Hormone-Conditional Activation of Bcr-Abl Kinase Highlights Stat5 Anti-Apoptotic Pathway Prior to Promoting Cell Growth.

Abstract 3380

Bcr-Abl protein elicits a diverse array of downstream signals and is responsible for development of Philadelphia chromosome (Ph)-positive leukemias. In this fusion protein, disruption or deletion of a N-terminal coiled-coil (CC) region of Bcr results in substantially decreased tyrosine kinase activity and defective cellular transformation, indicating the essential role of Abl oligomerization in its constitutive kinase activity. Fusion of the estrogen receptor (ER) ligand binding domain (LBD) to the C-terminus of Abl generates a ligand-activated tarnsforming version, while additional sequences in Bcr may also be required for oncogenic competence since Bcr-Abl mutants containing just the extreme CC region cannot transform fibroblasts.

To revisit the mechanism of Bcr-Abl-induced leukemogenesis and especially to dissect early signaling events upon Bcr-Abl activation, we applied this fusion technology to construct p190DccER, a p190^Bcr-Abl mutant including ER-LBD at the C-terminus but not CC region at the N-terminus. GM-CSF-dependent human TF-1 cells were virally transduced with p190DccER as well as wild-type p190 (WT), p190Dcc and vector control, respectively, and a series of transformants were subjected to biological assays as well as biochemical analysis

During a few days after switch from GM-CSF to 4-HT, viable cell number of p190DccER-transformed TF-1 cells was not increased but maintained, and thereafter proliferated at the comparable rate to GM-CSF-supported cells. Their growth was dose-dependent on 4-HT unless not more than 1.0mM. The effect of 4-HT on TF-1/p190DccER cells was easily canceled by imatinib in a dose-dependent manner. The profile of phosphotyrosine containing proteins quite resembled between 4-HT-treated TF-1/p190DccER cells and TF-1/p190WT cells. Unexpectedly, stable detection of autophosphorylated p190DccER required as long as several hours or beyond a day after 4-HT stimulation and so did phospho-CrkL. This can be explained by the observation that ligand-free p190DccER was highly unstable, and upon 4-HT binding, its stability increased in a time-dependent manner. Such a stabilizing mechanism might be adapted to substrate proteins including CrkL, which are directly bound to and phosphorylated by Bcr-Abl. On the contrary, 4-HT-induced tyrosine phosphorylation of Stat5 could be observed within 10min, suggesting its dominant role in the initial anti-apoptotic phase triggered by p190DccER.

Next, we investigated gene expression profiling of TF-1/p190DccER cells using custom-made oligonucleotide DNA microarray and found a small number of genes differentially expressed before and after 4-HT treatment. Quantitative real-time polymerase chain reaction (QR-PCR) analysis confirmed that seven genes (BCL-XL, HIF-1A, HSPA1A, WT1, PRAME, BAG3 and GATA2) were significantly upregulated by 4-HT in a time-dependent manner. To identify Stat5 target genes among these candidates, we created a doxycycline (DOX)-inducible lentiviral expression system for constitutively active Stat5 mutant (mStat5A1*6). Then, selective activation of Stat5 in TF-1 cells resulted in suppression of apoptosis after GM-CSF withdrawal, and significantly upregulated five of seven candidate genes (BCL-XL, HIF-1A, HSPA1A, WT1, PRAME).

These results suggest that the Bcr-Abl/Stat5 pathway is likely to integrate multiple effector molecules to prevent apoptosis, and that they are potential molecular targets in Ph-positive leukemias. The present experimental system helps us to perform functional dissection of signal transducers activated by Bcr-Abl kinase.