Published OnlineFirst January 12, 2010; DOI: 10.1158/1078-0432.CCR-09-1937 Published Online First on January 12, 2010 as 10.1158/1078-0432.CCR-09-1937 Cancer Therapy: Preclinical Clinical Cancer Research Romidepsin and Belinostat Synergize the Antineoplastic Effect of Bortezomib in Mantle Cell Lymphoma Luca Paoluzzi1, Luigi Scotto3, Enrica Marchi3, Jasmine Zain3, Venkatraman E. Seshan2, and Owen A. O'Connor3 Abstract Purpose: Romidepsin and belinostat are inhibitors of histone deacetylases (HDACI). HDACIs are known to induce cell death in malignant cells through multiple mechanisms, including upregulation of death receptors and induction of cell cycle arrest. They are also known to be prodifferentiating. Mantle cell lymphoma (MCL) is an aggressive subtype of non–Hodgkin lymphoma characterized by the t(11;14) (q13;q32) translocation leading to the overexpression of cyclin D1. Experimental Design: Assays for cytotoxicty including mathematical analysis for synergism, flow- cytometry, immunoblottings, and a xenograft severe combined immunodeficient beige mouse model were used to explore the in vitro and in vivo activity of romidepsin and/or belinostat alone or in combination with the proteasome inhibitor bortezomib in MCL. Results: In vitro, romidepsin and belinostat exhibited concentration-dependent cytotoxicity against a panel of MCL cell lines. Both HDACI showed strong synergism when combined with the proteasome inhibitor bortezomib in MCL. An HDACI plus bortezomib also induced potent mitochondrial membrane depolarization and apoptosis, whereas no significant apoptosis was observed in peripheral blood mono- nuclear cells from healthy donors with the combination. These events were associated with a decrease in cyclin D1 and Bcl-XL, and an increase in accumulation of acetylated histone H3, acetylated α-tubulin, and Noxa in cell lines. In a severe combined immunodeficient beige mouse model of MCL, the addition of belinostat to bortezomib enhanced efficacy compared with either drug alone. Conclusions: Collectively, these data strongly suggest that HDACI such as romidepsin or belinostat in combination with a proteasome inhibitor could represent a novel and rationale platform for the treatment of MCL. Clin Cancer Res; 16(2); 554–65. ©2010 AACR. Mantle cell lymphoma (MCL) represents a distinct sub- ubiquitin E3-ligase that targets p27 for proteolytic degra- type of aggressive lymphoma, characterized by gross cell dation, may contribute to the loss of cell cycle inhibition cycle dysregulation. The pathognomonic lesion in the dis- (3). The collective importance of these genetic lesions has ease is the reciprocal translocation t(11;14)(q13;q32) lead- been supported by additional lines of data showing the ing to the juxtaposition of the cyclin D1 gene downstream importance of proliferation in MCL. Data by Rosenwald of the immunoglobulin heavy chain gene promoter (1). and colleagues (4) identified a “proliferation signature” This genetic event leads to constitutive overexpression of in MCL, in which the patients with the most highly prolif- cyclin D1. In addition, many forms of MCL exhibit loss erative disease experienced the poorest prognosis, whereas of the cyclin-dependent kinase (cdk) inhibitors p27 and patients with the least proliferative disease exhibited a su- p21, leading to further dysregulation of cell cycle control perior survival. This biology has also been validated by Ki- (2). Although the precise mechanism explaining the loss 67 immunohistochemical staining, which also correlated of p27 remains to be clarified, several lines of evidence overall survival with the proliferative index in MCL (5) suggest that overexpression of Skp2, a component of the These data suggest that by merely “lowering” the prolifer- ative signature of the disease, it might be possible to alter the natural history and overall prognosis of the disease. Authors' Affiliations: 1Herbert Irving Comprehensive Cancer Center, In addition to the lesions involved in cell cycle control, Columbia University; 2Biostatistics Shared Resources, Herbert Irving Comprehensive Cancer Center; and 3NYU Cancer Institute, NYU it is also clear that aberrant expression of various Bcl-2 Langone Medical Center, New York, New York family members, including Mcl-1, and deficiencies in Note: Supplementary data for this article are available at Clinical Cancer BH3-only proteins such as Bim and Noxa may render Research Online (http://clincancerres.aacrjournals.org/). these cells relatively resistant to apoptosis (6). Pharmaco- Corresponding Author: Owen A. O'Connor, NYU Langone Medical Cen- logic strategies that directly address these specific lesions ter, 522 First Avenue, Smilow Room 1101, New York, NY 10016. Phone: represent an innovative strategy for targeting “targeted 212-263-9884; Fax: 212-263-9210; E-mail: owen.o'[email protected]. therapy” to a specific disease context. doi: 10.1158/1078-0432.CCR-09-1937 The proteasome inhibitor bortezomib was approved for ©2010 American Association for Cancer Research. the treatment of relapsed or refractory MCL based on the 554 Clin Cancer Res; 16(2) January 15, 2010 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst January 12, 2010; DOI: 10.1158/1078-0432.CCR-09-1937 HDAC Inhibitors in Mantle Cell Lymphoma through, vorinostat, a hydroamic acid analogue, was the Translational Relevance first and, to date, only HDACI approved for the treatment of cancer (cutaneous T-cell–lymphoma; refs. 24–26). Al- Mantle cell lymphoma (MCL) is a relatively rare, al- though it has been difficult to ascribe a singular mecha- – though challenging, form of non Hodgkin's lympho- nism of action to the HDACI in any specific biological ma. Recently, several promising new drugs with context, the biological effect of these drugs has been important single-agent activity have begun to teach shown to include cell cycle arrest, terminal differentiation, us about the unique biology of this disease. The com- and induction of apoptosis, depending on the concentra- bination of a proteasome inhibitor and HDAC inhibi- tion of drug and the cell model studied. tors represents a novel and theoretically highly targeted The immediate rationale for combining these two clas- drug combination suited to address the unique patho- ses of drugs in MCL revolves around recent observations genetic lesions indicative of MCL. In this article, we that HDACIs, such as vorinostat and LBH-589, may be show the remarkable synergy these two classes of drugs able to “turn off” cyclin D1. Ellis et al. (27) showed in a have in MCL, establishing the fact they are able to phase 1 experience that LBH-589 consistently produced “ ” “ ” turn off cyclin D1 expression and turn on NOXA downregulation of cyclin D1 in punch biopsies of patients in vitro in vivo in a manner that is synergistic and . These with cutaneous T-cell lymphoma treated with LBH-589 by data establish the class effects of these agents providing gene expression array. Similarly, vorinostat, trichostatin A, a clear-cut rational for moving these combinations in- and even the aliphatic acids have been shown to reduce to clinical trials for patients with MCL. In addition, intracellular cyclin D1 protein in MCL cell lines, but not they provide insights into possible biomarkers of activ- in cell lines of acute myeloid leukemia. These observations ity that could be explored in future clinical studies. were shown to be dependent on concentrations of drugs that also induced accumulation of acetylated histone H3 (28, 29). Although the precise mechanism of this downre- gulation remains to be established, the observation creates consistent observations from independent phase 2 studies a unique opportunity to both turn off cyclin D1 and “turn (7–10) including one registration-directed phase 2 study on” cdk inhibitors such as p21 and p27 in a disease char- (11). In addition, bortezomib has been shown to comple- acterized by these very lesions. Belinostat and romidepsin ment a host of other targeted drugs including Bcl-2– are both pan-HDACIs that now in advanced stage clinical targeted agents such as obatoclax mesylate, ABT-737, and trials. Given the activity of proteasome inhibitors in MCL, oblimersen sodium (7, 12–14). Although there are a mul- and its ability to enforce accumulation of cdk inhibitors, titude of biological effects induced by inhibition of cellu- we sought to explore the merits of this combination in lar proteasome, accumulation of the cell cycle–dependent models of MCL as well as in normal peripheral blood kinase inhibitors such as p27, p21, p53; inhibition of NF- mononuclear cells (PBMC) from healthy donors. κB; and accumulation of proapoptotoic proteins such as Noxa have been consistently shown in many cell line Materials and Methods models (15). The acetylation and deacetylation of histones regulate Cells and cell lines. HBL-2, Jeko-1, and Granta-519 are transcriptional activation by mediating chromatin conden- well-characterized MCL lines (30, 31). Mononuclear cells sation and decondensation (16). The acetylation status of from PBMC samples of healthy donors were purchased histones and nonhistone proteins is determined by the from Allcells. All cell lines were grown as described previ- balance between histone acetyl transferase (HAT) and his- ously (12, 13). tone deacetylase (HDAC) activity. In addition to histone, Materials. All reagents for Western blotting were ob- at least 50 nonhistone proteins have been documented to tained from Bio-Rad