Threshold levels of ABL tyrosine kinase inhibitors retained in chronic myeloid leukemia cells determine their commitment to apoptosis

Thomas O'Hare, Christopher A. Eide, Anupriya Agarwal, Lauren T. Adrian, Matthew S. Zabriskie, Ryan J. MacKenzie, Dorian H. La Tocha, Kara J. Johnson, Huihong You, Jenny Luo, Steven M. Riddle, Bryan D. Marks, Kurt W. Vogel, Dennis Koop, John Apgar, Jeffrey Tyner, Michael W. Deininger, Brian Druker

Research output: Contribution to journalArticle

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Abstract

The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCRABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKIinduced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies.

Original languageEnglish (US)
Pages (from-to)3356-3370
Number of pages15
JournalCancer Research
Volume73
Issue number11
DOIs
StatePublished - Jun 1 2013

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Myeloid Cells
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Protein-Tyrosine Kinases
Apoptosis
Phosphotransferases
Pharmaceutical Preparations
Tandem Mass Spectrometry
Liquid Chromatography
Half-Life
Flow Cytometry
Cell Death
Cell Line

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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Threshold levels of ABL tyrosine kinase inhibitors retained in chronic myeloid leukemia cells determine their commitment to apoptosis. / O'Hare, Thomas; Eide, Christopher A.; Agarwal, Anupriya; Adrian, Lauren T.; Zabriskie, Matthew S.; MacKenzie, Ryan J.; La Tocha, Dorian H.; Johnson, Kara J.; You, Huihong; Luo, Jenny; Riddle, Steven M.; Marks, Bryan D.; Vogel, Kurt W.; Koop, Dennis; Apgar, John; Tyner, Jeffrey; Deininger, Michael W.; Druker, Brian.

In: Cancer Research, Vol. 73, No. 11, 01.06.2013, p. 3356-3370.

Research output: Contribution to journalArticle

O'Hare, T, Eide, CA, Agarwal, A, Adrian, LT, Zabriskie, MS, MacKenzie, RJ, La Tocha, DH, Johnson, KJ, You, H, Luo, J, Riddle, SM, Marks, BD, Vogel, KW, Koop, D, Apgar, J, Tyner, J, Deininger, MW & Druker, B 2013, 'Threshold levels of ABL tyrosine kinase inhibitors retained in chronic myeloid leukemia cells determine their commitment to apoptosis', Cancer Research, vol. 73, no. 11, pp. 3356-3370. https://doi.org/10.1158/0008-5472.CAN-12-3904
O'Hare, Thomas ; Eide, Christopher A. ; Agarwal, Anupriya ; Adrian, Lauren T. ; Zabriskie, Matthew S. ; MacKenzie, Ryan J. ; La Tocha, Dorian H. ; Johnson, Kara J. ; You, Huihong ; Luo, Jenny ; Riddle, Steven M. ; Marks, Bryan D. ; Vogel, Kurt W. ; Koop, Dennis ; Apgar, John ; Tyner, Jeffrey ; Deininger, Michael W. ; Druker, Brian. / Threshold levels of ABL tyrosine kinase inhibitors retained in chronic myeloid leukemia cells determine their commitment to apoptosis. In: Cancer Research. 2013 ; Vol. 73, No. 11. pp. 3356-3370.
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abstract = "The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCRABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKIinduced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies.",
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AU - Eide, Christopher A.

AU - Agarwal, Anupriya

AU - Adrian, Lauren T.

AU - Zabriskie, Matthew S.

AU - MacKenzie, Ryan J.

AU - La Tocha, Dorian H.

AU - Johnson, Kara J.

AU - You, Huihong

AU - Luo, Jenny

AU - Riddle, Steven M.

AU - Marks, Bryan D.

AU - Vogel, Kurt W.

AU - Koop, Dennis

AU - Apgar, John

AU - Tyner, Jeffrey

AU - Deininger, Michael W.

AU - Druker, Brian

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