Effects of the tyrosine kinase inhibitor AG957 and an anti-Fas receptor antibody on CD34+ chronic myelogenous leukemia progenitor cells

Carmelo Carlo-Stella, Ester Regazzi, Gabriella Sammarelli, Simona Colla, Daniela Garau, Aviv Gazit, Barbara Savoldo, Daniela Cilloni, Antonio Tabilio, Alexander Levitzki, Vittorio Rizzoli

Research output: Contribution to journalArticle

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Abstract

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 μmol/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 μmol/L; P = .008), burst-forming unit-erythroid (BFU-E; 29 v 89 μmol/L; P = .004), colony-forming unit-granulocyte-macrophage (CFU-GM; 34 v 85 μmol/L; P= .004), and LTC-IC (43 v 181 μmol/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 μmol/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 μmol/L) and CH11 (1 μg/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.

Original languageEnglish
Pages (from-to)3973-3982
Number of pages10
JournalBlood
Volume93
Issue number11
Publication statusPublished - Jun 1 1999

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CD95 Antigens
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Protein-Tyrosine Kinases
Stem Cells
Antibodies
Granulocyte-Macrophage Progenitor Cells
Erythroid Precursor Cells
Chromosomes
Cell culture
Cell Culture Techniques
Macrophages
anti-Fas monoclonal antibody
Growth
tyrphostin AG957
Tyrphostins
Apoptosis
Therapeutics
Purging
Chromosomes, Human, Pair 22
Philadelphia Chromosome

ASJC Scopus subject areas

  • Hematology

Cite this

Carlo-Stella, C., Regazzi, E., Sammarelli, G., Colla, S., Garau, D., Gazit, A., ... Rizzoli, V. (1999). Effects of the tyrosine kinase inhibitor AG957 and an anti-Fas receptor antibody on CD34+ chronic myelogenous leukemia progenitor cells. Blood, 93(11), 3973-3982.

Effects of the tyrosine kinase inhibitor AG957 and an anti-Fas receptor antibody on CD34+ chronic myelogenous leukemia progenitor cells. / Carlo-Stella, Carmelo; Regazzi, Ester; Sammarelli, Gabriella; Colla, Simona; Garau, Daniela; Gazit, Aviv; Savoldo, Barbara; Cilloni, Daniela; Tabilio, Antonio; Levitzki, Alexander; Rizzoli, Vittorio.

In: Blood, Vol. 93, No. 11, 01.06.1999, p. 3973-3982.

Research output: Contribution to journalArticle

Carlo-Stella, C, Regazzi, E, Sammarelli, G, Colla, S, Garau, D, Gazit, A, Savoldo, B, Cilloni, D, Tabilio, A, Levitzki, A & Rizzoli, V 1999, 'Effects of the tyrosine kinase inhibitor AG957 and an anti-Fas receptor antibody on CD34+ chronic myelogenous leukemia progenitor cells', Blood, vol. 93, no. 11, pp. 3973-3982.
Carlo-Stella, Carmelo ; Regazzi, Ester ; Sammarelli, Gabriella ; Colla, Simona ; Garau, Daniela ; Gazit, Aviv ; Savoldo, Barbara ; Cilloni, Daniela ; Tabilio, Antonio ; Levitzki, Alexander ; Rizzoli, Vittorio. / Effects of the tyrosine kinase inhibitor AG957 and an anti-Fas receptor antibody on CD34+ chronic myelogenous leukemia progenitor cells. In: Blood. 1999 ; Vol. 93, No. 11. pp. 3973-3982.
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abstract = "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 μmol/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 μmol/L; P = .008), burst-forming unit-erythroid (BFU-E; 29 v 89 μmol/L; P = .004), colony-forming unit-granulocyte-macrophage (CFU-GM; 34 v 85 μmol/L; P= .004), and LTC-IC (43 v 181 μmol/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 μmol/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 μmol/L) and CH11 (1 μg/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.",
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T1 - Effects of the tyrosine kinase inhibitor AG957 and an anti-Fas receptor antibody on CD34+ chronic myelogenous leukemia progenitor cells

AU - Carlo-Stella, Carmelo

AU - Regazzi, Ester

AU - Sammarelli, Gabriella

AU - Colla, Simona

AU - Garau, Daniela

AU - Gazit, Aviv

AU - Savoldo, Barbara

AU - Cilloni, Daniela

AU - Tabilio, Antonio

AU - Levitzki, Alexander

AU - Rizzoli, Vittorio

PY - 1999/6/1

Y1 - 1999/6/1

N2 - 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 μmol/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 μmol/L; P = .008), burst-forming unit-erythroid (BFU-E; 29 v 89 μmol/L; P = .004), colony-forming unit-granulocyte-macrophage (CFU-GM; 34 v 85 μmol/L; P= .004), and LTC-IC (43 v 181 μmol/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 μmol/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 μmol/L) and CH11 (1 μg/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.

AB - 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 μmol/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 μmol/L; P = .008), burst-forming unit-erythroid (BFU-E; 29 v 89 μmol/L; P = .004), colony-forming unit-granulocyte-macrophage (CFU-GM; 34 v 85 μmol/L; P= .004), and LTC-IC (43 v 181 μmol/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 μmol/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 μmol/L) and CH11 (1 μg/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.

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