ORIGINAL ARTICLE Preclinical activity of P276-00, a novel small-molecule cyclin-dependent kinase inhibitor in the therapy of multiple myeloma N Raje 1,2 , T Hideshima 1 , S Mukherjee 2 , M Raab 1 , S Vallet 1,2 , S Chhetri 1 , D Cirstea 1 , S Pozzi 1 , C Mitsiades 1 , M Rooney 1 , T Kiziltepe 1 , K Podar 1 , Y Okawa 1 , H Ikeda 1 , R Carrasco 1 , PG Richardson 1 , D Chauhan 1 , NC Munshi 1 , S Sharma 3 , H Parikh 3 , B Chabner 2 , D Scadden 2 and KC Anderson 1 1 Division of Hematologic Malignancies, Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; 2 Division of Hematology/Oncology, Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA and 3 Nicholas Piramal India Ltd, Mumbai, India Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. Here, we sought to identify the effects of targeting cyclin D in MM. We first confirmed cyclin D mRNA overexpression in 42 of 64 (65%) patient plasma cells. Silencing cyclin D1 resulted in 450% apoptotic cell death suggesting its validity as a potential therapeutic target. We next evaluated P276-00, a clinical-grade small-molecule cyclin- dependent kinase inhibitor as a way to target the cyclins. P276-00 resulted in dose-dependent cytotoxicity in MM cells. Cell-cycle analysis confirmed either growth arrest or caspase- dependent apoptosis; this was preceded by inhibition of Rb-1 phosphorylation with associated downregulation of a range of cyclins suggesting a regulatory role of P276-00 in cell-cycle progression through broad activity. Proliferative stimuli such as interleukin-6, insulin-like growth factor-1 and bone-marrow stromal cell adherence induced cyclins; P276-00 overcame these growth, survival and drug resistance signals. Because the cyclins are substrates of proteasome degradation, combi- nation studies with bortezomib resulted in synergism. Finally, in vivo efficacy of P276-00 was confirmed in an MM xenograft model. These studies form the basis of an ongoing phase I study in the treatment of relapsed/refractory MM. Leukemia (2009) 23, 961–970; doi:10.1038/leu.2008.378; published online 8 January 2009 Keywords: cyclin D; cyclin-dependent kinase; multiple myeloma; P276-00; small molecule Introduction Multiple myeloma (MM) is a hematological malignancy characterized by a clonal proliferation and infiltration of the bone marrow (BM) by plasma cells (PCs). 1,2 In 2008, an estimated 19 920 new cases of MM will be diagnosed in the United States and 10 790 deaths will be attributed to this disease 3 accounting for a disproportionate 2% cancer-related death rate. Despite currently available treatments including thalidomide, 4,5 bortezomib, 6,7 lenalidomide 8 and autologous transplantation 9 resulting in an improvement in overall survival of our patients, 10 MM remains incurable underscoring the need for a continued search for novel therapeutics. One major limitation to the development of anti-MM therapy is its heterogeneity. Recent studies of our understanding of the molecular pathogenesis of MM have resulted in a new proposed classification of MM subdividing patients into groups based on supervised gene expression profiles. 11–13 Chromosomal ab- normalities associated with MM are broadly classified into hyperdiploid, accounting for 50% of the tumors, and non- hyperdiploid (NHRD), including tumors hypodiploid, pseudo- diploid or subtetraploid. These NHRD tumors have been associated with a poorer prognosis. Five recurrent IgH translo- cations have been seen in MM including MMSET and FGFR3 (15%), cyclin D3 (3%), cyclin D1 (15%), c-maf (5%) and MAFB (2%) accounting for a prevalence of 40%. Recent evidence suggests that three of these five translocations are predominant in the NHRD tumors. 11,14,15 All MM tumor cells have high levels of cyclin D1, D2 and/or D3, including monoclonal gammopathy of unknown significance, suggesting an early unifying oncogenic event. 11–13,16,17 Dysregulation of the cyclin genes may be a result of known immunoglobulin G transloca- tions, or by presently unknown mechanisms. For example, mechanisms of cyclin D dysregulation include IgH transloca- tions that can directly affect cyclins D1 or D3, MAF or MAFB transcription factors that target cyclin D2 and biallelic dysregu- lation of the cyclin D1 gene. Cyclin D1 is specifically dysregulated by translocation (11q13) or biallelic dysregulation in over 50% of MM tumors. These molecular insights have allowed for a classification based on the five major transloca- tions and cyclin D overexpression in MM. Thus, cyclin D overexpression in nearly all MM patients is a critical common hallmark in this otherwise heterogeneous disease and may be involved in the progression of MM (Table 1). Given the crucial role played by the cyclins in MM, and specifically cyclin D1, in 50% of MM, we asked in the present study: (1) whether targeting cyclin D1 in MM was of potential therapeutic benefit; (2) whether P276-00, 18,19 a known small- molecule cyclin-dependent kinase (Cdk) inhibitor with activity against Cdk4/cyclin D1 had preclinical activity in MM and (3) whether combining P276 with other novel and conventional agents would provide improved efficacy in MM. Materials and methods Cell culture and reagents Dexamethasone (Dex)-sensitive (MM.1S) and Dex-resistant (MM.1R) human MM cell lines were kindly provided by Dr Steven Rosen (Northwestern University, Chicago, IL, USA). Doxorubicin-resistant (Dox40) and melphalan-resistant (LR5) RPMI-8226 human MM cells were kindly provided by Dr William Dalton (Moffitt Cancer Center, Tampa, FL, USA). OPM2 Received 25 August 2008; revised 24 November 2008; accepted 28 November 2008; published online 8 January 2009 Correspondence: Dr N Raje, Division of Hematology/Oncology, Massachusetts General Hospital Cancer Center, 55 Fruit Street, POB 216, Boston, MA 02114, USA. E-mail: nraje@partners.org Leukemia (2009) 23, 961–970 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu