Preclinical Models Of Human Cancer Research Articles

BET protein inhibitor JQ1 attenuates Myc-amplified MCC tumor growth in vivo.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumor of the skin currently with no cure. In this study, we have first demonstrated that c-Myc overexpression is common in MCC. By targeting c-Myc, bromodomain inhibitors have demonstrated antitumor efficacy in several preclinical human cancer models. Thus, we interrogated the role of c-Myc inhibition in MCC with c-Myc amplification by using the BET inhibitor JQ1. We have uncovered that c-Myc can be regulated by JQ1 in MCC cells with pathologic c-Myc activation. Moreover, JQ1 potently abrogates c-Myc expression in MCC cells and causes marked G1 cell-cycle arrest. Mechanistically, JQ1-induced cell-cycle arrest coincides with downregulation of cyclin D1 and upregulation of p21, p27, and p57, whereas JQ1 exerts no effect on apoptosis in MCC cells. Further knockdown of p21, p27, or p57 by shRNA partially protects cells from JQ1-induced cell-cycle arrest. In addition, c-Myc knockdown by shRNA generates significant cell-cycle arrest, suggesting that c-Myc overexpression plays a role in MCC pathogenesis. Most importantly, JQ1 significantly attenuates tumor growth in xenograft MCC mouse models. Our results provide initial evidence, indicating the potential clinical utility of BET protein inhibitors in the treatment of MCC with pathologic activation of c-Myc.

Abstract 1815: Efficacy of FASN-selective small molecule inhibitors in preclinical tumor models

Abstract Fatty acid synthase (FASN) expression increases with tumor progression and is associated with impaired response to chemotherapy and diminished patient survival in many solid and hematopoietic tumor types. Palmitate, the product of FASN enzymatic activity, functions in vital cellular processes including energy metabolism and cell growth, cellular membrane biosynthesis and architecture, and protein localization and function. 3-V Biosciences has discovered and developed a series of potent, selective, orally available, and reversible FASN inhibitors with excellent pharmaceutical properties. Single agent FASN inhibition has been shown to cause anti-tumor activities in preclinical models of human cancer, including inhibition of AKT and mTOR signal transduction, induction of tumor cell apoptosis, and tumor growth inhibition and regression in pancreatic, ovarian, and lung cancer xenograft tumor models of human cancer. Current studies are supporting the active clinical development of TVB-2640, 3-V Biosciences' first-in-class FASN inhibitor, as an oncology therapeutic. These studies have multiple objectives: (1) characterizing a wide range of tumor types for responsiveness to FASN inhibition using tumor cell line and Champions TumorGraftTM efficacy models; (2) investigating the benefit of FASN combination therapy with chemotherapeutic or targeted anti-cancer agents; (3) developing biomarkers that characterize efficacy response; and (4) further elucidating the mechanisms of action that emanate from the inhibition of palmitate synthesis and lead to tumor cell apoptosis and in vivo anti-tumor efficacy. The results of these investigations show that FASN inhibition causes tumor growth inhibition or regression in diverse tumor types; and moreover, that tumor growth inhibition additivity or synergy is observed when FASN inhibition is combined with chemotherapy agents. Genomics and directed analysis of mRNA, proteins, and lipids following FASN inhibition have identified biomarker candidates and provided insights into the tumor growth inhibition mechanisms of action. For example, genome-wide gene expression analysis shows FASN inhibition to significantly increase the expression of many genes in biosynthetic (sterol, lipid, etc.) and apoptosis pathways while significantly reducing the expression of many genes in cell growth and proliferation pathways (DNA replication, cell cycle progression, mitosis, etc.). These data strongly support the clinical development of TVB-2640 as a first-in-class single and combination oncology therapeutic and are advancing the discovery and validation of biomarkers to inform clinical utility and response. Citation Format: Timothy S. Heuer, Richard Ventura, Joanna Waszczuk, Kasia Mordec, Julie Lai, Russell Johnson, Lilly Hu, Haiying Cai, Allan Wagman, Douglas Buckley, Stanley T. Parish, Elizabeth Bruckheimer, George Kemble. Efficacy of FASN-selective small molecule inhibitors in preclinical tumor models. [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 1815. doi:10.1158/1538-7445.AM2014-1815

Muramyl tripeptide-phosphatidyl ethanolamine encapsulated in liposomes (L-MTP-PE) in the treatment of osteosarcoma.

Bacille Calmette-Guerin (BCG) has been used for decades as an immune stimulant to treat cancer. Early work by Fidler and Kleinerman identified muramyl dipeptide (MDP) as a critical component of the BCG cell wall which retained most of the immunostimulatory properties of the native BCG. Addition of a peptide to MDP resulted in muramyl tripeptide (MTP) which allowed incorporation into liposomal membranes. The resulting pharmaceutical, liposomal muramyl tripeptide phosphatidyl ethanolamine (L-MTP-PE or mifamurtide) showed activity in preclinical models of human cancers. Phase I studies documented the safety of the compound for human administration. These trials did not reach a maximally tolerated dose (MTD), and the dose chosen for phase II trials was a biologically optimized dose, not an MTD. Phase II studies showed decreased risk of further recurrence in patients who received mifamurtide after surgical ablation of metastatic osteosarcoma. A phase III prospective randomized trial demonstrated a statistically significant reduction in the risk of death from osteosarcoma when MTP was added to systemic chemotherapy for the treatment of localized osteosarcoma. The same trial allowed treatment of patients who presented with initially metastatic disease. While the overall and event-free survival was improved in patients with metastatic osteosarcoma who received L-MTP-PE, the sample size was small and the improvement did not achieve conventional statistical significance. From 2008 to 2012, patients with metastatic and recurrent osteosarcoma were given L-MTP-PE in an expanded access trial, and the results suggest a decreased risk of subsequent recurrence and death with the inclusion of L-MTP-PE in the treatment strategy for these high-risk patients.

Phospho-sulindac inhibits pancreatic cancer growth: NFATc1 as a drug resistance candidate.

Phospho-sulindac (P-S), a promising anticancer agent, is efficacious in pre-clinical models of human cancer and is apparently safe. Here, we studied the effect of P-S on pancreatic cancer growth. We found that P-S strongly inhibits the growth of human pancreatic cancer cells in vitro, is efficacious in inhibiting the growth of pancreatic xenografts in nude mice, and has an excellent safety profile. Microarray analysis revealed that P-S induced the expression of nuclear factor of activated T-cells, isoform c1 (NFATc1) gene. NFATc1, a calcineurin-responsive transcription factor associated with aggressive pancreatic cancer. The role of increased NFATc1 expression on the growth inhibitory effect of P-S on cancer growth was evaluated by silencing or by overexpressing it both in vitro and in vivo. We found that when the expression of NFATc1 was abrogated by RNAi, pancreatic cancer cells were more responsive to treatment with P-S. Conversely, over-expressing the NFATc1 gene made the pancreatic cancer cells less responsive to treatment with P-S. NFATc1 likely mediates drug resistance to P-S and is an unfavorable prognostic factor that predicts poor tumor response. We also demonstrated that NFATc1-mediated resistance can be overcome by cyclosporin A (CsA), an NFAT inhibitor, and that the combination of P-S and CsA synergistically inhibited pancreatic cancer cell growth. In conclusion, our preclinical data establish P-S as an efficacious drug for pancreatic cancer in preclinical models, which merits further evaluation.

Abstract B261: Characterization of FASN-selective small-molecule inhibitors in preclinical tumor models.

Abstract The purpose of this study is to characterize the activity of small-molecule FASN inhibitors in preclinical models of human cancer to evaluate their potential utility as novel cancer therapeutics. FASN-synthesized palmitate is required for vital cellular processes that include energy metabolism, cellular membrane biosynthesis, and protein localization and function. Many solid and hematopoietic tumors overexpress FASN. Moreover, tumor expression of FASN is increased in a stage-dependent manner that is correlated with patient survival. FASN activity can promote the tumorigenic capacity of cells by multiple mechanisms that include: activating cell growth and survival signal transduction pathways such as PI3K-AKT and enhancing cellular processes such as macromolecular biosynthesis, cellular stress response, and energy metabolism. 3-V Biosciences has discovered and developed a series of selective, reversible FASN inhibitors that have been optimized and characterized using in vitro and in vivo assays. These molecules are highly active in both in vitro and in vivo studies, and selected FASN inhibitors demonstrate excellent biochemical (< 10 nM) and cell-based (<100 nM) IC50 values. In vitro, inhibitory activity of cell-based palmitate synthesis aligns with activity in cell phenotypic (e.g. growth and viability) and mechanistic (e.g. AKT phosphorylation and PARP cleavage) assays in cell lines derived from pancreas, breast, lung, colon, and other tumor tissues. Moreover, pharmacologic supplementation of palmitate to cell culture medium rescues cells from cell death in FASN-inhibition cell viability assays. Together, these data show that in vitro FASN inhibition is associated with growth-inhibitory and apoptosis-inducing effects. These novel FASN inhibitors show oral bioavailability, dose dependent plasma exposure, and demonstrate excellent in vivo pharmacodynamic activity. Tumor xenograft studies in mice have been conducted with the FASN inhibitor TVB-3166, and the results demonstrate in vivo tumor growth inhibition both as a single agent and in combination with standard of care chemotherapy agents. TVB-3166 in combination with different chemotherapy agents shows synergistic or additive anti-tumor efficacy in these xenograft studies. In conclusion, these data support continued investigation of FASN inhibition as an approach for the development of a novel cancer therapeutic with the potential for anti-tumor activity in multiple tumor types. Further characterization of FASN inhibitor activity as a single agent and in combination with chemotherapeutics is ongoing using cell-based mechanistic and phenotypic assays as well as tumor xenograft studies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B261. Citation Format: Timothy S. Heuer, Minchao Chen, Richard Ventura, Joanna Waszczuk, Satya Yendluri, Julie Lai, Samnang Tep, Shirley Feng, Russell Johnson, Cristiana Zaharia, Douglas Buckley, George Kemble. Characterization of FASN-selective small-molecule inhibitors in preclinical tumor models. [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 B261.

A first-in-human phase I study of the CDK4/6 inhibitor, LY2835219, for patients with advanced cancer.

2500 Background: Cyclin dependent kinases 4 and 6 (CDK4/6) act with D-type cyclins to inactivate the retinoblastoma (Rb) tumor suppressor protein and enable cell cycle progression from G1 to S phase. LY2835219 is a selective inhibitor of CDK4/6 that shows antitumor activity in preclinical models of human cancer and also distributes efficiently to the brain. We performed a phase 1 study to evaluate safety, pharmacokinetics, pharmacodynamics, and antitumor activity of LY2835219. Methods: 3+3 dose escalation was followed by expansions in 5 tumor types (brain metastases permitted): non-small cell lung cancer (NSCLC), glioblastoma, breast cancer, melanoma, and colorectal cancer. LY2835219 was taken orally every 12 or 24 hours (in escalation) and every 12 hours (in expansions) on days 1-28 of a 28-day cycle. Results: 55 patients (pts) received LY2835219. In escalation, 33 pts received LY2835219 on 1 of 2 schedules: 50, 100, 150, 225 mg every 24 hours (Q24H) or 75, 100, 150, 200, 275 mg every 12 hours (Q12H). On the Q24H schedule, the maximum tolerated dose (MTD) was not identified. On the Q12H schedule, the MTD was 200mg Q12H with dose limiting toxicity of G3 fatigue at 200 mg (1/6 evaluable pts) and 275 mg (2/3 evaluable pts). At 200mg Q12H, the mean Cmax and AUC0-24hr at steady state were 285 ng/mL and 5502 ng-hr/ml, respectively. In skin, LY2835219 induced pharmacodynamic inhibition of both Rb phosphorylation and topoisomerase IIα expression. In the ongoing expansions, 22 pts have received LY2835219. Across the study, the most common related adverse events were diarrhea (52%, including 5% G3), nausea (30%, 4% G3), fatigue (21%, 7% G3), vomiting (18%, 2% G3), and neutropenia (16%, 7% G3). 15 pts have reached ≥4 cycles for stable disease or better with 3 pts achieving 8, 16, and 26 cycles. One pt with ovarian cancer had a durable CA-125 response with >50% decrease for 16 cycles. One pt with KRAS mutant NSCLC had a 27% decrease by RECIST. One pt with CDKN2A-/- NRAS mutant melanoma had a confirmed partial response. Early clinical activity has been observed in ovarian cancer, NSCLC, breast cancer, and melanoma. Conclusions: LY2835219 shows acceptable safety and early clinical activity as a single agent for patients with advanced cancer. Clinical trial information: NCT01394016.

Abstract LB-122: LY2835219, a selective inhibitor of CDK4 and CDK6, inhibits growth in preclinical models of human cancer.

Abstract Cell growth is regulated by the activity of certain cyclin-dependent kinases (CDKs), including CDK4 and CDK6 (hereafter CDK4/6), which specifically regulate cell cycle progression through the G1 restriction point. These CDKs act with the D-type cyclins to phosphorylate and inactivate the retinoblastoma (Rb) tumor suppressor protein, which in turn enables cell cycle progression from G1 to S phase. LY2835219 is a selective inhibitor of CDK4/6 with IC50 of 2 and 10 nM for CDK4 and CDK6, respectively. The current study evaluates the activity of LY2835219 in preclinical models of human cancer including non-small cell lung cancer (NSCLC), melanoma, mantle cell lymphoma (MCL), and ovarian cancer. A secondary focus of this study is to identify potential predictive biomarkers for response to LY2835219 in NSCLC, melanoma, and other cancers. For NSCLC, increased sensitivity to LY2835219 is associated both in vitro and in vivo with KRAS mutations. For established melanoma cell lines, similar levels of in vitro sensitivity are observed in different genetic subtypes. Accordingly, LY2835219 shows single-agent activity in the A375 melanoma xenograft model with daily oral dosing at 45 or 90 mg/kg. For MCL, durable growth inhibition of subcutaneously implanted xenografts is achieved with daily oral dosing at 50mg/kg. Finally, in an intra-peritoneal model that recapitulates clinical features of human ovarian cancer, LY2835219 not only inhibits tumor growth but also increases survival. These studies suggest that LY2835219 may have potential therapeutic application in the treatment of human cancers with specific molecular alterations. Citation Format: Jack A. Dempsey, Edward M. Chan, Teresa F. Burke, Richard P. Beckmann. LY2835219, a selective inhibitor of CDK4 and CDK6, inhibits growth in preclinical models of human cancer. [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 LB-122. doi:10.1158/1538-7445.AM2013-LB-122

Abstract 1398: Establishment and characterization of a clinically-relevant personalized hormone-dependent breast cancer tumor model.

Abstract Background: The START Patient Tumor Program was established to improve clinical outcomes for cancer patients by creating a biorepository of blood and cancer tissues for characterization and preclinical model development and testing. To date samples from over 1500 patients have been collected with over 400 models generated including 40 patient-derived breast tumor xenografts. These models can be used for personalized studies, particularly in situations where treatment options have been exhausted. In the current study a breast biopsy was collected from a 48 year old Hispanic female with recurrent, chemotherapy-refractory ER+/HER2+ metastatic breast cancer and implanted into an immune-deficient mouse. The resulting model was expanded and evaluated against eight FDA-approved and four investigational agents. Methods: ST340 ER+/HER2+ breast PDX model: The tumor sample from an inform-consented participant was implanted into nude mice and the established model confirmed by histologic analysis and linked with clinical treatment and outcome data. Activating mutation profiling was performed on fifty-two known cancer genes with extracted DNA using a PCR-based assay with customized primers. An efficacy study was performed testing various approved and investigational agents. Study endpoints included tumor volume and time from treatment initiation. Reported results included tumor growth inhibition or delay and regression. Results: Prior to treatment initiation, estradiol was withheld to confirm dependence and was found requisite for growth. Following treatment, results identified impressive sensitivity to the targeted therapy everolimus including tumor regressions and moderate sensitivity towards an aromatase inhibitor. Treatment with this combination regimen resulted in improved tumor markers and overall quality of life for an extended period of time. Following progression, subsequent treatment with a cytotoxic agent found active in the initial screen resulted in additional months of progression free disease. Conclusion: Participation in the START Patient Tumor Program provided beneficial data which improved clinical outcome for this patient demonstrating that personalized preclinical models of human cancer can predict useful agents for clinical treatment in select situations. Citation Format: Michael J. Wick, Teresa L. Vaught, Lizette Gamez, Jennifer Brown, Alyssa Moriarty, Anthony W. Tolcher, Drew W. Rasco, Amita Patnaik, Gladys Rodriguez, Ronald L. Drengler, Kyriakos P. Papadopoulos. Establishment and characterization of a clinically-relevant personalized hormone-dependent breast cancer tumor model. [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 1398. doi:10.1158/1538-7445.AM2013-1398

Assessment of the interstitial fluid pressure of tumors by dynamic contrast-enhanced magnetic resonance imaging with contrast agents of different molecular weights

Background. Cancer patients showing highly elevated interstitial fluid pressure (IFP) in the primary tumor may benefit from particularly aggressive treatment. There is some evidence that gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) may be a useful non-invasive method for providing information on the IFP of tumors. The purpose of this preclinical study was to investigate whether any association between DCE-MRI-derived parametric images and tumor IFP can be strengthened by using MR contrast agents with higher molecular weights than that of Gd-DTPA. Material and methods. A-07 human melanoma xenografts were used as preclinical models of human cancer. Three contrast agents were compared: Gd-DTPA (0.55 kDa), P846 (3.5 kDa), and gadomelitol (6.5 kDa). A total of 46 tumors were subjected to DCE-MRI and subsequent measurement of IFP. Parametric images of Ktrans (the volume transfer constant of the contrast agent) and ve (the fractional distribution volume of the contrast agent) were produced by pharmacokinetic analysis of the DCE-MRI series. Results. Significant inverse correlations were found between median Ktrans and IFP for Gd-DTPA (p = 0.0076; R2 = 0.46; n = 14) and P846 (p = 0.0042; R2 = 0.45; n = 16), whereas there was no correlation between median Ktrans and IFP for gadomelitol (p > 0.05; n = 16). Significant correlation between median ve and IFP was not found for any of the contrast agents (p > 0.05 for Gd-DTPA, P846, and gadomelitol). Conclusion. Ktrans images, but not ve images, derived by pharmacokinetic analysis of DCE-MRI data for low-molecular-weight contrast agents may provide information on the IFP of tumors. Any association between Ktrans and IFP cannot be expected to be improved by using contrast agents with higher molecular weights than those of Gd-DTPA and P846.

Tumor penetration and epidermal growth factor receptor saturation by panitumumab correlate with antitumor activity in a preclinical model of human cancer

BackgroundSuccessful treatment of solid tumors relies on the ability of drugs to penetrate into the tumor tissue.MethodsWe examined the correlation of panitumumab (an anti-epidermal growth factor [EGFR] antibody) tumor penetration and EGFR saturation, a potential obstacle in large molecule drug delivery, using pharmacokinetics, pharmacodynamics, and tumor growth rate in an A431 epidermoid carcinoma xenograft model of human cancer. To determine receptor saturation, receptor occupancy, and levels of proliferation markers, immunohistochemical and flow cytometric methods were used. Pharmacokinetic data and modeling were used to calculate growth characteristics of panitumumab-treated tumors.ResultsTreatment with panitumumab in vivo inhibited pEGFR, Ki67 and pMAPK levels vs control. Tumor penetration and receptor saturation were dose- and time-dependent, reaching 100% and 78%, respectively. Significant tumor inhibition and eradication (p < 0.05) were observed; plasma concentration associated with tumor eradication was estimated to be 0.2 μg/ml. The tumor inhibition model was able to describe the mean tumor growth and death rates.ConclusionsThese data demonstrate that the antitumor activity of panitumumab correlates with its ability to penetrate into tumor tissue, occupy and inhibit activation of EGFR, and inhibit markers of proliferation and MAPK signaling.

Use of PTEN status to determine the functional outcome of combined MEK and mTOR inhibition.

e13512 Background: PTEN is a lipid phosphatase couteracting the activity of the PI3K pathway. PTEN mutations and deficiencies are prevalent in many types of human cancers and are associated with poor prognosis and therapeutic resistance. MAPK is another key cellular network that works independently from, in parallel to, and/or through interconnections with PI3K to promote cancer development. Here we investigated whether a strategy combining MEK and mTOR inhibition may be effective in preclinical models of human cancer and the role of PTEN loss in determining sensivity/resistance to single and combined pathway inhibition. Methods: We employed in vitro assays (cell proliferation assays, cell cycle and apoptosis analysis, WB, and ELISA assays) to determine functional and molecular drug effects. Pharmacologic interactions between MEK and mTOR inhibitors were analyzed by conservative isobologram analysis using a fixed dose-ratio experimental design. Results: In cell lines with wt-PTEN (including melanoma, breast, lung, and colon cancer), combined MEK and mTOR blockade achieves synergistic effects at suboptimal drug concentrations but becomes frankly antagonistic in the presence of complete inhibition of MEK-to-ERK signaling (combination index - CI: 1.2-&gt;1000). This, in turn, led to the identification of a novel crosstalk mechanism by which MEK blockade restores PTEN expression and cross-inhibits the PI3K/AKT/mTOR pathway, thus bypassing the need for double pathway blockade. Conversely, in cancer cell lines with PTEN loss, combined MEK and mTOR blockade resulted in strongly synergistic effects (CI: 0.0005-0.4), due to cooperative induction of apoptosis. A similar synergistic growth inhibition was also observed in a patient-derived lung cancer stem cell line, which displayed low to undetectable PTEN protein levels. Conclusions: Our results suggest that combined MEK and mTOR inhibition exerts strongly synergistic effects in cancer models with PTEN loss. This notion may be helpful in selecting appropriate cellular (and possibly clinical) contexts for the design rational therapeutic strategies based on concomitant inhibition of the RAF/MEK/ERK and PI3K/AKT/mTOR pathways.

Abstract 3795: BubR1 function is required for the antiproliferative activity of the novel microtubule-targeting drug BAL27862 (active moiety of the prodrug BAL101553)

Abstract Background: BAL27862 is a novel small molecule that induces apoptosis in cancer cells through microtubule destabilization associated with a distinct microtubule phenotype. BAL27862 has broad antitumor activity in preclinical models of human cancer, and has recently entered Phase I clinical trials in advanced cancer patients administered as a highly soluble lysine prodrug (BAL101553). Methods: Combinatorial RNAi screening using HeLa cervical carcinoma cells and a siRNA library targeting the human kinome/phosphatome was followed by image analysis of cell number and fraction with normal morphology after optimal BAL27862 treatment (50nM, 24h). BubR1 siRNA effects were also assessed by immunoblotting (IB) and 48h crystal violet proliferation assays. Drug-resistant tumor lines were selected in vitro by incubation with increasing BAL27862 concentrations. Patient-derived tumors were maintained in mice and analyzed by immunohistochemistry (IHC). Results: Combinatorial RNAi screening identified BubR1 (BUB1B gene) as a protein required for the antiproliferative and phenotypic effect of BAL27862. This finding was confirmed using single independent siRNAs. BubR1, an evolutionary conserved protein kinase controlling cell division, is a core component of the spindle assembly checkpoint. Consistent with this, a more detailed siRNA analysis across a panel of lines derived from different histological backgrounds (breast, colon, lung, brain) confirmed the essential role of BubR1 in the in vitro response of diverse tumor lines to BAL27862 treatment. For example, non-targeting control siRNA (NTC)-treated MDA-MB-453 breast carcinoma and H460 NSCLC cells entered a cell death program following BAL27862 treatment (∼60 % &amp; ∼40 % cell death resp. vs. starting cell number); a response clearly attenuated by BubR1 down regulation (∼80% cell proliferation. vs. DMSO controls). Further IB analysis of tumor cell variants selected for stable drug resistance to BAL27862 (∼3-24-fold resistant), indicated that BubR1 protein expression was down regulated in 3 of 5 variants as compared to parental lines (SKOV3 ovarian, A549 &amp; H460 non-small cell lung cancer [NSCLC]). Hence, reduced BubR1 expression may modulate development of resistance to BAL27862 under treatment. Strikingly, IHC analysis of paired patient-derived tumors derived from gastric and NSCLC carcinomas, demonstrated that intrinsic resistance to BAL27862 treatment ex vivo can also be associated with reduced BubR1 tumor expression. Conclusions: Reduced BubR1 expression correlates with resistance to BAL27862 treatment in a number of experimental models. Further work to understand the functional significance of BubR1 to the mechanism of action of BAL27862 is ongoing to explore the potential of using this biomarker to support patient stratification efforts during the clinical development of BAL101553. 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 3795. doi:1538-7445.AM2012-3795

Abstract 2797: AMG 511, a potent and selective class I PI3K inhibitor, demonstrates anti-tumor activity in multiple xenograft models

Abstract Background: Constitutive activation of the phosphoinositide 3-kinases (PI3K) signaling pathway is a frequent event in many cancers and results in increased cell growth and survival. Therefore, PI3K is an attractive target for oncology. This study evaluated the correlation between the pharmacokinetics (PK) and pharmacodynamics (PD) of AMG 511, a potent and selective class I PI3K inhibitor, and anti-tumor activity in preclinical models of human cancer which harbor mutations in the PI3K pathway. Method: To determine the PD, PK, and efficacy relationship of AMG 511, U87 MG (PTEN null), HCT-116 (PI3K &amp; KRAS mutant), or BT474 (PI3K mutant, HER2 amplified) tumor bearing mice were treated with up to 10 mpk AMG 511 daily by oral gavage. For tumor PD time course and dose response studies, AKT phosphorylation (pAKT) was measured using an electrochemiluminescence detection assay after a single-dose of AMG 511 in U87 MG tumor bearing mice. To determine in vivo efficacy, mice bearing established U87 MG, HCT-116, or BT474 tumors were treated daily and measured 2x/week. The mechanism of action of AMG 511 was determined by examining effects on cell proliferation (BrdU incorporation) and apoptosis (caspase staining) using a combination of immunohistochemistry and flow cytometry analyses of ex-vivo U87 MG xenograft tumors following treatment with AMG 511. Results: Inhibition of pAKT in U87 MG tumor tissue was associated with increased plasma concentration of AMG 511 after a single dose (EC50=59 ng/mL). Treatment with 0.3, 1, and 3 mpk AMG 511 inhibited pAKT in tumor tissue by 39, 81, and 92 %, respectively. Treatment with 10 mpk AMG 511 significantly inhibited pAKT up to 16 hrs (p&amp;lt;0.01). A dose-dependent inhibition of xenograft growth was observed with AMG 511 versus vehicle control in multiple tumor xenograft models with PI3K pathway mutations (p&amp;lt;0.03). Flow cytometry analysis demonstrated that AMG 511 treatment resulted in a dose-dependent decrease of BrdU incorporation in tumor and CD31 positive endothelial cells at peak and trough plasma levels (p&amp;lt;0.05). Immunohistochemical analysis confirmed that AMG 511 treatment induced tumor cell apoptosis as measured by caspase staining in U87 MG tumor samples. Conclusion: AMG 511 is a potent pan-PI3K inhibitor, which has significant anti-tumor activity in human tumor xenograft models which harbor PI3K pathway mutations. Tumor growth inhibition is the result of an increase in AMG 511 exposure, a reduction in PI3K signaling, an inhibition of tumor cell and endothelial cell proliferation and an induction of tumor cell apoptosis. These data together provide evidence for the potential clinical investigation of AMG 511 in patients whose tumors harbor mutations in the PI3K pathway. 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 2797. doi:1538-7445.AM2012-2797

Abstract A224: CS-7017, a selective PPARγ agonist, attenuates the TGF-β/Smad-mediated enhanced cell motility of EGFR-tyrosine kinase inhibitor-resistant non-small cell lung cancer cells.

Abstract Background: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been shown to possess antitumor property in preclinical models of human cancers including non-small cell lung cancer (NSCLC). CS-7017, a novel selective PPARγ agonist of thiazolidinedione class, has been reported to induce morphological change and cellular differentiation as well as its antiproliferative effects. A phase II clinical trial of CS-7017 in combination with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib is currently ongoing in advanced NSCLC patients. Though EGFR-TKIs are effective for NSCLCs with EGFR-activating mutations, the majority of responders eventually develop acquired resistance. We have previously reported that EGFR-TKI-resistant NSCLC cells acquire enhanced cell motility via activation of TGF-β/Smad pathway. In this study, we aim to investigate the effects of CS-7017 in EGFR-TKI-resistant NSCLC cells with a focus on cell motility. Materials and Methods: Erlotinib-resistant PC-9/ER and gefitinib-resistant PC-9/ZD cells, established from PC-9 NSCLC cells with EGFR-activating mutation, were used for this study and constitutive PI3K/Akt activation and secondary T790M mutation have been previously shown to account for the acquired resistance, respectively. Growth-inhibitory effect of CS-7017 was evaluated by MTT assay. Wound closure assay and transwell assay were performed to evaluate the cell motility. Secretion level of TGF-β ligands was quantified by ELISA. Transcriptional activity of Smad and PPARγ was measured by luciferase assay. Effects of CS-7017 on the molecules relevant to EGFR, TGF-β and PPARγ pathways were observed by immunoblot analysis. Results: Both PC-9/ER and PC-9/ZD resistant cells showed enhanced cell motility compared with PC-9 parental cells. Significantly higher amount of secreted TGF-β2 was detected in both resistant cells than in PC-9 cells and induction of phosphorylated Smad2 was observed in the resistant cell lines. Activation of TGF-β/Smad pathway by the increased TGF-β2 secretion was suggested to confer the enhanced cell motility of the resistant cells. CS-7017 showed significant inhibitory effect on the cell motility of the resistant cells but showed no growth-inhibitory effect. Moreover, combined treatment with CS-7017 and erlotinib resulted in better inhibition of the cell motility of PC-9/ER cells. CS-7017 treatment was found to suppress the TGF-β2 secretion significantly and attenuated Smad2 phosphorylation was observed in the resistant cells. Smad-mediated transcriptional activity was also suppressed by CS-7017 treatment. CS-7017 treatment induced PPARγ-mediated transcriptional activity in PC-9 cells but not in PC-9/ER cells, suggesting that the inhibitory effect of CS-7017 on the cell motility of PC-9/ER cells may not be due to direct activation of PPARγ signaling by CS-7017. Conclusions: These results imply that CS-7017 may serve as a potential therapeutic agent to prevent metastasis in NSCLC patients who develop acquired resistance to EGFR-TKIs regardless of the types of resistance mechanisms. The mechanism of TGF-β2 downregulation by CS-7017 needs to be further elucidated. 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 A224.

Primary human tumor xenografted models (‘tumorgrafts’) for good management of patients with cancer

The treatment of cancer is continually improving as a result of a better understanding of oncogenesis and the development of new targeted compounds. Early clinical trials evaluating such candidate compounds require a large number of patients, and are expensive, time consuming, and expose patients to certain risks. To select the most effective molecules, preclinical investigation of antitumor compounds is an important step in the drug development process. Three main categories of preclinical cancer models are generally used in preclinical investigations, namely genetically engineered models, xenografts derived from human tumor cell lines, and human tumor fragments from patients implanted directly into immunodeficient mice, known as tumorgrafts. The establishment of tumorgrafts constitutes a long-term process consisting of various steps, in which the final objective is to show that the validated model accurately reproduces human cancer, with a high predictive value of therapeutic efficacy (regardless of the type of treatment), and closely mimics clinical situations frequently observed in patients with cancer, such as resistance to standard treatments, metastases, and relapse after initial therapies (involving residual tumor-initiating cells). The aim of this study is therefore to discuss the proposed criteria for the establishment and validation of preclinical models of human cancers that should be used for further pharmacological assessments.

Vandetanib inhibits both VEGFR-2 and EGFR signalling at clinically relevant drug levels in preclinical models of human cancer

Vandetanib is a multi-targeted receptor tyrosine kinase inhibitor that is in clinical development for the treatment of solid tumours. This preclinical study examined the inhibition of two key signalling pathways (VEGFR-2, EGFR) at drug concentrations similar to those achieved in the clinic, and their contribution to direct and indirect antitumour effects of vandetanib. For in vitro studies, receptor phosphorylation was assessed by Western blotting and ELISA, cell proliferation was assessed using a cell viability endpoint, and effects on cell cycle determined using flow cytometry. For in vivo studies, Western blotting, ELISA and immunohistochemistry (IHC) were used to assess receptor phosphorylation. Cell culture experiments demonstrated that anti-proliferative effects of vandetanib resulted from inhibition of either EGFR or VEGFR-2 signalling in endothelial cells, but were associated with inhibition of EGFR signalling in tumour cells. Vandetanib inhibited both EGFR and VEGFR-2 signalling in normal lung tissue and in tumour xenografts. In a lung cancer model expressing an activating EGFR mutation, the activity of vandetanib was similar to that of a highly selective EGFR inhibitor (gefitinib), and markedly greater than that of a highly selective VEGFR inhibitor (vatalanib). These data suggest that at the plasma exposures achieved in the clinic, vandetanib will significantly inhibit both VEGFR-2 and EGFR signalling, and that both inhibition of angiogenesis and direct inhibition of tumour cell growth can contribute to treatment response.

Radiotherapeutic Efficacy of 131I-CLR1404 in Preclinical Human Cancer Models

Preclinical PET imaging studies with 124I-CLR1404, a radioiodinated phospholipid ether analog, indicate selective tumor uptake and prolonged tumor retention in 52/54 solid cancers. The agent does not localize in either inflammatory sites or premalignant lesions and thus affords excellent promise for therapy of solid malignancies due to its prolonged tumor cell retention properties. The overall objective of this study was to evaluate the therapeutic efficacy of 131I-CLR1404 in a variety of solid human xenograft cancer models.

Phase I/II study of vandetanib for patients with recurrent malignant gliomas.

2083 Background: EGFR and VEGFR signaling have been implicated in the development and growth of glioblastoma, and simultaneous inhibition of both of these signal transduction pathways has the potential to be a more effective therapeutic approach than inhibiting a single pathway. Vandetanib is an inhibitor of VEGFR-2 and EGFR tyrosine kinase activities, which has previously been shown to have anti-tumor activity in histologically diverse pre-clinical models of human cancer, including subcutaneous and orthotopic models of human glioma. Methods: A phase I study evaluated the MTD of vandetanib in patients on enzyme-inducing antiepileptics (EIAEDs). Eligibility criteria were histologically proven glioblastoma (GBM), anaplastic glioma (AG), progressive low-grade glioma (LGG), or radiographically diagnosed brainstem gliomas, radiologic progression after RT, age ≥ 18, KPS ≥ 60, and adequate bone marrow and organ function. There was no limit on the number of prior relapses or chemotherapies. Doses were escalated on a 3 + 3 design, with dose levels of 300, 400, 500, 700, and 900 mg QD. MTD was defined as the highest dose with DLTs in 1/6 or fewer patients. In phase II, patients with histologically proven AG were eligible, with other eligibility criteria as outlined above. Patients on EIAEDs were excluded from the phase II study. Patients received vandetanib 300 mg QD on 4- week cycles. Primary endpoint was PFS6. Results: In phase I, 15 patients were evaluated (8 GBM; 5 AG, 2 LGG). Median age 44 (18-66); median KPS 90 (80-100); median prior chemotherapies 1 (1-5). The MTD was determined to be 400 mg. There was 1/6 DLT (grade 3 rash) at this dose. At 500 mg there were 2/6 DLTs; one grade 3 prolonged QTc, and one grade 4 DVT/PE. In the phase II trial, there were 32 patients with AG. Median age 41 (18-69), median KPS 90 (60-100), median prior chemotherapies 2 (0-6). There were 2 PRs, and 13 SDs. One patient remains progression-free on study for over 21 months. PFS6 was 13.2% (95% CI: 4.2-27.5%), and median PFS was 1.8 mo (95% CI: 0.89-2.66 mo). Median OS was 7.4 mo (95% CI: 3.9-12.5 mo). Conclusions: Vandetanib is well tolerated, with a MTD of 400 mg QD in patients taking EIAEDs. In patients not on EIAEDs, vandetanib 300 mg QD had only limited monotherapy activity in recurrent AG. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca

Correction: Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer

Correction: Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer

Abstract 4486: OSI-027, a selective dual mTORC1/TORC2 kinase inhibitor displays broad spectrum anti-tumor activity in preclinical models of human cancer

Abstract The mammalian target of rapamycin (mTOR) protein kinase plays a central role in regulating cell proliferation and cell survival. The PI3K/AKT signaling pathway that activates mTOR is frequently altered in many human cancers. mTOR exists as multi-protein complexes with distinct signaling functions. Complexes containing mTOR and RAPTOR (mTORC1) phosphorylate S6K and 4E-BP1 and are rapamycin sensitive. Whereas, complexes that contain mTOR and RICTOR (mTORC2) phosphorylate AKT and 4E-BP1 and are insensitive to rapamycin treatment. OSI-027 is a selective small molecule dual inhibitor of both mTORC1 and mTORC2 kinase activity with biochemical IC50 values of 22 nM and 65 nM, respectively. In the MDA-MB 231 breast carcinoma xenograft model, a single 65 mg/kg oral dose of OSI-027 resulted in significant inhibition of 4E-BP1 phosphorylation (&amp;gt;70%) up to 16 h in tumor tissue. At 24 h post-dose, 59% inhibition of phospho-4E-BP1 was observed with a corresponding plasma OSI-027 concentration of 2.2 μM. Consistent with its ability to inhibit mTORC2 in vitro, a single dose of 65 mg/kg also resulted in significant inhibition of phospho-AKT (Ser473) for up to 8 hours with slow recovery thereafter. Such extended target suppression at 65 mg/kg was associated with significant efficacy corresponding to 100% tumor growth inhibition (TGI) with 19% regression in the MDA-MB 231 xenograft model when dosed for 14 consecutive days. In comparison, a lower dose of 25 mg/kg resulted in only 39% TGI which corresponded with a shorter duration of pharmacodynamic effects in vivo. In particular, at the 25 mg/kg dose, inhibition of 4E-BP1 phosphorylation was observed only up to 8 h in tumor tissue with return to predose levels by 16 h which corresponded with plasma drug concentrations of &amp;lt;0.1 µM. These data suggest that maximal anti-tumor efficacy may require plasma levels sufficient to chronically suppress phospho-4E-BP1 in tumor tissue. In addition to significant efficacy in MDA-MB 231 breast carcinoma model, OSI-027 demonstrated robust anti-tumor activity in several different human xenograft models representing different histologies, including colon carcinomas, non-small cell lung carcinomas and ovarian carcinomas. These preclinical studies indicate that OSI-027, a dual mTORC1/2 kinase inhibitor, may show broad spectrum anti-tumor activity, including superior efficacy compared to mTORC1-selective rapalogs against some human cancers. OSI-027 is currently being evaluated in Phase I clinical studies. 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 4486.