Treatment with the Novel TOPK Inhibitor OTS514 Exhibits Potent Anti-Myeloma Activity in Pre-Clinical Models

Abstract

Multiple myeloma (MM) continues to be considered incurable, necessitating new drug discovery. TOPK, a mitotic kinase, is upregulated in many human cancers, including MM, and is associated with proliferation of tumor cells, maintenance of cancer stem cells, and poor patient prognosis. Gene expression profiling shows elevated TOPK transcript levels in the plasma cells (PC) of patients with MM relative to healthy individuals, but not in PC derived from patients diagnosed with MM precursor states MGUS or smoldering myeloma. Western blot analysis of cells from bone marrow aspirates (BMAs) from patients with frank MM shows strong TOPK protein levels in the enriched CD138+ population (MM PCs), but not in the CD138- fraction or in peripheral blood mononuclear cells (PBMCs). In addition, TOPK protein is robustly expressed among all human myeloma cell lines (HMCLs) tested. In this study, we targeted this candidate oncogene with the selective inhibitor OTS514 to assess the contribution of TOPK to MM cell survival.Treatment of HMCLs with the TOPK inhibitor OTS514 causes rapid cleavage of caspase 3 and PARP, and subsequent DNA fragmentation, indicative of a robust apoptotic response. Viability assays reveal potent cell death in a panel of 9 HMCLs, with 72-hour IC50s ranging from 11-29 nM. Importantly, no apparent resistance to TOPK inhibition was observed among six p53-mutant cell lines tested. OTS514 treatment of total marrow mononuclear cells derived from BMAs from patients with MM resulted in robust cell death of CD138+ PC, as measured by flow cytometric analysis of Annexin-V/propidium iodide-stained cells, whereas CD138- cell populations were largely protected from apoptotic death, corroborating the relative lack of TOPK expression in non-PC and suggesting a highly favorable therapeutic index. OTS514 treatment also prevented CD138+ cell outgrowth from MM patients' PBMCs stimulated with IL-3 and IL-6, suggesting TOPK may play a role in maintenance of the putative myeloma stem cell populations.TOPK inhibition causes time- and dose-dependent decreases in both TOPK auto-phosphorylation and total protein levels. Protein expression of known TOPK target FOXM1 is also lost, as well as expression of pro-survival factors, including phospho-Akt. Microarray analysis of H929 cells revealed elevated expression of 73 small nucleolar RNAs (snoRNA), which are involved in ribosome maturation, and this is likely the result of a nucleolar/ribosomal stress response caused by cell cycle perturbation. Gene Set Enrichment Analysis revealed reduced expression of E2F1 target genes and western blot analysis showed a time-dependent decrease in levels of E2F1 in response to OTS514 treatment, suggesting a role of TOPK at the G1 checkpoint.The anti-myeloma activity of OTS514 was also explored in combination with the immunomodulatory agent lenalidomide and found to be exquisitely synergistic; Chou-Talalay combination indices at IC50, IC75, and IC90 were <1 among all HMCL tested. Combination treatment with OTS514 and lenalidomide also led to additive loss of the key MM cell survival proteins IKAROS and IRF4.The modified TOPK inhibitor OTS964, which is more amenable to oral administration, was evaluated in an animal model. H929 cells were xenografted subcutaneously into NOD/SCID mice and daily drug gavage was given at 50 or 100 mg/kg for 14 days after tumors reached 100 mm3. After 14 days of treatment, average tumor volume was reduced by 81% in mice treated with the 100 mg/kg dose of OTS964 relative to controls. The drug was well-tolerated, with minimal and reversible body weight loss in the group receiving the highest dose.TOPK is emerging as a key oncogene in several cancer types. Our data strongly support further study of the mechanistic contribution of TOPK to the development of MM and its inhibition as a potential therapy for MM. DisclosuresPark:OncoTherapy Science, Inc.: Consultancy. Jakubowiak:Amgen Inc., BMS, Celgene, Janssen, Karypharm, Millennium-Takeda, Sanofi, SkylineDX: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Chicago: Employment.