The integrity of signaling pathways involved in cell cycle arrest, chromatin remodeling and DNA repair are critical for maintaining the fidelity of replicated DNA. Normal cells repair damaged DNA during G1 arrest; in contrast, leukemic cells often have a deficient G1/S checkpoint and depend on a functional G2/M checkpoint for DNA repair. Aurora Kinase A (AKA) is a key component of centrosome cycle and polar spindle assembly required for regulated progression from G2 to M and throughout M phase. It is frequently overexpressed in cancers, where it correlates with a poor prognosis. Notably, AKA overexpression is associated with genomic instability, one major trait of chronic myeloid leukemia (CML). Our hypothesis is that AKA, together with Polo like kinase 1 (Plk1), cooperates with the constitutive TK activity of the BCR-ABL1 fusion protein by increasing DNA damage, promoting the occurrence of additional genomic alterations and driving TKIs resistance and disease progression to blast crisis.
In this study, the effects of AKA and Plk1 inhibition were evaluated in K562 cells sensitive (K562-S) and resistant (K562-R) to imatinib.
Protein expression and activation was assessed by Western Blotting (WB). Danusertib and Volasertib were used to investigate the effects of AKA and Plk1 inhibition in K562-S and K562-R cells. Both cell lines were incubated with increasing concentrations of Danusertib and Volasertib (0.5-1 µM) or control medium for 24, 48, 72 and 96 hours to perform growth curve experiments and to identify the best sub-lethal drug doses in liquid medium. The percentage of apoptotic cells was quantified by annexin V/propidium iodide staining and flow cytometry. WB experiments were performed to evaluate AKA and Plk1 expression and activation before and after treatment with Danusertib and Volasertib alone or in combination with the Wee1 inhibitor MK1775. Further experiments were performed using polyclonal antibodies against cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 to confirm apoptosis activation. PARP cleavage and γH2AX expression and activation were also tested. The effects of Danusertib and Volasertib on cell cycle progression were evaluated by flow cytometry in K562-S and K562-R cells after incubation in medium containing 0.5 µM of each drugs for 24 h.
Both Danusertib and Volasertib (0.5 µM for 24h) were effective in cell growth inhibition in K562-S and K562-R by inducing cells to undergo cell cycle 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 (MK1775) 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 (Figure 1). MK1775 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, -8 and -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.
Our results provide support for MK1775 to be used in synergistic combination therapies to improve the outcomes of patients with CML in blast crisis or resistant to multiple lines of TKI therapy.
Supported by ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project.
Castagnetti: Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta: Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Rosti: Incyte: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Martinelli: Johnson&Johnson: Consultancy; Roche: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy. Soverini: Bristol-Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.
Asterisk with author names denotes non-ASH members.