Prognosis for patients with Philadelphia chromosome (Ph) positive ALL remains dismal. Ph+ ALL is characterized by the activation of several tyrosine kinases that provide the neoplastic clone with its proliferative capacity and survival advantage. We found that WP1066, a novel derivative of the tyrphostin AG490, inhibits the JAK-STAT pathway and cytokine-dependent and independent signaling pathways. Therefore, we sought to investigate the activity of WP1066 in Ph+ ALL. To do this, we first studied the effect of WP1066 on the Ph+ ALL cell lines Z-119 and Z-181 (

Estrov Z et al. J. Cell Physiol. 166(3):18, 1996
). Using the MTT assay we found that WP1066 inhibited the growth of both Z-119 and Z-181 cells in a concentration-dependent manner with only 8% and 4% of the cells surviving at a concentration of 4 mM, respectively. Similarly, the clonogenic growth of both Z-119 and Z-181 cells was effectively inhibited by WP1066 with more than 90% reduction in colony numbers at concentration of 4 mM. Using Western Immunoblott analysis of cell lysates, we found that 4 mM of WP1066 induced caspase-3 cleavage in a time- and dose-dependent manner in both Z-119 and Z-181 cells. In addition, WP-1066 downregulated uncleaved poly (adenosine diphosphate-ribose) polymerase (PARP) and upregulated cleaved PARP protein levels in a time-dependent manner after 2 hours of exposure to 4 mM. We further evaluated induction of apoptosis using the annexin V-FITC assay and showed a dose dependent increase of the fraction of apoptotic cells in both Z-119 and Z-181 cells. After 24 hour of exposure to 4 mM of WP1066 the fraction of apoptotic cells increased by 23% and 43%, respectively. To elucidate the mechanisms by which WP1066 induces growth inhibition and apoptosis in Ph+ ALL cells, we investigated the effect of this agent on the phosphatidylinositol 3-kinase (PI-3K) pathway because the PI-3K pathway is constitutively activated in Ph+ leukemias. We found that WP1066 inhibited the phosphorylation of AKT in a time-dependent fashion in both cell lines and that this inhibitory effect lasted for 24 hours.

In conclusion, our data suggest that exposure to WP1066 induces caspase-dependent apoptosis, is associated with PI3-K inhibition and reduces the growth of the Ph+ cell lines Z-119 and Z-181. The activity of WP1066 in Ph+ ALL should be further studied.

Topics:
1-phosphatidylinositol 3-kinase, chromosomes, poly(adp-ribose) polymerases, adenosine, annexins, caspase-3, caspases, cytokine, diphosphates, fluorescein-5-isothiocyanate

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2005, The American Society of Hematology
2005
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