Abstract
Juvenile myelomonocytic leukemia (JMML) is a rare and fatal early childhood hematological malignancy. Somatic SHP2 mutation accounts for 30-40% of JMML, with poor prognosis and lack of effective therapy methods. Aurora kinases play important roles in chromosome alignment, segregation and cell division during mitosis. Malignant hematopoietic cell diseases, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), as well as chronic myeloid leukemia (CML), aberrantly express Aurora kinase A or B, which predict a poor overall prognosis. Aurora kinase represents a promising therapeutic target. However, to date there is no study on the relationship of aurora kinase and SHP2 mutant JMML.
HCD57 cells are murine erythroleukemia cells that solely depend on exogenic erythropoietin (EPO) for survival. We constructed SHP2/D61Y and SHP2/E76K transformed HCD57 cells through retroviral transfection and selection. The two generated transformed cell lines (HCD57-SHP2/D61Y, HCD57-SHP2/E76K) were dependent on mutated-SHP2-mediated signaling. Based on these three cell lines, we screened 12 inhibitors, including aurora kinase A inhibitors, aurora kinase B inhibitors and pan-aurora kinase inhibitors. The selectivity and efficacy of the lead drugs were further evaluated on SHP2 mutant HCD57 cells and patient-derived PBMC cells by cell viability, flow cytometry, shRNA mediated gene silencing, western blotting and colony formation assays.
Previous studies have shown that our established SHP2-mutant HCD57 cell line is a leukemia cell line representing the molecular signature of signaling pathways in SHP2 mutant JMML. The Aurora kinase B/C inhibitor GSK1070916 selectively inhibited SHP2-mutant HCD57 cells through drug screening. Cell viability assay demonstrated a dose-dependent inhibition pattern of HCD57-SHP2/D61Y and HCD57-SHP2/E76K cells and had no significant inhibition effect on parental HCD57 cells. GSK1070916 induced cell cycle arrest at G2/M phase and cell apoptosis in HCD57-SHP2/D61Y and HCD57-SHP2/E76K cells. Although HCD57 cells were also arrested in G2/M phase after GSK1070916C treatment, the cells did not show significant apoptosis. Western blot analysis showed that the protein level of anti-apoptotic protein Bcl-xL demonstrated a time-dependent increase, while caspase-3 and PARP proteins had no obvious cleavage in HCD57 cells after treatment of GSK1070916. However, in HCD57-SHP2/D61Y and HCD57-SHP2/E76K cells, protein level of Bcl-xL was not significantly increased, while the cleaved form of caspase-3 and PARP significantly increased. This result demonstrated that SHP2-mutant cells were less capable in generating pro-survival signal and was sensitive to the inhibition of AURKB. Furthermore, the phenotype induced through silencing Aurkb by shRNA was similar to the inhibition effect of GSK1070916. We further investigated the ex vivo inhibitory effect of GSK1070916 on JMML patient-derived PBMC cells (mutation type: SHP2/E76A). The colony-forming ability of primary JMML cells was completely inhibited under treatment of GSK1070916 (500 nM).
In conclusion, aurora kinase B can be used as a promising therapeutic target for SHP2-mutant JMML patients, and the aurora kinase B/C inhibitor GSK1070916 could be a candidate for the treatment of JMML.
Disclosures
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
