Contributions
Abstract: PB1892
Type: Publication Only
Background
Polo-like kinases (PLKs) and Aurora kinases (AKs) act as key cell cycle regulators in healthy human cells. In cancer, these protein kinases are often overexpressed and deregulated, thus contributing to uncontrolled cell proliferation, growth and consequent genomic instability. Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem cell. Despite the striking success of tyrosine kinase inhibitor (TKI) therapy, a small but significant proportion of CML patients may still progress from the chronic phase (CP) to the accelerated phase (AP)and eventually to the blast crisis (BC). This is likely due to cooperating molecular events additional to the initial t(9;22) translocation. Even in the TKi era, CML patients who progress to BC have a poor outcome, hence the urgent need to identify novel therapeutic strategies, targeting alternative signaling pathways important for leukemic cell proliferation are required.
Aims
In this study, a new therapeutic strategy based on AKA or Plk1 inhibition with PHA-739358 (Danusertib) or BI6727 (Volasertib), associated with Wee1 inhibition with AZD1775 was evaluated in K562 cells sensitive (K562-S) and resistant (K562-R) to imatinib and in primary cells from CML patients in BC.
Methods
Protein expression and activation was assessed by Western Blotting. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Cell cycle progression were evaluated by flow cytometry. Drug cytotoxicity in ex vivo experiments was evaluated in clonogenic assays in 1 healthy donor and in 3 CML patients in BC.
Results
Both Danusertib and Volasertib (0.5 µM for 24h) showed cytostatic and cytotoxic effects in CML cells by inducing G2/M-phase arrest and apoptosis. Moreover, they caused a dose- and time-dependent reduction of the G0/G1 cell fraction and an increase of the G2/M fraction. Cell cycle arrest was associated with increased levels of phospho (p)-Chk1 and p-Chk2, p-cyclin B1, p-cdc2 and p-Wee1. Using a Wee1 inhibitor (AZD1775) after 24h treatment with Danusertib and Volasertib 0.5 µM, when cells were arrested in G2 phase and Wee1 was overexpressed and hyper-activated, resulted in a synergistic inhibition of cell viability in both K562-S and -R. AZD1775 combined with either Danusertib or Volasertib caused a time-dependent increase of annexin-V-positive cells by activating the mitochondrial apoptotic pathway as reflected by an increment of Bax expression and induction of the cleavage of caspase-3, -9 and PARP. Moreover, both drug combinations induced a significant increase of the DNA double-strand break marker γH2AX, suggesting that Wee1 inhibition promotes mitosis and propagates genomic instability by forcing the cells through successive replication cycles, ultimately resulting in apoptosis from mitotic catastrophe. Finally, clonogenic assays performed by using CD34+ progenitors from three BC CML patients, showed that PLK1 or AKs inhibition, associated with Wee1 inhibition, reduce the clonogenic activity of the CD34+ compartment in a synergistic way compared to the same drugs administrated alone.
Conclusion
Taken together, our findings indicate that PLK1 and AKs inhibitors associated with WEE1 inhibition display the potential for being further explored in innovative clinical trials aimed to improve the outcomes of patients with CML in blast crisis, or resistant to multiple lines of TKI therapy.
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): Apoptosis, Cell cycle progression, CML blast crisis, Targeted therapy
Abstract: PB1892
Type: Publication Only
Background
Polo-like kinases (PLKs) and Aurora kinases (AKs) act as key cell cycle regulators in healthy human cells. In cancer, these protein kinases are often overexpressed and deregulated, thus contributing to uncontrolled cell proliferation, growth and consequent genomic instability. Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem cell. Despite the striking success of tyrosine kinase inhibitor (TKI) therapy, a small but significant proportion of CML patients may still progress from the chronic phase (CP) to the accelerated phase (AP)and eventually to the blast crisis (BC). This is likely due to cooperating molecular events additional to the initial t(9;22) translocation. Even in the TKi era, CML patients who progress to BC have a poor outcome, hence the urgent need to identify novel therapeutic strategies, targeting alternative signaling pathways important for leukemic cell proliferation are required.
Aims
In this study, a new therapeutic strategy based on AKA or Plk1 inhibition with PHA-739358 (Danusertib) or BI6727 (Volasertib), associated with Wee1 inhibition with AZD1775 was evaluated in K562 cells sensitive (K562-S) and resistant (K562-R) to imatinib and in primary cells from CML patients in BC.
Methods
Protein expression and activation was assessed by Western Blotting. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Cell cycle progression were evaluated by flow cytometry. Drug cytotoxicity in ex vivo experiments was evaluated in clonogenic assays in 1 healthy donor and in 3 CML patients in BC.
Results
Both Danusertib and Volasertib (0.5 µM for 24h) showed cytostatic and cytotoxic effects in CML cells by inducing G2/M-phase arrest and apoptosis. Moreover, they caused a dose- and time-dependent reduction of the G0/G1 cell fraction and an increase of the G2/M fraction. Cell cycle arrest was associated with increased levels of phospho (p)-Chk1 and p-Chk2, p-cyclin B1, p-cdc2 and p-Wee1. Using a Wee1 inhibitor (AZD1775) after 24h treatment with Danusertib and Volasertib 0.5 µM, when cells were arrested in G2 phase and Wee1 was overexpressed and hyper-activated, resulted in a synergistic inhibition of cell viability in both K562-S and -R. AZD1775 combined with either Danusertib or Volasertib caused a time-dependent increase of annexin-V-positive cells by activating the mitochondrial apoptotic pathway as reflected by an increment of Bax expression and induction of the cleavage of caspase-3, -9 and PARP. Moreover, both drug combinations induced a significant increase of the DNA double-strand break marker γH2AX, suggesting that Wee1 inhibition promotes mitosis and propagates genomic instability by forcing the cells through successive replication cycles, ultimately resulting in apoptosis from mitotic catastrophe. Finally, clonogenic assays performed by using CD34+ progenitors from three BC CML patients, showed that PLK1 or AKs inhibition, associated with Wee1 inhibition, reduce the clonogenic activity of the CD34+ compartment in a synergistic way compared to the same drugs administrated alone.
Conclusion
Taken together, our findings indicate that PLK1 and AKs inhibitors associated with WEE1 inhibition display the potential for being further explored in innovative clinical trials aimed to improve the outcomes of patients with CML in blast crisis, or resistant to multiple lines of TKI therapy.
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): Apoptosis, Cell cycle progression, CML blast crisis, Targeted therapy