Page 277 - Haematologica March 2020

Kinome profiling to target Multiple Myeloma
A
B
Figure 6. Conventional multiple myeloma therapies are potentialized by selected kinase inhibitors. Co-treatment with selected kinase inhibitors at IC20 and Melphalan or Lenalidomide. (A) Apoptosis induction was analyzed using Annexin V APC staining by flow cytometry. (B) DNA damage induction was analyzed meas- uring ΥH2AX levels; Results are representative of four independent experiments. CI: calculated combination index. Statistical significance was tested using a Student T-Test for pairs. P-value: *<0.05; **<0.01; ***<0.001. #Significantly different of each individual treatment.
cell lines, PLK4i and CDC7-DBF4i demonstrated a signifi- cantly higher toxicity in the XG7 Mres cell line (Figure 7B) but not in XG2 Mres HMCL (Online Supplementary Figure S13B). Sublethal IC20 of CHK1i, PBKi and CDC7-DBF4i overcame Melphalan resistance of both cell lines tested (Figure 7C and Online Supplementary Figure S13C), while the other inhibitors tested did not show a significant effect. It should however be underlined that the inhibitors alone are active on both resistant and sensitive cell lines as shown in Figure 7B and Online Supplementary Figure S13B. Thus, our results highlight the therapeutic interest of CHK1i, MELKi, CDC7-DBF4i and SRPK1i used alone or in combination with conventional therapies, even in case of acquired resistance.
Discussion
Here we identified 36 kinases associated with a prog- nostic value in three independent cohorts of MM patients, allowing the creation of a kinase-related gene expression profile (GEP) risk score KI. Among them, CHK1, CDC7- DBF4, and MELK were identified as being of therapeutic interest in MM.31–33 PLK4, SRPK1, MPS1/TTK and PBK rep- resent new therapeutic targets in MM. Using inhibitors of these seven kinases, we validated their therapeutic inter- est to target MM cells alone or in combination with con- ventional therapies. In addition, we also highlighted a list of protein kinases for which no inhibitor is currently avail- able and which represent promising new therapeutic tar- gets at least in MM.
Our approach differs from a previous study exploiting a
RNAi library to target the human kinome in six myeloma cell lines.8 Surprisingly, only one kinase, AURKA, was selected in both studies. This discrepancy could reflect the fact that our study relies on the analysis of primary MM cells from patients and not on HMCL as in previous stud- ies. Since a large number of kinase (135/661) are differen- tially expressed between primary MM cells and HMCL (Online Supplementary Table S3), we believe that our study provides a relevant analysis of the protein kinases impor- tant for the survival of MM cells.
Our KI is strikingly enriched in kinases involved in the progression through mitosis (PBK, PLK4, MELK, MPS1) and in the replication stress response (CHK1, CDC7- DBF4, SRPK1). These kinases are also enriched in prolifer- ation34 and proliferation GEP-based signatures, which rep- resent also powerful risk factors in MM.10,35 The 36 genes of the KI only have a limited overlap with these signatures indicating that KI does not simply reflect a higher cell pro- liferation index.
Among the inhibitors against targets validated here (CHK1, MELK, PLK4, SRPK1, CDC7-DBF4, MPS1/TTK and PBK), the CHK1 inhibitor AZD7762 was of particular interest due to its ability to act alone or in combination with other drugs. Our results differ from two earlier stud- ies reporting a limited toxicity of AZD7762 on HMCL at doses equivalent of our calculated IC50, but at high Melphalan concentration, when combined with this drug.31,36 These discrepancies could reflect differences in culture conditions, as in our hands, the drug sensitivity of HMCL depended exquisitely on the confluency status at seeding and on the treatment protocol. Furthermore, we validated the therapeutic interest of CHK1i using primary
haematologica | 2020; 105(3)
791