Page 275 - Haematologica March 2020

Kinome profiling to target Multiple Myeloma
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Figure 4. Selected kinases inhibition induces human primary multiple myeloma (MM) cell death and toxicity on 5TMM murine cells. A) Mononuclear cells from five patients with MM were treated or not with CHK1i, MELKi and PLK4i. At day 4 of culture, the viability and total cell counts were assessed and the percentage of CD138+ viable plasma cells and bone marrow non-myeloma cells were determined by flow cytometry. Results are median values of the numbers of myeloma cells in the culture wells. Results were compared with a Student T-Test for pairs. B) Murine myeloma cell (5T33vv) viability was monitored by CTG after 24 and 48 hours treatment with CHK1i, MELKi and PLK4i. Results are representative of three independent experiments C) Apoptosis and Signaling pathways targeted by CHK1i, MELKi and PLK4i. Proteins accumulations were monitored after 48h treatment on AMO1 human myeloma cell lines (HMCL) using proteome profiler array. Relative amount was calculated as the mean of pixel density. P-value: *<0.05; **<0.01; ***<0.001.
and Online Supplementary Figure S8A). For the immunomodulatory agent Lenalidomide, no significant effect was observed with the tested combinations in two Lenalidomide resistant HMCL: XG1 and XG21. However, the effect of Lenalidomide was significantly potentialized in two other HMCL (AMO1 and OPM2) in combination with the CHK1, MELK or PBK inhibitors. Remarkably, addition of CHK1i, MELKi or PLK4i could overcome Lenalidomide resistance of the AMO1 cell line (Figure 5B and Online Supplementary Figure S8B). Conversely, we could not observe any synergy or even additivity for the co-treatment with Velcade, regardless of the cell line test- ed or the kinase inhibitor used (Online Supplementary Figure S9A). Altogether these results demonstrate the therapeutic
interest of CHK1i, MELKi, CDC7-DBF4i and PBKi in com- bination with Melphalan and IMiDs in MM (Online Supplementary Figure S9B).
To characterize the mechanisms involved, we moni- tored apoptosis after co-treatments of kinases inhibitors with Melphalan or Lenalidomide in AMO1 and OPM2 cells. A sub-lethal dose of Melphalan or Lenalidomide was used in combination with the calculated IC20 of the kinase inhibitors. CHK1i, MELKi and CDC7-DBF4i increased cell death via apoptosis when cells were co- treated with Melphalan or Lenalidomide. In addition, PLK4i co-treatment only potentialized cell death with Lenalidomide (Figure 6A and Online Supplementary Figure S10A). As expected from cell growth analyses, SRPK1i and
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