Thrombotic microangiopathy as a cause of cardiovascular toxicity from the BCR-ABL1 tyrosine kinase inhibitor ponatinib

Abstract

The third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events is unknown. We hypothesized that ponatinib produces an endothelial angiopathy involving excessive endothelial-associated von Willebrand factor (VWF) and secondary platelet adhesion. In wild-type mice and ApoE-/- mice on a Western diet, ultrasound molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Ibα was performed to quantify endothelial-associated VWF and platelet adhesion. After treatment of wild-type mice for 7 days, aortic molecular signal for endothelial-associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham therapy (P < .001), whereas dasatinib had no effect. In ApoE-/- mice, aortic VWF and platelet signals were two- to fourfold higher for ponatinib-treated compared with sham-treated mice (P < .05) and were significantly higher than in treated wild-type mice (P < .05). Platelet and VWF signals in ponatinib-treated mice were significantly reduced by N-acetylcysteine and completely eliminated by recombinant ADAMTS13. Ponatinib produced segmental left ventricular wall motion abnormalities in 33% of wild-type and 45% of ApoE-/- mice and corresponding patchy perfusion defects, yet coronary arteries were normal on angiography. Instead, a global microvascular angiopathy was detected by immunohistochemistry and by intravital microscopy observation of platelet aggregates and nets associated with endothelial cells and leukocytes. Our findings reveal a new form of vascular toxicity for the TKI ponatinib that involves VWF-mediated platelet adhesion and a secondary microvascular angiopathy that produces ischemic wall motion abnormalities. These processes can be mitigated by interventions known to reduce VWF multimer size.

Conflict of interest statement

Conflict-of-interest disclosure: B.J.D. is a member of the scientific advisory board (SAB) for Aileron Therapeutics, ALLCRON, Cepheid, Gilead Sciences, Vivid Biosciences, Celgene, and Baxalta (inactive); is an SAB member and owns stock in Aptose Biosciences, Blueprint Medicines, β Cat, GRAIL, Third Coast Therapeutics, and CTI BioPharma (inactive); is a scientific founder of and owns stock in MolecularMD; is a member of the board of directors and owns stock in Amgen; is a member of the board of directors for Burroughs Wellcome Fund, CureOne; is a member of the Joint Steering Committee at Beat AML LLS; and receives clinical trial funding from Novartis, Bristol-Myers Squibb, and Pfizer, and royalties from patent 6958335 (Novartis exclusive license) and Oregon Health & Science University and Dana-Farber Cancer Institute (one Merck exclusive license). The remaining authors declare no competing financial interests.

© 2019 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Survival and blood pressure according to treatment assignments. Kaplan-Meier curves illustrate survival after initiation of ponatinib (30 mg/kg per day) or vehicle (sham treatment) in wild-type C57Bl/6 mice (A) and ApoE−/− mice on a WSD (B). Tail-cuff systolic blood pressure was measured in awake wild-type (C) and ApoE−/− mice on a WSD (D) in animals that were acclimatized to the procedure prior to initiation of therapy. *P < .05 vs vehicle. BP, blood pressure.

Figure 2.

Molecular imaging of aortic endothelial phenotype in wild-type and ApoE−− mice. (A) Mean (± standard error of the mean [SEM]) signal enhancement measured from the proximal thoracic aorta on CEU molecular imaging using tracers targeted to platelet GPIbα, VWF A1-binding domain, or control agent, in wild-type mice treated for 1 week with ponatinib (30 mg/kg per day), dasatinib (20 mg/kg per day), or vehicle. *P < .01. (B) Illustrative images from a ponatinib-treated wild-type mouse showing 2-dimensional (2-D) ultrasound (14 MHz) of the proximal thoracic aorta and origin of the brachiocephalic artery (outlined), and background-subtracted color-coded (scales at right) CEU molecular imaging with control or targeted contrast agents. (C) Mean (± SEM) signal enhancement on CEU molecular imaging of the proximal thoracic aorta in ApoE−/− mice on a WSD treated for 1 week with ponatinib (30 mg/kg per day) or vehicle. *P < .05. (D) CEU molecular imaging in ponatinib-treated ApoE−/− mice on a WSD showing effects of either daily coadministration of NAC (600 mg/kg per day) or IV rADAMTS13 (5 μg) given 1 hour prior to imaging. *P < .05; **P < .01. IU, international unit.

Figure 3.

Echocardiographic detection of LV dysfunction. (A) Echocardiography in the parasternal long-axis plane at end-diastole and end-systole illustrating an inferoapical WMA (arrow, see online videos for examples of WMAs). (B) Proportion of animals with segmental LV wall motion abnormalities after 1 week of ponatinib therapy. (C-D) Echocardiographic measurement of stroke volume (bars represent mean ± standard deviation) at baseline and after treatment (vehicle or ponatinib) in wild-type (C) and ApoE−/− mice on a WSD (D). ns, not significant.

Figure 4.

Coronary artery and LV coronary microvascular anatomy. (A) Ex vivo epifluorescent illumination of microspheres in sequential ventricular short-axis sections illustrating normal perfusion, and focal regions of myocardial perfusion defect (MPD) (arrows). (B) CT coronary angiography in the left lateral projection illustrating lack of arterial occlusion in 2 ponatinib-treated mice with WMAs. (C) Fluorescent confocal microscopy of the LV myocardium from ponatinib-treated wild-type mice showing regions with and without WMAs and from a sham-treated mouse. Staining was performed with isolectin (green) for microvessels, Hoechst stain for nuclei (blue), and platelet CD41 immunohistochemistry (red). The bottom rows illustrate the red channel alone to better display platelets. Scale bars, 100 μm. (D) Higher-magnification image from the ponatinib-treated animal with WMA.

Figure 5.

Microvascular platelet and leukocyte recruitment on intravital microscopy. Box-whisker plots for intravital microscopy data from the cremasteric microcirculation of vehicle (sham) or ponatinib-treated wild-type and ApoE−/− mice illustrating the number of platelets adhering to the microvascular endothelium (A), the number of platelet strings or nets (B), the area of adherent platelet strings or nets (C), and the number of leukocytes adhered in postcapillary venules (D). *P < .05; **P < .01. (E-F) Histograms illustrating the distribution of leukocyte rolling velocities in cremasteric venules for ApoE−/− mice on a WSD for animals treated with vehicle (E) or ponatinib (F). Rolling velocity was slower for ponatinib-treated mice (P < .01 by Mann-Whitney rank-sum test). ns, not significant.

Figure 6.

Dose-dependent effects of ponatinib on endothelial cell ROS production and viability. (A) ROS generation by SVEC4-10 endothelial cells in culture measured by mean (± SEM) H2DCFDA fluorescence after 24 hours of ponatinib treatment. Positive control data are shown for phorbol 12-myristate 13-acetate (PMA)–treated cells. (B) Examples of SVEC4-10 cells exposed for 24 hours to ponatinib (0.1 to 2.5 μM) by fluorescent imaging of H2DCFDA and DAPI (nuclear staining) illustrating dose-dependent increase in ROS, and low-magnification bright-field (LM-BF) microscopy illustrating loss of cell confluence at high concentrations. (C) Mean (± SEM) cell viability after 24 hours of exposure to ponatinib (0.1 to 2.5 μM) determined by the proportion of cells staining positive for PI. *P < .05 vs all other conditions by post hoc testing after correction for multiple comparisons. (D) Cell viability at various concentrations of ponatinib (Pon) with and without NAC. *P < .05. (E-F) Mean (± SEM) area for externalized VWF on HUVECs in a microfluidic chamber after 24 hours of exposure to ponatinib (0.5 μM), TNF-α (10 ng/mL), or control serum, and examples of fluorescent microscopy for each condition. *P < .01. AU, arbitrary unit.