Abstract
T lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy with poor outcome, especially for relapse/refractory disease. Early T- cell precursor acute lymphoblastic leukaemia (ETP-ALL) is a recently identified subtype of T-ALL with worse treatment outcome compared with other subtypes of T-ALL and treatment options are limited. T-ALL frequently harbors genetic aberrations leading to cell cycle dysregulation and it is one of the major molecular pathogenesis of T-ALL. WEE1 is a protein kinase that is responsible for inhibiting mitosis with unrepaired damaged DNA via inactivating CDK1. WEE1 is highly express in adult T-ALL and its overexpression is associated with adverse prognosis in various cancers. Inhibiting WEE1 expression is a novel approach of therapy. Bortezomib is a 26S proteosome inhibitor and it is FDA approved for treating plasma cell myeloma and mantle cell lymphoma. Bortezomib had been demonstrated therapeutic efficacy in clinical setting for relapse/refractory paediatric T-ALL and B-ALL when combined with chemotherapy. Despite its therapeutic efficacy in clinical studies, the mechanism of action of Bortezomib in T-ALL remain uncertain. The role of Bortezomib in cell cycle modulation had not been established in T-ALL. Moreover, it had not been demonstrated that the effect of Bortezomib in WEE1 expression in T-ALL. Here, we present our study that demonstrated the therapeutic efficacy of Bortezomib in treating T-ALL via cell cycle modulation and downregulation of WEE1 by Bortezomib.
T-ALL cell lines including MOLT16, MOLT4, LOUCY and CEM were used in the study. Cell viability was measured by trypan blue. Apoptosis and cell cycle analysis were measured by flow cytometry. Western blot of WEE1, p53, cyclin B1, p21 and p27 were performed.
Our result showed that Bortezomib reduce the cell viability of T-ALL cell lines in dose and time-dependent manner. Bortezomib was also sensitive towards LOUCY, a T-ALL cell line with ETP-ALL phenotype. It implied that Bortezomib could be a promising therapy for ETP-ALL. Bortezomib also triggered apoptosis in various T-ALL and the effect of apoptosis was more pronounced after 72 hours of treatment when compared with 24-hour. Again, Bortezomib was able to induce apoptosis in LOUCY cell line. G2/M cell cycle arrest was observed in various T-ALL upon treatment of Bortezomib. The effect on cell cycle modulation was also observed in LOUCY cell line. The protein expression of p21 and p27 were increased after the treatment of Bortezomib. The level of cyclin B1 was increased also. There was upregulation of p53 after Bortezomib treatment. Strikingly, the protein expression level of WEE1 was reduced. The findings of WEE1 downregulation by Bortezomib is a novel findings. We also showed that Bortezomib downregulate WEE1 mRNA expression by quantitative PCR.
Our study showed that Bortezomib is active against T-ALL cell lines, including ETP-ALL cell line, LOUCY and modulates cell cycle with G2/M arrest. Bortezomib had been shown to increase the level of p21, p27 and cyclin B1 and induced G2/M cell cycle arrest in glioblastoma cells. However, studies on cell cycle modulation by Bortezomib in T-ALL are scarce. Here, we demonstrated Bortezomib stabilized p21, p27 and upregulation of cyclin B1 in T-ALL as well, which could account for the G2/M cell cycle arrest. We first showed that downregulation of WEE1 after treatment with Bortezomib, in protein level as well as in mRNA level. Recent study showed that inhibition of WEE1 is a novel target of therapy in T-ALL. WEE1 is upregulated in T-ALL to prevent entry of mitosis with unrepaired damaged DNA. The downregulation of WEE1 by Bortezomib as showed by our study could reverse its effect and leads to apoptosis of leukaemic cells.
In summary, our study provides the insight on mechanism of action of Bortezomib in modulating cell cycle in T-ALL. Moreover, it is the first study to demonstrate WEE1 downregulation by Bortezomib in T-ALL. These findings not only enhance our understanding of mechanism of action of Bortezomib in T-ALL, but also rationalized the use of certain synergistics combination therapy with Bortezomib in treating T-ALL, e.g., chemotherapeutic agents, PARP inhibitors which could damage DNA of leukaemic cells. Further research is needed to explore those combination therapy in T-ALL and molecular mechanism of downregulation of WEE1 by Bortezomib in T-ALL.
Figure 1 Figure 1.
Disclosures
No relevant conflicts of interest to declare.
UCN-01 induces S and G2/M cell cycle arrest through the p53/p21waf1or CHK2/CDC25C pathways and can suppress invasion in human hepatoma cell lines