Abstract
Background:
Despite significant progress in the treatment of multiple myeloma (MM) through the introduction of immunomodulatory drugs and proteasome inhibitors, therapeutic progress is limited for high-risk patients. Therefore, it is essential to define and validate novel drug targets in myeloma to implement personalized treatment options, predict drug activity and finally improve treatment outcome for all subgroups of MM patients. In the current study we aimed to analyse the prognostic value of maternal embryonic leucine zipper kinase (MELK) in MM and investigated the activity of a small molecule inhibitor of MELK (OTSSP167).
Methods:
MELK expression levels were analysed in two large cohorts of publically available gene expression (GEP) datasets (GSE2658 and GSE9782; n=551 and n=264, respectively) and 8 human myeloma cell lines (HMCLs). The utility of MELK as potential drug target in MM was investigated by using a selective small molecule inhibitor against MELK (OTSSP167). HMCLs were treated at varying concentrations (0-1000 nM) and analysed for viability, apoptosis and cell cycle status. Regulatory networks involved in the mechanisms of OTSSP167 were revealed by quantitative PCR (qPCR) and proteomic profiling of HMCLs after short-term (5 hours) treatment with OTSSP167 at 25-50 nM.
Results:
The prognostic impact of MELK was studied in two publically available GEP-datasets (GSE2658 and GSE9782). Interestingly, MELK expression was significantly elevated in the GEP-defined high-risk proliferation associated molecular subgroup (P<0.001). High levels of MELK expression were accordingly associated with poor prognosis. We observed significantly reduced overall survival in patients with high compared to low MELK levels treated within the total therapy 2 (P=0.0003), total therapy 3 (P=0.04) and the APEX trial protocol (P=0.002).
These results pointed to a role for MELK as potential drug target in high-risk patients. To test this, we used a highly selective inhibitor of MELK (OTSSP167). In line with their proliferative character, MELK expression was detected in 8 of 8 HMCLs. Treatment with OTSSP167 led to a dose-dependent reduction of viability in all HMCLs tested (median IC50: 10.16 nM, range: 7.6 - 15.2 nM). Importantly, we also detected synergistic and additive drug activity of OTSSP167 in combination with pomalidomide and carfilzomib.
OTSSP167 induced apoptosis in all HMCLs investigated, verified by annexin V/7-AAD staining, detection of cleaved PARP and mitochondrial membrane depolarization. The apoptotic effects might be attributed to a significant downregulation of IRF4 (up to 0.75±0.27 fold reduction, P=0.009) and MCL-1 (up to 0.51±0.09 fold reduction, P=0.002) expression. We also observed accumulation of MM cells in the G2 phase of the cell cycle (OPM-2: 24% vs 42%, MM.1S: 23% vs 38.8% of cells in G2 phase without or with OTSSP167 at 25 nM for 48 hours, P<0.001). This was associated with downregulation of central genes involved in the propagation of the cell cycle, including PLK-1, cyclin B1 and aurora kinase A (up to 0.82±0.08, 0.93±0.04 and 0.88±0.08 fold reduction, respectively; P<0.0001). We also detected downregulation of cyclin D1 (up to 0.41±0.3 fold reduction, P=0.03). These results were confirmed at the protein level by using proteomic profiling of three HMCLs after 5 hours of treatment with OTSSP167 at 50 nM. Again, we observed deregulation of proteins involved in the initiation of mitosis (cyclin B1, aurora kinase A, nucleolin). Of note, we also detected upregulation of several proteins involved in protein folding and stabilization (e.g. Csp, HSP90 alpha and HSP90 beta) as well as glycolysis (e.g. ENO1, ALDOA, G3P2, TPI1), suggesting that these factors might regulate potential drug resistance mechanisms.
Conclusion:
Our findings reveal MELK expression as a novel poor prognostic marker in newly diagnosed and relapsed MM, depicting a group of high-risk patients. Inhibition of MELK with OTSSP167 led to the induction of apoptosis and cell cycle arrest by targeting central genes of the MM signaling network (e.g. MCL-1, cyclin D1, aurora kinase A). Moreover, combination of OTSSP167 with established anti-MM drugs showed synergistic activity. These results emphasize to initiate further pre-clinical and clinical testing of MELK inhibition as a novel drug target in MM, especially for patients at high-risk.
Disclosures
Ludwig: Takeda: Research Funding; Celgene Corporation: Honoraria, Speakers Bureau; Onyx: Honoraria, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Janssen Cilag: Honoraria, Speakers Bureau.
The Greatwall kinase: a new pathway in the control of the cell cycle