Identification of molecular endpoints as a guide pefi clinical decision making in STI571-treated chronic myelogenous leukemia patients

K. Karamlou, L. Lucas, Brian Druker

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The Bcr-Abl oncogene is a constitutively activated tyrosine kinase and the causative agent of chronic myelogenous leukemia (CML). STI571, a tyrosine kinase inhibitor specific for the Abl tyrosine kinase, is currently in clinical trials for the treatment of CML knd shows significant activity with minimal toxicity. In the Phase I trials, a maximum tolerated dose (MTD) for STI571 has not been reached. Although responses in chronic phase patients have been durable, relapses in blast crisis patients have been common. A niore rational approach to dose selection for a molecularly targeted agent would be to evaluate maximal inhibition of the target rather than using the MTD. Similarly, to evaluate mechanitms of relapse, one would want to first determine whether or not the intended target was inhibited. To achieve this goal, we have been developing tools to monitor Abl kirjase inhibition. For this purpose, we have chosen three proteins that are known toi be phosphorylated in CML patient samples. These proteins include Bcr-Abl itself, Crkl bid STATS. Major sites of tyrosine phosphorylation of these proteins have been mapped pnd phosphospecific antibodies to these specific tyrosine residues have been generated; I the Abl and Crkl antisera by our laboratory and the STATS antibody from a commercial source. To validate these reagents, K562 cells (a human CML cell line) were incubated with l (IM STI571 for various period of time and lysates were analyzed by immunoblouing with the phosphospecific antibodies. A dramatic decrease in the phosphorylation of BcrAbl is seen following a 1 hour incubation with ST1571. At 48 hours, there is a complete absence of Bcr-Abl phosphorylation. The phosphospecific Crkl antisera detecteld a significant decrease in the phosphorylation of Crkl within 1 hour of treatment with STI571 and an 80% reduction in its phosphorylation at 48 hours. Using the phosphospetjific STAT5 antibody, a complete loss of STATS phosphorylation was noted within 24 ruburs of exposure of the cells to STI571. In conclusion, we have developed specific reagents hat enable us to detect kinase inhibition in a CML cell line treated with the kinase inhitlitor STI571. These reagents will be tested on CML patients undergoing treatment with STIB71 and should allow for direct assessment of kinase inhibition in a clinical setting. This

Original languageEnglish (US)
JournalBlood
Volume96
Issue number11 PART I
StatePublished - 2000

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Phosphorylation
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Decision making
Phospho-Specific Antibodies
Protein-Tyrosine Kinases
Phosphotransferases
Maximum Tolerated Dose
Tyrosine
Immune Sera
Cells
abl Genes
Patient treatment
Blast Crisis
Recurrence
Cell Line
Proteins
K562 Cells
Antibodies
Clinical Decision-Making
Imatinib Mesylate

ASJC Scopus subject areas

  • Hematology

Cite this

Identification of molecular endpoints as a guide pefi clinical decision making in STI571-treated chronic myelogenous leukemia patients. / Karamlou, K.; Lucas, L.; Druker, Brian.

In: Blood, Vol. 96, No. 11 PART I, 2000.

Research output: Contribution to journalArticle

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abstract = "The Bcr-Abl oncogene is a constitutively activated tyrosine kinase and the causative agent of chronic myelogenous leukemia (CML). STI571, a tyrosine kinase inhibitor specific for the Abl tyrosine kinase, is currently in clinical trials for the treatment of CML knd shows significant activity with minimal toxicity. In the Phase I trials, a maximum tolerated dose (MTD) for STI571 has not been reached. Although responses in chronic phase patients have been durable, relapses in blast crisis patients have been common. A niore rational approach to dose selection for a molecularly targeted agent would be to evaluate maximal inhibition of the target rather than using the MTD. Similarly, to evaluate mechanitms of relapse, one would want to first determine whether or not the intended target was inhibited. To achieve this goal, we have been developing tools to monitor Abl kirjase inhibition. For this purpose, we have chosen three proteins that are known toi be phosphorylated in CML patient samples. These proteins include Bcr-Abl itself, Crkl bid STATS. Major sites of tyrosine phosphorylation of these proteins have been mapped pnd phosphospecific antibodies to these specific tyrosine residues have been generated; I the Abl and Crkl antisera by our laboratory and the STATS antibody from a commercial source. To validate these reagents, K562 cells (a human CML cell line) were incubated with l (IM STI571 for various period of time and lysates were analyzed by immunoblouing with the phosphospecific antibodies. A dramatic decrease in the phosphorylation of BcrAbl is seen following a 1 hour incubation with ST1571. At 48 hours, there is a complete absence of Bcr-Abl phosphorylation. The phosphospecific Crkl antisera detecteld a significant decrease in the phosphorylation of Crkl within 1 hour of treatment with STI571 and an 80{\%} reduction in its phosphorylation at 48 hours. Using the phosphospetjific STAT5 antibody, a complete loss of STATS phosphorylation was noted within 24 ruburs of exposure of the cells to STI571. In conclusion, we have developed specific reagents hat enable us to detect kinase inhibition in a CML cell line treated with the kinase inhitlitor STI571. These reagents will be tested on CML patients undergoing treatment with STIB71 and should allow for direct assessment of kinase inhibition in a clinical setting. This",
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N2 - The Bcr-Abl oncogene is a constitutively activated tyrosine kinase and the causative agent of chronic myelogenous leukemia (CML). STI571, a tyrosine kinase inhibitor specific for the Abl tyrosine kinase, is currently in clinical trials for the treatment of CML knd shows significant activity with minimal toxicity. In the Phase I trials, a maximum tolerated dose (MTD) for STI571 has not been reached. Although responses in chronic phase patients have been durable, relapses in blast crisis patients have been common. A niore rational approach to dose selection for a molecularly targeted agent would be to evaluate maximal inhibition of the target rather than using the MTD. Similarly, to evaluate mechanitms of relapse, one would want to first determine whether or not the intended target was inhibited. To achieve this goal, we have been developing tools to monitor Abl kirjase inhibition. For this purpose, we have chosen three proteins that are known toi be phosphorylated in CML patient samples. These proteins include Bcr-Abl itself, Crkl bid STATS. Major sites of tyrosine phosphorylation of these proteins have been mapped pnd phosphospecific antibodies to these specific tyrosine residues have been generated; I the Abl and Crkl antisera by our laboratory and the STATS antibody from a commercial source. To validate these reagents, K562 cells (a human CML cell line) were incubated with l (IM STI571 for various period of time and lysates were analyzed by immunoblouing with the phosphospecific antibodies. A dramatic decrease in the phosphorylation of BcrAbl is seen following a 1 hour incubation with ST1571. At 48 hours, there is a complete absence of Bcr-Abl phosphorylation. The phosphospecific Crkl antisera detecteld a significant decrease in the phosphorylation of Crkl within 1 hour of treatment with STI571 and an 80% reduction in its phosphorylation at 48 hours. Using the phosphospetjific STAT5 antibody, a complete loss of STATS phosphorylation was noted within 24 ruburs of exposure of the cells to STI571. In conclusion, we have developed specific reagents hat enable us to detect kinase inhibition in a CML cell line treated with the kinase inhitlitor STI571. These reagents will be tested on CML patients undergoing treatment with STIB71 and should allow for direct assessment of kinase inhibition in a clinical setting. This

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