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. 2016 Aug 19;6(8):e460.
doi: 10.1038/bcj.2016.71.

Anti-myeloma activity of MELK inhibitor OTS167: effects on drug-resistant myeloma cells and putative myeloma stem cell replenishment of malignant plasma cells

A T Stefka  1 , J-H Park  1 , Y Matsuo  2 , S Chung  2 , Y Nakamura  1   3 , A J Jakubowiak  1 , S Rosebeck  1
Affiliations

Anti-myeloma activity of MELK inhibitor OTS167: effects on drug-resistant myeloma cells and putative myeloma stem cell replenishment of malignant plasma cells

A T Stefka et al. Blood Cancer J. .
No abstract available

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Conflict of interest statement

YN is a stock holder and a scientific advisor of OncoTherapy Science, Inc. JP is a scientific advisor of OncoTherapy Science, Inc. YM and SC are employees of OncoTherapy Science, Inc. AJJ is a consultant, member of the advisory board and receives honoraria from Bristol-Myers Squibb, Celgene, Janssen Pharmaceuticals, Karyopharm Therapeutics, Millennium/Takeda Pharmaceuticals, Onyx/Amgen Pharmaceuticals, Sanofi-Aventis and SkylineDx. The remaining authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression and inhibition of MELK in MM cells. (a) Gene expression analysis of MELK mRNA expression was performed using publically available data sets, which include CD138+ PC from normal donors (nPC), monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (sMM) or newly diagnosed MM. Statistical significance of differences (set at P<0.05) was determined using one-way analysis of variance with Tukey's multiple comparison test in GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Adjusted P-values are indicated. (b) A panel of HMCL was exposed to increasing doses of OTS167 (OncoTherapy Science, Inc., Kawasaki, Japan) and cell viability was determined by MTT assay after 72 h. (inset) IC50 values were calculated in GraphPad Prism 6. (c) Western blot analysis of MM1S and U266 cells for PARP cleavage after treatment for 2–24 h with OTS167 at 100 nm. The vertical line indicates the merger of two scanned film images of different exposure times to depict comparable levels of PARP. (d) Total bone marrow samples were treated for 24 h with OTS167 at 10 and 100 nm, followed by staining with APC-CD138 (clone DL-101; BioLegend, San Diego, CA, USA) or control mouse IgG1 κ isotype (clone MOPC-21; BioLegend), Annexin fluorescein isothiocyanate and propidium iodide to monitor apoptosis in total bone marrow cells, CD138+ MM PC only, or cells of the marrow not marked by CD138 expression (CD138− cells). Data from representative experiments performed in duplicate are depicted. The percentage of live, early or late apoptotic and necrotic cells is shown. (e) MM1S and U266 cells were treated with increasing doses of OTS167 as indicated for 24 h before lysis and western blot analysis for the indicated proteins.
Figure 2
Figure 2
MELK inhibition overcomes drug resistance in the recapitulated bone marrow microenvironment and impairs putative myeloma stem cell repopulation of malignant MM PC. (a) The indicated cell lines were cultured alone or together as indicated and treated with OTS167 in increasing concentrations for 48 h before measuring metabolically active cell numbers using the CellTiter-Glo Luminescent Cell Viability Assay on a Glomax 96 microplate luminometer (Promega, Madison, WI, USA). Statistical significance of differences (set at P<0.05) was determined using two-way analysis of variance with Tukey's multiple comparison test in GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). Adjusted P-values are indicated. Raw luciferase units are reported. Results shown are of two experiments performed in triplicate. *P<0.0001; ▪P=0.0063; •P=0.0015; ΔP=0.0167; □P=0.0053; ○P=0.0035; ♦ P=0.0024. (b) Western blot analysis of MM1S and HS-5 stromal cells cultured alone or together with increasing concentrations of OTS167 for 48 hours demonstrates preferential apoptosis in myeloma cells. (c) Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll gradient separation and 2.0–2.5 × 106 PBMC/well in six-well plates were cultured as indicated with recombinant human interleukin-3 (IL-3) and IL-6 (both at 5 ng/ml), purchased from R&D Systems (Minneapolis, MN, USA) (catalog number 203-IL and 206-IL, respectively), or OTS167 (10 nm) alone or in combination for 6 days. Cultures were collected and subjected to immunomagnetic positive selection to separate CD138+ plasma cells from CD138− cells. Trypan blue-negative cells were counted and the fold change in each population in response to each treatment is depicted. Data represent six independent determinations from six separate blood samples from MM patients. Statistical significance of differences (set at P<0.05) was determined using one-way analysis of variance with Tukey's multiple comparison test in GraphPad Prism 6 (GraphPad Software). P-values are indicated. Effects on CD138− populations are not significant (NS).
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