Cell Lung Cancer Xenograft Model Research Articles

Aerosol azacytidine inhibits orthotopic lung cancers in mice through Its DNA demethylation and gene reactivation effects.

We devised an aerosol based demethylation therapy to achieve therapeutic efficacy in premalignant or in situ lesions of lung cancer, without systemic toxicity. Optimum regimens of aerosolized azacytidine (Aza) were designed and used in orthotopic human non-small cell lung cancer xenograft models. The therapeutic efficacy and toxicity of aerosol Aza were compared with intravenously administered Aza. We observed that 80% of the droplets of the aerosol Aza measured ∼0.1–5 microns, which resulted in deposition in the lower bronchial airways. An animal model that phenocopies field carcinogeneisis in humans was developed by intratracheal inoculation of the human lung cancer cells in mice, thus resulting in their distribution throughout the entire airway space. Aerosolized Aza significantly prolonged the survival of mice bearing endo-bronchial lung tumors. The aerosol treatment did not cause any detectable lung toxicity or systemic toxicity. A pre-pharmacokinetic study in mice demonstrated that lung deposition of aerosolized Aza was significantly higher than the intravenous route. Lung tumors were resected after aerosol treatment and the methylation levels of 24 promoters of tumor-suppresser genes related to lung cancer were analyzed. Aerosol Aza significantly reduced the methylation level in 9 of these promoters and reexpressed several genes tested. In conclusion, aerosol Aza at non-cytotoxic doses appears to be effective and results in DNA demethylation and tumor suppressor gene re-expression. The therapeutic index of aerosol Aza is >100-fold higher than that of intravenous Aza. These results provide a preclinical rationale for a phase I clinical trial of aerosol Aza to be initiated at our Institution.

Abstract 4293: Whole-body bio-distribution of anti-5T4-mcMMAF (anti-5T4-ADC) using fluorescence molecular tomography (FMT) imaging in a non-small cell lung cancer mice model

Abstract Introduction: Currently bio-distribution of biologic drugs is evaluated by PET imaging, autoradioraphy using radio-labeled molecules or ex vivo methods. Advances in optical probes and non-invasive imaging technologies have given us an opportunity to conduct such studies without the use of radio-labeled materials or by traditional pharmacokinetic (PK) studies. 5T4 (also known as TPBG or oncofetal antigen) is a transmembrane glycoprotein expressed highly on tumor-initiating cells. Anti-5T4-mcMMAF used in these studies is an anti-5T4-antibody drug conjugate (ADC) that reacts to human, cyno and marmoset orthologs of 5T4. Previously, we showed the efficacy of anti-5T4-ADC in pre-clinical models (Sapra et al.,). In this study we show the utility of Fluorescence Molecular Tomography (FMT) imaging in bio-distribution studies with this ADC using a H1975 non-small cell lung cancer (NSCLC) xenograft model. Methods: For the H1975 xenograft model, five million cells in 50% matrigel were injected into the subcutaneous flanks of the female nu/nu mice, and a biodistribution study was initiated when the tumors reached ∼500 mm3. The anti-5T4-ADC and a control ADC (non-binding) were conjugated with near-IR fluorophore VivoTag680XL. The labeling efficiency and quality was determined by Nanodrop-8000 spectrophotometer and binding assays. FMT imaging was performed longitudinally at 5min, 6hr, 24hr, 48hr, 96hr and 240hrs post injection of labeled ADCs. Ex vivo imaging of organs was performed at intermittent time points after perfusing with PBS. Data was analyzed using TrueQuant software. Plasma and tissues were collected at various time points and analyzed by GyrolabTM workstation and LCMS methods. Results: VivoTag680XL conjugation was efficient and achieved degree of labeling between 2-3. Three-dimensional quantitative analysis of FMT data showed significant specific targeting of anti-5T4-ADC to the tumors (ex vivo and in vivo comparison), relative to the control non-binding ADC. The peak accumulation in tumor was observed at 48hrs post injection and the concentration decreased in later time points. Liver was the major organ for the non-specific accumulation of these antibodies/ADCs, followed by kidneys and lung. Both 5T4-ADC and Control-ADC showed similar accumulation at 48hrs and 96hrs in liver, whereas it decreased significantly at 240hrs. The FMT imaging data was comparative and correlated with the traditional plasma PK profile data. Conclusion: These results show that anti-5T4-ADC targets the tumor better than non-binding ADC. This study also shows the utility of FMT in bio-distribution studies of biologics. Since the fluorophore can be conjugated to any protein/peptide, this novel approach can become a platform technology in conducting biodistribution studies of all biologic drugs. Citation Format: Anand Giddabasappa, Rand Norberg, Mauricio Leal, David Paterson, Kush Lalwani, Ted Levkoff, Stella Rapa, Puja Sapra, Michael Ritche, Joann Wentland, Brian Rago, Jeetendra Eswaraka. Whole-body bio-distribution of anti-5T4-mcMMAF (anti-5T4-ADC) using fluorescence molecular tomography (FMT) imaging in a non-small cell lung cancer mice model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4293. doi:10.1158/1538-7445.AM2014-4293

Antitumor Activity of Bevacizumab Combined with Erlotinib in T790M Resistance Mutation Positive Non-Small Cell Lung Cancer Xenograft Models

ABSTRACT Aim: Erlotinib (ERL), a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, benefits patients with non-small cell lung cancer (NSCLC), especially those with EGFR active mutations. However, progressive disease occurs when resistance is acquired by T790M mutation or MET amplification. Bevacizumab (BEV), a humanized anti-vascular endothelial cell growth factor monoclonal antibody, has been demonstrated to be effective in combination with standard chemotherapies for advanced NSCLC patients. We previously reported the promising efficacy of combining ERL and BEV in mouse models of NSCLC harboring EGFR exon 19 deletion. In the present study, we examined the antitumor activity of BEV combined with ERL in NSCLC models harboring T790M resistance mutation. Methods: BALB-nu/nu mice were subcutaneously inoculated with NSCLC cell lines, NCI-H1975 (L858R + T790M mutation) or HCC827-EPR (exon19 deletion + T790M mutation). After randomization on Day 1, BEV (5 mg/kg) was intraperitoneally administered once a week, and ERL (75 mg/kg) was orally given daily. Antitumor activity was evaluated by tumor growth inhibition (TGI) with measuring tumor volume. In tumor tissues, phosphorylations of EGFR signaling molecules were evaluated by western blot analysis, and microvessel density was evaluated by CD31 immunohistochemistry. Results: In the NCI-H1975 model, the TGI on Day 11 of ERL, BEV and combination was 8%, 45% and 35%, respectively. NCI-H1975 tumors were resistant to ERL, and the combination of ERL and BEV did not enhance the antitumor activity of BEV monotherapy. In the HCC827-EPR model, the TGI on Day 50 of ERL, BEV and combination was 96%, 63% and 111%, respectively. HCC827-EPR tumors exhibited significant sensitivity to ERL (p ≤ 0.05) in vivo, and combining the two agents showed significantly higher antitumor activity than each monotherapy (p ≤ 0.05). The phosphorylation levels of EGFR, AKT or ERK in HCC827-EPR tumors were suppressed by ERL. The microvessel density was significantly decreased by BEV in both models. Conclusions: The combination treatment of ERL and BEV has the potential to be effective for ERL-responding NSCLC models with T790M resistance mutation. Disclosure: T. Mitsudomi: Dr.Mitsudomi has received research funds from Astra Zeneca, Chugai Pharma, Boehringer Ingelheim, Roche, Pfizer. All other authors have declared no conflicts of interest.

Abstract B52: Dual mTORC1/2 and PI3K inhibitor VS-5584 preferentially targets cancer stem cells.

Abstract Cancer stem cells (CSCs) constitute a subpopulation of cancer cells with tumor-initiating capability. Because CSCs are generally resistant to chemotherapy and drive tumor metastasis and recurrence, they represent an important challenge in the clinical treatment of cancer. In a high-throughput screen for compounds that preferentially target CSCs, several inhibitors of the PI3K/mTOR pathway were identified, suggesting the importance of this signaling pathway for CSC biology. VS-5584 is a highly potent and selective mTORC1/mTORC2/PI3K inhibitor with equipotency against all four human Class I PI3K isoforms and the mTOR kinase. We demonstrate that VS-5584 preferentially targets CSCs in vitro and in vivo in multiple orthogonal assays based on known characteristics of CSCs. Cancer stem cells express higher levels of aldehyde dehydrogenase (ALDH) than bulk tumor cells, thus an Aldefluor assay which measures ALDH enzymatic activity is used to quantify CSCs. VS-5584 decreased the percentage of Aldefluor-positive cells across multiple breast cancer cell lines. In SUM159 triple negative breast cancer cells, VS-5584 dose-dependently induced apoptosis preferentially in Aldefluor-positive cells as measured by both Annexin V and Caspase 3/7 assays. In contrast, paclitaxel induced more apoptosis in Aldefluor-negative cells and as a consequence increased the proportion of CSCs. Another characteristic of CSCs is enhanced drug efflux compared to non-CSCs, thus a Hoechst 33342 dye-exclusion assay, also known as a side population (SP) assay, is used to identify CSCs. In the H69 small cell lung cancer model, VS-5584 effectively eliminated the CSC side population, while the cytotoxic agents cisplatin and etoposide increased the CSC side population. To further evaluate the effect of VS-5584 on CSCs, we utilized ex vivo culture of surgically excised primary cancer tissue. Ex vivo treatment of breast tumors with VS-5584 decreased the proportion of Aldefluor-positive and CD44hi/CD24lo CSCs. Similarly, VS-5584 reduced the proportion of Aldefluor-positive and CD44hi/CD117hi CSCs in primary ovarian cancer tumor specimens. Significantly, oral dosing with VS-5584 was found to substantially reduce CSCs in vivo in the MDA-MB-231 triple negative and MCF-7 ER+ breast cancer xenograft models as evidenced by decreases in the percentage of Aldefluor-positive cells, tumorsphere-forming efficiency, and tumor-initiating capability in an in vivo limiting dilution re-implantation assay. Consistent with its strong suppression of CSCs, VS-5584 effectively impeded tumor regrowth following cisplatin treatment in a H69 small cell lung cancer xenograft model. The preferential targeting of CSCs thus provides a strong rationale for the clinical development of VS-5584 with the goal of achieving a more durable response in cancer patients. Phase 1 testing of VS-5584 is planned to initiate in the fourth quarter of 2013. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B52. Citation Format: Vihren N. Kolev, Quentin G, Wright, Christian M. Vidal, Jennifer E. Ring, Irina M. Shapiro, David T. Weaver, Mahesh V. Pavdal, Jonathan A. Pachter, Qunli Xu. Dual mTORC1/2 and PI3K inhibitor VS-5584 preferentially targets cancer stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B52.

Establishment of patient-derived non-small cell lung cancer xenograft models with genetic aberrations within EGFR, KRAS and FGFR1: useful tools for preclinical studies of targeted therapies

BackgroundPatient-derived tumor xenograft models have been established and increasingly used for preclinical studies of targeted therapies in recent years. However, patient-derived non-small cell lung cancer (NSCLC) xenograft mouse models are relatively few in number and are limited in their degree of genetic characterization and validation. In this study, we aimed to establish a variety of patient-derived NSCLC models and characterize these for common genetic aberrations to provide more informative models for preclinical drug efficacy testing.MethodsNSCLC tissues from thirty-one patients were collected and implanted into immunodeficient mice. Established xenograft models were characterized for common genetic aberrations, including detection of gene mutations within EGFR and KRAS, and genetic amplification of FGFR1 and cMET. Finally, gefitinib anti-tumor efficacy was tested in these patient-derived NSCLC xenograft models.ResultsTen passable patient-derived NSCLC xenograft models were established by implantation of NSCLC specimens of thirty-one patients into immunodeficient mice. Genetic aberrations were detected in six of the models, including one model with an EGFR activating mutation (Exon19 Del), one model with KRAS mutation, one model with both KRAS mutation and cMET gene amplification, and three models with FGFR1 amplification. Anti-tumor efficacy studies using gefitinib demonstrated that the EGFR activating mutation model had superior sensitivity and that the KRAS mutation models were resistant to gefitinib. The range of gefitinib responses in the patient-derived NSCLC xenograft models were consistent with the results reported from clinical trials. Furthermore, we observed that patient-derived NSCLC models with FGFR1 gene amplification were insensitive to gefitinib treatment.ConclusionsTen patient-derived NSCLC xenograft models were established containing a variety of genetic aberrations including EGFR activating mutation, KRAS mutation, and FGFR1 and cMET amplification. Gefitinib anti-tumor efficacy in these patient-derived NSCLC xenografts containing EGFR and KRAS mutation was consistent with the reported results from previous clinical trials. Thus, data from our panel of patient-derived NSCLC xenograft models confirms the utility of these models in furthering our understanding of this disease and aiding the development of personalized therapies for NSCLC patients.

A novel bispecific EGFR/Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity

Simultaneous targeting of epidermal growth factor receptor (EGFR) and Met in cancer therapy is under pre-clinical and clinical evaluation. Here, we report the finding that treatment with EGFR inhibitors of various tumor cells, when stimulated with hepatocyte growth factor (HGF) and EGF, results in transient upregulation of phosphorylated AKT. Furthermore, EGFR inhibition in this setting stimulates a pro-invasive phenotype as assessed in Matrigel-based assays. Simultaneous treatment with AKT and EGFR inhibitors abrogates this invasive growth, hence functionally linking signaling and phenotype. This observation implies that during treatment of tumors a balanced ratio of EGFR and Met inhibition is required. To address this, we designed a bispecific antibody targeting EGFR and Met, which has the advantage of a fixed 2:1 stoichiometry. This bispecific antibody inhibits proliferation in tumor cell cultures and co-cultures with fibroblasts in an additive manner compared with treatment with both single agents. In addition, cell migration assays reveal a higher potency of the bispecific antibody in comparison with the antibodies' combination at low doses. We demonstrate that the bispecific antibody inhibits invasive growth, which is specifically observed with cetuximab. Finally, the bispecific antibody potently inhibits tumor growth in a non-small cell lung cancer xenograft model bearing a strong autocrine HGF-loop. Together, our findings strongly support a combination treatment of EGFR and Met inhibitors and further evaluation of resistance mechanisms to EGFR inhibition in the context of active Met signaling.

18F-EF5 PET Imaging as an Early Response Biomarker for the Hypoxia-Activated Prodrug SN30000 Combined with Radiation Treatment in a Non–Small Cell Lung Cancer Xenograft Model

Hypoxia is a significant therapeutic problem for solid tumors because hypoxic cells are treatment-resistant and more aggressive. Hypoxia-activated prodrugs such as SN30000 use a mechanism of activation in hypoxic cells similar to that of 2-nitroimidazole hypoxia PET tracers. Therefore, we have evaluated the usefulness of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-(18)F-pentafluoropropyl)-acetamide ((18)F-EF5) PET to monitor and predict tumor response to SN30000 plus radiation treatment (RT). Human non-small cell lung cancer xenografts (H460) in athymic rats were imaged with (18)F-EF5 PET before and after treatment with SN30000 (90 mg/kg), with or without 15-Gy RT. The feasibility of imaging early changes in hypoxia in response to SN30000 was examined 24 h after treatment, followed by ex vivo γ-counting and immunohistochemical examination to study drug-induced apoptosis. Subsequently, the therapeutic effects of SN30000 with or without RT were evaluated in tumor growth delay studies and compared with early treatment-induced changes observed by (18)F-EF5 PET. Changes in tumor hemoglobin oxygen saturation as a function of time after treatment measured by optical spectroscopy were compared with PET data. The uptake of (18)F-EF5 was significantly lower in SN30000-treated tumors than in saline controls 24 h after treatment (mean standardized uptake value, 0.44 ± 0.08 vs. 0.56 ± 0.08 for control group; P < 0.05). Apoptosis was significantly higher in SN30000-treated tumors than in controls. Early treatment-induced changes in (18)F-EF5 uptake were indicative of tumor response in growth delay studies at the group level. SN30000 plus RT significantly decreased (18)F-EF5 uptake relative to baseline and resulted in complete tumor remission in 5 of 7 animals. SN30000 alone decreased (18)F-EF5 uptake, generally in tumors with high initial standardized uptake values, and showed a minor tumor growth delay effect. The changes induced by SN30000 with or without RT in (18)F-EF5 uptake correlated with baseline hypoxia levels. RT caused significant increases in tumor oxygen concentration and hemoglobin oxygen saturation. A hypoxia PET imaging agent can measure changes in tumor hypoxic fraction in response to SN30000. These results suggest the utility of (18)F-EF5 PET for monitoring early response to tumor treatment with SN30000 plus RT in the clinical development of this novel hypoxia-activated prodrug.

Abstract 5619: Preclinical development of anti-SLC34A2 antibody drug conjugate as a therapeutic for non-small cell lung and ovarian cancers.

Abstract SLC34A2, also known as type II Na/Pi co-transporters NaPi2b or NaPi3b, is a member of solute carrier protein family SLC34. SLC34A2 is highly expressed on cancers of the lung, ovary and thyroid as well as on normal lung pneumocytes, and is a promising target for antigen-directed therapy. Antibody drug conjugates (ADCs) specify the delivery of a cytotoxic drug to cells expressing an accessible antigenic target. Here we have appended monomethyl auristatin E (MMAE) to an antibody recognizing SLC34A2. Anti-SLC34A2-vc-MMAE inhibits tumor growth in multiple ovarian and non-small cell lung cancer xenograft models that express SLC34A2. Anti-SLC34A2 antibody and its conjugate cross-react with both human and monkey, and toxicity studies in monkeys are conducted to identify both target- and MMAE-related effects. Anti-SLC34A2-vc-MMAE was well tolerated in monkeys at efficacious doses and produced dose-related bone marrow suppression and increased apoptosis and mitotic figures consistent with MMAE-related pharmacology. Despite high levels of expression in normal lung of non-human primate, the cross-reactive ADC exhibited an acceptable safety profile with a dose-limiting toxicity unrelated to normal tissue expression. Overall, our preclinical results suggest that the antibody drug conjugate targeting the SLC34A2 may provide an effective new therapy for the treatment of NSCLC and ovarian cancer. Citation Format: Kedan Lin, Crystal Zhang, Eric Harstad, Hajime Hiraragi, Willy Solis, Siao Ping Tsai, Keyang Xu, Maria Hristopoulos, Rayna Venook, Bonnee Rubinfeld, Paul Polakis. Preclinical development of anti-SLC34A2 antibody drug conjugate as a therapeutic for non-small cell lung and ovarian cancers. [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 5619. doi:10.1158/1538-7445.AM2013-5619

Abstract 2776: Establishment of patient non-small cell lung cancer derived models for test of anticancer drugs.

Abstract BACKGROUND Human tumor xenograft models established by transplantation of human tumor cell lines into immunodifficient mice have been routinely used for preclinical test of anticancer agents. But tumor cell lines have a relatively low transplantability and resulted in a limited number of tumor models available for selection of right models for testing novel agents based on their antitumor mechanism. Recently, we have developed a large number of patient primary non-small cell lung cancer (NSCLC) xenograft models by transplanting patients’ fresh tumor tissues into nude mice, which have been employed for preclinical test of anticancer agents. METHODS The fresh NSCLC tissues were collected from local hospitals. The tumor fragments of 1-2 mm were subcutaneously implanted in the flanks of the nude mice by trocar needle. Sixteen tumor fragments were grafted into four mice from one patient tumor tissue (passage 0). All therapeutic efficacy experiments used female mice bearing passage 5 xenografts. The test drugs included cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, erlotinib, gefitinib, and pemetrexed. RESULTS A total of 213 patient primary NSCLC samples were implanted into nude mice, and 107 primary tumor models have been established with a tumor taking rate of 50% for the first passage. The tumor taking rates were higher (80-100) in the later passages. The therapeutic efficacy of the test drugs in these models is consistent with their clinical findings. The histopathology and gene sequence of the established primary tumor xenografts were analyzed; their architecture, histopathological morphology, and genomic mutation status from five generations of xenografts retained the patients’ original tumor characteristics. CONCLUSIONS The patient primary NSCLC models can be established in a large number for right models selection in preclinical setting, and have been employed for test of standard of care drugs and novel anticancer agents. The primary tumor models retain a similarity in histology and genomic mutation status to their patients’ original tumors. They may predict more relevant clinical response rate and higher correlation with clinical findings than use of traditional xenograft models established from long-term cultured cancer cell lines. Especially, they have advantages for test of target-oriented therapeutics in new drugs discovery and development programs. Citation Format: Changnian Liu, Connie Sun, Wenwei Li, Wen Zhou, Yong Liu, Rui Zhou, Fang He, Chang Bai. Establishment of patient non-small cell lung cancer derived models for test of anticancer drugs. [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 2776. doi:10.1158/1538-7445.AM2013-2776

Aflibercept: A Novel VEGF Targeted Agent to Explore the Future Perspectives of Anti-Angiogenic Therapy for the Treatment of Multiple Tumors

Angiogenesis is the process of formation of new blood vessels due to over expression of VEGF (vascular endothelial growth factor) which plays a critical role in the growth and development of all solid tumor types. With the advancement in understanding of tumor angiogenesis and VEGF, there have been a number of agents developed to target VEGF for the treatment of cancer. These targeted agents can affect downstream VEGF signal transduction by unique mechanisms at different cellular and extracellular levels. FDA has recently approved Aflibercept or VEGF-Trap in August 2012 for the treatment of colorectal cancer. It is a recombinant, decoy receptor fusion protein, rationally designed to block angiogenesis by targeting VEGF-A, VEGF-B and placental growth factor. VEGF-Trap exerts its antiangiogenic effects through regression of tumor vasculature, remodelling or normalization of surviving vasculature and inhibition of new tumor vessel growth. In this review, pre-clinical and clinical data have been summarized for aflibercept alone and in combination with chemotherapy to explore its efficacy and benefits in ovarian cancer, breast cancer, non-small cell lung cancer, pancreatic cancer, glioblastoma, adenocarcinoma and renal cell cancer xenograft models.

18F-EF5 PET Imaging as an Early Response Biomarker for the Hypoxia-Activated Prodrug SN30000 Combined with Radiation Treatment in a Non-Small Cell Lung Cancer Xenograft Model

18F-EF5 PET Imaging as an Early Response Biomarker for the Hypoxia-Activated Prodrug SN30000 Combined with Radiation Treatment in a Non-Small Cell Lung Cancer Xenograft Model

Carbon Irradiation Exerts Potent Antiangiogenic Effects via Downregulation of Vascular Endothelial Growth Factor in Lung and Prostate Cancer Models

Among the numerous pro-angiogenic factors discovered, the Vascular Endothelial Growth Factor (VEGF) pathway was found to play a central role in regulation of tumor angiogenesis. Therefore, VEGF has emerged as a key target in the field of antiangiogenic cancer therapy. It has been reported that tumors evade the antiangiogenic/anti-cancer effects of photon therapy by compensatory expression of different factors including VEGF. Here, we studied the regulation of VEGF after carbon ion radiation therapy at HIT using the androgen independent human prostate adenocarcinoma- (PC3) and the non-small cell lung cancer (NSCL, A549) xenograft models.

Lentivirus‐Mediated RNAi Knockdown of NUPR1 Inhibits Human Nonsmall Cell Lung Cancer Growth In Vitro and In Vivo

NUPR1 (nuclear protein 1) was found to play a key role in the development of several malignancies including pancreas, breast, and prostate cancers. However, the functional role of NUPR1 in nonsmall cell lung cancer (NSCLC) progression and development is little known. Here, lentivirus-mediated small interfering RNA (siRNA) was employed to downregulate endogenous NUPR1 expression to study the function of NUPR1 in growth of nonsmall cell lung cancer. A lentivirus-mediated RNAi technology was used to specifically knock down the expression of NUPR1 in H1299 cells. Quantitative real-time reverse transcriptase polymerase chain reaction, flow cytometry, western blot and cell count assays were studied to characterize NUPR1 expression in vitro. Furthermore, nonsmall cell lung cancer xenograft models in nude mice were established to investigate whether knockdown of NUPR1 reduces the tumor growth in vivo. We found that downregulation of NUPR1 expression significantly inhibited nonsmall cell lung cancer H1299 cells proliferation and colony formation in vitro. Moreover, the specific downregulation of NUPR1 arrested cells in G0 phase of cell cycle and increased apoptosis rate. Silencing of NUPR1 also suppressed tumor growth by tail vein injection of lentivirus encoded shRNA against NUPR1 in vivo. Our findings revealed that the NUPR1 gene represents a promising target for gene silencing therapy in nonsmall cell lung cancer.

Abstract 2343: Establishment of patients’ primary non-small cell lung cancer xenograft models for test of anticancer drugs

Abstract BACKGROUND Human xenograft tumor models established by transplantation of human tumor cell lines into immunodifficient mice have been routinely used for preclinical test of anticancer agents. But tumor cell lines have a relatively low transplantability and resulted in a limited number of tumor models available for selection of right models for preclinical testing novel agents based on their antitumor mechanism. Recently, we have developed a large number of patient primary non-small cell lung cancer (NSCLC) xenograft models by transplanting patients’ fresh tumor tissues into nude mice, which have been employed for preclinical test of anticancer agents. METHODS The fresh NSCLC tissues were collected from local hospitals. The tumor fragments of 1-2 mm were subcutaneously implanted in the flanks of the nude mice by trocar needle. Sixteen tumor fragments were grafted into four mice from one patient tumor tissue (passage 1). All therapeutic efficacy experiments used female mice bearing passage 5 xenografts. The test drugs included cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, tarceva, and iressa. RESULTS A total of 142 patient primary NSCLC samples were implanted into nude mice, and 71 primary tumor models were established with a tumor taking rate of 50% for the first passage. The tumor taking rates were higher (80-100) in the later passages. The therapeutic efficacy of the test drugs in these models is consistent with their clinical findings. The histopathology and gene sequence of the established primary tumor xenografts were analyzed; their architecture, histopathological morphology, and genomic mutation status from five generations of xenografts retained the patients’ original tumor characteristics. CONCLUSIONS The patient primary NSCLC models have been employed for preclinical test of standard clinically used and novel anticancer agents. The primary tumor models retain a similarity in histology and genomic mutation status to their patients’ original tumors. They may predict more relevant clinical response rate and higher correlation with clinical findings than use of traditional xenograft models established from long-term cultured cancer cell lines. Especially, they have advantages for test of target-oriented therapeutics in new drugs development programs. 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 2343. doi:1538-7445.AM2012-2343

Abstract 3787: Pharmacokinetics and pharmacodynamics of AV-203, a humanized anti-ERBB3 antibody

Abstract ERBB3 is widely expressed in human carcinomas, and its over expression is associated with poor prognosis in patients with various carcinomas, (i.e., breast, ovarian, prostate, colorectal, pancreatic, gastric, and head and neck cancers). The presence of ERBB3 correlates with local to distal metastasis in lung, gastric, and colorectal cancers as well as bone invasion in prostate cancer. Activation of ERBB3 is also implicated in the development of resistance to current cancer treatments. Due to its lack of kinase activity, the activation of the ERBB3 receptor is dependent on heterodimerization with active receptor tyrosine kinases (RTKs). The recruitment of ERBB3 into active, heterodimer complexes is mediated by its ligand Neuregulin-1 (NRG-1) or by amplified, over expressed RTKs in a ligand independent manner. Therefore, ERBB3 can crosstalk with most major receptors involved in cancer development and maintenance such as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and c-MET. AV-203 is a potent, humanized anti-ERBB3 antibody that inhibits both ligand-dependent and independent activation of ERBB3 both in vitro and in vivo. Pharmacokinetics and pharmacodynamics of AV-203 were characterized in mice using the A549 non-small cell lung cancer xenograft model. AV-203 administered IV in mice exhibits acceptable pharmacokinetics supporting preclinical efficacy studies. AV-203 administered IV in A549 NSCLC xenograft tumor bearing mice had lower serum AUC than naïve mice, demonstrating that the presence of human ErbB3 may alter the PK parameters of AV-203. In evaluating pharmacodynamics in vivo, AV-203 was able to down regulate total ERBB3 and ERBB3 signaling in A549 tumors in a time-dependent manner. Inhibition of ERBB3 signaling correlated with significant dose-dependent tumor growth inhibition in the A549 xenograft model. Dose scheduling studies with the constant AV-203 dose of 2.5 mg/kg revealed that the most efficacious schedule is the more frequent dosing at Q3D. In comparing the total dose of 10 mg/kg per 14 day cycle, at varying dose per injection and frequency, AV-203 resulted in significant tumor growth inhibition at all dose schedules. These data conclude that the efficacy of AV-203 is driven by total drug exposure and that AV-203 is not dependent on Cmax for its antitumor activity. 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 3787. doi:1538-7445.AM2012-3787

Abstract 4075: Aerosol azacytidine inhibits the orthotopic lung cancer in mice by its demethylation function but not cytotoxic effect

Abstract Introduction: Silencing of key tumor suppressor genes by promoter hypermethylation plays an important role in lung cancer carcinogenesis. Reversing the hypermethylation in the early lung cancer cells adjacent to the airway surface may have a potential to inhibit lung cancer development. This study was to explore the therapeutic potential of aerosol administered demethylating agent azacytidine (Aza) for the treatment of bronchial epithelial premalignancy or lung cancer. Methods: The demethylation and gene reactivation functions of Aza administered by aerosol were detected by qPCR methylation array and western blotting assay in the tumor tissues from the orthotopic human non-small cell lung cancer (NSCLC) xenograft models in nude mice. The therapeutic efficacy and toxicity of the aerosol Aza were also evaluate in the same lung cancer models. An intravenous administered Aza with clinical equivalent dose and schedule was used as a control. Results: The aerosol Aza is an appropriate pharmaceutical inhalation formulation with about 80% of droplets size distributing between 0.1 to 5 micron. This aerodynamic size range ensures the aerosol droplets depositing in the lower airway. In animal studies, aerosol administration of Aza significantly prolonged the survival of nude mice with intratracheally inoculated human NSCLC tumors. The %-increased life span was 5- to 10-fold higher than that of a systemic treatment of Aza at a clinical equivalent dose. The aerosol treatment did not cause any detectable lung toxicity or myelosuppression (systemic toxicity of Aza), since the treatment dose was &amp;gt;100-fold lower than the toxic dose. The lung tumors were resected after the aerosol Aza treatment and the methylation levels of the 24 promoters that drive the lung cancer related tumor suppressor genes (TSG) were detected. We found that aerosol Aza significantly reduced the methylation level in 9 of these 24 promoters. The dementhylating aerosol also resulted in the gene reactivation in the protein level of several tested genes. Conclusions: Aerosol administration of Aza has significant therapeutic advantages over the systemic Aza. It can be given at a non-cytotoxic dose, thus its main therapeutic efficacy is the demethylation function of Aza and the TSG reactivation. In our case, the therapeutic index of aerosol Aza is &amp;gt;800-fold higher than that of IV Aza. The clinical trial of aerosol Aza is worth to be conducted. Acknowledgement: Supported by a NIH (NCI) grant 5R01CA154755-02 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 4075. doi:1538-7445.AM2012-4075

FDG-PET as a pharmacodynamic biomarker for early assessment of treatment response to linifanib (ABT-869) in a non-small cell lung cancer xenograft model

Linifanib (ABT-869) is a multitargeted receptor tyrosine kinase inhibitor. This work aims to evaluate F-fluorodeoxyglucose-positron emission tomography (FDG-PET) as a pharmacodynamic (PD) biomarker for linifanib treatment utilizing the Calu-6 model of human non-small cell lung (NSCLC) cancer in SCID-beige mice. Animals received either vehicle or 12.5mg/kg linifanib orally twice a day for the duration of the study. Imaging was performed at -1, 1, 3, and 7days after beginning treatment (n=12-14 per group). Linifanib inhibited tumor growth and suppressed tumor metabolic activity. Changes in tumor FDG uptake were observed as early as 1day after beginning linifanib treatment and were sustained for the duration of the study. This study confirms that linifanib is efficacious in this xenograft model of human NSCLC and confirms FDG-PET is a potential PD biomarker strategy for linifanib therapy.

Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.

Abstract 5045: The Hh inhibitor IPI-926 inhibits relapse of a non-small cell lung cancer xenograft model following treatment with an EGF-R targeted tyrosine kinase inhibitor

Abstract The Hedgehog (Hh) pathway plays a key role in multiple types of cancer, either by mutational activation of the pathway in tumor cells or by ligand-dependent signaling to the tumor cells or paracrine signaling between tumor cells and the tumor microenvironment. We have previously shown in a primary model of SCLC that the Hh pathway inhibitor IPI-926 prevents tumor relapse after regression of the tumor with chemotherapy (Travaglione et al., AACR 2009). To investigate whether tumor regression with a targeted agent could elicit a similar response as that seen with chemotherapy, two NSCLC lines (H1650 and HCC827), with activating mutations in the EGFR were evaluated. NSCLC cell lines bearing mutant EGFR are exquisitely sensitive to EGFR kinase inhibitors. Upon treatment with gefitinib in vitro, HCC827 increased the expression of Indian and Sonic hedgehog ligands 10-fold, as measured by qRT-PCR. When grown in mice as subcutaneous xenografts, H1650 tumors regressed following treatment with the EGFR inhibitor gefitinib (40 mg/kg every day) for 7 days. The gefitinib-treated mice were then administered either vehicle or IPI-926 at 40 mg/kg every other day for three weeks, and in this setting IPI-926 treatment led to a 65% decrease (p=0.0104) in tumor volume compared to vehicle-treated mice. Conversely, tumors that had not been regressed with the EGFR TKI showed no response to the Hh pathway inhibitor, similar to our previous results in SCLC with chemotherapy. Studies are ongoing to define the mechanism of action of IPI-926 in this setting. When grown in mice as subcutaneous xenografts, these tumor cells produce Shh ligand in conjunction with stromal Gli-1 expression in the murine stroma. Stromal Gli-1 expression is inhibited by treatment with IPI-926, indicating that Hh ligand produced by NSCLC tumor cells signals to stroma in a paracrine manner. Together with our previous results in SCLC, these data suggest that therapeutic inhibition of the Hh signaling pathway might be an important strategy to extend progression free survival in patients who initially respond to therapy but later relapse, and that this paradigm is applicable to multiple cancer types and treatment modalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5045.

Abstract 5390: QBI-139, a human RNase variant in a phase I trial, works in combination

Abstract The EVade™ Ribonucleases (RNases) are a new class of anti-cancer drugs that target the RNA in cancer cells. These EVade™ RNases are variants of native mammalian RNases that have been converted to anti-cancer agents by diminishing their binding to their natural inhibitor protein. One of these EVade™ RNases, called QBI-139, has advanced to human clinical trials. QBI-139 shares 95% sequence identity with the wild type human RNase 1. QBI-139 has shown tumor growth inhibition against a variety of cancers, including: colon, non-small cell lung, ovarian, pancreatic and prostate cancers. QBI-139 is currently in a Phase I clinical trial in patients with solid tumors. A frog RNase called Ranpirnase (Alfacell) that shares only 20% sequence identity with human RNase 1 has been in clinical trials and responses have been seen in esophageal, non-small cell lung and breast cancers as well as unresectable mesothelioma. The next stage of clinical development of QBI-139 will require selection of a specific indication as well as an existing treatment regimen to be used in combination with QBI-139. The Phase I trial may provide insights about sensitive tumor types. However, mouse models of cancer will be used to select the combinations for development. QBI-139 has shown efficacy against non-small cell lung cancer (A549 cell line) in xenograft models. Cisplatin is a standard of care treatment for non-small cell lung cancer in patients. The ability of QBI-139 and cisplatin to work together to cause tumor growth inhibition was determined in a non-small cell lung cancer xenograft model (A549 cell line). Cisplatin (3 mg/kg) and QBI-139 (15 mg/kg) were used as controls. The combination of cisplatin and QBI-139 (3 and 15 mg/kg, respectively) was also tested. Cisplatin treatment resulted in tumor growth inhibition (TGI) of 60% while QBI-139 caused 76% TGI. The combination of the two drugs resulted in a TGI of 96%, strongly supporting the use of QBI-139 in combination with cisplatin for the treatment of lung cancer. Even the combination of the two drugs exhibited minimal impact on the health of the animals. EVade™ Ribonucleases are novel agents that provide a new way to target the RNA in cancer cells and are an innovative new tool for oncologists. QBI-139 has a variety of benefits, including: a novel mechanism of action with clinical support, broad in vivo anti-tumor activity alone and in combination with standard of care therapies and a strong tolerability profile. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5390.