Single Agent and Combination Studies of Pralatrexate and Molecular Correlates of Sensitivity
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central British Journal of Cancer (2011) 104, 272 – 280 & 2011 Cancer Research UK All rights reserved 0007 – 0920/11 www.bjcancer.com Single agent and combination studies of pralatrexate and molecular correlates of sensitivity 1 2 1 3 2 1 1 *,1 M Serova , I Bieche , M-P Sablin , GJ Pronk , M Vidaud , E Cvitkovic , S Faivre and E Raymond 1 INSERM U728, RayLab, and Departments of Medical Oncology, Beaujon University Hospital, Assistance Publique – Hoˆpitaux de Paris, Paris 7 Diderot, 2 100 boulevard du Ge´ne´ral Leclerc, Clichy 92110, France; Laboratory of Molecular Genetics, Beaujon University Hospital, Paris 7 Diderot, Clichy, France; 3 Allos Therapeutics, Inc., Westminster, CO, USA BACKGROUND: Pralatrexate is a dihydrofolate reductase (DHFR) inhibitor with high affinity for reduced folate carrier 1 (RFC-1) and Translational Therapeutics folylpolyglutamate synthetase (FPGS), resulting in extensive internalization and accumulation in tumour cells. Pralatrexate is approved in the US for the treatment of relapsed or refractory peripheral T-cell lymphoma and is being investigated in various malignancies. Here, we evaluated molecular correlates of sensitivity to pralatrexate and explored combinations with a variety of anticancer agents. METHODS: Antiproliferative effects of pralatrexate were evaluated in 15 human-cancer cell lines using the MTT assay. Gene expression was evaluated using qRT–PCR. RESULTS: Pralatrexate and methotrexate had a similar pattern of cytotoxicity, pralatrexate being more potent. Pralatrexate potentiated the effects of platinum drugs, antimetabolites and EGFR inhibitors. Dose- and time-dependent cytotoxicity of pralatrexate correlated with high mRNA expression of FPGS. Acquired resistance to pralatrexate was associated with decreased RFC-1 expression, whereas methotrexate resistance correlated with increased DHFR expression, suggesting different mechanisms of acquired resistance. CONCLUSION: Pralatrexate was more potent than methotrexate in a panel of solid tumour lines. Our findings support the further clinical development of pralatrexate in combination with certain cytotoxics and targeted therapies, and suggest that RFC-1, FPGS and DHFR may be potential biomarkers of outcome. British Journal of Cancer (2011) 104, 272–280. doi:10.1038/sj.bjc.6606063 www.bjcancer.com Published online 21 December 2010 & 2011 Cancer Research UK Keywords: methotrexate; antimetabolites; antifolates; drug resistance; combination chemotherapy; folate transporters The folate pathway has a key role in cell growth and proliferation activity as compared with methotrexate (DeGraw et al, 1993; (Appling, 1991; Odin et al, 2003). Folic acid (folate) enters cells Sirotnak et al, 1998). The cytotoxicity of pralatrexate was shown to through reduced folate carrier 1 (RFC-1), is polyglutamated by be correlated with RFC-1 mRNA expression in human lymphoma folylpolyglutamate synthetase (FPGS), and is reduced to dihydro- cells (Wang et al, 2003). folate, which is further converted to tetrahydrofolate (THF) by Pralatrexate is undergoing clinical evaluation as a single agent or dihydrofolate reductase (DHFR). The different enzymes and in combination in several tumour types, including lymphoma transporters involved in this pathway are targets for an important (O’Connor et al, 2007; Marneros et al, 2009), and has received class of cytotoxic agents, antifolates. Methotrexate was one of the accelerated approval by the US Food and Drug Administration first agents of this class and was first used in the treatment of (FDA) for patients with refractory or relapsed peripheral T-cell childhood acute lymphoblastic leukaemia (Farber et al, 1948). lymphoma. Since then, methotrexate has been widely used in haematologic In this study, we have compared the antiproliferative activity of and solid cancers, and new generations of antifolates have been pralatrexate with that of methotrexate and other antimetabolites in rationally designed to exploit multiple aspects of the folate several human solid tumour cell lines. In addition, we further pathway (e.g., raltitrexed in colorectal cancer (Cocconi et al, evaluated molecular mechanisms of action of pralatrexate, and 1998), and pemetrexed in malignant pleural mesotheliomas screened for markers of sensitivity and acquired resistance to (Vogelzang et al, 2003) and non-small-cell lung carcinomas pralatrexate, knowledge of which could be of potential use for (NSCLC) (Hanna et al, 2004)). designing future clinical trials. Finally, possible sequence-depen- Pralatrexate ((RS)-10-propargyl-10-deazaaminopterin) is a syn- dent synergy or additive effects of pralatrexate with various thetic anti-DHFR antifolate, rationally designed to have greater cytotoxic and targeted agents were also investigated. affinity for RFC-1 and FPGS, resulting in increased cytotoxic *Correspondence: Dr E Raymond; E-mail: [email protected] MATERIALS AND METHODS This work was presented in part at the 2009 Annual Meeting of the Cell lines American Association for Cancer Research. Revised 22 November 2010; accepted 30 November 2010; published A panel of colon (HT29, HCT116, COLO205 and HCC2998), breast online 21 December 2010 (MCF7), melanoma (MDA-MB-435, formerly designated as a Pralatrexate activity in cancer cells M Serova et al 273 breast cancer line), NSCLC (HOP62 (adenocarcinoma) and HOP92 (Becton Dickinson, Le Pont de Claix, France). Apoptosis induction (large cell carcinoma)), ovarian (OVCAR3, IGROV1), prostate was evaluated using an Annexin V-FITC Apoptosis Detection Kit (DU145, PC3) and head and neck (SCC61, HEP2 and SQ20B) (BD Biosciences, Le Pont de Claix, France). cancer cell lines was purchased from the ATCC (Rockville, MD, USA) and National Cancer Institute collections. Cells were grown Gene expression analysis by RT–PCR as monolayers in RPMI medium supplemented with 10% fetal calf serum (Invitrogen, Cergy-Pontoise, France), 2 mM glutamine, The theoretical and practical aspects of quantitative real-time PCR À1 À1 100 U ml penicillin and 100 mM ml streptomycin. In this (qRT–PCR) using the ABI Prism 7900 Sequence Detection System study, we used unpurified media potentially containing glycine, (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) have hypoxanthine and thymidine, which may have theoretically been described in detail elsewhere (Bie`che et al, 2001). Results reduced drug activity. However, considering the high levels of were expressed as fold differences in target gene expression resistance developed in our cell lines and taking into account that relative to the TBP gene (an endogenous RNA control) and relative both parental and derived resistant counterpart were grown in to a calibrator (1 Â sample), consisting of the cell line sample from similar media, it remains unlikely that this would have severely our tested series that contained the smallest amount of target gene impact on data. mRNA. Experiments were performed in duplicate. Cytotoxicity assays Western blot analysis Cell viability was determined using the MTT assay, which was Cells were lysed in buffer containing 50 mM HEPES (pH 7.6), carried out as described previously (Hansen et al, 1989). Briefly, 150 mM NaCl, 1% Triton X-100, 2 mM sodium vanadate, 100 mM cells were seeded in 96-well plates at a density of 2 Â 103 cells NaF and 0.4 mg mlÀ1 phenylmethylsulfonyl fluoride. Equal per well. Cells were incubated for 120 h and then 0.4 mg mlÀ1 of amounts of protein (20–50 mg per lane) were subjected to SDS– MTT dye (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium PAGE and transferred to nitrocellulose membranes. Membranes bromide; Sigma, Saint-Quentin Fallavier, France), was added for were probed with anti-cleaved PARP, anti-cleaved caspase 3, 4 h at 371C. Media was removed and the monolayer suspended in anti-caspase 9 (Cell Signaling, Saint Quentin Yvelines, France), 0.1 ml of DMSO, after which the absorbance at 560 nm was anti-DHFR (Abcam, Paris, France), anti-b-actin (Sigma-Aldrich, measured using a microplate reader (Thermo, Saint-Herblain, Saint-Quentin Fallavier, France)-specific primary antibodies, France). The control value corresponding to untreated cells was followed by peroxidase-linked secondary antibodies and visualisa- defined as 100% and the viability of treated samples was expressed tion by chemiluminescence. as a percentage of the control. IC50 values were determined as concentrations that reduced cell viability by 50%. For single agent studies, cells were treated with increasing RESULTS concentrations of pralatrexate, methotrexate, pemetrexed, 5-FU and 50-DFUR for up to 72 h. Then, the cells were allowed to recover Single-agent antiproliferative effects in compound-free medium for 48 h before determination of growth The antiproliferative effects of pralatrexate were examined in 15 Translational Therapeutics inhibition using the MTT assay. cancer cell lines as displayed in Table 1. Time course experiments For combination studies of pralatrexate with other anticancer showed that optimal antiproliferative effects were achieved when drugs, sequential (pralatrexate for 24 h, followed by the second cells were exposed to pralatrexate for 72 h (Figure 1A). Pralatrexate agent for 24 h or the inverse schedule) or simultaneous (exposure IC50 values ranged from 0.01±0.002 mM for the prostate cancer cell to both agents for 24 h) schedules were used. Cells were treated line PC3 to 4350 mM for the MDA-MB-435 cell line. Interestingly, with various concentrations