Page 274 - Haematologica March 2020

H. de Boussac et al.
AB
C
Figure 3. Selected kinases inhibition induces human myeloma cell toxicity. (A) Selection of seven kinases for biological investigations based on citation report in Pubmed and the availability of inhibitors. (B) IC50 of the different drugs in four human myeloma cell lines (HMCL), and calculated IC20 for the AMO1 HMCL; C) Kinase inhibitors induce apoptosis (annexin V and PARP cleavage) in AMO1 MM cell line at concentrations close to the calculated IC20 and IC50. Annexin, and PARP cleav- age, were monitored by flow cytometry after four days of treatments. Results are representative of four independent experiments. Statistical significance was tested using a Student T-Test for pairs. P-value: *<0.05; **<0.01; ***<0.001.
Finally, using a proteome array we examined the path- ways involved in apoptosis and cell cycle following treat- ments in AMO1 cells and in OPM2 cells that are p53 mutated.25 For all three tested treatments we observed in AMO1, but as expected not in OPM2, an increased p53 phosphorylation on S15 (DNA damage response), S46 (apoptosis) and S392 (growth inhibition) (Figure 4C and Online Supplementary Figure S7). Other apoptotic markers including caspase 3 cleavage, p27, cytochrome C, HSP60, TRAIL, BAD and BCL-X were also induced. Upon CHK1i treatment in AMO1, we also observed a decrease in Claspin and Survivin levels, two proteins involved in cell cycle and replication that have been linked to the CHK1 pathway. Indeed Claspin is a co-activator of CHK1,26,27 whereas Survivin degradation depends on the XAF1/XIAP128 a pro-apoptotic complex involved in CHK1 degradation.29 Those effects were not observed in OPM2 cells although we observed an increase of the pro-apoptot- ic proteins Diablo and FADD and a decreased in the pro- liferation related proteins TOR and P70 S6 kinases.30 Heterogeneity of the cell lines regarding the p53 status
could explain these differences. However, in both tested cell lines anti-, and pro-apoptotic signals were deregulat- ed. Altogether, these data demonstrate the pro-apoptotic and anti-proliferative effects of these three molecules in MM cells and highlight the potential of these kinases as new therapeutic targets in high-risk MM patients.
Conventional MM therapies are potentialized by selected kinase inhibitors
We then investigated the therapeutic interest of combin- ing these kinase inhibitors with therapeutic drugs com- monly used in MM (e.g. Melphalan, Lenalidomide, Velcade). Combining sub-lethal IC20 for all the kinase inhibitors with increasing concentrations of standard agents allowed us to identify a significant potentialization of Melphalan toxicity by CHK1, MELK, PBK and CDC7- DBF4 inhibitors in at least two out of the four HMCL investigated. However, no significant effect on the calcu- lated IC50 was noticed for the co-treatment of Melphalan with PLK4, MPS1 and SRPK1 inhibitors with a potential calculated antagonism of the two molecules (Figure 5A
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