Threshold Levels of ABL Tyrosine Kinase Inhibitors Retained in Chronic Myeloid Leukemia Cells Determine Their Commitment to Apoptosis

Abstract

The imatinib paradigm in chronic myeloid leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKIs). 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 BCR-ABL 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 TKI-induced 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 FACS 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 demonstrated 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.

Sustained inhibition of STAT5, but not JAK2, is essential for TKI-induced cell death in chronic myeloid leukemia

“…This has subsequently been confirmed by other groups postulating that reduction of pSTAT5 is a sensitive marker for the presence of residual/low concentrations of TKI, rather than reduced CrkL phosphorylation. 26,27 In this study, we have used the optimal washout (OPT wash) method as a model of the effect of TKI availability on Bcr-Abl activation and evaluated the critical factors involved in commitment to TKI-induced cell death. Accordingly, the duration of potent TKI exposure required to commit BCR-ABL1 þ cells to death was investigated in the setting of transient Bcr-Abl kinase inhibition.…”

Prolonged cellular midostaurin retention suggests potential alternative dosing strategies for FLT3-ITD-positive leukemias

“…Recently, several groups have demonstrated that BCR–ABL transformed cells can undergo apoptosis through prolonged intracellular drug accumulation and retention upon high-dose TKI pulse exposure. 1 , 2 , 3 , 4 , 5 This suggests that a given TKI might still be present at active concentrations in the target cells, although the drug has already been cleared from the patient's plasma. Our group has pioneered the measurement of intracellular TKI accumulation using both direct drug detection and functional read outs.…”

KIT Signaling Governs Differential Sensitivity of Mature and Primitive CML Progenitors to Tyrosine Kinase Inhibitors

“…Surprisingly, shKIT significantly enhanced the effects of PPY-A on CML CD34 + colony formation in the absence of SCF (Fig 3B, upper panel). This could reflect persistence of a low level of BCR-ABL1 kinase activity not detected by pCRKL immunoblots (29) or constitutive KIT activation that is independent of BCR-ABL1 kinase activity. Additionally, the band corresponding to pKIT Y721 was not completely abolished by BAW667 or imatinib (Fig.…”