bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B12: The antitumor and antiangiogenic activity of brivanib, a dual inhibitor of VEGFR-2 and FGFR-1 kinases
Authors: Fargnoli Joseph , Lewin Anne , Henley Benjamin , Jeyaseelan Robert , Wautlet Barri , Hunt John T.
Published: June 2012

Angiogenesis plays a central supporting role in tumorigenesis in a wide variety of different cancer types. This complex and highly regulated process has been shown to be driven by various proangiogenic factors. The vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family of growth factors in conjunction with their associated tyrosine kinase receptors exert a major influence on tumor growth and dissemination, and potentially work synergistically to promote angiogenesis. Brivanib alaninate (BMS-582664) which is currently in phase III clinical trails is the orally active prodrug of brivanib (BMS-540215), a selective dual inhibitor of VEGF and FGF signaling. We demonstrate here that in vivo brivanib provides antitumor activity in a broad range of xenograft models over multiple dose levels, and that brivanib alaninate demonstrates dose-dependent efficacy equivalent to brivanib. Brivanib alaninate also reduces tumor cell proliferation as determined by a significant reduction in Ki-67 staining as well as a decrease in tumor vascular density as reflected by a reduction in CD34 staining, a cellsurface marker of endothelial cells. Additionally, using Matrigel™ plug assays in athymic mice we demonstrated that brivanib alaninate inhibits angiogenesis driven by VEGF or bFGF alone as well as when both of these cytokines are combined to drive angiogenesis in this model. Moreover, brivanib was more effective in inhibiting angiogenesis in this model system under all these conditions when compared to either bevacizumab, an antibody directed at the human VEGF cytokine or DC101, an antibody directed at the murine form of VEGF receptor-2. Dynamic contrast-enhanced magnetic resonance imaging, used to assess the effects of brivanib alaninate on tumor microcirculation, demonstrated a marked decrease in contrast agent uptake at an efficacious dose level, with a reduction in area under the plasma concentration-time curve from time 0 to 60 min (AUC60) at 24 h and 48 h of 54% and 64%, respectively. These results demonstrate that brivanib alaninate is an effective antitumor agent in preclinical tumor and angiogenesis models across a range of doses, and that efficacy is accompanied by changes in cellular and vascular activities.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B12.
Abstract B127: Single or combined in vivo therapies of cancer with h224G11, a humanized antibody targeting the c-Met receptor
Authors: Goetsch Liliane , Gonzalez Alexandra , Geronimi Fabien , Fabre-Lafay Stéphanie , Broussas Matthieu , Lepecquet Anne-Marie , Wurch Thierry , corvaia Nathalie , Bailly Christian
Published: June 2012

c-Met is the prototypic member of a sub-family of receptor tyrosine kinases (RTK) which also includes RON and SEA. The c-Met RTK family is structurally distinct from other RTK families and is the only known highaffinity receptor for the hepatocyte growth factor (HGF), also called scatter factor (SF). While the controlled regulation of c-Met and HGF is essential for in mammalian development, tissue maintenance and repair, their deregulation is implicated in the progression of cancers. Aberrant signalling of c-Met can arise by ligand-dependent and independent mechanisms such as over-expression of c-Met, paracrine or autocrine activations, or through gain-of-function mutations. However, an oligomerization of the c-Met receptor, either in the presence or absence of the ligand, is required to regulate the binding affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide substrates. An inappropriate c-Met activation plays a crucial role in tumorigenesis and metastasis. We have generated, a humanized anti-c-Met IgG1 monoclonal antibody (h224G11) that display fully antagonist activities. The h224G11 Mab exhibits a high affinity for the receptor, blocks HGF binding, inhibits c-Met phosphorylation and c-Met dimerization. These effects are presumably responsible for the inhibition of the major functions of c-Met including cell proliferation, migration, invasion but also cell scattering and angiogenesis, observed in vitro with different cell lines. In addition to these in vitro properties, h224G11 dramatically inhibits in vivo growth of autocrine (U87-MG), partially autophosphorylated (NCI-H441) and c-Met amplified cell lines (MKN45, EBC1 and Hs746T). A dose effect experiment showed that a single i.v. injection of h224G11 resulted in a significant tumor inhibition with an in vivo EC50 about 9 mg/kg. Ex-vivo experiments on the Hs746T gastric tumor, demonstrated that h224G11 down-regulates c-Met, inhibits cell proliferation, induces a massive apoptosis of tumor cells and abrogates c-Met phosphorylation. In addition to this powerful effect as a mono-therapy, an almost complete inhibition of NCI-H441 (NSCLC) tumor growth was observed when mice were treated with the anti-c-Met antibody combined with the tubulin-binding vinca-alkaloid vinorelbine (Navelbine®). Likewise, our results showed that a combined h224G11/temozolomide bi-therapy significantly improved in vivo growth inhibition of U87-MG tumors compared to the corresponding mono therapies. Taken together the in vitro and in vivo data suggest that the h224G11 antibody targeting c-Met is a promising candidate for the treatment of ligand-dependent and ligand-independent tumors as a single or combined therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B127.
Abstract B141: Effects of AZD8055, a novel mTOR kinase inhibitor, in human cancer cells developing resistance to rapamycin
Authors: Serova Maria , Koumaravel Jacqueline , Bieche Ivan , Vidaud Michel , Guichard Sylvie , Raymond Eric , Faivre Sandrine
Published: June 2012

Background: Cancer cells are thought to adapt to rapamycin, an inhibitor of mTORC1, by activating AKT via mTORC2 and others mTORC1-independent survival pathways. In this study, we explored the effects of AZD8055, an ATP competitive inhibitor of mTOR, inhibiting both mTORC1 and mTORC2, in cancer cells with intrinsic and developing resistance to rapamycin.

Materials and Methods: Antiproliferative effects and cellular growth inhibition of AZD8055 and rapamycin were evaluated in a panel of 20 human cancer cell lines by MTT assay. Baseline and phosphorylated protein levels were assessed usingWestern blot. mRNA expression was studied by qRT-PCR.

Results: AZD8055 and rapamycin (48h exposures) displayed antiproliferative effects in our cell panel at IC50s ranging 0.005–>3µM and 0.04–20µM, respectively. CAKI1 and SK-HEP1 cells expressed high mRNA levels of mesenchymal markers (vimentin, N-cadherin, ZEB1, SIP1, SLUG, ACTA2, and HMGA2) and low levels of E-cadherin associated with intrinsic resistance to several receptor tyrosine kinase inhibitors. AZD8055 induced antiproliferative effects in CAKI1 and SK-HEP1 cells at concentrations <0.1µM. In CAKI1 and SK-HEP1 cells exposed to 0.1µM rapamycin for 5–24h, we observed the inhibition of p-S6 Ser235/236 but an activation of p-AKTser473. Consistent with its mechanism of action, exposure to 0.1µM AZD8055 led to the inhibition to p-S6, and p-4EBP1Ser65, without p-AKT ser473 activation. Downstream to mTORC2, PKC Ser657 was transiently activated by both rapamycin and AZD8055 in CAKI1 cells but not in SK-HEP1 cells. To study the acquired resistance to rapamycin, SK-HEP1 cells were grown with increased concentrations of rapamycin for >6 months, making these cells tolerant to ≥4-fold IC50 concentrations. In SK-RAP cells, we observed the transcriptional activation of IGF1R, TGFB2, the anti-apoptotic factors BCL2 and CDKN1Ap21, and the multidrug resistance ABCB1mdr1. In SK-RAP cells, sustained exposure to rapamycin increased pAKTser473 and decreased p-S6 and p-PKC. Consistent with the inhibition of mTOR kinase activity, exposure to AZD8055 for 5h almost completely abrogated rapamycin-induced AKTser473 activation in SK-RAP cells. Furthermore, SK-RAP cells were more sensitive to AZD8055 than to the parental SK-HEP1 cells (IC50: 0.1 versus >3µM, p=0.044), possibly due to the extended functions of AZD8055 against mTORC1 and inhibition of mTORC2.

Conclusions: AZD8055, by inhibiting mTOR kinase activity, decreases mTORC1 substrates pS6 and p-4EBP1Ser65 and mTORC2 substrate AKTser473. AZD8055 decreases cancer cell proliferation in cell lines expressing mesenchymal markers displaying intrinsic and developing resistance to rapamycin, abrogating the hyperactivation of AKT observed in these cells after rapamycin exposure.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B141.
Abstract B151: Epithelial-to-mesenchymal transition (EMT) in resistance to the antiproliferative effects of elisidepsin (Irvalec™), a novel marine-derived depsipeptide, in colon cancer cells
Authors: Serova Maria , Bieche Ivan , Emami Shahin , Gespach Christian , Berx Geert , Vivier Sabrina , Vidaud Michel , Aracil Miguel , Faivre Sandrine , Raymond Eric
Published: June 2012

Background: Elisidepsin (PM02734) belongs to Kahalalide F family and displays antiproliferative effects as single agent and in combinations with both cytotoxic and targeted therapies. This study aimed evaluating the role of EMT in resistance to elisidepsin.

Materials and Methods: Antiproliferative effects of elisidepsin were evaluated in a panel of colon (HT29, HCT116, COLO205, HCC2998), breast (MCF7, MDA-MB-435, SKBR3), ovarian (OVCAR3, IGROV1), lung (Hop62, Hop92), prostate (PC3, DU145), head&neck (SCC61, SQ20B, HEP2), and pancreatic (MiaPaCa2, CAPAN1) cancer cell lines after 72 hour exposure using MTT assay. Expression of several genes was evaluated using quantitative RT-PCR.

Results: Elisidepsin displayed antiproliferative effects against cancer cells with IC50s ranging 0.4’9 µM. As previously reported, cancer cells with high mRNA levels of Erb-B3 also displayed sensitivity to elisidepsin. Consistently, we found that cancer cells displaying an epithelial phenotype, characterized by high levels of mRNA expression of E-cadherin (CDH1), were more sensitive to elisidepsin than mesenchymal cancer cells showing high mRNA and protein levels of vimentin and N-cadherin (CDH2). To further evaluate the role of EMT in resistance to elisidepsin, we studied the antiproliferative effects of elisidepsin in the epithelial colon Colo205-S cells and its derived mesenchymal counterpart Colo205-R cells characterized by the overexpression of the transcription factors SIP1, ZEB1, SNAIL, SLUG, TWIST, and TGFβ. Interestingly, Colo205-S cells were at least 8-fold more sensitive to elisidepsin than Colo205-R cells, IC50s being 0.5 and 4.0µM, respectively. To confirm the role of EMT on resistance to elisidepsin, we used another colon cancer model with inducible SNAIL expression (DLD-1TR21-hSNAIL). In this model, SNAIL induction leads to a mesenchymal phenotype that coincides with loss of aggregation and induction of invasion (EMT). In this model, more than 100-fold SNAIL increase was associated with 12 and 6-fold decrease of E-cadherin and Erb-B3 expression, respectively. Consistent with our hypothesis, we found that the cytotoxicity of elisidepsin was at least 7-fold higher in parental epithelial cells than in SNAIL-induced cells in this model.

Conclusions: Sensitivity to elisidepsin correlates with high Erb-B3 and E-cadherin expressions in colon cancer cells. These data suggest that sensitivity to elisidepsin may be dependent of cancer cell differentiation, better results being observed in cancer cells retaining an epithelial phenotype.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B151.
Abstract B153: Small molecule modulators of tumor-stromal cell interactions, new natural compounds from fungi
Authors: Manabu Kawada , Arakawa Masayuki , Momose Isao , Someno Tetsuya , Ikeda Daishiro
Published: June 2012

Tumor microenvironment is now recognized as one of attractive strategies for cancer therapy. We have been studying molecular targets for tumor-stromal cell interactions and searching for small molecules that modulate the interactions. We have previously reported that angiogenin and IGF-I play critical roles in the development of prostate cancer. Because small molecules that interfere with these targets could suppress tumor growth through tumor-stromal cell interactions, we have designed assay systems and conducted random screening to find active small molecules. Here we report new natural compounds that can modulate the interactions. Terrein glucoside, a new terrein, was isolated from Aspergillus sp. PF1381 as a specific inhibitor of angiogenin secretion. Angiogenin is one of angiogenic factors that confer high tumorigenicity in human prostate cancer LNCaP cells. Terrein glucoside inhibited angiogenin secretion from a tumorigenic LNCaP subline without effect on VEGF secretion and also inhibited tube formation of HUVEC. On the other hand, NBRI23477 A and B, new atpenins, were isolated from Penicillium atramentosum PF1420 by another assay system. Human prostate stromal cells (PrSC) increased the growth of human prostate cancer DU-145 cells in the coculture of both cells through IGF-I secreted from PrSC. NBRI23477 A and B inhibited the growth of DU-145 cells in the coculture with PrSC more strongly than that of DU-145 cells cultured alone suggesting that these compounds indirectly suppress the tumor growth through modulation of tumor-stromal cell interactions. We are now continuing these screening programs to find more potent compounds.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B153.
Abstract B184: HSP90 inhibitor STA-9090 potently suppresses heterogeneous KIT kinase-domain mutations responsible for gastrointestinal stromal tumor progression during imatinib therapy
Authors: Fletcher Jonathan A. , Debiec-Rychter Maria , Swank Stephen , Morgan Jennifer , Liegl Bernadette , Rege Tanya A. , Heinrich Michael C. , Corless Christopher C. , Donsky Rachel , Fletcher Christopher D.M. , Raut Chandrajit P. , Ou Wenbin , Foley Kevin , Wang Yuexiang , Demetri George D.
Published: June 2012

Background: Most GISTs express mutant KIT or PDGFRA oncoproteins, which are targets of tyrosine kinase inhibitors (TKIs), such as front-line imatinib (IM) or second-line sunitinib (SU). GIST clinical resistance to IM or SU is commonly associated with the acquisition of heterogeneous secondary mutations in the KIT/PDGFRA ATP-binding pocket (ABP) or activation loop (AL), which maintain the constitutively activated state of these kinases. We therefore asked whether the heterogeneous IM-resistant KIT oncoproteins in GIST are uniformly HSP90 clients, and whether they can be inhibited by STA-9090, a synthetic small molecule HSP90 inhibitor that is structurally unrelated to the first-generation natural product-derived ansamycin HSP90 inhibitor 17-AAG.

Methods: KIT and PDGFRA were genotyped in up to 15 metastases from each of 10 patients whose metastatic GIST had progressed after IM therapy. Kinase mutants were biochemically profiled for IM and STA-9090 sensitivity in: 1. Ba/F3 cells transformed by mutant KIT constructs; 2. GIST cell lines (IM-sensitive and IM-resistant); and 3. a novel assay measuring inhibition of kinase phosphorylation after drug treatment in GIST48B (KIT-negative) GIST cells transfected with mutant KIT constructs. Drug effects on proliferation, apoptosis and cell cycle were evaluated in five GIST cell lines, including a KIT-dependent GIST subline (GIST882B) that is resistant to 17-AAG.

Results: As many as 8 different secondary KIT IM-resistance mutations (both ABP and AL) were detected in individual patients whose GISTs progressed after IM therapy. All mutations were sensitive to STA-9090. STA-9090 was 5-15 fold more potent than 17-AAG against these IM-resistant KIT secondary mutations, and was at least as effective against the primary + secondary (IM-resistant) mutations in combination, as compared to the primary IM-sensitive mutation alone (Table). STA-9090 also potently inhibited the 17-AAG resistant GIST882B cell line.

Conclusions: STA-9090 was more potent than 17-AAG against a panel of KIT mutations found in TKI-resistant GISTs. STA-9090 potency was undiminished against combination activating KIT mutations (including secondary kinase-domain resistance mutations), as typically occur in IM-resistant GIST clones. Based on these results, we hypothesize that STA-9090 might have broad clinical activity against IM-resistant GIST.

Table: IC50 (nM) for GIST viability: HSP90-inhibition vs. imatinib GIST Line STA-9090 17-AAG Imatinib GIST882: KIT Ex 13 40 200 300 GIST882B 35 >1000 300 GIST430: KIT Ex 11 + V654A (ABP) 20 300 >1000

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B184.
Abstract B188: Inhibition of Notch signaling for the treatment of cancer
Authors: Patel Bharvin K. R. , Bender Mark H. , Capen Andrew R. , Hanson Jeff C. , Credille Kelly M. , Heinz-Taheny Kathleen M. , Pomeroy Julie , Uhlik Mark T. , Perkins Everett J. , Reynolds Vincent L. , Audia James E.
Published: June 2012

The Notch pathway is a highly conserved signaling system that plays an important role in development and tissue homeostasis. While Notch mutations are well characterized and implicated in hematological malignancies such as T-cell acute lymphoblastic leukemia, in solid tumors ligand or receptor over-expression may lead to enhanced/sustained Notch function, triggering increased tumor cell proliferation/survival, angiogenesis and tumor formation. In order to delineate an oncogenic role of activated Notch in tumors of epithelial origin, we carried out a series of in-vitro and in-vivo studies. We demonstrated that the activated Notch1 receptor (a - secretase-dependent Notch1 E with the transmembrane domain and a-secretase-independent constitutively activated Notch1 intracellular fragment) can transform normal rat cells, RK3E. These transformed cells formed colonies in soft agar, confirming their anchorage-independent growth potential, and when implanted subcutaneously, formed tumors in athymic nude mice. Inhibition of Notch signaling through a small molecule inhibitor of -secretase, a key regulator of Notch processing, may provide an attractive targeted cancer therapeutic strategy. We have identified and characterized a novel small molecule that is an exquisitely potent inhibitor of Notch signaling in tumor cell lines and endothelial cells with an IC50 ranging from 0.005 nM to 20 nM. The Notch inhibitor meets all pharmacokinetic criteria in pre-clinical species. In a xenograft tumor model, the novel compound inhibited Notch cleavage in a dose-dependent manner at 6 hours after a single oral dose. This inhibition of Notch cleavage resulted in the induction of apoptosis (as measured by activated caspase-3 levels) that was statistically significant at 24 hours after a single oral dose of 3 mg/kg. Analysis of tumors from animals treated with the Notch inhibitor revealed inhibition of angiogenesis through formation of leaky vasculature which may also contribute to observed anti-tumor activity. Furthermore, Notch inhibition produced tumor regression in the Notch-dependent tumor models. Anti-tumor activity was also observed in several human xenograft tumors of epithelial origin. To mitigate mucoid gasteroentropathy due to Notch inhibition, PK/PD data were incorporated in devising dosing strategies that identified an optimal intermittent dosing schedule without negatively impacting efficacy. Furthermore, the mucoid gastroentropathy was also mitigated by the prophylactic administration of dexamethasone without negatively impacting Notch inhibitor mediated efficacy. In summary, we have characterized an orally bio-available small molecule Notch inhibitor that may provide therapeutic benefit to cancer patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B188.
Abstract B190: Efficacy of the specific ETA receptor antagonist zibotentan (ZD4054) in cancer cells and fibroblasts from colorectal cancer
Authors: Haque S. , Heetun M. , Farooqui N. , Loizidou M. , Dashwood M. , Shiwen X. , Abraham D.
Published: June 2012

Endothelin-1 (ET-1) contributes to growth and progression of solid cancers, mainly through endothelin receptor A (ETAR). Therefore endothelin receptor antagonism is emerging as a potential treatment for neoplasms. We evaluated the efficacy of the specific ETAR antagonist zibotentan (ZD4054) in blocking ET-driven cellular effects in colorectal cancer (CRC). CRC cell lines (HT29, SW620) and primary normal fibroblast strains isolated from human colorectal tissues (CF36, CF56, CF65, CF75) were incubated in ET-1 with/without BQ123, zibotentan (ETAR antagonists), BQ788 (ETBR antagonist). Resultant cell growth was measured by the colourimetric methylene blue assay; migration by a modified monolayer scratch assay; contraction in collagen gels; downstream effectors by western blotting. ET-1 driven growth (18%–45% above control) was significantly inhibited (p<0.01) by ETAR (not ETBR) antagonism (BQ123=zibotentan CRC; fibroblasts). ET-1 driven fibroblast migration and contraction were blocked by ETAR and ETBR antagonism (zibotentan=BQ123). CRC cells did not migrate or contract. ET-1-stimulated expression of downstream effectors was driven by ETAR or ETBR, eg: (1) connective tissue growth factor (CTGF) was blocked by ETAR antagonism (zibotentan>BQ123; CRC and fibroblasts); (2) collagen XI was blocked by ETAR>ETBR antagonism (zibotentan>BQ123; fibroblasts). The specific ETAR antagonist zibotentan is at least as efficacious as BQ123 in blocking ET-1 driven growth, migration and contraction both in CRC cells and colorectal fibroblasts, which form the supporting tumor stroma. Zibotentan is a strong candidate for adjuvant treatment in CRC.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B190.
Abstract B207: Prostate specific membrane antigen is a diagnostic marker of response to allosteric androgen receptor inhibition
Authors: Evans Michael J. , Smith-Jones Peter , Wongvipat John , Larson Steven M. , Sawyers Charles L.
Published: June 2012

Molecular imaging in oncology aims to complement the growing repertoire of chemotherapies with noninvasive diagnostic tools to measure pharmacodynamics and improve predictions of disease response. In prostate cancer, several chemotherapeutic strategies have been developed to inhibit androgen receptor (AR) function, and currently show promise in treating advanced, castration resistant disease. Among these, competitive inhibitors of steroid agonist/receptor interactions have been effectively staged with fluorine-18-fluoro-5α-dihydrotestosterone, an AR agonist compatible with quantitative PET imaging. However, some allosteric inhibitors of AR do not impact agonist/receptor interaction (or AR stability) and currently cannot be evaluated with a quantitative imaging technology. We address this problem here by presenting evidence that prostate specific membrane antigen is a diagnostic marker of allosteric AR inhibition. In culture or in xenograft models of LNCaP, 22Rv1, LAPC4, and VCaP, PSMA is repressed by androgen treatment. Ablation of AR with siRNA inhibits the hormone-mediated suppression of PSMA, directly implicating AR in the mechanism. We further show that PSMA suppression is antagonized by several competitive and allosteric AR inhibitors. Finally, we conducted a pilot study with 22Rv1 xenografts to demonstrate that 64Cu-DOTA-J591, a monoclonal antibody designed for clinical PET imaging of PSMA, quantitatively detects hormone-mediated PSMA suppression. Collectively, these results promote the use of PSMA imaging reagents in the clinic to stage allosteric AR inhibitors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B207.
Abstract B217: Annexin A2 is a novel molecular target for TM601, a peptide with specific tumor targeting and antiangiogenic properties
Authors: Kesavan Kamala , Ratliff Judson , Johnson Eric , Dahlberg William , Jacoby Douglas B.
Published: June 2012

In this study we sought to identify the cell surface target for TM601, a peptide with tumor-targeting and anti-angiogenic effects. The 131I-radiolabeled form of TM601 is currently in clinical trials for having been shown to specifically bind several human tumor types including malignant glioblastoma, colorectal, pancreatic, prostate and non-small cell lung carcinomas. We used a biochemical approach of surface cross-linking followed by affinity-binding assays in conjunction with mass spectrometry and siRNA knock-down to identify and validate the target for TM601 on tumor and vascular endothelial cell. We identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and HUVEC vascular endothelial cells. We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line, Panc-1 is dependent on the expression of annexin A2. Annexin A2 is over-expressed in several human cancers and has functions in angiogenesis by binding to tissue plasminogen activator (tPA) and regulating plasminogen activation on vascular endothelial cells. In HUVEC cells, TM601 inhibits both VEGF and bFGF induced tPA activation which is in turn required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits PDGF-CC induced trans-well migration of both HUVEC vascular endothelial and U373-MG glioma cells. In conclusion, we have identified annexin A2 as a novel cell surface target for TM601 that might mediate both the tumor-targeting and anti-angiogenic effects of the peptide. Importantly, our results also support a role annexin A2 as a molecular target in cancer therapeutics.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B217.
Abstract B221: Expression of EGFR ligands in non-small cell lung carcinoma xenografts as potential biomarkers for classical or targeted therapies
Authors: Fichtner Iduna , Rivera Maria , Rolff Jana , Merk Johannes
Published: June 2012

Increased levels of the epidermal growth factor receptor (EGFR) and its cognate ligands are common in non-small cell lung cancer (NSCLC) and correlate with a poor prognosis. In the last years targeted therapies against the EGFR were developed, but results of clinical trials were rather disappointing. The identification of biomarkers in NSCLC as predictive factors for an individualized therapy is urgently needed.

Twenty six xenografts were established from fresh surgical material from NSCLC patients and characterized with regard to their sensitivity to four cytostatics (Carboplatin, Etoposide, Paclitaxel, Gemcitabine) and two EGFR-inhibitors (Cetuximab, Erlotinib). The expression of EGFR and its ligands epidermal growth factor (EGF), transforming growth factor alpha (TGFα), epiregulin and amphiregulin were examined at mRNA and protein level. Three ligands, which bind exclusively to EGFR (EGF, TGFα and amphiregulin) showed correlations to the mRNA expression of the receptor. Also the mRNA-levels of amphiregulin and epiregulin correlated with each other. The responsiveness towards Etoposide correlated negatively with the EGF-protein (r=-0.479, P<0.05) and with the EGFR-mRNA (r=-0.663, P<0.01) expression. This could mean that the expression of the EGFR-pathway related molecules is able to protect tumor cells from the induction of apoptosis through a cytostatic drug. The response rate to Gemcitabine showed a negative correlation with the TGFα-mRNA level (r=-0.525, P<0.01). Controversially, the amphiregulin-mRNA level correlated positively with the responsiveness to Gemcitabine (r=0.553, P<0.01). A high level of TGFα-mRNA correlated with the resistance to Erlotinib (r=-0.456, P<0.05). The epiregulin-mRNA level showed a positive correlation concerning the sensitivity towards Cetuximab (r=0.476, P<0.05). High EGFR levels seem to be indicative for the resistance to Erlotinib (r=0.636, P<0.05) in squamous cell carcinomas and for the sensitivity to Cetuximab (r=-0.888, P<0.05) in adenocarcinomas.

The results of the present study showed the role of the EGFR activated pathways in the dynamics of tumor progression in NSCLC on the one hand and reflect the high level of redundancy and interrelationships of the EGFR signalling pathway on the other hand. The patient-derived NSCLC models are suitable tools for the identification of biomarkers and their regulation mechanisms in a clinically related and standardized way.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B221.
Abstract B225: Use of RNAi to identify targets for further improving the antitumor activity of gemcitabine and nab-paclitaxel combination therapy in patients with advanced pancreatic cancer
Authors: Xie Lifang , Zhao Yu , Von Hoff Daniel , Han Haiyong
Published: June 2012

Pancreatic cancer remains the fourth leading cause of cancer-related deaths in the United States. Gemcitabine has been used as a standard first-line therapy for more than a decade for this deadly disease. However, the benefit of single-agent gemcitabine in advanced pancreatic cancer is small. Combination regimens including gemcitabine have provided only modest improvement. In a Phase I/II clinical trial gemcitabine plus nab-paclitaxel had significant clinical activity (Von Hoff, et al. 2009, J. Clin. Oncol. 27 (15s): Abstract #4525). To further improve the response of gemcitabine plus nab-paclitaxel therapy, we performed a high throughput RNAi (HT-RNAi) screen with a kinase-focused siRNA library consisting of siRNA oligos against 588 genes (2 siRNAs/gene) in the MIA PaCa-2 pancreatic cancer cell line. Eighty eight siRNAs targeting 82 genes were identified as positive hits (≥2 fold decrease in IC50 values). Several commercially available small molecule inhibitors of the positive hits were obtained and tested in combination with gemcitabine and nab-paclitaxel, both alone or in combination. Among the small molecule inhibitors tested, the CHK1 inhibitor, SB 218078, showed the most significant synergistic effect with gemcitabine and nab-paclitaxel, gemcitabine alone, or nab-paclitaxel alone in two pancreatic cancer cell lines, MIA PaCa-2 and PANC-1. The mechanism of SB 218078 induced sensitivity of pancreatic cancer cells to the gemcitabine and nab-paclitaxel are currently under investigation. In summary, using an HT-RNAi screen we have identified a list of gene targets that, when down-regulated by siRNA oligos, sensitize pancreatic cancer cells to the gemcitabine plus nab-paclitaxel combination therapy. Inhibitors of these targets may potentially enhance the anticancer activity of gemcitabine and nab-paclitaxel. (This work was supported by NIH/NCI Program Project grant CA109552).

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B225.
Abstract B23: Notch-induced Hrb suppresses apoptosis in bone marrow stem cells
Authors: Khwaja Shariq S. , van Deursen Jan , Bram Richard J.
Published: June 2012

Notch is activated by somatic mutation in more than 50% of cases of T-cell acute lymphoblastic lymphoma/leukemia (T-ALL). Exuberant notch signaling drives several oncogenic phenotypes, including enhanced proliferation, improved survival, and impaired differentiation. Inhibition of Notch is likely to yield improved treatment strategies for this disease and certain other malignancies, as well. However, because global inhibition of Notch may have intolerable toxicities, efforts are underway to identify the key downstream mediators of its neoplastic effects. We find that the clathrin-mediated receptor uptake adapter Hrb (HIV-rev binding protein) is a downstream target of Notch1. Hrb protein was induced to high levels by retroviral-mediated expression of constitutively active Notch (ICN1) in bone marrow stem cells and T cell precursors in vitro. To explore a potential role for Hrb downstream of Notch, we have examined the effects of ICN1 on bone marrow progenitor cells (BMPCs) from Hrb-deficient mice. Wild type BMPCs transduced with ICN1 grew to higher density in culture than did Hrbdeficient cells. There was no difference in distribution in cell cycle phases, and proliferative rates appeared to be the same. However, we found a significant increase in apoptosis in Hrb-deficient BMPCs in comparison to cells from littermate control mice. This increase in apoptosis could be reversed by reexpression of Hrb in the mutant cells, thus verifying that Hrb provides an enhanced survival signal to ICN1-expressing bone marrow cells. Potential downstream mediators of this survival signaling cascade are currently being identified. Because resistance to apoptosis is a key feature of leukemic cells, we hypothesize that the pro-survival effect of Hrb provides a portion of the oncogenic properties of Notch. Thus, because Hrb-knockout mice have no evident phenotype, other than male sterility, Hrb may be a useful molecular target for therapy of Notch-dependent malignancies, such as T-ALL.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B23.
Abstract B233: c-Met antagonist Avimer™ polypeptide that exhibits potent in vivo and in vitro inhibitor activity
Authors: Cho Song , Smith Richard , Duguay Amy , Li Peng , Bakker Alice , Alba Ben , Thomas Melissa , To Wayne , Silverman Josh , Swimmer Candace , Moore Kevin , Strout Patrick , Bobofchak Kevin , McCarthy Mike , Pemmaraju Kalyani , Lacourciere Gerard , Zhou Qin , Damschroder Melissa , Wu Herren , Coats Steve , Kamal Adeela
Published: June 2012

c-Met is a receptor tyrosine kinase (RTK) that is activated upon binding to its only known ligand, hepatocyte growth factor (HGF). Although essential in development, aberrant c-Met signaling can lead to oncogenesis by promoting proliferation, survival, metastasis, and angiogenesis. Deregulated expression/activation of cMet is found in many neoplasms, and the linkage to disease severity and poor prognosis make this RTK an attractive candidate for targeted anticancer therapy. Challenges of inhibiting c-Met/HGF signaling with a classic antibody therapeutic approach have included: (1) the inhibition of receptor activation required more than one antibody, and (2) bivalent antibodies caused receptor activation. Using a novel non-antibody protein technology platform, we have identified potent polypeptide inhibitors of HGF-mediated c-Met activation. This non-antibody class of proteins, termed Avimer (avidity multimer) polypeptides, are based on naturally occurring domain (e.g., A-domain) containing proteins and can be generated using a phage display library and expressed in E. coli. c-Met antagonist Avimer polypeptides potently inhibit HGF-mediated receptor activation, migration, and proliferation in in vitro assays. Improvement in pharmacokinetic profile gained through attaching polyethylene glycol molecules to avimers enabled in vivo studies. In multiple preclinical tumor models (U87MG glioblastoma, KP-4 pancreatic, U118 glioblastoma), Avimer-treated animals exhibited reduced cMet activation in xenografts and significantly decreased tumor growth. These data indicate that the c-Met antagonist Avimer polypeptides are potent c-Met inhibitors and offer an alternative to small molecules or antibodies in targeting cMet/HGF pathway for anticancer therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B233.
Abstract B25: Apoptotic signaling in AML cells after therapy with the monoclonal antibody-therapeutic gemtuzumab ozogamicin
Authors: Haag Petra , Lindberg Marita Lagergren , Kanter Lena , Lewensohn Rolf , Stenke Leif , Viktorsson Kristina
Published: June 2012

The aim of this study was to identify essential signaling molecules involved in apoptotic cell death in acute myeloid leukemia (AML) cells after treatment with the targeted drug gemtuzumab ozogamicin (GO).

New effective therapeutic drugs are highly desirable in the treatment of AML. Although a majority of treated patients initially respond to conventional treatment, most patients will relapse within 1–2 years. Also, the treatment is associated with severe off-target effects. GO is a novel therapeutic approach, consisting of a monoclonal CD33 antibody coupled to a DNA damaging toxin, calicheamicin. GO enters the cell after binding to the CD33 antigen, preferentially expressed on myeloid cells, and cause DNA double strand breaks (DSB). We have previously demonstrated that activation of Bak and Bax is critical for GO-induced apoptotic signaling, followed by downstream mitochondrial depolarization and caspase-3 activation. As a continuation, we have now examined potential proapoptotic signaling events induced by GO upstream of mitochondria in HL60 AML cells in vitro. We identified caspase-2 and the pro-apoptotic BH3-only protein Bid as two candidates. By analyzing caspase-2 by western blotting after GO treatment we found that GO caused cleavage of caspase-2 after 24h, which was prior to mitochondria-mediated signaling. At the same time, full-length Bid was cleaved to its truncated, active form tBid. The importance of caspase-2 activation in the signaling cascade after GO treatment was demonstrated using z-VDVAD-fmk, a caspase 2 inhibitor. Preincubation with this inhibitor reduced caspase-3 activation with around 50%, suggesting that caspase-2 is an important molecule, located upstream of caspase-3, in the signaling between DNA double stranded breaks and apoptosis signaling in AML cells. In conclusion, we demonstrate that GO, at clinically applicable concentrations (100–1000ng/ml), induce pro-apoptotic signaling which involve activation of caspase-2, and cleavage of the BH3-only protein Bid to the truncated, active protein tBid. Blocking caspase-2 activity reduced caspase-3 activation to only half. This suggests multiple signaling pathways capable of activating caspase-3. The caspase-2 pathway appears central for downstream caspase-3 activity, after GO treatment in AML. Our findings may highlight possible resistance mechanisms in AML, which might have profound therapeutic implications.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B25.
Abstract B29: SMAC-mimetic induced apoptosis of SKOV3 cells requires NIK and activation of canonical NFκB signaling
Authors: Sullivan John F. , Stegmeier Frank , Porter Dale , Zawel Leigh
Published: June 2012

By stimulating the autoubiquitination and proteosome mediated degradation of CIAP1, Smac mimetics hypersensitize tumor cells to TNFα mediated apoptosis. Indeed, Smac mimetics induce the production of TNFα in sensitive but not insensitive tumor cell lines. The precise mechanism of NFκB activation and subsequent TNFα induction is not well understood, and identifying the mechanism by which LBW242 induces TNFα may provide potential biomarkers for patient stratification. Paradoxically, these agents have been reported to both activate and inhibit the canonical NFκB pathway and to activate the non-canonical pathway.

To elucidate this mechanism, we knocked down nodes of the canonical and non-canonical NFκB pathways and assessed activation of an NFκB/p65-reporter, induction of TNFα expression and cell death after treatment with the Smac mimetic compound LBW242. In the canonical pathway, knockdown of IKKB and p65 ablated TNF induction and rescued SKOV cells from LBW242-mediated cell death, suggesting that activation of the canonical pathway is required for LBW242-mediated cell death. In the noncanonical pathway, knockdown of NIK, but surprisingly not RelB, rescued SKOV cells from LBW242-mediated cell death. This data suggests a role for NIK in LBW242-mediated activation of the canonical but not the non-canonical NFκB pathway. Consistent with this model, we find overexpression of NIK is sufficient to activate the p65 reporter. Moreover, activation of the non-canonical NFκB pathway was robustly observed in both sensitive and insensitive tumor cell lines, further invalidating the contribution of the non-canonical arm to Smac mimetic induced TNF production. We propose NIK-mediated activation of the canonical NFκB signaling is responsible for Smac mimetic induced TNFα production.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B29.
Abstract B60: Combination of tivozanib (AV-951) and temsirolimus in patients with renal cell carcinoma (RCC): Preliminary results from a phase 1 trial
Authors: Fishman Mayer N. , Srinivas Sandy , Hauke Ralph J. , Amato Robert J. , Esteves Brooke , Dhillon Reena , Cotreau Monette , Al-Adhami Mohammed , Bhargava Pankaj , Kabbinavar Fairooz F.
Published: June 2012

Background: Based on preclinical data, a combination of VEGF and mTOR inhibitors (mTORi) may be active in RCC and other tumors. However, it has been difficult to combine VEGF tyrosine kinase inhibitors (TKIs) and mTORi due to toxicity. Clinical trials have demonstrated that VEGF TKI and mTORi can only be combined after reducing the doses of both agents, which may also result in reduced anti-tumor activity of this drug combination. Tivozanib, a potent VEGF receptor 1, 2 and 3 TKI, has demonstrated antitumor activity in a phase 2 study in RCC and may be easier to combine with mTORi due to its selective adverse event profile (Proceedings of ASCO 2009, Abstract no. 5032). The combination of tivozanib and temsirolimus was tested in a phase 1 study.

Methods: Patients (pts) with advanced RCC (with clear cell component) who had failed up to one prior VEGF-targeted therapy received oral tivozanib (3 wks on, 1 wk off) and intravenous temsirolimus (once weekly). Dose levels tested (tivozanib/temsirolimus) were: 0.5mg/15mg, 1.0mg/15mg, 1.5mg/15mg, 1.5mg/25mg. The goals of the study were to determine the maximum tolerated dose (MTD), safety, dose-limiting toxicity (DLT), pharmacokinetics (PK) and clinical activity of this drug combination.

Results: As of September 1, 2009, 15 pts had been enrolled: 15 males/ 0 females, median age 63 yrs (range 43–70), KPS 100% (11 pts) or 90% (4 pts). Nine (60%) pts had received prior VEGF targeted therapy. The median duration of treatment was 8 wks (range 0–40 wks). Based on preliminary data, treatment-related AEs seen in ≥20% pts (No. of pts with all grades/grade 3 toxicity) were thrombocytopenia (5/2), fatigue (5/1), mucositis (4/0), rash (3/0) and stomatitis (3/0). There were no DLTs and no grade 4 toxicities. The MTD of this combination was: tivozanib 1.5 mg and temsirolimus 25 mg. Available data suggest lack of PK interaction between the two agents. One pt had a confirmed PR, and 7 pts had stable disease for ≥ 8 wks.

Conclusions: The combination of tivozanib with temsirolimus is well tolerated in patients with advanced RCC. Tivozanib is the first VEGF TKI that can be combined with temsirolimus at full doses of both agents. Additional patients are being enrolled in the MTD cohort for an expanded safety assessment, and updated results will be available for presentation. An all oral combination of tivozanib and the mTORi everolimus is also being developed for the treatment of RCC and other tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B60.
Abstract B61: Multicenter phase I clinical trial of an insulin like growth factor (IGF-R) inhibitor (IMC-A12) in combination with a mammalian target of rapamycin (mTOR) inhibitor (CCI-779) to overcome resistance to mTOR inhibition
Authors: Naing Aung , Kurzrock Razelle , Chen Helen , Hong David S. , Falchook Gerald , Doyle Laurence , Iawhorn Kristie , Burger Angelika M. , Ng Chann , Amin Hesham , LoRusso Patricia M.
Published: June 2012

Mammalian target of rapamycin (mTOR) inhibitors mediate AKT activation through a type I insulin-like growth factor receptor (IGF-1R)-dependent mechanism, resulting in resistance to mTOR inhibition. Combining an mTOR inhibitor with an IGF-1R antibody/inhibitor is expected to enhance mTORtargeted anticancer therapy. This NCI-CTEP sponsored multicenter phase I clinical trial funded by R-21 mechanism was aimed to define the maximum tolerated dose (MTD), biologic effects, and metabolic activity, individually and in combination, of IMC-A12, a fully humanized IgG1 monoclonal antibody directed at the IGF-IR, and temsirolimus, an inhibitor of mTOR.

Methods: A standard “3+3” design used a fixed dose of 25 mg IV weekly of temsirolimus with an escalating weekly dose of IV IMC-A12 at 3, 4 and 6 mg/kg with no intrapatient dose escalation; 32 patients with advanced cancer (13 male, 19 female) were enrolled across 3 dose cohorts with the current dose cohort at 6 mg/kg weekly IV of IMC-A12 and 25 mg weekly IV of temsirolimus. The MTD has not been reached. Two out of three patients with Ewing sarcoma had more than 20% tumor reduction. One patient with adenocortical carcinoma and one patient with Ewing sarcoma had stable disease for more than 6 months and are still receiving treatment.

Conclusions: Preliminary correlatives data show downregulation of pAKT and pS6K and suggest that the combination is well tolerated, warranting further investigation with dose escalation.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B61.
Abstract B67: Thapsigargin sensitizes multidrug-resistant human ovarian carcinoma cells to doxorubicin
Authors: Lim Chuan-Bian , Ky Nung , Hamza Mohamed Sabry , Zhao Yan
Published: June 2012

Chemoresistance to anticancer drugs is a common and troublesome occurrence in ovarian cancer patients. Despite advances in surgical and chemotherapeutic modalities, survival rates remain alarmingly low. Hence, it has been suggested that strategies for reversing multidrug resistance (MDR) are top priorities for reducing cancer mortality. Thapsigargin (TG), a sesquiterpene lactone isolated from the roots of Thapsia garganica, has been demonstrated to possess anticancer properties. The impact of TG, however, on chemoresistance has not been fully elucidated. Here, we report the effects of combining TG with doxorubicin (DOX), since DOX forms part of the standard treatment for women with advanced ovarian cancer. Multidrug-resistant A2780/MDR human ovarian carcinoma cells were treated with TG in combination with a clinically relevant concentration of DOX (10 µM) and assessed for cell proliferation using the MTT assay. A2780/MDR cells were 100-fold more resistant to DOX than parental A2780 cells on the basis of MTT assay. The IC50 of DOX decreased from 8.02 µM to 0.07 µM, in the presence of 1 µMTG, after 24 h of continuous drug combination exposure, producing a 115-fold reversal of MDR. Flow cytometric analysis of cells treated for 24 h with 10 µM DOX with and without 5 µMTG demonstrated significantly increased apoptosis in the presence of TG. TG by itself had little effect on the viability of A2780/MDR cells, as evident by less than 5% cell death after 24-h TG treatment. The activation of caspases-3 and -9 and PARP cleavage were enhanced in combination therapy relative to single agent treatments. Combined treatment with TG and DOX also increased JNK phosphorylation in A2780/MDR cells and pretreatment with JNK inhibitor SP600125 partially rescued apoptosis. Our data demonstrated that addition of TG at subtoxic concentration restored the sensitivity to DOX-induced apoptosis in A2780/MDR cells via JNK pathway. Taken together, these data support the concurrent use of DOX and TG in ovarian cancer therapeutics.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B67.