Preclinical Validation of a Clinical Grade Novel Specific Small Molecule Cyclin D1 Inhibitor, P276-00 for the Treatment of Multiple Myeloma.

Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. We sought to identify the effects of targeting cyclin D1 for the treatment of MM. We first studied gene expression of cyclin D1 in MM cells from 64 patients and normal plasma cells from healthy volunteers as controls (n=24). Cyclin D1 was over-expressed in 42/64 MM patients (65%). To assess the importance of targeting cyclin D1, we next used shRNA lentiviral constructs to silence cyclin D1 in INA6 and MM.1S MM cells. Our data demonstrated > 50% of apoptotic cell death in cyclin D1 shRNA versus control transfected MM cells, indicating that small molecule cyclin dependent kinase inhibitors may provide a valuable therapeutic tool to specifically target cyclin D1. We next evaluated a clinical grade small molecule cyclin D1 specific inhibitor P276-00 in MM. Its specificity has been confirmed in an in vitro kinase assay by potent inhibitory activity for Cdk4-D1 as compared to Cdk2-E. Additionally, in vitro kinase assays against a broad range of other kinases have also confirmed its specificity at nanomolar concentrations for D1 and B cyclins. P276-00 treatment of MM cell lines and patient derived cells induced both time and dose dependent in vitro growth inhibition in a broad range of MM cells sensitive and resistant to conventional agents including dexamethasone, doxorubicin, and melphalan, with IC₅₀ ranging from 400–800nM. Cell cycle analysis confirmed that P276-00 induced either growth arrest or apoptosis in MM cells. Apoptosis was, at least in part, caspase-dependent, since cytotoxicity was partially reversed by Z-VAD Fmk. P276-00, in a dose and time dependent fashion inhibited Rb-1 phosphorylation as early as 6 hours associated with down-regulation of cdk4, suggesting a regulatory role of P276-00 in cell cycle progression. These changes preceded growth arrest and apoptosis in MM cells. Proliferative stimuli such as IL-6, IGF-1, and adherence to bone marrow stromal cells induced cyclin D1 and B cyclins, contributing to the development of drug resistance; P276-00 was able to overcome these proliferative signals and induce apoptosis in MM cells. Next we evaluated in vivo efficacy of P276-00 in NOD-SCID mice bearing GFP+ MM xenografts. Mice were treated with either control PBS or P276-00 intraperitoneally at 25 mg/kg three times a week for 3 weeks. Our data confirmed in vivo anti-tumor activity of P276-00, evidenced by a significant decrease in biluminesence of GFP+ MM cells (p<0.05) and a decrease in tumor volume, as well as an improvement in overall survival of treated mice. Finally, in vitro combination studies with bortezomib showed strong synergism associated with down-regulation of the anti-apoptotic protein MCL-1. These studies form the basis of an ongoing Phase I study in the treatment of relapsed/refractory MM.