Van Vuong

Patupilone Acts as Radiosensitizing Agent in Multidrug-Resistant Cancer Cells In vitro and In vivo

Interference with microtubule function is a promising antitumoral concept. Paclitaxel is a clinically validated tubulin-targeting agent; however, treatment with paclitaxel is often limited by taxane-related toxicities and is ineffective in tumors with multidrug-resistant cells. Patupilone (EPO906, epothilone B) is a novel non-taxane-related microtubule-stabilizing natural compound that retains full activity in multidrug-resistant tumors and is clinically less toxic than paclitaxel. Here we have investigated the effect of combined treatment with ionizing radiation and patupilone or paclitaxel in the P-glycoprotein-overexpressing, p53-mutated human colon adenocarcinoma cell line SW480 and in murine, genetically defined E1A/ras-transformed paclitaxel-sensitive embryo fibroblasts. Patupilone and paclitaxel alone and in combination with ionizing radiation reduced the proliferative activity of the E1A/ras-transformed cell line with similar potency in the sub and low nanomolar range. SW480 cells were only sensitive to patupilone, and combined treatment with low-dose patupilone (0.1 nmol/L) followed by clinically relevant doses of ionizing radiation (2 and 5 Gy) resulted in a supra-additive cytotoxic effect. Inhibition of the drug efflux protein P-glycoprotein with verapamil resensitized SW480 cells to treatment with low doses of paclitaxel alone and in combination with IR. In tumor xenografts derived from SW480 cells a minimal treatment regimen with patupilone and fractionated irradiation (1 × 2 mg/kg plus 4 × 3 Gy) resulted in an at least additive tumor response with extended tumor growth arrest. Analysis by flow cytometry in vitro revealed an apoptosis- and G2-M-independent mode of radiosensitization by patupilone. Interestingly though, a transient accumulation of cells in S phase was observed on combined treatment.Overall, patupilone might be a promising alternative in paclitaxel-resistant, P-glycoprotein-overexpressing tumors for a combined treatment regimen using ionizing radiation and a microtubule inhibitor.

Current Concepts for the Combined Treatment Modality of Ionizing Radiation with Anticancer Agents

In current applied radiobiology, there exists a tremendous effort in basic and translational research to identify novel treatment modalities combining ionizing radiation with anticancer agents. This is mainly due to the highly improved molecular understanding of intrinsic radioresistance and the profiling of cellular stress responses to irradiation during recent years. Ionizing radiation not only damages DNA but also affects multiple cellular components that induce a multi-layered stress response. The treatment responses can be restricted to the individual cell level but might also be part of an intercellular stress communication network. Both DNA damage-induced signaling (which results in cell cycle arrest and induction of the DNA-repair machinery) and also ionizing radiation-induced signal transduction cascades, which are generated at cellular sites distant from and independent of DNA-damage, represent interesting targets for anticancer treatment modalities to sensitize for ionizing radiation. Due to the lack of molecular knowledge classic radiobiology assembled the cellular and tissue responses into four groups (4 Rs of radiotherapy) which describe biological factors influencing the treatment response to fractionated radiotherapy. These classic 4 Rs are Repair, Reassortment, Repopulation and Reoxygenation. With the tremendous progress in molecular oncology we now begin to understand theses factors on the molecular level. At the same time this classification may guide modern molecular radiobiologists to identify novel pharmaceuticals and antisignaling agents which can modulate the treatment response to irradiation. In this review we describe current approaches to sensitize tumor cells with novel anticancer agents along the lines of these 4 Rs.

Role of the microenvironment for radiosensitization by patupilone

Purpose: The combined treatment modality of ionizing radiation (IR) and the clinically relevant microtubule-stabilizing compound patupilone (epothilone B, EPO906) is a promising approach for anticancer therapy. Here, we investigated the role of the tumor microenvironment for the supra-additive in vivo response in tumor xenografts derived from patupilone-sensitive and patupilone-resistant non-small cell lung cancer cells. Experimental Design: The treatment response to a combined regimen of patupilone and IR was investigated in vitro and in tumor xenografts derived from wild-type A549 and A549.EpoB40 cells, which are resistant to patupilone due to a β-tubulin mutation. Results: In both A549 and A549.EpoB40 cells, proliferative activity and clonogenicity were reduced in response to IR, whereas patupilone, as expected, inhibited proliferation of the mutant cell line with reduced potency. Combined treatment with patupilone and IR induced a cytotoxic effect in vitro in an additive way in A549 cells but not in the tubulin-mutated, patupilone-resistant A549.EpoB40 cells. A supra-additive tumor growth delay was induced by combined treatment in xenografts derived from A549 cells but not in xenografts derived from A549.EpoB40 cells. Histologic analysis revealed a significant decrease in tumor cell proliferation (Ki-67) and microvessel density and a treatment-dependent change of tumor hypoxia in A549 but not A549.EpoB40 xenografts. Conclusions: Using a genetically defined patupilone-sensitive and patupilone-resistant tumor model, we here showed that the major cytotoxic effect of the combined treatment modality of IR and patupilone is directed against the tumor cell compartment. The induced antiangiogenic effect derives indirectly from the tumor cell.

Temperature sensitivity of phospho-Ser473-PKB/AKT

The phospho-PKB/Akt status is often used as surrogate marker to measure activation of the PI3K/Akt/mTOR signal transduction pathway. Though, inconsistencies of the p-Ser(473)-PKB/Akt status have raised doubts in the validity of p-Ser(473)-PKB/Akt phosphorylation as endpoint. Here, we determined that p-Ser(473)-PKB/Akt but not p-Thr(308)-PKB/Akt phosphorylation is highly temperature sensitive. p-Ser(473)-PKB/Akt phosphorylation was rapidly reduced to levels below 50% on exposure to 20-25 degrees C in murine and human cell lines including cells expressing constitutively active PI3K or lacking PTEN. Down-regulation of p-Ser(473)-PKB/Akt was reversible and re-exposure to physiological temperature resulted in increased p-Ser(473)-PKB/Akt phosphorylation levels. Phosphatase activity at low temperature was sustained at 75% baseline level and phosphatase inhibition prevented p-Ser(473)-PKB/Akt dephosphorylation induced by the low temperature shift. Interestingly temperature-dependent deregulation of the p-Ser(473)-PKB/Akt status was also observed in response to irradiation. Thus our data demonstrate that minimal additional stress factors deregulate the PI3K/Akt-survival pathway and the p-Ser(473)-PKB/Akt status as experimental endpoint.

Hypoxia modulation and radiosensitization by the novel dual EGFR and VEGFR inhibitor AEE788 in spontaneous and related allograft tumor models

Concomitant inhibition of ErbB1/2- and VEGF receptor-signaling synergizes when used in combination with DNA-damaging agents. Here, we investigated for the first time the combined treatment modality of the novel dual specific receptor tyrosine kinase inhibitor AEE788 with ionizing radiation and analyzed treatment-induced end points in situ as indicators for a potential sensitizing mechanism. Furthermore, we assessed tumor hypoxia in response to different antiangiogenic and antiproliferative treatment modalities. The combined treatment effect was investigated in a spontaneously growing mammary carcinoma model and against Her-2/neu-overexpressing mammary carcinoma allografts. In tumor allografts derived from murine mammary carcinoma cells of mouse mammary tumor virus/c-neu transgenic mice, a minimal treatment regimen with AEE788 and fractionated irradiation resulted in an at least additive tumor response. Treatment response in the corresponding spontaneous tumor model strongly exceeded the response induced in the isogenic allografts. Treatment-induced changes of tumor proliferation, apoptosis, and microvessel density were similar in the two tumor models. Treatment with AEE788 alone or in combination with IR strongly improved tumor oxygenation in both tumor models as determined by the detection of endogenous and exogenous markers of tumor hypoxia. Specific inhibition of the VEGF-receptor tyrosine kinase versus Erb1/2-receptor tyrosine kinase indicated that it is the antiproliferative and not the antiangiogenic potency of AEE788 that mediates the hypoxia-reducing effect of this dual kinase-specific inhibitor. Overall, we show that concomitant inhibition of ErbB- and VEGF-receptor signaling by AEE788, in combination with ionizing radiation, is a promising treatment approach, especially in hypoxic, oncogenic ErbB-driven tumors. [Mol Cancer Ther 2007;6(9):2496–504]

Metabolism of tumors under treatment: mapping of metabolites with quantitative bioluminescence.

BACKGROUND AND PURPOSE The metabolic switch to aerobic glycolysis (Warburg effect) and enhanced lactate production is characteristic for aggressive tumor cells and is a co-determining factor for tumor response and treatment outcome. Thus analysis of the metabolic status under treatment is important to understand and improve treatment modalities. MATERIALS AND METHODS Metabolite concentrations were determined by the immersion of tumor sections in an ATP, lactate or glucose-depending luciferase-containing buffer system. Integrated light output is detected in a bioluminescent detection system. RESULTS Mice carrying tumor xenografts derived from A549 lung cancer cells were treated with the microtubule stabilizing agent patupilone, ionizing radiation or in combination. Lactate levels were significantly reduced and glucose levels drastically increased in comparison to untreated tumors. Interestingly, these changes were only minimal in tumors derived from patupilone-resistant but otherwise isogenic A549EpoB40 cells. ATP levels of all tumors tested did not change under any treatment. When compared with histological endpoints, basal and treatment-dependent changes of lactate levels in the different tumors mainly correlated with the proliferative activity and the tumor growth response to treatment. CONCLUSIONS This study shows that the tumor metabolism is responsive to different treatment modalities and could eventually be used as an early surrogate marker for treatment response.

Combined Treatment Strategies for Microtubule Stabilizing Agent-Resistant Tumors

BACKGROUND Resistance to microtubule-stabilizing agents is a major hurdle for successful cancer therapy. We investigated combined treatment of microtubule-stabilizing agents (MSAs) with inhibitors of angiogenesis to overcome MSA resistance. METHODS Treatment regimens of clinically relevant MSAs (patupilone and paclitaxel) and antiangiogenic agents (everolimus and bevacizumab) were investigated in genetically defined MSA-resistant lung (A549EpoB40) and colon adenocarcinoma (SW480) tumor xenografts in nude mice (CD1-Foxn1, ICRnu; 5-14 per group). Tumor growth delays were calculated by Kaplan-Meier analysis with Holm-Sidak tests. All statistical tests were two-sided. RESULTS Inhibition of mTOR-kinase by everolimus only minimally reduced the proliferative activity of β tubulin-mutated lung adenocarcinoma cells alone and in combination with the MSA patupilone, but everolimus inhibited expression and secretion of vascular endothelial growth factor (VEGF) from these cells. mTOR-kinase inhibition strongly sensitized tumor xenografts derived from these otherwise MSA-resistant tumor cells to patupilone. Tumors treated with the combined modality of everolimus and patupilone had statistically significantly reduced tumor volume and stronger tumor growth delay (16.2 ± 1.01 days) than control- (7.7 ± 0.3 days, P = .004), patupilone- (10 ± 0.97 days, P = .009), and everolimus-treated (10.6 ± 1.4 days, P = .014) tumors. A combined treatment modality with bevacizumab also resensitized this MSA-refractory tumor model to patupilone. Treatment combination also strongly reduced microvessel density, corroborating the relevance of VEGF targeting for the known antivasculature-directed potency of MSA alone in MSA-sensitive tumor models. Resensitization to MSAs was also probed in P glycoprotein-overexpressing SW480-derived tumor xenografts. Different bevacizumab regimens also sensitized this otherwise-resistant tumor model to clinically relevant MSA paclitaxel. CONCLUSIONS A treatment combination of MSAs with antiangiogenic agents is potent to overcome tumor cell-linked MSA resistance and should be considered as strategy for MSA-refractory tumor entities.

Dynamics of Tumor Hypoxia in Response to Patupilone and Ionizing Radiation

Tumor hypoxia is one of the most important parameters that determines treatment sensitivity and is mainly due to insufficient tumor angiogenesis. However, the local oxygen concentration in a tumor can also be shifted in response to different treatment modalities such as cytotoxic agents or ionizing radiation. Thus, combined treatment modalities including microtubule stabilizing agents could create an additional challenge for an effective treatment response due to treatment-induced shifts in tumor oxygenation. Tumor hypoxia was probed over a prolonged observation period in response to treatment with different cytotoxic agents, using a non-invasive bioluminescent ODD-Luc reporter system, in which part of the oxygen-dependent degradation (ODD) domain of HIF-1α is fused to luciferase. As demonstrated in vitro, this system not only detects hypoxia at an ambient oxygen concentration of 1% O2, but also discriminates low oxygen concentrations in the range from 0.2 to 1% O2. Treatment of A549 lung adenocarcinoma-derived tumor xenografts with the microtubule stabilizing agent patupilone resulted in a prolonged increase in tumor hypoxia, which could be used as marker for its antitumoral treatment response, while irradiation did not induce detectable changes in tumor hypoxia. Furthermore, despite patupilone-induced hypoxia, the potency of ionizing radiation (IR) was not reduced as part of a concomitant or adjuvant combined treatment modality.

Ionizing radiation and inhibition of angiogenesis in a spontaneous mammary carcinoma and in a syngenic heterotopic allograft tumor model: a comparative study

BackgroundThe combined treatment modality of ionizing radiation (IR) with inhibitors of angiogenesis (IoA) is a promising treatment modality based on preclinical in vivo studies using heterotopic xeno- and allograft tumor models. Nevertheless reservations still exist to translate this combined treatment modality into clinical trials, and more advanced, spontaneous orthotopic tumor models are required for validation to study the efficacy and safety of this treatment modality.FindingsWe therefore investigated the combined treatment modality of IR in combination with the clinically relevant VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787 in the MMTV/c-neu induced mammary carcinoma model and a syngenic allograft tumor model using athymic nude mice. Mice were treated with fractionated IR, the VEGFR-inhibitor PTK787/ZK222584 (PTK787), or in combination, and efficacy and mechanistic-related endpoints were probed in both tumor models. Overall the treatment response to the IoA was comparable in both tumor models, demonstrating minimal tumor growth delay in response to PTK787 and PTK787-induced tumor hypoxia. Interestingly spontaneously growing tumors were more radiosensitive than the allograft tumors. More important combined treatment of irradiation with PTK787 resulted in a supraadditive tumor response in both tumor models with a comparable enhancement factor, namely 1.5 and 1.4 in the allograft and in the spontaneous tumor model, respectively.ConclusionsThese results demonstrate that IR in combination with VEGF-receptor tyrosine kinase inhibitors is a valid, promising treatment modality, and that the treatment responses in spontaneous mammary carcinomas and syngenic allografts tumor models are comparable.

Abstract C233: BAL27862: A unique microtubule‐targeted agent with a potential for the treatment of human brain tumors

Background: BAL27862 is a synthetic small molecule, potently inducing apoptosis in cancer cells through tubulin depolymerization characterized by a unique microtubule phenotype. BAL27862 has demonstrated a broad in vitro anti‐proliferative activity against a range of human tumor histotypes (low nM IC50s). Significant antitumor responses occur in animal models of human cancer after oral or intravenous (i.v.) administration, including tumors refractory to conventional treatments. The purpose of this study was to investigate the tissue distribution of BAL27862 in tumor‐bearing mice. Tumor and brain penetration, together with anti‐proliferative activity in glioblastoma (GBM) cell lines, is described. Materials and Methods: Anti‐proliferative activity and induction of tumor cell death in vitro were analyzed using the YO‐PRO® assay (48h incubation). CD1 nu/nu female mice were implanted with human colon carcinoma SW480 cells. For pharmacokinetic studies, tumor‐bearing (approx. 150 mm 3 ) mice were administered i.v. with either 2 mg/kg 14 C‐BAL27862 (n=6, single dose) or 8 mg/kg cold BAL27862 (n=33, once‐weekly during 4 weeks). Animals were culled at serial time points. Radioactivity was measured in slices, with Bio‐Imaging Analyzer read‐outs of the exposed phosphor imaging plates quantified with Aida software. Cold BAL27862 was determined in plasma, brain and tumors using a specific LC‐MS/MS method after sample homogenization and protein precipitation. Results: BAL27862 elicited a potent anti‐proliferative activity in 6 GBM cell lines (IC50 range: 10–20nM), which was independent of PTEN status. Strikingly, at optimal concentrations, a dramatic loss of cell viability was observed (% cell death at 50nM BAL27862: >15% in 5 lines, >30% in 2 lines), demonstrating that BAL27862 potently drives GBM cells into a cell death program..In mice, after i.v. administration of 14 C‐BAL27862, radioactivity was distributed to all organs: notably brain and tumor. A delayed peak was observed in slowly perfused organs such as skin, tumor and fat‐tissue. There was no tissue‐specific retention of radioactivity, as halflives were comparable between tissues and blood. 48h after administration, radioactivity was almost undetectable in most tissues. Following administration of 8 mg/kg i.v. cold BAL27862 to mice, the excellent brain and tumor penetration was confirmed. Specifically, similar levels of BAL27862 were found in brain and plasma (Cmax of 6.86 µg/g and 7.34 µg/mL, respectively). The ratio tumor/plasma was also around 1. There was no brain or tumor accumulation over time, as concentrations paralleled those in plasma. Conclusions: BAL27862 is efficiently distributed to tissues and tumor in a mouse model of human cancer. Significant brain penetration, coupled with cytotoxic activity in GBM tumor cell lines, would support further evaluation of BAL27862 for the treatment of human brain cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C233.