Molecular Cancer Therapeutics 1249 Peroxisome proliferator-activated receptor ; and retinoid X receptor ligands are potent inducers of differentiation and apoptosis in leukemias Marina Konopleva,1 Elena Elstner,4 (i.e., all trans-retinoic acid) enhanced differentiating and Teresa J. McQueen,1 Twee Tsao,1 growth-inhibitory effects. 2-Cyano-3,12-dioxooleana-1,9- Andrey Sudarikov,1 Wei Hu,1 Wendy D. Schober,1 dien-28-oic acid induced differentiation and apoptosis Rui-Yu Wang,1 David Chism,1 Steven M. Kornblau,1 with much greater potency than the other PPAR; ligands Anas Younes,2 Steven J. Collins,5 H. Phillip Koeffler,6 in established cell lines and primary chronic lymphocytic and Michael Andreeff1,3 leukemia samples. Exposure to 2-cyano-3,12-dioxo- oleana-1,9-dien-28-oic acid induced mitochondrial depo- Section of Molecular Hematology and Therapy and Departments larization and caspase activation, which was associated of 1Blood and Marrow Transplantation, 2Lymphoma, and 3Leukemia, University of Texas M.D. Anderson Cancer Center, with apoptosis induction. In Bcl-2-overexpressing chronic Houston, Texas; 4Department of Medicine (Charite´), Division lymphocytic leukemia cells, the small-molecule Bcl-2 of Hematology/Oncology, Humboldt University, Berlin, Germany; inhibitor HA14-1 sensitized these cells to 2-cyano-3,12- 5Fred Hutchinson Cancer Research Center, Seattle, Washington; 6 dioxooleana-1,9-dien-28-oic acid–induced apoptosis. and Division of Hematology/Oncology, Cedars-Sinai Medical ; Center/University of California at Los Angeles School of Medicine, These results suggest that PPAR ligation alone and in Los Angeles, California combination with retinoids holds promise as novel therapy for leukemias by activating the transcriptional activity of target genes that control apoptosis and differentiation in Abstract leukemias. [Mol Cancer Ther 2004;3(10):1249–62] The peroxisome proliferator-activated receptor ; (PPAR;) is a member of the nuclear receptor family that forms heterodimers with retinoid X receptor. These heterodimers Introduction bind to DNA and activate the transcription of target genes. Recent progress in the molecular and cellular biology of the Here, we report that the PPAR; receptor protein is nuclear steroid receptor superfamily has identified certain expressed in primary myeloid and lymphoid leukemias members as molecular targets for cancer therapy (1). They and in lymphoma and myeloma cell lines. In this study, we include estrogen receptors, retinoic acid receptors, retinoid compared the activity of several PPAR; ligands including X receptors (RXR; the RXR-specific ligands are termed BRL49653 (rosiglitazone), 15-deoxy-#12,14-prostaglan- ‘‘rexinoids’’), and the vitamin D receptor (the vitamin D– specific ligands are termed ‘‘deltanoids’’). These nuclear din J2, and the novel triterpenoid 2-cyano-3,12-dioxo- oleana-1,9-dien-28-oic acid on leukemia cells. Exposure to receptors are putative cancer therapy targets because they these PPAR; ligands induced apoptosis in myeloid (U937 function as transcription factors that control the expression and HL-60) and lymphoid (Su-DHL, Sup-M2, Ramos, Raji, of many genes related to cell differentiation (1, 2). The Hodgkin’s cell lines, and primary chronic lymphocytic strongest evidence for the therapeutic potential of this leukemia) cells. A similar exposure to these PPAR; ligands approach comes from the efficacy of retinoic acid receptor a induced the differentiation of myeloid leukemic cells. A activation in the treatment of acute promyelocytic leukemia combination of PPAR; ligands with a retinoid X receptor (3). Over 90% of patients with acute promyelocytic agonist (i.e., LG100268) or a retinoic acid receptor agonist leukemia achieve complete remission following treatment with the naturally occurring retinoid all trans-retinoic acid (ATRA; ref. 4). The peroxisome proliferator-activated receptor g (PPARg) is a recent addition to the nuclear steroid receptor Received 7/14/03; revised 7/2/04; accepted 7/22/04. superfamily that is believed to be involved in the regulation Grant support: Leukemia and Lymphoma Society (M. Konopleva), of lipid metabolism. Like other nuclear steroid receptors, National Cancer Institute grants RO1 CA89346 and 1 P50 CA100632-01, (M. Andreeff) Deutsche Forschungsgemeinschaft grant and J-Carreras the dysregulation of PPARg has been implicated in (E. Elstner). aberrant differentiation. PPARg is an important mediator The costs of publication of this article were defrayed in part by the of the differentiation of preadipocytes to adipocytes and is payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to a target in the treatment of diabetes, where receptor indicate this fact. activation increases sensitivity to endogenous insulin. To Requests for reprints: Michael Andreeff, Department of Blood and Marrow activate transcription, PPARg must form a heterodimer Transplantation, University of Texas M.D. Anderson Cancer Center, with RXRa (5). The PPARg/RXR heterodimers can then be 1515 Holcombe Boulevard, Unit 448, Houston, TX 77030. Phone: 713-792-7260; Fax: 713-794-4747. activated by the ligation of PPARg or RXR ligands (6), but E-mail: [email protected] the simultaneous ligation of both PPARg and RXR max- Copyright C 2004 American Association for Cancer Research. imizes their activation. PPARg can be activated by Mol Cancer Ther 2004;3(10). October 2004 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. 1250 PPARg and RXR Ligands in Leukemias naturally occurring ligands such as the long-chain fatty (27), a selective PPARg antagonist, was synthesized and acids 15-deoxy-D12,14-prostaglandin J2 (15-d-PGJ2) and 9- kindly provided by Dr. Stephen H. Safe (Texas A&M and 13-cis-hydroxyoctadecadienoic acid (7–9). They are University, College Station, TX). HA14-1 (ethyl 2-amino-6- also activated by synthetic ligands including the antidia- bromo-4-[1-cyano-2-ethoxy-2-oxoethyl]-4H-chromene-3- betic agents troglitazone, BRL49653 (rosiglitazone), and carboxylate), a novel nonpeptidic ligand of the Bcl-2 pioglitazone; L-Tyr-based PPARg ligands (10); Fmoc-L-Leu BH3-binding pocket (28, 29) that inhibits Bcl-2 function, was amino acids (11); and certain triterpenoids (12). kindly provided by Dr. Ziwei Huang (University of Illinois Although PPARg ligands have mainly been used for the at Urbana-Champaign, Urbana, IL). The Fas signaling anti- treatment of diabetes, they have also been shown to induce body CH11 was purchased from Immunotech (Miami, FL) lipogenic differentiation of malignant cells in vivo in and tumor necrosis factor–related apoptosis-inducing li- patients with liposarcomas (13). In hematopoiesis, PPARg gand (TRAIL) was purchased from Alexis (San Diego, CA). expression is increased during differentiation of monocytes Cell Lines to macrophages and the activation of PPARg/RXR-regu- HL-60, U937, Jurkat, Daudi, Raji, Ramos, 8226, and IM-9 lated genes induces macrophage differentiation (9). PPARg cell lines were obtained from the American Type Culture expression has been detected in human breast, lung, Collection (Rockville, MD). HL-60 is a myeloid leukemia bladder, colon, prostate, and pancreatic cancer cells and cell line; U937 is a monocytic leukemia cell line; Daudi, the ligand-induced activation of PPARg/RXR induced Ramos, and Raji are human Burkitt lymphoma cell lines; g apoptosis in these cell types (14–21). PPAR /RXR signal- Jurkat is a T-lymphoblast cell line; 8226 and IM-9 are ing is involved in the induction of apoptosis in T myeloma cell lines. The Sup-M2 and Su-DHL-1 cell lines lymphocytes (22) and myeloid leukemias (23). Interesting- were derived from CD30+ anaplastic large-cell lymphomas ly, a recent report showed that synthetic PPARg agonists and carry the t(2;5) chromosomal translocation (30). The used at high concentrations inhibited the translation human Hodgkin and Reed-Sternberg–derived cell lines initiation of eukaryotic initiation factor-2 by phosphoryla- HD-MYZ, HD-LM-2, L-428, and KM-H2 were provided by tion in PPARg-deficient embryonic stem cells, suggesting the German Collection of Microorganisms and Cell the existence of a receptor-independent antiproliferative Cultures (Braunschweig, Germany). The phenotypes and mechanism (24). genotypes of these cell lines have been published previ- In this study, we investigated the effects of structurally ously (31). The HL-60-derived subline CDM-1, which different PPARg ligands on a variety of myeloid and expresses low levels of PPARg, was kindly provided by lymphoid leukemic cell lines and primary leukemia Dr. Peter Davies (University of Texas, Houston, TX; ref. 23). g samples. The PPAR ligands decreased viability and Subjects induced apoptosis, with the novel triterpenoid 2-cyano- Bone marrow or peripheral blood samples were obtained 3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) being the for in vitro studies from patients with chronic lymphocytic most potent cytotoxic agent. Concomitant RXRa ligation leukemia (CLL), acute lymphoblastic leukemia, and acute enhanced the effects of PPARg ligands and the combination myelogenous leukemia (AML). Samples were obtained of a PPARg ligand with ATRA induced maturation of during routine diagnostic procedures after informed myelomonocytic cells. Furthermore, the proapoptotic effects of CDDO could be further enhanced by inhibition consent was obtained in accordance with regulations and of Bcl-2. Hence, the apoptotic and differentiation-inducing