A Novel Orally Active Small Molecule Potently Induces G 1 Arrest in Primary Myeloma Cells and Prevents Tumor Growth by Specific Inhibition of Cyclin-Dependent Kinase 4/6 Linda B. Baughn, 1 Maurizio Di Liberto, 1 Kaida Wu, 4 Peter L. Toogood, 5 Tracey Louie, 1 Rachel Gottschalk, 3 Ruben Niesvizky, 2 Hearn Cho, 2,3 Scott Ely, 1 Malcolm A.S. Moore, 4 and Selina Chen-Kiang 1,3 Departments of 1 Pathology and 2 Medicine and 3 Graduate Program in Immunology and Microbial Pathogenesis, Weill Medical College of Cornell University; 4 Memorial Sloan-Kettering Cancer Center, New York, New York; and 5 Pfizer Global Research and Development, Ann Arbor, Michigan Abstract Cell cycle deregulation is central to the initiation and fatality ofmultiplemyeloma,thesecondmostcommonhematopoietic cancer,althoughimpairedapoptosisplaysacriticalroleinthe accumulation of myeloma cells in the bone marrow. The mechanism for intermittent, unrestrained proliferation of myeloma cells is unknown, but mutually exclusive activation of cyclin-dependent kinase 4 (Cdk4)-cyclin D1 or Cdk6-cyclin D2 precedes proliferation of bone marrow myeloma cells in vivo . Here, we show that by specific inhibition of Cdk4/6, the orally active small-molecule PD 0332991 potently induces G 1 arrest in primary bone marrow myeloma cells ex vivo and prevents tumor growth in disseminated human myeloma xenografts. PD 0332991 inhibits Cdk4/6 proportional to the cycling status of the cells independent of cellular transforma- tion and acts in concert with the physiologic Cdk4/6 inhibitor p18 INK4c . Inhibition of Cdk4/6 by PD 0332991 is not accom- panied by induction of apoptosis. However, when used in combination with a second agent, such as dexamethasone, PD 0332991 markedly enhances the killing of myeloma cells by dexamethasone. PD 0332991, therefore, represents the first promising and specific inhibitor for therapeutic targeting of Cdk4/6inmultiplemyelomaandpossiblyotherB-cellcancers. (Cancer Res 2006; 66(15): 7661-7) Introduction Multiple myeloma, the second most common hematopoietic cancer, represents a clinically defined collection of plasma cell neoplasms in which malignant plasmacytoid cells are arrested at various stages of plasma cell differentiation (1). Unlike normal plasma cells, which are permanently withdrawn from the cell cycle (2, 3), multiple myeloma cells retain their self-renewing potential (4, 5). During the stable phase of the disease, myeloma cells accumulate in the bone marrow mainly due to impaired apoptosis (6). However, among the noncycling cells, there are self-renewing myeloma cells, which reenter the cell cycle and divide without restraint during relapse and drug resistance. As multiple myeloma is invariably fatal, it is imperative to define the mechanism of cell cycle deregulation in multiple myeloma. Reentry and progression through the G 1 phase of the cell cycle is driven by cyclin-dependent kinases (Cdk) in cooperation with cyclins and inhibited by Cdk inhibitors (CKI; ref. 7). Phosphor- ylation of the retinoblastoma protein p105 (Rb) by Cdk4 or Cdk6 together with cyclin D in early G 1 and by Cdk2 in conjunction with cyclin E in late G 1 leads to the release of E2F transcription factors and S-phase entry (8). In turn, the Cdk4 and Cdk6 activities are attenuated by CKIs of the INK4 family, whereas that of Cdk2 is inhibited by CKIs of the Cip/Kip family (7). Inhibition of Cdk6 by p18 INK4c (p18) of the INK4 family (9, 10) is required for G 1 arrest and differentiation of antibody-secreting, end-stage plasma cells from antigen-activated B cells in vivo and in vitro (2, 3). In the absence of p18, plasmacytoid cells expressing CD138 (syndecan-1), a proteoglycan present on both normal and malignant plasma cells (11), are generated, but they continue to cycle and are rapidly eliminated by apoptosis (3). Attenuation of Cdk4/6 by p18, therefore, is critical for normal plasma cell differentiation. The mechanism that underlies cell cycle deregulation in myeloma is largely unknown. Overexpression of D cyclins has been implicated in promoting myeloma progression based on chromosomal translocation of cyclin D1 and cyclin D3 genes to immunoglobulin loci and elevation of D cyclin RNA in myeloma cells by microarray analysis (12–14). Paradoxically, cyclin D1 overexpression was associated with a more favorable clinical outcome (15–17). Analysis of Cdk4/6-specific Rb phosphorylation as an indicator of progression through early G 1 in primary bone marrow myeloma cells in vivo and ex vivo has now shown that overexpression of cyclin D1 alone is insufficient to promote cell cycle progression (18). Instead, aberrant coactivation and pairing of Cdk4-cyclin D1 and Cdk6-cyclin D2 precedes proliferation of myeloma cells and is enhanced in advanced disease, regardless of the treatment history or the clinical heterogeneity (18). The mutually exclusive pairing of Cdk4-cyclin D1 and Cdk6-cyclin D2 reveals a previously unappreciated specificity and complexity of cell cycle deregulation in myeloma and identifies Cdk4 and Cdk6 as key determinants in the loss of cell cycle control in myeloma. These findings further suggest that Cdk4/6 may be effective targets for therapeutic intervention. To test this possibility, we inhibited Cdk4 and Cdk6 in primary bone marrow myeloma cells using PD 0332991, an orally active, water-soluble cell-permeable pyridopyrimidine, which potently inhibits recombinant Cdk4 and Cdk6 (IC 50 = 0.011-0.016 Amol/L) by competing for the ATP- binding sites (19). Unlike other Cdk4/6 inhibitors that have been Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). L.B. Baughn and M. Di Liberto contributed equally to this work. Requests for reprints: Selina Chen-Kiang, Department of Pathology, Cornell University Medical College, 1300 York Avenue, C-338, New York, NY 10021. Phone: 212- 746-6440; Fax: 212-746-7996; E-mail: sckiang@med.cornell.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1098 www.aacrjournals.org 7661 Cancer Res 2006; 66: (15). August 1, 2006 Research Article