The central role of the control of apoptosis in the pathophysiology of Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) has been recently reinforced in genetic and pharmacological studies. The inhibitor of apoptosis protein (IAP) family has eight members and plays an important role in apoptosis, the most studied ones being survivin (BIRC5) andX-linked inhibitor of apoptosis (XIAP). YM155 is a small molecule with antineoplastic potential that has been described as a suppressant of survivin and XIAP.

BIRC5 and XIAP mRNA expression data from blood samples of healthy donors and MPN patients were obtained from GEOR2. HEL and SET2 JAK2V617F cells were used. YM155 with potential antineoplastic activity was used. Ruxolitinib was used as a reference drug. Cell viability was assessed by MTT assay, apoptosis by annexin V/propidium iodide labeling and flow cytometry (FC), clonogenicity by autonomous colony formation, cell cycle by propidium iodide and FC, protein expression by Western Blot analysis and gene expression by quantitative PCR.

BIRC5 expression was significantly increased in primary myelofibrosis patients compared to healthy donors (p < 0.05). On the other hand, XIAP expression was reduced in MPN patients (all p < 0.05). In cellular models with JAK2V617F mutation, BIRC5 expression was higher in HEL and SET2 cell lines when compared to normal hematopoietic cells. In a time-concentration manner, HEL cells showed great sensitivity compared to SET2 cells when exposed to the drug. IC50 values for HEL cells were 48.4, 1, and 0.6 μM, and for SET2 cells were 31.4, 3.8, and 3.3 μM for 24, 48, and 72h, respectively (p < 0.05). In both cell lines, synergic, additive, or antagonistic effects were not observed in the combination of YM155 and ruxolitinib. In HEL and SET2 cells, YM155 induced apoptosis in a dose-dependent manner being the less concentration responsible for inducing apoptosis (0.32 μM) in both cells line (p < 0.05). As observed in the apoptotic effect after a low exposure of YM155, both cells had a decrease in colony formation and an increase in cell death after being exposed to a higher concentration of YM155 as seen in the cell cycle where increased cells in the subG1 population were observed (p < 0.05). Genes involved in cell cycle progression, autophagy, DNA damage, and apoptosis were modulated in HEL and SET2 cells (all p < 0.05). YM155 induced modulation in apoptosis, autophagy, and DNA damage proteins. In both cell lines, ULK1, SQSTM1/p62, and LC3BII (markers of autophagy) cleaved PARP1 (marker of apoptosis) and yH2AX (marker of DNA damage) were modulated.

Our results indicate that survivin/BIRC5 and XIAP are differently expressed in MPN and YM155 has multiple antineoplastic effects in MPN cell models, suggesting that IAP proteins may be a point of pharmacological intervention for the treatment of these diseases.

Supported by FAPESP, CNPq, and CAPES.