Preclinical Models Of Lung Cancer Research Articles

Stromal and antitumor effects of BIBF 1120 (nintedanib) in a preclinical lung cancer model.

e13563 Background: BIBF 1120 is an angiogenic receptor tyrosine kinase inhibitor that potently inhibits VEGFR, PDGFR and FGFR kinase activity in in vitro enzymatic assays. This study investigated the effect and mechanism of BIBF 1120, in vitro and in vivo, as a single agent and in combination with chemotherapy in preclinical models of lung cancer. Methods: Anti-tumor effects of BIBF 1120 in vitro were assessed using cell proliferation assays on five lung cancer lines (A549, Calu-3, Calu-6, H1993, H1703) with BIBF 1120 as a single agent and in combination with gemcitabine and cisplatin. To demonstrate anti-tumor activity in vivo, NOD/SCID mice bearing subcutaneous A549 grafts were treated daily with BIBF 1120 or BIBF 1120 plus chemotherapy. Perfusion studies were conducted using labeled dextrans. Ex vivo tumor tissues were assessed for architecture, microvessel density, pericyte coverage, proliferation, and apoptosis. Results: In vitro, BIBF 1120 did not show anti-proliferative effects at pharmacologically achievable concentrations (IC50>20μM) as a single agent; nor did it sensitize tumor cells to chemotherapy. However, BIBF 1120 inhibited primary tumor growth as a single agent and in combination therapy in subcutaneous endpoint studies with A549 xenografts. There was no significant difference in tumor architecture between BIBF 1120 and control-treated animals. Microvessel density (CD31,endomucin) and pericyte-covered vessels (NG2) were significantly decreased in BIBF 1120-treated animals compared to the control and chemotherapy only groups. Proliferation (phospho-histone 3) was decreased and apoptosis (cleaved caspase 3) was increased in BIBF 1120-treated animals compared to the control and chemotherapy only groups. Finally, BIBF 1120 significantly decreased perfusion in A549 xenografts. Conclusions: BIBF 1120 demonstrated potent anti-tumor and anti-angiogenic activity in a preclinical model of lung cancer. Because A549 was previously demonstrated to be an anti-VEGFR therapy resistant cell line, this study highlights the importance of FGFR and PDGFR pathways in the treatment of lung cancer.

Abstract 1876: Preclinical efficacy of fibroblast growth factor ligand trap HGS1036 in lung carcinoma models with genomic amplification of FGFR1

Abstract Lung cancer is the most common cause of cancer-related death in industrialized countries. In 2010, it is estimated that cancer of the lung and bronchus will be responsible for 157,300 deaths in the US (Jermal et al. 2010). A number of recent publications have reported the genomic amplification of the fibroblast growth factor receptor 1 gene (FGFR1) in lung cancer (Weiss et al. 2010; Voortman et al. 2010). Subtype analysis indicates that approximately 22% of squamous non-small cell lung cell carcinomas (NSCLC) and 27-33% of small cell lung cancer (SCLC) have amplification of the FGFR1 gene. Amplification of the FGFR1 gene in lung cancer cell lines is associated with over-expression of the FGFR1 receptor protein and increased signaling through the FGFR1 pathway resulting in increased cell proliferation and tumorigenesis. HGS1036 (formerly FP-1039) is a soluble fusion protein consisting of the extracellular domain of human FGFR1 isoform α-IIIc linked to the Fc region of human immunoglobulin G1 (IgG1). HGS1036 acts as a ligand “trap” that sequesters multiple fibroblast growth factor (FGF) family ligands, blocking their ability to bind to and activate multiple FGF receptors. We investigated the preclinical efficacy of HGS1036 in preclinical lung cancer models with genomic FGFR1 amplification as a novel therapeutic strategy. A panel of lung cancer cell lines (n=5) were identified that displayed genomic FGFR1 amplification on chromosome 8. Two of the cell lines, DMS53 and DMS114, were derived from the tumors of patients with SCLC. The other three cell lines, NCI-H1581, NCI-H520 and NCI-H1703, were derived from the tumors of patients with NSCLC. Examination of in vitro drug sensitivity indicated that all 5 cell lines were sensitive to HGS1036. Treatment of NCI-H1581, NCI-H520, NCI-H1703, DMS53 and DMS114 cells with HGS1036 in vitro resulted in a 52%, 71%, 46%, 47% and 82% reduction in cell proliferation, respectively, as determined by CellTiterGlo. Sensitivity to HGS1036 in vitro generally correlated with FGFR1 copy number and total FGFR1 protein expression. HGS1036 treatment of mice bearing FGFR1 amplified lung cancer xenografts resulted in tumor growth inhibition in all five FGFR1-amplified models examined (p=<0.01). The degree of tumor growth inhibition, as assessed by area-under-the-curve analysis, varied from 31-84% depending on the FGFR1 amplified lung model examined. These experiments demonstrate that lung tumor cell lines with genomic FGFR1 amplification can be sensitive to FGF-ligand blockade by HGS1036 and provide preclinical rationale for the clinical study of HGS1036 in the treatment of lung cancer. 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 1876. doi:1538-7445.AM2012-1876

Abstract 881: Leveraging the molecular heterogeneity of patient-derived tumor samples to simulate clinical trial settings and model combination treatments

Abstract Combination therapy, with rare exception, is a requirement for successfully treating cancer patients. Advances in genetic characterization coupled with a growing understanding of tumor heterogeneity are poised to revolutionize future strategies for combination treatments. Historically, working out the best combinations has been challenging and largely through iterative cycles of clinical trial and error due to gross limitations of preclinical models to accurately predict activity. Current strategies for developing new anticancer drugs rely heavily on preclinical testing in cancer cell lines and their derived in vivo xenograft models. These models are fraught with significant limitations including highly passaged cancer cell lines grown on plastic that ignores the microenvironment and tempered representation of the complex heterogeneity of cancer. Tumor cell microenvironment has significant impact on growth kinetics, cell signaling and response to drug treatments. 3D models attempt to recapitulate elements of the microenvironment, are more biologically relevant models compared to 2D models and have gained preference among cancer researchers and drug developers. The purpose of this study is to investigate the utility of molecularly characterized patient derived lung tumor cells (PTCs) for steering decisions for effective combination treatments in the clinical setting. We have established preclinical lung cancer models using PTCs grown in a 3D culture system. Here we present a retrospective study in lung PTCs evaluating single agents and combinations of molecularly targeted as well as cytotoxic agents including erlotinib, crizotinib, etoposide, cisplatin, carboplatin, gemcitabine, paclitaxel, vinorelbine, topotecan and irinotecan. We utilized a range of methods (PCR, FISH, IF, WB) to determine the genetic and molecular features of the PTCs prior to performing drug treatments. We used high content imaging to evaluate subpopulations within PTCs, colony morphology and proliferative endpoints. Our results identified responder and non-responder populations and emphasize the need to base combination treatment strategies on subpopulation analysis of the tumor. Patient-derived tumor cell models grown in 3D culture conditions coupled with molecular characterization are essential for conducting hypothesis driven studies. This approach offers an informed starting point for subsequent in vivo studies from which data can guide personalized medicine decisions toward true translation in the clinic. 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 881. doi:1538-7445.AM2012-881

Osteopontin induces growth of metastatic tumors in a preclinical model of non-small lung cancer

Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity anti-OPN monoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvβ3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvβ3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvβ3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a KrasG12D-LSLp53fl/fl subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.

Itraconazole inhibits angiogenesis and tumor growth in non-small cell lung cancer.

The antiangiogenic agent bevacizumab has been approved for the treatment of non-small cell lung cancer (NSCLC), although the survival benefit associated with this agent is marginal, and toxicities and cost are substantial. A recent screen for selective inhibitors of endothelial cell proliferation identified the oral antifungal drug itraconazole as a novel agent with potential antiangiogenic activity. In this article, we define and characterize the antiangiogenic and anticancer activities of itraconazole in relevant preclinical models of angiogenesis and lung cancer. Itraconazole consistently showed potent, specific, and dose-dependent inhibition of endothelial cell proliferation, migration, and tube formation in response to both VEGF- and basic fibroblast growth factor-mediated angiogenic stimulation. In vivo, using primary xenograft models of human NSCLC, oral itraconazole showed single-agent growth-inhibitory activity associated with induction of tumor hypoxia-inducible factor 1 alpha expression and marked inhibition of tumor vascularity. Itraconazole significantly enhanced the antitumor efficacy of the chemotherapeutic agent cisplatin in the same model systems. Taken together, these data suggest that itraconazole has potent and selective inhibitory activity against multiple key aspects of tumor-associated angiogenesis in vitro and in vivo, and strongly support clinical translation of its use. Based on these observations, we have initiated a randomized phase II study comparing the efficacy of standard cytotoxic therapy with or without daily oral itraconazole in patients with recurrent metastatic NSCLC.

Bioluminescent Orthotopic Mouse Models of Human Localized Non-Small Cell Lung Cancer: Feasibility and Identification of Circulating Tumour Cells

BackgroundPreclinical models of non-small cell lung cancer (NSCLC) require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC.MethodsAthymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models.ResultsGroup 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours.ConclusionsTranspleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.

The therapeutic potential of anaplastic lymphoma kinase inhibitors in lung cancer: rationale and clinical evidence

In the last few years it has been demonstrated that approximately 4% of non-small-cell lung cancer is driven by an ALK gene rearrangement. When such a rearrangement occurs an oncogene addicted state appears to then exist within the cancer cell. Crizotinib, an anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor, is effective in preclinical models of ALK-rearranged lung cancer, and has demonstrated high response rates in Phase I/II clinical trials in this sub-population. This review summarizes ALK biology, recent developments in techniques for the detection of ALK rearrangements, clinical characteristics of this population and relevant clinical trial results. Following filing for accelerated approval, crizotinib may soon be licensed for the treatment of ALK-positive non-small-cell lung cancer in the USA as well as worldwide.

Abstract 1336: Nicotine does not promote tumor growth in preclinical mouse models of lung cancer

Abstract Nicotine replacement therapy (NRT) is a popular and effective regimen for the cessation of smoking. Expanded use of NRT is controversial based on preclinical studies that show that nicotine activates signaling pathways that promote cellular survival and proliferation. To address the tumor promoting effects of nicotine in mouse models of lung cancer, nicotine was administered in the drinking water and tested in a tobacco carcinogen-driven model (NNK), a genetically engineered model (KRasLA2), and syngeneic mouse xenografts. Pharmacokinetic analysis showed that mice had normal water consumption and achieved an average serum cotinine level of 135 ng/ml, a level comparable to that observed in NRT users. In the NNK model, nicotine was administered after 3 weekly intraperitoneal injections of NNK, and was continued for 12 wk. At the time of sacrifice, nicotine did not increase lung tumor multiplicity or size, and did not activate the Akt/mTOR pathway. In the KRasLA2 model, mice were placed on nicotine at the time of weaning. At 6 wk, there was no effect on tumor multiplicity, size, histology, or tumor biomarkers. Studies assessing the effects of nicotine on overall survival of K-RasLA2 mice are ongoing. Similar negative results were observed with nicotine administration in xenograft studies with isogenic NNK-transformed lung adenocarcinoma cell lines. To assess whether the response to nicotine might be dose-dependent, we assessed activation of the PI3K/Akt/mTOR, MEK/ERK, and JAK/STAT pathways in these cell lines using immunoblotting. Stimulation was only observed at concentrations higher than that observed with NRT. These results suggest nicotine at doses comparable to NRT does not promote lung tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1336. doi:10.1158/1538-7445.AM2011-1336

CD4+ T cells are critical to the therapeutic efficacy of TAA-based cancer vaccines and control of recurrences (48.11)

Abstract The role of CD4+ T cells remains ambiguous in cancer immunotherapy. We herein addressed this issue using a novel form of soluble 4-1BBL (SA-4-1BBL) costimulatory molecule as an effective adjuvant component of HPV E7 and survivin as TAA-based vaccines in E7 expressing TC-1 cervical and survivin over expressing 3LL lung cancer preclinical models, respectively. Vaccination of C57BL/6 mice on day 6 having established tumors with SA-4-1BBL admixed with E7 or survivin TAAs resulted in the eradication of TC-1 and 3LL tumors in 70% and 80% of mice, respectively, along with generation of tumor specific long-term memory that prevented tumor recurrence on second challenge. The in vivo depletion of CD4+ T cells one day before tumor challenge significantly compromised vaccine efficacy in both TC-1 (25%) and 3LL (12.5%) models. In marked contrast, the depletion of CD4+ T cells one day prior to vaccination did not significantly alter the therapeutic efficacy of the vaccines. However, these mice had compromised immunological memory since a second challenge of 3LL on day 60 resulted in tumor development in the majority (85.7%) of mice. Taken together, this study demonstrates an indispensable role of CD4+ T cells in priming immune responses against cancer as well as establishment of long-term memory.

142 GDC-0941 PI3K inhibitor activity in preclinical lung cancer models

We have recently reported the discovery of pyrrolo[3,2-d]pyrimidine derivatives 1a and 1b as potent triple inhibitors of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and Tie-2 kinases. To identify compounds having strong inhibitory activity against fibroblast growth factor receptor (FGFR) kinase, further modification was conducted using the co-crystal structure analysis of VEGFR2 and 1b. Among the compounds synthesized, urea derivative 11l having a piperazine moiety on the terminal benzene ring showed strong inhibitory activity against FGFR1 kinase as well as VEGFR2 kinase. A binding model of 11l complexed with VEGFR2 suggested that the piperazine moiety forms additional interactions with Ile1025 and His1026.

Actinomycin D Decreases Mcl-1 Expression and Acts Synergistically with ABT-737 against Small Cell Lung Cancer Cell Lines

ABT-737, which blocks the function of Bcl-2 and Bcl-X(L) but not Mcl-1, has shown single-agent activity in preclinical models of small cell lung cancer (SCLC). Elevated expression of Mcl-1 induces resistance to ABT-737 in SCLC. Based on the short half-life of Mcl-1 mRNA and protein, we hypothesized that the actinomycin D could reverse Mcl-1-induced resistance to ABT-737. The dose-response of multiple SCLC cell lines to actinomycin D in the absence and presence of ABT-737 was followed by the assessment of Bcl-2 family expression and poly ADP ribose polymerase cleavage by Western blot, viability by tetrazolium dye reduction and clonogenic assay, and cell cycle kinetics by flow cytometry. Actinomycin D decreased Mcl-1 expression and resulted in a cell line-dependent increase in Noxa expression. Clinically relevant concentrations of actinomycin D from 0.4 to 4 ng/mL showed single-agent activity across a panel of SCLC cell lines. When combined with low micromolar doses of ABT-737, near complete loss of viability was seen with synergistic combination indices of 0.5 to 0.7. Exposure to 4 ng/mL actinomycin was only required for the first 24 hours of the combined incubation, mimicking a clinically achievable area under the curve, but the presence of ABT-737 was required for an additional 48 hours to obtain maximal effect. Clinically relevant concentrations of actinomycin D act synergistically with ABT-737 to induce SCLC apoptosis, which can be at least partially attributed to the actinomycin D-induced decrease in Mcl-1 and increase in Noxa expression. Taken together, these data suggest the feasibility of combining actinomycin D with BH3-mimetic drugs in the clinical setting.

Abstract 2637: Hsp90 inhibitor STA-9090 enhances the activity of standard of care therapies in erlotinib-sensitive and -resistant NSCLC models

Abstract BACKGROUND: Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the folding, stability and activity of many kinases and signal transduction proteins that are critical for the proliferation and survival of cancer cells. These Hsp90 client proteins include wild type and drug-resistant forms of such clinically important drug targets as: BCR-ABL, BRAF, EGFR, HER2, KIT, MET and VEGFR1. STA-9090 is a highly potent and less toxic, next-generation Hsp90 inhibitor that is structurally unrelated to first-generation agents such as 17-AAG and IPI-504. STA-9090 induces proteasome-mediated degradation of Hsp90 client proteins preferentially in cancer versus normal cells, resulting in cell cycle arrest and apoptosis. STA-9090 is currently being evaluated in multiple Phase 1/2 clinical trials in sold tumor and hematologic malignancies, including an open-label, multi-center Phase 2 trial in patients with advanced NSCLC harboring wild type or activated forms of EGFR or KRAS. In anticipation of conducting clinical trials combining STA-9090 with therapeutic agents commonly employed to treat NSCLC, we have examined the ability of STA-9090 to enhance the in vitro and in vivo activity of paclitaxel, docetaxel, erlotinib and bevacizumab in preclinical models of lung cancer. RESULTS: In vitro cytotoxicity studies were conducted with the erlotinib-sensitive HCC827 (expressing EGFRdel746–750) and erlotinib-resistant NCI-H1975 (expressing EGFRL858R/T790M) NSCLC cell lines. Using the median-effect method of Chau and Talalay, STA-9090 was found to significantly enhance the in vitro cytotoxicity of the taxanes, paclitaxel and docetaxel, with combination index (CI) values ranging from 0.3-0.7, which is indicative of synergy rather than additive or antagonistic effects. Similarly, STA-9090 also displayed synergy (0.4-0.8 CI) with the EGFR inhibitor erlotinib against erlotinib-sensitive cells, but as expected, not against erlotinib-resistant cells. Consistent with these results, STA-9090 enhanced the in vivo efficacy of concomitantly dosed paclitaxel and docetaxel in the HCC827 and NCI-H1975 xenograft models. Further, STA-9090 also enhanced the in vivo efficacy of the anti-VEGF monoclonal antibody bevacizumab in these models. These combinations did not result in significant additional toxicity relative to the single agents alone, and pharmacokinetic analyses demonstrated that these findings were not due to drug-drug interactions. CONCLUSIONS: Our results demonstrate that STA-9090 potently synergizes with paclitaxel, docetaxel, erlotinib and bevacizumab, which are widely employed to treat NSCLC in the clinic. Hsp90 inhibition using STA-9090 represents a novel and promising approach to combination therapy for NSCLC. 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 2637.

Abstract 1380: Antitumor activity of motesanib alone and in combination with chemotherapy in xenograft models of human non-small cell lung cancer

Abstract Introduction: Angiogenesis is required for tumor growth. Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) play a critical role in the development of neovascularization. Motesanib is an orally administered small-molecule inhibitor of angiogenesis with direct antitumor activity, targeting VEGFR1, 2, and 3; platelet-derived growth factor receptor (PDGFR), and stem cell factor receptor (c-kit). In this study, we examined the effects of motesanib in preclinical models of human non-small cell lung cancer (NSCLC), both as a single agent and in combination with docetaxel or cisplatin. Methods: The ability of motesanib to inhibit the growth of NSCLC cell lines in vitro was assessed using the ATPlite cell viability assay. Five human NSCLC cell lines were evaluated, including A549 (adenocarcinoma), Calu-6 (anaplastic carcinoma), NCI-H358 (bronchioalveolar carcinoma), NCI-H1299 (large cell carcinoma) and NCI-H1650 (adenocarcinoma). To determine in vivo efficacy, mice bearing established xenografts were treated orally with motesanib BID or QD at doses ranging from 7.5 to 75 mg/kg. For combination experiments, cisplatin (4 or 5 mg/kg) or docetaxel (5, 15 or 30 mg/kg) administered IP once weekly were given with motesanib. Tumors were collected for assessment of blood vessel area (anti-CD31 staining) and viable tumor area (hematoxylin staining). Tumor burden was calculated as the viable fraction multiplied by the respective terminal tumor weight. Results: Motesanib had no effect on the in vitro growth of any of the lung cancer cell lines tested (IC50 >5 µM). In contrast, treatment with motesanib in vivo resulted in a dose-dependent inhibition of tumor growth in all five lung cancer models. In combination studies, treatment with motesanib and cisplatin significantly enhanced (p <0.05) the antitumor activity in the NCI-H358, NCI-H1650, and Calu-6 xenograft models. Treatment with motesanib in combination with docetaxel significantly enhanced (p <0.05) the anti-tumor activity in the Calu-6 and A549 models, compared with either single agent alone. All treatments were well tolerated. The factors responsible for the differential sensitivity of these tumors to motesanib are currently being explored. Histological analyses of NCI-H358 tumors revealed a reduction in blood vessel area after treatment with motesanib alone and in combination with cisplatin, as well as a significant decrease in viable tumor burden after combination therapy (p <0.05). Together, these data suggest that motesanib prevented tumor growth by inhibiting angiogenesis, and combination of motesanib with chemotherapy resulted in a significantly enhanced reduction in tumor growth. Conclusion: The antitumor activity of motesanib in multiple preclinical models of NSCLC supports development of this antiangiogenic agent for the management of NSCLC in combination with standard of care chemotherapy. 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 1380.

Abstract 5687: Grape seed extract induces apoptosis via reactive oxygen species-dependent mechanism in human non-small-cell lung cancer cells

Abstract Lung cancer is the most frequently diagnosed malignancy and leading cause of cancer-related deaths worldwide. The 5-year survival rate of 14% has shown little improvement over the last 30 years even with the development of molecularly targeted therapies. Non-small-cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers, suggesting that additional approaches and strategies are needed to control this malignancy. For the last few decades, major emphasis has also been placed on identifying natural dietary and/or non-dietary products for both prevention and intervention of several malignancies including lung cancer. One such naturally occurring polyphenolic product is grape seed extract (GSE); which is especially rich in proanthocyanidins. In recent studies, GSE has shown promising cancer chemo-preventive and anti-cancer effects against various epithelial cancers. Accordingly, here we evaluated the efficacy of GSE against a panel of human NSCLC cell lines, namely A549, H460, H1299 and H322 cells. GSE (50-100 µg/ml) treatment for 24 and 48 hrs significantly suppressed cell proliferation and induced cell death in all the four cell lines studied; however, H460 and H322 cells were more sensitive to GSE treatment even at comparatively lower doses (20-50 µg/ml). Additional studies showed that GSE causes a dose- and a time-dependent apoptotic cell death in A549, H460, H322 and H1299 cells which was confirmed by the Annexin V-PI staining, activation of caspase-3 and −9, and cleavage of PARP. In detailed mechanistic studies, to determine the cause of GSE-induced apoptotic cell death, pre-treatment of cells with N-acetylcysteine (NAC) strongly inhibited GSE-caused apoptotic cell death, suggesting the involvement of reactive oxygen species (ROS). Interestingly, GSE treatment also resulted in a strong dose- and time-dependent phosphorylation of ERK1/2 in these cell lines, which was inhibited by pre-treatment of cells with NAC, further supporting the role of GSE-caused oxidative stress in the activation of ERK1/2. Based on these results, next we assessed whether GSE causes oxidative stress in these NSCLC cell lines. Dihydroethidium staining followed by flow cytometry analysis of cells showed that indeed GSE treatment results in ROS generation. Next we assessed the intracellular reduced glutathione levels, which were decreased by 82% and 60% in A549 and H460 cells, respectively, after GSE treatment. Together, these results confirm that GSE generates ROS causing oxidative stress that leads to apoptotic death in NSCLC cells. Based on these findings, more studies are needed in future to evaluate both anti-cancer and chemopreventive efficacy of GSE in relevant pre-clinical lung cancer models to establish its potential usefulness against human lung cancer. 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 5687.

The polyamine analog PG11047 potentiates the antitumor activity of cisplatin and bevacizumab in preclinical models of lung and prostate cancer

PG11047 is a polyamine analog currently in Phase I trials for advanced cancer as a monotherapy and in combination with a number of approved anti-cancer agents. The use of polyamines as a target for antiproliferative therapy is based on findings that cells synthesize polyamines excessively when induced to grow and that polyamine metabolism is frequently dysregulated in cancer. A selective polyamine transport system provides access for PG11047 into rapidly dividing cells to inhibit polyamine biosynthetic enzymes, to induce the polyamine catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO) which could subsequently induce reactive oxygen species that contribute to tumor cell responses to PG11047, and to function as a polyamine with altered function when it binds to natural polyamine binding sites. The objective of the present study was to assess the antitumor effects of PG11047 alone and in combination with approved anti-cancer agents. The antitumor efficacy of PG11047 as a single agent, and in combination with cisplatin and bevacizumab, was tested in models of lung (A549) and prostate (DU-145) cancer, respectively. PG11047 significantly inhibited tumor development in both lung and prostate cancer models when administered as a single agent. In the lung cancer model, PG11047 potentiated the antitumor effect of cisplatin. Although potent activity was observed with PG11047 and bevacizumab when administered as single agents in the prostate cancer model, the combination arm significantly enhanced antitumor activity compared with either agent alone. In all experiments, PG11047 was well tolerated with no adverse effects on bodyweight gain. The preclinical data support the rationale for the current Phase I trials which are assessing PG11047 as a monotherapy and in combination with a number of approved anti-cancer agents including cisplatin and bevacizumab.

Circulating Biomarkers of Cell Death After Treatment with the BH-3 Mimetic ABT-737 in a Preclinical Model of Small-Cell Lung Cancer

This study evaluated epithelial cell death ELISAs that measure circulating cytokeratin 18 in mice bearing small-cell lung cancer xenografts treated with a proapoptotic dose of the BH-3 mimetic ABT-737. H146 tumor-bearing and non-H146 tumor-bearing severe combined immunodeficient (SCID)/bg mice were treated with ABT-737 or vehicle control. Plasma collected before and 2 to 360 hours after treatment was analyzed by M30 (caspase-cleaved cytokeratin 18) and M65 (intact and cleaved cytokeratin 18) ELISA. In parallel, tumors were interrogated for cleaved caspase-3 and cleaved cytokeratin 18 as biomarkers of apoptosis. ABT-737-treated tumors regressed by 48 hours (P < 0.01) compared with controls, correlating with increased cleaved cytokeratin 18 (P < 0.01; 6 and 24 hours) and increased intact cytokeratin 18 (P < 0.01; 24 hours). Cleaved cytokeratin 18 levels decreased below baseline between 72 and 360 hours for ABT-737-treated and control mice whereas intact cytokeratin 18 decreased below the level of detection at 8 and 15 days in ABT-737-treated mice only. Apoptosis in tumors reflected changes in circulating cytokeratin 18 (cleaved caspase-3, P < 0.05 at 2 hours and P < 0.001 at 6, 12, and 24 hours; caspase-cleaved cytokeratin 18, P < 0.05 at 15 days, for drug treated versus controls). ABT-737 caused tumor regression by apoptosis in H146 xenografts that mapped to a drug-specific, early increase in circulating cleaved cytokeratin 18 that subsequently declined. Circulating, intact cytokeratin 18 levels correlated with tumor burden. Cleaved caspase-3 and caspase-cleaved cytokeratin 18 in tumor correlated with treatment (P < 0.05, 2 hours; P < 0.001, 6, 12, and 24 hours; cleaved caspase-3, P < 0.05, 15 days; caspase-cleaved cytokeratin 18), indicating that events in plasma were tumor derived. These circulating biomarker data will be translated to clinical trials wherein serial tumor biopsies are rarely obtained.

Abstract PL01-04: The role of regulatory T cells in K-Ras driven lung cancer

Abstract PL01-04 Background K-Ras mutations occur in up to 30% of human lung adenocarcinomas (Salgia and Skarin 1998) and occur almost exclusively in patients with a history of smoking. These mutations confer resistance to commonly used cytotoxic chemotherapies as well as targeted agents (Eberhard, Johnson et al. 2005). In preclinical mouse models of lung cancer, K-Ras mutations are necessary for tobacco carcinogen-driven lung tumorigenesis and are sufficient to cause lung adenocarcinomas in transgenic mice (Johnson, Mercer et al. 2001). Inflammation is characteristic of smoking-related lung cancer and preclinical models of K-Ras-driven lung tumorigenesis. Specific components of the inflammatory response such as regulatory T cells (Treg) regulate inflammation and are thought to contribute to tumorigenesis. Because inhibitors of mTOR such as the immunosuppressant rapamycin can prevent K-Ras mediated murine lung tumorigenesis (Wislez, Spencer et al. 2005; Granville, Warfel et al. 2007), we hypothesized that the anti-tumor effects of rapamycin might be related to alterations in lung-associated inflammation. Methods and Findings Lung tumorigenesis was studied in three different murine models that depend on mutant K-Ras; a tobacco carcinogen-driven model, a syngeneic inoculation model and a transgenic model. Splenic and lung-associated T cells were studied using flow cytometry and immunohistochemistry. Exposure of A/J mice to the tobacco-carcinogen NNK that causes K-Ras mutations (Belinsky, Devereux et al. 1989) tripled the fraction of lung-associated Treg prior to tumor development. Induction of lung tumors and induction of lung associated Foxp3+ cells by NNK was dose dependent. When administered to achieve trough levels comparable to that in humans, rapamycin prevented the induction of lung associated Foxp3+ cells by NNK, coincident with a 90% decrease in lung tumors. In A/J mice inoculated with syngeneic lung adenocarcinoma cells that were resistant to the anti-proliferative effects of rapamycin, rapamycin neither inhibited tumor growth nor decreased Treg. In contrast, depletion of Treg in inoculated mice using an anti-CD25 antibody prevented lung tumorigenesis by 80%. Transgenic mice that express mutant K-Ras (K-RasLA2) also develop lung tumors with infiltrating Foxp3+ cells, and when K-RasLA2 mice were crossed to genetically engineered mice that bear a loss of function mutation in Foxp3, lung tumorigenesis was inhibited by 75%. Conclusions These studies show that Treg are required for K-Ras-mediated lung tumorigenesis, and provide a strong rationale to clinically evaluate rapamycin or other agents that decrease Treg, to determine if decreasing Treg will inhibit K-Ras driven lung cancer. Citation Information: Cancer Prev Res 2008;1(7 Suppl):PL01-04.

BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models

Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.

Phase 1/2 Dose Escalating Study of Twice-Monthly Pemetrexed and Gemcitabine in Patients with Advanced Cancer and Non-small Cell Lung Cancer

Pemetrexed is synergistic with gemcitabine in preclinical models of non-small cell lung cancer (NSCLC). The optimal dose and utility of gemcitabine and pemetrexed was evaluated in a dose-escalating study. The phase 1 study included patients with advanced tumors, whereas the phase 2 study included patients with locally advanced or metastatic NSCLC. Gemcitabine was infused over 30 minutes, followed by pemetrexed administered over 10 minutes on day 1 of a 14-day cycle. Treatment continued for 12 cycles or until disease progression. All patients received folic acid, Vitamin B12, and steroid prophylaxis. Maximum tolerated dose was gemcitabine 1500 mg/m, followed by pemetrexed 500 mg/m. Fifty-three patients (29 male, 24 female) were enrolled in the phase 2 study. Response rate was 20.8% (95% CI: 0.108-0.341), and the clinical benefit rate (CR + PR + SD) was 64.2%. Median time to disease progression was 4.6 months (95% CI: 2.79-6.18), median survival was 10.1 month (95% CI: 5.95-14.09, censorship = 20.75%), and 1-year survival was 41.0%. Common grade 3 or 4 adverse events (% of patients) were neutropenia (28.3%), fatigue (22.6%), and febrile neutropenia (9.4%). Twice-monthly gemcitabine and pemetrexed was well tolerated, with overall survival and clinical benefit indicating disease activity in NSCLC patients.

ARLTS1 – a novel tumor suppressor gene

ARLTS1 (ADP-ribosylation factor-like tumor suppressor gene 1) is a member of the ARF family of the Ras superfamily of small GTPases that are known to be involved in multiple regulatory pathways altered in human carcinogenesis. Here, we review recent work that has provided insights into the role of this small gene as an emerging player in both familial and sporadic cancers of several histotypes. ARLTS1 is a low penetrance gene that is primarily dysregulated in sporadic lung cancer by promoter hypermethylation. Two ARLTS1 polymorphisms are also associated with familial cancer risk. Down-regulation of ARLTS1 is seen in all lung cancer cell lines studied and in a significant proportion (37%) of primary lung tumors. Restoration of ARLTS1 expression in ARLTS1-deficient lung cancer cell lines by either demethylation or adenoviral transduction leads to apoptosis via caspase-dependent mechanisms. Furthermore, ARLTS1 re-expression induces an in vivo decreased tumor growth in preclinical models of lung cancer. Microarray analysis of gene expression patterns in cells transduced with ARLTS1 demonstrates that various pathways involved in cell survival, proliferation and development mediate its pro-apoptotic effects.