Résumé
Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite introduction of novels agents that have significantly improved clinical outcomes, most patients relapse and develop drug resistance. Therefore, novel therapeutic strategies to overcome chemotherapy resistance are needed in clinical settings.
MM is characterized by genomic instability and high level of replicative stress that arise during the disease's pathogenesis. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways that include the kinases ATR, Chk1 and Wee1. Chk1 is activated by ATR after DNA damage or replicative stress and promote cell cycle arrest through the regulation of S and G2 checkpoints. Wee1 kinase plays a central role in the proper timing of cell division cycle controlling entry into mitosis and DNA replication during S phase. Interestingly, experimental studies have identified that Chk1 inhibitors are strongly synergistic with Wee1 inhibitors in various cancer models. Furthermore, Chk1 and Wee1 inhibitors are currently under clinical investigation.
Here, we investigated the therapeutic interest of Chk1 and Wee1 in MM.
First we investigated the prognostic value of Chk1 and/or Wee1 gene expression in MM patients. Both Chk1 and Wee1 were significantly overexpressed in human myeloma cell lines (HMCLs, n= 40) compared to bone marrow plasma cells (BMPCs, n=5) (P<001 and P<001 respectively). Chk1 and Wee1 expression were significantly higher in the poor prognosis “proliferation” MM subgroup (P<001 and P<001 respectively).
High Chk1 expression and high Wee1 expression could predict for shorter overall survival (OS) in 4 independent cohorts of newly-diagnosed patients (P<0.001 and P=0.007 in the HM cohort (N=206), P<0.001 and P=0.008 in UAMS-TT2 cohort (N=345), P<0.001 and P<0.001 in Hovon cohort (N=282) and P=0.002 and P<0.001 in UAMS-TT3 cohort (N=186)). Same results were obtained for event free survival (EFS). Moreover, Chk1 and Wee1 high expression are associated with a poor prognosis in a cohort of 242 patients treated by Bortezomib monotherapy (Mulligan Cohort, P<0.001 and P=0.001 respectively).
Interestingly, combined high expression of Chk1 and Wee1 are associated to significantly shorter survival compared to high expression of Chk1 or Wee1 alone in the 4 independent cohorts investigated. Same results were obtained for event free survival (EFS).
According, to these results, we investigated the therapeutic interest of Chk1 and Wee1 inhibitors (AZD7762 and MK-1775) alone and in combination in MM.
Chk1 inhibitor (AZD7762) induced a dose dependent cell growth inhibition in 13 HMCLs with a median IC50 of 179nM (range 69-366nM). A correlation between HMCLs sensitivity and P53 mutation was observed (r =0.7, P<0.01).
Wee1 inhibitor (MK-1775) induced also a dose dependent inhibition of cell growth in all investigated HMCLs (n=10) with a median of IC50 of 450nM (range 100-800nM).
Combination treatment of Wee1 inhibitor IC20 with various doses of Chk1 inhibitor was performed in 4 HMCLs. We identified a high synergy (combination index range 0.4-0.7) associated with a significant and remarkable reduction of Chk1 inhibitor (AZD7762) IC50 (198nM to 20nM for XG6; 166nM to 25nM for AMO1; 386nM to 18nM for XG7 and 163nM to 21nM for OPM2) in all the tested HMCLs. Chk1 and Wee1 inhibitors combination demonstrated a significant increase in apoptosis induction, proliferation inhibition and DNA damage induction compared to Chk1i or Wee1i alone in 3 HMCLs.
We investigated the effects of Chk1i, Wee1i and Chk1i/Wee1i combination on primary MM cells of patients (n = 5) co-cultured with their bone marrow microenvironment. After 4 days of treatment, cells were enumerated and the fraction of viable myeloma cells (CD138+) and non-myeloma cells (CD138-) were determined by flow cytometry. Chk1i/Wee1i combination significantly and specifically induced a higher toxicity on myeloma cells of patients compared to Chk1i or Wee1i alone. This toxicity is specific of MM cells since normal bone marrow cells were significantly less affected by the combination treatment (P < 0.01).
Taken together, these data suggest that association of Chk1i and Wee1i may represent a promising therapeutic approach in high-risk MM patients characterized by high Chk1 and Wee1 expression.
Bruyer:Diag2tec: Employment. Martin:Diag2tec: Employment. Cartron:Celgene: Consultancy, Employment; Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Hose:EngMab: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding.