bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B142: PKI-402, a dual PI3K/mTOR inhibitor
Authors: Mallon Robert G. , Holland Irwin , Feldberg Larry , Lucas Judy , Abraham Robert , Gibbons Jay , Dehnhardt Christoph , Venkatesan Aranapakam , Santo Efren Delos , Chen Zecheng , Santos Oswaldo dos , Ayral-Kaloustian Semiramis , Brooijmanns Natasja , Mansour Tarek
Published: June 2012

We have developed small molecule inhibitors of the PI3K/mTOR signaling pathway. An example is PKI-402. This compound is a reversible, ATP competitive, and equipotent inhibitor of class I PI3Ks, including the E545K and H1047R mutant forms of PI3K- , and mTOR (IC50 vs PI3K- = 1nM). Selectivity of PKI-402 was established in a screen against 236 diverse human kinases. PKI-402 caused in vitro growth inhibition of human tumor cell lines derived from breast, brain (glioma), pancreas, and non small cell lung cancer (NSCLC) tissues. In many cases IC50 values were <100 nM. In vitro, PKI-402 suppressed phosphorylation of PI3K and mTOR effector proteins, particularly p-Akt at threonine 308 (T308), at concentrations that closely matched those that inhibited tumor cell growth. In MDA361, a breast tumor line with elevated levels of Her2 receptor, and mutant PI3K- (E545K), 30 nM PKI-402 induced cleaved PARP, a marker for apoptosis. This occurred after an exposure time of 4 h. In vivo, PKI-402 displayed anti-tumor activity when administered by IV route in glioma (U87MG, PTEN), NSCLC (A549; K-Ras, STK11), and breast (MDA361: Her2+, PIK3CA [E545K]) xenograft models. A key question about PI3K/mTOR signaling inhibitors is: can they cause tumor regression in pre-clinical models? At 25, 50, and 100 mg/kg PKI-402 caused regression of MDA361 tumors. PKI-402 effect was most pronounced at 100 mg/kg (daily for 5 days [dx5], 1 round), which reduced an initial tumor volume of 260 mm3 to 129 mm3, and prevented tumor re-growth for 70 days. Tumor re-growth occurred between days 16–20 when PKI-402 was administered at 25 and 50 mg/kg (dx5, 2 rounds). However, when PKI-402 was re-administered at 100 mg/kg (dx3, 1 round) at day 37 (25 mg/kg group), large tumors (∼600 mm3) were reduced in volume by 69%. In MDA361 tumor tissue PKI-402 at 100 mg/kg (single dose) caused sustained suppression of Akt phosphorylation at both the T308 and S473 sites, and induced cleaved PARP. Suppression of p-Akt was complete at 8 h, and still evident at 24 h; while cleaved PARP was evident at both 8 h and 24 h. In vivo biomarker analysis of heart and lung tissue showed minimal effect on p-Akt and no induction of cleaved PARP by PKI-402 at 100 mg/kg. Preferential accumulation of PKI-402 in tumor tissue was observed. It may be that complete and sustained suppression of p-Akt is needed to cause tumor regression in the MDA361 and other tumor xenograft models. We are testing whether dual PI3K/mTOR inhibitors can durably suppress p-Akt, induce cleaved PARP, and cause tumor regression in a diverse set of human tumor xenograft models.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B142.
Abstract B144: NVP-BEZ235, a dual inhibitor of PI3K and mTOR, induces MAPK phosphorylation in HER2-positive cells
Authors: Elizalde Violeta Serra , Eichhorn Pieter JA , Scaltriti Maurizio , Prudkin Ludmila , Rodriguez Olga , Guzman Marta , Baselga José
Published: June 2012

The PI3K pathway is highly deregulated in human cancer with the most common alterations being amplification of the receptor tyrosine kinases (RTKs) EGFR and HER2 and mutations in PI3K itself or inactivation of the tumor suppressor PTEN. Thus, the rationale exists to develop therapeutic agents targeting the PI3K/AKT pathway. Several small molecule inhibitors targeting PI3K, AKT and mTOR are now in clinical trials. However, these novel compounds may interfere with regulatory feedback loops. One such example involves the activation of AKT following rapamycin analogues treatment, such as everolimus, an allosteric mTOR inhibitor. Blocking mTOR signaling also alters the regulation of other signaling pathways, like the MAPK cascade. The aim of this study was to investigate the mechanism/s by which inhibition of the PI3K pathway leads to activation of MAPK kinase signaling in breast cancer cell lines and its relevance in tumor treatment. We have observed an increased MAPK phosphorylation in HER2 positive BT474, SKBR-3, MDA-361 and MCF-7HER2 cells when treated with NVP-BEZ235, a dual inhibitor of both mTOR and p110, the catalytic component of PI3K. The increase of P-MAPK was observed by western blot and correlated with hyperphosphorylation of several RTKs. PD325901, a MEK1/2 inhibitor, or lapatinib, a HER1/2 tyrosine kinase inhibitor, reverted the NVP-BEZ235-induced MAPK phosphorylation. On the other hand, NVP-AEW541, an IGFR-1 tyrosine kinase inhibitor, did not prevent NVP-BEZ235-dependent HER2 phosphorylation and downstream MAPK activation. Activation of MAPK upon administration of NVP-BEZ235 was also observed in vivo. We are currently investigating the possible involvement of known regulatory feedback loops as well as the individual RTK contribution. Activation of parallel pathways in a condition of PI3K inhibition may reduce potential benefits of these anti-cancer treatments. For this reason, we propose the combination of an anti-HER2 therapy together with PI3K inhibitors for the treatment of HER2 positive tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B144.
Abstract B162: Differential stress-induced CXC-chemokine expression, signaling and responses in PTEN-deficient prostate tumors
Authors: Waugh David J. , Maxwell Pamela , McClatchey Ashleigh
Published: June 2012

We have shown that expression of the proinflammatory CXC chemokine, interleukin-8 (IL-8) and its receptors CXCR1 and CXCR2 is elevated in malignant prostate cancer (CaP) epithelium. Published studies confirm that hypoxia and/or chemotherapy-induced stresses underpin AP-1, HIF-1 and NFκB-mediated transcription-driven increases in IL-8, CXCR1 and CXCR2 expression in CaP cells. The current study determines the relevance of PTEN, a commonly mutated or deleted tumor suppressor gene in CaP, in regulating the induction of CXC-chemokine signaling and the cellular response of stressed CaP cells. Time-dependent increases in CXCL8, CXCR1 and CXCR2 mRNA were observed in PTEN-deficient LNCaP and PC3 cells. ELISA confirmed increased IL-8 secretion following hypoxia, while immunoblotting confirmed elevated CXCR1 and CXCR2 expression in both cells. In contrast, CXCL8, CXCR1 and CXCR2 expression was only marginally up-regulated in PTEN wild-type DU145 and 22Rv1 cells under hypoxia. Subsequently, PTEN status was shown to regulate the magnitude and duration of CXC-chemokine-promoted signaling and altered gene expression profiles. For example, CXCL8 administration increased expression of HIF-α and increased the activity of this transcription factor in PTEN-deficient LNCaP and PC3 cells but not in PTEN wild-type cells. Furthermore, expression of HIF-1 target genes (VEGF, TGF) was also induced following CXCL8 stimulation in PTEN deficient but not PTEN wild-type cells. Attenuation of PTEN in the DU145 and 22Rv1 cells using siRNA revealed the CXCL8-induced responses including the increase in HIF-1 expression and activation. Functionally, the transcription-mediated elevation in IL-8 signalling underpins an increased survival of hypoxic prostate cancer cells to DNA-damage-based chemotherapy. Additionally, CXCL8 signaling was shown to induce cysteine cathepsin and serine protease expression in CaP cell lines and stromal cells, indicating a capacity to promote tumor invasion. In summary, our studies suggest that the magnitude of CXC-chemokine signaling and its subsequent effects are enhanced in PTEN-deficient prostate tumors, promoting proliferation, survival, chemoresistance, androgen-independent transition and changes in the tumor microenvironment.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B162.
Abstract B170: Downregulation of Yes-associated protein 1 (YAP1) expression reduces cell proliferation and clonogenicity of pancreatic cancer cells
Authors: Diep Caroline H. , Zucker Kelly , Watanabe Aprill , Hostetter Galen , Munoz Ruben , Von Hoff Daniel D. , Han Haiyong
Published: June 2012

Recent genetic studies in Drosophila have shown the Hippo pathway to be a potential tumor suppressor pathway that could play vital role(s) in mammalian cancer development. The Hippo pathway regulates organ size by inhibiting cell proliferation and promotes cell apoptosis upon its activation. The Yes Associated Protein 1 (YAP1) is a nuclear effector that promotes cell growth as a transcription co-activator. In human cancer, YAP1 has been shown to be a candidate oncogene as the chromosomal region containing YAP1 (11q22) has been reported as amplified and over-expressed in several tumor types, such as colon, liver, and lung (Overholtzer et al. 2006 PNAS, 103: 12405–12410). To determine the role of YAP1 in the tumorigenesis and progression of pancreatic cancer, here we examine YAP1 protein expression in pancreatic tumors and investigate the phenotypic effects of YAP1 down-regulation in cultured pancreatic cancer cells. Initially, we evaluated YAP1 expression in pancreatic tumors through immunohistochemistry (IHC) using tissue microarrays (TMA) to determine relative protein abundance and localization at the tissue and cellular level. YAP1 staining intensities were moderate to strong in the nucleus and cytoplasm of the cells, whereas the companion normal pancreas tissue showed negative to weak staining. In cultured cells, YAP1 expression and localization was modulated by cell densities. YAP1 total protein expression and Ser127 phosphorylation increased concurrently with cell density in whole cell lysates and cytosolic fractions, but does not in nuclear fractions. Additionally, transfection of YAP1-targeting siRNA oligonucleotides within pancreatic cancer cell lines, BxPC-3 and PANC-1, significantly decreased cell viability and reduced cell proliferation in vitro. An additional observation was the diminished anchorage-independent growth on soft agar of BxPC-3 and PANC-1 cells upon transfection with YAP1 siRNA oligonucleotides. The number of colonies formed were approximately 5% in BxPC-3 (p<0.007) and 20% (p<0.04) in PANC-1 compared to the negative siRNA control. Furthermore, the reduction of YAP1 protein expression by siRNA oligonucleotides correlates with the reduced growth on soft agar. The silencing of YAP1 abated clonogenicity of pancreatic cancer cells, suggesting a role of YAP1 in tumorigenesis. In summary, we demonstrate that YAP1 is overexpressed in pancreatic cancer and potentially plays an important role in the tumorigenesis and growth of pancreatic cancer. (This work was supported by a grant from the National Foundation for Cancer Research.)

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B170.
Abstract B175: Lactadherin/MFGE8 favors bladder tumor progression by promoting tolerogenic immune responses
Authors: Bernard-Pierrot Isabelle , Sugano Gael , Laé Marick , Battail Christophe , Allory Yves , Stransky Nicolas , Krumeich Sophie , Donnadieu Marie-Helene , Abbou Claude , Benhamou Simone , Lebret Thierry , Sastre-Garau Xavier , Amigorena Sebastian , Radvanyi François , Thery Clotilde
Published: June 2012

MFG-E8/lactadherin is a secreted integrin-binding protein that promotes elimination of apoptotic cells by phagocytes leading to tolerogenic immune responses, and VEGF-induced angiogenesis, two important processes for cancer development. Here, by transcriptomic analysis of 229 biopsies of bladder carcinomas, we observed increasing expression of lactadherin with tumor progression, suggesting a protumoral role of lactadherin in this cancer. Consistently, using a mouse model of bladder carcinoma development due to carcinogen exposure, we showed that lactadherin-expressing mice developed more invasive tumors than lactadherin-deficient animals. We demonstrated that the proangiogenic function of lactadherin was not responsible for its protumoral role, since angiogenesis was similar in carcinogen-treated lactadherin-expressing or -deficient bladders, and since genes involved in VEGF-mediated angiogenesis were not co-regulated with lactadherin in human tumors. By contrast, we could ascribe the protumoral activity of lactadherin to its protolerogenic role, by showing that lactadherin expression did not promote tumor progression in mice devoid of adaptive immune system, and that lactadherin-overexpressing human tumors were invaded with immunosuppressive macrophages and regulatory T lymphocytes. Thus, lactadherin favors bladder tumor progression in human and mouse through its tolerogenic, rather than proangiogenic, function, and represents a promising therapeutic target in invasive bladder carcinomas.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B175.
Abstract B177: Detection and quantification of ligand-receptor interactions in formalin-fixed paraffin embedded samples by a novel proximity-based assay
Authors: Dua Rajiv , Zhang Jianhuan , Parry Gordon , Penuel Elicia
Published: June 2012

Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) sections, a format used to preserve human tissue samples. This is the only format for readily available tissues from clinical trials and patient disease management. However, phosphorylation is generally less stable and consequently, less reproducible which can, in a clinical setting lead to ambiguous data. Here, we describe an alternative, novel approach to detect and quantify activation of receptors in FFPE samples. We used the ligand/receptor, HGF/c-MET as our model system. HGF is the only known ligand of c-MET and ligand binding causes activation of the c-MET receptor. Using, the VeraTag technology, a proximity-based assay, validated antibodies were used to detect and quantify total c-MET and total HGF in FFPE samples as well as to detect and quantify ligand stimulated and endogenous HGF/c-MET interactions in FFPE cell line pellets. The detection was validated in human non-small cell lung and stomach carcinoma FFPE samples. Our data demonstrate a novel approach to detect and quantify ligand-receptor interactions by VeraTag proximity-based assays that can be widely applied to measure activation of receptors in archived FFPE human tissue samples.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B177.
Abstract B186: Preclinical antileukemic activity of F14512, a novel targeted cytotoxic agent
Authors: Kruczynski Anna , Recher Christian , Pourtau Sandrine , Cartron Valérie , Marty Fabien , Vandenberghe Isabelle , Chansard Nathalie , Gallay Nathalie , Pillon Arnaud , Pesnel Sabrina , Le Pape Alain , Créancier Laurent , Menon Yoann , Gomes Bruno , Blanchet Jean-Christophe , Ricome Christel , Guminski Yves , Imbert Thierry , Payrastre Bernard , Bailly Christian , Demur Cécile , Guilbaud Nicolas
Published: June 2012

Despite advances in the therapy of acute myeloid leukemia (AML), the majority of patients die from their disease. Therefore, the lack of effective therapy mandates the development of novel compounds to improve the outcome of patients with relapsed and refractory leukemias. F14512 is a potent spermine-epipodophyllotoxin conjugate exploiting the polyamine transport system for tumor cell delivery. In this study, we report the in vivo antitumor activity of F14512 against experimental models of AML cell lines and of patient AML samples. F14512 markedly reduced the growth of HL-60 and U937 cell lines in an in vivo xenotransplantation model, resulting in a highly significant increase of survival of leukemia-bearing mice. Etoposide evaluated concurrently demonstrated only moderate in vivo activity against these models. F14512 induced in vivo apoptosis of HL-60 cells, as shown by caspase-3 activation and PARP cleavage. In an effort to mimic the human disease, we injected approximately 106 AML cells collected from a patient into NOD/SCID mice and allowed them to establish as xenografts for 8 weeks. Subsequent treatment with F14512 was carried out for 2 or 3 weeks followed by the analysis at the end of treatment and 1 week after the end of treatment. Two human AML samples were analyzed. Multiple i.v. administrations of F14512 at 0.32 mg/kg, induced an extensive reduction of the number of leukemic cells in mouse bone marrow and blood (97–99%), assessed by flow cytometry analysis, quantitative RT-PCR and histology. To identify leukemic cells expressing an active polyamine transport system, we developed a functional method based on the measurement of the cellular uptake of a nitrobenzoxadiazole fluorescent probe (F96982) combining the same spermine moiety as F14512. The level of fluorescence emitted by the probe F96982 was high in HL-60 cells as well in the 2 patient AML samples that proved to be sensitive to F14512 in vivo. Collectively, these results demonstrated that F14512 exhibits a marked in vivo antileukemic activity, supporting its clinical development. Phase I clinical trials in onco-hematology are now initiated with this novel promising drug candidate.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B186.
Abstract B194: Enhanced antitumor activity of the HIF-1 inhibitor PX-478 combined with radiation and chemotherapy in pancreatic cancer
Authors: Powis Garth , Von Hoff Daniel , Bankson James , Iemos Robert , Hostetter Galen , Demeure Michael , Schwartz David
Published: June 2012

Growing tumors respond to hypoxic stress through increased expression of the HIF-1α transcription factor resulting in an adaptive switch to glycolytic metabolism, angiogenic signaling, survival, and metastasis. HIF-1α expression is associated with tumor resistance to cytotoxic therapy and inferior clinical outcomes. Pancreatic cancer is frequently associated with extensive desmoplasia, hypovascularity, and hypoxia, and is resistant to chemotherapy and radiation treatment. We have observed that HIF-1α is increased compared to normal pancreatic tissue in 24/32 (75%) of patent pancreatic ductal carcinoma samples, while 43% of the surrounding stromal tissue also showed elevated HIF-1α. Distal stroma showed no increase in HIF-1α. We have investigated the effects of the novel selective HIF-1α inhibitor PX-478 currently in Phase I clinical trial, on the in vitro and in vivo radiation response of human pancreatic cancer models. In pancreatic cancer cell lines, inhibition of HIF-1α by PX-478 was associated with increased cell killing by radiation with a radiation enhancement factor at 0.2 surviving fraction (SF2) in hypoxia (1% O2) of between 1.3 and 1.4. In mice with Panc-1 xenografts, 1 Gy QD alone × 5 yielded a tumor inhibition T/C at 70 days of 65% (with no regression), PX-478 20mg/kg po QD × 5 yielded a T/C of 14% (with 41% tumor regression) and the combination of 20mg/kg po PX-478 given daily 4 hr before 1 Gy × 5 provided a T/C of 8% (with 81% tumor regression). In CF-PAC-1, or SU.86.86 sc pancreatic xenografts, concurrent administration of 10 mg/kg PX-478 potentiated the antitumor effects of fractionated radiation and 5FU 20 mg/kg iv QD × 5 with a T/C of up to 3%, and with 66% tumor regression. In Panc-1 and SU.86.86 xenografts 20 mg/kg PX-478 potentiated the antitumor effects of radiation in combination with gemcitabine 120 mg/kg ip Q3D given one week later with a T/C of up to 8%, and with 80% tumor regression. Early tumor responses to combined PX-478/radiation treatment could be rapidly quantified by clinically relevant vascular imaging biomarkers. DCE-MRI and ultrasound imaging reproducibly discriminated tumor responses to PX-478 alone or combined with radiation as early as 10 days post-treatment, before differences in anatomic tumor size could be detected. Therefore, PX-478 is a mechanistically appealing and potentially clinically relevant enhancer of pancreatic cancer radiosensitivity, inhibiting tumor and stromal HIF-1 pro-angiogenic signaling and reducing the innate radiation resistance of hypoxic tumor cells. Supported by CA CA109552-05.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B194.
Abstract B20: In vitro and in vivo antitumor activities of the dual PI3K/mTor inhibitor NVP-BEZ235. Implications for patient stratification strategies
Authors: Maira Michel , Brachmann Saskia , Fritsch Christine , Hofmann Irmgard , Schnell Christian , Garcia-Echeverria Carlos
Published: June 2012

The abnormal activation of the PI3K pathway is a recurrent feature observed in human tumors. Epidemiological and experimental studies have clearly underlined the role of this pathway for the maintenance of the tumorigenic state. A number of the intracellular components of this pathway have been targeted as anticancer drug discovery activities leading to clinical candidates like NVP-BEZ235. This compound, which is a dual pan-PI3K/mTOR, is currently undergoing Phase I clinical trials.To identify cancer patient populations that would benefit from NVP-BEZ235 treatment, the compound was profiled pre-clinically in a panoply of tumor cell lines, either in vitro or in vivo settings. Although not predictive from the in vitro antiproliferative data, the observed in vivo antitumor effects could be classified in three groups: strong, medium and low responders. Interestingly, 2 out of the 4 strong tumor xenograft responders presented genetic alterations of EGFR members. Constitutively active mutants or amplification of EGFR members is frequently observed in breast, ovarian and in non small cell lung cancer. Follow-up in vitro studies using a panel of over 40 breast cancer cell lines showed that a better understanding of tumor cell sensitivity to BEZ235 treatment could be obtained when cell death was used as a readout. ErbB2 amplification and/or PIK3CA activating mutations correlated well with cell death induction, but not PTEN deletion or mutations. An in depth analysis of death markers revealed that the cell death observed with BEZ235 could be recapitulated with other PI3K inhibitors and that this event is linked with active PARP cleavage reflective of an apoptotic process. Moreover, the effect seemed to be independent of Caspase-9 executioner and mitochondrial activated caspase, suggesting an alternate route for apoptosis induction by this class of compound. The exact mechanism is currently being elucidated. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer Phase II trials for BEZ235

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B20.
Abstract B222: MUC16/CA125 synthetic lethal kinase screen for novel ovarian cancer targets
Authors: Eng Stephanie , Robin Manson , Zhou Qin C. , Thapi Dharmarao , Roslaes Nestor , Iasonos Alexia , Spriggs David R.
Published: June 2012

Background: MUC16 is a tethered, cell surface mucin which is over expressed on ovarian cancer cells and encodes the CA125 antigen. The CA125 antigen is present in the serum of women with ovarian cancer and appears to be an independent adverse marker of prognosis. Elements of the MUC16 molecule appear to promote invasion, alter gene expression and will induce malignant transformation in NIH 3T3 cells. We have previously demonstrated that MUC16 expression alters the expression of invasion/metastasis genes and genes linked to drug resistance. We hypothesize that the forced expression of MUC16 induces changes that might be relected in altered kinase activities and provides an opportunity to specifically target the MUC16+ ovarian cancer cell.

Methods: A human kinome siRNA library (2 siRNA's/well) was purchased from Qiagen. A2780 cells with and without MUC16 expression were labeled with GFP or mCherry Fluorescence proteins and plated in 96 well plates at 25000 cells/well. Each siRNA was plated into six replicate wells and analyzed for inhibition compared to negative controls and a proprietary “All Star Death” siRNA mix. If the candidate siRNA inhibited the proliferation of MUC16+ cells statistically (p<0.05) more than MUC16 negative cells at 72hrs, that siRNA was scored as a “hit”.

Results: To date, we have screened siRNA's for a total of 160 of the planned 714 kinases. From the initial screen, we identified 28 kinase candidates for additional evaluation. Two additional siRNA's per kinase were obtained for confirmatory screening. From this initial group of kinases, inhibition of 3 kinases (CHEK1, ABL1 and CDK4) were significantly more inhibitory for MUC16+ cells than the syngeneic MUC16- comparator. Eight additional kinases (including CSK, CHUK, ILK and LTK) were more inhibited in the MUC16+ cells for 3 of 4 tested siRNA's for the target kinase. Although CDK4 protein levels were not altered in MUC16 transformed A2780 cells, CDK4 inhibitors were differentially active between the MUC16 +/- syngeneic pair and additional testing with other small molecule inhibitors is ongoing.

Conclusions: 1)MUC16 expression alters the biologic behavior of ovarian cancer cells and 2)MUC16 targeted, syngeneic screening of kinase siRNA libraries can identify potential clinical target candidates for ovarian cancer selected for MUC16.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B222.
Abstract B226: Induction of growth arrest DNA damage-inducible gene 45 beta (GADD45b) expression contributes to sorafenib-induced apoptosis in hepatocellular carcinoma (HCC) cells
Authors: Ou Da-Liang , Shen Ying-Chun , Chen Kuen-Feng , Wang Ching-Tzu , Fan Hsiang-Hsuan , Yu Sung-Liang , Hsu Chiun , Cheng Ann-Lii
Published: June 2012

GADD45b, which is associated with cellular stress response and apoptosis regulation, was frequently under-expressed in HCC. In a previous study we found GADD45b belongs to a group of sorafenib-regulated genes that are independent of mitogen-activated protein kinase-extracellular signal-regulated kinase (MEK)/ extracellular signal-regulated kinase (ERK) signaling pathway. The present study explored the role of sorafenib-induced GADD45b expression in HCC cells. HCC cell lines tested included HepG2, Hep3B, Huh-7, and Huh-7R, a sorafenib-resistant cell line established by continuously exposure of Huh-7 cells to sorafenib. GADD45b expression was examined by quantitative real-time PCR and western blotting. Control of GADD45b transcription was examined by luciferase reporter assay using a series of reporter plasmids with deletions or site-directed mutants of the 5′-flanking region of GADD45b promoter. Apoptosis was analyzed by measuring the subG1 fraction and annexin V staining using flow cytometry. Both the mRNA and the protein levels of GADD45b was increased after sorafenib but not after the MEK inhibitor CI-1040 or U0126 treatment, suggesting that GADD45b induction was independent of cellular MEK/ERK activity. GADD45b induction was more prominent in sorafenib-sensitive HCC cells (Huh-7 and HepG2, IC50 6–7 µM) but less so prominent in sorafenib-resistant HCC cells (Hep3B and Huh-7R, IC50 12–15 µM). Knockdown of GADD45b expression by siRNA partially abrogated the pro-apoptotic effects of sorafenib in Huh-7 and HepG2 cells but not Hep3B cells. The region −339/−267 in the 5′-flanking region of GADD45b promoter, which contained AP-1 (−298/−292) and Sp1 (−285/−277) binding sites, was found crucial for GADD45b induction by sorafenib. Binding of c-Jun and Sp1 to GADD45b promoter was confirmed by chromatin immunoprecipitation. Sorafenib also increased the phosphorylation of JNK in sorafenib-sensitive but not in sorafenib-resistant HCC cells. Sorafenib-induced GADD45b expression can be alleviated by treatment with the specific JNK inhibitor SP600125. Our data indicated that induction of GADD45b expression, which is mediated by cellular JNK/c-Jun signaling pathway but not MEK/ERK signaling, contributes to sorafenib-induced apoptosis in HCC cells. Failure of GADD45b induction may confer sorafenib resistance. GADD45b may be used as a predictive biomarker for sorafenib sensitivity in HCC. (Supported by grants NSC97-3112-B-002-012, NSC98-3112-B-002-007, NSC98-3112-B-002-037)

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B226.
Abstract B228: A hepatocyte growth factor antagonist engineered by targeted disruption of heparan sulfate binding
Authors: Cecchi Fabiola , Pajalunga Deborah , Rabe Daniel , Fowler Andrew C. , MacDonald Nicholas , Blackman Davida K. , Stahl Stephen J. , Byrd Andrew R. , Bottaro Donald P.
Published: June 2012

Hepatocyte growth factor (HGF) stimulates cell proliferation, motility, and morphogenesis upon binding to the receptor tyrosine kinase Met and cell surface heparan sulfate (HS) glycans. NK1 is a truncated HGF isoform consisting of the N-terminal (N) and first kringle (K1) domains of full-length HGF that stimulates all major HGF biological activities. Within NK1, the N domain contains the HS binding site, while K1 contains the primary determinants of Met binding. To further define the role of HS in ligand-receptor interaction and biological signaling, we identified the primary HS binding residues in N domain (K60, K62, R73) and generated opposite charge mutations at these positions in NK1. Nuclear magnetic resonance analysis of recombinantly expressed mutant and wild type (WT) proteins revealed that both were folded normally. Mutant proteins displayed diminished binding to immobilized HS relative to WT, and the triple mutant protein (3M) also lacked intrinsic motogenic and mitogenic activity. Interestingly, 3M NK1 maintained Met binding and competitively inhibited DNA synthesis stimulated by WT NK1 as well as full-length HGF in normal cells. Expression plasmids encoding 3M and WT NK1 proteins were transfected into the glioblastoma cell U87 MG, which is dependent on HGF/Met autocrine signaling for tumorigenicity in mice. 3M/U87 MG cells displayed a substantially lower rate of growth relative to WT/U87 MG or the parental cell line, suggestive of growth antagonism by 3M NK1. As anticipated, expression of WT NK1 significantly accelerated the growth of U87 MG tumor xenografts in mice, which reached maximal volume (1 ml) by 22 days post-implantation, compared to 45 days required by the parental cell line. In contrast, tumor growth in animals injected with 3M/U87 MG was dramatically impaired: at 60 days post implantation, average tumor volume for this group was < 0.3 ml. Because both WT and 3M transfectants expressed equal amounts of their respective recombinant proteins, the HS binding site mutations delayed measurable tumor growth from one week to two months. Thus HS binding is critical for HGF-driven mitogenesis and tumor growth, and inhibition of these activities by 3M NK1 validates a novel strategy for developing antagonists of the HGF pathway and potentially those of other HS-binding ligands

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B228.
Abstract B234: Dual targeting of the tyrosine kinase receptors EGFRvIII and c-Met in glioblastoma multiforme
Authors: Pillay Vinochani , Scott Andrew M. , Johns Terrance G.
Published: June 2012

The most common mutation of the epidermal growth factor receptor (EGFR) in glioblastoma multiforme (GBM) is an extracellular truncation of 267 amino acids known as EGFRvIII (or de2-7 EGFR). While the EGFRvIII is unable to bind any known ligand, it displays a low level of constitutive activation that is enhanced by its inability to undergo downregulation. HGF/SF is the ligand for the receptor tyrosine kinase c-Met, both of which are often co-expressed in GBM. The presence of the HGF/c-Met axis or expression of EGFRvIII each independently enhances GBM growth, angiogenesis capacity, migration and invasiveness. Using tyrosine kinase array technology, we now show that expression of EGFRvIII in U87MG glioblastoma cells (U87MG. 2-7 cells) leads to co-activation of several receptor tyrosine kinases, including PDGFR and c-Met. A fully human neutralizing antibody directed to HGF (AMG 102) did not inhibit this EGFRvIII mediated activation of c-Met in vitro, demonstrating that it is ligand independent. We examined if this transactivation of c-Met influenced the efficacy of therapies targeting the HGF:c-Met axis. Treatment of parental U87MG xenografts with low doses of AMG 102 resulted in significant inhibition of tumor growth (see Burgess et al. 2006), while U87MG. 2-7 xenografts (see Johns et al 2007) were profoundly resistant to AMG 102 treatment. Treatment with Panitumumab, a fully human antibody directed to the EGFR and EGFRvIII, was able to inhibit the EGFRvIII mediated activation of c-Met and significantly reversed the resistance to AMG 102 observed in U87MG. 2-7 xenografts. Indeed, combination therapy with Panitumumab and AMG 102 resulted in a marked increase in tumor cell apoptosis, especially in the central parts of the xenograft. An EGFRvIII molecule with an active kinase but incapable of autophosphorylation at the five major autophosphorylation sites (tyrosine changed to phenylalanine) involved in signal transduction was also able to mediate resistance to AMG 102, suggesting that the activation of c-Met by EGFRvIII occurs via a direct interaction rather than through these docking sites. These studies show that expression of EGFRvIII leads to the promiscuous activation of several kinases, causing resistance to ligand inhibitory based strategies. Since Panitumumab can reverse this process, the combination of this antibody with AMG 102 may be an effective treatment for GBM patients positive for EGFRvIII.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B234.
Abstract B261: A novel small molecule inhibitor of protein kinase D blocks pancreatic cancer growth in vivo
Authors: Ireson Christopher R. , Jamieson Stephen , Stribbling Stephen , Sutherland Rachel , Raynham Tony , Williams Juliet , Charles Mark , Bagherzadeh Azadeh , Farooq Muddasar , Harikumar Kuzhuvelil B. , Kunnumakkara Ajaikumar B. , Deorukhkar Amit , Tong Zhimin , Maru Dipen , Diagaradjane Parmeswaran , Matsuo Yoichi , Krishnan Sunul , Gelovani Juri , Aggarwal Bharat , Guha Sushovan
Published: June 2012

Background: Protein kinase D (PKD) is a novel family of serine-threonine kinase with diverse biological functions including cell proliferation and growth. Pancreatic Cancer (PaCa) is a devastating disease with few therapeutic options. We showed earlier that PKD signaling pathways promote mitogenesis in multiple PaCa cell lines. However, nothing is known about targeting biological functions of PKD in PaCa. Our PKD inhibitor discovery program yielded CRT0066101 that specifically blocks PKD activation.

Aim: The aim of our study was to determine the effects of CRT0066101 in PaCa, both in vitro and in vivo.

Methods and Results: Our immunohistochemical analysis showed that activated PKD (pS916PKD1) is significantly upregulated in PaCa as compared to normal ducts (91% vs 22%; p<0.001). Using Panc-1 as a model system, we demonstrated that CRT0066101 blocked proliferation and BrdU incorporation with an IC50 of 1 µM. We showed that CRT0066101 given orally (80 mg/kg/day) for 4 weeks significantly abrogated growth in a subcutaneous Panc-1 xenograft model (n=8; p<0.01). The expression of activated PKD (pS916PKD1) in the treated tumor explants was significantly inhibited (p<0.05) with peak concentration (12 M) of CRT0066101 achieved within 6 hours of oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/day) for 21 days in an orthotopic model potently blocked Panc-1 tumor growth (n=7; p<0.01). CRT0066101 significantly reduced Ki-67+ proliferation index (p< 0.01), increased apoptosis (measured by in situ TUNEL assay) of PaCa tumors (p<0.05) and potently abrogated expression of multiple proliferative and pro-survival proteins including Cyclin D1, survivin, Bcl-2, Bcl-xL, activated PKD1 (pS916PKD1), and activated PKD2 (pS876PKD2).

Conclusion: Our results demonstrate for the first time that the PKD-specific small molecule inhibitor CRT0066101 blocks PaCa growth both in vitro and in vivo. Thus, PKD is a novel therapeutic target in PaCa.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B261.
Abstract B273: A phase I trial of topotecan and sorafenib for patients with recurrent, platinum-resistant epithelial ovarian cancer
Authors: Matei Daniela E. , Schilder Jeanne , Perkins Susan , Khan Mohammad , Calley Cynthia , Menning Nancy , Sutton Gregory
Published: June 2012

Background: The objectives of the study were to determine the maximum-tolerated dose (MTD), the dose-limiting toxicities (DLTs) and objective response rate to combination therapy with sorafenib and topotecan in patients with platinum resistant epithelial ovarian cancer (EOC).

Methods: This multi-institutional phase I trial used a fixed dose of weekly topotecan and escalating doses of sorafenib. Eligible patients had recurrent platinum-resistant EOC, measurable or detectable disease, and up to three prior cytotoxic treatments. Prior anti-angiogenic therapy was not allowed. Sorafenib was administered orally daily and topotecan was given iv weekly at a fixed dose of 4 mg/m2, 3 weeks on and 1 week off. Each treatment cycle was 28 days. A standard 3 + 3 dose escalation design was used, testing two sorafenib dose levels: 400 mg daily (level 1) and 800 mg daily (level 2). The topotecan dose was de-escalated to dose level −1 (3.5mg/m2 weekly). The primary endpoints of the phase I study were to determine the MTD and the toxicity profile of this combination.

Results: 16 patients were enrolled between 12/12/07 and 11/13/08. Median number of prior regimens was 2 (range 1–7), median age was 52.5 (range 41–66), and all 16 patients had EOC. Distribution of patients per dose level and DLTs encountered are summarized in the table below. Five patients were inevaluable because of intercurrent illness or withdrawal of consent. Grade 3–4 toxicities during cycle 1 included: thrombocytopenia (3), neutropenia (2), febrile neutropenia (1), nausea and vomiting (2), rash (2), abdominal pain (1) and abnormal liver function tests (1). Grade 3–4 toxicities occurring after cycle 1 included: neutropenia (2), thrombocytopenia (1), anemia (2), nausea and vomiting (1), anorexia (1), and insomnia (1). Clinical activity was an exploratory endpoint. There was 1 partial response by RECIST lasting for over 12 months, but no complete responses. Six patients had stable disease.

Conclusions: Sorafenib can be safely combined with topotecan in EOC patients. The dose recommended for phase II investigation is sorafenib 400mg daily with topotecan 3.5 mg/m2 weekly.

Toxicities during dose escalation Topotecan dose (mg/m2) Sorafenic dose (mg) No patients No patients DLT/Decision DLT/Decision 4 400 6 2; not tolerated 3.5 400 4 0, tolerated, phase 2 3.5 800 6 2, not tolerated

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B273.
Abstract B43: Determination of biomarkers responsible for sensitivity and resistance of RGB-286638 action in vitro
Authors: Loferer Hannes , Amon Peter , Ivanov Igor
Published: June 2012

Understanding of molecular markers responsible for activity/resistance profiles of anti-cancer agents is essential for successful drug development. Here we demonstrate an approach to identify genes selectively regulated in either sensitive or resistant cell lines following treatment with RGB-286638, a multi-targeted protein kinase inhibitor currently in Phase I development. In order to assess whether the experimental conditions for in-vitro treatment of cancer cell lines with RGB-286638 used in this study resembles the mouse in-vivo situation, we adopted an in vitro assay based on the time growth curve rather than dose response curve. We found that the time growth curves were very similar to “standard” tumor growth curves observed in the mouse xenograft models. Next, a range of concentrations (25–100 nM of RGB-286638) was selected in which the shape of the in vitro growth curves closely resembled the shape of the in vivo growth curves. In addition to the similarity of growth curves, the pharmacodynamic markers PARP cleavage and RB-phosphorylation were used to confirm similarity of responses observed in xenograft models and in vitro cell culture. Using eight concentrations within the selected range and 6 cell lines with different responsiveness to RGB-286638, we analyzed gene expression profiles following RGB-286638 treatment for 24 hours. We could identify genes that were specifically deregulated in sensitive or resistant cells. In addition, some genes were down-regulated upon treatment independent of responsiveness of the cell lines to RGB-286638 treatment. Surprisingly, some of these genes are involved in cell cycle, e.g. regulation of cyclin B1, and were previously considered as pharmacodynamic markers but now appear to be nonspecific for the action of RGB-286638.

These results facilitate the selection of relevant biomarkers and can provide a rational for future combination studies.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B43.
Abstract B46: Collagen XXIII: A biomarker for non-small cell lung cancer
Authors: Spivey Kristin A. , Banyard Jacqueline , Solis Luisa M. , Wistuba Ignacio I. , Barletta Justine A. , Gandhi Leena , Feldman Henry A. , Rodig Scott J. , Chirieac Lucian R. , Zetter Bruce R.
Published: June 2012

Purpose: To determine the protein expression of collagen XXIII in tumor tissues from a variety of cancers and in tumor tissues and urine samples from non-small cell lung cancer (NSCLC) patients and to assess collagen XXIII's utility as a biomarker for NSCLC.

Experimental Design: A multi-cancer tissue microarray (TMA) was used to immunohistologically examine collagen XXIII protein expression in a variety of cancers. Subsequently, collagen XXIII expression was analyzed in three separate cohorts using TMAs with representative tumor and normal control lung tissues from patients with NSCLC. In addition, urine samples from NSCLC patients were analyzed for the presence of collagen XXIII via Western blot.

Results: Collagen XXIII staining was present in tissue samples from a variety of cancers. Within lung cancer tissues, collagen XXIII staining was enriched in the NSCLC subtypes. Tissues from 296 of 335 (81%) lung adenocarcinoma and 99 of 135 (73%) squamous cell carcinoma (SqCC) patients had detectable collagen XXIII. In urine, collagen XXIII was present in 23 of 29 (79%) NSCLC patient samples and only 15 of 54 (28%) of control samples. There was a significant association between collagen XXIII positivity in tissue or urine and presence of NSCLC (p<0.001, Fisher's exact test).

Conclusions: We demonstrate that collagen XXIII staining is increased in a variety of carcinomas. Specifically, collagen XXIII is expressed in a large subset of NSCLC patient tissue and urine samples, suggesting its utility as a potential NSCLC biomarker.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B46.
Abstract B58: Decreasing the time to activate clinical trials: Initial data from the Multiple Myeloma Research Consortium (MMRC) model
Authors: Wear Sandra , Kelley Susan , Quinn-Young Anne , Lonial Sagar , Kumar Shaji , Jakubowiak Andzej , Anderson Kenneth
Published: June 2012

Background: Activation of oncology trials is a lengthy and complex process. Recent literature has identified numerous opportunities to improve activation timelines1, 2. The Multiple Myeloma Research Consortium (MMRC) has implemented several processes to identify and overcome myeloma clinical trial activation barriers within the consortium network of 14 North American cancer centers. We examined the impact of these processes preand post- implementation and also compared activation times with published clinical trial metrics data.

Methods/Results: 12 MMRC-facilitated trials had sufficient trial data available for review within the study period of May 2006 to March 2009. Activation time was defined as the time from a center's receipt of the final clinical trial protocol from the sponsor, to the time the first patient was enrolled into a trial at any MMRC participating center. The earlier group (EG) of MMRC trials (n=7) required a mean of 257 calendar days for activation, comparable to the mean published data of 251 calendar days1. The more recent group (RG) of MMRC trials (n=5) required a mean of 158 calendar days for activation, representing a 38% shortening of activation time. Additionally, implementation of standardized processes and rigorous project management resulted in a reduction in activation times for all MMRC centers in a given trial from a mean of 7.5 months to a mean of 5.1 months. Improving the mean time of several several specific steps in the process including time to initial protocol review submission from receipt (EG=37 vs. RG=19 days), scientific review committee time (EG=98 vs. RG=55 days), and the time from trial opening to first patient dosed (EG=74 vs. RG=27 days) contributed to the overall decrease in activation times. The EG metrics data are comparable to published data1 for each process step.

Conclusion: The MMRC member institutions have seen substantial improvements in trial activation times and are in the process of implementing additional processes to further improve protocol development and trial conduct metrics. The efficiencies and process improvements realized from application of rigorous MMRC project management resources and the standardization of contracting and other key activities may be applied in other areas of oncology clinical trials beyond multiple myeloma. With increasing numbers of new oncology agents to be evaluated and greater pressure on trial sponsors to introduce efficiencies into trial timelines, overcoming such barriers is of critical importance.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B58.
Abstract B79: Antitumor activity of lapatinib as a single agent and in combination with capecitabine in a HER2-amplified human gastric tumor model
Authors: Liu Li , Avrutskaya Anna , Godfrey Beverly , Britt Carmella C. , Meshaw Alan , Mullin Robert J. , Gilmer Tona
Published: June 2012

HER2 has been reported to be amplified or over-expressed in approximately 20% of gastric tumors and is an attractive target for treatment of gastric cancer. Lapatinib, a potent, oral, small-molecule, dual inhibitor of EGFR and HER2 tyrosine kinases, is approved in combination with capecitabine for the treatment of advanced HER2-positive metastatic breast cancer. It inhibits the phosphorylation of EGFR, HER2, HER3, down stream signaling pathways, and cell growth in HER2-amplified gastric tumor cells in vitro. The preclinical in vivo efficacy of lapatinib monotherapy and the combination of lapatinib with capecitabine, an oral prodrug of 5-fluorouracil was evaluated in the HER2-amplified N87 gastric xenograft model established in female SCID mice. Anti-tumor activities were determined from analyses of tumor growth inhibition (TGI), defined as the decrease in median tumor volume for treated versus vehicle control mice and tumor growth delay (TGD), defined as the increase in median time to end point in treated versus vehicle control mice. Twice daily oral administration of lapatinib resulted in 57% and 62% TGI and 111% and 113% TGD at 50 mg/kg and 65 mg/kg, respectively. Capecitabine monotherapy (dosed once daily at 350 mg/kg for 7 consecutive days, followed by 7 days off and repeated for 2 cycles) inhibited tumor growth by 57% and produced 159% TGD. The combinations of lapatinib (both 50 mg/kg and 65 mg/kg) with capecitabine (350 mg/kg) were well tolerated and resulted in significantly greater tumor volume reduction (71% and 74% TGI, respectively) and tumor growth delay (203% and 179%, respectively) compared to either single agent alone. These data demonstrate that lapatinib is highly efficacious as a single agent and combination of lapatinib with capecitabine is beneficial in HER2-positive gastric tumors, and support ongoing clinical trials of lapatinib, oxaliplatin, and capecitabine in patients with HER2-amplified gastric cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B79.