bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A206: Clinically relevant perspectives of the clonal nature of cancer
Authors: Barrett Michael T. , Savage Stephanie , Lenkiewicz Elizabeth , Kiefer Jeff , Chow Donald , Yin Holly , Ruiz Christian , Bubendorf Lukas , Mousses Spyro , Demeure Michael J. , Von Hoff Daniel
Published: June 2012

It has been hypothesized and subsequently shown that tumors arise as a result of an acquired genomic instability and the subsequent evolution of clonal populations of neoplastic cells. Thus the pattern and behavior of distinct genomically aberrant clones may underlie multiple clinical phenotypes in a variety of cancers. A fundamental hypothesis of cancer genome studies is that genes and cellular networks targeted by selected aberrations represent vulnerabilities that can be exploited for effective personalized therapies. However, the heterogeneity of neoplastic cells and the presence of admixtures of genomically normal cells make it difficult to comprehensively define cancer genomes in patients in vivo. By applying single parameter and multi parameter DNA content based flow cytometry of nuclei to isolate clonal populations from solid tumor biopsies, and coupling this strategy with array CGH, we obtained high definition genomic profiles of clonal populations from pancreatic adenocarcinomas (PA), adrenal cortical carcinomas (ACC), and prostate carcinomas (PC). The genomes of clonal populations in these disparate cancers have distinct patient-specific sets of aberrations with clinical relevance. For example, our clonal analyses detected unique gene-specific homozygous deletions converging on NOTCH signaling and multiple focal amplicons that target cell migration in PA, distinguished divergent clones within single biopsies, and identified genomic aberrations specific to therapeutically sensitive and resistant clones arising during the evolution of androgen independent metastatic PC. Application of these technologies in combination with next generation sequencing allows for elucidation of the unique clonal identities of cancer genomes in patients in vivo, which in turn can help guide diagnoses and tailor approaches to treatment.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A206.
Abstract A228: Noninvasive magnetic resonance spectroscopic PD markers of a minor-groove interstrand cross-linking agent (BN2629) in human colon carcinoma and melanoma xenografts
Authors: Chung Yuen-Li , Troy Helen , Stubbs Marion , Thurston David E. , Judson Ian R. , Leach Martin O. , Griffiths John R.
Published: June 2012

BN2629 (also known as SJG-136) is a novel pyrrolobenzodiazepine dimer analogue that binds covalently in the minor groove of DNA, inducing little or no distortion of the double helix. Hence, it evades the p53-mediated detection and up-regulation of nucleotide excision repair mechanisms but induces apoptosis. It has just completed Phase 1 clinical trials in the USA (NCI) and UK (CR-UK), and has been approved by the NCI for Phase II evaluation.

The aim of this work was to develop a robust and non-invasive surrogate marker for tumor response following BN2629 treatment. We carried out an in vivo 31phosphorus magnetic resonance spectroscopy (31P MRS) study of BN2629 (80 g/kg via ip for 3 days) and vehicle in HT29 human colon carcinoma and LOX IMVI melanoma xenograft models. In vitro 31P MRS were performed on tumor extracts.

Significant growth delays (p<0.0001) were observed in both BN2629-treated HT29 and LOX IMVI xenografts when compared with controls. In vivo31P MRS of the HT29 xenografts showed an increase in the phosphomonoester/total phosphorus signals (PME/TotP) (p=0.04) and PME/ - NTP ratios (p<0.02) after 3 days of BN2629 treatment. No significant changes were observed in the control group. In vitro 31P MRS of extracts from BN2629-treated HT29 tumors showed significant increases in phosphoethanolamine (PE; p<0.01), glycerophosphocholine (GPC; p<0.01) and glycerophosphoethanolamine (GPE; p<0.03) when compared with controls.

In vivo 31P MRS of the LOX IMVI xenografts showed an increase in - NTP/TotP ratio ( -NTP/TotP) (p<0.01) after 3 days of BN2629 treatment. No significant changes were observed in the control group. In vitro 31P MRS of extracts from BN2629-treated LOX IMVI tumors showed significant increases in GPC (p<0.001), GPE (p<0.01) and ATP (p=0.04) when compared with controls.

Treatment with BN2629 resulted in tumor growth delay and altered bioenergetic and phospholipid metabolism in vivo, which are consistent with metabolic changes observed following treatment with agents such as 5-FU [1] and cyclophosphamide [2]. The phospholipid changes may be associated with alteration in membrane turnover following treatment, and these changes may have potential as surrogate non-invasive markers for determining tumor response following treatment with BN2629.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A228.
Abstract A238: The effects of foretinib on MET phosphorylation, gene expression, cell proliferation and tumor growth in MET-amplified tumor cells in vitro and in vivo
Authors: Liu Li , Liu Yuan , Hong Shi , Greger James , Martin Anne-Marie , Gilmer Tona
Published: June 2012

The MET receptor tyrosine kinase (RTK) is frequently deregulated in many types of cancer and is a promising target for cancer drug development. Foretinib is a multikinase inhibitor with potent activity against MET and is currently in phase II clinical trials. In these studies, we determined the anti-tumor activity of foretinib in MET-amplified tumor cells and the MKN45 gastric xenograft tumor model in nude mice. We also measured the effect of foretinib on MET phosphorylation by immunoprecipitation and western blot and evaluated gene expression by Affymetrix microarrays in these models. In vitro foretinib demonstrated the most potent cell growth inhibitory activity in MET-amplified tumor cell lines MKN45 (IC50 = 9 ± 2 nM), HS746T (IC50 = 8 ± 2 nM), SNU5 (IC50 = 2 ± 0.2 nM) and NCI-H1993 (IC50 = 69 ± 5 nM). Foretinib inhibited the phosphorylation of MET, suppressed the phosphorylation of AKT and ERK, and reduced the expression of cyclin D1 in a dose dependent manner in these cells. Furthermore, foretinib given orally showed durable and near complete inhibition of MET phosphorylation (>90%) and dose dependent anti-tumor activity against MKN45 MET-amplified gastric xenograft tumors in nude mice. Tumor stasis and significant tumor growth delay were observed for foretinib dosed at 10 mg/kg and PF-02341066, a MET selective inhibitor, dosed at 50 mg/kg in the same study. In addition, foretinib or PF-02341066 showed similar gene alterations on both MKN45 cells and xenograft tumors. These genes are involved in RTK cross-talk and MET signaling pathways (e.g., HER2, HER3, MET, PIK3IP1, DUSP4, DUSP6), cell motility (e.g., CDH1, TIMP2, CLDN3, MMP1), cell proliferation (e.g., CCND1, TP53INP1, TYMS, CCNB1) and apoptosis (e.g., TNFSF10, CASP8). These results demonstrate that foretinib is a potent MET inhibitor with associated changes in gene expression which correlate with anti-tumor activity in MET-amplified tumor cells both in vitro and in vivo. This gene signature warrants further investigation for its relevance as a MET-inhibitor mediated pharmacodynamic marker.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A238.
Abstract A239: MPS1 (TTK) kinase inhibition selectively kills aneuploid cells
Authors: Rawjee Teeara , Rowlands Martin , McAndrew Craig , Stubbs Mark , Lord Christopher J. , Reis-Filho Jorge S. , Ashworth Alan , Aherne Wynne , Workman Paul , Blagg Julian , Linardopoulos Spyridon
Published: June 2012

Monopolar spindle 1 (MPS1, also known as TTK) is a dual-specificity, cell cycle-regulated kinase required for the establishment and maintenance of the spindle assembly checkpoint during mitosis. Meta-analysis on expression array datasets showed increased MPS1 expression strongly correlated with time to relapse from breast cancer and this was significantly associated with breast tumors of a basal-like phenotype. However, the issue of whether inhibition of MPS1 in human tumor cells compromises their viability has not yet been fully addressed. We show here, using an RNA interference (RNAi) approach, that inhibition of MPS1 can indeed reduce the viability of human tumor cells and that this effect is selective; aneuploid tumor cell lines are sensitive to MPS1 inhibition whereas non-cancerous cells remained resistant. To identify chemical inhibitors of MPS1, a high-throughput screen of a library of small molecules using the Homogeneous Time-Resolved Fluorescence (HTRF) assay was performed. This strategy successfully identified a hit compound that selectively reduces viability in human breast tumor cell lines. These results further validate MPS1 as a therapeutic target and identify a proof-of-principle small molecule inhibitor.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A239.
Abstract A243: SCH727965, a novel potent cyclin dependent kinase inhibitor, inhibits pancreatic tumor growth and Ras-mediated tumorigenic signaling
Authors: Mishra Anjali , Feldmann Georg , Garrido-Laguna Ignacio , Dadon Tikva , Strock Christopher J. , Bannerji Rajat , Hidalgo Manuel , Maitra Anirban , Nelkin Barry D.
Published: June 2012

We have previously shown activation of cyclin-dependent kinase 5 (CDK5), a kinase associated with neuronal migration, in human pancreatic cancer. Abrogation of CDK5 function using a dominant-negative protein, RNA interference, or pharmacological disruption significantly mitigated invasion, migration and anchorage independent growth in vitro, and orthotopic tumor formation and systemic metastases in vivo. CDK5 blockade resulted in inhibition of RalA, a key effector pathway downstream of oncogenic Ras, central to tumorigenesis in pancreatic cancer. Restitution of Ral function rescued the effects of CDK5 inhibition in pancreatic cancer cells. Thus, CDK5 is important for Ras signaling in pancreatic cancer, suggesting that CDK5 inhibition may be an effective therapeutic strategy in pancreatic cancer. To begin to explore the therapeutic potential of CDK5 inhibitors in pancreatic cancer, we have examined the effect of the semi-selective CDK5 inhibitor SCH727965 (Schering-Plough). SCH727965 is a potent inhibitor of CDK1, CDK2, CDK5 and CDK9, with in vitro IC50 values of 3, 1, 1 and 4 nM, respectively. In this study, we evaluated the effect of SCH727965 on pancreatic tumor growth and migration. MTT assay results demonstrated that the IC50 for 2 pancreatic tumor cell lines, MiaPaCa-2 and Panc1.98, was 12nM and 24nM respectively. Migration and anchorage independent growth were significantly more sensitive to SCH727965. Thus, Boyden chamber assay, wound-healing assay and soft agar assays indicated 50% inhibition of migration and soft agar growth inhibition at 5nM, and complete inhibition at 10nM drug concentration — concentrations at which the effects on anchorage dependent growth were relatively minor. Tumors from low passage pancreatic tumor xenografts, or from pancreatic cell line orthotopic xenografts, showed 40–75% reduction of tumor growth after treatment with SCH727965, with significant reduction of metastases. SCH727965 treatment resulted in inhibition of RalA activation. Ral activity could be restored by enforced RalA activation, via expression of Rgl2-CAAX, a constitutively active form of a RalGEF (Ral guanine exchange factor) gene.

Our results show that SCH727965 is very effective against pancreatic cancer growth and metastasis. These results also suggest that SCH727965 blocks pancreatic tumor growth, migration and metastasis, at least in part by inhibition of dysregulated Ras signaling, by inhibiting CDK5.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A243.
Abstract A248: Substituted 4-amino-1H-pyrazolo[3,4-d]pyrimidines as multitargeted inhibitors targeting insulin-like growth factor-1 receptor (IGF1R) and members of ErbB-family receptor tyrosine kinases
Authors: Wang Gary T. , Mantei Robert A. , Hubbard Robert D. , Wilsbacher Julie L. , Zhang Qian , Tucker Lora , Hu Xiaoming , Kovar Peter , Johson Erick F. , Osterling Donald J. , Bouska Jennifer , Wang Jieyi , Bell Randy L. , Davidsen Steven K. , Sheppard George S.
Published: June 2012

Insulin-like growth factor-1 receptor (IGF1R) and members of ErbB family (eg. EGFR, ErbB2) are receptor tyrosine kianses that play significant roles in cancer and have been pursued as drug targets for cancer therapy. There is substantial evidence for the existence of reciprocal cross-talk between IGF1R axis and ErbB axis of cellular signaling. Acquired resistance to EGFR inhibitors gefitinib and erlotinib or ErbB2 inhibitor trastuzumab has been linked to activation of IGF1R signaling through induced overexpression of IGF1R or loss of IGF binding- proteins. Additionally, synergy between IGF1R inhibition and ErbB inhibition has been demonstrated in cancer cell growth inhibition and in xenograft models. Thus, molecules targeting both IGF1R and ErbB-family might have superior anti-tumor efficacy. This poster describes the lead discovery, optimization and biological characterization of a series of substituted 4-amino-1H-pyrazolo[3,4-d]pyrimidines as potent inhibitors of IGF1R, EGFR and ErbB2. The leading compound, A-923573, showed IGF1R IC50 of 12 nM, EGFR (L858R) IC50 of 31 nM and ErbB2 IC50 of 11 nM, potent activity in cellular functional assays and anti-proliferation assays, as well as in an in vivo pharmacodynamic assay.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A248.
Abstract A25: Establishment and characterization of primary human Biomerk Tumorgraft™ Models: Application to oncology drug development
Authors: Strawn Steven J. , Wick Michael , Nieves Francis , Sidransky David , Bruckheimer Elizabeth M.
Published: June 2012

One of the key challenges facing oncology drug development is the high attrition rates of compounds that enter the drug development pipeline, where very few achieve successful approval and marketing. Champions Biotechnology, in an effort to enhance the value of preclinical compounds and accelerate oncology drug development, has developed a novel preclinical platform derived from Biomerk Tumorgraft™ models; an innovative approach that utilizes the implantation of primary human tumors in immune-deficient mice in a manner that preserves the biological properties of the original human tumor. Biomerk Tumorgrafts™ differ from traditional xenograft models in that they are not passaged in tissue culture, and are instead exclusively passaged in vivo. Additionally, the Biomerk Tumorgraft™ models: (a) maintain the fundamental genotypic features of the original cancer, (b) represent the genetic heterogeneity of the cancer, and (c) do not change over several passages. Champions has established a bank of over 200 Tumorgraft models encompassing all major solid tumor types, all of which have been extensively characterized. Here we describe a panel of NSCLC lung, colon, and pancreatic tumorgraft models including gene expression profiling, mutational analyses and responses to standard of care agents. Of the NSCLC lung models, 6/8 demonstrated resistance to Erlotinib with 2/8 demonstrating an intermediate response, 2/6 also possessed a mutant K-ras or a mutant EGFR. Similarly, 3/6 K-ras mutant colon Tumorgraft models demonstrated sensitivity to irinotecan. In the case of the pancreatic models, 7/8 showed resistance to Erlotinib with the remaining model showing only a modest response. Through Champions' Personalized Oncology Services, we have also shown a high correlation between the Tumorgraft response and the clinical response. Together, these results demonstrate that each model differs with respect to its genetic composition and response to standard agents, and thus represents the efficacy of a compound on an individual patient. Given the diversity of models, the maintenance of the genomic and phenotypic characteristics of the original patient tumor, and the correlation between Tumorgraft and clinical responses, Champions Biomerk Tumorgraft™ models represent a novel preclinical in vivo platform capable of optimizing drug regimens for cancer patients and predicting the clinical effectiveness of preclinical drug candidates. Overall, the application of Biomerk Tumorgraft™ models has the potential to accelerate and enhance oncology drug development.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A25.
Abstract A253: Acquired Bcr-Abl kinase domain mutations are not responsible for persistence of dasatinib-refractory disease in murine Ph+ ALL
Authors: Boulos Nidal , Calabrese Christopher R. , Sherr Charles J. , Williams Richard T.
Published: June 2012

Despite the dramatic efficacies of imatinib and second-generation BCR-ABL kinase inhibitors (dasatinib and nilotinib) in chronic myelogenous leukemia (CML), these drugs fail to provide durable therapeutic benefit in patients with Philadelphia-chromosome positive (Ph+) BCR-ABL-induced acute lymphoblastic leukemias (ALLs). In contrast to chronic-phase CML, deletion of the CDKN2A (INK4A-ARF) tumor suppressor locus occurs in 65% of Ph+ ALL cases. Accordingly, introduction of cultured Arf−/− p185BCR-ABL-expressing (p185+) pre-B cells, but not their Arf+/+ counterparts, into healthy syngeneic recipient mice induces fulminant B cell leukemia; virtually every p185+, Arf−/− cell is a leukemia-initiating cell (LIC). We have now monitored disease progression and dasatinib-responsiveness in vivo by following the fate of p185+ Arf−/− luciferase-expressing LICs. In animals bearing high leukemic burdens (simulating the human clinical condition at diagnosis), two weeks of dasatinib therapy induced dramatic reductions in luminescent signals, but all animals harbored persistent, measurable deposits of drug-refractory cells. The vast majority of BCR-ABL alleles from these residual cells were free of kinase domain (KD) mutations, but rare leukemic clones harboring the T315I KD mutation, known to confer near-complete drug resistance to imatinib, dasatinib and nilotinib, were detectable in a subset of recipients. Following several weeks of continued dasatinib therapy, all mice developed clinical relapse preceded by dramatic increases in luminescent signals, both in the hematopoietic compartment and central nervous system. Many of these drug-resistant leukemic cells now harbored the T315I KD mutation. Animals that had been maintained in remission with 4 weeks of continuous dasatinib therapy quickly relapsed upon therapy discontinuation, almost always without evidence of KD mutations. In this clinically-relevant Ph+ ALL model, several factors including Arf loss-of-function, disease burden, intensity of therapy, and length of drug exposure interact to determine therapeutic outcome and to trigger confluent mechanisms of drug resistance. Critically, while continuous dasatinib therapy efficiently selects for and maintains cells harboring drug-resistant KD mutations that mediate eventual clinical relapse, most drug-refractory leukemic cells survive in hematopoietic tissues in the absence of KD mutations during maintenance therapy, implying that mutation-independent factors sustain LIC survival. We propose that Arf inactivation in Ph+ ALL (but not in CML) enhances the biological ‘fitness’ of leukemic cells and diminishes the efficiency with which targeted therapy can successfully eradicate drug-refractory disease. This facilitates the subsequent emergence of cell-intrinsic drug resistance, most frequently manifested as BCR-ABL KD mutations.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A253.
Abstract A255: Activity of VEGFR inhibitor tivozanib as a single agent or in combination with EGFR inhibitor erlotinib in engineered lung adenocarcinoma models
Authors: Sun Xiaojian , Bressel Angela , Rideout William M. , Zhou Yinghui , Heyer Joerg , Chiu Isabel , Robinson Murray O. , Lin Jie
Published: June 2012

Tivozanib (AV-951 or KRN951) is an ATP competitive small molecule VEGFR antagonist that exhibits picomolar inhibitory activity against all three VEGF receptors. It has demonstrated robust clinical activity in renal cell carcinoma and is currently being evaluated in multiple clinical studies including a monotherapy phase 1b/2a trial in NSCLC. NSCLC is a highly heterogeneous disease, in which tumor histology and genetics have defined sensitivities and settings to targeted therapeutics. To model and explore potential clinical settings, we created genetically engineered lung adenocarcinoma models driven by EGFRL858R, EGFRL858R/T790M or KRAS G12V, propagated these models in vivo both subcutaneously and orthotopically and evaluated tumor inhibitory activity of tivozanib as a single agent or in combination with EGFR inhibitor erlotinib. To further explore activity of metastatic lesions to the lung, we also evaluated the activity of tivozanib using an engineered breast tumor metastatic model.

Treatment of subcutaneous lung KRAS tumors or lung EGFRL858R/T790M tumors with tivozanib alone resulted in complete tumor growth inhibition. Histological analysis revealed substantial central necrosis with proliferating (Ki67+) tumor cells present only at the tumor periphery, typical of an anti-angiogenesis mechanism. When tivozanib was given to treat lung EGFRL858R+T790M tumors in an orthotopic setting in the lungs after tumors were well established, significant antiangiogenic and antitumor effects, as demonstrated by histology and extended survival period, were observed. However, the necrotic lung lesions showed no sign of resorption or elimination after 6 weeks of treatment. Similar results were also observed in a HER2 driven breast tumor lung metastatic model. This prompted us to further explore the activity of tivozanib in an orthotopic lung tumor minimal disease model. Tivozanib treatment in this setting did not alter tumor lesion numbers but effectively blocked the progression of micro pre-angiogenic lesions. Finally, we explored the combined activity of tivozanib and erlotinib in a model constructed to reflect the emergence of erlotinib resistance by implanting tumor material comprising both EGFRL858R and EGFRL858R/T790M tumor cells. In this setting, the combination of tivozanib and erlotinib resulted in significantly greater control of tumor progression than erlotinib alone.

Our results suggest that tivozanib may have activity as a single agent in lung tumors with KRAS or EGFR L858R/T790M mutations and that combinations with other targeted lung cancer drugs such as erlotinib may provide meaningful clinical benefit.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A255.
Abstract A38: Members of the noncanonical WNT pathway confer resistance to the MEK 1/2 inhibitor AZD6244 in colorectal cancer cell lines
Authors: Tentler John J. , Nallapareddy Sujatha , Tan Aik Choon , Spreafico Anna , Flanigan Sara A. , Pitts Todd M. , Eckhardt S. Gail
Published: June 2012

Background: The ERK pathway is constitutively activated in several human cancers including CRC. The MAPK/ERK kinase, MEK, occupies a central role in the ERK pathway and therefore, MEK inhibitors constitute a promising class of novel targeted cancer therapies. AZD6244 (ARRY-142886) is a potent, selective MEK1/2 inhibitor with nanomolar potency against cell lines and activity in several in vivo models of CRC. Despite initial enthusiasm for this class of agents in tumors with a high incidence of RAS or BRAF mutations, clinical studies indicate that resistance to MEK inhibitors remains a problem, and therefore, improved patient selection strategies are needed. The goal of this study was to identify and target predictive markers of resistance to AZD6244 in order to develop a rational basis for patient selection and combination therapy in CRC.

Methods and Results: In order to identify predictive markers of responsiveness to AZD6244, 27 CRC cell lines were exposed to the agent, of which five highly sensitive (S) or resistant (R) cell lines were selected with IC50 values of <0.1 and >1.0 µM, respectively, and subjected to Affymetrix U133 plus 2.0 gene array analysis. ANOVA comparison of gene expression profiles between S and R cell lines revealed over 100 differentially expressed genes with a global p-value of <0.04, whereas 64 transcripts met the p<0.001 criteria. Of note, two genes, frizzled homolog 2 (FZD2) and wingless integrated 5B (WNT5B) were overrepresented in AZD6244 R cell lines by 17 and 7.5-fold, versus the S cells, respectively. These two genes comprise a receptor-ligand pair that has been shown to signal via the non-canonical, β-catenin-independent WNT signaling pathway. To determine the functional significance of over-expression of the non-canonical WNT pathway in resistance to AZD6244, we silenced FZD2 or WNT5B expression with stably transfected shRNAs and assessed the responsiveness of SW480 R cells to AZD6244. We observed a greater than 50% reduction in the IC50 to AZD6244 in the FZD2 knock-down cells and greater than 65% reduction in the WNT5B knock-down compared to wild-type and scrambled shRNA controls. Finally, we tested the effects of combining cyclosporine A (CsA) and tacrolimus, inhibitors of the non-canonical WNT pathway, with AZD6244. We observed synergistic inhibition of proliferation of SW480 cells treated with the combination of either inhibitor with AZD6244.

Conclusions: Members of the non-canonical WNT pathway confer functional resistance to AZD6244 and thereby may represent not only predictive biomarkers of responsiveness to AZD6244, but also the basis for rational combination partners. These biomarkers and combination strategies are currently being validated in vivo with cell line and patient-derived CRC xenograft models.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A38.
Abstract A43: Detection of tumor-associated KRAS mutations in the plasma and tumor tissue of colorectal cancer patients
Authors: Jeffers Michael , Pena Carol , Henderson David , Wilhelm Scott , Christensen Olaf , Lathia Chetan
Published: June 2012

The mutational evaluation of tumor-derived genetic material is becoming increasingly important in the era of personalized medicine. DNA harvested from a variety of sources (e.g., tumor biopsies, plasma, serum, urine and stool) may be useful to determine the mutational status of a tumor. In addition, a variety of methods for performing mutational analyses are available. In the present study, DNA derived from tumor biopsies and plasma was evaluated for KRAS mutational status by two different methodologies: Sequenom and BEAMing. In an initial experiment, KRAS mutations were identified in 36/64 (56%) of colorectal tumors analyzed by the Sequenom method. Matched plasma samples corresponding to a subset of the patients with KRAS-mutant tumors (n = 18) were subsequently evaluated for KRAS mutational status by Sequenom and BEAMing methodologies. Each of these methods detected the expected tumor-associated KRAS mutation in approximately 70% of the plasma samples analyzed. An additional set of 38 plasma samples from late stage metastatic colorectal cancer patients was then evaluated by both Sequenom and BEAMing, which lead to the identification of KRAS mutations in 9/38 (24%) and 19/38 (50%) of the samples analyzed by the respective technologies. Biopsies from 8 of these 38 patients were available and evaluated for KRAS mutational status by Sequenom. In one case, a KRAS mutation was found in the tumor that had not been detected in the corresponding plasma sample, and in another case a KRAS mutation that had been detected in a plasma sample was not detected in the corresponding tumor. This study indicates that the Sequenom method can be successfully used to determine the KRAS mutational status in both tumor biopsies and plasma. The BEAMing method was also successfully used to determine KRAS mutational status in plasma, and appears to be more sensitive than the Sequenom method. Since the frequency of KRAS mutation detected in the plasma of 38 metastatic colorectal cancer patients using BEAMing (i.e., 50%) is similar to that reported in the literature for this patient population when using tumor biopsies for mutational analysis, the assessment of tumor-associated mutations in plasma may represent a viable option in this patient population. However, since the KRAS mutational status in plasma and tumor do not always correspond, data obtained from these two sources should be independently evaluated in the clinical setting.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A43.
Abstract A65: MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT
Authors: Sun Hong , Zhang Zhan , Grandori Carla
Published: June 2012

Intratumoral hypoxia is well known as a hallmark of most solid tumors. Here, we studied expression level of about 200 microRNAs in a panel of cancer cell lines from different tissues, and identified a microRNA miR210, its expression was prominently up-regulated by hypoxia. Over expression of miR210 in cancer cell lines partially reversed the hypoxic gene expression signature and allowed cells to escape from hypoxia-induced cell-cycle arrest. We then identified MNT, a known MYC antagonist, as a key miR-210 target. miR-210 directly down-regulated MNT transcript and protein via recognition sites in the transcript 3′ UTR. Consistent with a role for MNT as a miR-210 target, RNA interference-mediated knockdown of MNT phenocopied miR-210 over expression cell cycle effect. Furthermore, loss of MYC abolished the ability of miR-210 to override hypoxia-induced cell-cycle arrest. Thus, miR-210 could function as a modulator in cancer cells in response to hypoxic conditions through Myc network.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A65.
Abstract A66: Potent CDK4 inhibitor in human breast cancer therapy
Authors: Sun Yuan
Published: June 2012

Deregulated cell cycle is a common feature in many human cancers. Overexpression of cyclinD1 oncogene and highly activation of the cyclinD1/Cdk4Cdk6 kinase modulate the tumor initiation, tumor progression and the chemo-sensitivity of cancer cells. Targeting G1 phase of cell cycle leading cancer cell into senescence or quiescence stage or causing cell death has become a promising approach in cancer treatment. Here, we report the in vitro anti-tumor functions of the synthetic Cdk4 inhibitor, Naphtho [2, 1-α] pyrrolo [3, 4-c] carbazole-5, 7 (6H, 12H)-dione (NPCD) in a panel of human breast cancer cell lines. NPCD is able to inhibit cancer cell growth, arrest cancer cells at G1 phase and cause cell death as results of diminished Cdk4Cdk6 phosphorylation of the Rb protein without disrupting the cyclinD1/Cdk4Cdk6 complex formation or affecting G1 phase proteins intracellular localization. The sensitivity to NPCD is associated with the intracellular cyclinD1 and Cdk4 or Cdk6 proteins level. Upon NPCD treatment, the changes in several G1-S phase proteins levels differ between cell lines which could because different cell lines develop different signaling networks responding to same stimuli. The dramatically decreased p27kip protein is a common feature between cell lines we studied upon NPCD treatment; it could be either a result of NPCD induced G1 arrest and inactivation of cyclinD1/Cdk4Cdk6 or could be an upstream mediator to cause Cdk4Cdk6 inactivation and G1 arrest. In recent years, the functions of p27kip protein have been extended involving in cell cycle regulation, embryonic development, tumor genesis and tumor progression, consequently, pursuing the mechanisms resulting rapidly decreased p27kip protein in responding to NPCD may help us to better understand the function of this protein in deregulated cell cycle system.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A66.
Abstract A89: Synthesis and structure-activity relationship of 3-O-acylated (−)-epigallocatechins as 5α-reductase inhibitors
Authors: Huang Jiann-Jyh , Lee Ying-Shuan , Hiipakka Richard A. , Liao Shutsung
Published: June 2012

A series of 3-O-acylated (−)-epigallocatechins were synthesized and their inhibition of steroid 5α-reductase was studied. They were prepared by the reaction of EGCG with tert-butyldimethylsilyl chloride followed by reductive cleavage of the ester bond. The resultant (−)-epigallocatechins penta-O-tert-butyldimethylsilyl ether was esterified with different fatty acids using DCC and DMAP and then desilylated with aqueous HF to provide the corresponding products. For 3-O-acylated (−)-epigallocatechins with saturated fatty acids, the 5α-reductase inhibitory activity increased with the increasing numbers of carbon atoms at the C3-O position, reaching maximum for 16 carbon atoms (compound 4h, with palmitic acid) with an IC50 of 0.53 µM, which was ∼12-fold more potent than EGCG (IC50 = 6.29 M). Introduction of monounsaturated fatty acid to the C3-O position of (−)-epigallocatechins enhanced the activity, providing the most potent compound 6 (IC50 = 0.48 µM, with palmitoleic acid), which showed moderate anti-tumor activity in a PC-3 prostate cancer xenograft model. Replacement of the ester moiety at C3-O with carbamate, or permethylation or peracylation of hydroxyl groups diminished 5α-reductase inhibitory activity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A89.
Abstract A94: Preventive effects of naproxen and NO-naproxen in carcinogen induced models of urinary bladder, colon, and mammary cancers
Authors: Grubbs Clinton J. , Steele Vernon , Boring Daniel , Lubet Ronald , Rao Chinthalapally , Zhang Yuting , Patlolla Jagan
Published: June 2012

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been highly effective in preventing colon, urinary bladder, and skin cancer preclinically; and also in clinical trials of colon adenoma formation. However, certain NSAIDs cause gastrointestinal (GI) ulceration and may increase cardiovascular (CV) events. Naproxen appears to cause the lowest CV events of the common NSAIDs other than aspirin. NO-naproxen was tested based on the finding that adding a nitric oxide (NO) group to NSAIDs may help alleviate GI toxicity as well as the expectation that the production of NO itself might be therapeutically effective. When examined in the hydroxybutyl(butyl)nitrosamine (OH-BBN) rat urinary bladder cancer model, naproxen at doses of either 400 or 200 ppm in the diet greatly decreased the development of large invasive bladder cancers when treatment was initiated shortly after the last OH-BBN dosing. In a second study, naproxen at 133 ppm or NO-naproxen at 183 or 550 ppm caused striking decreases in the incidence of large tumors. Surprisingly, if treatments were started three months after the last OH-BBN (when multiple microscopic lesions already exist) both NO-naproxen (550 ppm) and naproxen (400 ppm) were still highly effective (91–96% decreases); implying that most of their effects are late. In the azoxymethane (AOM)-induced rat colon aberrant crypt foci (ACF) model, naproxen administered at 200 and 400 ppm reduced mean ACFs in the colon by approximatley 45–60%. NO-naproxen administered at roughly equimolar doses (300 and 600 ppm), reduced total ACF by 20–40%, respectively. In contrast, in the methylnitrosourea (MNU)-induced ER+ mammary cancer model in rats (where most NSAIDs are relatively ineffective) neither NO-naproxen nor naproxen showed significant inhibition at doses of 550 and 400 ppm. These data show that both naproxen and NO-naproxen are effective agents against urinary bladder and colon (but not mammary) carcinogenesis. Additional data showing the effects of sulindac and NO-sulindac in the urinary bladder model will also be presented. Data also imply that both naproxen and NO-naproxen are effective, but that the NO-derivative does not appear to be more effective.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A94.
Abstract B102: Zibotentan (ZD4054), a specific antagonist of the endothelin A receptor, suppresses the metastatic phenotype of bladder cancer cells in vivo
Authors: Thomas Christopher Y. , Dunlap-Brown Marya , Brautigan David L. , Theodorescu Dan
Published: June 2012

There is mounting evidence that endothelin-1(ET-1) / endothelin receptor axis contributes to the pathogenesis of urothelial bladder cancer. In a previous study, reduced expression of the metastasis suppressor protein RhoGDI2 in bladder cancer cell lines and tumors was associated with increased expression of ET-1 (Titus B et al, Ca Res 65:7320, 2005). This suggests that ET-1 acts down-stream of repressed RhoGDI2 expression to promote the metastatic phenotype. If so, then blocking activation of endothelin receptor A (ETAR) by ET-1 would suppress this phenotype. To test this hypothesis, Zibotentan, a specific inhibitor of ETAR (IC50 = 21 nM), was evaluated for anti-tumor activity in a xenograft metastasis model. To screen for toxicity, 40 athymic nu/nu female received 12.5 to 50 mg/kg/d of zibotentan by gastric lavage for 97 days. The drug was well tolerated at all dose levels and produced no significant changes in body weight, behavior, or general physical condition. In a separate experiment, T24T bladder cancer cells (106 cells suspended in 0.1 mL of medium) were inoculated into the tail veins of a second group of 40 mice. One week later, 20 of animals were treated with zibotentan 50 mg/Kg/d and 20 controls received vehicle only. After 90 days, the animals were euthanized and the lungs were removed, fixed with Bouin's solution, stained, and examined by microscopy to determine the number and size of metastasis (large ≥3mm, small <3mm). Metastases were found in 12/20 (60%) controls and 3/20 (15%) of treated mice (p=0.024); overall, 89 metastases were identified in the controls and 19 in the treated animals (p=0.012). Although the drug-treated mice had fewer metastases, 13/19 (68%) of these were classified as large tumors as compared to 20/89 (22%) in the control group. These results suggest that inhibition of ETAR signaling by zibotentan reduced the frequency and number of metastases, most likely by suppressing metastatic colonization rather than the growth of established metastases. The latter is consistent with earlier data that inhibition of ETAR does not block the proliferation of T24T cells in vitro. Taken together, these and the previous observations provide a rationale for clinical trials to determine if treatment with zibotentan will prevent or delay the development of metastatic disease in patients with clinically localized but high-risk bladder cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B102.
Abstract B11: Preclinical and preliminary phase 1 trial results of JI-101: A novel, oral tyrosine kinase inhibitor that selectively targets VEGFR2, EphB4, and PDGFR
Authors: Velleca Mark , Brittelli David , Elkin Lisa , Klein Udo , Mitchell Scott , Qian Dapeng , Qian Xiaobing , Srinivasa Sreesha , Stephan Thomas , Govindarajan Raman , Stern Warren , Gordon Michael , Sharma Sunil , Miller Linda
Published: June 2012

Introduction: JI-101 is a highly selective and potent angiogenesis inhibitor with unique EphB4 activity that distinguishes it from other agents in clinical development. We report the preclinical pharmacology and early clinical experience with this compound.

Procedures: Lead optimization was used to identify cgi1842 (now JI-101) with a targeted activity profile that is selective and unique. JI-101 was tested in binding, enzymatic, and cell-based assays for activity against kinase and non-kinase targets. Preclinical in vivo testing for PK, PD, efficacy, and safety pharmacology was performed in mouse, rat, guinea pig, and dog. A Phase I clinical trial is underway: 9 patients, 3/cohort, are being treated with 28-day oral daily dosing at 100 mg, 200 mg, and 400 mg. PK, PD, and imaging analyses are being performed.

Data: JI-101 has a MW of 466D and high (<100 nm) potency against VEGFR2, EphB4, and PDGFR in enzymatic and cell-based assays. Broad kinase cross-screening (Ambit KinomeScan, 445 kinases) reveals that JI-101 is highly selective for angiogenic kinases with only 23 kinases demonstrating a Kd 15). JI-101 was co-administered with paclitaxel in a MDA-MB-231 mouse xenotransplant model, resulting in greater efficacy and no increased toxicity. Based on 28-day GLP toxicology studies conducted in rat and dog, a 100 mg qd starting dose (2 × 50 mg capsules) was chosen for Phase I studies. JI-101 appears to be very well-tolerated to date with no dose limiting toxicities. Patients have received 1–8 cycles and 5 remain on study at 100 mg (1 pt), 200 mg (1 pt), and 400 mg (3 pt). Manageable hypertension has been seen in all cohorts indicating a possible PD effect. Further clinical details of the PK, PD, tolerability and efficacy of JI-101 will be presented.

Conclusions: JI-101 potently inhibits 3 critical angiogenic kinases (VEGFR2, EphB4, and PDGFR) and is the only such “triple” angiogenesis inhibitor currently in development. JI-101 shows robust preclinical and clinical pharmacology and is well-tolerated to date in human trials. In tumors where EphB4 may play an oncogenic role, such as head and neck cancer, JI-101 may have anti-proliferative effects in addition to anti-angiogenic effects and should be amenable to combination therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B11.
Abstract B13: Identification of a metalloprotease-chemokine signaling system in the ovarian cancer microenvironment: Implications for antiangiogenic therapy
Authors: Agarwal Anika , Tressel Sarah , Kaimal Rajani , Bala Marianthi , Iam Francis , Covic Lidija , Kuliopulos Athan
Published: June 2012

Ovarian cancer is a lethal gynecologic malignancy with poor survival, hence it would benefit from therapies that block key signaling pathways involved in tumor-stromal interactions and tumor vascularity. We previously showed that Protease Activated Receptor 1 (PAR1) is an important regulator of angiogenesis, ascites and metastasis in peritoneal mouse models of ovarian cancer. In this study we tested the hypothesis that matrix metalloprotease1 (MMP-1) activates PAR1 which stimulates angiogenesis and tumorigenesis in ovarian cancer through its paracrine control of the endothelial CXCR1/2 receptors. We demonstrated that MMP-1-PAR1 stimulation leads to a 2–5 fold increase in several angiogenic factors including IL-8 and GRO from ovarian cancer cells. These secreted factors then act on endothelial CXCR1/2 receptors to cause endothelial cell proliferation, tube formation and migration. A cell penetrating pepducin, PZ-206, that targets the third intracellular loop of CXCR1 and CXCR2 receptors (also known as X1/2pal-i3) can significantly inhibit both endothelial cell proliferation and tube formation. Avastin-the VEGF blocking antibody was unable to inhibit IL-8 and GRO- stimulated tube formation. To validate our in vitro observations we injected matrigel plugs containing MMP-1 stimulated/unstimulated ovarian cancer (OVCAR-4) conditioned media into the flanks of nude mice. After 7 days, the matrigel plugs were harvested and stained for CD31 and whole mount confocal microscopy was done in a blinded manner. Mice injected with MMP-1 stimulated, OVCAR-4 CM showed an 8 fold increase in blood vessel formation which was significantly inhibited by injecting mice with CXCR1/2 inhibitor PZ206 (5mg/kg/d) as compared with control pepducin or vehicle. These studies will help us validate novel therapeutic targets such as CXCR1/2 pepducins for ovarian cancer that block angiogenesis and tumor progression to improve survival in this lethal disease.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B13.
Abstract B133: SEA technology: A novel strategy for enhancing antibody effector function
Authors: Alley Stephen C. , Sussman Django R. , Jeffrey Scott C. , Torrey Lindsay , Burke Patrick J. , Klussman Kerry , Okeley Nicole M. , Senter Peter D. , Benjamin Dennis R.
Published: June 2012

The activity of monoclonal antibodies (mAbs) can be enhanced by a number of chemical and genetic strategies. We describe a novel strategy for enhancing antibody-dependent cellular cytotoxicity (ADCC) through modification of the mAb carbohydrate during expression, designated Sugar Engineered Antibody (SEA) technology. A series of small molecule fucose analogs were added to mAb-expressing Chinese hamster ovary (CHO) cells, with the resulting mAbs showing a significant reduction in their carbohydrate fucosylation. We demonstrate that these mAbs show a substantial increase in ADCC activity and an improvement in CD16 binding. We find that the mechanism of action is inhibition of GDP-mannose dehydratase (GMD), the first enzyme in de novo synthesis of GDP-fucose, leading to a depletion of substrate for the fucosyltransferase responsible for mAb carbohydrate fucosylation. We also demonstrate that this strategy yields mAbs with significantly reduced fucosylation in large scale CHO cell culture as well as in additional expression systems such as hybridomas and transient transfectants. Since genetic modification of the mAb-producing cell line is not required, SEA technology can be readily applied from the mAb screening to manufacturing stage to generate effector function enhanced therapeutic antibodies.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B133.
Abstract B140: In vitro sensitivity profiling identifies selectively responsive tumor types and molecular subtypes to the PI3K inhibitor GSK2126458
Authors: Greshock Joel , Hardwicke Mary Ann , Conway Theresa , Degenhardt Yan , Cartee Leanne , Moy Christopher , Lager Joanne , Wooster Richard , Bachman Kurtis
Published: June 2012

The phosphoinositide 3-kinase (PI3K) signalling pathway is activated in a broad spectrum of human cancers. Activation of this pathway often occurs indirectly by somatic aberrations of receptor tyrosine kinases, or directly by mutations in PI3K genes, such as PIK3CA. GSK2126458 is a novel, orally administered inhibitor of wild type phosphoinositide 3-kinase alpha (PI3K) and the common activation mutants of p110 found in human cancers that has recently entered Phase I clinical trials. In order to determine histological and genetic profiles of patients that are more likely to gain clinical benefit from GSK2126458, the compound was tested in a 3-day continuous exposure proliferation assay on a panel of 198 genetically characterized human cancer cell lines derived from a diversity of tissue types. Cells originating from bladder, breast, and ovarian cancers are most responsive based on growth inhibition metrics (gIC50's). Breast cancer cell lines harboring activating mutations of PIK3CA are associated with increased levels of response (p = 0.027); an observation which was validated in an independent cohort of 53 breast cell lines (p = 0.021). While colon cancers exhibit an intermediate level of sensitivity compared to other tumor types, specific exon 5 G12/13 hotspot mutations of the KRAS gene are predictive of decreased cellular response (p = 0.025). Additionally, a comparison of these cell line proliferation data to a similarly-assayed panel of 19 compounds of diverse target class shows that while the overall pattern of cellular response to GSK2126458 resembles other inhibitors or the PI3K/AKT/mTOR pathway, distinctions emerge that correlate with pathway targeting strategy. For example, while breast cancers are preferentially responsive to GSK2126458 and other inhibitors of this pathway, cell lines harboring PTEN mutations are less responsive to GSK2126458 than to compounds directly targeting AKT. Collectively these data suggest a unique profile for GSK2126458 among compounds that inhibit the PI3K/AKT/mTOR pathway. Positive predictors of response, such as PIK3CA mutations in breast cancer, may identify patients who are most likely to benefit from GSK2126458. The use of this predictor to select patients has the potential to reduce the size of a clinical registration trial for GSK2126458 by approximately 25%, thereby limiting cost and accelerating the development of this compound.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B140.