Preclinical Pharmacologic Evaluation of Pralatrexate and Romidepsin

Published OnlineFirst February 12, 2015; DOI: 10.1158/1078-0432.CCR-14-2249 Cancer Therapy: Preclinical Clinical Cancer Research Preclinical Pharmacologic Evaluation of Pralatrexate and Romidepsin Confirms Potent Synergy of the Combination in a Murine Model of Human T-cell Lymphoma Salvia Jain1, Xavier Jirau-Serrano2, Kelly M. Zullo2, Luigi Scotto2, Carmine F. Palermo3,4, Stephen A. Sastra3,4,5, Kenneth P. Olive3,4,5, Serge Cremers3, Tiffany Thomas3,YingWei6, Yuan Zhang6, Govind Bhagat3, Jennifer E. Amengual2, Changchun Deng2, Charles Karan8, Ronald Realubit8, Susan E. Bates9, and Owen A. O'Connor2 Abstract Purpose: T-cell lymphomas (TCL) are aggressive diseases, NOG mouse model of TCL were used to explore the in vitro and in which carry a poor prognosis. The emergence of new drugs for vivo activity of pralatrexate and romidepsin in combination. TCL has created a need to survey these agents in a rapid and Corresponding mass spectrometry–based pharmacokinetic and reproducible fashion, to prioritize combinations which should be immunohistochemistry-based pharmacodynamic analyses of prioritized for clinical study. Mouse models of TCL that can be xenograft tumors were performed to better understand a mech- used for screening novel agents and their combinations are anistic basis for the drug:drug interaction. lacking. Developments in noninvasive imaging modalities, such Results: In vitro, pralatrexate and romidepsin exhibited con- as surface bioluminescence (SBL) and three-dimensional ultra- centration-dependent synergism in combination against a panel sound (3D-US), are challenging conventional approaches in of TCL cell lines. In a NOG murine model of TCL, the combination xenograft modeling relying on caliper measurements. The recent of pralatrexate and romidepsin exhibited enhanced efficacy com- approval of pralatrexate and romidepsin creates an obvious pared with either drug alone across a spectrum of tumors using combination that could produce meaningful activity in TCL, complementary imaging modalities, such as SBL and 3D-US. which is yet to be studied in combination. Conclusions: Collectively, these data strongly suggest that the Experimental Design: High-throughput screening and multi- combination of pralatrexate and romidepsin merits clinical study modality imaging approach of SBL and 3D-US in a xenograft in patients with TCLs. Clin Cancer Res; 1–11. Ó2015 AACR. Introduction sone–based chemotherapy (2). Although advances have been made to improve the outcome, a host of factors have contrib- The peripheral T-cell lymphomas (PTCL) represent a hetero- uted to poor outcomes. Intrinsic drug resistance, rapid acqui- geneous group of lymphoma's with a poor prognosis (1). Only sition of acquired drug resistance, and the use of regimens 10% to 15% of patients will experience long-term survival with extrapolated from B-cell lymphomas are among the most standard cyclophosphamide–doxorubicin–vincristine–predni- commonly cited explanations for the observed differences in outcome between B-cell lymphoma and TCL. Since 2009, four 1 Beth Israel Deaconess Medical Center, Harvard Medical School, Bos- new drugs have been approved for patients with relapsed ton, Massachusetts. 2Center for Lymphoid Malignancies, Columbia University Medical Center, New York, New York. 3Department of or refractory PTCL, including pralatrexate, romidepsin, and Pathology and Cell Biology, Columbia University Medical Center, New belinostat for PTCL, and brentuximab vedotin for patients York, New York. 4Department of Pathology and Cell Biology, Columbia with CD30-positive anaplastic large cell lymphoma. All drugs University Medical Center, and Irving Institute for Clinical and Trans- lational Research, New York, New York. 5Department of Medicine, have overall response rates ranging from 26% to 41%, in Columbia University Medical Center, New York, New York. 6Herbert roughly similar populations, all with similar durations of Irving Comprehensive Cancer Center, Columbia University Medical benefits (3–5). Interestingly, these single agents seem to pro- 7 Center, New York, New York. Department of Biostatistics, Columbia duce benefits in excess of what one might expect for conven- University Medical Center, New York, New York. 8Department of Sys- tems Biology High-Throughput Screening Facility, Columbia Univer- tional chemotherapy, suggesting that their differing mechan- sity Medical Center, New York, New York. 9Center for Cancer Research, isms of action may overcome acquired drug resistance. The National Cancer Institute, National Institutes for Health, Bethesda, collective experience with these drugs to date suggests that these Maryland. agents may have lineage-specificactivityinTCL. Note: Supplementary data for this article are available at Clinical Cancer The prospect to improve the outcomes of patients with PTCL Research Online (http://clincancerres.aacrjournals.org/). will rely on our ability to identify agents with potentially selective Corresponding Author: Owen A. O'Connor, Columbia University Medical Center, activity in TCL, and to explore the potential merits of their 51 West 51st Street, New York, NY 10019. Phone: 212-327-5720; Fax: 212-326- combinations. A major challenge in the context of TCLs has been 5725; E-mail: [email protected] the limitation of reasonable preclinical models that can be used doi: 10.1158/1078-0432.CCR-14-2249 for validation of novel therapeutic approaches. In vitro studies of Ó2015 American Association for Cancer Research. novel agents have been hindered by the fact that neoplastic T cells www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst February 12, 2015; DOI: 10.1158/1078-0432.CCR-14-2249 Jain et al. Combination cHTS procedure Translational Relevance H9 and HH cell lines were plated at optimal density into 384- Four new drugs have been approved in T-cell lymphomas well tissue culture plates (Greiner 781080) at 50 mL per well, (TCL), including 2 histone deacetylase inhibitors, an antibody incubated for 24 hours before drug addition (18). A total of 10 drug conjugate, and pralatrexate. The recent approval of these concentrations per drug in the combination were added in three agents creates the prospect that drugs with lineage-specific plate replicates with DMSO (0.2%). Drugs were added using HP activity can be potentially combined, creating a new platform D300 Digital Dispenser. Twenty-five microliter of Cell Titer Glo of care. Based on single-agent activity of pralatrexate and (Promega) was added, and viability was measured at 24, 48, and romidepsin, we systematically explored the activity of these 72 hours. The standard reference model of Bliss independence was agents in combination, employing new imaging techniques. employed. Bliss predicts the combined response C for two single Detailed pharmacologic analysis of drug disposition in these compounds with effects A and B as C ¼ A þ B À A Â B, where each models, including intratumoral concentrations, reveals that effect is expressed as fractional inhibition between 0 and 119. The pralatrexate and romidepsin are markedly synergistic in vivo, difference between Bliss expectation and observed growth inhi- and that even lower doses achieve highly synergistic concen- bition induced by the combination of agents A and B at the same trations in plasma and tumor, resulting in marked remissions dose is the "Bliss excess" (18, 19). and an overall survival benefit for the combination. These observations have led to a phase I–II clinical trial, now actively RNA analyses accruing. We believe this will hasten the prioritization of Total RNA was isolated with Trizol reagent (Invitrogen). RNA promising drug combinations to be studied in patients with (1 mg) was reverse transcribed using a commercially available peripheral TCL. cDNA synthesis kit (Bioline). Quantitative PCR was performed on RNA that was reverse transcribed with random primers (Invitro- gen) and amplified in a LightCyclerThermocycler using probes from the Roche Universal ProbeLibrary (Roche Diagnostics). Primers and probe sets are shown in Supplementary Table S1. are difficult to grow in culture, and deriving cell lines from primary Each experiment was repeated 3 to 4 times. Induction of each tumors is challenging. Moreover, in certain diseases like mycosis mRNA was expressed relative to the untreated control, after fungoides, skin lesions contain both malignant and reactive normalization to rRNA. benign T cells, and discriminating between the two populations can be difficult (6). Mouse models that can be used for rapid Transfection of cell lines noninvasive spatiotemporal tracking of tumor responses to new The H9 and HuT-78 cell lines were transfected with the previ- drugs and combinations are lacking in TCL research, and thus ously described pGLCherryluciferase plasmid using the transfec- have hampered advances in the field (7, 8). New developments in tion reagent, Effectene (12). in vivo imaging using dual-functioning reporters that are both fluorescent and bioluminescent (BLI) provide maximum exper- Analysis and sorting of transfected cells by flow cytometry imental flexibility enabling unique biologic applications (9–12). Cells were harvested 48 hours after transfection, washed, and More precise and reproducible techniques like three-dimensional resuspended in PBS. The cells were analyzed for mCherry expres- ultrasound (3D-US) imaging and BLI are increasingly being used sion and sorted using the MoFlo Legacy cell sorter (Beckman

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