Romidepsin Synergizes with Lenalidomide in T Cell Lymphoma Cell Lines By Increasing Reactive Oxygen Species and Modulating PI3K/AKT and MAPK/ERK Signaling Pathways
Authors: Cosenza Maria , Civallero Monica , Fiorcari Stefania , Pozzi Samantha , Marcheselli Luigi , Sacchi Stefano 
Published: October 2019

Abstract
Background and purpose.T-cell lymphoma (TCL) is an heterogeneous and aggressive group of lymphoproliferative disorders which commonly relapse with current chemotherapy regimens and then difficult to treat. Therefore, novel treatment approach are urgently needed. Romidepsin is a potent and specific inhibitor of class I and II HDAC inhibitor that has shown remarkable activity in the treatment of TCL in preclinical studies and early-phase clinical trials. Lenalidomide (IMiD), is an immunomodulatory agent highly effective for the treatment of a wide range of hematological malignancies with a low toxicity profile. In this study, we sought to define the potential anticancer effect of romidepsin (Celgene) in combination with lenalidomide (Celgene) in TCL preclinical model.
Methods . The TCL cell lines Hut-78 and Karpas-299 were treated with increasing concentrations of romidepsin (0,5-25 nM) and lenalidomide (1-100 µM) as single agents from 24-72h to identify the IC50 of each drug. The interaction between romidepsin (0,5nM, 1nM, 2,5nM) and lenalidomide (2µM, 4µM, 10µM) was evaluated using the Chou-Talalay method. The cell proliferation was determined using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit. The cell cytotoxicity was quantified by MTT. Clonogenic survival was studied with the methylcellulose clonogenic assay. Apoptosis, reactive oxygen species (ROS), cell cycle analysis and Bcl-2 protein expression were measured by flow cytometry, whereas caspase activation, AKT/PI3K and MAPK/ERK pathways were analyzed by Western blot.
Results. Treatment with romidepsin alone resulted in time- and dose-dependent increase in cytotoxicity in Hut-78 and Karpas-299 cell lines with an IC50 at 24h of 5.87 nM and 6.36 nM for Hut-78 and Karpas-299, respectively. Lenalidomide alone did not inhibit cells viability up to72 h of treatment in the two TCL cell lines, as we previously described in other cell lines probably due to a more pleitropic (or cytostatic) effects. However, the combination of lenalidomide (10µM) with a low dose of romidepsin (2.5 nM) showed a strong synergistic interaction with combination index (CI) of 0.14 in Hut-78 cells and an additive effect in Karpas-299 cells, (CI of 1.08) after 24h. In Hut-78 cells sequential treatment with romidespin for 6h followed by washout and the next addition of lenalidomide for 24h enhanced the cytotoxic effect of romidepsin and confirmed its irreversible effect. Treatment with romidepsin and lenalidomide for 24 h reduced clonogenic survival to approximately 15 % in HUT-78 and to 28 % in Karpas-299. The combination did not trigger relevant decreased in the viability of normal peripheral blood mononuclear cells and suppressed cell proliferation of TCL cell lines when co-cultured with bone marrow mesenchymal stromal cells in a system that mimics the bone marrow microenvironment. Romidepsin and lenalidomide in combination induced apoptosis with little effect on cell cycle. Interestingly, the treatment with the caspase inhibitor z-VAD-fmk allowed detection of cell cycle arrest in G0/G1 phase and a decrease of S phase. The induction of apoptosis by drugs combination was associated with an increase in ROS production. The co-administration of the antioxidant N-acetyl cysteine (NAC) blocked the increased ROS generation and apoptosis observed in the drugs combination compared with control or single-agent treatments. Cell death was associated with the activation of caspases -3, -9 -8 and PARP and was mediated by Bim, Bax, Bad, Bcl-xL and Mcl-1 but did not modify Bcl-2 expression. The drugs combination induced up-regulation of cell cycle protein p21, p27 and a slight decrease of cyclin E and cyclin D. These events were associated with the dephosphorylation of PI3K/Akt and MAPK/ERK pathways. Finally, the induction of histone acetylation (H3) and acetylated alpha-tubulin was confirmed.
Conclusion . The combination of romidepsin with lenalidomide triggered cell death through interacting mechanisms including ROS generation, caspase activation and down-regulation of PI3K/AKT and MAPK/ERK pathways. These results provided a rational basis for the clinical development of the combination of romidepsin and lenalidomide for the treatment of TCL

Disclosures
No relevant conflicts of interest to declare.