Complete Inhibition Of Tumor Growth Research Articles

A multi-stimuli responsive gold nanocage–hyaluronic platform for targeted photothermal and chemotherapy

Noninvasive and pinpointed intracellular drug release that responds to multiple stimulus is still a formidable challenge for cancer therapy. Herein, we reported a multi-stimuli responsive platform based on drug loaded gold nanocages @ hyaluronic acid (AuNCs-HA) for pinpointed intracellular drug release. These well-prepared nanohybrids could specifically recognize cancer cells via HA-CD44 interactions and be efficiently endocytosed by receptor-mediated process. Subsequently, the coated HA molecules could be degraded in lysosomes, resulting in the release of encapsulated drug. In addition, by taking advantage of the excellent photothermal properties, the AuNCs could accelerate the release of encapsulated drug and induce a higher therapeutic efficacy upon near-infrared (NIR) irradiation. In vitro results confirmed that the encapsulated drug could only be pinpointedly released in intracellular environments, which permitted high therapeutic efficacy against cancer cells and minimized the side effects. Importantly, as compared to that of the two therapies independently, a complete inhibition of tumor growth treated with the combination of chemotherapy and photothermal therapy was observed in vivo. Taken together, our present study provides new insights into developing pinpointed, multi-stimuli responsive intracellular drug release systems for synergistic cancer therapy.

PLK1shRNA and doxorubicin co-loaded thermosensitive PLGA-PEG-PLGA hydrogels for osteosarcoma treatment

Combination cancer therapy has emerged as crucial approach for achieving superior anti-cancer efficacy. In this study, we developed a strategy by localized co-delivery of PLK1shRNA/polylysine-modified polyethylenimine (PEI-Lys) complexes and doxorubicin (DOX) using biodegradable, thermosensitive PLGA-PEG-PLGA hydrogels for treatment of osteosarcoma. When incubated with osteosarcoma Saos-2 and MG-63 cells, the hydrogel containing PLK1shRNA/PEI-Lys and DOX displayed significant synergistic effects in promoting the apoptosis of osteosarcoma cells in vitro. After subcutaneous injection of the hydrogel containing PLK1shRNA/PEI-Lys and DOX beside the tumors of nude mice bearing osteosarcoma Saos-2 xenografts, the hydrogels exhibited superior antitumor efficacy in vivo compared to the hydrogels loaded with PLK1shRNA/PEI-Lys or DOX alone. It is noteworthy that the combination treatment in vivo led to almost complete suppression of tumor growth up to 16 days, significantly enhanced PLK1 silencing, higher apoptosis of tumor masses, as well as increased cell cycle regulation. Additionally, ex vivo histological analysis of major organs of the mice indicated that the localized treatments showed no obvious damage to the organs, suggesting lower systemic toxicity of the treatments. Therefore, the strategy of localized, sustained co-delivery of PLK1shRNA and DOX by using the biodegradable, injectable hydrogel may have potential for efficient clinical treatment of osteosarcoma.

Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970.

Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients.

Cutting Edge: Hypoxia-Induced Nanog Favors the Intratumoral Infiltration of Regulatory T Cells and Macrophages via Direct Regulation of TGF-β1

Emerging evidence suggests a link between tumor hypoxia and immune suppression. In this study, we investigated the role of hypoxia-induced Nanog, a stemness-associated transcription factor, in immune suppression. We observed that hypoxia-induced Nanog correlated with the acquisition of stem cell-like properties in B16-F10 cells. We further show that Nanog was selectively induced in hypoxic areas of B16-F10 tumors. Stable short hairpin RNA-mediated depletion of Nanog, combined with melanocyte differentiation Ag tyrosinase-related protein-2 peptide-based vaccination, resulted in complete inhibition of B16-F10 tumor growth. Nanog targeting significantly reduced immunosuppressive cells (regulatory T cells and macrophages) and increased CD8(+) T effector cells in tumor bed in part by modulating TGF-β1 production. Additionally, Nanog regulated TGF-β1 under hypoxia by directly binding the TGF-β1 proximal promoter. Collectively, our data establish a novel functional link between hypoxia-induced Nanog and TGF-β1 regulation and point to a major role of Nanog in hypoxia-driven immunosuppression.

Contribution of ADAMTS1 as a tumor suppressor gene in human breast carcinoma. Linking its tumor inhibitory properties to its proteolytic activity on nidogen-1 and nidogen-2.

The extracellular protease ADAMTS1 (A disintegrin and metalloprotease with thrombospondin repeats 1) has been described as an anti-angiogenic molecule and its role as a putative tumor protective molecule has also been suggested. Here, we have used a tumor xenograft model to determine the role of ADAMTS1 in tumor growth and angiogenesis. Increasing levels of the protease led to the complete inhibition of tumor growth. In an attempt to elucidate the mechanism of action of this protease, we focused our attention on its proteolytic activity on nidogens, one of the main components of the vascular basement membrane. The increased expression of ADAMTS1 was accompanied by increased proteolysis of nidogen-1 and -2 and their almost complete removal from vascular structures, together with major morphological alterations of tumor blood vessels and a decreased vessel density. The clinical relevance of this work is supported by our observations that ADAMTS1 expression is decreased in breast tumor specimens when compared with healthy tissue. Our studies also reveal that the cleavage of nidogen-1 and -2 is partially inhibited in human tumor samples. Moreover, the deposition of both nidogens surrounding vascular structures is drastically altered, implying a possible reduction in the maintenance of vessel integrity. Our studies reflect the requirement to explore the functional interactions between proteases and specific substrates in cancer biology.

Abstract 2337: Efficacy of the RAF/PI3Kα/anti-EGFR triple combination LGX818 + BYL719 + cetuximab in BRAFV600E colorectal tumor models.

Abstract Selective RAF inhibitors have a significant role in the treatment of patients with metastatic melanoma whose tumors express BRAFV600E with the majority of patients experiencing significant tumor regression. However in patients with colorectal cancer (CRC) whose tumors express BRAFV600E, response-rates appear to be much lower, with only a few patients reported to experience a partial tumor response to vemurafenib (Kopetz et al., 2010) or the combination of dabrafenib plus trametinib (Corcoran et al., 2012). LGX818 is a highly potent RAF inhibitor with selective anti-proliferative activity in cells expressing BRAFV600E. A distinguishing feature of LGX818 is its very long dissociation half-life from BRAFV600E which leads to strong and sustained target inhibition even following drug wash-out. In addition, LGX818 has a very wide therapeutic index, with tumor regression observed at doses as low as 3 mg/kg bid and excellent tolerability up to 300 mg/kg bid in melanoma xenograft models. In the CRC cell line Colo205 (BRAFV600E), LGX818 inhibits proliferation with an EC50 = 0.005 μM and in vivo leads to significant tumor regression at doses as low as 20 mg/kg. However, other BRAFV600E CRC cell lines do not appear to be as sensitive (EC50 = 0.018 to >2.7 μM) and this intrinsic resistance translates in vivo in xenograft models generated from these cell lines. Based on clinical and preclinical data, it is clear that combination strategies will be required if RAF inhibitors are going to play a significant role in the treatment of CRC. Recent publications have implicated feedback-mediated activation of EGFR in BRAFV600E CRC cells treated with RAF and MEK inhibitors and this led us to test combinations of LGX818 with anti-EGFR therapies such as cetuximab and erlotinib. In vitro and in vivo studies indicated that the combination of LGX818 with cetuximab or erlotinib can be highly synergistic, leading to enhanced cell killing. In vivo the combination of LGX818 + cetuximab led to complete inhibition of tumor growth at doses where no single-agent activity was observed. Since PIK3CA mutations often co-occur with BRAFV600E in CRC tumors, we also tested the alpha-selective PI3K inhibitor BYL719 in combination with LGX818 and cetuximab as a triple combination. In vitro, this triple combination produced synergistic anti-proliferative effects and in vivo resulted in tumor regression. These preclinical data led to the initiation of an ongoing Phase I/II trial to evaluate the triple combination of LGX818, cetuximab and BYL719 in BRAFV600E CRC. Based on the preclinical results described above it is anticipated that the combination of a RAF inhibitor with anti-EGFR therapy and a PI3K inhibitor will have superior efficacy to single-agent RAF inhibitor in BRAFV600E CRC. Citation Format: Giordano Caponigro, Z A. Cao, Xiaobin Zhang, Hui Q. Wang, Christine M. Fritsch, Darrin D. Stuart. Efficacy of the RAF/PI3Kα/anti-EGFR triple combination LGX818 + BYL719 + cetuximab in BRAFV600E colorectal tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2337. doi:10.1158/1538-7445.AM2013-2337

Abstract 85: Endocrine resistant luminal breast cancer xenografts are powerful models for the analysis of sensitivity to endocrine and everolimus treatments .

Abstract Background. Activation of the PI3K pathway is associated with resistance to endocrine treatments (ET) in luminal breast cancer (LBC). Everolimus, an mTOR inhibitor, has been approved in combination with exemestane in endocrine resistant breast tumors. However, no definite signature of ET resistance or predictive factors of sensitivity to everolimus (EVE) treatment have been evidenced so far. In order to further decipher these molecular patterns, we used patient derived LBC xenografts (LBC-PDX) treated with various ET and EVE based combinations. Methods. 3 LBC-PDX (named HBCx-3, HBCx-21 and HBCx-34) were treated with ET during several months to establish hormono-resistant (HR) xenografts. Both parental and HR models were treated by tamoxifen, estrogen deprivation, fulvestrant, EVE alone, and EVE combined with all three ET modalities. Tissue Micro Arrays from control and treated tumor samples were generated for IHC analyses. RT-PCR analyses were conducted on genes related to proliferation, ER, PI3K, IGF-1R and angiogenesis pathways. Results. In primary PDX, ET showed a poor level of sensitivity for HBCx-3 and a high and sustained efficacy of ER targeting and/or ER deprivation for HBCx-21 and HBCx-34 tumors. In vivo resistance to ET was confirmed in all HR variants with tumor growth rates faster than parental xenografts. ER expression was conserved in HR tumors, but expression of the ER-responsive genes PS2, PR and MYB was strongly decreased, indicating impaired ER transcriptional activity. Acquired resistance to tamoxifen and ovariectomy was also associated to a strong decrease of IGF-1R signaling in HBCx-21, and was related to higher P-AKT expression, although P-pS6 was highly expressed in both sensitive and resistant tumors. Furthermore, in vivo experiments showed that EVE alone was highly efficient in all models independently of P-AKT expression/PTEN loss. EVE combined with fulvestrant yielded the most complete and durable tumor growth inhibition. In HBCx-3, proliferation and pS6 levels were markedly decreased only by the EVE-fulvestrant combination. In HBCx-21 and HBCx-34 HR models, EVE-based treatments decreased proliferation, expression of angiogenesis markers, as well as P-pS6 expression with no variation in (P-)AKT nor (P-)mTOR levels. A high IGF-1R protein expression was found only in the HBCx-34 TAM-R tumor, which was strongly decreased in the fulvestrant-treated tumors. Conclusions. LBC-PDX with confirmed HR status have been established and may serve as models to study alternate therapies. Of note, in the HBCx-21 and -34 models, activation of the PI3K pathway is poorly correlated with baseline sensitivity to ET, suggesting other pathways of resistance. EVE alone is highly efficient in all settings, and combination with fulvestrant is promising at the in vivo and molecular levels. Proteomic, genomic and gene expression studies are ongoing. Citation Format: Paul H. Cottu, Thomas Bagarre, Jean-Jacques Fontaine, Franck Assayag, Sophie Richon, Sophie Chateau-Joubert, Aurélie Thuleau, Patricia de Cremoux, Khemaies Slimane, Didier Decaudin, Anne Vincent-Salomon, Ivan Bièche, Elisabetta Marangoni. Endocrine resistant luminal breast cancer xenografts are powerful models for the analysis of sensitivity to endocrine and everolimus treatments . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 85. doi:10.1158/1538-7445.AM2013-85

A recombinant adenovirus vector encoding the light chain of human coagulation factor VII and IgG1 Fc fragment to targeting tissue factor for colorectal cancer immunotherapy in the mouse model

Over expression of tissue factor (TF) occurs in more than 50 % of colorectal cancer (CRC). Therefore, TF represents an attractive target antigen for immunotherapy in CRC. Here, we assessed the safety and efficacy of a recombinant adenovirus vector encoding the light chain of human coagulation factor VII (hfVII-LC) and human IgG1 Fc fragment (hIgG1-Fc), termed "benc vector," by targeting TF in the mouse model with colon cancer. Benc vector was administered intravenously or intratumorally in SCID mice with TF over-expressing HT-29 colon cancer. The safety and efficacy of benc vector were observed during animal experiments. Complete inhibition of tumor growth (5/5) was observed not only in the intravenously injection of benc vector group but also in the intratumorally of benc vector group. We also observed a precautionary effect on lung metastases of HT-29 cells by intratumoral injection of benc vector. In the control group of animals given empty control vector, all animals (5/5) developed lung tumors and exhibited a higher number of nodules after injection with HT-29 cells via the tail vein. In contrast, only three animals (3/5) in the treatment group receiving benc vector had any observable lung metastases and a lower number of nodules. No animals died and no bleeding was observed both in treatment groups and control groups. Moreover, only moderate liver damage was detected in mice receiving benc vector by intravenous injections. Benc vector encoding hfVII-LC and hIgG1-Fc can effectively inhibit tumor growth and metastases in SCID mice with TF over-expressing colon cancer and shows promise as an agent for CRC immunotherapy.

Combined near-IR photothermal therapy and chemotherapy using gold-nanorod/chitosan hybrid nanospheres to enhance the antitumor effect.

Motivated by the enhanced cytotoxicity of some chemotherapeutic agents at temperature rise, multifunctional chitosan nanospheres which co-carried gold nanorods and cisplatin were prepared. These hybrid nanospheres were characterized by dynamic light scattering, zeta potential, UV-Vis absorption spectra and transmission electron microscopy. It was demonstrated that these 120 nm-sized hybrid nanospheres could be selectively accumulated at the tumor site with about 11% injection dose per g of tumor, and produced local hyperthermia to an average temperature of 49 °C in tumor tissue after near-IR irradiation for 10 min. Due to the enhanced cytotoxicity of cisplatin at elevated temperatures, cisplatin-loaded hybrid nanospheres showed about one-second lower IC50 values than hybrid nanospheres alone in vitro and almost complete tumor growth inhibition in vivo. Compared with chemotherapy or photothermal treatment alone, the combined photothermal therapy and chemotherapy had a significantly synergistic effect and improved the therapeutic efficacy, which was supported by immunofluorescence staining and in vivo apoptosis imaging.

A T Cell-Engaging CD3 Recruit-Tandab Potently Kills CD19+ Tumor B Cells

Abstract 3721 Background:CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies. The CD3 RECRUIT-TandAb AFM11, a humanized bispecific tetravalent antibody with two binding sites for both CD3 and CD19, is a novel therapeutic for the treatment of NHL that harnesses the cytotoxic nature of T cells. Methods:We engineered a bispecific anti-CD19/anti-CD3e tetravalent TandAb with humanized and affinity-matured variable domains. The TandAb's binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. In vivo efficacy was evaluated in a murine NOD/scid xenograft model reconstituted with human PBMC. Results:AFM11 mediates highly potent CD19+ tumor cell lysis in cytotoxicity assays performed on a panel of cell lines (JOK-1, Raji, Nalm-6, MEC-1, VAL, Daudi) and primary B-CLL tumors: EC50 values are in the low- to sub-picomolar range and do not correlate with the expression density of CD19 on the target cell lines. The cytotoxic activity of tetravalent AFM11 is superior to that of alternative bivalent antibody formats possessing only a single binding site for both CD19 and CD3. High affinity binding of AFM11 to CD19 and to CD3 is essential for efficacious T cell recruitment. Both CD8+ and CD4+ T cells mediate cytotoxicity however the former exhibit much faster killing. We observe that AFM11 displays similar cytotoxic efficacy at different effector to target ratios (from 5:1 to 1:5) in cytotoxicity assays; this suggests that T cells are engaged in the serial killing of CD19+ target cells. In the absence of CD19+ target cells in vitro, AFM11 does not elicit T cell activation as manifested by cytokine release (from a panel of ten cytokines associated with T cell activation), their proliferation, or their expression of activation markers. AFM11 activates T cells exclusively in the presence of its targets and mediates lysis of CD19+ cells while sparing antigen-negative bystanders. In the absence of CD19+ target cells, AFM11 concentrations in excess of 500-fold over EC50 induce down-modulation of the CD3/TCR complex. Yet, AFM11-treated T cells can be re-engaged for target cell lysis. All of these features of AFM11-induced T cell activation may contribute additional safety without compromising its efficacy.In vivo AFM11 demonstrates a robust dose-dependent inhibition of subcutaneous Raji tumors in mice. At 5 mg/kg AFM11 demonstrates a complete suppression of tumor growth, and even at 5 ug/kg tumor growth is reduced by 60%. Moreover, we observe that a single administration of AFM11 produces inhibition of tumor growth similar to that of 5 consecutive administrations. Conclusions:In summary, our in vitro and in vivo experiments with AFM11 demonstrate the high potency and efficacy of its anti-tumor cytotoxicity. Thus, AFM11 is a novel highly efficacious drug candidate for the treatment of B cell malignancies with an advantageous safety profile. Disclosures:Zhukovsky:Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment.

Novel Alginate-Enclosed Chitosan–Calcium Phosphate-Loaded Iron-Saturated Bovine Lactoferrin Nanocarriers for Oral Delivery in Colon Cancer Therapy

To develop polymeric-ceramic nanocarriers (NCs) in order to achieve oral delivery of the anticancer neutraceutical iron-saturated bovine lactoferrin (Fe-bLf) protein. Fe-bLf or paclitaxel (Taxol®) were adsorbed onto calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate. The Fe-bLf or Taxol-loaded NCs indicated as AEC-CP-Fe-bLf or AEC-CP-Taxol NCs, respectively, were made by combination of ionic gelation and nanoprecipitation. Size distribution, morphology, internalization and release profiles of the NCs were studied along with evaluation of in vitro and in vivo anticancer activities and compared with paclitaxel. AEC-CP-Fe-bLf NCs obtained spherical morphology and showed enhanced endocytosis, transcytosis and anticancer activity in Caco-2 cells in vitro. AEC-CP-Fe-bLf NCs were supplemented in an AIN 93G diet and fed to mice in both prevention and treatment human xenograft colon cancer models. AEC-CP-Fe-bLf NCs were found to be highly significantly effective when given orally, as a pretreatment, 1 week before Caco-2 cell injections. None of the mice from the AEC-CP-Fe-bLf NC-fed group developed tumors or showed any signs of toxicity, while the mice fed the control AIN 93G diet showed normal tumor growth. Fe-bLf or Taxol, when given orally in a diet as nanoformulations post-tumor development, showed a significant regression in the tumor size with complete inhibition of tumor growth later, while intratumoral injection of Taxol just delayed the growth of tumors. The pharmacokinetic and bioavailability studies indicated that nanoformulated Fe-bLf was predominantly present on tumor cells compared to non-nanoformulated Fe-bLf. Fe-bLf-loaded NCs were found to help in absorption of iron and thus may have utility in enhancing the iron uptake during iron deficiency without interfering with the absorption of calcium. With the promising results of our study, the future potential of NC-loaded Fe-bLf in chemoprevention and in the treatment of human colon cancer, deserves further investigation for translational research and preclinical studies of other malignancies.

Thermal Targeting of an Acid-Sensitive Doxorubicin Conjugate of Elastin-like Polypeptide Enhances the Therapeutic Efficacy Compared with the Parent Compound In Vivo

Elastin-like polypeptides (ELP) aggregate in response to mild hyperthermia, but remain soluble under normal physiologic conditions. ELP macromolecules can accumulate in solid tumors because of the enhanced permeability and retention effect. Tumor retention of ELPs can be further enhanced through hyperthermia-induced aggregation of ELPs by local heating of the tumor. We evaluated the therapeutic potential of ELPs in delivering doxorubicin in the E0771 syngeneic mouse breast cancer model. The ELP-Dox conjugate consisted of a cell-penetrating peptide at the N-terminus and the 6-maleimidocaproyl hydrazone derivative of doxorubicin at the C-terminus of ELP. The acid-sensitive hydrazone linker ensured release of doxorubicin in the lysosomes/endosomes after cellular uptake of the drug conjugate. ELP-Dox dosed at 5 mg doxorubicin equivalent/kg, extended the plasma half-life of doxorubicin to 5.5 hours. In addition, tumor uptake of ELP-Dox increased 2-fold when hyperthermia was applied, and was also enhanced compared to free doxorubicin. Although high levels of doxorubicin were found in the heart of animals treated with free doxorubicin, no detectable levels of doxorubicin were found in ELP-Dox-treated animals, indicating a correlation between tumor targeting and reduction of potential cardiac toxicity by ELP-Dox. At an optimal dose of 12 mg doxorubicin equivalent/kg, ELP-Dox in combination with hyperthermia induced a complete tumor growth inhibition, which was distinctly superior to free drug that only moderately inhibited tumor growth. In summary, our findings show that thermal targeting of ELP increases the potency of doxorubicin underlying the potential of exploiting ELPs to enhance the therapeutic efficacy of conventional anticancer drugs.

Abstract 1919: Pharmacological characterization of BI 847325, a dual inhibitor of MEK and Aurora kinases

Abstract The RAS-dependent MAP kinase signaling pathway plays an important role in the regulation of cell proliferation and survival. RAS genes are frequently mutated in human cancer; however, it has not been possible to date to design direct inhibitors of RAS proteins. Inhibitors of the downstream kinase MEK are active against a subset of KRAS-mutant cancers in preclinical studies, but have shown limited success to date in clinical trials. We have identified a compound that potently inhibits MEK as well as Aurora kinases, a family of serine/threonine kinases involved in the regulation of mitosis. In enzymatic assays, BI 847325 inhibited the activity of Aurora A, B and C with IC50 values of 3, 25 and 15 nM, respectively; IC50 values of 25 and 4 nM were determined for MEK1 and MEK2, respectively. To determine whether the enzyme profile translates into cellular activity we used Western blot and FACE-ELISA assays of phospho-histone H3 (pHH3) and phospho-ERK (pERK) levels in cells treated with BI 847325 and observed EC50 values of 44 nM (NCI-H460 cells, KRAS and PI3Kα mutant) and 37 nM (A375 cells, BRAF mutant), respectively. In vitro profiling in a panel of 240 cell lines with diverse tissue origin and genetic background demonstrated that BI 847325 is a potent inhibitor of cell proliferation (gm GI50 = 28 nM) and induces cell death in a subset of cell lines. In vitro potency significantly correlated with mutations in RAS or BRAF. For comparison, a significant correlation of mutation status and sensitivity was also observed for a selective MEK inhibitor, AZD6244, but not for a selective Aurora inhibitor, BI 811283. In vivo efficacy was studied in nude mouse xenograft models of NSCLC (Calu-6, mutant KRAS) and cutaneous melanoma (A375, mutant BRAF). A daily oral dose of 10 mg/kg resulted in complete inhibition of tumor growth in the Calu-6 model (TGI = 102%, regression in 4/7 animals) and the A375 model (TGI = 116%, regression in 7/7 animals). Inhibition of Aurora B and MEK was monitored ex vivo by determining the phosphorylation state of histone H3 and ERK1/2 in tumor tissue. Immunohistochemical analyses confirmed a significant reduction of both pERK and pHH3 levels in the A375 tumors of treated animals compared to controls. To further profile the mode of action of the compound, the efficacy of BI 847325 in the MIAPaCa2-pancreatic adenocarcinoma model (mutant KRAS) was directly compared with that of the MEK inhibitor GSK 1120212. BI 847325 effectively induced tumor regression (TGI on day 22 = 126%) that was maintained in all animals on day 43. In contrast, treatment with GSK 112021 resulted in initial tumor regression, followed by re-growth after 3-4 weeks (TGI on day 22 = 129%; regrowth on day 43 in 5/7 animals). Inhibition of Aurora kinase in addition to MEK blockade thus may prevent/delay onset of resistance. Additional xenograft models with diverse genetic background are currently under investigation. Clinical development of BI 847325 has been initiated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1919. doi:1538-7445.AM2012-1919

Abstract LB-264: Axitinib improves radiotherapy for murine xenograft lung tumors

Abstract Biological agents are currently being tested to improve the outcome of chemo-radiotherapy for locally advanced non-small cell lung cancer (NSCLC). The anti-angiogenic drug axitinib (AG-013736, Pfizer), is a small molecule receptor tyrosine kinase (RTKs) inhibitor. Axitinib selectively targets VEGF receptors 1, 2 and 3 (VEGFR), has a high potency for VEGFR-2 and inhibits the signal transduction induced by VEGF binding to VEGFR. Axitinib used in a phase II trial for advanced NSCLC demonstrated a promising one-year survival rate with manageable toxicities. We have tested the combination of axitinib with radiotherapy in a pre-clinical model of orthotopic A549 model of human lung carcinoma xenografts in nude mice. Mice-bearing established A549 lung tumor nodules were treated orally and daily with axitinib at 25mg/kg/day concomitant with daily fractionated radiation for 5 consecutive days. Radiation was administered to the left lung at a dose 2 Gy per day and then, axitinib treatment was continued for 5 days per week for 4 more weeks. A mild decrease in overall weight was observed in mice treated with axitinib or axitinib and radiation indicating that axitinib was safe and well tolerated, probably as a result of its short-half life (2-5h). No systemic effect on the vasculature of other organs including the heart, kidney and liver was observed by histology. Axitinib caused decreased tumor cellularity in lung nodules associated with a heavy infiltration of inflammatory cells. Mice treated with axitinib and radiation had a significant decrease in the size and number of lung tumor nodules which contained degenerating giant tumor cells and inflammatory infiltrates. Morphometric measurements of tumor nodules confirmed that the size of tumor nodules was significantly smaller in the irradiated left lung and was further reduced by combination with axitinib compared to that of mice treated with single modality (p<0.05). Ki-67 staining showed a marked decrease in the number of proliferating tumor cells. In the surrounding lung tissue, scattered intra-alveolar hemorrhage was estimated to cover 10 to 20% of the lung surface in treated mice but it was comparable to that observed in untreated mice. Axitinib caused mild chronic interstitial pneumonia and combined axitinib and radiation treatment was associated with moderate interstitial pneumonia. In independent experiments, treatment of lung tumors with a single dose of radiation (10 Gy) administered to the lungs and 10 weeks of axitinib showed a complete inhibition of tumor growth in the lungs. Histology showed the presence of distinctive lymphohistiocytic nodules with no detectable viable tumor cells. This treatment was well tolerated with minimal weight loss. These findings suggest a potential novel approach for the treatment of locally advanced NSCLC by combining radiotherapy with the anti-angiogenic drug axitinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-264. doi:1538-7445.AM2012-LB-264

Abstract 2718: REGN1400, a fully-human ERBB3 antibody, potently inhibits tumor growth in preclinical models, both as a monotherapy and in combination with EGFR or HER2 blockers

Abstract ERBB3 is a member of the ERBB family of receptor tyrosine kinases, that includes EGFR and HER2, well-established drivers of tumorigenesis. Recent preclinical studies indicate that both EGFR and HER2 cooperate with ERBB3 to activate the phosphatidylinositol-3 kinase/Akt survival pathway and to promote tumor cell survival. Furthermore, ERBB3 activation has been demonstrated to mediate resistance to EGFR inhibitors in non-small cell lung cancer cell lines and to HER2 inhibitors in breast cancer cell lines. Consistent with these findings, anti-ERBB3 monoclonal antibodies have been shown to inhibit the growth of human tumor xenografts, demonstrating the importance of ERBB3 for tumor growth in vivo. REGN1400 is a fully-human anti-ERBB3 monoclonal antibody that binds human ERBB3 with high affinity (KD ∼50pM) and potently inhibits binding of the ERBB3 ligand neuregulin 1 (IC50 ∼30pM). REGN1400 inhibited phosphorylation of ERBB3 and Akt in multiple human tumor cell lines in vitro, including A431 (epidermoid carcinoma), MDA-MB-175-VII (breast cancer) and FaDu (head and neck cancer). In addition, REGN1400 strongly inhibited the growth of these cell lines in vitro. Consistent with its potent effects on tumor cell growth in vitro, REGN1400 strongly inhibited the growth of A431 and FaDu tumor xenografts in a dose-dependent manner, providing 70% and 97% tumor growth inhibition, respectively. Inhibition of FaDu tumor growth by REGN1400 was accompanied by a significant decrease in tumor ERBB3 phosphorylation, as assessed by both western blot and immunohistochemistry, confirming target inhibition in vivo. While treatment of FaDu tumors with single agent REGN1400 provided almost complete tumor growth inhibition, but not regression, the combination of REGN1400 plus anti-EGFR antibody caused significant tumor regression. Furthermore, combination treatment with REGN1400 plus the anti-HER2 antibody trastuzumab inhibited the growth of BT474 breast tumor xenografts more potently than either single agent. These findings indicate that ERBB3-directed therapies might be more effective when combined with agents that inhibit other ERBB family members. Finally, gene profiling studies in REGN1400-treated tumor cells revealed upregulation of several genes that could promote resistance to REGN1400, e.g., ERBB3 itself and ERBB4. Further analysis of tumor gene expression changes induced by REGN1400 could provide insight into potentially useful combination regimens. In summary, REGN1400, a novel anti-ERBB3 antibody, exhibits significant antitumor activity in preclinical models, suggesting the possibility that it could provide benefit to patients with multiple types of cancer, either as a single agent or when combined with therapies targeting other ERBB family members. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2718. doi:1538-7445.AM2012-2718

A thermosensitive liposome prepared with a Cu2+ gradient demonstrates improved pharmacokinetics, drug delivery and antitumor efficacy

Here we report the development of an enhanced thermosensitive formulation composed of DPPC and Brij78, loaded with doxorubicin (DOX) using a Cu2+ gradient and post-inserted with an additional amount of Brij78. This optimal formulation (HaT-II: Hyperthermia-activated cytoToxic) displayed significantly improved stability in serum at 37°C, and enhanced drug release rates at 41–42°C, compared to LTSL (lyso-lipid temperature sensitive liposomes, DPPC/MSPC/DSPE-PEG2000=86/10/4, pH gradient drug loading). HaT-II released 100% DOX within 15–40s at 40–42°C, with only 5% drug leakage at 37°C after 30min in serum, while LTSL lost 30% of its drug content at 37°C and exhibited ~2-fold decreased release rate constants at 41–42°C under the same conditions. The pharmacokinetics of DOX was significantly improved in non-heated HaT-II treated healthy mice with 2.5-fold increased area under the curve and 2-fold prolonged circulation half life compared to LTSL. This led to 2-fold improved drug delivery to the heated tumor by HaT-II (~20% injected dose/g tissue), relative to LTSL and significantly enhanced antitumor efficacy with complete inhibition of tumor growth after a single dose of HaT-II. Finally, HaT-II exhibited little toxicity in mice, inducing no body weight loss and no abnormality in the blood chemistry (10mg DOX/kg).

Abstract IA17: Functional genomics, small RNAs and cancer

Abstract Efforts to sequence cancer genomes have begun to uncover comprehensive lists of genes altered in cancer. Unfortunately, the complexity and sheer volume of data that emerges from these efforts has made dissecting the underlying biology of cancer difficult. In addition, the number and types of candidate targets that require functional validation in vivo represent a significant barrier to using this information as a starting point for drug development programs, since many do not represent targets amenable to traditional small molecule or antibody-based therapeutic approaches. To address this problem, we have combined comprehensive analyses of cancer genomes with novel materials science to develop a platform that enables the ready in vivo validation of cancer targets. Specifically, we have developed a screening platform to perform loss of function screens at genome scale and have applied this in a massively parallel manner to screen several hundred full characterized cancer cell lines. We have combined the outputs of these screens with data emerging from the study of cancer genomes to identify candidate oncogenes. To credential these targets in vivo, we have helped develop novel nanocomplexes formed from siRNA bound to tandem peptides containing tumor-penetrating, endosomal escape and siRNA-binding domains that permit deep penetration in tumor. We use these nanoparticles to target a hitherto undiscovered ovarian cancer oncogene, ID4, an undruggable transcriptional regulator, and show that these tumor-penetrating nanocomplexes induced complete suppression of tumor growth and significant improvement of survival. Taken together, these studies provide a platform for the discovery and initial validation of candidate cancer targets. Citation Format: William C. Hahn. Functional genomics, small RNAs and cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr IA17.

Abstract A121: Direct SH2 domain-targeting inhibitors of Stat3: Potent anticancer agents and mitigators of drug resistance.

Abstract As a master regulator of cell signaling and tumorigenesis, signal transducer and activator of transcription 3 (Stat3) protein has emerged at the forefront of anti-cancer drug development. Abnormal Stat3 activity has been demonstrated in a wide variety of human cancers including leukemia, lymphoma, multiple myeloma, glioblastoma and cancers of the pancreas, breast, prostate, and ovary. Constitutive Stat3 activation interferes with normal cell cycling and causes the accumulation of anti-apoptotic proteins. This renders malignant cells resistant to naturally occurring apoptotic cues and allows them subject to proliferate rapidly. Cancer cells become reliant on increased levels of Stat3 activity are vulnerable to therapeutic intervention through Stat3 inhibition. In healthy cells, Stat3 activity is transient and non-essential, thus, inhibiting Stat3 presents an avenue for the development of novel cancer therapeutic agents. Our approach involves the interruption of several critical Stat3 functions by occupation of the SH2 domain with small molecule inhibitors. Stat3's SH2 domain is a key component in the Stat3 signaling pathway as it not only facilitates activation of monomeric Stat3 but also moderates the formation of the transcriptionally active Stat3:Stat3 homodimer. Our research groups have conducted a thorough structure-activity relationship on a known Stat3-SH2 domain binder (S3I-201) and have discovered several more potent and more drug-like Stat3 inhibitors. Most recently, we have utilized a tetrapodal scaffold that has allowed more complete occupation of the SH2 domain and resulted in greatly improved binding affinity. These novel compounds effectively displace an SH2 domain-binding peptide probe, prevent Stat3 phosphorylation in cell line models and suppress Stat3 target gene expression at near-nanomolar concentrations. Our latest Stat3 inhibitors are effective across a wide variety of human cancers and exhibit a 10–20-fold improvement in cellular EC50 values over the parent compound, S3I-201. Remarkable activity is observed in mouse xenograft models of human breast cancer where nearly complete inhibition of tumor growth is observed at a dosing of 3 mg/kg daily. Furthermore, recent experiments demonstrate the same potent activity when the drug is administered by oral gavage, with plasma drug concentrations reaching 20 μM. Preliminary investigations have also shown that our lead compounds can re-sensitize malignant cells that are resistant to conventional chemotherapeutics agents. We present our newest library of Stat3 inhibitors, which holds great promise in the fight against cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A121.

Abstract B137: Pharmacokinetics, antitumor activity, and therapeutic index of Nampt inhibitor MPC-8640 in mice.

Abstract Background: MPI-0487316 is a potent and orally bioavailable small molecule inhibitor of nicotinamide phosphoribosyltransferase (Nampt), an enzyme which catalyzes the rate-limiting step for synthesis of NAD from nicotinamide. Inhibition of Nampt by MPI-0487316 results in cell death as a consequence of NAD depletion and inhibition of ATP synthesis. We have previously reported that MPI-0487316 can induce regressions in a xenograft model. MPC-8640, a prodrug of MPI-0487316, was developed to increase solubility for improved formulation. Myrexis has recently initiated preclinical development of this prodrug and here we present data on the pharmacokinetic and anti-tumor activity of MPC-8640 in mice. Methods: MPI-0487316 concentration in plasma was measured using LC-MS/MS. For xenograft studies, HT1080 human fibrosarcoma cells were implanted subcutaneously into nude mice and mice were administered vehicle or MPC-8640 by oral gavage at the times and doses indicated. Results: Oral administration of MPC-8640 to mice resulted in substantial plasma concentrations of the active moiety MPI-0487316 with increasing AUC and Cmax over a wide range of doses. MPC-8640 concentration itself was negligible in plasma when dosed orally at 300 mg/kg. MPC-8640 demonstrated strong activity in the HT1080 human fibrosarcoma xenograft model when dosed qd or bid for one to two weeks. After bid dosing for one week, complete tumor growth inhibition (TGI) was observed at 6 mg/kg and substantial regression at 10 mg/kg. There was no difference between responses after seven or 14 doses bid. For qd dosing, complete tumor growth inhibition required 20 mg/kg MPC-8640 and ≥24 mg/kg for tumor regression. The anti-tumor response seen at the end of seven days of qd dosing was subsequently maintained for at least one week. TGI was observed with three or four doses qd, but with lesser potency than for five or more consecutive qd doses. In studies to determine maximum tolerated dose, 98% of mice survived up to 90 mg/kg MPC-8640 qd for one week, whereas only 60% survived doses >90 mg/kg. Conclusions: Oral MPC-8640 is an effective prodrug in mice for systemic delivery of its active moiety Nampt inhibitor MPI-0487316. The lack of significant plasma concentrations of MPC-8640 indicates that the prodrug is effectively converted to active moiety in the gut or immediately after absorption and that anti-tumor activity of MPC-8640 against subcutaneous xenografts is through its active moiety. A one week on/one week off, daily dosing schedule appears optimal, since one week of dosing, either qd or bd, was maximally effective and the anti-tumor response was sustained for at least one week after the end of dosing. Tumor regressions were induced at doses of MPC-8640 well below its maximum tolerated dose, providing promise that MPC-8640 may have anti-tumor activity in the clinic at well-tolerated doses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B137.

Site-specific PEGylation endows a mammalian ribonuclease with antitumor activity

Mammalian ribonucleases are emerging as cancer chemotherapeutic agents. Their cationicity engenders cell permeability, and their enzymatic activity destroys the biochemical information encoded by RNA. The pharmacologic potential of ribonucleases is, however, obviated by their high sensitivity to a cytosolic inhibitor protein (RI) and their small size, which limits their residence in serum. We reasoned that site specific conjugation of a poly(ethylene glycol) (PEG) chain could both reduce sensitivity to RI and increase serum half-life. We found that appending a PEG moiety can enable bovine pancreatic ribonuclease (RNase A) to evade RI, depending on the site of conjugation and the length and branching of the chain. Although a pendant PEG moiety decreases antiproliferative activity in vitro, PEGylation discourages renal clearance in vivo and leads to nearly complete tumor growth inhibition in a mouse xenograft model. These data demonstrate that a pendant PEG moiety can be beneficial to the action of proteins that act within the cytosol, and that strategic site-specific PEGylation can endow a mammalian ribonuclease with potent antitumor activity.