Pralatrexate Is Synergistic with the Proteasome Inhibitor Bortezomib in in Vitro and in Vivo Models of T-Cell Lymphoid Malignancies
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Published OnlineFirst May 25, 2010; DOI: 10.1158/1078-0432.CCR-10-0671 Published OnlineFirst on July 13, 2010 as 10.1158/1078-0432.CCR-10-0671 Cancer Therapy: Preclinical Clinical Cancer Research Pralatrexate Is Synergistic with the Proteasome Inhibitor Bortezomib in In vitro and In vivo Models of T-Cell Lymphoid Malignancies Enrica Marchi1, Luca Paoluzzi1, Luigi Scotto1, Venkatraman E. Seshan2, Jasmine M. Zain1, Pier Luigi Zinzani3, and Owen A. O'Connor1 Abstract Purpose: Pralatrexate (10-propargyl-10-deazaaminopterin) is an antifolate with improved cellular up- take and retention due to greater affinity for the reduced folate carrier (RFC-1) and folyl-polyglutamyl synthase. Based on the PROPEL data, pralatrexate was the first drug approved for patients with relapsed and refractory peripheral T-cell lymphoma. Bortezomib is a proteasome inhibitor that has shown some activity in patients with T-cell lymphoma. Experimental Design: Assays for cytotoxicity including mathematical analysis for synergism, flow cy- tometry, immunoblotting, and a xenograft severe combined immunodeficient-beige mouse model were used to explore the in vitro and in vivo activities of pralatrexate alone and in combination with bortezomib in T-cell lymphoid malignancies. Results: In vitro, pralatrexate and bortezomib exhibited concentration- and time-dependent cytotoxi- city against a broad panel of T-lymphoma cell lines. Pralatrexate showed synergism when combined with bortezomib in all cell lines studied. Pralatrexate also induced potent apoptosis and caspase activation when combined with bortezomib across the panel. Cytotoxicity studies on normal peripheral blood mononuclear cells showed that the combination was not more toxic than the single agents. Western blot assays for proteins involved in broad growth and survival pathways showed that p27, NOXA, HH3, and RFC-1 were all significantly modulated by the combination. In a severe combined immunodeficient-beige mouse model of transformed cutaneous T-cell lymphoma, the addition of pralatrexate to bortezomib enhanced efficacy compared with either drug alone. Conclusion: Collectively, these data suggest that pralatrexate in combination with bortezomib repre- sents a novel and potentially important platform for the treatment of T-cell malignancies. Clin Cancer Res; 16(14); 3648–58. ©2010 AACR. Malignancies derived from mature (post-thymic) T cells grams are still the most commonly used regimens despite and natural killer cells, collectively referred to as peripher- the suboptimal outcomes. Overall, CHOP-based chemo- al T-cell lymphomas (PTCL), encompass a variety of rare therapies achieve overall response rates of 30% to 60% and often challenging diseases. PTCLs represent 10% to with an overall survival at 5 years of approximately 15% 15% of all non–Hodgkin's lymphomas worldwide (1), to 20%. Efforts to improve on these approaches with more accounting for 6,000 to 9,000 cases annually in the United dose-intense combination chemotherapy regimens (2, 3) States. CHOP (cyclophosphamide, doxorubicin, vincris- have failed to show a benefit (62% versus 56%, respec- tine, and prednisone) and CHOP-like chemotherapy pro- tively). Given the often dismal results seen in patients with PTCL compared with other subtypes of non–Hodgkin's lymphoma, there is an urgent need to identify new agents Authors' Affiliations: 1New York University Cancer Institute, NYU Langone Medical Center; 2Department of Epidemiology and with demonstrable activity in these T-cell neoplasms. Over Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New the past few years, a number of promising new drugs have York; and 3Institute of Hematology and Medical Oncology “L. & A. emerged, which seem to have marked single-agent activity Serágnoli,” University of Bologna, Bologna, Italy in T-cell lymphomas, including gemcitabine (4–7), alemtu- Note: Supplementary data for this article are available at Clinical Cancer zumab (8, 9), and the histone deacetylase (HDAC) inhibi- Research Online (http://clincancerres.aacrjournals.org/). tors (10). More recently, pralatrexate, a novel antifol with Corresponding Author: Owen A. O'Connor, Division of Hematologic Malignancies and Medical Oncology, New York University Cancer Insti- improved cellular uptake and retention, has become the tute, NYU Langone Medical Center, New York, NY 10016. Phone: 212- first drug approved by the U.S. Food and Drug Administra- 263-9884; Fax: 212-263-9210; E-mail: owen.o'[email protected]. tion for the treatment of relapsed/refractory PTCL. doi: 10.1158/1078-0432.CCR-10-0671 The results of the PROPEL registration study based on ©2010 American Association for Cancer Research. 109 evaluable patients showed an overall response rate 3648 Clin Cancer Res; 16(14) July 15, 2010 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst May 25, 2010; DOI: 10.1158/1078-0432.CCR-10-0671 Pralatrexate-Based Combination in T-Cell Malignancies of 29% in patients with very heavily treated disease.4 and CEM (18) are T-acute lymphoblastic leukemia (T- Subgroup analyses have shown that the activity of pra- ALL) lines resistant to γ-secretase inhibitors, and KOKT- latrexate was equivalent irrespective of the amount of K1 (19), DND-41, and HPB-ALL (20) are T-ALL lines prior therapy, age, prior autologous stem cell transplant, sensitive to γ-secretase inhibitors. All leukemic cell lines or subtype of PTCL. Pralatrexate belongs to the class of were provided by the laboratory of Dr. Adolfo Ferrando folate analogues known as 10-deazaaminopterins, which (Columbia University, New York, NY). Peripheral blood have shown markedly superior antitumor effects com- mononuclear cells (PBMC) from healthy donors were pared with methotrexate in severe combined immunode- purchased from Allcells. All cell lines were grown as ficient (SCID)-beige xenograft models of lymphoma (11, previously described (21). 12). Pralatrexate was designed to have greater affinity than methotrexate for the natural folate and antifolate Materials principal transporters reduced folate carrier-1 (RFC-1; All reagents for Western blotting were obtained from an oncofetal protein) and folyl-polyglutamyl synthase, Bio-Rad Laboratories and Pierce Biotechnology, Inc.; leading to enhanced intracellular accumulation and DMSO was obtained from Sigma. Pralatrexate was from polyglutamylation in tumor cells. Allos Therapeutics, and bortezomib from Millennium. Recent preclinical studies by our group have established that pralatrexate synergizes with other agents active in Cytotoxicity assays PTCL, including gemcitabine. These preclinical data (13) For all in vitro assays, cells were counted and resus- have been confirmed in a phase I clinical trial exploring pended at an approximate concentration of 3 × 105 per the schedule-dependent activity of pralatrexate and gemci- well in a 96-well plate (Becton Dickinson Labware) and tabine in patients with non–Hodgkin's lymphoma. In ad- incubated at 37°C in a 5% CO2 humidified incubator dition to PTCL, pralatrexate seems to be very active in for up to 72 hours. Pralatrexate was added at concentra- patients with drug-resistant cutaneous T-cell lymphoma tions from 100 pmol/L up to 200 μmol/L, whereas borte- (CTCL), producing an overall response rate of 55% in pa- zomib was tested at concentrations from 1 to 100 nmol/L tients who received a median of 6 prior regimens (range, to determine growth inhibition curves for all cell lines. In 1-25; ref. 14). Collectively, these data suggest that prala- combination experiments, pralatrexate was added at con- trexate exhibits marked activity across different subtypes centrations of 2 to 5.5 nmol/L, and bortezomib at 3 to of T-cell neoplasms and could combine favorably with a 6 nmol/L. These concentrations were selected to approxi- host of other agents known to be active in T-cell lympho- mate the IC25-50 (inhibitory concentration of 25-50% cells ma. The observation that bortezomib has now been for each drug) for up to 72 hours. Following incubation at shown to exhibit significant activity across both CTCL 37°C in a 5% CO2 humidified incubator, 100 μLfrom and PTCL raises the interesting prospect that these agents each well were transferred to a 96-well opaque-walled could also be combined in an effort to define a new plat- plate; CellTiter-Glo reagent (Promega Corporation) was form for these diseases that is not CHOP dependent. For used according to the manufacturer's instruction. The example, Zinzani et al. (15) have reported that bortezo- plates were allowed to incubate at room temperature for mib produces an ORR of 67% in patients with relapsed 10 minutes before recording luminescence with a Synergy or refractory CTCL and PTCL, including a complete re- HT Multi-Detection Microplate Reader (Biotek Instru- sponse rate of 17%. ments, Inc.). Each experiment was done in triplicate and Given the relatively poor prognosis of patients with T- repeated at least twice. cell malignancies and the relatively poor outcomes asso- ciated with conventional chemotherapy regimens such as Flow cytometry for apoptosis CHOP, there is now a strong rationale to begin to com- H9, HH, P12, and PF382 cells were seeded at a density of bine and integrate these targeted agents into novel com- 3×105/mL and incubated with pralatrexate (2-5.5 nmol/L) bination treatment regimens for patients with T-cell and bortezomib (2-6 nmol/L) alone or in combination for malignancies that are not CHOP based. Based on this 48 or 72 hours.