Effect of ag9s7, a bcr-abl-specific tyrosine kinase inhibitor, on chronic myelogenous leukemia progenitor

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by a chimeric BCR/ABL gene giving rise to a 210-kD fusion protein (p210BCR'ABL) with increased protein tyrosine kinase (PTK) activity. AG957 is a tyrphostin known to specifically inhibit p210clABL activity. We evaluated the effects of AG957 on the in vitro growth of CML-derived mulripotent (CPUMix), erythroid (BFU-E), granulocyte-macrophage (CFU-GM) and long-term culture-initiating cell (LTC-IC) progenitors. Preincubation (30 min) of CD34CML cells with AG957 (1-100 \M) induced a dose-dependent suppression of colony growth. AG957 doses inducing 95% (IDM) growth inhibition of CPU-Mix, BFU-E and CFU-GM were 78, 86, and 85 |lM, respectively. AG957 (>50 (J.M) exerted a significant growth suppression of CML LTC-IC. Normal progenitors were significantly less inhibited than CML progenitors. DNA electrophoresis demonstrated that preincubation with AG957 (100 jiM, 24 hours) of the BCR-ABL-transfected Mo316 cell line induced apoptosis. To increase the apoptotic effect of AG957, exposure of CML CD34cells to AG957 was followed by incubation with Fas ligand (1 μg/ml). This treatment resulted in a significant increase of colony suppression. In CML patients at diagnosis, individual colonies were analyzed for the presence of BCR/ABL mRNA by reverse transcription polymerase chain reaction (RT-PCR). Preincubation with AG957 (50 nM) markedly reduced the percentage of CFU-GM expressing the hybrid BCR/ABL mRNA (control samples: 100%, AG957-treated samples: 33%). In conclusion, our data demonstrate that: (a) AG957 strongly inhibits CML, but not normal, primitive and committed progenitors; (b) the apoptotic effect of AG957 is significantly enhanced by Fas ligand; (c) AG957-induced destruction of BCR/ABL positive progenitors suggests the use of this tyrphostin for in vitro purging.