Effects of the tyrosine kinase inhibitor AG957 and an Anti-Fas receptor antibody on CD34(+) chronic myelogenous leukemia progenitor cells.
The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR gene on chromosome 22 with c-ABL sequences translocated from chromosome 9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase (PTK) activity exerting a key role in malignant transformation. Targeting the tyrosine kinase activity of BCR/ABL or using agents capable of triggering apoptosis might represent attractive therapeutic approaches for ex vivo purging. AG957, a member of the tyrphostin compounds, exerts a selective inhibition of p210(BCR/ABL) tyrosine phosphorylation. We report here that preincubation of CML or normal CD34(+) cells with graded concentration of AG957 (1 to 100 micromol/L) resulted in a statistically significant, dose-dependent suppression of colony growth from multipotent, erythroid, and granulocyte-macrophage progenitors as well as the more primitive long-term culture-initiating cells (LTC-IC). However, AG957 doses causing 50% inhibition (ID50) of CML and normal progenitors were significantly different for multilineage colony-forming units (CFU-Mix; 12 v 64 micromol/L; P =.008), burst-forming unit-erythroid (BFU-E; 29 v 89 micromol/L; P =.004), colony-forming unit-granulocyte-macrophage (CFU-GM; 34 v 85 micromol/L; P =.004), and LTC-IC (43 v 181 micromol/L; P =.004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by reverse transcription-polymerase chain reaction showed that AG957 at 50 micromol/L significantly reduced the mean (+/-SD) percentage of BCR/ABL-positive progenitors (92% +/- 10% v 33 +/- 5%; P =.001). Because AG957 treatment resulted in significantly higher percentages of apoptotic cells (30% v 9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93 cells (BCR/ABL-negative), we investigated the combined effects of AG957 with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that triggers apoptosis. As compared with AG957 alone, the sequential treatment of CML CD34(+) cells with AG957 (1 micromol/L) and CH11 (1 microgram/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the treatment of normal CD34(+) cells with AG957 and CH11 failed to enhance AG957-induced colony growth inhibition. We conclude that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived colony formation by both primitive LTC-IC as well as committed CFU-Mix, BFU-E, and CFU-GM; (2) this growth inhibition is associated with the selection of a substantial amount of BCR/ABL-negative progenitors; and (3) the antiproliferative effect of AG957 is dramatically increased by combining this compound with the anti-Fas-R antibody CH11. These data may have significant therapeutic applications.
Carlo-Stella
C
C
Department of Hematology, University of Parma, Parma, Italy. ccs@ipruniv.cce.unipr.it
Regazzi
E
E
Sammarelli
G
G
Colla
S
S
Garau
D
D
Gazit
A
A
Savoldo
B
B
Cilloni
D
D
Tabilio
A
A
Levitzki
A
A
Rizzoli
V
V
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy / Q000276:immunology / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000276:immunology / Q000473:pathology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D018124:Receptors, Tumor Necrosis Factor / Q000276:immunology*; D014407:Tumor Cells, Cultured; D020032:Tyrphostins / Q000494:pharmacology* / Q000627:therapeutic use; D019014:fas Receptor / Q000276:immunology
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D018124:Receptors, Tumor Necrosis Factor; D020032:Tyrphostins; D019014:fas Receptor; C104965:tyrphostin AG957; D011505:Protein-Tyrosine Kinases