Targeting the Mitotic Checkpoint in Myeloma with OSU-13, a Novel Mps1/Ttk Inhibitor

Despite recent advances, there is still a lack of treatment options for patients with high-risk multiple myeloma (MM), warranting the need for novel therapeutic targets. Monopolar spindle 1 (Mps1), also known as TTK protein kinase, is localized at the kinetochores and centromeres essential for the mitotic spindle checkpoint and centrosome duplication. In cancer cells, high levels of Mps1 help to support aneuploidy, a common malignant trait. In fact, high MPS1 expression correlates to unfavorable prognosis in colon and breast cancers. Here, we report the importance of Mps1 in MM and perform functional characterization of a novel Mps1 inhibitor, OSU-13.

To assess the relevance of Mps1 in MM prognosis, we analyzed the CoMMpass database and examined the association of MPS1 expression with clinical outcome and genetic alterations in MM patients (n=769). Elevated MPS1 expression correlates with decreased overall survival (p-value = 0.0001) and decreased event-free survival (p-value < 0.0001). In addition, MPS1 expression is higher in high-risk MM with specific genetic alterations, such as deletion of 17p (p-value = 0.0003), Myc translocation t(8;14) (p-value = 0.02), and gain of 1q21 (p-value = 0.0001).

We therefore compared MPS1 expression in eight different MM cell lines and primary CD138 ^- and CD138 ⁺ cells isolated from bone marrow (BM) of four recently diagnosed, untreated MM patients. Gene expression levels assessed by quantitative qPCR were normalized to 18S mRNA internal control, and relative quantification was performed using the ΔΔCt method. MPS1 expression was higher in all MM cell lines compared to the average expression level in primary CD138 ⁺ BM plasma cells, with a minimum of 3.2-fold increase in L363 and a maximum of 16.2-fold increase in MM1S. In addition, MPS1 expression was 2-fold higher in CD138 ⁺ MM cells compared to CD138 ^- counterparts from the BM of the same patient, suggesting its selectivity as a target.

Next, we characterized the effects of OSU-13, a novel Mps1 inhibitor. In an Mps1 target engagement assay (NanoBRET™) in HEK293 cells, we measured the relative levels of OSU-13-mediated inhibition of Mps1-NanoLuc binding to a fluorescent tracer in comparison to MM clinical candidates. OSU-13 showed EC ₅₀=10 nM, almost 10-fold lower than other agents tested.

Endpoint measurement by MTT (3-[4,5- dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate cytotoxicity of OSU-13. MM cell lines were treated with increasing doses of OSU-13 for 72h and cell viability was measured. OPM-2 was the most sensitive cell line (IC ₅₀ = 610 nM), followed by H929 (IC ₅₀ = 1440 nM), RPMI-8226 (IC ₅₀ = 4196 nM), and U266 (IC ₅₀ = 5071 nM).

To investigate the kinetics and the mechanisms used by OSU-13 to cause cell death, we treated H929 and OPM-2 cell lines with 500 nM of OSU-13 for 24h, 48h, 72h, 96h, and 120h and assessed both Annexin V-FITC and Zombie-Aqua staining by flow cytometry. Treatment with OSU-13 caused an increase in Annexin V staining as early as 24h in H929 (2.1-fold) and OPM-2 (1.6-fold). Maximum increase of apoptotic cells (Annexin V single positive) was observed after 72h, with a 5-fold increase in OPM-2 and 8.6-fold increase in H929 cell line. Cell death peaked at 120h, with Zombie staining 74% of OPM-2 and 60% of H929 cells. The activity of caspases 3/7 was further assessed in H929 myeloma cells at five time points after treatment with different concentrations of OSU-13. Caspases 3/7 activation started after 48h with 500nM OSU-13, and peaked after 72h post-treatment with the same dose, in agreement with the Annexin staining kinetics results.

Finally, we evaluated the in vivo effect of OSU-13 in tumor growth in a H929 MM subcutaneous xenograft model in CB.17 SCID mice. Mice were treated with 5 mg/kg or 10 mg/kg daily dose of OSU-13 per oral gavage for 21 days. Treatment with 10 mg/kg daily dose of OSU-13 produced a 22% tumor growth delay (p<0.05).

Collectively, we show here that elevated MPS1/TTK expression correlates with poorer prognosis in MM, and OSU-13, a novel Mps1/TTK inhibitor, induces apoptosis and cell death in MM cell lines, and decreases tumor growth in mice containing H929 cell xenografts. Together, our findings reveal that targeting the mitotic checkpoint using OSU-13 is a potential novel strategy, particularly for high risk MM.