Pre-Clinical Evaluation Of A Small Molecule Inhibitor Of Cdk1 As Potential Therapy For Myc Expressing Multiple Myeloma Tumors

Abstract Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor’s protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.