Thrombotic Microangiopathy As a Cause of Cardiovascular Toxicity from the BCR-ABL1 Tyrosine Kinase Inhibitor Ponatinib
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From www.bloodjournal.org by guest on April 19, 2019. For personal use only. Regular Article VASCULAR BIOLOGY Thrombotic microangiopathy as a cause of cardiovascular toxicity from the BCR-ABL1 tyrosine kinase inhibitor ponatinib Yllka Latifi,1,* Federico Moccetti,1,* Melinda Wu,1,2 Aris Xie,1 William Packwood,1 Yue Qi,1 Koya Ozawa,1 Weihui Shentu,1 Eran Brown,1 Toshiaki Shirai,3 Owen J. McCarty,3 Zaverio Ruggeri,4 Javid Moslehi,5 Junmei Chen,6 Brian J. Druker,7 Jose A. Lopez,´ 6 and Jonathan R. Lindner1,8 1Knight Cardiovascular Institute, 2Doernbecher Children’s Hospital, and 3Department of Biomedical Engineering, Oregon Health & Science University, Portland, OR; 4Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA; 5Cardiovascular Division, Vanderbilt University, Nashville, TN; 6Bloodworks NW, Seattle, WA; 7Knight Cancer Institute, Oregon Health & Science University, Portland, OR; and 8Oregon National Primate Research Center, Oregon Health & Science University, Portland, OR KEY POINTS The third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events l Ponatinib therapy can result in VWF- is unknown. We hypothesized that ponatinib produces an endothelial angiopathy in- mediated platelet volving excessive endothelial-associated von Willebrand factor (VWF) and secondary adhesion to the platelet adhesion. In wild-type mice and ApoE2/2 mice on a Western diet, ultrasound microvascular molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Iba endothelium. was performed to quantify endothelial-associated VWF and platelet adhesion. Af- l Ponatinib-related ter treatment of wild-type mice for 7 days, aortic molecular signal for endothelial- microangiopathy can associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham reduce segmental P < 2/2 left ventricular therapy ( .001), whereas dasatinib had no effect. In ApoE mice, aortic VWF and dysfunction. 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 ApoE2/2 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. (Blood. 2019;133(14):1597-1606) Introduction at one point temporarily halted, by the US Food and Drug Ad- ministration, because of high rates of vascular toxicity manifest as Tyrosine kinase inhibitors (TKIs) have revolutionized the treat- acute thrombotic occlusive events within months of initiating ment of patients with chronic myelogenous leukemia (CML). In therapy, the cause of which is unknown.6,7 CML, the t(9;22) chromosomal translocation results in production of the oncogenic tyrosine kinase BCR-ABL1. TKIs targeted to BCR-ABL1 inhibit the proliferation and survival of malignant In this study, in vivo imaging methods unique in their ability to cells and result in long-term disease-free survival rates of nearly investigate events at the endothelial-blood pool interface, in- 80%.1,2 Ponatinib is a third-generation BCR-ABL1 TKI with a cluding in vivo ultrasound molecular imaging and intravital mi- broad range of activity against other TKs (vascular endothelial croscopy, were used to assess endothelial alterations that underlie growth factor, fibroblast growth factor, and platelet-derived the vascular complications of ponatinib. These approaches have growth factor receptors) and is the only TKI that is approved recently been applied to elucidate sudden high-risk endothelial for treating CML rendered drug resistant by the T315I mutation.3-5 changes that occur globally after focal myocardial ischemia.8 However, the use of ponatinib in patients has been curtailed, and We applied these techniques to investigate whether ponatinib © 2019 by The American Society of Hematology blood® 4 APRIL 2019 | VOLUME 133, NUMBER 14 1597 From www.bloodjournal.org by guest on April 19, 2019. For personal use only. produces an arterial and microvascular endothelial angiopathy recombinant human ADAMTS13 (a disintegrin and metal- involving excessive endothelial-associated von Willebrand fac- loprotease with thrombospondin type I repeats-13) (5 mg IV) tor (VWF) and secondary glycoprotein-Iba (GPIba)–mediated 1 hour prior to imaging. ADAMTS13, which is a regulatory platelet adhesion. Because myocardial infarction is one of the protease that cleaves VWF at the A2 domain, was used to most frequent vascular complications, we also assessed whether reduce ultralarge VWF multimer size after surface expression these endothelial changes result in ischemic left ventricular (LV) by endothelial cells.15,16 wall motion abnormalities (WMAs). With regards to potential mitigation strategies, we tested whether the platelet-related Targeted microbubble contrast agent preparation angiopathy could be partially prevented by N-acetylcysteine (NAC), which can potentially reduce VWF multimer size through for CEU molecular imaging fl direct effects on self-association and secondary effects on en- Lipid-shelled deca uorobutane microbubbles bearing a bifunctional dogenous protease activity.9 conjugation moiety consisting of a hydrophobic domain and a biotin domain at the end of a polyethylene glycol spacer were prepared by sonication of a gas-saturated aqueous suspension Materials and methods of distearoylphosphatidylcholine (2 mg/mL), polyoxyethylene- Animals 40-stearate (1 mg/mL), and distearoylphosphatidylethanolamine- PEG(2000)biotin (0.4 mg/mL). Conjugation of biotinylated The study was approved by the Animal Care and Use Committee ligand to the microbubble surface was performed with biotin- of Oregon Health & Science University. We studied wild-type streptavidin bridging as previously described.11,17 Ligands used C57Bl/6 mice and hyperlipidemic mice with susceptibility to for targeting were dimeric murine recombinant A1 domain of atherosclerosis produced by gene deletion of the apolipoprotein- VWF A1 (mature VWF amino acids 445 to 716) for targeting E(ApoE2/2)feda“Western-style” diet (WSD) consisting of 42% of platelet GPIba, and a cell-derived peptide representing the calories from fat (Teklad TD.88137, Envigo) for 6 weeks prior N-terminal 300 amino acids of GPIba for targeting the VWF to the study. Mice were studied at 15 to 20 weeks of age, which A1 domain, which is exposed by shear on ultralarge multimers.18 for ApoE2/2 mice on a WSD is expected to represent a stage at Control microbubbles were prepared with isotype control anti- which there are early atherosclerotic lesions in the aortic root and body (R3-34, BD Biosciences). Microbubble concentrations and proximal aorta.10,11 For all studies except intravital microscopy, size distributions were measured by electrozone sensing (Multi- mice were anesthetized with 1.0% to 2.0% inhaled isoflurane, and sizer III; Beckman Coulter, Brea, CA). euthermia was maintained. A jugular cannula was placed for IV injection of contrast agents or drugs when necessary. For intravital Contrast ultrasound molecular imaging microscopy, mice were anesthetized with intraperitoneal injection CEU molecular imaging of the ascending thoracic aorta and (10-15 mL/g) of a solution containing ketamine hydrochloride proximal arch were imaged using a right parasternal window with (10 mg/mL), xylazine (1 mg/mL), and atropine (0.02 mg/mL). a 15L7 linear-array probe (Sequoia; Siemens, Mountain View, TKI drug therapy and study design CA). Multipulse phase-inversion and amplitude-modulation imaging was performed to specifically detect the nonlinear For TKI therapy in mice, either ponatinib (30 mg/kg) or dasatinib signals generated at the fundamental and harmonic frequencies (20 mg/kg) was dissolved in a solution of citrate titrated to achieve by microbubble cavitation.19,20 Imaging was performed at a a pH of 2.75 and administered as a daily oral gavage. Doses of transmission frequency of 7 MHz and a dynamic range of 55 dB. TKI were selected a priori based on studies demonstrating The mechanical index was set at 1.0. Images were acquired therapeutic response in murine models of malignancy.12-14 NAC 8 minutes after IV injection of targeted or control microbubbles was administered by daily gavage at the same time as ponatinib at (1 3 106) performed in random order. Signal from retained a dose (600 mg/kg) that was weight adjusted to be in slight excess microbubbles alone was determined as previously described by to that used to treat acetaminophen poisoning. Mice were treated acquiring the first ultrasound frame and then digitally subtracting for 7 days prior to being studied with contrast-enhanced ultra- several averaged frames obtained after complete destruction of sound (CEU) molecular imaging, intravital microscopy,