Abstract
Uncontrolled proliferation and development of drug resistance in multiple myeloma (MM) cells are consequences of the numerous genetic aberrations which are further stimulated in the context of bone marrow microenvironment. Thus, inhibition of complementary pro-survival signaling and transcriptional networks rather than individual pathway is required for induction of optimal cytotoxicity in MM cells. Recent studies have shown that cyclin-dependent kinases inhibitors (CDKIs), designed to block cell cycle progression through inhibition of CDK/cyclin complexes, block transcription through suppression of RNA polymerase II phosphorylation at its C-terminal domain (CTD), resulting in downregulation of cell proliferation (cyclins: D, A, B1, pRb) and anti-apoptotic proteins (i.e. Mcl-1, survivin, XIAP). Here we examined the anti-MM activity of RGB 286638, a novel multi-targeted small molecule inhibitor, originally designed to induce broad cell cycle suppression via multiple CDK inhibition. Treatment with RGB 286638 triggered a dose-dependent cytotoxicity in conventional drug-sensitive (MM.1S, RPMI 8226, U266, OPM2), and resistant (MM.1R, Dox-40, LR5) MM cell lines, as well as primary tumor cells from MM patients. Induction of apoptosis was evidenced by Annexin V/PI staining, and confirmed by PARP and caspase cleavage. Additionally, RGB 286638 overcame the proliferative advantage conferred by MM patient-derived bone marrow stromal cells (BMSCs) and cytokines (IL-6, IGF-1) on MM cells. To determine molecular mechanisms responsible for RGB 286638-induced cytotoxicity, we assessed the cell cycle profile, which revealed G2/M arrest followed by increased sub-G1 phase. Importantly, RGB 286638 inhibited phosphorylation of RNA polymerase II in a dose- and a time-dependent fashion, followed by suppression of CDK1/cyclin B, CDK4, 6/Cyclin D1, D3, and CDK2/Cyclin E complexes associated with rapid down-regulation of Rb phosphorylation. Interestingly, RGB 286638 effectively reduced IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation (Tyr705) and Janus kinase 2 (JAK2) phosphorylation (Tyr 1007/1008), suggesting that RGB 286638 is a possible JAK2 inhibitor. Based on sufficient in vitro cytotoxicity, we examined anti-tumor activity of RGB 286638 in vivo using a human MM cell xenograft model in SCID mice and demonstrated that RGB 286638 inhibited tumor growth and prolonged survival. In conclusion, our data demonstrate preclinical activity and provide the rational to test RGB 286638 in the treatment of MM.
TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma
