Cancer Therapy: Preclinical Dual Targeting of the Cyclin/Rb/E2F and Mitochondrial Pathways in Mantle Cell Lymphoma with the Translation Inhibitor Silvestrol

Purpose: During cell-cycle progression, D-cyclins activate cyclin-dependent kinases (CDKs) 4/6 to inactivate Rb, permitting E2F1-mediated S-phase gene transcription. This critical pathway is typically deregulated in cancer, and novel inhibitory strategies would be effective in a variety of tumors. The protein synthesis inhibitor silvestrol has potent activity in B-cell leukemias via the mitochondrial pathway of apoptosis, and also reduces cyclinD1 expression inbreast cancer and lymphoma cell lines.Wehypothesized that this dual activity of silvestrolwouldmake it especially effective inmalignancies drivenby aberrant cyclin D1 expression. Experimental Design:Mantle cell lymphoma (MCL), characterized by elevated cyclin D1, was used as a model to test this approach. The cyclinD/Rb/E2F1pathwaywas investigated in vitrousingMCL cell lines and primary tumor cells. Silvestrol was also evaluated in vivo using an aggressive model of MCL. Results: Silvestrol showed low nanomolar potency both inMCL cell lines and primaryMCL tumor cells. D-cyclins were depleted with just 10 nmol/L silvestrol at 16 hours, with subsequent reductions of phosphorylated Rb, E2F1 protein, and E2F1 target transcription. As showed in other leukemias, silvestrol causedMcl-1 depletion followedbymitochondrial depolarization and caspase-dependent apoptosis, effects not related to inhibition of CDK4/6. Silvestrol significantly (P < 0.0001) prolonged survival in a MCL xenograft model without detectable toxicity. Conclusions: These data indicate that silvestrol effectively targets the cyclin/CDK/Rb pathway, and additionally induces cytotoxicity via intrinsic apoptosis. This dual activity may be an effective therapeutic strategy in MCL and other malignancies. Clin Cancer Res; 18(17); 4600–11. 2012 AACR. Introduction Innormal cells, the progression fromG1to S-phaseof the cell cycle is tightly controlled by a conserved mechanism involving cyclins D1, D2, and/or D3, cyclin-dependent kinases (CDK) 4 and/or 6, CDK inhibitory proteins of the INK4 family, the tumor suppressor Rb, and transcription factors of the E2F family. In nondividing cells, hypophosphorylated Rb binds E2F proteins to suppress their activity. Upon appropriate signaling, D-cyclins bind and activate CDK4/6 to phosphorylate and inactivate Rb, freeing E2F to form a complex with other factors to drive the transcription of genes required for cell-cycle progression and DNA synthesis (reviewed in ref. 1). Nearly all tumors are defective in some aspect of this pathway, for example, through cyclin overproduction, INK4 mutations, or Rb inactivation, providing tumor cells a strong growth advantage and escape from normal mitotic control. Components of this pathway are proposed to constitute valuable therapeutic targets (2, 3), and considerable efforts are underway to develop specific pharmacologic inhibitors. As an example, the CDK4/6specific inhibitor PD-0332991 (4) has efficacy in a variety of tumor models (5–9), and is currently undergoing clinical testing (10, 11). However, as a single agent PD-0332991 was reported to be cytostatic rather than cytotoxic, although it sensitizes cells to cytotoxic agents (6). Owing to the near universal dysfunction of the cyclin/Rb pathway across cancer types, a dual strategy to block the cyclin D/CDK4,6/Rb pathway, while concurrently activating apoptosis has the potential to provide broad therapeutic benefit. Authors' Affiliations: Department of Internal Medicine, College of Medicine; Center for Biostatistics; and Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio R. A. Baiocchi and D. M. Lucas have made equal contributions to this article. Corresponding Authors: David M. Lucas, 410 West 12th Avenue, Room 455, Wiseman Hall, Columbus, OH 43210. Phone: 614-2921551; Fax 614-292-3312; E-mail: [email protected] and Robert A. Baiocchi, 410 West 12th Avenue, Room 455, Wiseman Hall, Columbus, OH 43210. Phone: 614-292-1551; Fax 614-292-3312; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-12-0839 2012 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 18(17) September 1, 2012 4600 on April 14, 2017. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst July 12, 2012; DOI: 10.1158/1078-0432.CCR-12-0839