. We identified two library members (#3 and #59), which conferred to E. coli sensitivity to 5FC equivalent to wildtype CD but only in the presence of coexpressed HIF-1α. In the absence of HIF-1α, #3 conferred a 5-fold higher LC50 to 5FC and #59 did not confer any sensitivity to 5FC. Both proteins were purified and characterized in vitro for their activity in the presence and absence of HIF-1a. Protein #3 exhibited a 4-fold increase in its CD activity in the presence of HIF-1α, whereas protein #59's activity did not have a significant dependence on HIF-1α. Currently, the two genes are being examined in human cancer cell lines for their ability to confer sensitivity to 5FC in the presence of HIF-1α.

Using advanced protein engineering techniques, we have created two proteins that confer sensitivity to 5FC in the presence of the tumor-marker, HIF-1α. We believe that these genes have therapeutic potential for the treatment of cancers that exhibit elevated HIF-1α levels. Our strategy circumvents a limitation of GDEPT by not requiring selective delivery of the gene to cancer cells.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C245.
Abstract C250: The pericyte as a novel stromal element and therapeutic target in sarcoma
Authors: Morozov Alexei , Downey Robert , Healey John , Moreira Andre L. , Lou Emil , Maki Robert , Moore Malcolm A.S.
Published: June 2012

Background: Mesenchymal Stem Cells (MSCs) have been proposed as the cell of origin of sarcoma. In soft tissues, the microvascular pericyte has been recently shown to have MSC properties (Crisan et al, Cell Stem Cell 3:301). We have previously reported the isolation of benign mesenchymal cultures from sarcoma surgical samples. These cells have the immunophenotype (CD45-CD31- CD73+CD105+CD90+) and in vitro differentiation potential characteristic of MSCs. We hypothesized that these sarcoma-associated MSCs (SA-MSCs) are derived from pericytes associated with the sarcoma vasculature.

Methods: We examined whether benign SA-MSCs have surface markers characteristic of pericytes and cooperate with endothelial cells in tube formation assays. We also examined expression of CD146, a pericyte marker, and CD105, an MSC marker, in sarcoma archived tissue by IHC.

Results: Benign SA-MSCs indeed demonstrated properties of pericytes, such as characteristic cytoplasmic projections at low density, surface expression of pericyte markers (CD146, PDGFR-beta, NG2 and endosialin) and cooperation with endothelial cells in tube formation assays in matrigel. To demonstrate the existence of pericytes in sarcoma archived tissue, we used CD146 that has been shown to be restricted to the vasculature in sarcoma, but thought to be an endothelial marker (Shih et al, CCR 2:569). We demonstrate for the first time the existence of CD146+ pericytes in diverse sarcoma subtypes. In addition, we examined sarcoma archived material for expression of CD105 (endoglin), an MSC marker and a known tumor-specific endothelial marker in carcinoma. We found that CD105 was expressed in sarcoma endothelium similarly to its expression on carcinoma endothelium. In addition, by three-color immunofluorescence, CD105 was expressed in a subset of sarcoma pericytes. It was not expressed in normal surrounding tissues.

Conclusion: We show that SA-MSC cultures have properties of pericytes. Using a novel combination of markers we demonstrate that pericytes are abundant in sarcoma. Being in contact with both the endothelial cells and the malignant sarcoma cells, pericytes represent a novel stromal element in sarcoma and a potential therapeutic target. Based on these findings we are evaluating endosialin (CD248) and CD105 antibodies, both of which are in clinical development, for effectiveness in preclinical models of sarcoma.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C250.
Abstract C251: Heparanase mechanistically links chronic colitis and tumorigenesis
Authors: Lerner Immanuel , Zcharia Eyal , Bensoussan Esther , Sherman Yoav , Vlodavsky ISrael , Elkin Michael
Published: June 2012

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is closely associated with colon cancer. Here we report that heparanase enzyme acts as an important mediator of colitis-associated tumorigenesis. Heparanase is an only known mammalian enzyme that cleaves heparan sulfate, the major polysaccharide of the extracellular matrix, and plays multiple roles in inflammation and cancer progression. Applying histological specimens from UC patients and a mouse model of dextran sulfate sodium (DSS)-induced colitis, we found that heparanase is constantly overexpressed and activated during the course of the disease, both in the active and inactive phases of inflammation. Employing heparanase-overexpressing transgenic mice in the model of colitis-associated cancer, induced by carcinogen azoxymethane followed by repeated DSS administration, we demonstrated that heparanase overexpression markedly increased the incidence and severity of colitis-associated colonic tumors, enabling faster tumor take, angiogenic switch and enhanced tumor progression. Notably, DSS-induced colitis alone (without azoxymethane pretreatment) lead to formation of colonic tumors in heparanase-transgenic, but not wild type mice, positioning heparanase as important physiological determinant in inflammation-driven colon carcinoma, replacing the need for carcinogen. Investigating molecular mechanisms underlying heparanase induction in colitis, we found that TNF-alpha is responsible for continuous overexpression of heparanase by chronically-inflamed colonic epithelium. Moreover, our results suggest the occurrence of heparanase-driven vicious cycle that powers colitis and associated tumorigenesis: heparanase activity in inflamed colon, acting synergistically with the local cytokine milieu, stimulates macrophage activation, and the activated macrophages secrete TNF-alpha which stimulate further production of heparanase by colonic epithelium. In addition, activated macrophages secrete cathepsin L - a cysteine protease responsible for proteolytic activation of latent heparanase enzyme. Altogether, our results identify heparanase as a key factor in pathogenesis of colitis-associated cancer and attest the inhibition of heparanase as a promising mean to disrupt the vicious cycle that fuels chronic colitis and the associated tumorigenesis.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C251.
Abstract C254: Suppression of E-cadherin function leads to deregulation of FAK and Src to direct squamous cell carcinoma progression
Authors: Alt-Holland Addy , Sowalsky Adam G. , Szwec-Levin Yonit , Shamis Yulia , Feig Larry A. , Garlick Jonathan A.
Published: June 2012

Advanced stages of epithelial carcinogenesis are linked to loss of intercellular adhesion, but it remains unclear how alterations in cell-cell and cell-matrix adhesions are coordinated to promote the early stages of cancer development. To address this, we used 3D tissue models that mimic human premalignant disease, and studied the impact of E-cadherin suppression in early stage, epithelial tumor cells (HaCaT-II-4) on the expression and activity of Focal Adhesion Kinase (FAK) and Src kinase and on tumor cell motility and invasiveness. Suppression of E-cadherin function triggered elevated expression of FAK, increased tyrosine phosphorylation of FAK and Src, and redirected these protein tyrosine kinases to a perinuclear distribution. Pharmacological inhibition of FAK or Src, by either Tyrphostin AG1007 or PP2, reduced their phosphorylation, and reversibly inhibited tumor cell motility in 2D, monolayer cultures. Decreased FAK or Src expression by lentivirus-mediated shRNA restored E-cadherin expression and cell-cell adhesion and inhibited cell migration. 3D, bioengineered tissues harboring E-cadherin-deficient-II-4 cells in which FAK or Src were depleted, demonstrated increased membrane localization of E-cadherin, normalization of tissue phenotype and suppression of tumor cell invasion into the underlying matrix. When these 3D tissues were grafted to the dorsum of immunocompromised mice, down-regulation of FAK or Src resulted in suppression of tumor growth in vivo. Specifically, decreased Src expression reversed the tumor phenotype from high-grade, aggressive and infiltrative SCC to low-grade, well-differentiated and demarcated tumor islands. Collectively, our findings demonstrate that E-cadherin suppression directs the biological behavior of early stage, E-cadherin-deficient tumor cells in vitro and SCC progression in vivo, through modulation of FAK and Src expression and/or activity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C254.
Abstract C26: Nuclear factor I/B regulates cell proliferation of ER negative breast cancer
Authors: Kim Jeong-Ah , Han Wonshik , Jung Eun-Mi , Hwang Ki-Tae , Noh Dong-Young
Published: June 2012

Previously, we reported that DNA copy number of NFIB is amplified in triple negative breast cancer (TNBC; estrogen(−)/progesterone(−)/HER2(−)). The transcriptional activator NFIB plays wide roles in regulation of gene transcription, cell proliferation, viral DNA replication and development. But the functional role of NFIB in breast cancer cell has not been elucidated. In order to understand the role of upregulated NFIB in breast cancers, we examined mRNA and protein level of NFIB in 20 breast cancer tissues. In ER negative breast cancer subtype, expression of NFIB is up-regulated and correlated with Ki67 expression. And intracellular localization of NFIB is restricted in nucleus. In ER negative cell line, knock-down of NFIB leads to reduced cell proliferation by cell cycle arrest. Cell cycle arrest is accompanied by S phase arrest and alteration of S phase regulating protein expression. Take together, transcription activator of NFIB regulates cancer cell proliferation in ER negative breast cancer and it may act as a therapeutic marker.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C26.
Abstract C33: A two-arm phase 1 clinical trial with NPI-0052, a novel proteasome inhibitor
Authors: Price Timothy , Millward Michael , Mainwaring Paul , Harrison Simon , Catley Laurence , Townsend Amanda , Sukumaran Shawgi , Longenecker Angie , Palladino Michael A. , Lloyd G. Kenneth , Neuteboom Saskia , Padrik Peeter , Spear Matthew A. , Spencer Andrew
Published: June 2012

Background: The bi-cyclic structure of NPI-0052 differentiates it from other proteasome inhibitors which are structurally polypeptide based, leading to its differential properties of rapid, broad and prolonged inhibition of all 3 proteolytic sites, and unique proteasome inhibition, toxicology and efficacy profiles. Preclinical studies indicated an increased therapeutic ratio with activity in hematologic and solid tumor malignancies leading to the initiation of Phase 1 clinical trials. These biologic differences also led to the question of whether twice weekly administration schedule, as is typical for other proteasome inhibitors, or more convenient once weekly schedule would be preferable.

Materials and Methods: Patients with solid tumor, lymphoma, leukemia or myeloma diagnoses failing after standard therapies were given NPI-0052 intravenously on a weekly or twice weekly arm in the dose escalation portion of this study. Subsequently 10 patient Recommended Phase 2 dose Cohorts of patients with lymphomas, CLL and myeloma are enrolled. Proteasome inhibition and pharmacokinetics are also assayed in whole blood, and/or peripheral blood mononuclear cells (PBMC) on Day 1 and multiple subsequent time points.

Results: 44 patients have been enrolled into this study. Notable adverse events have been fatigue, parosmia/dysgeusia, transient peri-infusion site pain, lymphopenia, headaches, dizziness / unsteady gait, closed-eye visuals, cognitive changes. Peripheral neuropathy, neutropenia and thrombocytopenia do not appear to be induced. Pharmacokinetic data indicate a short T1/2 and large Vss. Proteasome inhibition assays demonstrated inhibition of chymotrypsin-like activity of up to 88% Day 1 and 100% Day 15. Inhibition accumulates initially in whole blood presumably secondary to the inability of RBC to synthesize new proteasomes, whereas inhibition recovers over several days in PBMC further leading the question as to an optimal dosing regimen. Stable disease, regression or response, have been reported in mantle cell lymphoma, myeloma, Hodgkin's lymphoma, cutaneous marginal zone lymphoma, follicular lymphoma, sarcoma, prostate carcinoma and melanoma.

Conclusions: NPI-0052 produces expected or greater levels of proteasome inhibition in patient blood samples, with a toxicity profile that is dissimilar to what is reported with other proteasome inhibitors. The profile of the proteasome inhibition, toxicity and efficacy profiles that have been revealed during this clinical trial and correlative studies reveals further basis and hypothesis as to what an optimal dosing regimen might be, which is being assessed in this study.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C33.
Abstract C37: A Phase 1 open-label study evaluating pharmacokinetics, safety, and tolerability of linifanib in Japanese patients with solid tumors
Authors: Tamura Tomohide , Yamamoto Noboru , Nokihara Hiroshi , Yamada Yasuhide , Asahina Hajime , Shibata Takashi , Goto Yasushi , Tanioka Maki , Tamura Yosuke , Seki Yoshitaka , Chiu Yi-Lin , Gupta Neeraj , Carlson Dawn
Published: June 2012

Background: Linifanib (ABT-869) is a novel orally active and selective inhibitor of vascular endothelial growth factor and platelet derived growth factor families of receptor tyrosine kinases. Linifanib demonstrated antitumor activity in a variety of advanced solid tumor pts enrolled in early-phase clinical trials. Preliminary data from an international phase 1 multicenter study (M04-710) showed specific single-agent antitumor activity with a recommended tolerated dose of 0.25 mg/kg and a maximum tolerated dose of 0.30 mg/kg. Efficacy was observed in 3 phase 2 studies in pts with hepatocellular carcinoma, non-small cell lung cancer and renal cell carcinoma.

Methods: This is a phase 1, open-label, dose-escalating study evaluating the PK, safety/tolerability of linifanib in Japanese pts with solid tumors. Pts were assigned to 4 dosing cohorts (0.05, 0.10, 0.20 and 0.25 mg/kg) of linifanib given orally once daily (QD) on Cycle (C) 1 Day (D) 1 under fasting conditions for 21 days. PK samples were collected at various time points after single dose on C1D1 and multiple doses on C1D15. CT scans were performed at baseline and at D1 of every second cycle; efficacy was evaluated using RECIST criteria. Adverse events (AEs) were graded by NCI CTCAE V3.0.

Results: As of August 2009, 15 pts have received linifanib in this study: 3 at 0.05 mg/kg; 6 at 0.10 mg/kg; 3 at 0.20 mg/kg and 3 at 0.25 mg/kg. Pts had NSCLC (5), lung carcinoid (1) sarcoma (4), breast cancer (3), thymic carcinoma (1) and angioendothelioma (1). Partial response was noted in 1 breast cancer pt in the 0.20 mg/kg cohort (32% reduction from baseline). 15 pts had ≥ Grade 2 AEs; hypertension (12); neutropenia (4), thrombocytopenia (3) and hand foot syndrome (2). Among all pts, 2 experienced a dose limiting toxicity; 1 each at 0.10 mg/kg (Grade 3 ALT increase) and at 0.25 mg/kg (ECG T wave inversion). 4 pts had AEs leading to dose interruptions (3 pts at C3 and 1pt at C1) and 1 pt had an AE leading to dose reduction in C5. 5 pts have discontinued study at the time of this analysis; 2 due to radiographic progressive disease (PD), 1 due to clinical PD and 2 due to AEs. At 0.25 mg/kg dose, the exposures (AUC24) after single and multiple doses were 6.2±1.2 µg*hr/mL (mean±SD) and 10.7±3.1 µg*hr/mL, respectively. These multiple dose exposures are approximately 2-fold higher than efficacious exposures predicted based on observed results in preclinical animal models. The estimation of effective half-life based on observed accumulation was approximately 1 day.

Conclusion: Preliminary ongoing results show that linifanib is tolerated in Japanese pts at ≤ 0.25 mg/kg and has antitumor activity as a single-agent in 1 pt with breast cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C37.
Abstract C4: TLN-4601 is a novel tumor selective pro-apoptotic agent acting independently of Bax, Bak or Bcl-2 expression
Authors: Bertomeu Thierry , Gourdeau Henriette
Published: June 2012

Background: TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through Thallion's proprietary DECIPHER® technology platform. The compound has demonstrated broad in vitro cytotoxicity and in vivo tumor growth inhibition. Phase I clinical testing has shown the compound to be safe and well tolerated, with early signs of anti-tumor activity. Although considerable research efforts have focused on the ability of TLN-4601 to inhibit the Ras/MAPK signalling pathway, the compound has also been shown to induce apoptosis through caspase activation. The objectives of the present work were to further elucidate the molecular events, pathways and temporal patterns associated with TLN-4601-induced apoptosis.

Material and Methods: Protein extracts from Jurkat cells (a human T cell lymphoblast-like cell line), Jurkat cells with caspase 8 knocked-down, Jurkat cells over-expressing Bcl-2, mouse embryonic fibroblasts isolated from Bax/Bak double knock-out mice, and both quiescent and PHA-L stimulated human peripheral blood lymphocytes (PBLs) treated with TLN-4601 over different exposure times were analyzed by Western blot to evaluate the induction of apoptotic markers, including cytochrome c release, caspase activation and PARP cleavage. Apoptosis was assessed by DNA laddering and sub-G1 DNA content. The effect of TLN-4601 on cell viability were measured using commercial Caspase-3/7 activity and ATP assays.

Results: TLN-4601 caused rapid apoptotic cell death in Jurkat cells as characterized by mitochondrial cytochrome c release, caspase activation, loss of cellular ATP and DNA laddering. Equal sensitivity of the wild-type and caspase 8 knocked-down Jurkat cells to TLN-4601 suggests that the extrinsic apoptotic pathway was not involved in mediating this activity. Interestingly, TLN-4601 exposure resulted in cytochrome c release independently of the apoptosis-inducing proteins Bax and Bak. Furthermore, the over-expression of the anti-apoptotic protein Bcl-2 did not affect the ability of TLN-4601 to induce cell death. Finally, a short exposure of TLN-4601 exhibited a preferential selectivity for triggering apoptosis in Jurkat cells as compared to either quiescent and PHA-L stimulated PBLs.

Conclusions: TLN-4601 mediates rapid cell death via the intrinsic apoptotic pathway independent of Bax, Bak or Bcl-2 expression. Moreover, the differential sensitivity to TLN-4601 observed between Jurkat cells and PBLs highlights the potential for a usable therapeutic margin between cancer and normal cells. Taken together, these properties represent a very promising profile for an anti-cancer agent and suggest that further investigation of TLN-4601 in hematological malignancies is warranted.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C4.
Abstract C42: QBI-139, a human RNase variant in a phase I trial, has broad in vivo efficacy
Authors: Strong Laura E. , Kink John A. , Mei Baigen , Shahan Mark N. , Pensinger Dan , Raines Ronald T.
Published: June 2012

The RNA in cancer cells is the target of a new class of anti-cancer drugs: the EVade™ Ribonucleases (RNases). The EVade™ RNases are variants of native RNases that have been modified to diminish their binding to their natural inhibitor, allowing the EVade™ RNases to kill cancer cells. These RNase variants are broadly efficacious, competitive with approved drugs. A lead candidate, called QBI-139, has been selected for clinical devleopment and is currently in a Phase I clinical trial in patients with solid tumors. The next stage of development requires selection of a specific indication for a Phase II trial of QBI-139. In addition to the Phase I trial, mouse models of cancer will be used to select the tumor type for future development. The efficacy of QBI-139 in in vivo models of human cancer (e.g. lung, colon and prostate) has been examined relative to FDA approved cancer therapies. QBI-139 was tested at different doses in nude mouse xenograft models using a variety of tumor types and multiple cell lines within the different tumor types (e.g. prostate cancer cell lines DU145 and PC3). Each model compared the efficacy of QBI-139 with control groups which included: vehicle, and an effective commercial chemotherapeutic specific for each tumor type (e.g. docetaxel, 5-fluorouracil and cisplatin). The human cancer cells were grown in culture and approximately 5 ×106 cells were implanted subcutaneously into the flanks of (nu/nu) nude mice. The tumors are allowed to grow to ∼75 mm3 prior to treatment and all drugs were administered intraperitoneally (ip) once weekly. Calculated tumor volumes in mm3 and animal body weights (g) were recorded twice weekly. The percent tumor growth inhibition (TGI) used as a measure of efficacy was determined by comparing final and starting tumor volumes of groups treated with drug relative to the vehicle.

A favorable response was seen for QBI-139 against prostate cancer (DU145) compared to docetaxel. The TGI of QBI-139 was 83% compared to 84% for docetaxel. Treatment with QBI-139 did not lead to observable toxicity and the mice all gained body during the course of treatment. In contrast, docetaxel treatment resulted in death for one mouse after 4 treatments (day 42) and another after 6 treatments (day 52). In colon cancer (HT-29), the efficacy of QBI-139 was 58%, similar to the results for 5-fluorouracil (54%). Additional results will be provided, including for multiple cell lines of human non-small cell lung cancer.

QBI-139 is a broadly efficacious agent that provides a new way to attack the RNA in cancer cells and an innovative new tool for oncologists. The results above will be considered with the results of the Phase I trial to select an indication for a Phase II clinical trial.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C42.
Abstract C47: Dual kinase inhibition of EGFR and HER2 can overcome resistance to an EGFR-targeting agent
Authors: Quesnelle Kelly M. , Joyce Sonali C. , Grandis Jennifer R.
Published: June 2012

EGFR and HER2 expression are correlated with poor prognosis in several cancers including bladder cancer. The EGFR-targeting agent cetuximab is currently in phase II clinical trials for the treatment of bladder cancer, but has only been modestly effective as a single agent in most solid tumor types. In colon cancer, lack of cetuximab efficacy has been attributed to compensatory activating mutations in Kras. The incidence of Kras mutations is rare in bladder carcinomas. There is a need for preclinical models with which to study resistance to EGFR-targeting agents. Here, we generated four cetuximab-resistant clones of a cetuximab-sensitive transitional bladder cancer cell line in vivo by exposing cetuximab-sensitive bladder cancer cell line xenografts to slowly increasing concentrations of cetuximab. We verified this resistance in vivo by demonstrating growth of cetuximab-resistant xenografts in the presence of high doses of cetuximab, as well as establishing differential sensitivity to cetuximab between sensitive and resistant xenografts in vivo. We validated the cetuximab-resistant phenotype of these cells in vitro by showing decreased response to cetuximab in an invasion assay. We examined the levels of total and phosphorylated EGFR, HER2, HER3, AKT, MAPK, and the total protein levels of epithelial-to-mesenchymal transition markers E-cadherin and Vimentin in the isogenic cetuximab-sensitive and cetuximab-resistant cells. Increased phosphorylation of the ERBB family members HER2 and HER3 was detected in cetuximab-resistant clones compared with their cetuximab-sensitive parental cell lines. Treatment of the cetuximab-resistant cells with an irreversible dual kinase inhibitor targeting EGFR and HER2 (BIBW2992), successfully inhibited their growth. These data suggest that activation of HER2 and HER3 may contribute to cetuximab resistance where targeting these other HER family members may enhance the efficacy of EGFR blockade.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C47.