Targeting the Cyclin D - CDK4/6 - Rb Axis in Mantle Cell Lymphoma with the Novel Translation Inhibitor Silvestrol,

Abstract 3498

During cell cycle progression, D class cyclins activate cyclin dependent kinases (CDK) 4 and 6 to phosphorylate and inactivate Rb, allowing E2F-1 mediated transcription of additional cell cycle genes including cyclin E to drive S phase entry. This critical pathway is nearly universally dysregulated in cancer, providing tumor cells a strong growth advantage and escape from normal mitotic control. Substantial research is being directed toward targeting this pathway in many cancer types, with some preliminary successes being achieved with pharmacologic inhibitors of CDK4/6. However the development of alternative strategies to block this pathway could potentially provide broad therapeutic benefit.

A prime example of a tumor with a disrupted cyclin D axis is Mantle Cell Lymphoma (MCL), in which the t(11;14) translocation places CCND1, the gene for cyclin D1, under the control of an immunoglobulin promoter. This results in sustained cyclin D1 expression in tumor cells and concomitant Rb inactivation, S phase entry and cell division. MCL is a relatively uncommon subset of Non-Hodgkin Lymphoma, but accounts for a disproportionate number of deaths. Treatments are limited and relapse is nearly universal; thus, new treatment strategies are essential for this disease.

Silvestrol is a structurally unique, plant-derived cyclopenta[b]benzofuran with potent in vitro and in vivo anti-tumor activity in several model systems including B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). Silvestrol inhibits the initiation step of translation by preventing assembly of eIF4A and capped mRNA into the eIF4F complex, leading to selective loss of short half-life proteins such as Mcl-1 and cyclin D1. We therefore hypothesized that silvestrol, through the depletion of cyclin D1, would demonstrate efficacy in MCL. Silvestrol showed low nanomolar IC50 values in the JeKo-1 (13 nM), Mino (17 nM) and SP-53 (43 nM) MCL cell lines at 48 hr (MTS assay; cell death confirmed by propidium iodide flow cytometry). This potency was similar in primary MCL tumor cells. Longer exposure times substantially improved the cytotoxicity of silvestrol assessed at 48 hr (approximately 50% effect achieved with a 16 hr exposure vs. 80% effect with a 24 hr exposure), suggesting that the cellular impacts of this agent increase with exposure time. Cyclins D1 and D3 were dramatically reduced in MCL cell lines with just 10 nM silvestrol at 16 hr (cyclin D2 was undetectable in these cells), with subsequent loss of Rb phosphorylation as well as cyclin E mRNA and protein, culminating in G1 cell cycle arrest. Similar to what we previously showed in CLL and ALL cells, silvestrol treatment under these conditions also caused loss of Mcl-1 protein with concurrent mitochondrial depolarization, although the exact mechanism of silvestrol-mediated cytotoxicity in these cells is still under investigation. In an aggressive xenograft mouse model of MCL, silvestrol produced a highly significant improvement in survival [median survival of vehicle vs. silvestrol treated mice (1.5 mg/kg every 48 hr) = 27 vs. 38 days; P<0.0001] without detectable toxicity. Together, these data demonstrate that the translation inhibitor silvestrol has promising in vitro and in vivo activity in MCL preclinical models. Furthermore, as the cyclin D/CDK/Rb axis is disrupted in most tumor types, this strategy may be broadly effective in other cancers as well.