Targeting Cyclin D1 in the Treatment of Multiple Myeloma: Preclinical Validation of a Novel Specific Small Molecule Cyclin D1 Inhibitor, P276-00.
Authors: Raje Noopur , Hideshima Teru , Vallet Sonia , Chhetri Shweta , Mitsiades Constantine , Rooney Melissa , Yasui Hiroshi , Kiziltepe Tanyel , Podar Klaus , Okawa Yutaka , Ikeda Hiroshi , Chauhan Dharminder , Munshi Nikhil C. , Sharma Somesh , Parikh Himanshu , Chabner Bruce , Anderson Kenneth C. 
Published: October 2019

Abstract
Either an overexpression or dysregulation of cyclin D1, D2, or D3, has been reported in the majority of multiple myeloma (MM) tumors, suggesting a possible early unifying event in MM pathogenesis. This proposed critical role of cyclin D dysregulation in myeloma pathogenesis makes the cyclins, specifically cyclin D1, an attractive therapeutic target. We have evaluated a specific small molecule cyclin D1 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 specificity for D1 and B cyclins at nanomolar concentrations. P276-00 has been tested against a wide range of cancer cell types in both in vitro and tumor xenograft models. Based on these data, it is undergoing phase I clinical testing in North America. We have observed both time and dose dependent in vitro activity against a broad range of MM cells sensitive and resistant to conventional agents like dexamethasone, doxorubicin, and melphalan with IC50 ranging from 400–800nM. Spectral karyotyping confirmed t(11;14) (q13;q32) in KMS 12 MM cells which were sensitive to P276-00. Importantly, it has demonstrated activity in primary patient derived tumor cells. Cell cycle analysis confirmed that P276-00 induced either growth arrest or apoptosis in MM cells depending on the cell line. Apoptosis was in part caspase dependent suggested by partial reversal of cytotoxicity by Z-VAD Fmk. P276-00 inhibited Rb-1 phosphorylation as early as 6 hours in most of the MM cell lines tested associated with a decrease in cdk4 suggesting a regulatory role of P276-00 in cell cycle progression. These changes preceeded growth arrest and apoptosis of MM cells on cell cycle analysis. Ongoing studies are using SiRNA to Cyclin D1 to confirm this regulatory role of P276-00. As cyclin D1 dysregulation or overexpression can render MM cells more susceptible to proliferative stimuli such as IL-6, IGF-1, and the bone marrow microenvironment, we tested the effects of P276-00 in the presence of these cytokines and bone marrow stromal cells (BMSCs). Our data confirms that 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. Animals were treated with either control PBS or P276-00 intraperitoneally at 25 mg/kg three times a week for 3 weeks. Our data confirms in vivo anti-tumor activity of P276-00 as suggested by a significant decrease in biluminesence of GFP+ MM cells (p<0.05) and a decrease in tumor volume. Immunohistochemistry on tumor tissue from P76.00 treated, and control animals validates our in vitro studies and will be presented. In vitro combination studies with bortezomib have been completed suggesting synergism. P276-00 and bortezomib combination is currently being tested in our in vivo model. These studies confirm cyclin D1 to be an important therapeutic target in MM and form the basis of a phase I/II study of P276-00 alone and in combination in the treatment of MM.